Book cover of Introduction to Food Chemistry

Vassilis Kontogiorgos

Introduction to Food Chemistry

2nd ed. 2024

The Springer logo.

Vassilis Kontogiorgos

Associate Professor in Food Chemistry, University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC, Canada

ISBN 978-3-031-53557-4e-ISBN 978-3-031-53558-1

https://doi.org/10.1007/978-3-031-53558-1

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021, 2024

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About This Book

Vassilis Kontogiorgos

The positive response and feedback from food science students have been instrumental in shaping the evolution of this textbook. A commitment to excellence in education and a passion for food chemistry has driven the journey from the first edition to this second iteration. Building on the first edition’s success, I have refined all chapters to facilitate a more engaging and effective learning experience. In the second edition, certain parts were restructured, the descriptions and language were improved, and entirely new sections were created to provide a more thorough understanding of the subject matter.

Key features of the second edition include:

A new section on the glass transition in foods

A new section on the chemistry of proteins used in foods

A new section on sweeteners and food additives

Revamped colour and flavour chemistry chapters

Revamped learning activities

All figures and schemes have been redrawn and enhanced

25 new figures and 12 new tables

As previously, some prerequisite knowledge is necessary to make the best use of the book. The reader must have knowledge of general chemistry and general organic chemistry to be able to follow the concepts presented in this textbook. The book is organised into eight chapters covering structures, chemistry, and functionality of all components found in food. Concepts from the physical chemistry of foods and food analysis are also introduced and explored in each chapter. At the end of each chapter, there are learning activities to help revise important concepts.

I want to sincerely thank the students who used the first edition. Your valuable feedback has been integral to the improvements made in this edition, and I hope the updates and enhancements will further contribute to your educational journey. I look forward to hearing what you think about the second edition and how I can further improve it.

Once more, let’s start with water and explore why it is so special for food stability.

Contents

1 Water1

1.​1 Introduction1

1.​2 Water and Ice Structure1

1.​3 Hydrogen Bonding of Water2

1.​4 Interactions of Water with Food Constituents2

1.​5 Colligative Properties4

1.​6 Water Activity6

1.​7 The Glass Transition10

1.​7.​1 Glass Formation in Food Processing13

1.​7.​2 Factors Influencing Glass Transition14

1.​7.​3 Storage of Glassy Foods14

1.​8 Review Questions15

1.​8.​1 Multiple Choice Questions15

1.​8.​2 Short Answer Questions16

2 Carbohydrates19

2.​1 Introduction19

2.​2 Monosaccharide Structure19

2.​3 Monosaccharide Reactions24

2.​4 Disaccharides-Oligosaccharides​30

2.​5 Polysaccharides31

2.​5.​1 Starch32

2.​5.​2 Carrageenan36

2.​5.​3 Alginates37

2.​5.​4 Pectin38

2.​5.​5 Cellulose39

2.​5.​6 Galactomannans39

2.​5.​7 Gum Arabic40

2.​5.​8 Xanthan40

2.​5.​9 Chitin40

2.​5.​10 Dietary Fibre41

2.​6 Review Questions42

2.​6.​1 Multiple Choice Questions42

2.​6.​2 Short Answer Questions44

3 Proteins-Enzymes47

3.​1 Introduction47

3.​2 Amino Acids48

3.​3 Proteins52

3.​3.​1 Protein Classification52

3.​3.​2 Protein Structure54

3.​3.​3 Changes in Protein Structure:​ Solubility, Denaturation and Hydrolysis57

3.​3.​4 Functional Properties of Proteins60

3.​3.​5 Proteins of Major Food Commodities62

3.​4 Enzymes68

3.​4.​1 Mechanism of Enzymatic Reactions69

3.​4.​2 Enzyme Kinetics71

3.​4.​3 Enzyme Nomenclature and Classification74

3.​4.​4 Food Enzymes75

3.​5 Review Questions80

3.​5.​1 Multiple Choice Questions80

3.​5.​2 Short Answer Questions81

4 Lipids85

4.​1 Introduction85

4.​2 Fatty Acid Nomenclature and General Characteristics85

4.​3 Triacylglycerols​ (TAGs)88

4.​4 Lipid Oxidation91

4.​4.​1 Autoxidation (Triplet Oxygen Oxidation)91

4.​4.​2 Photo-Oxidation (Singlet Oxygen Oxidation)93

4.​4.​3 Enzymatic Oxidation94

4.​5 Fat Crystallisation95

4.​5.​1 Crystal Formation95

4.​5.​2 TAG Conformation and Polymorphism96

4.​5.​3 Other Properties of Fats99

4.​6 Review Questions101

4.​6.​1 MultipleChoice Questions101

4.​6.​2 Short Answer Questions102

5 Browning Reactions103

5.​1 Introduction103

5.​2 Enzymatic Browning103

5.​2.​1 Reactions and Substrates104

5.​2.​2 Deactivation Strategies106

5.​3 Non-enzymatic Browning106

5.​3.​1 Caramelisation106

5.​3.​2 Ascorbic Acid Browning109

5.​3.​3 Maillard Reaction109

5.​4 Review Questions113

5.​4.​1 Multiple Choice Questions113

5.​4.​2 Short Answer Questions114

6 Vitamins-Minerals-Additives117

6.​1 Introduction117

6.​2 Fat-Soluble Vitamins118

6.​3 Water-Soluble Vitamins119

6.​4 Minerals123

6.​4.​1 Effect of Processing on Mineral Bioavailability124

6.​5 Additives125

6.​5.​1 Safety of Food Additives126

6.​6 Review Questions128

6.​6.​1 Multiple Choice Questions128

6.​6.​2 Short Answer Questions128

7 Colour Chemistry131

7.​1 Introduction131

7.​2 Interactions of Light with Food132

7.​3 Pigment Chemistry135

7.​3.​1 Myoglobin137

7.​3.​2 Chlorophyll138

7.​3.​3 Carotenoids139

7.​3.​4 Anthocyanins140

7.​3.​5 Betalains143

7.​3.​6 Other Natural Pigments143

7.​3.​7 Artificial Colours143

7.​4 Review Questions144

7.​4.​1 Multiple Choice Questions144

7.​4.​2 Short Answer Questions145

8 Flavour Chemistry147

8.​1 Introduction147

8.​2 Sweet Molecules147

8.​2.​1 Carbohydrate-Based Sweet Molecules149

8.​2.​2 Sweet Peptides and Proteins150

8.​2.​3 Sulfonamides150

8.​2.​4 Glycosides151

8.​2.​5 Saponins151

8.​2.​6 Sweetener Synergy and Suppression151

8.​3 Bitter Molecules152

8.​4 Salty, Sour and Umami Molecules152

8.​5 Trigeminal Perception Molecules154

8.​6 Flavour Characteristics and Sources155

8.​7 Flavour Delivery158

8.​8 Flavour Interactions and Stability160

8.​9 Review Questions161

8.​9.​1 Multiple Choice Questions161

8.​9.​2 Short Answer Questions162

Appendices163

Bibliography165

Index169

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024

V. KontogiorgosIntroduction to Food Chemistryhttps://doi.org/10.1007/978-3-031-53558-1_1

1. Water

Vassilis Kontogiorgos1  

(1)

Associate Professor in Food Chemistry, University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC, Canada

Vassilis Kontogiorgos

Email: v.kontogiorgos@ubc.ca

Learning Objectives

After studying this chapter, you will be able to:

Describe the structure of the water

Describe the hydrogen bond and its importance for foods

Describe the interactions of water with food constituents

Describe colligative properties and their importance in food processing

Discuss water activity and its significance in foods

Describe glass transition and its importance in foods

1.1 Introduction

Water is an essential constituent of foods, and its content varies significantly across different products. For example, foods like fruits or meat products have a water content of ~95% or ~70%. In contrast, there are foods with limited water, ranging between 10% and 20% (Table 1.1). As several chemical and enzymatic reactions need water to occur, it serves as a solvent of water-soluble ingredients and a medium for diffusion. Water is also essential for the growth of beneficial (e.g., lactic acid bacteria) and spoilage (e.g., Pseudomonas) microorganisms. During food processing, it is crucial to calculate the correct amount of water in the formulation to achieve the specified product’s consistency and optimum shelf-life stability. For instance, minor deviations in water content in bakery foods may result in quality losses and rejection of the final product. It is also of economic concern, as it adds weight to the product and increases shipping costs. It should be stressed that regardless of the processing, food will always contain a certain amount of water that is impossible to remove entirely. For example, wafers or crisps that are particularly dry foods have a remaining water content of ~7%. As a result, controlling and predicting water behaviour is very important in food processing. The first step to controlling water in foods is understanding its structure, chemical, and physical properties. Especially important are the mechanisms with which it interacts with the various food constituents, as it is most frequently the underlying reason for product stability. This chapter presents all the essential information needed to explore the broad area of water functionality in foods.

Table 1.1

Examples of the broad range of moisture content in foods

Food

Water content (%)

Fruits and vegetables

85–95

Meat

60–75

Sausages

30

Honey

20

Dried fruits

10–20

Noodles/Pasta

12

Food powders

5–6

1.2 Water and Ice Structure

When two hydrogen atoms form covalent bonds with an oxygen atom, they form water. Each hydrogen atom has a nucleus consisting of a proton surrounded by a negatively charged electron. The oxygen atom has eight electrons, and because of its higher electronegativity, oxygen attracts the shared electrons of the covalent bonds to a greater extent than hydrogen atoms. As a result, the oxygen atom acquires a partial negative charge (δ⁻), while the hydrogen atoms acquire a partial positive charge (δ⁺) (Fig. 1.1).

A ball and stick model of H 2 O molecule. The angle between hydrogen atoms is 104.5 degrees. The oxygen atom has 2 lone pairs. Each H atom carries a delta plus charge and each lone pair of the oxygen atom carries a delta minus charge.

Fig. 1.1

Structure and partial charges of water. The angle between hydrogen atoms is about 104.5°. The letter δ indicates the partial charge

Polarity is the separation of electric charges, making water a polar molecule. As a result of polarity, water molecules in the liquid phase interact with each other with short-lived (also known as transient) hydrogen bonds that result in unusual physical properties.

When the temperature decreases below 0 °C, water molecules arrange themselves in space in a hexagonal pattern, creating the so-called hexagonal ice that is the form of ice present in foods (Fig. 1.2). Hexagonal ice has an open structure and lower density than liquid water, and, as a result, ice floats. This expansion of water volume on freezing is a major cause of structural deterioration in frozen foods. As the ice expands, it essentially breaks down the structure, and, in most cases, it cannot be recovered on thawing.

Two ball and stick models of water molecules. A. Hexagonal pattern. B, Water molecules arranged in hexagons that are fused together in multiple layers.

Fig. 1.2

Hexagonal ice: (a) Water molecules arrange in a hexagonal pattern at sub-zero temperatures, and (b) structure of an ice cube. Dashed lines represent hydrogen bonds between water molecules

1.3 Hydrogen Bonding of Water

A hydrogen bond is a particular type of van der Waals attraction that results from attractive forces between a hydrogen atom covalently bonded to an electronegative atom of the first molecule (e.g., O, N or F) and an electronegative atom of a second molecule (Fig. 1.3). Hydrogen bonds are intermolecular, occurring between different molecules, or intramolecular when they occur within the same molecule. For example, both types are found in proteins or polysaccharides, influencing the structure and properties of these food components. Hydrogen bond (~5–20 kJ/mol) is weaker than covalent bond (~460 kJ/mol) but generally stronger than van der Waals attractions (~1.2 kJ/mol). It should be pointed out that the strength of hydrogen bonding increases with the lowering of temperature.

A ball and stick model. Numerous numbers of water molecules are scattered, and they are bonded by hydrogen bonds. The hydrogen bond is formed between a hydrogen atom of a water molecule and an oxygen atom of the adjacent water molecule.

Fig. 1.3

The dashed lines indicate hydrogen bonds between water molecules

As a result of hydrogen bonding, water presents abnormal properties compared to molecules with similar molecular weight or structure, thus influencing food processing (Table 1.2). For example, with its unusually high boiling point (100 °C), it requires large amounts of energy to remove during evaporation or dehydration processes. Additionally, volume increase in crystallisation damages food structure, which is very important in frozen products. Because of its abnormal physical properties, water creates technological difficulties in nearly all processes encountered in the food industry.

Table 1.2

Examples of the abnormal properties of water because of hydrogen bonding and relevance to food processing operations

Unusual property

Process

High melting point

Freezing

High boiling point

Evaporation-dehydration

High critical point

Supercritical fluid extractions

Liquid water exists at very low temperatures

Freezing

Volume increases on crystallisation

Freezing

Large volume changes from liquid to gas

Evaporation-use of steam

High specific heat capacity (twice that of ice)

Heating-cooling

High heat of vaporisation

Evaporation

High heat of sublimation

Freeze drying

1.4 Interactions of Water with Food Constituents

Water interacts with all substances found within food in different ways. The primary forms of interactions are dipole-ion, dipole-dipole, dipole-induced dipole, and hydrophobic interactions. The strength and extent of these interactions depend on the chemical nature of non-aqueous components, for example, salt concentration, pH, or temperature. A typical example of dipole-ion interactions is between water and NaCl. Water arranges itself around ions to partially neutralise ionic charges, separating positive and negative ions in aqueous solutions (Fig. 1.4).

Two diagrams. A sodium ion is surrounded by 5 water molecules. The oxygen atom has delta negative charge and each hydrogen atom has delta plus charge. A chlorine molecule is surrounded by some water molecules.

Fig. 1.4

Dipole-ion interactions between water and NaCl

The strength of water-ion interactions is usually greater than hydrogen bonding and varies widely between 40–600 kJ/mol, depending on the specific ions involved. As mentioned, water is a highly structured liquid because of the extensive network of hydrogen bonds. However, small ions with strong electric fields are structure-forming. They cause water molecules to rearrange themselves in the hydration shell around ions and exhibit a larger average density than pure water (e.g., Li³⁺, Na⁺ or Ca²⁺). In contrast, large ions with weak electric fields are structure-breaking with the opposite effect (e.g., K⁺, Cl⁻ or I⁻). Although this may seem to be a mere scientific detail, it has enormous consequences for food fabrication and stability, as the ability of ions to influence protein denaturation is linked to how they interact with water (see Sect. 3.​3.​3, Hofmeister series). Dipole-dipole interactions refer to interactions between water and non-ionic hydrophilic solutes in food. For instance, molecules that carry hydroxyl (monosaccharides), amino (proteins), or carbonyl (flavours) groups may form hydrogen bonds with water (Fig. 1.5). The strength of these hydrogen bonds is similar to that of water molecules.

Three representations. A, Dipole dipole interaction between the oxygen atom of a carboxyl group and H of an amino group. B, Dipole dipole interaction between oxygen atom of a carboxyl group and H of H 2 O. C, Dipole dipole interaction between oxygen atom of H 2 O and H of an amino group.

Fig. 1.5

Dipole-dipole interactions between (a) amino and carboxyl groups, (b) water and carboxyl group, and (c) water and amino group

A dipole-induced dipole interaction is a weak attraction that results when water induces a temporary dipole in a non-polar (hydrophobic) molecule by disturbing the arrangement of its electrons. This attraction is usually weak but may lead to compounds being loosely adsorbed at interfaces that may influence emulsion or foam stability. Finally, hydrophobic interactions are the attractive interactions between hydrophobic molecules in aqueous environments. Molecules with hydrophobic groups tend to exclude water from their surroundings and interact with each other. For example, a common observation is when an oil droplet falls in water and forms a nearly perfect sphere (Fig. 1.6).

A photograph of the formation of oil droplets on water. A zoomed-in view of the droplets presents the molecules of triglycerides surrounded by water molecules.

Fig. 1.6

Triglycerides in the oil interact closely with each other through hydrophobic attractive forces to exclude water molecules from their vicinity. The shape that minimises interactions with water is that of a sphere

The reason is that the oil triglycerides (see Chap. 4) interact closely with each other through hydrophobic attractive forces to exclude water molecules from their vicinity. The most favourable shape is that of a sphere because the molecules have the least interactions with water and the most with each other (Fig. 1.6). Hydrophobic interactions are critical for the physical properties of proteins, lipids, and surfactants commonly used in food formulations. Hydrophobic interactions are also responsible for gelation, such as heat-induced gelation of methylcellulose or proteins (e.g., egg boiling). Hydrophobic interactions are relatively weaker (~4–10 kJ/mol) than hydrogen bonds, and their strength increases with temperature. Table 1.3 summarises the main types and properties of water-solute interactions that influence food processing and stability.

Table 1.3

Types of water-solute interactions

Species involved

Type of force

Examples

Strength (kJ/mol)

Temperature dependency

Ion – Polar molecule

Ion-Dipole

Water-NaCl

40–600

No dependency

Two polar molecules

Dipole-Dipole

Water-−OH

5–30

Stronger as T decreases

Polar and nonpolar molecule

Dipole-Induced dipole

Water-Hydrocarbons

Very low

No dependency

Two nonpolar molecules

Hydrophobic

Hydrocarbons- Hydrocarbons

4–10

Stronger as T increases

1.5 Colligative Properties

Colligative properties are the properties of solutions that depend on the number of molecules in a given volume of water and not on their chemical nature. Colligative properties are the lowering of vapour pressure, elevation of boiling point, depression of freezing point, and osmotic pressure. Colligative properties may influence various food manufacturing processes (e.g., freezing or evaporation) or can be used for analytical purposes (e.g., examining for milk adulteration using cryoscopy). Sugars (e.g., glucose or sucrose) and salts (e.g., NaCl or CaCl₂) are frequently found in food formulations and can influence processing conditions. For example, during the concentration of fruit juice, sugar concentration continuously increases with water evaporation. As a result, the boiling point of the remaining concentrated juice increases, requiring further heating to maintain evaporation. Another example is the freezing of ice-cream mix, where sugars in formulation increase in concentration due to freeze-concentration in the unfrozen mix and the melting point is depressed. As a result, the temperature needs to be lowered even more to maintain ice cream structure formation. These two examples may be extrapolated to foods that undergo high- or low-temperature processing. Ultimately, the presence of solutes determines the processing cost and the market price of food, as it controls energy expenditure. It becomes evident that colligative properties and how they influence food processing must be well understood.

The underlying fundamental cause of the above events is the lowering of the vapour pressure of the solution due to solute-water interactions. Solutes immobilise water and cannot escape from the surface as vapour, and, as a result, the higher the solute concentration, the lower the vapour pressure of a solution. The vapour pressure of a solution is proportional to the number of particles in the solution, as described in Raoult’s law. The vapour pressure of a solution is always lower than that of water. Freezing point depression (ΔΤf = Κf m) and boiling point elevation (ΔΤb = Κb m) are proportional to the molality (m) of the solution (i.e., amount of substance (moles) in a specified amount of mass of water (kg), m = molessolute/masssolvent) with Kf or Kb being the cryoscopic or ebullioscopic constants, respectively. Finally, osmosis is the diffusion of water through a semi-permeable membrane from a solution of low solute concentration (hypotonic) to a solution of high solute concentration (hypertonic) (Fig. 1.7a). During this physical process, water moves without energy input across a semi-permeable membrane (permeable to the water but not to the solute), separating the two solutions of different concentrations (forward osmosis). This process is essential in cells in both animal and plant tissues and controls the movement of water between the cytoplasm and the surrounding layers of the cells. Osmotic pressure is the pressure we must apply to the hypertonic solution to prevent osmosis (Fig. 1.7b). The solutions are called isotonic when the concentrations on either side are equal. Now, suppose the applied pressure exceeds the pressure required to prevent osmosis. In that case, the water flow is reversed, i.e., from the high-concentration solution to the low-concentration solution (reverse osmosis) (Fig 1.7c). Forward osmosis is used in the osmotic dehydration of some fruits. In contrast, reverse osmosis is a membrane separation technology used in water desalination (i.e., removal of NaCl from seawater). In addition, in foods that need syrup or brine (e.g., canned fruits or tuna), the solid concentration needs adjustment to the correct levels to prevent osmotic dehydration of the product that may adversely affect quality.

4 schematic diagrams. A, A cubic chamber. Hypotonic solution is separated from hypertonic solution by a semi permeable membrane. Glucose and water are kept. Due to osmosis, isotonic solutions are formed. B, Osmotic pressure is applied on molecules by a piston. C, Flow is reversed from concentrated solution.

Fig. 1.7

(a) Osmosis, (b) osmotic pressure, and (c) reverse osmosis. (See text for description)

1.6 Water Activity

Water content or moisture content is the quantity of water in food (g of H₂O/100 g of food). One may be eager to draw conclusions about the long-term shelf-life stability based on water content. However, various types of food with the same water content differ significantly in perishability. For example, marmalade and bread with a moisture content of ~33% w/w have quite different shelf lives, with bread losing its physical stability within hours and microbiological stability within a week. In contrast, marmalade may be stable for years after processing. As a result, water content is not a reliable predictor of microbial growth, chemical reactions, or structure stability of foods. The term water activity (aw, dimensionless number, ranges from 0 to 1) accounts for the intensity with which water associates with various non-aqueous constituents and is the ratio of the vapour pressure of water above food (p) over the vapour pressure of pure water (po), at the same temperature.

In Fig. 1.8, water is in a sealed container that does not allow vapours to escape. Pure water at a specific temperature T reaches a dynamic equilibrium where the amount of water molecules that evaporate (liquid to gas) is the same as the number of water molecules that condense (gas to liquid) (Fig. 1.8a). Once equilibrium is established, vapours will exert on the container pressure po and aw = 1. The inclusion of solutes, e.g., NaCl (green spheres, Fig. 1.8b), restricts the evaporation of water, and fewer molecules transfer to the gaseous state, creating pressure p. As a result, pressure p is always smaller than po and aw < 1. The same principle exists in foods, but the number of water molecules that escape to the gaseous state depends on the specific interactions of water with the food constituents (Fig. 1.8c). These interactions strongly depend on the product’s chemical composition; the stronger the interactions, the lower the aw.

Three schematic diagrams. A, A rectangle pot. Pure water. There is an equilibrium of gas and liquid at P 0, T. B, A rectangle pot. Aqueous solution. There is an equilibrium of gas and liquid at P, T. C, A rectangle pot. Foods. There is an equilibrium of gas and foods at P, T.

Fig. 1.8

Illustration of dynamic equilibrium: (a) water, (b) aqueous solution, and (c) food. (See text for description)

At this juncture, it is necessary to emphasise the difference between water content and aw. While water content may differ substantially between foods, the water activity may remain the same. Table 1.4 shows that while water content ranges between 5–20%, water activity remains at ~0.7. The foods in this table have diverse chemical compositions, resulting in different capacities to interact with water molecules. For instance, sucrose interacts strongly with water and has a high capacity to decrease aw, whereas lipids do not interact strongly and have a low capacity to decrease aw. Frequently, small changes in food formulation that are not perceivable by consumers allow a considerable increase in shelf life because of the changes in the strength of water interactions at the molecular level.

Table 1.4

Water activity depends on the strength of interactions between food components. Essentially, the chemical composition controls aw

Food

Water content (%)

aw

Main chemical constituents

Nuts

5

0.7

Lipids, protein

Milk powder

7

0.7

Protein, lipids, lactose

Cocoa powder

10

0.7

Fibres, protein

Dried vegetables

15

0.7

Fibres

Dried fruits

20

0.7

Fibres, sugars

Using aw as a predictor of microbiological, chemical, and physical stability, we can distinguish three classes of foods: dry foods with aw < ~0.5, intermediate moisture foods (IMF) with aw between ~0.5 and ~0.8 and high moisture foods with aw > 0.8. These three classes have different preservation requirements, and although the specific preservation details of each product vary depending on the composition, the following generalisations usually apply: dry foods are stable without refrigeration but need rehydration before consumption (e.g., pasta), IMF can be stored without refrigeration for prolonged periods but do not need rehydration before consumption (e.g., salami) whereas high moisture foods need refrigeration and no rehydration is necessary (e.g., yoghurt). In addition, the packaging requirements of these foods are different. Dry foods require packaging entirely impermeable to atmospheric moisture to maintain quality, as shelf life shortening is usually associated with a failure in the packaging rather than the food itself. The other two classes of products usually require less strict packaging requirements concerning the moisture barrier properties of the packaging materials.

Moisture and Water Activity Determination

The total moisture content of high-moisture foods is determined by drying (e.g., oven), where water is removed by heat and moisture is calculated from the weight loss. Karl Fischer titration is a method for water determination in low-moisture foods such as dried fruits and vegetables, confectionery products, chocolate, coffee, and other low-moisture foods high in fats, sugar or protein. The method is based on the reaction between sulphur dioxide and iodine in the presence of water, imidazole (catalyst) and alcohol (solvent). Alcohol reacts with sulfur dioxide (SO₂) and base to form an intermediate alkyl sulfite salt, which is then oxidised by iodine to an alkyl sulfate salt. This oxidation reaction consumes water and iodine in a 1:1 ratio. Once all of the water is consumed, the presence of excess iodine is detected titrimetrically. The amount of water is calculated based on the concentration of iodine consumed in the titration.

Water activity is measured with instruments designed for this purpose only. The principle of the method is based on measuring the equilibrium relative humidity surrounding the sample’s atmosphere at a constant temperature.

A plot of water content vs water activity is known as moisture sorption isotherm (MSI) and shows how water activity changes with addition (absorption) or removal (desorption) of water from food at a specific temperature. Food engineers use this curve to design dehydration processes, but we will explore it from a different angle. The shape of the moisture sorption isotherm is unique for each product. Foods where water uptake occurs readily are called hygroscopic. Small changes in moisture in these foods result in considerable changes in water activity. Conversely, compounds that absorb large amounts of water with a minimal increase in water activity are called anticaking agents. Most foods lie between these two extremes, and their moisture sorption isotherm has a sigmoid shape. The shape of the moisture sorption isotherm is unique for each product. In this plot, three zones are distinguished, corresponding to water with different physical properties (Fig. 1.9a).

Four multiline graphs of moisture content versus a W. A, It presents an ascending S-shaped curve between low and high moisture. B, It presents two ascending S-shaped curves for cold and warm. C, It presents two curves that indicate an increase in sorption and a decrease in desorption. D, An ascending S-shaped curve.

Fig. 1.9

Moisture sorption isotherms of foods, (a) the three zones of water with different mobility, (b) influence of temperature on MSI. Storage at high temperatures increases aw, (c) influence of addition or removal of water on MSI, and (d) texture changes above critical aw. Poor control of critical aw leads to texture breakdown

In Zone 1, or low moisture regime, water has the least molecular mobility (i.e., the ability of water molecules to move and diffuse) and corresponds to a product with the highest shelf-life stability. In this region, water molecules are in direct contact with food components and interact strongly with each other. As a result, they form a monomolecular layer of water with entirely different properties than regular water (Fig. 1.10). This water cannot be removed from food by dehydration, and it does not freeze under normal industrial freezing processes (~ −40 °C). Additionally, it cannot act as a solvent, support chemical reactions, or sustain microbial growth. Even the driest of foods, for example, pasta, crisps, or food powders, always have a residual water content in the range of ~5–10% w/w. Frozen foods stored at −25 °C or − 18 °C have substantial amounts of water in the liquid state. In Zone 2, additional layers of water accumulate on top of the first layer, forming multiple layers of water with intermediate molecular mobility (Fig. 1.10). This water is easier to remove or freeze than Zone 1 water. IMFs such as dried fruits, hard cheeses or cured meat products are typical foods containing multilayer water. Finally, water in Zone 3 has the highest molecular mobility, and its physical properties do not differ substantially from the properties of pure water (Fig. 1.10). As a result, it can act as a solvent and influence the rate of reactions and microbial growth, affect food structure, and cause changes in the overall stability and quality of foods.

A schematic diagram. A line of food ingredient surface that first increases, then decreases to its lowest position, and then again increases. The water molecules form a monolayer, multiple layers, and a secondary layer on the food ingredient surface. Monolayers form due to low water mobility.

Fig. 1.10

Water layering corresponding to different zones of an MSI. Zone I – monolayer, Zone II – secondary layer, and Zone 3 – multiple layers

It should be emphasised that aw depends on temperature. An increase in temperature increases aw for the same moisture content due to higher water mobility, resulting in loss of stability (Fig. 1.9b). Consequently, the temperature should always be maintained during storage to avoid chemical, physical and microbiological changes in the product. Moisture sorption isotherms prepared by adding water (rehydration) to a dehydrated sample differ from those prepared by water removal (dehydration). This phenomenon, called hysteresis, occurs due to permanent structural changes in food during drying and may affect the texture and other sensory properties of products on rehydration and consumption (Fig. 1.9c). When aw exceeds a critical value, food usually loses its structural stability. For example, crispiness in dry bakery products or the ability of powders to flow is compromised if aw exceeds a specific value, usually ~0.30 (Fig. 1.9d, Table 1.5).

Table 1.5

Approximate critical aw values for the onset of chemical, physical or microbiological changes

Critical aw

Event

~0.2–0.3

Lipid oxidation increases

~0.2–0.3

Reactions requiring water do not occur

~0.35–0.4

Powder agglomeration

~0.4–0.5

Loss of crispiness when moisture is absorbed

~0.5

Onset of hardening when moisture is lost

~0.6

Onset of microbial growth

~0.85

Onset of pathogen growth

Moisture migration in multicomponent foods influences physical stability, as migration from a high (component A in Fig. 1.11) to a low (component B in Fig. 1.11) moisture region occurs continuously until a thermodynamic equilibrium with the surrounding environment is established. Techniques to slow this process or prevent it altogether from occurring include the addition of edible layers between food components to separate the regions with different moisture contents physically (e.g., a chocolate layer on the inside of an ice cream cone to reduce the moisture migration and maintain crispness during storage) or matching the aw of food components (e.g., freeze-dried fruits in breakfast cereals).

A multiline graph of moisture content versus a W. It presents two ascending S-shaped curves for M S I of component A and M S I of component B. For component A, initial a w is at point a w, A on the horizontal line. For component B, initial a w of B is at point a w, B on the horizontal line.

Fig. 1.11

Moisture sorption isotherms of multicomponent foods. (See text for description)

Figure 1.12 shows a convenient plot with the reaction rates of important chemical, physical and microbiological changes as a function of aw. From this plot, some generalisations may be drawn: (a) the rates of microbial growth, enzymic activity, and hydrolytic reactions decrease at low aw, (b) Maillard reactions show a peak at aw ~ 0.7, (c) lipid oxidation increases at aw < 0.3 (i.e., in dehydrated foods), and (d) loss of crispiness, powder stickiness and structure collapse occur at aw between 0.3 and 0.5. Although the exact relationship between the reaction rates and the changes outlined in Fig. 1.12 are highly specific for each food, this plot presents helpful guidelines worth memorisation.

A multiline graph of reaction rate, moisture content versus a W presents variations for lipid oxidation, Maillard reaction, enzymic activity, moisture sorption isotherm, and microorganisms between zone 1 and zone 3. The critical a W for structural change occurs at approximately (0.29, 0).

Fig. 1.12

Dependency of the rate of chemical, physical and microbiological changes on aw

1.7 The Glass Transition

The glass transition is a state transition strongly linked to water content and is important for the physical properties and stability of low-moisture foods, including dehydrated foods, high-solids confectionary products (e.g., hard candy), food powders, and frozen foods. Glass transition is not relevant for high-moisture foods, IMFs or foods with high-fat content.

A distinction between crystalline and amorphous solids is essential in discussing glass transition. Solids can be either crystalline or amorphous. The crystal structure is highly organised, and atoms or molecules have specific locations in space that repeat themselves periodically, i.e., at regular intervals. In amorphous materials, molecules are disordered, meaning they do not have an exact position in space, as they are randomly positioned with no repeating pattern in the structure (Fig. 1.13). The simplest food example is table sugar. Table sugar exists as both crystalline or amorphous solid. As bought from the market, table sugar is crystalline, and the molecules have specific positions within each sugar grain. If we add a little water, it will dissolve and form a thick syrup. Cooling this thick syrup creates a hard candy. Even though the hard candy consists of the same sugar molecules, it will now be amorphous and not crystalline, as the sucrose molecules are now randomly positioned within the candy (Fig. 1.13). With this example in mind, we will explore the glass formation in foods and its implications for shelf-life stability.

Two schematic diagrams of molecular arrangements of crystalline and amorphous substances. In crystalline substances, the molecules are systematically arranged. In amorphous, the molecules are scattered. 2 photographs of crystalline sucrose and glassy sucrose are also presented.

Fig. 1.13

Solids can be either crystalline or amorphous. Sucrose is present in both states, depending on the processing. (See text for description)

In thermodynamics, a phase is the region of matter that exhibits uniform chemical composition and physical properties. A phase transition occurs when a substance transforms from one physical state to another without changing its chemical composition. In a single-component system (e.g., water), the pressure or temperature may be changed and induce a phase transition (e.g., lowering the temperature below zero creates ice). In food systems, temperature and concentration control phase changes, as most processing occurs at atmospheric pressure. In contrast, pressure may be relevant in high-pressure processing (HPP) and extrusion. Phase transitions of relevance to food chemistry are shown in Table 1.6. These transitions occur abruptly at a specific temperature and change the state of the matter (e.g., ice turns to liquid water at precisely 0 °C).

Table 1.6

Phase transitions encountered in food processing

Type of transition

Name

Solid → Liquid

Melting

Liquid → Solid

Crystallisation

Liquid → Gas

Evaporation

Gas → Liquid

Condensation

Solid → Gas

Sublimation

The glass transition refers to a specific type of transition that occurs in amorphous materials (i.e., not crystalline). More generally, the transformation of foods when they are cooled from a liquid state to a solid state without undergoing crystallisation is known as the glass transition. To contrast this idea, the softening of the hard glassy sucrose shown in Fig. 1.13 with heating is a glass transition, but the softening of chocolate with heating is melting because cocoa butter in chocolate is a crystalline solid. The underlying physics of these transitions are distinct and lead to different approaches when extending the shelf life of amorphous or crystalline foods. The glass transition is one of the most important physicochemical characteristics of non-crystalline, amorphous food solids, as it controls the functionality and stability of the final product. Amorphous materials are also known as glassy, and the glass formation process is termed vitrification. In practice, the glass transition in foods is mostly relevant to carbohydrates and proteins. These compounds show strong interactions through hydrogen bonding with water, a controlling factor during glass transition. Lipids do not exhibit a glass transition under any circumstances, and other small molecules are found in too low concentrations to be considered significant in this context.

In contrast to melting, which occurs at a specific temperature (e.g., for water at 0 °C), the glass transition occurs over a temperature range and requires several degrees to complete. This temperature range is known as the glass transition zone (Fig. 1.14), where most deteriorative changes occur, especially a decline in hardness resulting in textural losses or stickiness. The temperature at the onset of the glass transition zone is essential for food stability. The onset is usually known as the glass transition temperature and is indicated as Tg or Tgonset (Fig. 1.14, yellow marker). For instance, a food stored below its Tg retains its hardness throughout its shelf life, whereas storage at temperatures near Tg or within the glass transition zone results in rapid structural deterioration (Fig. 1.14). The glass transition is reversible, meaning that the food regains its hardness with cooling. However, heating and cooling multiple times due to improper handling during transportation or storage changes the internal structure of the amorphous glass, and the original structure cannot be regained. Consequently, sensory characteristics or water dispersibility in powders will be negatively affected.

A graph of hardness versus temperature. It plots a logistic decay curve. The three zones are glassy state, critical zone, and flow zone. From point, T g, on the curve, the critical zone starts. Below T g, the structure is stable. Above T g, the complete loss of structure occurs.

Fig. 1.14

The glass transition zone and changes in the hardness of the dry food with temperature. Tg indicates the onset of the glass transition

Molecular motion in the glass transition zone is also known as α-relaxation, and it is due to cooperative motions of the molecules or biopolymer chains (e.g., sugars, polysaccharides or proteins). Below Tg, molecules are immobilised in their positions (only rotations and vibrations may be observed but not translational motion), resulting in hard or brittle food (e.g., potato crisps or breakfast cereals) (Fig. 1.14). As a reminder, translational motion is the motion in which molecules move uniformly in the same direction. When a glassy food is heated above its Tg, molecules gain translational mobility, i.e., they can now move and slide one past another. Conversely, the amorphous food vitrifies by lowering the temperature below the glass transition.

Once the glassy food has been formed and is stored below its Tg, the molecular motion, although restricted, persists. This mobility is known as β-relaxation and is linked to the rotation of lateral groups (such as -OH or -NH) or changes in the conformation of the main chain in the case of proteins and polysaccharides. With storage near Tg or due to β-relaxations, the glassy phase changes in time in a process called physical ageing. Because of physical ageing, the food is also subject to microstructural rearrangements that may affect its stability. This, in practice, results in textural changes in the food, which may occur after prolonged storage at inappropriate temperatures (see below for storage of glassy foods).

Several theories have been put forward to describe the molecular basis of glass transition. The free volume concept is widely accepted because of its simplicity. In this model, rigid spheres replace the atoms, and the space not covered by these hard spheres is free volume. The free volume concept states that the mobility of particles when they are closely packed depends on the degree of packing, or in other words, on the free volume. With an increasing degree of packing, the mobility decreases as the spheres do not have sufficient space to move. At a critical degree of packing, the mobility falls to zero. Another theoretical approach that provides a better understanding of the underlying physics of the glass transition is the mode-coupling theory, which establishes a route to predict the flow behaviour of the glassy state.

1.7.1 Glass Formation in Food Processing

Food glasses are formed in two ways: rapid cooling or water removal by dehydration or freezing. Rapid cooling is usually applicable to liquid sugar formulations in the confectionery industry. In this case, syrups high in sugar, generally >80% solids, are cooled until they solidify to the desired structure. As cooling is fast, molecules do not have enough time to arrange themselves into a crystal lattice and crystallise (see Sect. 1.7.2). As a result of rapid cooling, sugar molecules vitrify and are arrested into the positions they had in the liquid syrup.

Ice formation results in a freeze-concentrated matrix in foods with high water content undergoing freezing. Once all ice formation has been completed, the maximally freeze-concentrated matrix undergoes a glass transition at sub-zero temperatures. The main changes in frozen foods during storage are chemical (e.g., oxidation), biochemical (e.g., enzymatic) or physicochemical (e.g., recrystallisation of ice) that proceed slowly and largely depend on the storage temperature. Knowledge of the glass transition temperature of frozen foods helps improve the freezing process and their frozen-storage stability. The optimum stability is usually achieved when the storage temperature is below the Tg of the freeze-concentrated matrix. In practical terms, this may not always be achievable as the Tg may be well below −35 °C, with most commercial freezers operating between −25 °C and −18 °C. These conditions are usually above the Tg of the freeze-concentrated matrix, and the reactions may proceed, albeit slowly, thus shortening the product’s shelf-life. Typical products influenced by these changes are ice cream and frozen desserts, frozen fruits high in sugars, frozen fruit concentrates, frozen vegetables, and frozen dough. In contrast, ice formation and recrystallisation in frozen meat storage are considered to be the main deteriorative factors.

Dehydration of foods is carried out by air drying, spray drying or freeze-drying, depending on the processing objective. In all these technologies, water is removed rapidly from the food, and the remaining molecules undergo vitrification. For example, spray drying of skimmed milk flash-evaporates water from the droplets. The rapid dehydration creates delicate, glassy structures consisting of protein and lactose. Similar glassy structures are created with freeze drying, where the food maintains its original shape. For example, freeze-dried strawberries will maintain their resemblance to a strawberry, as the entire strawberry is glassy after drying. In this case, the strawberry structure, mainly consisting of a cellulose matrix, has undergone a glass transition.

Extrusion of many cereals and starch-based foods also results in glassy structures. Starch is gelatinised inside the extruder and subjected to high temperatures and pressures. At the die (the exit point of the extruder), pressure is suddenly lowered to atmospheric, resulting in flash evaporation of water. This rapid water release and cooling results in the vitrification of the amorphous starch mixture and the formation of crispy glassy structures (e.g., cheese puffs). In cold extrusion, e.g., in pasta or noodle manufacturing, the wet and pliable dough undergoes a glass transition during drying and changes to the familiar stiff, brittle structure associated with these products.

Finally, baking is another process leading to glassy foods, most notably bakery products. For instance, biscuit baking removes water from the dough and immobilises the gluten proteins and starch network into a glassy state. Other glassy bakery products include ice cream wafers, waffles, and the bread crust after baking.

1.7.2 Factors Influencing Glass Transition

The glass transition is mainly influenced by cooling rate, water content, and food composition, including the components’ molecular weight and the presence of plasticisers. Water is the most critical determinant of the glass transition in foods, and even tiny changes in its content (e.g., through absorption from the atmosphere with improper packaging) may result in softening or powder agglomeration. This softening is termed plasticisation, and water is the most common food plasticiser responsible for undesirable, in most cases, softening (see below for mechanism). Increasing water content generally decreases the food’s Tg. Other common plasticisers are monosaccharides (e.g., glucose) or polyols (e.g., sorbitol or glycerol). Plasticisers interact through hydrogen bonding with polysaccharides or proteins and can be viewed as “lubricants” that lubricate molecules and facilitate their translational movement.

In most food processing technologies, food is heated to the desired processing temperature and then cooled. The cooling rate is of great significance as it determines the structure of the glassy food. Slow cooling allows more time for the molecules to come together, making the structure dense. Slow cooling sometimes results in crystallisation, which may be undesirable in many products. In contrast, fast cooling arrests the molecules further apart, resulting in open structures. Open structures are more prone to collapse than dense structures in prolonged storage. Consequently, the cooling rate determines the structure and stability of the dry product. For instance, consider two biscuits made with the same formulation and processing conditions, the only difference being their cooling rates after baking. The two products will have distinct microstructures with different textural characteristics. The control of the cooling rate is a powerful tool in food product development as it may result in new products with minimum investment.

The molecular weight of some components also influences glass transition; for example, increasing the molecular weight of certain ingredients (e.g., maltodextrins) increases the Tg. For example, maltodextrin is essential in artificial sweetener powders, where 99% of the formulation is maltodextrin, and only 1% is the artificial sweetener. In this example, the molecular weight of maltodextrin controls shelf-life stability and dispersibility in water.

1.7.3 Storage of Glassy Foods

In general, the effect of inappropriate storage of glassy foods usually manifests in three ways: softening (e.g., softening of a wafer), agglomeration (e.g., stickiness of powders) or structural collapse (e.g., collapse of a freeze-dried strawberry) and it is always linked to α- and β-relaxation processes. The objective is to store glassy foods at a storage temperature Tstorage < Tg throughout their shelf life and avoid moisture uptake. As a guideline, glassy foods should be stored at temperatures 10 °C lower than their Tgonset (Tstorage ≦ Tgonset – 10 °C), which is generally considered sufficient to limit molecular mobility, minimise structural changes and extend the shelf life. We illustrate this with an example of a glassy food in Fig. 1.15.

A multiline graph of hardness versus temperature. It plots three logistic decay curves. The points of T g shift to lower temperatures with moisture uptake, T g of the original product, and T g shifts to higher temperatures with reformulation are pointed on the curves.

Fig. 1.15

Changes in the onset of Tg with moisture uptake or reformulation. See text for description

When exiting the production line (orange curve, Fig. 1.15), the freshly made food has a Tg = 27 °C. The food should be stored at Tstorage = 27 °C – 10 °C = 17 °C or lower throughout its shelf life to maintain optimum stability, as this temperature keeps the food in its glassy state and is sufficiently low to minimise the molecular mobility in the product. Suppose the same product is stored at Tstorage = 30 °C (purple curve, Fig. 1.15) above its Tg. Since Tstorage > Tg, prolonged storage at this temperature is not sustainable, as the molecular mobility is high. If the product needs to be shipped to places with ambient temperatures exceeding 27 °C, it must be reformulated to withstand high storage temperatures. This can only be achieved by changing ingredients or processing conditions, and it is not a trivial task. Finally, suppose the product is stored incorrectly, resulting in moisture uptake (blue curve, Fig. 1.15). The excess water plasticises macromolecules, shifting Tg to 17 °C, making it unstable at this temperature since molecules now have adequate mobility to cause structural damage due to water plasticisation. This product (blue curve) must be stored at a new Tstorage = 17 °C – 10 °C = 7 °C to avoid microstructural damage and maintain its characteristics.

1.8 Review Questions

1.8.1 Multiple Choice Questions

1.

Water in foods is important because:

(a)

It is a solvent and medium for diffusion and reactions

(b)

It is a substrate for microbial growth

(c)

It influences storage stability

(d)

All of the above

2.

The polarity of water is primarily because of:

(a)

Oxygen electropositivity

(b)

Hydrogen electronegativity

(c)

Oxygen electronegativity

(d)

Hydrogen electropositivity

3.

Ice crystal structure is:

(a)

Tetragonal

(b)

Orthorhombic

(c)

Trigonal

(d)

Hexagonal

4.

Hydrogen bonding is formed between

(a)

Water and molecules containing an electropositive atom and hydrogen atom

(b)

Water and molecules containing an electronegative atom and hydrogen atom

(c)

Water and molecules containing nitrogen, as in proteins

(d)

Water and between atoms in proteins and polysaccharides

5.

Hydrogen bond is

(a)

Always an attractive electrostatic force

(b)

Always a repulsive electrostatic force

(c)

Attractive between water molecules but repulsive in proteins

(d)

Repulsive between water molecules but attractive in proteins

6.

Arrange the following bonds in order of increasing bond strength

(a)

(stronger) Covalent > van der Waals > Hydrogen (weaker)

(b)

(stronger) Covalent > Hydrogen > van der Waals (weaker)

(c)

(stronger) Hydrogen > van der Waals > Covalent (weaker)

(d)

(stronger) van der Waals > Hydrogen > Covalent (weaker)

7.

Colligative properties of solutions are:

(a)

elevation of boiling point, elevation of freezing point and lowering of water activity

(b)

depression of boiling point, elevation of freezing point, osmosis, and lowering of the vapour pressure

(c)

elevation of boiling point, depression of freezing point, osmosis and lowering of the vapour pressure

(d)

depression of boiling point, elevation of freezing point and elevation of water activity

8.

The importance of colligative properties is that:

(a)

the presence of sugars and salt controls the processing properties of foods

(b)

Controlling of boiling point extends shelf life

(c)

Minimises spoilage from bacteria

(d)

Controls water activity, especially in high-moisture foods

9.

Water activity and water content values of food are:

(a)

always the same

(b)

always different

(c)

cannot be compared as they are different properties

(d)

always different ONLY in dry foods

10.

Drying of foods creates products with water activity:

(a)

< 0.3

(b)

0.3 < aw < 0.5

(c)

0.5 < aw < 0.9

(d)

>0.9

11.

Glass transition is only relevant for:

(a)

IMF foods

(b)

Dry foods

(c)

High-moisture foods

(d)

Lipids

12.

In foods, the compounds that undergo glass transition are mostly:

(a)

Carbohydrates

(b)

Proteins

(c)

Carbohydrates and proteins

(d)

Lipids

13.

The glass transition is also known as:

(a)

α-relaxation

(b)

β-relaxation

(c)

γ-relaxation

(d)

δ-relaxation

14.

Molecular motion below Tg is known as:

(a)

α-relaxation

(b)

β-relaxation

(c)

γ-relaxation

(d)

δ-relaxation

15.

Food glasses can be formed during processing by

(a)

Spray drying

(b)

Rapid cooling

(c)

Air drying

(d)

All of the above

1.8.2 Short Answer Questions

1.

Discuss the abnormal physical properties of water because of hydrogen bonding and its relevance to food processing.

2.

Discuss the molecular interactions that occur between water and food constituents. Give one example of each interaction and its importance in food processing.

3.

Describe osmosis and define osmotic pressure. Use a drawing to support your answer.

4.

Describe the dynamic equilibrium between vapour and liquid water above food in a closed container at a constant temperature.

5.

Draw a moisture sorption isotherm and identify the three zones of water. Discuss the type of water and the strength of interactions between water and food constituents in each zone.

6.

An apple pie has three components: pastry, jam, and apple pieces. The initial formulation resulted in a soggy product with a short shelf life because of the differences in aw of the components. Discuss how moisture migration occurs in multicomponent foods with different water activities. What steps could you take to minimise moisture migration and extend the shelf life of this product?

7.

Describe the dependency of the rate of chemical, physical and microbiological changes on aw (Fig. 1.12).

8.

Online activity-Advanced Search online for the Clausius-Clapeyron relation and discuss its relevance to water activity.

9.

Using the glass transition concept, describe what happens to the stability of skimmed milk powder as the humidity increases.

10.

Describe what happens to the stability of hard candy as the temperature increases using the glass transition concept.

11.

Discuss why it is important to keep dry foods below their glass transition temperature.

12.

Discuss how glassy foods are formed in the food industry. Name five glassy foods.

13.

The Tgonset of a product is 29 °C. What storage temperature would you recommend for this product?

14.

Online activity: Find online the terms “first-order transition” and “second-order transition”. Identify the differences between them and give two examples for each.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024

V. KontogiorgosIntroduction to Food Chemistryhttps://doi.org/10.1007/978-3-031-53558-1_2

2. Carbohydrates

Vassilis Kontogiorgos1  

(1)

Associate Professor in Food Chemistry, University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC, Canada

Vassilis Kontogiorgos

Email: v.kontogiorgos@ubc.ca

Learning Objectives

After studying this chapter, you will be able to:

Describe the structure of monosaccharides

Discuss monosaccharide reactions

Identify the different types of glycosidic bonds in polysaccharides

Explain the changes that occur in starch during processing

Identify food polysaccharides based on their chemical structure

2.1 Introduction

Carbohydrates are structural components of living organisms, e.g., cellulose in plants or chitin in arthropods and a significant energy source through oxidation for both plants and animals. In foods, they may be used as sweeteners, emulsion stabilisers, gelation agents, or to retain water in food formulations. Chemically, carbohydrates are polyhydroxy aldehydes or polyhydroxy ketones and are classified into three groups. Monosaccharides are the simplest form of carbohydrates and the building blocks of the rest of carbohydrate-based molecules. Linking monosaccharides creates larger molecules such as disaccharides consisting of two monosaccharides or oligosaccharides comprised of between 3 and 20 monosaccharides. Finally, polysaccharides consist of more than 20 monosaccharide units, most frequently more than 200 and have distinct properties from the other two classes. This chapter introduces the chemical structure of carbohydrates relevant to foods and how their interactions with other molecules may influence product stability and quality.

2.2 Monosaccharide Structure

Monosaccharides are distinguished as aldoses (e.g., glucose) or ketoses (e.g., fructose) when they carry an aldehyde or ketone functional group. Fructose is the only relevant ketose from a food chemistry perspective discussed in this textbook. Monosaccharides have several carbon atoms, usually between three and six, but sometimes even more (e.g., seven). Those relevant to foods have six (e.g., glucose or fructose) or five (e.g., arabinose or xylose) carbon atoms. Monosaccharides with more than six carbon atoms are outside the scope of food chemistry. The six most important monosaccharides are glucose, mannose, galactose, arabinose, xylose, and fructose. A few more relevant for specialised applications (e.g., erythrose or ribose) or an in-depth description of polysaccharide structures (e.g., gulose or rhamnose) are mentioned only when necessary.

The first thing to know in the study of carbohydrates is the numbering of carbon atoms, as understanding chemical reactions, polysaccharide linkage, and other carbohydrate properties is problematic without correct numbering. Numbering starts from the carbonyl group, called “C1”, and proceeds sequentially to the sixth, “C6” (Fig. 2.1). In the case of fructose, the carbonyl group is “inside” the structure (Fig. 2.1). In that case, C1 will be the nearest terminal carbon to the carbonyl group.

3 chemical structures. A. (2 R, 3 S, 4 R, 5 R), 2, 3, 4, 5, 6 pentahydroxyhexanal. B. (2 R, 3 S, 4 S), 2, 3, 4, 5 tetrahydroxypentanal. C. (3 S, 4 R, 5 R), 1, 3, 4, 5, 6, pentahydroxyhexan 2 one.

Fig. 2.1

Glucose, arabinose (aldoses) and fructose (ketose). The numbering of carbon atoms starts from the carbon atom with the carbonyl group

Because of the intricacy of their structure, carbohydrates have several isomeric forms (Fig. 2.2). Specifically, monosaccharides show a form of isomerism termed stereoisomerism, where atoms are connected in the same order but differ in spatial arrangement (i.e., 3D arrangement). Stereoisomerism is divided into enantiomerism and diastereoisomerism. In enantiomerism, the structures are non-superimposable mirror images of each other, and the isomers are termed enantiomers. In diastereoisomerism, the isomers are not mirror images of each other and are termed diastereoisomers. Diastereoisomers are further divided into epimers and anomers. Epimers differ in the orientation of a -OH at only one stereocenter other than the anomeric carbon. Anomers differ at a new chiral carbon atom formed on ring formation (see below for a detailed explanation of the terms).

A tree diagram classifies the isomers into constitutional isomers and stereoisomers. The stereoisomers are further classified into enantiomers and diastereoisomers. The diastereoisomers are divided into anomers and epimers.

Fig. 2.2

Different forms of isomerism encountered in monosaccharides

Carbohydrates show such complex isomeric forms because of multiple chiral carbons in their structure. A carbon atom of a molecule with four different groups attached to it is called chiral carbon and indicated as C*. In the simplest case, a molecule with four different substituent groups will have two isomers where one structure is the reflection of the other (Fig. 2.3). These structures are called enantiomers and are non-superimposable mirror images of each other. Non-superimposable means that when one structure is placed over the other, they do not overlap. Sugars contain one or more chiral carbon atoms, so they exhibit 2n different arrangements of atoms, with n being the number of chiral carbons. For example, glucose with four chiral carbons has 2⁴ = 16 different isomers. Figure 2.3a shows the simplest sugar (glyceraldehyde) with three carbons and the arrangement of the various groups. All sugars with the hydroxyl group on the highest numbered chiral carbon atom positioned on the right-hand side are called d-sugars. Those with a hydroxyl group positioned on the left are called l-sugars (Fig. 2.3b). d- and l- monosaccharides are enantiomers, as they are mirror images of each other. Carbohydrates naturally occur in the d-form.

A. 3 D and Fischer projections of D-glyceraldehyde and L-glyceraldehyde. B. Fischer projections of D-glucose and L-glucose.

Fig. 2.3

(a) The d- and l- enantiomers of glyceraldehyde. Chiral carbons are indicated with a “*”. The structures are non-superimposable mirror images of each other. The top structures show the 3D arrangement of atoms, and at the bottom are the Fischer projections, (b) d- and l-glucose. The highest-numbered chiral carbon atom in glucose is carbon number 5 (C5, in blue). C1 and C6 are not chiral. At C5, the -OH group is positioned on the right for d- and the left for l-glucose

A property of chiral molecules, including monosaccharides, is their ability to rotate the plane of polarised light. This is known as optical activity, and the compounds are called optically active. The rotation units are degrees (o), for example, 45° rotation, and the angles are measured using a polarimeter. If polarised light passes through a monosaccharide solution, the plane of light will be rotated to the left or right. Enantiomers rotate the polarisation plane equally but in opposite directions, i.e., clockwise (to the right) or anticlockwise (to the left). The enantiomer is called dextrorotatory for clockwise rotation and is designated as “d-” or “+” or levorotatory for anticlockwise rotation and is designated as “l-” or “−”. An important distinction is that the capital letters d- and l- indicate the position of the -OH group as described above, whereas the small letters d-(+) and l-(−) indicate optical rotation and cannot be used interchangeably. For example, glucose can be written as d-(+)-glucose, which is dextrorotatory and because of this, glucose is also known as dextrose. In contrast, l-(−)-glucose is levorotatory. Apart from monosaccharides, many food compounds are optically active, including amino acids (e.g., l-(+)-alanine) or flavours (e.g., d-(+)-carvone). This property is most notably exploited in sugar industries to quantify sugar in raw materials (e.g., sugar cane) and the syrup industry (e.g., invert sugar). It can also be used to monitor and control the production process of monosodium glutamate, monitor alcoholic fermentations and ensure product quality by measuring the concentration and purity of compounds (e.g., maltose, fructose, honey, etc.) in sugar-based foods and syrups.

Carbohydrates are frequently depicted using the Fischer projection, a 2D representation of 3D organic molecules by projection, commonly used in carbohydrate chemistry. Monosaccharides have three dimensions in space (length, width, and height). However, we must draw monosaccharides on paper with only two dimensions (length and width). In that case, to obtain the Fischer projection, the substituent groups of carbons are mirrored onto a 2D plane (paper) using straight lines (Fig. 2.4). The monosaccharide 2D image formed by joining the substituent groups on the plane of the paper is termed projection. This structure is obtained by placing the carbonyl group on top and the rest of the carbon chain at the bottom (Fig. 2.3b). Fischer projection is also used to draw amino acids (see Chap. 3).

A 2 D representation of 2, 3 dihydroxypropanal obtained by projection of the dashed and wedge form.

Fig. 2.4

Drawing of Fischer projection of monosaccharides on paper. The same process is also used for amino acids

Monosaccharides carry aldehyde and alcohol groups in their structure, and as a result, they may react intramolecularly to form hemiacetals. Further reaction of hemiacetals with alcohols results in acetal (Fig. 2.5).

A chemical reaction presents the reaction of an aldehyde with alcohol to produce hemi-acetal followed by acetal.

Fig. 2.5

Hemiacetal and acetal formation. The reaction of an aldehyde with alcohol results in hemiacetal formation. The reaction of hemiacetals with alcohols results in acetal formation

This reaction forms a cyclic hemiacetal (for aldehydes, e.g., glucose) or hemiketal (in ketones, e.g., fructose) and a new chiral carbon. Monosaccharides can form 6- or 5-membered rings called pyranose rings (e.g., glucose) or furanose rings (e.g., fructose). The new chiral carbon is called anomeric carbon, which results in two stereoisomers that differ only in their configuration at the anomeric carbon. A Haworth projection is another type of projection used to represent the cyclic structures of monosaccharides. In this projection, carbon and hydrogen atoms sometimes are not depicted, and the thicker bonds indicate that the atoms are closer to the observer (a-d-glucose in Figs. 2.6 and 2.8). In the Haworth projection, the alpha- (α-) anomer of d-monosaccharides have the -OH group below the plane of the ring, whereas beta- (β-) anomers have the -OH group above the plane of the ring (Fig. 2.6). It is important to note that for the l- sugars the opposite is true. Numbering starts from the carbon atom next to the ring oxygen atom and proceeds clockwise, with C1 being the anomeric carbon (green marker in Fig. 2.6).

A transition chart. It begins with Fischer projection of glucose leading to a horizontal structure in which groups on the right face down and intramolecular hemiacetal formation to result in Haworth projections of glucose. In alpha form, one O H lines below the plane of the ring, whereas in beta form it lies above.

Fig. 2.6

Formation of Haworth projection and anomers through intramolecular hemiacetal formation. Starting from the Fischer projection, the structure is rotated 90 degrees clockwise. The -OH group of C5 reacts intramolecularly with the aldehyde group (highlighted in blue). Cyclisation produces a new chiral centre (green circle), forming α- and β-anomers. The positioning of hydroxyl groups (highlighted in yellow) is the only difference between the structures

Carbohydrates that differ in the orientation of the -OH group at only one chiral carbon other than the anomeric carbon are called epimers (Fig. 2.7). Epimers are non-superposable, non-mirror images with each other contrasting to enantiomers that are non-superposable, mirror images with each other. d-Mannose and d-galactose are both epimers of d-glucose. However, d-mannose and d-galactose vary at multiple chiral centres and are diastereomers but not epimers.

3 chemical structures. a. (3 S, 4 S, 5 S, 6 R), 6, hydroxymethyl, oxane, 2, 3, 4, 5 tetrol. b. (2 R, 3 S, 4 R, 5 R), 2, 3, 4, 5, 6, pentahydroxyhexanal. c. (3 R, 4 S, 5 R, 6 R), 6, hydroxymethyl, oxane, 2, 3, 4, 5, tetrol.

Fig. 2.7

Mannose and galactose are epimers of glucose at different carbon atoms (epimer at C2 or C4, respectively). The coloured boxes indicate the different orientations of hydroxyl groups in each case

The Haworth projection is not a realistic representation of monosaccharide structure in space because -OH groups and -H atoms interact and repel each other to minimise interactions between them. The 3D arrangement of atoms in space is termed conformation, and molecules may convert from one form to another by rotation about individual single bonds. The various structures resulting from the different arrangements of the substituent groups in space are called conformers. Because of geometrical restrictions collectively termed steric hindrances (e.g., bond length, size of substituent groups, etc.), the available number of 3D shapes is limited. The two most common conformers of pyranoses are the so-called chair and boat conformers (Fig. 2.8). The most stable is the chair, whereas the boat conformer is seen only in derivatives with very bulky substituents.

a and b are chair and boat conformations of pyranoses.

Fig. 2.8

Conformation of pyranoses, (a) chair, and (b) boat. The thicker bonds indicate that the atoms are closer to the observer

Chair conformation is a common way to depict carbohydrates. There are two positions for the substituent groups for each carbon atom in these structures: the axial and the equatorial (Fig. 2.10a). Axial bonds are parallel (red bonds), whereas equatorial bonds are perpendicular (blue bonds) to the axis of symmetry of the ring (Fig. 2.9b). Each carbon has one axial and one equatorial bond. Bulky groups are in equatorial positions, whereas hydrogen atoms are in axial positions, as this positioning is less crowded. Two types of chair conformations may be formed depending on the positioning of C1 and C4. When C4 is raised above the ring plane with C1 below the ring plane, then this conformation is designated as ⁴C₁. When the opposite is true, it is designated ¹C₄ (Figs. 2.9b, c and 2.10).

a. Chair conformation of (2 R, 3 R, 4 S, 5 S, 6 R), 6, hydroxymethyl, oxane, 2, 3, 4, 5, tetrol. b. The same structure with a vertical axis of symmetry. Plane of symmetry is also marked. c. C 4 and C 1 are marked.

Fig. 2.9

Chair conformations of β-d-glucopyranose. (a) equatorial and axial bond positions on a pyranose ring, (b) ⁴C₁, and (c) ¹C₄

Left. 4 C 1. C 4 above the plane of the ring, and C 1 below the plane of the ring. Right. 1 C 4. C 1 above the plane of the ring, and C 4 below the plane of the ring.

Fig. 2.10

Depending on the positioning of C1 and C4 relative to the plane of the ring, we may have ⁴C₁ or ¹C₄ conformations. “C” stands for “chair”, not “carbon”

When monosaccharides are dissolved in water, it results in an equilibrium mixture of open chain, five- and six-membered rings where the configuration of the anomeric carbons may be either α- or β-. The anomers interconvert (i.e., α ⇌ β) through their open form until an equilibrium is reached. This process is termed mutarotation because of the continuous change in the optical rotation of linearly polarised light until equilibrium is established (Fig. 2.11). For example, the equilibrium mixture of β-d-glucopyranose is ~64% of β-d-glucopyranose and ~36% of α-d-glucopyranose. In practical terms, it means that when we dissolve monosaccharides in water, we never have only one structure in the solution but a mixture of various forms depending on the monosaccharide and the surrounding environment (e.g., pH, temperature, etc.).

A reaction presents the interconversion of alpha-D-glucopyranose to beta-D-glucopyranose and vice versa.

Fig. 2.11

When pure anomers are dissolved in water, they undergo mutarotation, interconverting from one anomer to another until an equilibrium is reached

Tautomers are isomers of compounds that interconvert from one form to another after intramolecular proton transfer from carbon to oxygen. This isomerisation reaction, termed tautomerisation, is catalysed by acid or base. It requires carbonyl compounds having H-atoms on their α- carbons (i.e., carbons next to the carbonyl carbon), as in monosaccharides. Tautomers exist in pairs, with the keto-enol pair being most relevant to food reactions. Keto-enol tautomerism refers to interconversion between a keto form (ketone or aldehyde) and an enol (unsaturated alcohol) (Fig. 2.12). The interconversion is very fast, with the keto-form being the most stable. In the base-catalysed keto-enol tautomerism of d-glucose, the interconversions involve the movement of a proton and the shifting of bonding electrons. Consequently, when d-glucose is dissolved in water in alkaline conditions, the solution also contains d-mannose and d-fructose! Keto-enol tautomerism occurs in the non-enzymic browning of foods, as it is involved in Maillard reactions.

A chemical reaction presents the interconversion of keto-tautomer to enol-tautomer.

Fig. 2.12

Keto-enol tautomerism is the equilibrium between a ketone or an aldehyde (keto form) and alcohol (enol form). The keto and enol forms are tautomers of each other

2.3 Monosaccharide Reactions

Reactions of monosaccharides are crucial in food processing and preservation and influence the quality and shelf-life of products. Monosaccharide reactions involve both hydroxyl and carbonyl groups. Alcohol (hydroxyl group) may react to form esters and ethers or can be oxidised to form acids. Ketones can be reduced to secondary alcohols. In esterification, the reaction of alcohol and acid and hydroxyl groups of monosaccharides may be esterified. The reaction of alcohol with halides (compounds containing F, Cl, Br, or I) results in ether formation (Fig. 2.13). These two reactions are essential in the commercial production of modified starches or cellulose derivatives and the manufacturing of emulsifiers (e.g., sorbitan esters) used in various food applications.

2 chemical reactions. Top. Chair structure of glucose reacts with pyridine and C H 2 C O twice O to form a structure with H in O H replaced with C double bond O, C H 3. Bottom. Chair structure of glucose reacts with C H 3 I and A g 2 O to form a structure with H in O H replaced with C H 3.

Fig. 2.13

Esterification (top) and etherification (bottom) reactions of carbohydrates

The aldehyde group can be oxidised to the corresponding carboxylic acid to form aldonic acid. When an aldose is converted to aldonic acid, the suffix “-onic” is used. For example, the acid of glucose is gluconic acid (Fig. 2.14a). After isomerisation, ketoses can also be oxidised to form glucose or mannose via an intermediate, enediol, through keto-enol tautomerism described earlier. Aldonic acids may also cyclise through an interesterification reaction between the carboxyl group and the -OH at C-5 to form lactones. The lactone of glucose is termed glucono delta-lactone (GdL) and is a common food additive. GdL hydrolyses slowly to gluconic acid, ensuring a progressive decrease of pH that resembles the acidification produced in bacterial fermentations. GdL is used in many foods, including meat, dairy, soy, and confectionery. Under stronger oxidising conditions, the aldehyde and the terminal -OH groups oxidise to carboxylic acids to form aldaric acids. In this case, the suffix “-aric” is used, for example, the acid of glucose is glucaric acid (Fig. 2.14b). The final scenario is when oxidation occurs in only the terminal carbon to a carboxylic acid, with the aldehyde group remaining unaffected. These molecules are called uronic acids and have both carbonyl and carboxylic acid functional groups available for reactions (Fig. 2.14c). In this case, the suffix “-uronic acid” is used, for example, the uronic acid of glucose is glucuronic acid. Uronic acids are naturally found in foods and are important constituents in various polysaccharides such as in pectin (galacturonic acid, i.e., uronic acid of galactose) or alginates (mannuronic and guluronic acids, i.e., uronic acids of mannose and gulose).

3 chemical reactions. A. Oxidation of D-glucose to D-gluconic acid. B. Oxidation of D-glucose to D-glucaric acid. C. Oxidation of D-glucose to D-glucuronic acid.

Fig. 2.14

Oxidation of glucose to (a) aldonic acid, (b) aldaric acid, and (c) uronic acid

When monosaccharides oxidise (e.g., glucose, fructose, or galactose), another compound is reduced. Because of this property, monosaccharides are also known as reducing sugars (Fig. 2.15a). Some disaccharides (e.g., lactose or maltose), oligosaccharides and polysaccharides that have the anomeric carbon free to react (free aldehyde or ketone group) are also reducing. In contrast, sucrose, as we will see in the next section, is not a reducing sugar because the anomeric carbon is involved in the glycosidic bond between glucose and fructose. Oxidation is also used for reducing sugar determination using the Fehling solution.

3 chemical structures. A. Chair structure of glucose with anomeric carbon marked. B. A reaction presents the conversion of D glucose to D sorbitol.

Fig. 2.15

(a) The anomeric carbon C1 is free to react in monosaccharides. All monosaccharides are reducing sugars, and (b) reduction of monosaccharides forms alditols. In this case, glucose forms sorbitol

In reduction, carbonyl groups (aldehydes or ketones) are reduced to sugar alcohol and are termed alditols using the suffix “-itol” (Fig. 2.15b). For example, sorbitol (from glucose, also known as glucitol), mannitol (from mannose) or xylitol (from xylose) are commonly used as non-calorific sweeteners in the confectionery industry to create sugar-free products (e.g., sugar-free chewing gums).

A reaction of central importance in carbohydrate chemistry is the glycosidic bond formation that joins two monosaccharides or, very frequently, a monosaccharide to other molecules. In glycosidic bond formation, the hemiacetal in the anomeric carbon (C-1) reacts with alcohol to form an acetal called glycoside. The group is termed aglycone if the -OH does not belong to another monosaccharide (Fig. 2.16). Glycosides are most frequently linked via an oxygen atom (O-glycosides), but they may also link through a nitrogen (N-glycosides) or sulfur (S-glycoside) atom. Many natural food compounds are glycosides, such as flavonoids, steroids, or coumarins. Steviol glycosides are responsible for the sweet taste of stevia, which is used as a non-calorific sweetener. Hesperidin, naringin, and rutin are flavonoids that frequently taste bitter and may need to be removed from the product (e.g., grapefruit juice).

A chemical reaction presents the reaction of chair structure of glucose with methanol to form glucoside methyl glucose.

Fig. 2.16

Glucose reacts with methanol to form the glycoside methyl-glucose. In this case, the methyl group is the aglycone

Glycosidic bond notation involves three elements: the anomeric form of the sugar that carries the hemiacetal group, the number of the carbon atoms involved in the bond from the first monosaccharide and the number of the carbon atom that is involved in the bond from the second monosaccharide. When a disaccharide or polysaccharide is shown as having α-(1 → 4) links, the constituent monosaccharides are in the α anomeric form. Additionally, the first monosaccharide connects to the second with the C1 carbon, and the second monosaccharide connects to the first with the C-4 carbon (Fig. 2.17). In the case of β-(1 → 4), the only difference is that the monosaccharides are in the β anomeric form. An α-(1 → 6) indicates that the link in the second monosaccharide occurs at C6, and this linkage is responsible for the formation of branching in polysaccharides. Another equivalent way of writing glycosidic bonds is α-(1,4), i.e., a comma is used instead of an arrow. Although other bonds may be possible, for example, a-(1 → 3), β-(1 → 2), or β-(1 → 3), the three bonds α-(1 → 4), α-(1 → 6), β-(1 → 4) are by far the most prominent. Glycosidic linkage can be hydrolysed using specific enzymes or heat and acid to yield monosaccharides.

Top. A chemical reaction presents the formation of a glycosidic bond between 2 glucose molecules. Bottom. The labeling of the glycosidic bond is alpha, 1 to 4. Alpha is labeled anomeric form, and 1 and 4 are labeled carbons involved in bonding from first and second monosaccharides.

Fig. 2.17

Glycosidic bond formation between two glucose molecules and notation of the bond. In this example, it is an α-(1 → 4) glycosidic bond

Monosaccharides may also rotate around the glycosidic bonds until they reach a state of minimum energy. Angle φ is located between the anomeric carbon and the oxygen of the glycosidic linkage of the first monomer, and ψ between the oxygen of the glycosidic linkage and the non-anomeric carbon of the second monomer (Fig. 2.18). The introduction of branching at C6 gives one more possible angle of rotation (ω) about the C5 and C6 bond. Branching and rotation are more critical as the molecule gets bigger, as in oligosaccharides and polysaccharides. In these molecules, rotation around bonds is critical, as it frequently controls their functional properties.

A chemical structure presents how monosaccharides rotate around the glycosidic bonds until they reach a state of minimum energy. Omega, phi, and psi are marked.

Fig. 2.18

Rotation of monosaccharides around glycosidic bonds. φ, ψ, ω are the possible angles of monosaccharide rotation

Carbohydrate Determination

In the phenol-sulfuric acid method, sugars react with concentrated sulfuric acid to produce furfural derivatives. Adding phenol results in products with a yellow colour that can be determined colourimetrically.

A chemical reaction. Sugars react with sulfuric acid to produce furfural derivatives. Adding phenol results in a yellow color product.

The m-hydroxy diphenyl method used to determine total uronic acids is also based on the reaction of sugars in concentrated sulphuric acid followed by the formation of pink-coloured complexes with m-hydroxy diphenyl reagent that can be determined colourimetrically.

A chemical reaction. Sugars react with sulfuric acid to produce furfural derivatives. Adding m-hydroxy diphenyl results in a pink color product.

Reducing sugars can be determined with Fehling’s or Benedict’s solutions that contain Cu²⁺, which can be reduced by reducing sugars to Cu⁺. The reactions can be used in both qualitative and quantitative (with suitable modifications) analysis of reducing sugars. Polysaccharides are determined by quantifying monosaccharides that result after their complete hydrolysis, followed by an appropriate chromatographic method.

2.4 Disaccharides-Oligosaccharides

As has already been mentioned, the result of joining two monosaccharides is the formation of a disaccharide (Fig. 2.19). Maltose consists of two glucose molecules while retaining the reducing hemiacetal at the C1 that is not involved in the glycosidic bond (see above for explanation). Lactose that consists of glucose and galactose also retains the anomeric C1. Lactose-intolerant individuals cannot break the glycosidic bond of lactose during digestion, resulting in gastrointestinal discomfort. Lactose-free milk production involves hydrolysis of lactose, most frequently using the enzyme lactase to produce a mixture of glucose and galactose. Sucrose consists of glucose and fructose linked by an α-(1 → 2) glycosidic bond. Hydrolysis of sucrose to its constituent sugars (i.e., glucose and fructose) using the enzyme invertase or heat-and-acid produces a syrup known as invert sugar. Invert sugar is sweeter than table sugar and is more difficult to crystallise, making it an ideal sweetener for the confectionery and baking industry. It is called “invert sugar” because the mixture of glucose and fructose that is produced during hydrolysis shifts the optical rotation to the opposite direction than that of sucrose from +66.5° (pure sucrose) to −19.7° (for fully hydrolysed sucrose).

3 chemical structures of disaccharide compounds. Maltose consists of two glucose molecules, lactose consists of glucose and galactose, and sucrose consists of glucose and fructose.

Fig. 2.19

Common disaccharides found in food. Sucrose is a non-reducing disaccharide

Oligosaccharides have between two and twenty monosaccharides linked with glycosidic bonds, and very few occur naturally (Fig. 2.20). Raffinose (tri-saccharide), stachyose (tetrasaccharide) and verbascose (pentasaccharide) are some commonly occurring oligosaccharides of beans that are non-digestible and responsible for flatulence. Fructo-oligosaccharides are naturally occurring and consist of multiple fructose units, frequently termed fructans. Foods rich in fructans are asparagus, banana, garlic, leek, onion, and Jerusalem artichoke. Commonly occurring fructans are inulin and levan with fructose units linked by β-(2 → 1) or β-(2 → 6). They can be used as prebiotics inducing the growth of beneficial bacteria in the gut, as fat replacers or texture modifiers.

A chart of oligosaccharides. Sucrose consists of glucose and fructose. Raffinose consists of sucrose and galactose. Stachyose consists of 2 molecules of galactose and 1 of sucrose. Verbascose consists of stachyose and galactose.

Fig. 2.20

Common oligosaccharides found in foods

2.5 Polysaccharides

Polysaccharides are monosaccharide polymers, as several monosaccharide units are linked together via glycosidic bonds. The large molecule, frequently termed polysaccharide chain, resembles a string of beads. The number of monosaccharides comprising the chain is termed the degree of polymerisation (DP). For example, when the DP is 200, the polysaccharide consists of 200 monosaccharides. Usually, the DP of polysaccharides is greater than 200 and frequently over 3000. Depending on the monosaccharide composition, polysaccharides are distinguished as homo-polysaccharides consisting of only one monosaccharide unit or hetero-polysaccharides consisting of two or more monosaccharides (Fig. 2.21). Polysaccharides may be linear when they have only one type of glycosidic bond or branched when multiple types of glycosidic bonds occur in the same molecule. Polysaccharides are obtained from plants with minimal processing (e.g., rice or potato starch) or because of the processing of agricultural wastes (e.g., pectin). Other sources include algae (e.g., alginates or carrageenan), processing of by-products of the shellfish industry (e.g., chitin), or microbial fermentation (e.g., xanthan or gellan).

5 illustrations of different polysaccharides. Linear homopolysaccharide, branched homopolysaccharide, linear heteropolysaccharide, and branched heteropolysaccharide.

Fig. 2.21

Types of different polysaccharide structures. (See the text for explanation)

Although there are numerous monosaccharides in nature, the number of those found in polysaccharides is relatively small. Common monosaccharides, including glucose and mannose, form the backbone of some of the most important commercial polysaccharides. Other sugars or sugar acids such as galactose, xylose, arabinose or galacturonic, guluronic and mannuronic acids are found in industrially relevant polysaccharides. However, the type of linkages, isomeric forms, functionalisation of sugars, branching, and periodicity of the monomers in the backbone result in significant structural variability and diversity in functionality. These modifications are, for example, methylation or acetylation at various positions, the presence of sulfate or other functional groups or differences in the anomeric type of monosaccharides that make up the polysaccharide. A notable example is that of amylose and cellulose, which contain glucose. Glycosidic linkages between glucose units in amylose are α-(1 → 4), whereas, in cellulose, they are β-(1 → 4), resulting in different functional properties not only within the plant (source of energy vs structural) but also when they are used as food ingredients.

Polysaccharides are essential food ingredients, and it is challenging to find a food product that does not have a polysaccharide present either naturally or as an additive. Many terms in the literature describe polysaccharide functionality, such as bulking agents, emulsion stabilisers, fat replacers, gelation agents, thickening agents, viscosity enhancers, water-binding agents or texture modifiers. Despite application specifics, polysaccharide functionality falls into two main classes: structure formation and structure stabilisation (Table 2.1). When used as structure-forming agents, they form gels that hold a large amount of water and create a soft-solid three-dimensional structure. This functional property is used to hold the formulation parts together (e.g., jams, onion rings or batter in chicken nuggets) and provides essential textural characteristics to the product. When used as stabilisers, they increase viscosity, thus preventing physical separation (e.g., creaming or precipitation). This ensures the processing of a uniform formulation that otherwise would be impossible (e.g., ice cream mix before freezing) or prolongs the shelf life of products (e.g., suspension of particles in salad dressings). Frequently, polysaccharides have more than one role in the same formulation. For instance, in salad dressings, they may prevent phase separation of oil droplets and provide texture modification with an increase in viscosity. In most cases, they interact with other food ingredients (e.g., proteins, lipids, or cations), which makes food formulation with polysaccharides quite challenging. As a result, understanding polysaccharide chemical structure is the first step before application.

Table 2.1

Examples of polysaccharide functionality in foods

Property

Examples

Structure formation

Gelation

Gelation in onion-ring formation

Emulsification

Emulsification of flavour oils in beverages

Encapsulation

Conversion of oils into powder

Structure stabilisation

Viscosity enhancement

Prevention of phase separation in salad dressings

Water retention

Prevention of syneresis in dairy desserts

Texture modification

Changes thickness perception in low-fat formulations

2.5.1 Starch

Starch is the carbohydrate source in plants found in leaves, tubers, fruits, and seeds. It is widely used in food and non-food industries (e.g., paper, chemical or cosmetic). There is an extensive selection of starches from various sources, such as corn, wheat, rice, potato, or cassava (tapioca). Starch that is naturally found and has not been further processed is termed native. However, native starches should be physically, chemically, or enzymatically changed to obtain technological consistency and are called modified starches.

Starch is found in the starch granule and is a mixture of two polysaccharides: amylose and amylopectin. Amylose and amylopectin are glucose homopolysaccharides and chemically differ only in branching. Glucose units in amylose connect with α-(1 → 4) glycosidic bonds (Fig. 2.22). The positioning of glucose units gives amylose a helical shape. The core of this structure contains hydrogen atoms and is hydrophobic, whereas the -OH groups are positioned on the outer part of the helix, making it hydrophilic. Amylose may form complexes with hydrophobic molecules such as lipids or flavours entrapped in the molecule’s hydrophobic core. Iodine may also bind into the core and form the basis of amylose determination in starches.

Two molecular structures. A. Amylose. B. Amylopectin. Glucose units in amylose connect with alpha 1, 4 glycosidic bonds.

Fig. 2.22

Glycosidic bonds in (a) amylose and (b) amylopectin. n is the number of repeating units shown within the square brackets. In amylose, for example, if n = 100, the polysaccharide consists of 200 glucose molecules because within the brackets are two glucose molecules. Blue circle: glucose

Amylopectin is a large branched molecule having both α-(1 → 4) and α-(1 → 6) linkages (Fig. 2.22). The presence of α-(1 → 6) creates branches forming three types of chains. C-chain is the chain that carries the only reducing end (Fig. 2.23). Branches stemming from the C-chain are termed B-chains, whereas those originating from B-chains are called A-chains. A-chains do not have any branches. Starches with very little amylose made up almost exclusively of amylopectin are called waxy. Starch is located in the starch granule where amylopectin forms an ordered structure (crystalline) (Fig. 2.23). Amylopectin molecules are oriented radially around a point called hilum with the consequent formation of concentric regions of alternating amorphous and semi-crystalline structures (Fig. 2.23). Semi-crystalline regions consist of ordered parts of amylopectin chains, whereas the amorphous regions contain the branching points. The alternating arrangement of these regions results in the formation of growth rings. The hilum is important because water enters from this opening, resulting in starch granule swelling and facilitating gelatinisation. Starch granules have different shapes, sizes, and size distributions that determine their swelling capacity and other physical properties. Large (>10 μm) and lenticular granules (like a lens) are called A-type starch granules and are found in cereal endosperms. Small (<10 μm) and spherical are called B-type starch granules, have a lower swelling capacity, and are found in tubers and legumes (e.g., peas).

a. Structure of amylopectin. It has 3 chains, A to C, amorphous and semi-crystalline regions, and reducing and non-reducing ends. b. A schematic of the concentric contours with helium, an amorphous growth ring, and a crystalline growth ring. 2 micrographs, c and d, of the starch granule and fibrous.

Fig. 2.23

(a) Structure of amylopectin (see text for description), (b) schematic representation of a starch granule exhibiting growth rings consisting of alternating regions of semi-crystalline and amorphous regions, (c) microscopy image of growth rings in a starch granule, and (d) microscopy image of starch granules embedded in bread dough. The fibrous structures are gluten strands, and starch granules have different sizes. Microscopy image (c) is reproduced with permissions from Pilling and Smith (2003), Plant Physiology, 132, 365–371. Microscopy image (d) is reproduced with permissions from Kontogiorgos, Goff, Kasapis, (2008), Food Hydrocolloids, 22, 1135–1147

As mentioned earlier, industrial starch is classified into native and modified. Native starches exist naturally, as they are obtained from their source. The most common native starches are corn (maize), rice, wheat, potato, tapioca (cassava), and waxy maize. Waxy maize and other waxy native starches generally contain less than 2% amylose, whereas the rest contain somewhere between 15% and 30%. High amylose starches contain more than 30% amylose and have quite different properties. Native starches are also different in the crystal structure, pasting characteristics and minor components present within amylose and amylopectin, such as phosphate esters, phospholipids, or proteins. Because of these variations and the inconsistent nature of native starches, it is frequently problematic to use in food formulations, and therefore, we need to modify them. Modified starches are changed with a chemical or physical method to improve their functional properties. Native starches do not always have good pasting or gelling properties, whereas, with modification, starch functionality is improved, including resistance to heat treatment, shear, or acidic treatments. Modification of starches includes crosslinking, derivatisation, acid thinning (depolymerisation), pre-gelatinisation, oxidation or combining these methods. In crosslinked starch, linking reagents react with OH- groups from two adjacent molecules within the same granule and merge two chains. This process adds acid and heat stability to starches, improving viscosity and water-holding capacity. In derivatised starches, hydroxyl groups are modified, usually using an esterification or etherification reaction by adding bulky groups to the structure (see carbohydrate reactions in the previous section). Derivatisation interrupts linearity, reduces the association of chains, changes hydrophobicity, reduces retrogradation, and increases water binding capacity and the tendency of the paste to gel. Introducing hydrophobic groups imparts emulsification properties (e.g., esterification with octenyl succinic anhydride (OSA-starch)) and alters clarity or dispersions. Acid-thinned starches are hydrolysed using acid to produce smaller chains with a concomitant reduction in viscosity. Oxidised starches are produced by the reaction of starch with oxidising agents, usually hypochlorite. Oxidised starch exhibits low viscosity due to depolymerisation and improved stability of starch dispersions.

Native starch granules are not soluble in cold water but may absorb water through the hilum and swell. When heated in water, granules go through an event that is called gelatinisation. Gelatinisation is the disruption of the molecular order within the granules, resulting in loss of crystallinity, amylose leaching, and starch solubilisation. The initial temperature and the range over which it occurs are governed by factors such as starch type, starch-to-water ratio, pH, or concentration. It happens over a temperature range usually within ~55–95 °C, resulting in a starch paste. The above events are followed using a graph that shows the viscosity of starch dispersion as a function of temperature (heating and cooling) and is called the pasting curve (Fig. 2.24). During the swelling stage, the viscosity of the dispersion is initially low, but it increases rapidly as the starch granules swell. After peak viscosity has been reached, the granule bursts and viscosity drops rapidly. As mentioned, starch properties and other formulation factors control the paste’s viscosity. After gelatinisation has been completed, cooling of starch paste results in retrogradation, which refers to the recrystallisation of amylose and amylopectin. Gelatinisation is commonly confused with gelation but refers to entirely different physicochemical events. Gelatinisation occurs only in starch, as described above, but gelation is a general property of polysaccharides, proteins, lipids, or their mixtures to form a three-dimensional network (a gel) that can hold large amounts of water (i.e., a hydrogel) or oil (i.e., an oleogel).

A line graph of viscosity versus temperature. At the bottom of the graph heating, hold, and cooling stages are mentioned. At the top of the graph swelling, disruption, and aging stages are mentioned. The trend ascends to a peak under swelling, descends to a trough under disruption and ascends under ageing.

Fig. 2.24

Gelatinisation events and the pasting curve of starch. Starch granules swell and eventually burst, and viscosity decreases. In cooling, amylose and amylopectin aggregate in a process known as retrogradation

Amylose recrystallisation proceeds much faster than amylopectin and is the major reason for the observed quality defects in starch-containing products. For example, retrogradation is responsible for the staling of bread and bakery products and for the loss of viscosity and precipitation in soups and sauces that contain starch. Retrogradation is a common defect of gluten-free formulations that are heavily based on starch and results in rapid texture deterioration and short shelf life. Waxy starches, i.e., starches that contain very low amounts of amylose, do not show appreciable retrogradation. Utilisation of modified starches may eliminate or slow down retrogradation to such an extent that it has negligible impact on the quality and shelf life of the starch-containing product. Native starches do not form pastes in cold water and, therefore, require heating and go through gelatinisation, as described above. Pregelatinisation is a type of physical modification, i.e., without the use of chemical reagents that results in starch that can be dispersed in cold water. Pregelatinised starch is formed upon starch heating in water until complete gelatinisation, followed by drying and grinding to a fine powder. It usually forms paste in cold water at concentrations between 1–5%. This type of starch is used in instant puddings, pie fillings, soup mixes, salad dressings, confectionery and as a binder in meat products. The nutritional value is the same as that of the original starch. Resistant starch (RS) cannot be digested in the small intestine of healthy individuals. Depending on its properties, it is divided into four types, namely, RS 1 to 5. RS1 is physically inaccessible that digestive enzymes cannot come into contact, such as in the intact wholegrain unprocessed cereals, seeds and nuts (e.g., oats, rye, wheat, barley, semolina, corn, linseed, sesame) or legumes. RS2 is starch that digestive enzymes cannot break and occurs naturally, such as in uncooked potatoes, bananas, unripe fruits and legumes (e.g., lentils or beans). RS3 is starch formed when starch-containing foods are cooked and cooled again, as heating and cooling may increase the RS3 content due to retrogradation. RS4 starches are modified starches that resist digestion because digestive enzymes cannot cleave the glycosidic bonds. Finally, RS5 is starch complexed with lipids (V-type complexes) that may reduce starch digestibility.

A group of consumers are sensitive to a certain type of prolamins (e.g., coeliac disease) found mostly in the gluten of wheat flour, and consumption of wheat-based products results in gastrointestinal discomfort. The only treatment is to eliminate gluten from their diet. As a result, gluten-free bakery products require different approaches in their formulation. Most of them are formulated with a combination of starches supported by polysaccharides (e.g., xanthan or carboxymethyl cellulose) and plant proteins (e.g., pea or soy). The major problems of gluten-free products are low product volume, dense structure, and texture losses during storage. The first two result from the absence of gluten responsible for the desirable structure formation of bakery products. The third stems from the rapid retrogradation of starches used in the formulations. As a result, potato, tapioca, waxy maize, or modified starches are preferred, as they generally have low retrogradation rates. Including minor ingredients (e.g., lipids, salt, monosaccharides, enzymes etc.) is also used to retard retrogradation even further and improve texture and shelf life. Because of these formulation requirements, gluten-free products are usually higher in carbohydrates, fat, and NaCl than their regular counterparts.

Maltodextrins are oligosaccharides with up to 20 glucose units linked with α-(1 → 4) glycosidic bonds, whereas glucose syrups are starch hydrolysates with quite different properties. Both maltodextrins and glucose syrups are products of starch hydrolysis. The extent of hydrolysis is different and is measured in dextrose equivalents (DE). DE is a measure of the reducing sugars in the hydrolysate, and it ranges from 0–100%. Pure glucose has a DE of 100 (i.e., 100% reducing sugars), whereas starch has 0% (i.e., 0% reducing sugars). Maltodextrins with a DE of 10 would have 10% of the reducing power of glucose. We need to know the DE value, as it affects the functional properties of the resulting maltodextrins or glucose syrups (Fig. 2.25). Maltodextrins may be used as bulking or viscosity-enhancing agents or encapsulating various components (e.g., colours, flavours, or oils). Glucose syrups are used as sweeteners, texture modifiers or browning agents, and sugar crystallisation inhibitors in confectionery products.

A chart has a horizontal arrow with labels starch, maltodextrins, corn syrups and glucose. An inclined arrow from bottom to top is labeled low to high. Freezing point depression, osmolality, water solubility, hygroscopicity, water activity lowering, dextrose equivalents, browning, boiling point elevation and sweetness are marked.

Fig. 2.25

Changes in the functional properties of glucose-based ingredients with increasing dextrose equivalents (from starch to glucose). The intensity of each property increases from left (low) to right (high)

increasing.

2.5.2 Carrageenan

Carrageenan is a linear, sulphated galactan composed of alternating 3-linked β-d-galactose and 4-linked α-d-galactose or 4-linked 3,6-anhydro-α-d-galactose, thus forming its disaccharide repeating unit and is obtained from seaweeds (Fig. 2.26). The most industrially relevant types of carrageenan are κ-, ι-, and λ-carrageenan that have one, two, or three ester-sulphate groups (O-SO₃⁻) per repeating disaccharide unit, respectively. Carrageenan may form gels after heating to about 80 °C, followed by cooling to approximately 40–60 °C. They form chains resembling boiled spaghetti, termed random coils at high temperatures. On cooling, they undergo a process known as coil-to-double helix conformational transition, resulting in a gel. Potassium cations (K⁺) may help with helix-helix aggregation of adjacent chains containing sulphate groups. Gels are thermoreversible and melt on heating. Because of the presence of ester-sulfate that gives a negative charge to the polysaccharide at the pH of milk, carrageenan can interact strongly with positively charged amino acids of κ-casein of milk through ionic interactions. These interactions result in viscosity enhancement or structure formation that finds applications in many dairy products such as chocolate milk (e.g., cocoa particle stabilisation), dairy desserts or ice creams.

Structure of kappa carrageenan. Alpha D galactose, sulfate group on C 4, 3, 6 anhydro alpha D galactose and D enantiomer.

Fig. 2.26

Structure of κ-carrageenan. Yellow circle: α-d-galactose, 4S: sulfate group on C-4, 3,6 anh: 3,6-anhydro-α-d-galactose, D: d-enantiomer, n: number of repeating units. Structures of ι- and λ- carrageenan are very similar but with different amounts of ester-sulfate content. κ-Carrageenan has one, ι- two, and λ- three sulfate groups per disaccharide

2.5.3 Alginates

Alginates is the term used for the salts of alginic acid, but it also refers to all the derivatives of alginic acid and alginic acid itself. Alginates are structural components of brown seaweed present in the form of divalent salts of alginic acid and form the intercellular gel matrix. Alginates are linear polysaccharides that are composed of β-d-mannuronic acid (M) and α-l-guluronic acid (G) linked via 1 → 4 glycosidic linkages (Fig. 2.27). Accordingly, the alginate backbone consists of sequences of blocks of mannuronic acid (M-blocks) or guluronic acid (G-blocks) and regions of alternating sequences (e.g., MG, MMG, GGM). The M-blocks are linked via β-1 → 4 linkages adopting ⁴C₁ chair conformation that imparts flexibility to the chain. In contrast, G-blocks are rigid structures due to the ¹C₄ conformation of guluronate residues linked via α-1 → 4. As a result, the stiffness of the blocks on the backbone follows the order (from most to least rigid): GG > MM > MG. The most prominent physical property of alginates is the selective binding of divalent and multivalent cations, determining their ability to form gels. The egg-box model describes alginate gelation, according to which alginate chain–chain interactions are induced by cross-linking with divalent cations, most frequently Ca²⁺. Alginates may be used in dairy (e.g., creams and whipped creams, processed cheese) or in restructured food products (meat, fruits, vegetables, fish) as well as in the pharmaceutical industry (e.g., antacids or wound dressings).

Structure of alginates comprises the M-block, G-block, and M G block. Beta 4, alpha 4, n, D, and L are mentioned.

Fig. 2.27

Structure of alginates. Green/white: d-mannuronic acid, orange/white: l-guluronic acid, n: number of repeating units

Alginate Gelation

We have discussed that alginates may form gels with a mechanism described as the egg-box model. But how did we come up with this term? Guluronic acid units of two alginate chains that run antiparallel to each other create “holes” where Ca²⁺ can be embedded. This formation creates ionic cross-links between alginate chains as the positively charged calcium cations interact with the negatively charged carboxyl groups. A very similar mechanism also occurs in the gelation of LM-pectins. This chain formation resembles an egg box, with the calcium being the egg and the chains the box!

A chemical structure of alginate gel. It has guluronic acid units of two alginate chains with M and G blocks that run antiparallel to each other with C a 2 plus embedded in the holes. On the top right is a photo that presents spherical structures embedded in a series of spherical holes.

Polysaccharides have different ways of creating gels, but they are based on how chains interact. The forces that are involved are ionic (e.g., alginate gels), hydrogen bonds (β-glucan gels), or hydrophobic interactions (e.g., methylcellulose gels). Quite frequently, a combination of the above forces coincides. The quality of the final gel, such as its rigidity or water-holding capacity, is highly specific to the type of polysaccharide and depends on many factors, including the concentration of polysaccharide, pH, temperature, or molecular weight, to name a few.

2.5.4 Pectin

Pectin is a heteropolysaccharide belonging to a family of covalently linked galacturonic acid-rich polysaccharides. Pectin is obtained commercially from citrus fruits (e.g., oranges or lemons), apples and sugar beets. Pectin consists of two major structural classes, homogalacturonan (HG) and rhamnogalacturonan-I (RG-I), that are found in most pectins with the intra- and intermolecular interactions between these two segments controlling its functional properties (Fig. 2.28). Homogalacturonan is the most abundant and comprises long chains of linear 1 → 4 linked α-d-galacturonic acid residues (~200 units). Depending on plant species, some carboxyl groups are methyl-esterified at the C-6 position and acetyl-esterified at the O-2 and O-3 positions of the galacturonic acid. Rhamnogalacturonan-I (RG-I) is composed of the repeating disaccharide galacturonic acid and rhamnose [α-(1 → 2)-d-galacturonic acid–α-(1 → 4)-l-rhamnose]n where n can be greater than 100. RG-I segments usually have branches of galactose or arabinose that may also carry a significant amount of protein attached to them. Because of branching, the RG-I segment of the molecule is also known as the hairy region, whereas the HG segment that lacks branching is called the smooth region. Degree of esterification (DE) is the percentage of esterified galacturonic acid units out of the total number of galacturonic acid units present in pectin. Depending on DE value, pectins are divided into two commercially important groups with distinct functional properties: high methoxy pectins (HM) with DE >50% and low methoxy pectins (LM) with DE <50%. The most important distinguishing characteristic between HM and LM pectin is the mechanism of gel formation. HM pectin forms gels at pH <3.5 in the presence of high concentrations of sucrose. The gel is stabilised through hydrophobic interactions due to sucrose-induced dehydration of pectin chains. In contrast, LM pectin forms gels at a wider pH range; however, it does not require sucrose but needs Ca²⁺. This mechanism is similar to that of alginates, as Ca²⁺ forms ionic bridges between different pectin chains. Amidated pectin is a modified pectin with some galacturonic acid residues converted with ammonia to an amide. These pectins form gels with a similar mechanism to LM-pectin and are used in special applications. Pectin is used in many foods, including jams, bakery fillings, confectionery, tomato-based products, beverages, and low-pH milk products.

Structure of pectin comprises the homogalacturonan and rhamnogalacturonan 1. Alpha 4 and beta 4 bonds are mentioned.

Fig. 2.28

Structure of pectin. Yellow/white diamond: galacturonic acid, green triangle: rhamnose, green star: arabinose, yellow circle: galactose, OMe: methyl ester, OAc: acetyl ester, f: furanose

2.5.5 Cellulose

Cellulose is the major cell-wall component of plants and is composed of d-glucose connected via β-(1 → 4) glycosidic bonds (Fig. 2.29). Cellulose is insoluble in water and indigestible by human digestive enzymes, making it the major component of dietary fibre. Although cellulose is insoluble in water, it can be converted into a water-soluble polysaccharide with appropriate substitutions. These water-soluble polysaccharides are collectively known as cellulose derivatives. Examples are carboxy-methyl-cellulose (CMC), where a carboxymethyl group is attached after etherification and can be used to control ice crystal growth in ice creams. Methylcellulose (MC) is cold-water-soluble and forms thermoreversible gels on heating, making it insoluble in hot water. MC gels are stabilised by hydrophobic interactions between methyl groups belonging to different chains. Many vegetarian and vegan “burgers” cannot be formed in the absence of MC or similar cellulose derivatives. A plant-protein-based burger disintegrates on heating, leaving behind an undesirable structure with poor sensory attributes because plant proteins do not have the same structuring ability as those from meat (e.g., myosin and actin). In the presence of MC, a thermoreversible gel is formed on heating (i.e., cooking), holding the formulation together. On cooling to eating temperature (~30 °C), the gel melts and returns to a viscous liquid state, and the solid gel is not detectable during consumption. Using the same principle, MC is used in a range of products that are deep-fried before consumption (e.g., chicken nuggets, fish fingers, etc.) to preserve moisture and desirable textural properties. Microcrystalline cellulose (MCC) is partially depolymerised cellulose that is water-soluble and can be used as a non-caloric filler in various food products.

Structure of cellulose in linear form. Beta 4 bonds are mentioned.

Fig. 2.29

Structure of cellulose. The only difference between amylose and cellulose is that cellulose has glucose in the β anomeric configuration. This difference results in substantial chemical and physical properties between the two. Blue circle: glucose

2.5.6 Galactomannans

Galactomannans are seed polysaccharides commercially obtained mostly from guar and carob (locust bean) having a backbone of β-(1 → 4)-linked mannose with galactose branches linked with α-(1 → 6) glycosidic bonds (Fig. 2.30). The distinguishing feature between different galactomannans is the mannose-to-galactose ratio (M/G), which is characteristic of each source. The ratio ranges between 2:1 and 4:1 for guar or carob gum. Galactomannans form viscous solutions at low concentrations and can be used with xanthan or carrageenan to improve the stabilising or gelling properties of food formulations. They are commonly used in ice cream, where they prevent ice crystal growth on storage and slow meltdown, in salad dressings to improve viscosity and water holding capacity or in soft cheese products, where they improve spreading.

Structure of galactomannans in branched form. Beta 4 bonds are mentioned.

Fig. 2.30

Structure of galactomannans. Green: mannose, yellow: galactose

2.5.7 Gum Arabic

Gum Arabic (Acacia gum) is a tree exudate obtained from the Acacia tree species (A. senegal or A. seyal). It is a highly branched heteropolysaccharide with β-(1 → 3) galactose forming the backbone and branches at the C6 position consisting of galactose, arabinose, rhamnose and glucuronic acid. This structure is attached to a protein component, which is crucial to its functionality (Fig. 2.31). The presence of protein makes gum Arabic the most technologically important polysaccharide-based emulsifier. In particular, it is highly water-soluble and forms low-viscosity solutions even at very high concentrations (20–30%). Because of its low viscosity, it can emulsify flavour oils (e.g., orange oil) in the confectionery and beverage industry (e.g., fruit-flavoured beverages) without giving a slimy texture during beverage consumption.

A schematic presents a thread-like structure labeled protein wounded over oval-shaped structures of polysaccharides.

Fig. 2.31

Schematic representation of gum Arabic structure. Gum Arabic has a compact shape, allowing it to have low viscosity at high concentrations

2.5.8 Xanthan

Xanthan is a heteropolysaccharide produced by the bacterial fermentation of Xanthomonas campestris. It has a cellulose backbone, and every alternate glucose residue has a three-sugar side chain consisting of two mannose residues with a glucuronic acid residue between them. The mannose nearest the main chain may carry a C6 acetyl group, and the terminal mannose may carry a pyruvate group between C4 and C6 (Fig. 2.32). If this structure description seems complex, you can consider xanthan as cellulose with trisaccharide branches. This structure results in very stiff chains with limited capacity to fold when in solution. As a result of its stiff structure, the most important characteristic of xanthan is that it forms highly viscous solutions at very low concentrations, as low as 0.01%, depending on the formulation. It is used as a stabiliser in emulsions (e.g., mayonnaise, salad dressings, etc.) or bakery products in gluten-free formulations.

Structure of xanthan in branched form. Beta 4 and alpha 3 bonds are mentioned.

Fig. 2.32

Structure of xanthan. Blue circle: glucose, green circle: mannose, blue/white diamond glucuronic acid, Pyr: pyruvic acid, OAc: acetyl group

2.5.9 Chitin

Chitin, consisting of N-acetylglucosamine, is an amide and acetylated derivative of glucose (Fig. 2.33a). These carbohydrates are known as amino sugars, in which an amine group replaces a hydroxyl group, usually in position 2. Chitin (Fig. 2.33b) is the primary component of cell walls in the exoskeletons of arthropods, such as crustaceans (e.g., shrimp shells, squid pen) and insects. Deacetylation of chitin in alkaline environments removes acetyl groups from N-acetylglucosamine to form d-glucosamine, which results in chitosan formation. Chitosan is the only positively charged polysaccharide compared to the other polysaccharides that are either neutral or negatively charged. Chitin and chitosan belong to the group of polysaccharides known as GAGS (GlycosAminoGlycanS). Chitosan has very limited commercial uses in foods but finds applications in the pharmaceutical and biomedical industries.

Two chemical structures. A. N-acetyl glucosamine. B. Structure of chitin in linear form. It is composed of N-acetyl glucosamines bonded via beta 4 linkages.

Fig. 2.33

(a) Structure of N-acetyl glucosamine, and (b) structure of chitin. The white square is N-acetylglucosamine. Chitin has very limited food applications

2.5.10 Dietary Fibre

The term dietary fibre (DF) keeps changing as new scientific evidence is presented in chemistry, nutrition, and human physiology. Depending on the emphasis, they may be classified based on their ability to ferment in the large intestine or their solubility in aqueous buffers. The common characteristic of DF is that they are not hydrolysed by the endogenous enzymes in the small intestine of humans and exhibit beneficial health effects. Regardless of the specific definition, dietary fibres are the following compounds: cellulose, hemicelluloses, pectin, resistant starch, and lignin. The structure and major properties of cellulose, pectin and resistant starch have been described in the previous sections. Hemicelluloses are heteropolysaccharides with diverse structures and form part of the cell wall. The term “hemicelluloses” may create confusion, and one may attempt to find structural similarities with cellulose. However, hemicelluloses have a different structure than cellulose, and depending on the monosaccharide that forms the backbone of the molecule, they may be xylans (e.g., arabinoxylans), mannans (e.g., glucomannans), or glucans (e.g., β-glucan). Hemicelluloses can be linear (e.g., β-glucan) but frequently have branching points. For example, in arabinoxylans, the backbone consists of xylose and the branches of arabinose. Lignin is also part of the cell wall, but it is not a polysaccharide and is not fermented in the colon. Lignin is a highly heterogeneous polymer of oxygenated phenylpropane derivatives, including coniferyl, sinapyl and p-coumaryl alcohols (Fig. 2.34). Lignin has diverse structures and molecular weight and does not have a single chemical formula. Dietary fibres may be classified into two general groups based on their water solubility: soluble dietary fibre (SDF) and insoluble dietary fibre (IDF), with entirely different properties and challenges during food formulation. For example, SDFs increase the viscosity of the formulation in contrast to IDFs with limited capacity to enhance viscosity. Generally, formulating foods with ingredients enriched in IDF creates technological problems stemming from their limited hydration, inhomogeneous dispersion, or poor interactions with other food compounds. Frequently, final products have unacceptable textures and limited shelf-life stability compared to low IDF counterparts. Consequently, understanding the chemical structure of polysaccharides and how they interact with other food compounds is important in high DF food formulation.

4 bond line structures of the various lignan derivatives. It includes phenylpropane, p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, and lignins.

Fig. 2.34

Lignins are polymers of phenylpropane derivatives (e.g., coniferyl, sinapyl, and p-coumaryl alcohols)

Polysaccharides and Health

We have seen that the dietary fibres are chemically polysaccharides and lignin. While these polysaccharides cannot be digested, they can be fermented by our gut microbiota. Gut microbiota are the microorganisms that live in our gut, mainly in the colon. These microorganisms ferment dietary fibre into short-chain fatty acids (SCFAs), such as acetic or butyric acid, with many positive health benefits. For instance, butyric acid benefits colon health by providing energy for the cells in the colon lining and maintaining the integrity of the gut, thus reducing the risk of inflammatory bowel diseases and promoting overall gut health. The main microorganisms in our gut belong to the phyla Bacteroidota and Bacillota. Each of them has a different capacity and specificity in fermenting polysaccharides. The balance between these two classes in the gut depends on our diets and lifestyle, which differs among individuals. Besides providing health benefits as substrates for microbial fermentation, polysaccharides may benefit health through their physiochemical properties. For example, viscosity build-up from polysaccharides can slow gastric emptying (i.e., feeling fuller for longer) and physically improve bowel function. Additionally, they may bind bile acids in the small intestine, which may reduce fat absorption or regulate blood sugar levels by slowing down nutrient absorption in the small intestine.

2.6 Review Questions

2.6.1 Multiple Choice Questions

Monosaccharides

1.

An aldohexose is:

(a)

A monosaccharide with an aldehyde group and five carbons

(b)

A monosaccharide with a ketone group and six carbons

(c)

A disaccharide with an aldehyde group and five carbons

(d)

A monosaccharide with an aldehyde group and six carbons

2.

Two structures that have a chiral carbon in their structure are

(a)

Non super-imposable mirror images of each other

(b)

Non super-imposable mirror images of each other at the highest numbered chiral carbon

(c)

Super-imposable mirror images of each other

(d)

Super-imposable mirror images of each other at the highest numbered chiral carbon

3.

On a Fischer projection d-carbohydrates are those that:

(a)

On the last chiral carbon from the carbonyl carbon –OH group is positioned on the right

(b)

On the last chiral carbon from the carbonyl carbon –OH group is positioned on the left

(c)

On the last chiral carbon from the carbonyl carbon –OH group is positioned on the left ONLY in hexoses

(d)

On the last chiral carbon from the carbonyl carbon –OH group is positioned on the right BUT ONLY in those that have aldehyde group (i.e., glucose)

4.

In alpha anomers in a Howarth projection, the –OH group is:

(a)

below the plane of the ring in d-sugars

(b)

above the plane of the ring, in d-sugars

(c)

below the plane of the ring, in anhydro sugars

(d)

below the plane of the ring, in l-sugars

5.

The most stable conformation of a pyranose-forming sugar is:

(a)

The boat conformation

(b)

The chair conformation

(c)

The cable conformation

(d)

The coil conformation

6.

A uronic acid is

(a)

An acid that results from the oxidation of a monosaccharide with urea

(b)

An acid that results from the reduction of a monosaccharide with urea

(c)

An acid that results from the oxidation of terminal -OH of a monosaccharide

(d)

An acid that results from oxidation of -OH at C1 of a monosaccharide

7.

Aldonic acid is:

(a)

a carboxylic acid after oxidation of the ketone group of aldoses

(b)

a carboxylic acid after oxidation of the aldehyde group of aldoses

(c)

a carboxylic acid after oxidation of the aldehyde group of ketoses

(d)

a carboxylic acid after oxidation of the ketone group of ketoses

8.

In reducing sugars

(a)

The anomeric carbon is free to react, e.g., as in glucose

(b)

The anomeric carbon is free to react, e.g., as in sucrose

(c)

The chiral carbon is free to react, e.g., as in glucose

(d)

The anomeric carbon esterifies, e.g., as in glucose

9.

Reduction converts monosaccharides to

(a)

Alditols

(b)

Uronic acids

(c)

Esters

(d)

Ethers

10.

A glycosidic bond is the bond between two or more:

(a)

glucuronic acids

(b)

monosaccharides

(c)

reducing sugars ONLY in the presence of oxidative enzymes

(d)

monosaccharides ONLY when they exist in Howarth projection

Polysaccharides

1.

Homo- and hetero-polysaccharides are composed of:

(a)

One or several different monosaccharides, respectively

(b)

Several different or one monosaccharide, respectively

(c)

Linear or branched chains, respectively

(d)

Branched or linear chains, respectively

2.

Starch consists of:

(a)

amylose and pectin

(b)

polyamylose and glucose

(c)

amylose and amylopectin

(d)

amylase and amylopectinase

3.

Starch is made up of

(a)

Glucose connected with (1,4) and (1,6) glycosidic bonds

(b)

Glucose and fructose connected with (1,4) and (1,6) glycosidic bonds

(c)

Glucose connected with (1,4) glycosidic bonds

(d)

Glucose connected with (1,6) glycosidic bonds

4.

Modified starches can:

(a)

Slow retrogradation

(b)

Act as emulsifiers

(c)

Improve pasting characteristics

(d)

All of the above

5.

Starch gelatinisation is:

(a)

The disruption of the molecular order within starch granules

(b)

Formation of gel at high starch concentrations

(c)

Gelation of modified starches during heating

(d)

Formation of viscous solution that improves texture

6.

Starch retrogradation is mostly due to

(a)

recrystallisation of amylose during cooling

(b)

recrystallisation of amylopectin during cooling

(c)

recrystallisation of amylose during heating

(d)

recrystallisation of amylopectin during heating

7.

κ-carrageenan:

(a)

has ester sulfate content of ~50% and does not form gels

(b)

has ester sulfate content of ~25% and forms gels

(c)

has ester sulfate content of ~10% and does not form gels

(d)

has ester sulfate content of ~30% and forms gels

8.

The gel formation mechanism of alginates is known as:

(a)

Cardboard box model

(b)

Corrugated cardboard model

(c)

Eggbox model

(d)

Pizza box model

9.

Pectin

(a)

consists of guluronic acid and methylated guluronic acid

(b)

consists of galacturonic acid and amylopectin

(c)

consists of galacturonic acid and methylated galacturonic acid

(d)

consists of amylose, amylopectin and galacturonic acid

10.

Common cellulose derivative substituent groups are:

(a)

Carboxy-ethyl, ethyl, hydroxy-propyl, hydroxy-propyl-ethyl

(b)

Carboxy, methyl, hydroxy-, hydroxy-acetyl

(c)

Carboxy-methyl, methyl, hydroxy-propyl, hydroxy-propyl-methyl

(d)

Carboxy-acetyl, acetyl, hydroxy-acetyl, hydroxy-propyl-acetyl

2.6.2 Short Answer Questions

1.

Define all the different stereoisomers that occur in monosaccharides. Draw a pair of structures in each case and identify their differences.

2.

Draw the Fischer projection of the pairs d-glucose/d-mannose and d-ribose/d-arabinose. Number the carbons correctly, and identify the chiral carbons and the carbonyl groups.

3.

Write the reaction of hemiacetal formation. What is the consequence of this reaction in monosaccharides?

4.

Draw the Howarth projection of α-d-glucose and β-d-glucose. Number the carbon atoms from 1 to 6 correctly and identify the anomeric carbon. Try the same with α-l-glucose and β-l-glucose.

5.

Define the following terms: “optical rotation”, “dextrorotation”, and “laevorotation”. What is the difference between the prefixes d- and d- and l- and l-?

6.

Draw the chair conformation of α-d-glucose. Identify the axial and equatorial bonds and the plane of symmetry. What do ⁴C₁ and ¹C₄ notations mean? Check the structure of the α-d-glucose you have just drawn. Is it ⁴C₁ or ¹C₄?

7.

Draw the aldaric, glucaric and uronic acid or glucose and galactose in the Fischer projection.

8.

Draw two glucose molecules in their Howarth projection and link them with (a) 1 → 4, (b) 1 → 6, and (c) 1 → 2 glycosidic bonds.

9.

Draw raffinose and identify the type of glycosidic bonds in the structure.

10.

Draw xylitol and discuss the reaction used to form it.

11.

Draw the structure of maltose and sucrose and identify their anomeric carbon. Explain why sucrose is not a reducing sugar.

12.

Draw the structure of amygdalin, identify the type of bonds, and discuss its structure. Name three flavonoid glycosides.

13.

Online activity: Find the structure of steviol glycosides online and identify its aglycone.

14.

Online activity: Find online the terms “birefringence” and “Maltese cross” and their relationship to starch.

15.

Draw the structure of chitin and identify the acetyl group, the amine group, and the type of glycosidic bond in the structure.

16.

Draw and describe the structure of amylopectin. Identify the type of glycosidic bonds on the structure.

17.

Draw the structure of hydroxy-propylated starch and OSA-starch. Identify the functional groups and the positions where the substitution occurs.

18.

Name and describe the different types of resistant starch.

19.

Draw and describe the starch pasting curve.

20.

Draw and discuss the gelation mechanism of alginates and LM-pectin. Name two foods that use pectin gels.

21.

Name the major monosaccharides of guar gum, carrageenan, and xanthan. Name two food applications of each.

22.

What are maltodextrins, and what is dextrose equivalent?

23.

What are hemicelluloses? Draw the structure of arabinoxylan and identify the anomeric carbons.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024

V. KontogiorgosIntroduction to Food Chemistryhttps://doi.org/10.1007/978-3-031-53558-1_3

3. Proteins-Enzymes

Vassilis Kontogiorgos1  

(1)

Associate Professor in Food Chemistry, University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC, Canada

Vassilis Kontogiorgos

Email: v.kontogiorgos@ubc.ca

Learning Objectives

After studying this chapter, you will be able to:

Classify amino acids based on their chemical structure

Describe the pH-dependency of amino acid and protein charge

Classify food proteins based on their solubility and source

Discuss the different levels of protein structure

Discuss protein solubility, denaturation and hydrolysis

Discuss the functional properties of proteins in food processing

Discuss the proteins of the main food commodities

Describe the mechanism and the factors that influence enzymatic reactions

Describe the action of major food enzymes on their substrates

3.1 Introduction

Proteins are macromolecules with numerous biological functionalities in living tissues responsible, for example, for energy production, food digestion, or muscle contraction. From a nutritional point of view, they supply essential amino acids that are nutrients necessary to sustain the life and functionality of the body. Food proteins must be extractable in high yields to be further processed into ingredients or incorporated into food formulations. For example, while potato protein is generally of high biological value, as it is rich in essential amino acids, the amounts found in potatoes are negligible, making it difficult to isolate large quantities of commercial interest. In contrast, whey proteins, also rich in essential amino acids, can be easily collected during cheese manufacturing and processed to the appropriate form for further use (e.g., whey protein powder). Proteins used for food are of animal or plant origin. They can be consumed with minimal processing from their natural source (e.g., a glass of milk or a steak), extracted with suitable technologies and used as an ingredient in food formulations (e.g., whey protein in a cake mix), or processed into unique products (e.g., cheese, cold hams, or tofu). Animal protein sources are primarily meat, milk, eggs, and fish. Soy is the most common plant-based protein source, but other plant sources like peas, beans, or lentils are being explored. The need for high-quality protein with a suitable nutritional profile and functionality has resulted in intense research interest in identifying new protein sources such as those extracted from microorganisms (microbial protein), insects, algae or even lab-grown protein (cultivated meat).

Food-protein science is distinct from classic protein biochemistry encountered in other fields of science (e.g., human or animal biology), as it is mainly concerned with the properties of proteins from a material science perspective. For example, a food scientist should manipulate and control structures created with proteins in foods (e.g., gels or emulsions) to enhance shelf-life stability or improve textural properties. As a result, the tools and techniques needed to investigate protein functionality are broader than those encountered in classic biochemistry (e.g., amino acid sequencing, structure characterisation, etc.). They include techniques used in material sciences, for example, rheology, calorimetry or particle size distribution analysis. Another notable difference is the scale and purity of the protein. A biochemist is usually concerned with a few milligrams of pure protein, which has been isolated under precisely controlled conditions. In contrast, a food-protein scientist deals with kilograms of protein at a time that operates in highly complex formulations that may simultaneously include sugars, lipids, and other compounds. Regardless of the different approaches, the principles of protein science derived from fundamental biochemistry must be well-understood before further exploring protein functionality.

3.2 Amino Acids

Amino acids are the building blocks of proteins. They consist of an amino group (-NH₂), a carboxyl group (−COOH), a hydrogen atom, and a side chain (R-) linked to a carbon atom called α-carbon (Fig. 3.1a). Amino acids are chiral when R- is not hydrogen, which occurs only in glycine. As a result, amino acids are found in enantiomeric forms d- and l-, similarly to carbohydrates, and it is possible to draw them using a Fisher projection. In amino acids, the distinguishing characteristic in the Fisher projection is the positioning of the -NH₂ group when the −COOH group is placed at the top of the structure (Fig. 3.1b). If −NH₂ is located on the left of the structure, the amino acid is l-, whereas if it is on the right, it is d-. Unlike natural sugars found in a d-configuration, most natural amino acids are in an l-configuration.

a. A structure has a central carbon atom, which is double bonded to O and single bonded to O H and C H. C H is further bonded to C H 3 and H 2 N. b. 2 structures of D alanine and L alanine have a 2-carbon chain. C1 is double bonded to O and single bonded to O H. C2 is bonded to H, C H 3, and H 2 N.

Fig. 3.1

(a) Structure of α-amino acids. α-carbon has four different substituent groups and is chiral (*), and (b) Fischer projection of d- and l-alanine

The chemical properties of the side chains are used to classify amino acids. The most usual classification scheme is based on their capacity to interact with water, resulting in four classes, i.e., non-polar, polar, acidic, and basic. Non-polar amino acids (also called hydrophobic) contain mostly hydrocarbon R-groups and do not have either positive or negative charges (Fig. 3.2). Two types of hydrocarbon side chains are found in this group: aromatic (i.e., tryptophan and phenylalanine) and aliphatic (i.e., glycine, cysteine, methionine, alanine, valine, leucine, isoleucine, and proline). Non-polar amino acids interact poorly with water but strongly with each other through hydrophobic interactions. Notably, amino acids can be abbreviated using one- or three-letter codes, which is quite helpful when describing the amino acid sequence of proteins (Fig. 3.2).

10 bond line structures of amino acids. The compounds are glycine, alanine, valine, cysteine, proline, leucine, isoleucine, methionine, tryptophan, and phenylalanine.

Fig. 3.2

Non-polar amino acids. The one- and three-letter abbreviations are shown in parentheses

Polar amino acids (hydrophilic) have −OH or amide groups that may easily form hydrogen bonds with water (i.e., serine, threonine, tyrosine, asparagine, and glutamine) (Fig. 3.3). Acidic amino acids (i.e., aspartic acid, glutamic acid) are negatively charged, whereas basic amino acids (i.e., lysine, arginine, histidine) are positively charged at pH 7.0 and may interact with each other through ionic interactions (Fig. 3.4).

5 bond line structures of the amino acids. The compounds are serine, threonine, glutamine, tyrosine, and asparagine.

Fig. 3.3

Polar amino acids. The one- and three-letter abbreviations are shown in parentheses

5 bond line structures of the charged amino acids. The compounds are lysine, arginine, histidine, aspartic acid, and glutamic acid. The abbreviations of the compounds are L y s slash K, A r g slash R, His slash H, A s p slash D, and Glu slash E.

Fig. 3.4

Positively (Lys, Arg, His) and negatively (Asp, Glu) charged amino acids at pH 7.0. The one- and three-letter abbreviations are shown in parentheses

Amino acids may also be classified according to their nutritional significance into essential, non-essential (A, D, N, E, S) or conditionally essential. This classification may be necessary for some food formulations to create nutritionally balanced food. Essential amino acids are those that the body does not synthesise and must be obtained from the diet (H, I, L, K, M, F, T, W, V). Conditionally essential are those that are essential under certain circumstances, e.g., disease or early development (R, C, Q, G, P, Y). A limiting amino acid is an essential amino acid found in insufficient quantities. Most plant proteins have a limiting amino acid (e.g., lysine in grains, nuts, and seeds, threonine in grains, methionine in beans and tryptophan in corn).

An important characteristic of amino acids is that their ionisation state changes depending on the pH. For that reason, they are called amphoteric, which means they can react as acids or bases and form zwitterions (dipolar ions). The amino group is ionised at acidic conditions (low pH), and the amino acid carries a positive charge. In contrast, the carboxyl group is ionised at alkaline conditions (high pH), and the amino acid has a negative charge. The isoelectric point (pI) is the pH value at which amino acids have a zero net charge, as they exist primarily as zwitterions (Fig. 3.5). The exact pH at which the transition occurs (from positively to zwitterion to negatively charged) depends on its structure and the pKa values of each amino acid. Amino acids have up to three pKa values: one for the α-carboxyl group (pKCOOH), one for the α-amino group (pKNH3⁺) and one for the side chain (pKR). We can demonstrate the changes in amino acid ionisation using alanine with pKCOOH = 2.4 and pKNH3⁺ = 9.7 (Fig. 3.5). We start with alanine dissolved in a buffer with pH 1.0. At this pH, alanine is positively charged, as the pH is below pKCOOH and pKNH3⁺. We then add NaOH to increase pH to just above 2.4. At pH > 2.4, the hydrogen from the α-carboxyl group dissociates, and the amino acid acquires a negative charge in addition to the positive charge already in the structure (Fig. 3.5). We then keep adding NaOH until the pH > 9.7. Above this pH, the dissociation of hydrogen from the α-amino group results in the loss of the positive charge and the amino acid acquires a net negative charge. At pH values between 2.4 and 9.7, alanine is a zwitterion, and the net charge is nearly zero. The isoelectric point of amino acids can be calculated using the following equations:

Using the first equation and the pKa values shown above, the isoelectric point of alanine is 6.0. The pKCOOH pKNH3⁺ and pKR values of each amino acid can be easily found online. The relationship between pH and ionisation of amino acids is very important, as it also affects the properties of proteins and needs to be well understood before proceeding any further.

A reversible reaction of the amnio acid. The acidic compound reacts in the presence of an N a O H addition to yield an isoelectric point, which further reacts to form alkaline conditions. The alkaline forms the acid with the addition of H C l.

Fig. 3.5

Amphoteric nature of amino acids. The charge depends on pH. The isoelectric point is the pH at which the net charge is zero

The α-carboxyl and α-amino groups of amino acids have similar chemical reactivity and undergo the reactions common to these functional groups (e.g., esterification or reactions with ammonia). The reactivity of side chains is, however, a more important characteristic. The reason is that when amino acids are incorporated into proteins, the α-carboxyl and α-amino groups have already reacted in the formation of the peptide bond, and they are not available for further reactions (see below for peptide bond formation). As a result, only groups on the side chains are available for reactions. Reactivity is important in food formulation because amino acids react with other components and modify food properties during processing and storage. Peptide bond, disulfide bond, and Schiff base formation are the most relevant reactions.

Amino acids are linked via a bond called peptide bond. This bond is formed between the α-carboxyl and α-amino groups of two amino acids (same or different) and proceeds via a condensation reaction (i.e., loss of water) (Fig. 3.6). Following peptide bond formation, one end of the newly formed molecule will carry the −NH₂ group (N-terminal) and the other the −COOH group (C-terminal). By convention, the N-terminal is drawn on the left and the C-terminal on the right.

A chemical reaction of the amino acids. 2 amino acids react with each other to form a polypeptide with the byproduct of H 2 O.

Fig. 3.6

Peptide bond formation between two amino acids

When two amino acids are linked through a peptide bond, the product is called a dipeptide, three a tripeptide, etc. As a guideline, when up to ~50 amino acid residues are linked, the chain formed is termed polypeptide, whereas when it consists of more than ~50, it results in protein. Peptides are commonly formed during food fermentations with desirable or undesirable effects on food flavour. In some instances, bitter peptides may form (e.g., the tripeptide leucine-leucine-leucine), resulting in detrimental sensory attributes and product losses. Bioactive peptides may also be formed during fermentation or be naturally present in foods that may provide health benefits (e.g., casein hydrolysates). Protein hydrolysates resulting in complex peptide composition are commonly used as flavour enhancers (see Chap. 8). Some peptides, such as the dipeptide aspartame, may taste sweet and are used as artificial sweeteners. Bacteria produce bacteriocins that are peptides or small proteins with antibiotic properties. Nisin is an approved bacteriocin that is frequently used as a food preservative.

The sulfhydryl group (-SH) of cysteine is highly reactive, and -SH groups of two cysteines oxidise easily to form cystine (Fig. 3.7). The bond between two -SH groups is called disulfide bond or disulfide bridge. It is important for the stability of proteins and various technological applications of food ingredients. For example, the bread-making capacity of certain flours is entirely controlled by the ability of flour proteins (gluten) to form disulfide bonds with each other.

A chemical reaction of the cysteines. 2 cysteines react with each other to form a cystine with the byproduct of 2 H plus + 2 e negative. The bond line structures of the compounds are given.

Fig. 3.7

Disulfide bond formation in cysteine. The resulting dipeptide is called cystine. Notice the spelling difference

Free amino groups of amino acids may react with aldehydes or ketones to form Schiff bases (imines) (Fig. 3.8). An imine is a chemical compound that contains a carbon-nitrogen double bond. Schiff bases are frequently encountered in foods, as amino acids and reducing sugars are almost guaranteed. They are intermediate products of Maillard reactions, resulting in compounds with intense colour and odour (see Chap. 6). In addition, the formation of lysine-Schiff bases during the Maillard reaction removes lysine from food and reduces its nutritional quality, as lysine is an essential amino acid. A final reaction worth mentioning is the reaction of amino acids with ninhydrin used to detect and quantify free amino acids.

A chemical reaction of the lysine. The carbonyl group reacts with the lysine amnio acid to form the Schiff base with the byproduct of H 2 O + H plus. The bond line structures of the compounds are given.

Fig. 3.8

Schiff base formation from lysine. Amino groups of amino acids may react with carbonyl groups to form compounds with a carbon-nitrogen double bond

3.3 Proteins

3.3.1 Protein Classification

Proteins

have the most diverse functionality compared to the other molecules encountered in food and may be classified depending on their functionality, solubility, composition or shape (Table 3.1). Other classification schemes may be possible, but these presented cover the main classes relevant to food chemistry.

Table 3.1

Protein classification relevant to foods based on different characteristics. See text for examples

Functionality

Solubility

Composition

Shape

Enzymes

Albumins

Glycoproteins

Globular

Structural proteins

Globulins

Haemoproteins

Fibrous

Contractile proteins

Glutelins

Lipoproteins

Storage proteins

Prolamins

Metalloproteins

Protective proteins

Phosphoproteins

Proteins can be classified based on their function in the living organism. Enzymes are important in food processing and preservation and are discussed in the following section. Structural proteins are proteins such as keratin or collagen in animals’ skin. Collagen, after appropriate processing, is converted to gelatin, which is a commonly used food ingredient. Contractile proteins such as myosin or actin are found in muscle tissues and are responsible for muscle contraction. They are the main proteins ingested during the consumption of products of animal origin. For example, the generic term “steak” refers to a type of meat that is sliced across the muscle fibres of the carcass, and it essentially consists of myofibrillar proteins (myosin and actin ~50%), sarcoplasmic proteins (enzymes and myoglobin ~30%) and connective tissues (e.g., collagen ~15%). Storage proteins (seed proteins or egg albumen) include most proteins used in the food industry. Protective proteins (toxins, allergens) may be of significance for allergic individuals (e.g., soy or gluten peptides) or in case they cause some form of disease (e.g., botulism). Finally, hormones (e.g., insulin or growth hormone), transfer proteins, and antibodies are proteins responsible for various metabolic activities in the body but do not have any functionality in food formulation.

Proteins are also classified depending on their solubility, known as the Osborne protein classification of storage proteins. This classification is important because it allows the fractionation and isolation of most food proteins. Using this scheme, proteins are classified into four groups. Albumins are soluble in neutral salt-free water, whereas globulins are in neutral salt solutions. Glutelins are soluble in dilute acid or alkaline solutions, and prolamins are soluble in ethanol. While most food proteins are generally soluble in aqueous solutions, glutenin and gliadin are prime examples of major food protein fractions from wheat (gluten) insoluble in water. Instead, they hydrate and form a strong gluten network transformed into bakery products with appropriate processing. However, glutenin is soluble in dilute acids (i.e., a glutelin), whereas gliadin is soluble in ethanol (i.e., a prolamin).

Proteins frequently, except amino acids, can also contain non-protein compounds known as prosthetic groups, which are essential for their functionality. These proteins are known as conjugated proteins, and although they are not used as food, they may have implications in processing, quality, or health. Glycoproteins are conjugated with carbohydrates (e.g., lactoferrin in milk). Those with high carbohydrate content or high molecular weight polysaccharides are also called mucoproteins (or mucins). For example, ovomucin is responsible for the viscosity of egg whites. Haemoproteins have haem, as a prosthetic group, which is responsible for the red colour in meat (see Chap. 7). Lipoproteins are conjugated with various lipids, including cholesterol (e.g., high- or low-density lipoproteins (HDL, LDL)) and have implications in cholesterol metabolism. Metalloproteins are metal-binding proteins binding mainly iron, zinc, calcium, molybdenum and copper. Many enzymes need metal to function, and removing metal is a strategy to restrict their activity (see PPO, Chap 5). Phosphoproteins contain phosphoric groups, with casein from milk being the most notable example.

Finally, proteins can also be classified based on their shape as globular or fibrous. Globular proteins are roughly spherical, whereas fibrous tend to have linear structures. Usually, globular proteins are water-soluble, whereas fibrous are water-insoluble.

Depending on the source, food proteins usually have a specific name associated with them, especially when they constitute a major component of that protein source (Table 3.2). For example, whey protein is not a single protein but a mixture of approximately 65% β-lactoglobulin, 25% α-lactalbumin, and 8% bovine serum albumin. Table 3.2 is not exhaustive, as there are other proteins that we may also consume or use as ingredients for specialised applications. However, for all practical purposes, these are most proteins we consume daily. For example, when foods have a complex protein profile, like a cake made of wheat flour, eggs, and milk, we essentially consume gluten, egg, and milk proteins.

Table 3.2

Names of proteins from major food sources. The proteins presented are essentially the proteins we ingest when consuming foods with simple (e.g., boiled egg or tofu) or complex (e.g., a cake) composition

Food source

Major proteins names

Meat/fish

Muscle tissue

Myosin, actin

Connective tissue

Collagen – Also processed to gelatin

Egg

White

Ovalbumin, ovotransferrin, ovomucoid, ovomucin, and lysozyme

Yolk

Low-density lipoprotein (lipovitellin), high-density lipoprotein, livetin, and phosvitin

Milk

Caseins

αs1-, αs2-, β-, and κ-casein

Whey

β-lactoglobulin, α-lactalbumin, bovine serum albumin and immunoglobulins

Gluten

Glutenin

High- and low- molecular weight subunits

Gliadin

α-, γ- and ω-gliadins

Soy

β-conglycinin (7S), glycinin (11S)

Beans, peas, lentils and other legumes

Legumin, vicilin

Nuts and insects

Not sufficiently characterised and classified

3.3.2 Protein Structure

In proteins, four levels of structure may be distinguished: primary, secondary, tertiary, and quaternary. The primary structure is the sequence of amino acids in the polypeptide chain (Fig. 3.9). Changing the order of the twenty common amino acids makes it possible to get more than 2 × 10¹⁸ combinations of different peptides with unique structures and chemical properties. The specific sequence of amino acids in the polypeptide chain and their interactions with each other determine the secondary structure of the protein.

a. An illustration of the lysozyme structure has a sequence of 129 amino acids. b. A bond line structure of the lysozyme. The structure is made up of amino acids, benzene ring, C H, C H 3, N H, N H 2, S H, and O groups.

Fig. 3.9

(a) The primary structure of lysozyme, a protein consisting of 129 amino acids (PDB code: 1GXV). The numbers in red indicate the position of the amino acid in the sequence. (b)The first ten amino acids of lysozyme with their complete structures are shown

Secondary Structure

refers to the polypeptide chain’s specific geometrical arrangement along one axis due to hydrogen bonding between amino acids. The secondary structure results in the folding of the polypeptide chain, leading to a unique 3D structure. The driving force for this arrangement is to minimise exposure of hydrophobic sites of protein to water and maximise hydrophilic site exposure. Major types of secondary structures are the α-helix and the β-pleated sheet. α-Helix is a spiral, rigid structure stabilised by hydrogen bonding parallel to the helix axis (Fig. 3.10). Hydrogen bonds form between the amino group of each amino acid and the carbonyl group of the amino acid four residues away. There are 3.6 amino acid residues per turn of the helix, and the pitch (the distance between corresponding points per turn) is 0.54 nm.

4 schematic diagrams exhibit the different views of the alpha helical structures. The structure is composed of alanine, isoleucine, and serine. Each turn of the helical structure has 3.6 residues.

Fig. 3.10

The α-helix. (a) Side view, (b) side view with the amino acid residues shown, (c) top view, and (d) close-up view of one turn. A, I, S are alanine, isoleucine, and serine. Each turn (peak-to-peak) has 3.6 amino acid residues distinguished by different colours

α-Ηelix has an amphiphilic nature, with one side of the helical surface being hydrophobic and the other hydrophilic. The amino acids on the side chains determine the overall hydrophobicity of the helix. If the side chains are hydrophobic, the helix usually avoids water and is positioned within the protein structure, whereas if the side chains are hydrophilic, the helix is exposed to the water. The R-groups of amino acids extend outward from the helix. This conformation minimises the contact of hydrophobic parts of the helix with water while maximising the contact of the exterior parts, thus stabilising the structure. All amino acids participate in an α-helical structure except proline, interrupting its continuity. Polypeptide chains can also be found in an extended zigzag configuration known as β-pleated sheets consisting of β-strands. A β-strand is a stretch of polypeptide chain typically 3–10 amino acids long. β-Sheets connected alongside each other to form the structure, in which the adjacent β-strands run in the same (parallel) or opposite (antiparallel) directions (Fig. 3.11). Hydrogen bonds hold together the adjacent protein chains.

3 schematic diagrams of the protein conformation of beta strands. a. It has 2 arrows in opposite directions with an H bond to represent the parallel conformation. b. It has 2 leftward arrows with an H bond. c. It has a thread like structure.

Fig. 3.11

(a) Parallel, (b) antiparallel β-strands and (c) a random coil protein conformation. The arrows represent the β-strands. A loop connects the β-strands. The two neighbouring β-strands interact with hydrogen bonds (black dotted lines). In this example, the three hydrogen bonds hold the structure together

Many proteins contain combinations of α-helices and β-sheets known as motifs. For example, a protein may have a βαβ unit: two β-pleated sheets connected by an α-helix segment. β-Turn is a secondary protein structure where the polypeptide chain changes its direction (called a loop) and usually connects two secondary structure elements (Fig. 3.11a, b). Proline and glycine are common amino acids that are found in β-turns. Other common motifs that may be observed are referred to as β-meander, β-barrel, αα-unit or the Greek key. Finally, random coils do not have structure, and the polypeptide chain is oriented randomly in space (Fig. 3.11c). Two examples of the enzyme papain and egg-white protein are shown in Fig. 3.12 where different secondary structures combine to form the entire protein.

2 three-dimensional simulation models of the protein complex. The complex is composed of the alpha helix, beta sheets, and coils.

Fig. 3.12

Structures of papain found in papaya (left) and egg ovalbumin (right). Secondary structures of α-helices, β-sheets and random coils that form the proteins are easily distinguished. PDB codes 9PAP and 1OVA

The three-dimensional structure of proteins, in which the polypeptide chains are tightly folded and packed into a compact form, like papain shown in Fig. 3.12, is termed tertiary structure. There are two common types of tertiary structures: fibrous and globular (Table 3.1). Fibrous proteins are rod-like structures and are relatively heat and pH-stable, whereas globular proteins form spherical structures. Tertiary structure is stabilised by hydrophobic interactions where hydrophobic amino acids interact to avoid contact with water, electrostatic interactions (salt bridges) between positively charged and negatively charged groups, hydrogen bonding, and disulfide bonds when two cysteines react with each other (Fig. 3.13).

A schematic representation of the tertiary structure of the protein complex. The complex includes hydrophobic interactions, ionic interactions, disulfide bonds, and hydrogen interactions.

Fig. 3.13

Forces that stabilise the tertiary structure of proteins. See text for description

Finally, proteins with more than one chain may have a quaternary structure, which is the number and arrangement of multiple protein subunits in a complex (Fig. 3.14). These proteins comprise oligomers that are macromolecular complexes composed of non-covalent assemblies of two or more monomer subunits. Monomer subunits interact with hydrophobic interactions to form dimers, trimers, or tetramers for two, three or four monomers. Examples of such proteins are β-conglycinin and glycinin from soybeans or β-lactoglobulin from milk.

2 three-dimensional models of the beta conglycinin complex. The complex includes several monomer subunits, forming dimers, trimers, and tetramers.

Fig. 3.14

Quaternary structures of β-conglycinin from soy (left) and phaseolin (right) from beans forming trimers (green, brown, and blue). Structures seem similar, as they are both vicilins, the storage proteins of soy or beans. Examination of their primary structures can reveal their differences. PDB codes 1UIK and 2PHL

3.3.3 Changes in Protein Structure: Solubility, Denaturation and Hydrolysis

Protein solubility in water is one of the main parameters of interest in food formulation as it controls its functionality. Solubility is the amount of protein that can dissolve into an aqueous solution with a correctly folded structure. Food proteins are partly or entirely soluble in water, depending on many factors directly related to the protein’s characteristics (e.g., amino acid composition, sequence and molecular weight) and environmental factors (e.g., pH and temperature). Proteins rich in hydrophobic amino acids tend to be less soluble in water than those rich in polar amino acids. It should be emphasised that their sequence is also important. Contiguous hydrophobic amino acids form hydrophobic moieties, making proteins less soluble. In Fig. 3.15, the two proteins have the same amino acids. The amino acids in red are all hydrophobic, and in the first protein (Fig. 3.15a), they are all next to each other, whereas in the second protein (Fig. 3.15b), they are interspersed in the structure. As a result of this hydrophobic moiety, the first protein will have a lower solubility. The size of the protein, i.e., its molecular weight, also plays a significant role, with large proteins having lower solubility.

2 schematic diagrams exhibit the sequence of amino acid. a. The sequence of amino acid is E D T Q R R M W F F I I K H. The sequence M W F F I I is labeled a hydrophobic moiety. b. The sequence is E F D M T Q I R R W I K H F.

Fig. 3.15

Amino acid sequence influences protein solubility. See text for description

pH is a determining factor of protein solubility as the degree of water solubility results from the balance between electrostatic and hydrophobic interactions between the proteins. Solubility increases if electrostatic repulsion between proteins is higher than hydrophobic attractions. As described earlier, the point at which proteins have fewer electrostatic interactions is at their pI. Most food proteins have their lowest solubility at a pH of around 4, depending on the protein and the environmental conditions (Fig 3.16a). Proteins have the highest solubility at pH > 7, so protein extraction is usually carried out at alkaline pH.

2 profiles plot solubility versus p H and temperature. a. A curve begins along the y axis, follows a decreasing trend, reaches a minimum peak for minimum solubility, and again follows an increasing trend for maximum solubility. b. 2 curves for most and heat stable proteins follow a decreasing trend.

Fig. 3.16

(a) Influence of pH and (b) temperature on protein solubility. See text for description

Temperature is another determinant factor, as heating influences protein structure. Generally, protein solubility increases with temperatures up to around 40 °C and at higher than 50 °C, solubility is diminished (Fig 3.16b). A class of proteins known as heat-stable proteins may operate at higher temperatures without changing their structure and can still maintain their functionality. For example, thermostable amylases are used in the syrup industry to hydrolyse starch and operate at ~90 °C. Thermostable proteins may also be of concern when they are toxins and do not denature at normal processing temperatures. For instance, toxins from Bacillus cereus and Staphylococcus aureus, once formed, cannot be denatured with regular processing, causing food poisoning.

Proteins may lose their structure due to various processing factors, storage or during food digestion. Denaturation is the loss of proteins’ secondary, tertiary, or quaternary structure, resulting in protein unfolding (Fig. 3.17). Denaturation does not involve peptide bond cleavage, and frequently (but not always), if peptide bonds and disulfide bridges are not broken, denaturation is reversible.

A schematic diagram exhibits the denaturation of the proteins. The native protein is converted into the denatured protein. The native protein has secondary, tertiary, and quaternary structures. The denatured protein has an unfolded structure.

Fig. 3.17

During denaturation, proteins unfold without peptide bond cleavage

Denaturation has various consequences on protein structure and functionality. Coagulation or gelation is the random aggregation of denatured protein molecules due to protein-protein interactions, most frequently due to high temperatures or changes in pH. As protein unfolds, hydrophobic amino acids buried in the structure are now exposed and can interact through hydrophobic interactions, which results in gel formation. Common examples include the coagulation of milk proteins during yoghurt manufacturing when pH is lowered and heat-induced denaturation of egg proteins when egg albumen is heated. At this point, it should be emphasised that temperature (heat) and pH changes are the two most common denaturation methods in food processing. Denaturation also results in loss of biological activity, which is important in enzymatic reactions. Inactivation of enzymes by heat treatment is a common processing step to increase the shelf life of various food products. For instance, blanching is a low-temperature heat treatment to denature enzymes naturally present in vegetables before freezing. If left uncontrolled, these enzymes have detrimental effects on the product’s quality during sub-zero temperature storage. Denatured proteins are easily digested by enzymes in the digestive tract. Denaturation also increases the bioavailability of amino acids and other compounds associated with that protein (e.g., iron). High-temperature processing of certain foods is a protein denaturation step to make proteins digestible and confer desirable sensory characteristics (e.g., canned tuna). Finally, unfolding and exposure of hydrophobic sites improve the emulsification capacity of proteins, making them sometimes irreplaceable in specific food formulations (e.g., salad dressings).

Protein Gelation

Proteins, similarly to polysaccharides, can form gels under the appropriate conditions. Gelation is one of the critical properties of proteins, as it is one of the main ingredients responsible for food structure, i.e., holding things together. Without protein gelation, most of our food would resemble a thick custard. The most common method to form food gels involves heating. Heat causes protein denaturation, exposing reactive groups and favouring intermolecular interactions between proteins. Once more, the involved forces are the same as in polysaccharide gels (hydrogen, ionic, and hydrophobic interactions). Hydrophobic interactions are more important than in polysaccharide gels due to hydrophobic amino acids present in most proteins. Protein gels are either fine-stranded or particulate. Particulate gels (e.g., yoghurt) are opaque and have a low water-holding capacity, whereas fine-stranded gels (e.g., gelatin) are transparent or translucent and have a high water-holding ability.

A flow diagram exhibits the steps involved in the preparation of the protein gelation. The native state protein is heated to form denatured proteins, which are further cooled to form a translucent structure. The denatured protein undergoes aggregation, followed by an opaque structure.

Denaturation is caused by physical or chemical agents (Table 3.3). Physical agents include heat, mechanical shear, pressure, or irradiation. Heat, the most common process in the food industry, causes protein denaturation, as vibrations at high temperatures break protein intramolecular interactions (do not break peptide bonds). Amino acid composition affects thermal stability, and heat-stable proteins usually contain a high proportion of hydrophobic amino acids. Water facilitates thermal denaturation, and proteins in high-moisture foods can be easily denatured, unlike those found in dry powders. Mechanical shear refers to agitation such as whipping, shaking, kneading, or mixing encountered in several food processing operations. The input of mechanical energy destabilises protein structure, leading to the stretching of protein molecules and denaturation. Denaturation due to whipping is critical in foam formation used to create unique textures in various products (e.g., meringues). Hydrostatic pressure and irradiation used in non-thermal (athermal) processing of foods also cause denaturation by breaking the forces that stabilise the tertiary structure. Pressure is important in high hydrostatic pressure processing (HHPP), where pressure is usually between 1–12 kbar. Denaturation during HHPP occurs due to the compressibility of protein structure and is mostly reversible with the removal of pressure. Depending on the dose, ionising irradiation may cause unfolding, coagulation, and even cleavage of peptide bonds. The effect on food protein is quite complex, and it depends on the specific food being irradiated and the objectives of the technique (i.e., dose). Generally, irradiation may improve or impair the functional properties of proteins. For example, while it improves the foaming properties of egg white, it increases the enzymatic coagulation time with the action of rennet of milk proteins. pH plays a critical role in protein properties. Proteins unfold at extreme pH values, and unfolding is greater at alkaline pH values than in harsh acidic environments. pH-induced denaturation is frequently reversible if there is no peptide bond cleavage. Changes in pH influence protein charge similarly to amino acid ionisation, as it affects electrostatic interactions between charged groups that belong to the protein. Proteins are most stable at their isoelectric point, where the net charge is zero. Organic solvents such as ethanol or acetone mainly affect the stability of hydrophobic interactions. Hydrophobic interactions are weakened, and protein unfolds and precipitates. Electrostatic interactions are generally enhanced, but the effect depends on the protein-solvent pair. Ethanol is the only edible organic solvent used in food processing or produced during the fermentation of various products. Ethanol induces protein denaturation, altering the product’s stability and sensory characteristics. In certain alcoholic beverages (e.g., Irish creams that are alcoholic beverages made with cream, cocoa, and Irish whiskey), the coexistence of protein and ethanol makes the formulation of such products challenging. Although ethanol is the only edible solvent, other solvents may also be used as processing aids that may influence protein functionality to a certain extent (e.g., hexane use in soybean processing operations). Low molecular weight molecules that can influence molecular interactions may be present in the food or used in a laboratory setting in protein-related investigations. For example, urea or guanidine hydrochloride weakens the strength of hydrogen bonding, causing denaturation that may be reversible after denaturant removal. Mercaptoethanol and dithiothreitol both break disulfide bridges, destabilising the tertiary protein structure. Detergents such as sodium dodecyl sulfate (SDS) are powerful denaturing agents that disrupt hydrophobic interactions, causing protein unfolding. Sugars (e.g., sucrose) tend to stabilise the proteins, whereas salts have a complex effect on protein denaturation. Salts may cause denaturation as they compete with protein for interactions with water molecules. Protein structure is influenced more by anions than by cations. At sufficiently high salt concentrations, few water molecules are available to interact with protein to hydrate it and as a result, protein aggregates and precipitates. Salt-induced protein precipitation is called salting out, whereas protein solubilisation, resulting from appropriate amounts of salt, is termed salting in. Not all ions have the same capacity to denature proteins. The Hofmeister series or lyotropic series of salts is a classification of salts in order of their ability to precipitate (or solubilise) proteins (Fig. 3.18).

Table 3.3

Agents that influence protein denaturation

Agent

Comments

Physical

Heat

Heat processing

Mechanical shear

Mixing, extrusion, homogenisation, high shear processing

Pressure

High hydrostatic pressure processing, reversible with pressure removal

Ionising radiation

Processing by irradiation

aw

Low aw enhances thermal denaturation stability

Chemical

Organic solvents

Weaken hydrophobic interactions

pH

Proteins unfold at alkaline pH, precipitate at pI. Influences electrostatic interactions.

Urea, guanidine hydrochloride

Weaken hydrogen bonds

Mercaptoethanol and dithiothreitol

Break disulfide bonds

Detergents (e.g., SDS)

Weaken hydrophobic interactions

Sugars

Enhance thermal denaturation stability

Salts

See Hofmeister series

2 Hofmeister series. SO4 2 negative greater than HPO4 2 negative greater than Acetate negative greater than Citrate negative greater than Cl negative greater than NO 3 negative greater than Br negative greater than I. NH4 + greater than K + greater than Li + greater than Mg2 + greater than Ca2 +.

Fig. 3.18

Hofmeister series of anions and cations relevant to food proteins

NaCl is the most commonly used salt in foods, which is in the middle of the series and has intermediate aggregation (or solubilisation) properties. Other widely used inorganic salts are NaNO₃, CaCl₂, or phosphates, while organic salts are also frequently used (citrates, acetates, benzoates or sorbates). A salt used for research purposes is ammonium sulfate ((NH₄)₂SO₄). With both ionic species (NH₄⁺ and SO₄²⁻) at the left of the scale, it exhibits a very strong capacity to precipitate proteins and is used in protein isolation processes.

In contrast to denaturation, where the breaking of peptide bonds does not occur, hydrolysis involves cleavage of peptide bonds, yielding smaller polypeptides or free amino acids (Fig. 3.19). It is achieved by the action of acid and heat (acid hydrolysis) or proteolytic enzymes (enzymic hydrolysis). The main difference between these two modes of action is that acid hydrolysis is a random process cleaving the protein uncontrollably. Hydrolysis products depend on the conditions (e.g., heat, time or type of acid), and changing conditions will most certainly change the end products. In contrast, enzymic hydrolysis cleaves only one bond, always resulting in the same products. Both hydrolysis types are useful depending on the objective of the process. For instance, protein hydrolysates used as flavourings may be produced with acid hydrolysis, resulting in a complex mixture of peptides and free amino acids with a strong, savoury taste. In contrast, rennin used in cheese manufacturing acts only between amino acid residues 105 (Phe) and 106 (Met) of κ-casein.

A schematic diagram exhibits the free amino acid formation. The protein chain undergoes hydrolysis to form the separate amino acids and peptides.

Fig. 3.19

Protein hydrolysis results in cleavage of the peptide bond and formation of peptides and free amino acids

3.3.4 Functional Properties of Proteins

Besides their nutritional value, proteins have important functional roles in food manufacturing. Most of their properties are similar to polysaccharides and are frequently exploited simultaneously (Table 3.4).

Table 3.4

Proteins functionality in foods

Property

Examples

Gelation

Boiled egg, yoghurt

Emulsification

Salad dressings

Foaming

Meringue, beer

Flavour binding

Retention of food flavours

Water retention

Prevention of water losses from the structure

Enzymic action

See next section

For example, proteins can also gel and hold water within the structure. However, depending on the specific food, we may need both in the formulation and for different purposes. For example, the structure of crème caramel (a dairy dessert) is formed because of milk protein gelation. Milk proteins are responsible for holding water and creating the structure of this product. However, prolonged storage leads to the collapse of the structure and expulsion of water (i.e., syneresis), accompanied by changes in the product’s texture and aesthetics, limiting its shelf life. Adding small amounts of polysaccharide helps retain water within the structure and prolong the shelf life. In this example, protein is responsible for structure formation, water retention, and flavour binding, whereas the polysaccharide is responsible for water retention and texture modification. Proteins may also modify viscosity, but polysaccharides are mainly used as viscosity modifiers in most food applications. Proteins also form foams and emulsions, as they have both hydrophobic and hydrophilic parts in their structure. A denaturation step is most frequently required, resulting in unfolding that exposes the hydrophobic amino acids towards the hydrophobic phase, allowing the protein to adsorb. In emulsions, proteins create a protective thin film around the oil droplets and keep them dispersed (Fig. 3.20a). In foams, they arrange at the air-water interface and create a bubble entrapping air (Fig. 3.20b). Emulsion and foam stabilisation achieved with proteins is critical in many foods. For example, the quality and sensory properties of salad dressings and sauces depend on emulsion characteristics. Meringues and many confectionery products rely on foam film stability. Ice cream needs both suitable emulsion and foam stabilisation during processing. In solid foams (i.e., sponge cakes), proteins stabilise air cell structure essential for the volume and texture of the products (e.g., egg proteins in cake products). As is evident, the emulsification and foaming capacity of proteins are principal technological properties, and optimisation of their performance is an active field of research. Finally, proteins can bind flavours, thus improving or modifying the sensory properties of products.

2 diagrams for the formation of the protective film. a. The specific site of the mayonnaise is zoomed-in, which has the agglomerated oil droplets. A single droplet is zoomed in, which is surrounded by protein. b. The selected site of the foam is zoomed-in, where the bubble is covered by protein.

Fig. 3.20

(a) Emulsification and (b) foaming properties of proteins. Proteins surround the oil droplet or bubble to form a protective film. The physicochemical properties of the film are crucial for the stability of both emulsions and foams

Protein Determination

The internationally accepted method for protein determination in foods is the Kjeldahl method. The method does not measure protein but the total organic nitrogen released from a sample and absorbed by acid. The Kjeldahl method has three steps. In digestion, the sample reacts with concentrated sulfuric acid, and the nitrogen released is converted to ammonium sulfate. Distillation involves adding excess base to ammonium sulfate to convert NH₄⁺ to NH₃, which is distilled into a boric acid solution. Titration quantifies the amount of ammonia in the receiving solution. A conversion factor is finally used to convert total nitrogen to protein content.

3 reactions. Digestion. Protein + sulfuric acid yields N H4 2 S O 4 + H 2 O + C O 2. Distillation. N H 4 2 S O 4 + 2 N a O H yields 2 N H 3 + N a 2 S O 4 + 2 H 2 O. N H 3 + H 3 B O 3 yields N H 4 + H 2 B O 3 + H 3 B O 3. Titration. 2 N H 4 H 2 B O 3 + H 2 S O 4 yields N H 4 2 S O 4 + H 3 B O 3.

When pure proteins are isolated, other methods are usually used to determine protein depending on the needs and purpose of the analysis, such as the Biuret method, Lowry assay, bicinchoninic acid (BCA) assay, absorption at 280 nm or Bradford assay, all with advantages and disadvantages.

3.3.5 Proteins of Major Food Commodities

3.3.5.1 Milk

Milk is the liquid food produced by the mammary glands of mammals to feed their offspring. Milk provides a high-quality protein and is a source of energy, vitamins and minerals. Many mammalian species are used to produce milk and milk products, but cows produce most milk. The dairy industry converts milk into various products, including butter, cheese, condensed, dry, and fermented milk. In most cases, when the term “milk” is used, it refers to that of cows, whereas if it comes from another mammal, it is usually specified, e.g., “goat milk” or “sheep milk”.

Two main protein fractions are distinguished in milk: caseins (~80%) and whey proteins (~20%). A protein is considered casein if it precipitates at pH 4.6, whereas what remains after precipitation is whey. Caseins are not globular and have very different primary structures. They are divided into three classes: α-, β- and κ-casein. γ-Caseins are also present, but they are considered to occur from hydrolysis of β-casein with the enzyme plasmin. A characteristic of casein proteins is that they are assembled into a spherical structure known as casein micelle (Fig. 3.21). The diameter of the casein micelles ranges between 30 and 300 nm, and the light scattering on them makes milk appear white. Because the chemical characteristics of micelles affect milk’s technological properties, considerable research is dedicated to its structure. The exact positioning of the proteins (α-, β-, and κ-caseins) is unknown, but the consensus is that κ-casein is located predominately at the surface, with α- and β-caseins situated in the interior of the micelle. It should be emphasised that micelles are not rigid spheres but soft and squashy and are found in a dynamic equilibrium with their surroundings. As a result, they may change in size and composition depending on the environment (e.g., temperature or pressure). Because of its complexity, many models are attempting to explain casein micelle structure, with the “submicelle”, “nanocluster”, and the “dual binding” models being the most dominant.

A microscopic image of the casein micelle sample. The morphology of the sample has spherical structures, and the surface of the spheres is surrounded by proteins.

Fig. 3.21

Micrograph of an individual casein micelle. The particle surface is uneven and is generally attributed to the presence of κ-caseins. The exact protein composition remains unknown. Microscopy image is reproduced with permissions from Dalgleish et al., (2004), International Dairy Journal, 14, 1025–1031

Whey is the second major fraction of milk proteins, mainly consisting of β-lactoglobulin (b-LG) and α-lactalbumin (α-La). Other proteins in whey are immunoglobulins, bovine serum albumin, and the proteose-peptone fraction. b-LG is the main protein and a typical example of a globular food protein (Fig. 3.22). An interesting behaviour of this protein is its ability to form tertiary structures depending on the pH of the solution. Below pH of 3.5 b-LG is a monomer. As the pH increases, it begins to combine to form dimers, and with a further increase between 3.7 and 5.4, the dimers join to form octamers. Further pH increase between 5.4 and 7.5 dissociates the octamers into dimers, and, finally, at pH above 7.5, the dimers dissociate back into monomers. α-La is a protein naturally high in all essential amino acids, making it nutritionally unique. It has higher heat stability than b-LG and does not denature even after extensive heat treatments at high temperatures. However, it does not form gels, and as a result, it has a limited technological significance compared to b-LG.

A three-dimensional structure of the milk protein molecule. It consists of beta lactoglobulin and alpha lactalbumin.

Fig. 3.22

β-Lactoglobulin structure, a typical globular food protein. PDB code 1BEB

Lipoprotein lipase (LPL) and alkaline phosphatase (AP) are other proteins of interest found in milk. LPL is a lipase that hydrolyses fats into glycerol and free fatty acids and is responsible for fat lipolysis. AP hydrolyses specific phosphoric acid esters into phosphoric acid and the related alcohols. Minimum pasteurisation temperatures destroy the enzyme, and the phosphatase test can be done to ensure proper pasteurisation of milk. If the enzyme is present after the test, pasteurisation has not been completed correctly, and action must be taken.

3.3.5.2 Plant Proteins

Seeds of plants in the Fabaceae family, also known as legumes, are the sources of plant proteins used for food. These include soybeans (Glycine max), common beans (Phaseolus vulgaris), lentils (Vicia lens), peas (Pisum sativum), chickpeas (Cicer arietinum), peanuts (Arachis hypogea), fava beans (Vicia faba), lupins (Lupinus sp.), cowpeas (Vigna unguiculata), and pigeon peas (Cajanus cajan). Legume proteins are deficient in methionine and tryptophan but a good source of lysine. In soybeans, the globulin fraction makes up most of the proteins (~90%) and can be divided into the 7S and the 11S fractions. In biochemistry, the unit Svedberg (S) measures protein size. It measures how fast a protein sediments to the bottom of a centrifuge tube under the influence of centrifugal forces. For example, the 11S proteins are larger than the 7S. The 7S and the 11S fractions are found in all legume proteins. The 7S is called the vicilin fraction, and the 11S is the legumin fraction. By convention, only for soy proteins, the 7S is called β-conglycinin and the 11S glycinin. Each fraction consists of various subunits, and other minor fractions may also be present (e.g., 2S or 15S). With processing, soy proteins may form gels that form the basis of tofu. Other products include texturised protein or various liquid beverages colloquially called “soy milk”.

3.3.5.3 Egg Proteins

Egg proteins can be divided into egg white and yolk proteins. Egg white, also known as albumen, consists mainly of the proteins ovalbumin (the main protein, ~50%), ovotransferrin (or conalbumin, ~12%), and ovomucoid (~10%). Other proteins are also found to a lower concentration, include ovomucin, which is responsible for the egg white’s viscosity; flavoprotein, which binds riboflavin; avidin, which binds and inactivates biotin; and lysozyme, which has antibacterial action. The albumen proteins are globular albumins because they are soluble in water (the difference in spelling is crucial). Egg albumen proteins are surface active, particularly at the air-water interface. This means they are excellent foaming agents and an indispensable ingredient in the food industry. Beyond simple egg white-based dessert meringues, nearly all bakery sponges (for cakes and other foods) contain egg white that contributes to creating and stabilising the desirable aerated structure. It should be mentioned that egg white protein denaturation starts at 61 °C (ovotransferrin) and finishes at ~84 °C (ovalbumin). This is technologically convenient because the pathogen Salmonella can be thermally inactivated at temperatures <60 °C. Consequently, egg white can be pasteurised without denaturing protein (i.e., cooking the egg).

The main proteins of egg yolk are lipovitellenin (or Low-Density Lipoproteins (LDL)), lipovitellins (or High-Density Lipoproteins, HDL), phosvitin, and livetins. LDL represents most of the yolk proteins and is responsible for its emulsifying ability. HDL consists of α- and β-lipovitellins, proteins that carry most lipids in egg yolk. Phosvitin (α- and β-) is a phospho-glycoprotein that contains about 10% phosphorus and is one of the most highly phosphorylated proteins. Most of the phosphorus found in egg yolk is bound to phosvitin. Finally, livetins are water-soluble proteins that account for a third of yolk proteins and can be further subdivided into α-, β-, and γ-livetin. It should be noted this description refers to chicken eggs, but eggs from other birds have a very similar protein composition and properties. It should be mentioned that egg yolk is rich in lecithin and, together with the proteins, makes it one of the most valuable emulsifiers in food formulation.

3.3.5.4 Cereal Proteins

Wheat gluten is a mixture of more than fifty proteins, and its fractions are classified according to their solubility in ethanol (gliadins) or dilute acid solutions (glutenins). Gliadins are divided into three classes: α-, γ- (sulfur-rich) and ω- (sulfur-poor) gliadins. They are monomeric proteins interacting primarily by non-covalent interactions (i.e., hydrogen bonding and electrostatic interactions), forming transient binding partners (i.e., the interaction points change with time). The glutenin fraction consists of high- and low-molecular-weight subunits (HMW, LMW, respectively) cross-linked by disulfide bonds. HMW subunits, in particular, have been associated with the bread-making performance of flour, and it is the main target in the technological improvement of flour.

Gluten proteins are unique as they are the only food proteins with a technological significance, insoluble in water. Instead, they hydrate and interact, forming the gluten network responsible for the structure of products based on wheat flour. At the atomic scale, gluten proteins interact to form gluten sheets with all the forces described in the previous sections. Gluten sheets are arranged in a way that creates nanocapillaries filled with water. The thickness of the nanocapillaries is ~5 nm. Further packing of the nanoporous structure leads to a three-dimensional microscopic network. This network can entrap starch granules and CO₂ during the proving of bread. The role of gliadins in the gluten network is to act as a plasticiser and reduce its elasticity, while glutenins are the main building blocks (Fig. 3.23).

A diagram exhibits the macroscale, microscale, nanoscale, and atomic scale of the dough. The microscale of the dough has starch granules and a gluten network. The nanoscale has layers of gluten sheets separated by water. The atomic scale of the sheet has the glutenin with bonds and interactions.

Fig. 3.23

Structural hierarchy of gluten network. See text for description. The image has been redrawn from Kontogiorgos (2001), Food Research International, 44, 2582–2586

The protein content of flour is critical and dictates its application. Flours with ~8–10% protein are used in biscuits, ~10–14% for bread, and flours with protein >15% for pasta. Other cereal proteins include zein (corn), hordeins (barley), secalins (rye), avenins (oats) and rice proteins. While these proteins are consumed routinely in our diet, they are of little technological significance as they cannot form structures similar to gluten.

3.3.5.5 Meat Proteins

From a technological point of view, meat proteins can be divided into muscle proteins and connective tissue proteins. Muscle proteins consist of myofibrillar and sarcoplasmic proteins, with the first being mainly responsible for meat’s technological and sensory properties, particularly tenderness and processing characteristics. Myofibrillar proteins are the main proteins of the skeletal muscles of animals and those used for food. Livestock and fish are the main sources of myofibrillar proteins, and although differences may exist between species, molecularly, they all consist of the same main proteins: myosin and actin. Myosin is a protein complex with three parts, known as the “head,” “neck,” and “tail” (Fig. 3.24a). The tail consists of two intertwined peptide chains called “heavy chains”, whereas the four proteins around the “neck” are called “light chains”. This structure allows for specific interactions to occur, leading to muscle contraction. Actin forms a filamentous structure when multiple monomers link with non-covalent interactions (Fig. 3.24b). The actin monomer is called G-actin, but when G-actin monomers interact, the filament formed is called F-actin, which intertwines to form a double helix. Other proteins in the complex are troponin and tropomyosin, which play a functional role during muscle contraction. Interactions between myosin and actin are frequently irreversible and determine meat tenderness and processing characteristics. Sarcoplasmic proteins are found in the sarcoplasm surrounding the myofibrils and contain mainly enzymes involved in cellular metabolism and myoglobin. These proteins do not contribute to meat tenderness but impact meat quality aspects such as colour and water-holding capacity.

2 structures of myosin and actin. a. It has heavy and light chains with a tail, neck, and head. b. The structure has G actin, F actin, and troponin bound together along with the tropomyosin.

Fig. 3.24

(a) Structure of myosin, (b) structure of actin. See text for description

The connective tissues hold and support the muscles onto the bones. The major protein of the connective tissue is collagen, which contributes significantly to the toughness of meat. Collagen consists of three protein chains forming a triple helix primarily composed of glycine, alanine, proline and hydroxyproline, making gelatin of very low nutritional value (Fig. 3.25). Gelatin is a protein obtained by partial hydrolysis of collagen and has numerous applications in the food industry, including gelation, emulsification and thickening. The most abundant sources for gelatin extraction are pig skin, bovine hide, and pig and cattle bones, with gelatin obtained from fish having only a minor contribution.

A schematic diagram exhibits the collagen structure, which has the 3-helix structure assembled together.

Fig. 3.25

Structure of collagen showing the triple helix structure. PDB code 1K6F

Depending on collagen treatment, two types of gelatin can be obtained: Type-A gelatin (pI ~8–9) with acidic or Type-B gelatin (pI ~4–5) with alkaline collagen treatments. Depending on the extraction conditions and raw material, the final gelatin mainly consists of three protein fragments: α-chains, β-chains consisting of two α-chains covalently linked, and γ-chains consisting of three α-chains covalently linked with other fragments of lower molecular weight also present (Fig. 3.26a). The main quality characteristics of gelatin that influence food texture are its gel strength and melting characteristics. These two characteristics are linked to the amino acid composition, which depends on the extraction source and conditions. The Bloom strength is a quantitative parameter that can be used to measure gelatin gel strength. The Bloom strength is the force in grams required to depress the surface of a gelatin sample with specific geometrical characteristics, and it is determined using a uniaxial compression method. The Bloom strength determination is used to classify gelatin into high-Bloom (200–300 g), intermediate-Bloom (100–200 g), and low-Bloom (50–100 g). A higher Bloom corresponds to gelatins with a stronger gel network and higher melting temperature. The second characteristic that makes gelatin an invaluable hydrocolloid in texture modification is the thermoreversibility of the gels at near-body temperatures. On cooling hot gelatin solutions, the α-chains form triple helices, creating a gel network (Fig. 3.26b). During consumption, heating the gel results in sharp melting, occurring in a narrow temperature range of around 35 °C. This gives mouth-melting characteristics to the gels, which is important in texture design, especially for confectionery products. Gelatin can be used in many foods as it has a broad range of functional properties, and new sources are continuously explored.

a. A schematic diagram presents the triple helix structure of the collagen, which undergoes hydrolysis, forming alpha chains, beta chains, and gamma chains. b. A schematic diagram exhibits the alpha, beta, and gamma chains, which undergo cooling to form the gel network.

Fig. 3.26

(a) Collagen is hydrolysed to give the three main protein fragments making up gelatin, (b) Gelatin’s gelation mechanism. The α-, β-, and γ- chains form a gel network on cooling. Heating the gel results in melting occurring near the body’s temperature at ~35 °C

Emerging Sources of Food Protein

New food protein sources are continuously explored as a potential solution to various problems spanning from sociological (e.g., consumer demands) to environmental (e.g., global warming). The main alternative sources with some promise are insect and microalgae (e.g., Spirulina or Chlorella) proteins. Insects are an excellent source of protein (>50% protein) and need few resources to grow, but it isn’t easy to scale up in quantities required for industrial use. In contrast, microalgae have greater yields and grow very fast. However, it is difficult to remove colour (usually green-blue) and “fishy” off-flavours, thus limiting their applicability. Another technological problem with both sources is their high polyunsaturated fatty acid content. Protein isolated from these sources naturally contains an amount of co-extracted fatty acids. This leads to rapid lipid oxidation in the protein powder during storage or the finished product, making them unsuitable for most formulations. Another emerging technology is cultured meat or lab-grown meat (these terms are used interchangeably), which involves cultivating myofibrillar cells in bioreactors to produce meat. Cultured meat is hoped to have similar sensory properties to conventional with the additional benefit that it does not need to involve raising farm animals for food. At the time of writing this paragraph, none of the above sources have demonstrated technological viability under actual industrial conditions and remain research areas of intense interest.

3.3.5.6 Protein Digestion

Plant are less digestible than animal proteins, mainly because of their differences in secondary structure. The secondary structure of plant proteins is characterised by a high β-sheet content and a relatively low α-helix amount compared to animal proteins. The high content in β-sheet conformation is likely related to its resistance to proteolysis in the gastrointestinal tract, as the hydrophobic β-sheet structure of plant proteins facilitates protein aggregation, resulting in decreasing digestibility. In addition, plant-based proteins used in the industry contain non-starch polysaccharides, impeding the access of enzymes to proteins and thus reducing protein digestibility. Furthermore, protease inhibitors in raw legumes, cereals, and other sources disrupt protein digestion by complexing digestive enzymes, such as trypsin and chymotrypsin, produced by the pancreas to hydrolyse proteins. Heat treatments (e.g., drying) reduce trypsin inhibitory activity and enhance the protein digestibility of treated plant-based products. Aqueous solubility also affects digestibility. For example, whey proteins, highly soluble in acidic conditions, pass through the stomach and rapidly hydrolysed in the duodenum, causing rapid absorption. Caseins coagulate and are thus characterised by prolonged absorption. Soy and other plant proteins are digested faster than casein but are slower than whey proteins, so they have an intermediate digestibility. Finally, gluten proteins have minimal water solubility and the lowest digestibility. Temperature significantly influences the myofibrillar protein digestibility. Heating between 40–85 °C generally increases digestibility, whereas temperatures > ~ 85 °C reduces it. This has important implications as commercial sterilisation of meat products is carried out at 121 °C, inevitably resulting in losses in their nutritional profile.

3.4 Enzymes

Enzymes

are proteins with catalytic activity (i.e., catalysts), speeding up chemical reactions. Although enzymes accelerate the reaction they catalyse, they are not consumed during the reaction. They are still in the same form when the reaction is complete and ready to participate in a new reaction cycle. Enzymes facilitate a reaction that sometimes would be impossible without their presence. They are naturally found in foods and are important in the food industry since they have both desirable and undesirable influences on the quality of the products. When natural enzymatic activity shortens shelf life, we may need to inactivate them to preserve the quality (e.g., browning of fruits and vegetables). On the other hand, enzymes may confer the desired product qualities, such as in fermented products (e.g., cheese, salami or pickles). Enzymes are also used as indicators of proper processing. For instance, the deactivation of alkaline phosphatase in milk indicates correct pasteurisation, and the deactivation of peroxidase and catalase in blanching before freezing ensures that the frozen product has optimum quality during storage. Similarly, lipoxygenase deactivation is also used as an index enzyme in vegetable processing (e.g., corn, peas, and green beans). Enzymes may also be used directly to manufacture a specific food ingredient, for example, amylases in glucose manufacturing from starch. Finally, enzymes may be used as analytical tools to measure another compound in a food product, e.g., measuring glucose using glucose oxidase.

3.4.1 Mechanism of Enzymatic Reactions

Molecules must meet each other (collide) for chemical reactions to occur. Even when molecules collide, this needs to happen successfully to create products, and reactants must overcome some energy barrier for a successful collision. The energy molecules must overcome for a chemical reaction to occur is called activation energy. If the barrier is too high, the reaction is difficult, meaning more molecular collisions are needed. Enzymes decrease the required activation energy, thus facilitating the reaction (Fig. 3.27).

A profile plots energy versus reaction progress. 2 distribution curves for reactions with and without enzymes begin along the y axis, reach the peak, and follow a right-skewed distribution. The reaction without enzyme has the highest peak, while the reaction with enzyme has the lowest peak.

Fig. 3.27

Activation energy of enzymic reactions. Enzymes reduce the activation energy needed for successful collisions, making the reaction easier

Enzymes act upon molecules called substrates and are so specific that they usually only catalyse one type of reaction. When a substrate is chemically different from what the enzyme requires, the reaction does not occur. However, some enzymes may be able to act upon more than one substrate (see specificity below). Enzymes with the same or similar enzymatic activities but different protein structures are called isoenzymes.

Enzyme-catalysed reactions occur at a particular location on the enzyme’s surface, called the active site (Fig. 3.28). The specific substrate can form relatively weak, temporary bonds to the enzyme at the active site. The specificity of the substrate-enzyme interaction can be better understood with the lock-and-key analogy. A key represents the substrate, whereas the lock is the enzyme. When the fit of the key to the lock is correct, then the door opens, i.e., a reaction occurs. When the key does not fit the lock, the door does not open, i.e., the reaction is impossible. In reality, the substrate often does not fit exactly on the active site. However, when the correct substrate approaches the enzyme’s active site, the conformation (shape) changes for a closer fit. This is a more realistic depiction of how enzymes accommodate the substrates and is known as the induced fit theory (Fig. 3.28). Generally speaking, an enzymatic reaction occurs in four steps. In the first step, the substrate (denoted as [S]) approaches and enters the active site of the enzyme ([E]). Following this, an enzyme-substrate complex ([ES]) is formed. It is termed “complex”, as a new temporary molecule is formed that is stabilised with transient interactions (e.g., hydrogen bonding). The reaction occurs during the residence of the substrate in the active site, and after completion, the new complex that is formed is termed enzyme-product complex ([EP]), as the products still reside within the active site. In the final stage, the product(s) ([P]) leave the active site, and the enzyme is ready to participate in a new reaction cycle (Fig. 3.28).

A flow diagram exhibits the steps involved in the enzymatic reactions. The substrate is approaching the active site, the enzyme and substrate complex, the enzyme and products complex, and the products are leaving the enzyme.

Fig. 3.28

Mechanism of enzymic reactions. See text for description

Enzymes may be composed exclusively of protein (single or multiple subunits) or have a more complex structure. In the latter case, a non-protein compound is usually required for the enzyme to conduct the reaction and is termed a co-factor (Fig. 3.29). Co-factors are divided into metal ions (e.g., Zn²⁺, Cu²⁺, or Mn²⁺) or small organic molecules (e.g., vitamin-B complex). The latter, also called co-enzymes, are primarily vitamins and other organic essential nutrients (e.g., co-enzyme-Q₁₀). Co-enzymes are further divided into prosthetic groups, where the co-enzyme is covalently bound to the protein and co-substrates that interact with the active site with transient interactions (e.g., hydrogen or hydrophobic interactions) and is not permanently bonded to the enzyme. In the absence of the co-factor, the enzyme is inactive and is called apoenzyme, while when the co-factor is in place, it is called holoenzyme (Fig. 3.29).

A schematic diagram exhibits the binding of the substrate to the unfit active site. The substrate binds to the active site of the enzyme, but the substrate is not fitted to the active site. The substrate binds with the help of a cofactor.

Fig. 3.29

When the substrate does not fit the active site, co-factors act as adapters to allow the attachment of substrates

Enzymes that require metal in their structure for activity are known as metalloenzymes. Metalloenzymes bind and retain their metal atom(s) under all conditions with very high affinity. Those with a lower affinity for metal ions are known as metal-activated enzymes. Many food enzymes need metals to exert their catalytic activity, and a common strategy, if we need to inactivate them, is to remove the metal from the structure. For example, polyphenol oxidase (see Chap. 5) needs copper, alkaline phosphatase requires zinc, and α-amylase needs calcium.

Two key terms that are associated with enzymic function are specificity and selectivity. Specificity is the ability of an enzyme to catalyse only one reaction. For example, a lipase hydrolyses lipids but cannot hydrolyse proteins. Specificity arises from the 3D structure of the enzyme’s active site, as the substrate fits correctly and is optimally aligned to react. Selectivity is the preference of an enzyme towards competing substrates with similar structures. For example, the isomerisation of glucose into fructose can be catalysed by xylose isomerase. However, the natural reaction for xylose isomerase is the isomerisation of d-xylose into d-xylulose. Still, the activity towards glucose is sufficient for the industrial production of high-fructose corn syrups.

3.4.2 Enzyme Kinetics

Enzyme Kinetics

is the study of the rate at which an enzyme converts the substrate to products. It is essential to know the speed of the reaction because it allows us to use enzymes in industrially important applications efficiently and profitably. If the enzymic reaction is too slow or the yields are low (substrate conversion to product) under the required processing conditions, the enzyme may not be suitable and must be replaced. In addition, using enzyme kinetics, we may study the factors that influence the reaction and discover how to accelerate or stop it. Factors that control reaction rate include all the factors that influence protein tertiary structure (see Sect. 3.3.3), enzyme type and concentration, substrate type and concentration, presence of inhibitors, and water activity. As a general principle, any factor that changes the enzyme structure in an undesirable way (e.g., by denaturation or hydrolysis) reduces the speed or eliminates the enzymic activity.

3.4.2.1 Effect of Substrate Concentration

The term used in enzymology to describe the speed at which a reaction occurs is reaction velocity (V), with units being concentration per unit time, e.g., mmol/min. Knowing the reaction’s initial velocity (Vo) and maximum velocity (Vmax) is necessary in enzymatic reactions. Initial velocity is the reaction rate at the beginning of the reaction when the enzyme and substrate have just been mixed. It is called “initial” because the enzyme starts using up the substrate, the speed decreases and eventually drops to zero as there is no more substrate to catalyse. The relationship between Vo and [S] at low substrate concentrations is linear, i.e., increasing [S] results in a proportional increase of Vo (if we double [S], we double Vo; if we triple [S], we triple Vo etc.) (Fig. 3.30). This occurs because the active site of the enzyme is not saturated. As a result, the substrate can easily find a free active site and be converted to a product. With a gradual increase of [S], the active sites eventually become saturated (yellow circle). With further increments in [S], the reaction speed stays constant once the enzyme is saturated. This occurs because the substrate does not have available active sites to be converted to a product. Consequently, it has “to wait” until a reaction cycle has been completed and an active site becomes available again (Fig. 3.30).

A profile plots reaction velocity versus substrate concentration. A curve begins at the origin, follows an increasing trend, and remains stable. The initial curve is labeled active center unsaturated. The increasing curve is labeled partially saturated. The stable region is labeled fully saturated.

Fig. 3.30

Effect of substrate concentration on reaction velocity. This plot is known as the saturation curve

The velocity of the reaction at which all active sites are saturated with substrate and Vo is independent of [S] is called maximum velocity (Vmax). Vmax is the fastest velocity at which a given amount of enzyme can operate. Michaelis-Menten equation describes the relationship between Vo and [S]:

with Km being the Michaelis-Menten constant. The Michaelis-Menten equation says that rection velocity V at any instant depends on Km, Vmax, and [S] of that moment. Km is the substrate concentration that gives half of the maximum velocity (Vmax/2) and is a measure of how fast V increases with substrate concentration (unit of Km is molarity (M)) (green circle, Fig. 3.30). Km is also a measure of the tendency of the enzyme to bind to its substrate (enzyme affinity). A low Km corresponds to a high affinity for the substrate, i.e., the enzyme “likes” the substrate and requires a low substrate concentration to achieve Vmax. Higher Km corresponds to a lower affinity for the substrate, i.e., the enzyme “dislikes” the substrate and requires a high substrate concentration to achieve Vmax. If an enzyme catalyses a reaction with two similar substrates (e.g., glucose and mannose), it prefers the substrate for which it has a lower Km value. It becomes evident that by using Km, we may decide if the enzyme is suitable for a particular reaction we need to accomplish under certain processing conditions.

Before non-linear curve-fitting on modern software was possible, estimating Km and Vmax from saturation curves was laborious. Scientists carried out various linear transformations of the Michaelis-Menten equation using the Lineweaver-Burk, the Eadie-Hofstee or the Hanes–Woolf plots. All these methods use linear regression to estimate the slope and intercept to calculate Km and Vmax. Although these methods are helpful to visualise data, they are now largely obsolete. In modern biology, the analysis focuses on calculating these parameters using non-linear regression with computer software, as this technique allows direct determination of Km and Vmax from untransformed data points.

3.4.2.2 Effect of Enzyme Concentration

When the substrate is in excess, reaction velocity is proportional to enzyme concentration up to a certain point (yellow circle, Fig. 3.31). Adding more enzyme does not influence velocity at concentrations higher than those corresponding to the yellow circle. The underlying principles of this behaviour are the same as those described in the previous section on the effect of substrate concentration.

A profile plots reaction velocity versus enzyme concentration. A curve begins at the origin and follows an increasing trend. A dot is marked on the curve, and the vertical line is drawn from the dot and ends along the x axis.

Fig. 3.31

Effect of enzyme concentration on the velocity of enzymic reactions

3.4.2.3 Effect of Temperature, pH and aw

Enzymic reactions speed up as temperature increases. However, each enzyme has an optimum temperature at which it works best. Below the optimum temperature, enzymic activity is slowed, whereas the enzyme starts to denature above the optimum temperature (Fig. 3.32). When the enzyme denatures completely, it loses its activity. It should be emphasised that thermal denaturation of naturally occurring enzymes is the first tool (e.g., blanching) that a food technologist will use to control enzymic activity before considering any other approach (e.g., changes in pH or additives). The optimum temperature depends on the type of enzyme and the reaction it catalyses. It is usually around 45 °C for all practical purposes, whereas, beyond 50 °C, the enzyme starts to denature. A class of enzymes, known as heat-stable enzymes, retain their activity at higher temperatures, creating problems for the stability of some foods (see peroxidases below) or may be used for specialised applications as, for example, the heat-stable α-amylase that retains activity at temperatures >90 °C.

A profile plots reaction velocity versus p H, temperature, or a w. A bell-shaped curve is plotted for the optimum p H or temperature. A curve for a w follows an increasing trend.

Fig. 3.32

Effect of pH, temperature, and water activity on enzymic reactions

Similarly, enzymes function best within a specific pH range (Fig. 3.32). Outside this optimum range, enzymatic activity decreases. The pH value where the enzyme is most active is known as the optimum pH, and it varies depending on the enzyme and the reaction it catalyses. For example, pepsin, which works in the stomach, functions best in strongly acidic environments, whereas lipase, found in the small intestine, works best in alkaline environments.

Lowering water activity decreases enzymatic reaction rates and is usually eliminated below ~0.3, as the enzyme cannot function (Fig. 3.32). Dry products and powders with aw < 0.2 are stable against enzymatic reactions. IMFs with aw in the range of 0.3–0.8 require low-temperature preservation to minimise undesirable enzymatic activity, especially when stored for extensive periods (6 months or more). Products with aw higher than 0.8 require the deactivation of enzymes with heating or other means to extend their shelf life.

3.4.2.4 Enzyme Inhibitors

Enzyme Inhibitors

are substances that alter the catalytic action of enzymes and consequently slow down or, in some cases, stop catalysis. Inhibitors bind to the enzyme either reversibly or irreversibly. The irreversible inhibitors (poisons) change the enzyme’s tertiary structure permanently, and the enzyme cannot function again. Reversible inhibitors do not bind permanently and can be removed from the enzyme (Fig. 3.33). Two major types of reversible inhibition can be distinguished: competitive and non-competitive. Competitive inhibition occurs when the substrate and the inhibitor compete against each other for the enzyme’s active site. Non-competitive inhibitors are substances that, when added to the enzyme, alter the active site’s structure so that it cannot accept the substrate (Fig. 3.33). Many enzymes contain sulfhydryl (-SH), hydroxyl (-OH), or carboxyl groups (−COOH) as part of their active sites. Any chemical which reacts with these groups acts as a non-competitive inhibitor. Examples include heavy metals, such as Hg²⁺ or Pb²⁺, with strong affinities for -SH groups. The non-competitive inhibitor binds to the enzyme near or away from the active site. It is known as allosteric inhibition if it is away from the active site.

2 diagrams exhibit inhibition binding. In competitive inhibition, the substrate binds to the active site of the enzyme along with the inhibitor. In non-competitive inhibition, the inhibitor binds to the active site of the enzyme. The active site changes conformation and does not fit.

Fig. 3.33

In competitive inhibition, the inhibitor binds to the active centre, and the substrate cannot fit. In non-competitive inhibition, the active site changes shape, and the substrate does not fit

3.4.3 Enzyme Nomenclature and Classification

Enzymes are commonly named by adding a suffix “-ase” to the name of their major substrate. For example, protease catalyses the hydrolysis of a protein, sucrase catalyses the hydrolysis of sucrose, whereas lactase catalyses the hydrolysis of lactose. There are exceptions to this rule, for example, pepsin, trypsin, or chymotrypsin. Food enzymes may be classified into endogenous when they are naturally present within food or exogenous when added for a particular technological purpose. Endogenous enzymes are responsible for various desirable or undesirable changes in raw and processed products, such as colour changes (polyphenol oxidase), flavour (lipase, lipoxygenase), or texture modification (proteases, amylases, polygalacturonases). Exogenous enzymes have only desirable properties if they are carefully controlled. Examples of applications of such enzymes are lactase for lactose hydrolysis, rennin in cheese manufacturing, papain for tenderisation and meat recovery or amylases for improving bread volume.

Enzymes are classified based on their reaction mechanism into seven main classes (Table 3.5). It should be noted that not all classes are important in food technology that is mainly concerned with hydrolases and oxidoreductases.

Table 3.5

Enzyme classification, reactions they catalyse and importance for foods. Their relative importance is indicated with stars (1-star low, 2-stars intermediate, 3-stars high importance), and X: Not important

Class

Reaction

Examples

Importance for foods

EC 1 Oxidoreductases

Catalyse oxidation or reduction reactions of substrates

Lipoxygenase, polyphenol oxidase, peroxidases

A symbol of a 2-star.

EC 2 Transferases

Catalyse the transfer of functional groups from one molecule to another

Transglutaminases

A symbol of a 1-star.

EC 3 Hydrolases

Catalyse the hydrolysis (i.e., breaking of bonds with the help of water) of substrates

Amylases, lipases, proteases

A symbol of a 3-star.

EC 4 Lyases

Catalyse the addition or removal of groups from substrates by non-hydrolytic mechanisms

–

A symbol of a cross mark.

EC 5 Isomerases

Intramolecular rearrangement of substrates

Glucose isomerase

A symbol of a 1-star.

EC 6 Ligases

Catalyse linking of substrates

–

A symbol of a cross mark.

EC 7 Translocases

Catalyse the movement of molecules across membranes

–

A symbol of a cross mark.

When writing this sentence, more than 6500 enzymes are known to science, making their classification challenging. The enzyme commission numbering system (EC number) classifies enzymes based on the chemical reactions they catalyse. It uses a unique four-digit numerical code separating each digit by a full stop, which provides a finer distinction of their catalytic activity (Fig. 3.34). For example, in the code EC 3.2.1.1, number 3 is the main class of the enzyme, a hydrolase. The second number is the subclass, a glycosylase that hydrolyses glycosidic bonds. The third number is the sub-subclass, a glycosylase that hydrolyses O- and S-glycosyl compounds. Finally, the fourth number is the serial number of the enzyme within its sub-subclass and identifies the name of the individual enzyme, which in this case is α-amylase. The complete list of all enzymes can be found on online databases, such as the ExPASy enzyme database.

A schematic diagram exhibits the numbering of the enzyme. The code reads, E C 3 dot 2 dot 1 dot 1. 3 is the main class, 2 is a subclass, 1 is a sub-subclass, and 1 is a serial number.

Fig. 3.34

Enzyme numbering according to the enzyme commission numbering system. This code is for α-amylase

Certain enzymes that hydrolyse proteins or polysaccharides may be endo- or exoacting enzymes (Fig. 3.35). Endoacting enzymes hydrolyse in the protein’s or polysaccharide’s interior, resulting in polypeptides or oligosaccharides. Exoacting enzymes hydrolyse at the end of the chains, resulting in free amino acids or monosaccharides. Combining both may lead to complete hydrolysis of the macromolecule, such as in glucose production from starch, where a cocktail of enzymes is used to hydrolyse starch completely.

A schematic diagram exhibits the hydrolyzed protein chain with exo enzymes and endo enzymes, resulting in the exo and endo enzyme products.

Fig. 3.35

Endo- and exoacting enzymes and their products

3.4.4 Food Enzymes

Most enzymes used for industrial purposes are of microbial origin and produced through controlled fermentation. Bakery, dairy, and beverage industries are the most prominent users of enzymes. Nevertheless, all sectors of the food industry are directly or indirectly concerned with enzymes. The most important enzymes used in food processing and their specific activity are discussed below.

3.4.4.1 Carbohydrate-Acting Enzymes

α-Amylases

are endoenzymes that hydrolyse randomly α-(1 → 4) linkages to liquify starch, but they do not hydrolyse α-(1 → 6) linkages (branching points). Products of α-amylase are maltose, glucose and dextrins. The difference between dextrins and maltodextrins that were described in the previous chapter is that dextrins are low-molecular-weight carbohydrates with α-(1 → 4) and α-(1 → 6) bonds, whereas maltodextrins have predominantly α-(1 → 4). As a result of branching, dextrins have quite different functional properties than maltodextrins. β-Amylases are exoenzymes that remove maltose units from non-reducing ends of amylose chains. In amylopectin, the cleavage stops a few units away from the α-(1 → 6)-branches. Glucoamylase (or amyloglucosidase) removes successive glucose units from the non-reducing ends of starch molecules by cleavage of the α-(1 → 4)-linkages and α-(1 → 6)-linkages. Unlike α-amylase, it hydrolyses linear and branched chains to form glucose. Pullulanasesand iso-amylases hydrolyse only α-(1 → 6) glycosidic bonds in starch, whereas α-glucosidases hydrolyse α-(1 → 4)-linkages from the non-reducing end to release glucose (Fig. 3.36). A typical application of amylases is in manufacturing glucose, maltose, or high fructose corn syrups. Hydrolysis starts with a corn starch suspension heated above the starch gelatinisation temperature (>70 °C). The pH of gelatinised starch is then adjusted to 6.5, and the paste is heated to 85 °C with the addition of α-amylase producing dextrins. Depending on the product, glucoamylase is added to make glucose syrup or a mixture of pullulanases and β-amylases to make maltose syrup. Finally, if high fructose corn syrup (HFCS) is desired, glucose isomerase is used to convert glucose to fructose. Amylases are used in the baking industry to mildly hydrolyse starch to glucose used by yeasts to produce CO₂ essential in bread proofing. They are also added to bakery products to minimise staling since they hydrolyse amylose, thus delaying retrogradation. Amylases also reduce dough viscosity and improve loaf volume, crumb softness and crust colour. In brewing, naturally occurring amylases in barley malt hydrolyse starch to give maltose for yeast to utilise and produce CO₂ and ethanol.

A schematic diagram exhibits the amylase chain with a non reducing end and a reducing end. The chain has the location of the alpha glucosidase, beta amylase, alpha amylase, glucoamylase, and pullulanase slash iso amylase.

Fig. 3.36

Location of action and products of the four major amylases. Refer to the text for a description of their action

Cellulases

are enzymes that catalyse the hydrolysis of cellulose. Endocellulases that randomly hydrolyse non-covalent bonds of crystalline cellulose to create free cellulose chains (i.e., not in crystalline form), exocellulases cleave cellulose producing disaccharides (cellobiose) or tetrasaccharides (cellotetraose) and finally β-glucosidase (cellobiase) hydrolyses cellobiose and cellotetrose into glucose (Fig. 3.37). Humans do not produce cellulases and cannot digest cellulose (see Sect. 2.​5.​10, Dietary Fibre). It may, however, be partially broken down through fermentation in the large intestine, producing short-chain fatty acids with beneficial health properties.

A flow diagram for the formation of glucose from cellobiose and cellotetrose. The crystalline cellulose undergoes endocellulases to form cellulose. The cellulose further undergoes exocellulases, forming cellotetraose and cellobiose, which are further processed with beta glucosidase to form glucose.

Fig. 3.37

Action of cellulases. Refer to the text for a description of their action

Pectic enzymes

break down pectin into oligosaccharides, galacturonic acid or de-esterify pectin. Pectin methyl esterase hydrolyses the methoxyl group from pectin to form low-methoxyl pectin and polygalacturonic acid. Polygalacturonase (pectinase) hydrolyses α-(1 → 4) linkages of polygalacturonic acid to produce oligo-galacturonans and galacturonic acid. Polygalacturonase has both endo- and exo-forms that hydrolyse pectin randomly or sequentially from the non-reducing ends (Fig. 3.38). Pectic enzymes are used in several food processing operations, such as fruit juice, wine, or tomato processing. In particular, tomato processing proceeds either by breaking tomatoes under high (~95 °C, hot-break) or low temperatures (~65 °C, cold-break). The main difference between these two processes is that the cold-break technology achieves a partial, whereas hot-break, a total deactivation of pectic enzymes. Hot-break results in a paste with high viscosity, as pectin is intact and enhances thickness, suitable for sauces, ketchup, or purées. Cold-break leads to low-viscosity pastes suitable for tomato juices and sauces, as pectin has been hydrolysed.

A schematic diagram exhibits the structure of pectinases. It has a chain with the locations of exo polygalacturonase, endo polygalacturonase, and pectin esterase.

Fig. 3.38

Location of action and products of the pectinases. Refer to the text for a description of their action

Invertase

hydrolyses sucrose to glucose and fructose to a product known as invert sugar. Invert sugar is used in the confectionery industry because it is sweeter than sucrose and less likely to crystallise (e.g., in soft candy fillings). Lactase (β-d-galactosidase) hydrolyses lactose to glucose and galactose and is used in the dairy industry to create lactose-free dairy products (milk, cream, etc.). Glucose oxidase is used in the egg industry to deplete egg whites of glucose and reduce the potential of Maillard browning upon dehydration and storage. It may also be used in flour to induce disulfide links in gluten and improve flour bread-making capacity.

3.4.4.2 Protein-Acting Enzymes

Proteases

are enzymes responsible for hydrolysing proteins to peptides or amino acids. They are essential for the quality and shelf life of foods, as they can influence the product at any stage if they are not carefully controlled. Proteases are naturally present in foods or may be added for a particular purpose. They may be classified according to their pH optima into acid, neutral, or alkaline proteases and may be endo- or exo-acting. They may also be classified based on the nature of the catalytic residues at their active site into aspartic- (e.g., pepsins, cathepsins, rennins), cysteine- (e.g., papain, bromelain, ficin, actinidin), serine- (e.g., trypsin, chymotrypsin), metallo- (e.g., carboxypeptidase A, thermolysin), threonine-, glutamic-, and asparagine- proteases. When proteases hydrolyse from the N-terminus of the protein, they are called aminopeptidases. When they hydrolyse from the C-terminus, they are called carboxypeptidases.

Proteases may be added as meat-tenderising agents on inferior meat cuts (e.g., papain). The enzyme hydrolyses primarily connective tissue proteins (i.e., collagen) and softens the muscle. Other tenderising enzymes are ficin (from figs), bromelain (from pineapple) and microbial proteases. Chymosin or rennin is found in the stomach of ruminants and is essential to manufacture cheese. Chymosin acts on κ-casein and cleaves at the peptide bond between amino acid residues 105 (Phe) and 106 (Met). This results in hydrophobic para-κ-casein, which remains on the casein micelle, and casein macropeptide, which is hydrophilic and transferred to the whey. In the presence of calcium, para-κ-casein and other molecules become insoluble and casein micelle remnants aggregate to form a gel. This cheese curd is the first step in many cheese manufacturing processes. Digestive proteases such as trypsin, chymotrypsin, and pepsin found in the animal pancreas may cause quality problems in muscle foods if contamination from intestines occurs (e.g., on minced products) that may lead to over-softening of meat. Proteases are also used to make protein hydrolysates with an intense savoury taste, usually used in snack products. Finally, they may partially break down wheat flour proteins to reduce mixing time and make them more extensible. Some are added to help with flavour and texture development and speed up fermentation of dairy products. Finally, various strains of proteolytic bacteria that produce beneficial proteases may also be used during food fermentations to give foods unique flavour and textural characteristics.

3.4.4.3 Lipid-Acting Enzymes

Lipases

are hydrolases that hydrolyse triacylglycerols to form diglycerides, monoglycerides, glycerol and free fatty acids (Fig. 3.39). Lipases dramatically impact the quality of food products, especially those that contain significant amounts of fat. Lipases may lead to hydrolytic rancidity, which is undesirable when free fatty acids are released in muscle foods and react with proteins to denature them and give a tough texture. Furthermore, if they are not inactivated in milk, they release short-chain fatty acids that are very volatile, leading to the generation of off-flavours. However, lipases may have desirable effects when used in fermented products, as they are critical in the ripening of cheese, salami, and other dry-meat products. Short-chain fatty acids released from milk fat produce the characteristic odour and flavour of dairy products, especially those with eight carbon atoms. Finally, lipases can be used to modify the properties of lipids, especially in the margarine industry, to give different textures and melting points or to produce mono- and di-glycerides for use as emulsifiers.

A schematic diagram presents the action mechanism of the lipid acting enzymes. The triglyceride is processed with lipase to form monoglycerides, glycerol, diglycerides, and fatty acids.

Fig. 3.39

Lipases hydrolyse triacylglycerols to form diglycerides, monoglycerides, glycerol and free fatty acids

3.4.4.4 Other Enzymes

Lipoxygenases, peroxidases and polyphenol oxidases (PPO)

are the most important representatives of oxidoreductases. Lipoxygenases are found in a wide variety of plants (primarily legumes) and have also been identified in animal tissues (e.g., in fish skin), and they catalyse the oxidation of unsaturated fatty acids (see next chapter for oxidation mechanisms). Three naturally occurring fatty acids are substrates: linoleic acid (2 double bonds), linolenic acid (3 double bonds), and arachidonic acid (4 double bonds). Lipoxygenases may have desirable outcomes when used for bleaching wheat and soybean flours. They also contribute to forming disulfide bonds in the dough, thus avoiding the addition of chemical oxidisers. In other products, if the activity is not controlled, it may create significant quality defects, as lipid oxidation and reactivity of the breakdown products (hydroperoxides) give off-flavours and off-odours, leading to textural problems. One of the purposes of blanching, an essential step before vegetable freezing, is to inactivate lipoxygenases and extend their shelf life.

Peroxidases

oxidise several phenolic substrates (e.g., p-cresol, catechol, caffeic and coumaric acids), ascorbic acid or aromatic amines. They are heat-stable enzymes responsible for bleaching chlorophylls and betalains, resulting in undesirable colour in canned vegetables. Reaction with guaiacol results in a brown colour, and the absence of colour indicates correct blanching. Polyphenol oxidases are discussed separately in detail in Chap. 5.

Phospholipases

are enzymes that hydrolyse phospholipids into fatty acids and other lipophilic compounds with various degrees of specificity. They are found in various animal-derived foods (e.g., eggs or dairy) and may contribute to quality decline. They may also be used as additives for specific processes (e.g., lecithin production from eggs).

Naringinase

is an enzyme complex with α-l-rhamnosidase and β-d-glucosidase activities. It is used as a debittering enzyme in citrus juice production, especially grapefruit, acting on naringin that gives grapefruit a bitter taste. α-l-Rhamnosidase first breaks naringin into prunin and rhamnose, followed by β-d-glucosidase, which breaks prunin into glucose and tasteless naringenin.

Transglutaminases

catalyse the formation of a peptide bond between a free amine group of a protein-bound lysine and the acyl group at the end of the side chain of protein-bound glutamine. Bonds formed by transglutaminase exhibit high resistance to proteolysis. The most typical commercial products made with transglutaminases are imitation crabmeat, also known as surimi, and fish balls. A relatively unsuccessful commercial name for transglutaminase preparations creating confusion to consumers and technologists is “meat glue”, as the chemistry of adhesion (i.e., glues) and enzymic catalysis are entirely different.

3.5 Review Questions

3.5.1 Multiple Choice Questions

Proteins

1.

The isoelectric point of an amino acid is:

(a)

pH value at which the amino acid is neutral

(b)

pH value at which the amino acid is positively charged

(c)

pH value at which the amino acid is negatively charged

(d)

pH value at which the amino acid is denatured

2.

In l-amino acids, the amino group is positioned to the left of the chiral carbon. This statement is:

(a)

False

(b)

True

(c)

It is true ONLY for non-polar and polar amino acids

(d)

It is true ONLY for charged amino acids

3.

A non-polar amino acid has

(a)

A hydrophilic R-chain

(b)

A hydrophobic R-chain

(c)

A negatively charged R-chain

(d)

A positively charged R-chain

4.

A peptide bond is

(a)

An amide bond between two amines

(b)

An amide bond between two amino acids

(c)

A glycosidic bond between two amines

(d)

A glycosidic bond between two amino acids

5.

The primary structure of a protein is:

(a)

The sequence of monosaccharides in the polypeptide chain

(b)

The sequence of fatty acids in the polypeptide chain

(c)

The sequence of amino acids in the polypeptide chain

(d)

None of the above

6.

The secondary structure of a protein is:

(a)

The specific geometrical arrangement of the polypeptide chain along one axis

(b)

Two primary structures connected

(c)

Two primary structures parallel to each other

(d)

The specific arrangement of α-helix along a horizontal axis

7.

Tertiary structure of proteins is stabilised by:

(a)

Hydrophobic interactions, ionic bonds, hydrogen bonds and disulfide bridges

(b)

Hydrophilic interactions, ionic bonds, hydrogenated bonds and trans bridges

(c)

Hydrophilic interactions, ionic bonds, hydrogen bonds and disulfide bridges

(d)

Hydrophobic interactions, ionic bonds, hydrogen bonds and glycosidic linkages

8.

Denaturation results in cleavage of peptide bonds”:

(a)

True for meat proteins ONLY

(b)

False for meat proteins ONLY

(c)

False for ALL proteins

(d)

True for ALL proteins

9.

Proteins usually unfold:

(a)

Near their isoelectric point

(b)

At extreme pH values, e.g., highly acidic or alkaline

(c)

Near their isoelectric point but in the presence of NaCl

(d)

At low to medium NaCl concentrations

10.

“Protein hydrolysis involves breaking peptide bonds yielding smaller peptide chains or amino acids”

(a)

True

(b)

False

(c)

True ONLY for fermented products

(d)

True ONLY for acidic products such as orange juice

Enzymes

1.

Enzymes, in general, act:

(a)

on specific substrates catalysing a specific reaction.

(b)

on carbohydrates and proteins only catalysing a specific reaction.

(c)

on lipases only catalysing a range of hydrolytic reactions.

(d)

on plant and animal organisms only catalysing metabolic reactions.

2.

Complete the sentence: “A cofactor is a non-protein molecule…”

(a)

that is bound to a polysaccharide and is required for hydrolysis

(b)

that is bound to a protein and is required for the enzymic action

(c)

that is bound to the substrate and is required for the enzymic action

(d)

that is bound to a protein and is required for concurrent reactions

3.

Enzymic specificity is the ability of an enzyme:

(a)

to catalyse two reactions at the same time

(b)

to catalyse one reaction in proteins and one in polysaccharides

(c)

to catalyse only one reaction

(d)

to catalyse one reaction in each group of macronutrients (i.e., carbohydrates, proteins, lipids)

4.

Enzymic selectivity is:

(a)

the ability of enzymes to select which reaction to carry out

(b)

the preference of an enzyme to catalyse peptide instead of glycosidic bonds

(c)

the industrial preference for an enzyme for a specific manufacturing process

(d)

the preference of an enzyme toward similar competing substrates

5.

Endo-acting enzymes:

(a)

Act randomly in the interior of the protein or polysaccharide molecule

(b)

Start hydrolysing from the outside of the protein or polysaccharide molecule

(c)

Act in the endocarp of certain fruits, leading to tissue softening

(d)

Act in the exocarp of certain fruits, leading to tissue firming

6.

α-amylases catalyse:

(a)

hydrolysis of ester bonds in triglycerides.

(b)

hydrolysis of α-1,4 linkages of starch.

(c)

hydrolysis of peptide bonds.

(d)

hydrolysis of α-1,6 linkages of starch.

7.

Pectin methyl-esterase hydrolyses the:

(a)

acetyl group of pectin to form low-methoxyl pectin and polygalacturonic acid.

(b)

methoxyl group of pectin to form high-methoxyl pectin and polygalacturonic acid

(c)

methoxyl group of pectin to form low-methoxyl pectin and polygalacturonic acid

(d)

methoxyl group of pectin to form low-methoxyl pectin and polyguluronic acid

8.

Lactase catalyses hydrolysis of.

(a)

Lactose to glucose and fructose.

(b)

Lactic acid to glucose and galactic acid.

(c)

Galactose to glucose and lactose.

(d)

Lactose to glucose and galactose.

9.

Proteases catalyse.

(a)

Hydrolysis of peptide bonds of protein to peptides and amino acids.

(b)

Hydrolysis of peptide bonds of polysaccharides to monosaccharides.

(c)

Synthesis of peptide bonds to form proteins.

(d)

Synthesis of peptide bonds to form polysaccharides.

10.

Lipases hydrolyse:

(a)

Fats to form diglycerides, monoglycerides and free fatty acids.

(b)

Lipose to glucose and liparose.

(c)

Fatty acids to triglycerides.

(d)

Triglycerides to cholesterol and fat-soluble vitamin D.

3.5.2 Short Answer Questions

1.

Draw and discuss the changes in the ionisation state of amino acids with changes in pH. What is the consequence of changes in pH for food proteins?

2.

Draw cysteine and identify the carboxyl and amino groups, the side chain, and the sulfhydryl group. What kind of special bonds is this amino acid likely to form?

3.

The amino acid sequence SRFRASHGDFRI is from the protein vicilin. Identify the amino acids in this sequence.

4.

The table below shows the dissociation constants of three amino acids. Calculate their pI using the correct equation.

5.

The tripeptide LLF is a bitter peptide in whey protein hydrolysates. Draw its structure.

6.

Classify proteins according to the Osborne classification scheme.

7.

The following protein structure is bovine α-lactalbumin:

A three-dimensional structure of the gamma lactalbumin protein. The structure has alpha helixes and beta sheets.

How many α-helixes and β-sheets can you count? Are β-sheets parallel or antiparallel? Identify a loop and a random coil.

8.

Name and discuss the forces that stabilise the tertiary structure of proteins.

9.

List and discuss the factors that affect protein denaturation.

10.

The following protein is hydrolysed with an enzyme that cleaves the bonds between R and F amino acids (shown in bold).

RRHKNKNPFHFNSKRFQTLFKNQYGHVRVL.

Write the sequence of the peptides that are formed.

11.

The protein shown below is hydrolysed with the action of acid and heat at the amino acid positions shown in bold.

RRHKNKNPFHFNSKRFQTLFKNQYGHVRVL.

Write the sequence of the peptides that are formed. Name the free amino acids that are released.

12.

A protein has the following primary structure:

LLVWVAVWEDRKH.

13.

You use this protein to make an oil-in-water salad dressing. Identify and explain which part of the protein will arrange towards the oil phase and which part towards the aqueous phase (Tip: find the hydrophobic amino acids in the sequence).

14.

Discuss how the pH and temperature influence protein solubility.

15.

Name the agents that influence protein denaturation.

16.

Name the main proteins found in milk’s casein and whey protein fraction.

17.

Name the family of plants used to extract plant proteins, five species and the main protein fractions of plant proteins.

18.

Name the proteins found in egg white and egg yolk.

19.

A company uses protein gelation technology to stabilise the structure of a milk-based dessert. Describe the steps and the chemical changes that occur in proteins during gelation.

20.

Discuss the differences between the terms “gluten”, “glutenin”, “gliadin”, “glutelin”, and “prolamin”.

21.

Describe the structure of myofibrillar proteins.

22.

Describe the structure of collagen. How is gelatin obtained, and what are the differences between Type-A and Type-B gelatins?

23.

Name and discuss the factors that control enzymic activity.

24.

A company wants to make tomato paste with low viscosity suitable for soup formulations. Immediately after tomato crushing, they pasteurise the pulp to 88 °C, and unfortunately, the paste has high viscosity, making it unsuitable for the end product. Explain what happened, which enzymes act on tomato pulp, and how you will resolve this problem.

25.

Identify the locations of action and products of α-amylase, β-amylase and glucoamylase. Describe the use of these enzymes in glucose syrup production.

26.

Describe the action of chymosin, invertase and lactase on their respective substrates. Give one food application of each enzyme.

27.

Visit the website https://​www.​expasy.​org/​resources/​enzyme and find the enzymes EC 3.4.22.2, EC 3.1.1.3, and EC1.13.11.12. Browse the information shown.

28.

Further reading: Find the review article “McClements, D. J. (2004). Protein-stabilized emulsions. Current Opinion in Colloid & Interface Science, 9(5), 305–313”.

Name the physical and chemical parameters that influence emulsion stability.

How do thermal processing, homogenisation, drying, and freezing influence emulsion stability?

What is the influence of pH and ionic strength on emulsion stability?

Bibliography

Review Articles

McClements DJ (2004) Protein-stabilized emulsions. Current Opinion in Colloid & Interface Science 9 (5):305–313. https://​doi.​org/​10.​1016/​j.​cocis.​2004.​09.​003Crossref

Dalgleish DG, Spagnuolo PA, Goff HD (2004) A possible structure of the casein micelle based on high-resolution field-emission scanning electron microscopy. International Dairy Journal 14: 1025–1031

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024

V. KontogiorgosIntroduction to Food Chemistryhttps://doi.org/10.1007/978-3-031-53558-1_4

4. Lipids

Vassilis Kontogiorgos1  

(1)

Associate Professor in Food Chemistry, University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC, Canada

Vassilis Kontogiorgos

Email: v.kontogiorgos@ubc.ca

Learning Objectives

After studying this chapter, you will be able to:

Describe the structure of lipids found in food

Describe the mechanisms of lipid oxidation

Discuss the factors that influence the rate of lipid oxidation

Describe fat crystal formation

Describe triacylglycerol conformation and crystal polymorphism

Discuss factors that influence the melting point of fat crystals

4.1 Introduction

Lipids are soluble substances in organic solvents but insoluble in water. They are major components of adipose tissue and the cell membranes of animals. Their unique physical and chemical properties make them essential contributors to the sensory properties, storage stability and generally the overall quality of foods, especially those rich in lipids. Lipids comprise eight groups with distinct physical, chemical, and physiological properties: fatty acids, triacylglycerols, phospholipids, sphingolipids, steroids, waxes, fat-soluble vitamins, and carotenoids. Fats and oils in foods are differentiated depending on their source and physical state at room temperature. Fats are solid at room temperature (~20 °C), while oils are liquid. The main reason is that fats are derived from animals and are primarily saturated. In contrast, oils are derived from plants and are mono- or polyunsaturated. Three exceptions are coconut and palm oils, derived from plants but are solid at room temperature, and fish oils, which are liquid at room temperature. Animal sources include meat, poultry, and dairy, whereas plant sources include nuts, seeds, avocado, olives, and coconut. Most fruits and vegetables contain very little fat. In the following sections, we will explore these molecules’ structure and properties and their relevance in food manufacturing, quality, and shelf life.

4.2 Fatty Acid Nomenclature and General Characteristics

Fatty acids are carboxylic acids with an aliphatic (straight) carbon chain (Fig. 4.1). Fatty acids differ in two ways: the length of the carbon chain and the degree of saturation. The number of carbon atoms determines the length of the chain, and in naturally occurring fatty acids, it is always an even number, usually between 4 and 22 carbon atoms. The one end of the structure is hydrophilic, as it contains the carboxylic group, whereas the hydrocarbon chain at the other end is hydrophobic (Fig. 4.1). The counting of carbon atoms starts from the carbon atom of the carboxyl group and proceeds to the left. The number of double bonds between carbon atoms determines the degree of saturation. The fatty acids are saturated in the absence of a carbon-carbon double bond. Fatty acids with one carbon-carbon double bond are called monounsaturated and polyunsaturated with two or more carbon-carbon double bonds. Saturated fatty acids are primarily found in animal sources such as meat, poultry, butter, egg yolk, or lard. Plant sources that contain saturated fatty acids are coconut and palm oils. Major sources of monounsaturated fatty acids are olive and avocado oils and peanut butter. Polyunsaturated fatty acids are predominantly found in plants (e.g., corn, canola, sunflower, flaxseed) and fish oils.

A general structure of fatty acids. It has a carbon chain hydrophobic end and a carboxyl group hydrophilic end. The single and double bonds in the hydrophobic carbon chain are marked.

Fig. 4.1

General structure of fatty acids. (See text for description)

Fatty acids can be named according to the systematic IUPAC nomenclature system (e.g., butanoic acid) or use empirical names that usually relate to its source, for example, butyric acid found in butter. A way to write fatty acids is by using numerical abbreviations showing the number of carbon atoms and double bonds (Table 4.1). For example, butyric acid is 4:0 because it has four carbon atoms and zero double bonds, whereas 18:1 represents oleic acid with eighteen carbon atoms and one double bond. Omega fatty acids (ω) are polyunsaturated fatty acids characterised by the presence of a double bond three (ω-3) or six (ω-6) atoms away from the terminal methyl group (Fig. 4.2 and Table 4.2). Examples of ω-3 are α-linolenic acid (C18:3 n-3) or eicosapentaenoic acid (C20:5 n-3), and examples of ω-6 are linoleic acid (C18:2 n-6) or arachidonic acid (C20:4, n-6). The letter n in the abbreviation indicates the carbon atom number where the first double bond is found, counting from the end of the structure. The notation Δx,y,... is also used to indicate the positions of double bonds. For instance, arachidonic acid is Δ5,8,11,14, indicating double bonds on carbons 5, 8, 11, and 14. For Δ notation, counting, in this case, starts from the carboxyl group (Fig. 4.2).

Table 4.1

Saturated fatty acids found in foods

Common name

Systematic name

Structural formula

Abbreviation

Sources

Butyric acid

Butanoic acid

CH₃(CH₂)₂COOH

C4:0

Butter

Caproic acid

Hexanoic acid

CH₃(CH₂)₄COOH

C6:0

Coconut, palm oil, butter

Caprylic acid

Octanoic acid

CH₃(CH₂)₆COOH

C8:0

Coconut, palm oil, butter

Capric acid

Decanoic acid

CH₃(CH₂)₈COOH

C10:0

Coconut, palm, nut oil, butter

Lauric acid

Dodecanoic acid

CH₃(CH₂)₁₀COOH

C12:0

Coconut, palm, nut oil, butter

Myristic acid

Tetradecanoic acid

CH₃(CH₂)₁₂COOH

C14:0

Animal and plant fats

Palmitic acid

Hexadecanoic acid

CH₃(CH₂)₁₄COOH

C16:0

Animal and plant fats

Stearic acid

Octadecanoic acid

CH₃(CH₂)₁₆COOH

C18:0

Animal fats

Arachidic acid

Eicosanoic acid

CH₃(CH₂)₁₈COOH

C20:0

Peanut oil

The number of carbon atoms is even

Chemical structure of (5 Z, 8 Z, 11 Z, 14 Z), icosa, 5, 8, 11, 14, tetraenoic acid. Omega end and delta 14, 11, 8 and 5 double bonds are marked.

Fig. 4.2

Example of notations in fatty acids using the unsaturated arachidonic acid. Its IUPAC name is 5,8,11,14-eicosatetraenoic acid

Table 4.2

Unsaturated fatty acids found in foods

ω-n

Common name

Abbreviation

Δn

Configuration

Sources

ω−3

α-Linolenic acid

C18:3

Δ9,12,15

cis

Flaxseeds, chia seeds, walnuts

ω−3

Eicosapentaenoic acid

C20:5

Δ5,8,11,14,17

cis

Cod liver, herring, mackerel, salmon, menhaden and sardine

ω−6

Linoleic acid

C18:2

Δ9,12

cis

Peanut oil, chicken fat, olive oil

ω−6

Linolelaidic acid

C18:2

Δ9,12

trans

Partially hydrogenated vegetable oils

ω−6

Arachidonic acid

C20:4

Δ5,8,11,14

cis

–

ω−7

Palmitoleic acid

C16:1

Δ⁹

cis

Macadamia nuts

ω−9

Oleic acid

C18:1

Δ⁹

cis

Olive oil, canola oil

ω−9

Elaidic acid

C18:1

Δ⁹

trans

Hydrogenated vegetable oils

ω−9

Erucic acid

C22:1

Δ¹³

cis

Mustard oil

Double bonds in fatty acids result in cis- and trans- isomerism. In the cis-configuration, hydrogens attached to carbon atoms are on the same side of the double bond, whereas in trans-configuration, they are on either side of the double bond (Fig. 4.3). Naturally occurring fatty acids are always in cis-configuration. Fatty acids in trans-configuration have been found to have negative health implications and are usually present because of specific processing steps (e.g., hydrogenation or frying). However, modern processing technologies have alleviated this problem significantly.

Chemical structures of cis and trans forms of octadecenoic acid.

Fig. 4.3

Cis-, trans- isomerism of fatty acids. In the blue box, the positioning of hydrogen atoms around the double bond is emphasised

A structural characteristic of some fatty acids is the conjugated double bond when at least one pair of double bonds is separated by a single bond (Fig. 4.4). The presence of conjugated double bonds is important as it is responsible for colour in carotenoids and the properties of the family of conjugated linoleic acids (more than 28 isomers) with beneficial health effects. Notably, most naturally occurring fatty acids are in cis-configuration, not conjugated, and with even-numbered carbon atoms.

Structure of the conjugated double-bond system. It presents 8 carbons with 3 double bonds. The double bonds are separated by the single bond.

Fig. 4.4

Single bonds separate double bonds in conjugated double bond systems

4.3 Triacylglycerols (TAGs)

Esterification is the reaction of an alcohol with a carboxylic acid to give ester and water. Fats and oils are chemically known as triacylglycerols (TAGs) and are formed by the reaction of a fatty acid with glycerol (Fig. 4.5).

2 chemical reactions with the molecular structures. A. It presents the addition of alcohol in the fatty acid. Esterification and hydrolysis produces ester and water. B. It presents the addition of glycerol with the fatty acid. Triacylglycerol and water are produced.

Fig. 4.5

(a) General description of esterification, and (b) esterification of glycerol with a fatty acid to form triacylglycerol

When three fatty acids are attached to glycerol, they form a triacylglycerol; two fatty acids form a diacylglycerol, whereas one fatty acid forms a monoacylglycerol. The fatty acids in the glycerol can be identical (simple triacylglycerol) or different (mixed triacylglycerol) (Fig. 4.6).

Chemical structures of monoacylglycerol, diacylglycerol, triacylglycerol, simple triacylglycerol and mixed triacylglycerol.

Fig. 4.6

Different glycerides formed from esterification of glycerol with fatty acids. The R group is the same in simple triacylglycerols, whereas in mixed triacylglycerols, it is different

When the middle carbon in glycerol has four different substituent groups, it becomes chiral with mono-, di-, and mixed TAGs with chiral carbon. Consequently, the carbon atoms of glycerol are numbered stereospecifically to distinguish the configuration of the fatty acids in TAGs. A Fischer projection must be drawn with the hydroxyl group at C2 to the left. The carbon atom at the top of the structure is C1, and at the bottom is C3. In this case, the prefix sn (stereospecific numbering) is used, and in that way, it is possible to differentiate the positioning of the fatty acid(s) (Fig. 4.7).

Fischer projection of a triacylglycerol.

Fig. 4.7

Fischer projection of a TAG. Stereospecific numbering of triglycerides is used to designate the configuration of triacylglycerides

For instance, the following names 1-oleoyl-2-stearoyl-3-palmitoyl-sn-glycerol, sn-1-oleo-2-stearo-3-palmitin, sn-glycerol-1-oleate-2-stearate-3-palmitate are all equivalent referring to the same TAG and allow to distinguish the position of the esterified fatty acid(s). In this example, the -OH group of glycerol at C-1 is esterified with oleic acid at C-2 with stearic acid and C-3 with palmitic acid. It is important to know the stereospecific numbering in reactions such as interesterification or when crystallisation of TAGs is considered, as the location of the fatty acid on glycerol plays an important role in the physical properties of the resulting TAG. The fatty acid composition of foods is quite variable and complex, but some general guidelines may be drawn. Vegetable oils from oilseeds are highly unsaturated with 18-carbon fatty acids. Oils high in oleic acid include olive and canola, high in linoleic acid are soybean and corn, whereas high in linolenic acid is linseed oil. On the other hand, plant oils high in saturated fatty acids are cocoa butter, coconut, and palm oils. The levels of saturated fats from animal sources vary in the order of milk fat > sheep > beef > pig > chicken > turkey > fish. Finally, TAGs from plants or animals have unsaturated or saturated fatty acids, respectively, in the sn-2 position.

In phospholipids, one hydroxyl group in glycerol is esterified by phosphoric acid at the sn3 position (Fig. 4.8). This structure makes phospholipids amphiphilic with one part of the structure soluble in the aqueous phase while its hydrophobic part is soluble in the oil phase.

Chemical structure of lecithin is composed of 2 fatty acids connected via glycerol, a phosphate group and choline.

Fig. 4.8

Structure of lecithin, a common food phospholipid

As a result, this molecule is surface-active (surfactant) and can arrange at the air-water or oil-water interfaces, creating foams or emulsions, respectively. Phospholipids are used in the food industry as emulsifiers in beverages, baked goods, salad dressings, and confectionery products. Foods that naturally contain phospholipids include egg yolks, liver, soybeans, wheat germ, and peanuts, with lecithin being the most characteristic example (Fig. 4.8).

Sphingolipids are a class of lipids containing a backbone of Sphingoid bases. Sphingosine is an amino alcohol that forms the structural basis of the most important sphingolipids (Fig. 4.9a). For example, ceramides are composed of sphingosine with an N-linked fatty acid at the sn-2 position (Fig. 4.9b). Derivatives of ceramides may be formed by reactions of the -OH group at sn-1. For example, sphingomyelin consists of a ceramide with phosphocholine esterified at sn-1 (Fig. 4.9c). When a single sugar residue (usually glucose or galactose) is attached at sn-1, a cerebroside is obtained, whereas when an oligosaccharide with one or more sialic acids is attached, a ganglioside is obtained (Fig. 4.10). These compounds play a role in signal transduction and cell regulation. Although they are not typically considered dietary lipids due to their low concentration, their role is continuously examined and re-evaluated. Foods rich in sphingolipids and their derivatives include dairy, meat, eggs, soy, and seafood (e.g., mussels, scallops, etc.). In contrast, fruits and vegetables generally have a low content of these compounds.

a. Chemical structure of 2 amino 4 trans octadecene 1, 3 diol. b presents fatty acid residue in connection with 2 amino 4 trans octadecene 1, 3 diol. c presents fatty acid residue in connection with 2 amino 4 trans octadecene 1, 3 diol, phosphate ester and choline.

Fig. 4.9

Structures of (a) sphingosine, (b) ceramide, and (c) sphingomyelin

2 chemical structures. a. It presents fatty acid residue in conjunction with monosaccharide and sphingosine. b. It presents fatty acid residue in conjunction with oligosaccharide and sphingosine.

Fig. 4.10

Structures of (a) cerebrosides and (b) gangliosides

Sterols consist of interconnected cyclohexane rings with various attached side chains (Fig. 4.11). Many sterols are essential in maintaining the human body, including cholesterol, bile, sex (i.e., testosterone, oestrogen) and adrenal (i.e., cortisol) hormones, and vitamin D. The dietary sterol of the greatest significance in foods is cholesterol, where elevated levels of cholesterol in the blood are associated with diseases such as atherosclerosis that increases the risk of heart attack or stroke. Major dietary sources of cholesterol include red meat, egg yolks, liver, and butter. Phytosterols are plant sterols or stanols (e.g., β-sitosterol or stigmasterol) that may compete with intestinal cholesterol absorption and reduce its levels in the blood. Products belonging to the broader group of functional foods are available in the market, incorporating phytosterols in the formulations (e.g., margarine or yoghurt drinks) to help with cholesterol reduction. Finally, waxes are mixtures of esters of fatty acids and long-chain alcohols but are not triacylglycerides. Carnauba wax, beeswax, and candelilla wax are typical examples of glazing agents and fruit waxing in the food industry.

5 chemical structures. a. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 hexadecahydro 1 H cyclopenta, a, phenanthren 3 ol. b. Cholest 5 en 3 beta ol. c. Vitamin D 3. d. Beta sitosterol. e. Overlapping of the structures.

Fig. 4.11

Structures of sterols. The last image shows a structure overlap to illustrate common structural aspects between them. The common characteristic is the gonane structure consisting of three molecules of cyclohexane (A, B, and C rings) and one of cyclopentane (D ring)

4.4 Lipid Oxidation

Lipid oxidation, also known as oxidative rancidity, is the reaction of lipids with oxygen and is generally detrimental to foods. Food eventually oxidises, and the objective of the food scientist is to halt oxidation until the intended shelf-life of the product. Three types of lipid oxidation may be distinguished: auto-oxidation (triplet O₂ oxidation), photo-oxidation (singlet O₂ oxidation) and enzymatic oxidation (lipoxygenase).

4.4.1 Autoxidation (Triplet Oxygen Oxidation)

Autoxidation of lipids proceeds by a free radical mechanism. Free radicals are molecules or atoms with unpaired electrons and can oxidise easily other compounds. Auto-oxidation occurs in three stages: initiation, propagation, and termination (Fig. 4.12).

8 chemical reactions present the stage of lipid oxidation. It presents the initiation, propagation, and termination stages. The initiation presents R H plus O 2 producing alkyl radical plus H in presence of energy and catalyst. Propagation includes peroxyl radical and new alkyl radical.

Fig. 4.12

The three stages of lipid oxidation. (See text for description)

Initiation of oxidation occurs when a hydrogen atom in the double bonds of unsaturated fatty acids is removed to form an alkyl radical (R•). Metal ions (usually Fe, Cu, Mg, or Ni), UV radiation (light), singlet oxygen, lipoxygenase, and high temperatures may cause or promote oxidation. Pro-oxidants accelerate oxidation by direct interaction with unsaturated fatty acids to form lipid hydroperoxides or by promoting the formation of free radicals. Propagation involves the addition of oxygen to the alkyl radical and the production of peroxyl radicals (ROO•). Peroxyl radicals react with another fatty acid to produce hydroperoxides (ROOH) and a new alkyl radical, beginning a new oxidation cycle. Termination occurs when free radicals interact with each other or with an antioxidant, giving unreactive molecules. Hydroperoxides decompose to a multitude of compounds with flavour (aldehydes, ketones, hydrocarbons, furans, acids) and biological significance, causing major quality deterioration, including generation of off-flavours in oils, changes in melting point in TAGs, formation of coloured products, formation of toxic compounds or loss of nutritional value. Water activity is an important factor influencing the rate of lipid oxidation. The relationship between aw and oxidation rate is complex, and each food should be regarded separately. Generally, the oxidation rate is fast at very low aw (<0.2), low at intermediate aw (0.2–0.5), and fast at high aw (>0.5) (see also Fig. 1.​12). Catalyst activity (e.g., Fe, Cu, Mg, or Ni) is high in the dry state of food, supporting fast reactions. As water increases, it hydrates catalysts, thus reducing their catalytic action, and the rate of lipid oxidation slows down. In addition, at intermediate aw, the water interacts with polar hydroperoxides (ROOH) produced in the propagation step and cannot react fast enough. The increase of oxidation rate at high aw (>0.5) (Fig. 1.​12) is usually due to the increased re-mobilisation of the catalysts and the reduced viscosity of the aqueous phase that both facilitate the reaction. The increased rate is not due to the increased oxygen concentration in the aqueous phase since the reaction occurs in the lipid phase. It should also be noted that the solubility of oxygen in lipids is multiple times greater than in water. Control of water activity and temperature is necessary in foods susceptible to lipid oxidation. Choosing a packaging film material with low water vapour permeability to prevent moisture migration to or from the environment during storage is important. Other methods, such as vacuum packaging or addition chelators (e.g., EDTA), antioxidants or oxygen scavengers in the packaging, may also be used depending on the food composition.

The type of fatty acids in TAGs influences the reaction rate, and generally, as the number of double bonds increases, the reactivity increases. For instance, oleic acid with eighteen carbons and one double bond (18:1) oxidises ~100 times faster than stearic acid with eighteen carbons and zero double bonds (18:0). In contrast, the rate of oxidation of α-linolenic acid with eighteen carbons and three double bonds (18:3) can be more than 2000 times faster. Fish and plant oils are sensitive to oxidation, and care must be taken during food formulation and storage. Furthermore, residual unsaturated fatty acids that remain in plant protein isolates after extraction (e.g., pea or soy protein) create oxidative stability problems for the long-term storage of products formulated with them. An increase in temperature, oxygen concentration, surface area (e.g., bulk oil vs emulsion or minced meat vs whole muscle), and light (UV and visible) increase the rate of lipid oxidation. Strategies that minimise food exposure to these factors generally improve oxidative stability.

Antioxidants can stop or slow down oxidation, but grouping them into one category is difficult, as they can delay oxidation by different mechanisms. Antioxidants exert their influence by controlling free radicals, pro-oxidants, or oxidation intermediates. Free radical scavengers slow oxidation by reacting faster and scavenging (removing) free radicals, inhibiting the initiation and propagation reactions. They donate hydrogen to peroxyl radicals, forming low-energy scavenger radicals that are inefficient in propagating oxidation. Control of pro-oxidant metal ions (e.g., Fe, Cu), singlet O₂ or lipoxygenases is achieved with the use of chelators (e.g., EDTA) for removing metal cations, the addition of carotenoids to quench singlet O₂, or heat treatments to deactivate enzymes (e.g., blanching), respectively. Antioxidants can be natural (e.g., tocopherol, ascorbic acid, or carotenoids) or synthetic (e.g., butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), or tertiary butylhydroquinone (TBHQ), Fig. 4.13).

Top. A chemical reaction. Peroxyl radicals react with antioxidants to produce hydroperoxide and low energy radicals. Bottom. Chemical structures of B H T, T B H Q, propyl gallate, ascorbic acid, and alpha-tocopherol are presented.

Fig. 4.13

General mechanism of action of free radical scavengers (top) and structures of some synthetic and natural antioxidants (bottom)

The choice of antioxidants depends on food composition, desired shelf-life, and cost. Generally, the ideal antioxidant must not have any harmful physiological effects or contribute to an objectionable flavour, odour, or colour to the product. It should be effective at low concentrations, soluble in the lipid phase, and remain unaltered during processing. Cost, availability, and marketing issues (e.g., “natural”) should also be considered when choosing the correct approach. Alternatives to the use of antioxidants could be the elimination of oxygen (e.g., packaging under nitrogen, vacuum or with an oxygen scavenger), elimination of the sensitive substrates (e.g., replacement of polyunsaturated oils with less unsaturated that are more stable), or by decreasing the rate of oxidation with physical methods (e.g., storage at low temperatures, storage in the dark, use of lipids that contain low levels of oxidation promoters or use of ingredients that are naturally rich in antioxidants). An overview of the factors influencing lipid oxidation is presented in Table 4.3.

Table 4.3

Summary of factors influencing lipid oxidation

Parameter

Comments

Oxygen

Triplet and singlet O₂. Generally, higher O₂ concentration accelerates oxidation

Double bonds

Higher number of double bonds accelerates oxidation

Pro-oxidants

Transition metals, ¹O₂, and lipoxygenase accelerate oxidation

Temperature

Higher temperatures accelerate oxidation

Surface area

Higher surface area facilitates oxidation, e.g., emulsion vs bulk oil

Water activity

Fast at very low aw (<0.2), low at intermediate aw (0.2–0.5), and fast at high aw (>0.5)

Antioxidants

Delay ³O₂ oxidation. Limited effect on ¹O₂ oxidation

Light

UV and visible light accelerate oxidation

Storage time

The longer we store the food, the more it will oxidise

4.4.2 Photo-Oxidation (Singlet Oxygen Oxidation)

The two types of O₂ are triplet (³O₂) and singlet (¹O₂) oxygen. The difference lies in the arrangement of electrons in the molecular orbitals of O₂. The full explanation is quite involved, and details on the different types of O₂ can be found in a general chemistry textbook. What is relevant to our discussion is that ¹O₂ is more reactive than ³O₂. Singlet O₂ oxidation requires light and the presence of photosensitisers. Photosensitisers are compounds that absorb light and transfer the energy to ³O₂, converting it to ¹O₂. Singlet oxygen then reacts directly with the double bonds of fatty acids, forming hydroperoxides (Fig. 4.14). Photosensitisers (e.g., chlorophyll and riboflavin) are naturally found in raw ingredients, and in most cases, it is not possible to remove them from food.

A chemical reaction. Fatty acid undergoes oxidation through photosensitizer to produce hydroperoxide.

Fig. 4.14

Hydroperoxide formation with singlet oxygen

The main differences between autooxidation and photo-oxidation are that ¹O₂ oxidation does not proceed through radical formation and does not have an induction period. Additionally, photo-oxidation is not particularly influenced by temperature, the degree of unsaturation of fatty acids, and the amount of oxygen dissolved in food. Photooxidation produces different hydroperoxides than autooxidation that decompose to distinct breakdown products (e.g., aldehydes). Analysis of breakdown products allows differentiation between the two mechanisms and identifies which type of oxygen is responsible for product oxidation. Antioxidants are not very effective against ¹O₂ oxidation. Packaging impermeable to light, vacuum packaging, removal of photosensitisers or addition of singlet oxygen quenchers (e.g., ascorbic acid, tocopherol, or carotenoids) are some strategies that can be employed to minimise photo-oxidation depending on product composition. Soybean oil and milk are two foods that need to be stored away from sunlight because they are prone to photooxidation.

4.4.3 Enzymatic Oxidation

Lipolysis is the hydrolysis of ester bonds in lipids by enzymic action or heat and moisture, which liberates free fatty acids and leads to hydrolytic rancidity (Fig. 3.​39). The main enzymes involved in lipid oxidation in foods are lipoxygenases, lipases, and phospholipases. To a lesser extent, peroxidases and various metalloenzymes may also be involved as they accelerate lipid oxidation with various mechanisms. The action of these enzymes has been described in the Sects. 3.​4.​4.​3 and 3.​4.​4.​4.

Fat Analysis

Fat analysis in foods proceeds by extracting the fat with a suitable organic solvent (e.g., petroleum ether), evaporating the solvent and finally measuring the weight of the fat residue. These are known as solvent extraction methods, with the Goldfish and Soxhlet methods being the two most common representatives. Lipid characterisation also proceeds with a fat extraction step with a suitable solvent, depending on food composition. Following extraction, many analytical techniques are available to characterise the properties of the extracted lipids depending on the purpose.

Method

Purpose

Smoke, flash, and fire points

Identification of temperatures at which the sample begins to smoke (smoke point), ignites (flash point) or combusts (continuous burning) (fire point). Higher smoke point makes oil suitable for frying applications.

Melting point

Identification of the temperature range at which fat melts. Important for texture or storage considerations.

Iodine value

A measure of the degree of unsaturation. A higher number indicates more unsaturated lipid.

Saponification value

A measure of the average molecular weight of triacylglycerols in the sample. Smaller values indicate longer average fatty acid chain length.

Free fatty acids (FFA) and acid values

A measure of fat acidity reflecting the number of fatty acids hydrolysed from triacylglycerols during refining or storage. High FFA values reflect poor oil quality.

Solid fat content

It measures the amount of crystalline fat (solid fat) in a sample at a specific temperature.

Total polar compounds (TPC)

A measure of deep-frying oil quality. When a frying oil has > ~27% of TPC, then it should be discarded.

Peroxide Value

A measure of the amount of peroxide or hydroperoxide groups. Higher values indicate advanced lipid oxidation (see also next row).

Anisidine Value and Totox Value

Anisidine value determines the number of aldehydes, whereas the Totox value indicates the total oxidation of a sample using both the peroxide and anisidine values. Higher values indicate advanced oxidation.

Thiobarbituric Acid Test (TBA)

Measures malonaldehyde, which is a secondary product of lipid oxidation. Higher malonaldehyde concentration indicates advanced oxidation.

4.5 Fat Crystallisation

4.5.1 Crystal Formation

As described in Chap. 1 crystal is a solid in which the atoms, molecules or ions are arranged in a repeating pattern that extends in all three dimensions. The repeating pattern of crystals is termed a unit cell. Most crystals are symmetric, which means they are invariant (do not change) when subjected to certain transformations (e.g., rotation). Several types of symmetry exist, for example, symmetry about a point (centre of symmetry), line (axis of symmetry), or plane (plane of symmetry). Unit cells are classified into one of the seven lattice systems regarding rotational symmetry. The seven lattice systems are cubic (NaCl), hexagonal (ice), tetragonal, rhombohedral, orthorhombic (TAGs), monoclinic (most sugars), and triclinic (TAGs). TAGs can be molecularly organised to form crystals, and depending on composition and processing conditions, they may form crystals into one or more of the lattice systems mentioned above. Crystals are often formed during food processing and storage, influencing food properties, mouthfeel, and physical stability. For instance, the presence and type of crystals may affect fracture properties, stickiness (when small fat crystals do not melt in the mouth), cooling sensation (crystal melting is an endothermic process creating a cooling effect during eating), or physical stability of some foods (e.g., formation and sedimentation of crystals in oils, phase separation or blooming of chocolate).

Crystals form through a process that is termed crystallisation. Crystallisation generally proceeds in two steps: nucleation and crystal growth (Fig. 4.15).

A flowchart with diagrams of the crystallization process. It starts with the achievement of appropriate conditions, followed by nucleation, crystal growth, and recrystallization. It presents small spheres, clusters, bunch of cylinders, and distanced cylinders.

Fig. 4.15

Crystallisation process of food components

During nucleation, the TAG molecules must become oriented into a fixed lattice and resist their tendency to redissolve. When a collision between molecules is unsuccessful, they redissolve, whereas a nucleus is formed when it is successful. A nucleus or embryo is a minute crystal that acts as a centre for further crystallisation (Fig. 4.15). The critical radius (rc) represents the minimum size of a stable nucleus. Nuclei smaller than rc dissolve, whereas larger than rc continue to grow. Nucleation occurs from solution (i.e., sugar or salt solution) or melt (pure compound, e.g., only TAGs). Irrespective of the nucleation mode, a sufficient thermodynamic force must be generated to drive the process. Nucleation is most frequently achieved with either a decrease in temperature (supercooling), an increase in solute concentration (supersaturation), or a suitable combination of both. The cooling rate plays a crucial role in the crystallisation and properties of TAGs. Slow cooling rates generate few big crystals with few impurities, whereas fast cooling rates generate many small crystals that grow fast and contain imperfections. Crystallisation occurs at higher temperatures with a slow cooling rate. In contrast, crystallisation occurs at lower temperatures (we need to decrease temperature more) with fast cooling rates. Control of the cooling rate may be employed to manipulate the physical properties of TAGs and improve the stability and sensory properties of various foods (e.g., chocolate or margarine). After nuclei have been formed, the crystal continues to grow until the solution is no longer supersaturated or all material has been crystallised (Fig. 4.15). Once the first nuclei have been formed, they act as the foundation for additional molecules to deposit (attach) on them, and the crystal increases in three dimensions in successive layers. The growth pattern usually resembles that of onion layers, with layers getting increasingly thinner due to the greater surface area of the growing crystal. Impurities (e.g., other food components present in formulation) may influence nucleation rate and crystal growth, usually interfering with molecule deposition on the newly formed nucleus. In some cases, they may enhance nucleation, acting as nucleation points. Impurities are very common in foods due to the complexity of formulations, and careful control of raw materials and processing conditions is required for reproducible products.

Once crystals have been formed, reorganisation takes place during storage. This process, termed recrystallisation, is defined as the change of crystal size, number, shape, or orientation in time (Fig. 4.15). It occurs rapidly at storage temperatures close to the melting point, temperature fluctuations during transportation, or improper storage conditions. Recrystallisation is influenced by the composition of the product and the initial crystal size distribution (i.e., cooling rate during formation). There are five types of recrystallisation: isomass rounding (changes in shape during storage), Ostwald ripening (large crystals grow, small disappear), accretion (crystals that are close together grow as one (stick together)), melt-refreeze (due to temperature fluctuations during storage), and polymorphic transitions (changes in polymorphism during storage, see below). Accretion and melt-refreeze are the two most important recrystallisation mechanisms for most foods, whereas polymorphic transitions are important in TAG-rich products only.

4.5.2 TAG Conformation and Polymorphism

The shape of TAGs in the solid phase is usually compared to either a tuning fork or a chair (Fig. 4.16). When TAGs crystallise, they align side-by-side to form a unit cell in a specific manner, depending on their chemical properties. A double chain length structure is formed when the chemical properties of the three fatty acids are the same or very similar. A triple chain length structure is formed when the chemical properties of one or two of the three fatty acids differ (Fig. 4.17).

a and b are sets of chemical structures of tuning fork and chair confirmations of triacylglycerols.

Fig. 4.16

(a) Tuning fork and (b) chair conformations of TAGs. The red dots indicate the carbons of glycerol

2 molecular structures of the unit cells. Both present fork-like structures. Left. Double chain length. Right. Triple chain length.

Fig. 4.17

TAG stacking in the unit cell

The way TAGs assemble in the crystal has a profound effect on the physical properties of fats. The most notable property is polymorphism, which is the occurrence of several different crystal forms originating from the same TAG. Polymorphic forms are crystals of the same chemical composition that differ in crystal structure but yield identical liquid phases upon melting. The most common polymorphic forms of TAGs are α, β΄, and β and they differ in their melting points and crystallographic properties. Other polymorphic forms (e.g., γ or α΄) with associated nuances in their physical properties are also possible. TAG stacking in α-polymorphs is usually vertical tuning forks, in β΄-polymorphs is tilted tuning forks, and in β-polymorphs is stacked chairs (Fig. 4.18).

3 diagrams of the polymorphic forms of fats. It depicts a fork-like structure. Left. It presents the vertical tuning fork, alpha polymorph, random packing, and lowest melting point. Middle. It depicts the tilted tuning fork at 70 degrees. Right. It depicts the stacked chair at 59 degrees.

Fig. 4.18

TAG stacking in different polymorphic forms of fats

In most cases, upon cooling a liquid fat, α-crystals form first because nucleation is easier in the α-form. In fats of relatively homogeneous composition, the α-form is short-lived and is transformed into β΄, which generally has a longer lifetime. At certain conditions (e.g., constant temperature), β΄-crystals may transform to β-form. The irreversible process of going from the less stable (α) to the more stable form (β) depends on time and temperature and is termed polymorphic transition (or transformation) of fats. The transition rate also depends on TAG homogeneity. Homogeneous TAGs change rapidly into the stable β-form. Fats with random TAG distribution may retain β΄-form indefinitely. Processing factors such as formulation, cooling rate, or shear forces may affect the number and type of crystals formed. Since fats are complex mixtures of TAGs, the different polymorphic forms usually coexist. Polymorphic transitions during storage may change fat properties and influence quality, and therefore, formulations and processing parameters should be carefully controlled.

TAG stacking in crystals and possible polymorphic transitions bring about remarkable differences in thermal, morphological or optical properties. Most importantly, the melting point, which is a crucial characteristic of fats, increases as α < β΄ < β. The properties of some products, such as chocolate or shortenings, are susceptible to the correct polymorphic form. The product obtains its desirable characteristics only when the correct crystal is formed. For example, cocoa butter has several crystal polymorphs, and good chocolate can only be made from β-crystals. Tempering is a precise time-temperature process to ensure the formation of cocoa butter in the correct crystal polymorph. All the undesirable polymorphic forms melt during tempering, and only certain β-crystals with the optimum melting characteristics are retained. During storage at an inappropriate temperature or after incorrect tempering, fat moves from the centre of the chocolate to the surface and crystallises. This is known as chocolate blooming (a white “mouldy” appearance at the surface) and is a major reason for product failure, even though it is not a health hazard.

Fat Crystal Hierarchy

Bulk fats have different levels of structure depending on the length scale of observation (how deep we zoom in on the structure). Molecularly, TAGs associate to form a lamella and stacking of multiple lamellas form crystalline domains. The thickness of the domains corresponds to the thickness of the nanoplatelets. The nanoplatelets assemble into spherulites that, with further aggregation, finally form the bulk fat (Figure redrawn with permissions from Tang and Marangoni, 2006, Adv. Colloid Interface Sci.128–130, 257–265).

7 diagrams. It begins with bulk fat exposed to present a network of spherulites, a nanoplatelet, a domain with parallel lines and lamella composed of TAG.

It should be emphasised that important technological properties of fat, such as spreading, melting, or sensory properties, depend on a combination of properties at the different structural levels. However, structures with sizes between 1 μm and 200 μm (mesoscale structure) and the interactions between each other are most responsible for the technological properties of fats. As a result, the objective of the technologist would be to modify the crystal properties of fats at this length scale to improve or modify the product’s physical properties or shelf-life stability.

Shortening is a fat that is used to make bakery products. The polymorphic structure of the shortening is responsible for the product’s quality. β΄-Crystals result in a large number of small air bubbles, whereas β-crystals result in a small number of large air bubbles. Consequently, β΄-crystal shortenings are preferred as they help to form small air bubbles in batters that result in good volume, texture and tenderness of baked goods. In addition, the melting point of shortening is critical for the mouth-melting effect in some bakery products. In margarine production, β΄-crystals are important for smooth texture and spreading that is achieved with β΄-stable fats such as hydrogenated cottonseed oil.

Margarine is a water-in-oil (w/o) emulsion in which water is dispersed as droplets in a continuous fat phase. The lipid phase is usually a blend consisting of different fats and oils. The ratio and type of fats and oils in the fat blend are decisive in achieving margarine with the desired characteristics (e.g., spreading). Emulsifiers (lecithin, mono-, di-glycerides), flavours, colours, and antioxidants may also be added to achieve the desired characteristics. In margarine manufacturing, β΄ polymorphs are usually preferred as they are smaller, giving the product the appropriate spreading and textural properties. In contrast, β polymorphs form large crystals at a high solids content, resulting in brittle, hard margarine, while at a low solids content, the product becomes oily. Spreadability and mouth melting are two textural properties of margarine that depend on solid fat content and its crystalline structure. Good spreadability requires that the product retains its plasticity from the refrigerator to room temperature (4–22 °C). However, desirable mouth-melting requires rapid melting at mouth temperature (35–37 °C) for prompt flavour release. It is evident that these two properties conflict with each other, and an in-depth understanding of crystal structure is required to fabricate margarine with desirable textural characteristics.

Apart from its crystal structure, melting point (Tm) is also influenced by the chain length and number of double bonds in fatty acids. Generally, the greater the chain length, the higher the Tm, while the increase in double bonds decreases Tm. Straight-chain fatty acids have higher Tm than branched fatty acids. Double bond configuration is also important as a trans double bond allows the formation of an almost straight chain, causing a smaller difference in melting properties than a single cis bond compared to its saturated counterpart. Furthermore, the position of the double bonds in the chain is important as two conjugated cis double bonds (− CH = CH – CH = CH -) can give a nearly straight chain than two non-conjugated ones (− CH = CH – CH₂ – CH = CH -) (Table 4.4).

Table 4.4

Summary of factors influencing melting point (Tm) of TAGs. An interplay between all these factors influences the actual melting point

Low Tm

High Tm

Short-chain length

Long-chain length

Unsaturated

Saturated

Branched-chain

Straight-chain

cis-

trans-

Non-conjugated

Conjugated

α-polymorph

β-polymorph

4.5.3 Other Properties of Fats

Natural fats are always mixtures of several different triglycerides. They, therefore, have a melting range, meaning that they melt over a range of temperatures rather than at a specific temperature, as ice at 0 °C. Food fats are rarely 100% solid at processing temperatures which means that a certain proportion will be liquid at a specific temperature. The ratio of solid to liquid fat at a given temperature is termed solid fat content (SFC) and is important for the functional properties of fats (Fig. 4.19). SFC is a function of temperature, temperature history, time, and composition. It influences the appearance and stability of products stored at refrigeration temperatures (e.g., salad dressings, spreading of margarine and butter), the mouth-melting profile of chocolates, the texture of baked products, and ease of processing.

A line graph of solid fat content versus temperature. It compares the heating and cooling trends. The trends descend in an S-shaped manner. Heating trend has higher values.

Fig. 4.19

Solid fat content changes as a function of temperature. Hysteresis is also observed between heating and cooling

Chemical manipulation of TAG composition is also possible to change their physical properties. Hydrogenation is the addition of hydrogen to double bonds to convert liquid oils to semisolid fats (e.g., margarine, shortenings), change the crystallisation behaviour, or improve the oxidative stability of oils. It is usually carried out with H₂ with Ni as a catalyst (Fig. 4.20). Partial hydrogenation may result in double bonds in the trans configuration, which causes concerns, as trans fatty acids have been associated with an increased risk of cardiovascular disease. However, the modern food industry is moving away from partially hydrogenated fats towards alternative technologies such as complete hydrogenation, interesterification or other modern technologies (tropical fat fractionation, modified hydrogenation processes, crystallisation, and emulsification strategies) that do not result in the formation of trans fatty acids.

A chemical reaction presents the hydrogenation of T A Gs. It presents a zigzag structure with 2 linoleic and 1 oleic structures. After adding H 2 and N i, it becomes 2 oleic and stearic structures.

Fig. 4.20

Partial hydrogenation of TAGs. The double bonds in blue have been saturated with the addition of hydrogen (green). As hydrogenation is partial, two bonds have remained unaffected (yellow)

Interesterification is the randomisation (“shuffling”) of fatty acids in triacylglycerols with the same objective as hydrogenation (i.e., manipulation of melting point and crystallisation) but without changing the fatty acid composition (Fig. 4.21). In addition, interesterification creates negligible amounts of trans fatty acids compared to hydrogenation. Interesterification consists of complex reactions that can be carried out either with catalysts or enzymes and are used to manufacture shortenings, margarine, or confectionery oils.

A chemical reaction presents the interesterification. It depicts the E-shaped T A G structures with the S and O at each end. S S S plus O O O is converted to S S S plus S O S plus S O O plus O S O plus O S S plus O O O.

Fig. 4.21

Interesterification of two TAGs results in a mixture of TAGs with random positioning of fatty acids. S: stearic acid, O: oleic acid

4.6 Review Questions

4.6.1 MultipleChoice Questions

1.

Triacylglycerols, phospholipids, sphingolipids, steroids, waxes, fat-soluble vitamins, and carotenoids are all lipids

(a)

All except fat-soluble vitamins

(b)

True. They are all lipids

(c)

All except fat-soluble vitamins and carotenoids

(d)

All except fat-soluble vitamins, carotenoids, and waxes

2.

A fatty acid written as 18:2 is a:

(a)

Fatty acid with eighteen calories (instead of 9) and zero trans

(b)

Fatty acid with eighteen carbon atoms and two unsaturated bonds

(c)

Omega-3 fatty acid derived from avocado oil

(d)

Fatty acids with eighteen double bonds and two trans bonds

3.

A characteristic of trans-isomers is that:

(a)

Fatty acids that can be transformed into glycerol

(b)

Can be transformed to polyunsaturated depending on the processing conditions

(c)

Hydrogens attached to carbon atoms are on the same side of the double bond

(d)

Hydrogens attached to carbon atoms are on either side of the double bond

4.

Triacylglycerols are formed by esterification of

(a)

Glycerol and three fatty acids

(b)

Glycerol two unsaturated fatty acid and one carotenoid

(c)

Glycerol and three monosaccharides

(d)

Glycerol and three amino acids

5.

Stereospecific numbering is:

(a)

A way to differentiate the degree of unsaturation of the fatty acid(s) on glycerol

(b)

A way to differentiate the stereoisomerism of the fatty acid(s) on glycerol

(c)

A way to differentiate the isomerism of the fatty acid(s) on glycerol

(d)

A way to differentiate the positioning of the fatty acid(s) on glycerol

6.

The occurrence of several different crystal forms for the same lipid compound is called:

(a)

Isomerism

(b)

Polyisomerism

(c)

Polymorphism

(d)

Crystal morphism

7.

The three most common different crystal forms of lipids are:

(a)

α, β΄, and β

(b)

α, β, and γ

(c)

α, β΄, and γ΄

(d)

α, γ, and α΄

8.

The shape of triacylglycerols in the solid phase compares to a:

(a)

Tuning fork or chair

(b)

Tuning fork or boat

(c)

Plate and egg box

(d)

Plate and spear

9.

Lipid oxidation has the following stages:

(a)

Initiation, propagation, termination

(b)

Activation, propagation, termination

(c)

Activation, preparation, termination, recovery

(d)

Activation, preparation, recovery

10.

Hydrogenation is:

(a)

Addition of hydrogen to double bonds so we can create margarine

(b)

Addition of hydrogen to double bonds so we can create butter

(c)

Addition of hydrogen to single bonds so we can create margarine

(d)

Addition of hydrogen to single bonds so we can create butter

4.6.2 Short Answer Questions

1.

Draw the structure of cis-linoleic acid (18:2 Δ9,12, both double bonds in cis configuration). Draw the structure of rumenic acid (18:2 Δ9,11) with cis at 9 and trans at 11.

2.

Online activity: Search for “conjugated linoleic acids” and explore their structure and potential health benefits.

3.

What is the difference between a simple and a mixed triacylglycerol?

4.

What is stereospecific numbering in triacylglycerides? Draw the structure of the triglyceride sn-1-oleo-2-stearo-3-palmitin.

5.

Draw the structures of phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. Compare their structures and search online the term “membrane lipids”.

6.

What is the difference between cholesterol and phytosterol? Name two phytosterols.

7.

A bakery makes vegan muffins using pea flour. Pea flour has about 1% fat and is rich in unsaturated fatty acids. The muffin gets a rancid off-flavour after only one day at room temperature. Identify the cause of this defect. Discuss the chemistry of the reaction and the factors that influence its rate. What will you propose to stop the reaction and extend the shelf life of the muffin?

8.

A chocolate manufacturer prepared a chocolate formulation with a melting point of ~26 °C, dull surface colour, and poor snap. What crystal polymorph has most likely been formed? How will you improve the physical properties of the chocolate? List the polymorphic forms of fats in order of increasing melting point.

9.

A potato-crisp manufacturer uses vegetable oil for frying. Deep-fat frying causes hydrolysis and oxidation of the oil. Identify the products of these reactions. What strategies will you suggest to minimise the extent of oil hydrolysis?

10.

Describe the crystallisation process of TAGs.

11.

What does “partially hydrogenated oil” mean in the ingredient list of food labels?

12.

Describe the mechanisms of lipid autooxidation and photooxidation

13.

What are the main factors influencing the rate of lipid oxidation?

14.

What is an antioxidant, and how do they control lipid oxidation?

15.

Describe tempering used in chocolate manufacturing.

16.

The following TAGs are randomly interesterified. Write all the possible new TAGs (Tip: they are 18).

A chemical reaction presents E-shaped T A G structures with S O S and P O P at the end from top to bottom.

17.

Describe the common methods used in fat analysis and their primary purpose.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024

V. KontogiorgosIntroduction to Food Chemistryhttps://doi.org/10.1007/978-3-031-53558-1_5

5. Browning Reactions

Vassilis Kontogiorgos1  

(1)

Associate Professor in Food Chemistry, University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC, Canada

Vassilis Kontogiorgos

Email: v.kontogiorgos@ubc.ca

Learning Objectives

After studying this chapter, you will be able to:

Describe the mechanism of enzymatic browning

Discuss deactivation strategies of enzymatic browning

Discuss the reactions of non-enzymatic browning

Distinguish between caramel flavour and caramel colour formation

Describe Maillard reactions and major flavour products

5.1 Introduction

Food browning is divided into two types: enzymatic and non-enzymatic. The reaction needs specific enzymes to proceed in the first type, but they are not required in the second. As we will see below, non-enzymatic browning can be further divided into reactions with distinct chemistry. The end-products of both reactions are dark brown pigments, but the chemical pathways to obtain them are quite diverse depending on the reactants. Browning reactions have significant implications for the food industry and can be desirable or undesirable. For example, enzymatic browning of fruit and vegetables results in up to 50% of production losses worldwide. Still, the reaction may benefit certain products, such as tea or raisins, where enzymatic reactions provide their characteristic colour and flavour. Most of the time, non-enzymatic browning is a beneficial reaction when the processing conditions are well controlled, although occasionally, it may have negative implications. For instance, the browning of cookies during baking is necessary, and it is not possible to think of appealing cookies without the appropriate amount of browning. In addition, non-enzymatic browning reactions may be used in new product development. For example, by modifying the roasting conditions of coffee beans, we can produce a broad range of coffee products even though we may start from the same raw material. As it becomes evident, depending on the specific food and purpose, we need to control the reactions and take advantage of their outcome for a particular technological reason. This chapter presents the chemistry behind the reactions and strategies to control them.

5.2 Enzymatic Browning

Enzymatic browning is one of the most important colour reactions affecting fruits, vegetables, and seafood. It is catalysed by the enzyme polyphenol oxidase (PPO), widely distributed in plants, fungi, arthropods, and mammals (Fig. 5.1). It is also referred to as phenoloxidase, phenolase, monophenol oxidase, diphenol oxidase, catechol oxidase or tyrosinase. The enzymes found in plants, animals, or fungal tissues frequently differ in their primary structure, molecular weight, or substrate specificity, but they fundamentally catalyse the same reaction (isoenzymes).

4 diagrams present the structures of polyphenol oxidases. A. It depicts the ribbon-like structures. It presents the structures from grapes with a marked portion. B. It presents the structures of polyphenol oxidases from mushrooms. The close-up views of the marked portions are presented at the bottom.

Fig. 5.1

Structures of polyphenol oxidases from (a) grapes and (b) mushrooms. The zoom-in of the structures shows the copper ion required for activity. Although protein structures are different, they both have similar enzymatic activity. PDB codes 2P3X and 5M6B

5.2.1 Reactions and Substrates

The reaction requires the simultaneous presence of the enzyme, substrate, and oxygen. In addition, PPO is a metalloenzyme, as it contains copper in its active site, which is necessary for its enzymatic activity. If one of the abovementioned factors is absent, the reaction cannot proceed. Polyphenol oxidase catalyses two reactions: (i) the hydroxylation of a mono-phenol to the o-position adjacent to an existing hydroxyl group of the phenolic substrate (monophenol oxidase activity), and (ii) the oxidation of a di-phenol to o-benzoquinones (di-phenol oxidase activity) (Fig. 5.2). Both reactions utilise molecular oxygen as a co-substrate. The product is melanins, which are dark brown water-insoluble compounds. Laccase is a copper-containing polyphenol oxidase with the unique ability to oxidise p-diphenols, in contrast to PPO, which oxidises o-diphenols. They have low specificity and oxidise a wide variety of substrates. Laccases may be used to crosslink arabinoxylans through ferulic acid oxidation and improve the dough’s baking performance.

Top. Monophenol oxidase pathway. Alkyl hydroxybenzene reacts with O 2 to form alkyl dihydroxy benzene. Bottom. Diphenol oxidase pathway. 1, 2 dihydroxy benzene reacts with O 2 to form 1, 2 diketone benzene.

Fig. 5.2

Reactions catalysed by polyphenol oxidase. (See text for description)

PPO substrates are phenolic compounds that contain an aromatic ring bearing one or more hydroxyl groups (e.g., tyrosine, catechol, chlorogenic acid, coumaric acid or catechins) (Fig. 5.3). Enzymatic browning does not occur in intact plant cells since phenolic compounds located in the vacuole are separated from PPO found in various plastids (e.g., chloroplasts). As a result, to observe browning, the tissues must be ruptured by physical damage (e.g., cutting or bruising) or senescence (ageing) that brings together PPO and phenolic substrates. In crustaceans (e.g., shrimps), PPO is mainly located in the carapace (the “head”), whereas the substrates are released post-mortem from proteolysis of the digestive tract or other tissues. In the most commercially important mushroom, Agaricus bisporus, and in other mushrooms, the enzyme can be found throughout the tissues. The delicate structure of mushrooms makes them particularly susceptible to browning. The polyphenolic composition of commodities varies with species, cultivar, degree of ripening, environmental conditions of growth, and the substrate specificity of polyphenol oxidases will vary with the source of the enzyme. In addition to serving as substrates, phenolic compounds act as PPO inhibitors through competitive inhibition.

Chemical structures of benzoic acid, cinnamic acid, catechin, gallic acid, sinapic acid, quercetin, syringic acid, coumarin, kaempferol, chlorogenic acid, and tyrosine.

Fig. 5.3

Substrates of polyphenol oxidase (a) benzoic and cinnamic acid derivatives and (b) flavonoids and catechins

5.2.2 Deactivation Strategies

The strategies to deactivate PPO focus on eliminating one or more of the essential components for its activity (i.e., oxygen, enzyme, copper, or substrate) from the reaction. There are generally five approaches that may be followed to control PPO activity:

1.

Inhibition targeted toward the substrate includes oxygen elimination from the cut surface of fruits and vegetables that can be achieved with vacuum packaging or immersion in water, syrup, or brine, depending on the product.

2.

Action on the enzyme may include chelating agents that remove copper from the active site (e.g., EDTA) or denaturation of the enzyme. The latter is most frequently accomplished with heat treatments (e.g., blanching), but irradiation may be successful when available.

3.

Low-temperature preservation, such as refrigeration or freezing, reduces the rate of enzymatic reactions, but they do not eliminate activity.

4.

Dehydration creates a low aw environment that limits or stops enzymatic activity. However, this is impractical as the time during dehydration is adequate for PPO to catalyse the browning reaction. As a result, an intermediate step is needed that targets the reaction products (see next).

5.

Inhibition can be targeted toward the reaction products with chemical compounds that react with the products of PPO activity and inhibit the formation of colour. Reducing agents such as ascorbic acid and sulphites interfere with the reactions that lead to melanin formation, as they reduce pigment precursors (o-quinones) to colourless, less-reactive o-diphenols (Fig. 5.4). Ascorbic acid is commonly used in the fruit juice industry to maintain the colour of juices, purees, or during dehydration of fruit slices (e.g., banana chips or dried mango).

A chemical reaction. 1, 2 dihydroxy alkylbenzene reacts with O 2 to form 1, 2 diketone alkylbenzene.

Fig. 5.4

Reducing agents reduce o-quinones to colourless o-diphenols

5.3 Non-enzymatic Browning

Caramelisation, ascorbic acid browning, and Maillard reactions are the three types of non-enzymatic browning. As the term implies, no enzymes are involved in any of these reactions, and they all have different chemistry.

5.3.1 Caramelisation

Caramelisation refers to two distinct reactions that both involve sugars. The first class of reactions results in caramel colours, and the other in caramel flavours. Caramel colours are only colouring agents and do not influence the product’s flavour profile at the concentrations used. In contrast, caramel flavours are produced during food processing, resulting in a characteristic flavour profile most frequently associated with the product. For example, the caramelisation of malt in some beer-making processes impacts the beer’s flavour profile. However, adding caramel colour to certain beers does not influence their flavour. In another example, cookie-baking results in caramelisation of the top layer of dough that gives off volatiles, characteristic of cookies. In contrast, adding caramel colour in the cookie dough to maintain colour uniformity does not influence the product’s flavour. We should clarify that the food additive “caramel” (150a, 150b, 150c, and 150d) always refers to a caramel colour.

5.3.1.1 Caramel Flavours

Caramelisation in the context of flavour creation is the controlled thermal degradation (>100 °C) of sugars using acid or base or starting from a crystal. The reaction does not proceed rapidly at neutral pH (~7.0) and requires strong acidic (pH <3.0) or alkaline (pH >9.0) conditions. The reaction is poorly understood due to its complexity, but it generally involves four stages, as described below, using sucrose as an example:

Stage 1: Melting (~190 °C): Sucrose crystals melt at ~190 °C. At temperatures lower than ~190 °C, no reaction is observed.

Stage 2: Dehydration (~200 °C): This is the step where the reaction begins as, after crystal melting, heat cleaves the glycosidic bond between glucose and fructose, leading to the formation of 1,6-anhydro-β-d-glucose and fructose.

Stage 3: Formation of intermediates (~200–400 °C): The resulting molecules continue to decompose under the influence of heat. These compounds react with each other, forming furans, aldehydes, and ketones, producing the characteristic caramel aroma. Furfural and maltol derivatives are the most important classes of these chemicals. Specifically, hydroxymethyl furfural (HMF), hydroxy acetyl furan (HAF), hydroxy dimethyl furanone (HDF), maltol and hydroxy maltol contribute to the aroma profile of many products, especially hard caramel, coffee, dried fruits, dark beers, and bakery products (Fig. 5.5). Additionally, diacetyl is also produced with a buttery flavour frequently found in fermented products, such as beer or yoghurt.

Stage 4: Polymerisation (> ~400 °C): Finally, these compounds polymerise, forming large water-insoluble polymers with yellow colour. Further, heating > ~500 °C results in a black colour due to the complete pyrolysis of the molecules leaving carbon behind.

The caramelisation intensity and the exact final products vary depending on the type of sugar and reaction conditions. Fructose has the lowest caramelisation onset temperature (~110 °C), followed by galactose, glucose, and sucrose (~160 °C) and finally by maltose (~180 °C). As a note, the butane flame commonly used in the confectionery industry to create caramels has a temperature of ~1400 °C. Hence, the reactions occur rapidly and are usually difficult to control.

Chemical structures of 5, hydroxymethyl, furan 2 carbaldehyde, 3 hydroxy 2 methyl 4 H pyran 4 one, 2 acetyl furan, hydroxy dimethyl furanone and 3, 5 dihydroxy 2 methylpyran 4 one.

Fig. 5.5

Structures of caramelisation products with a strong odour

5.3.1.2 Caramel Colours

Caramel colours are among the most common colouring agents used in the food industry and are produced via caramelisation reactions. Caramel colours are divided into four classes depending on the presence or absence of ammonium or sulfite compounds. In Class I or plain caramel (caustic caramel), ammonium or sulfite compounds are not used during the reaction. In Class II (caustic sulfite caramel), the reaction is carried out in the presence of sulfite compounds but no ammonium compounds. Class III caramels (ammonia caramel) are produced with ammonium compounds but without sulfite compounds. Finally, Class IV (sulfite ammonia caramel) is made with sulfite and ammonium compounds (Table 5.1). The way caramel is produced influences its charge (positive or negative) and colour, making it a critical factor in the choice of caramel for a specific food application. For example, positively charged caramel colours may precipitate in the presence of negatively charged molecules in food, which is undesirable. Caramel colour for soft drinks and beverages (e.g., cola or coffee flavoured drinks) must carry a strong negative charge, whereas beer caramel colour should carry positive charges. In another example, caramel colours used in tea beverages need to show good stability in the presence of tannins so they do not form complexes and precipitate.

Table 5.1

General characteristics of different classes of caramel colours

Class I (150a)

Plain caramel

Class II (150b)

Caustic sulfite caramel

Class III (150c)

Ammonia caramel

Class IV (150d)

Sulfite ammonia caramel

Colour

Color code of class 1.

Color code of class 2.

Color code of class 3.

Color code of class 4.

Sulphite

No

Yes

No

Yes

Ammonium

No

No

Yes

Yes

Charge

Slightly negative

Negative

Positive

Strongly negative

Stability in:

 Ethanol

+

+

-

-

 Tannins

-

+

-

+

 Acid

-

+

+

+

Applications

Distilled spirits, cereals, baked products, pet food

Tea, liqueurs (cognac, vermouth brandy)

Beer, sauces, gravies

Soft drinks carbonated beverages

5.3.2 Ascorbic Acid Browning

Ascorbic acid browning occurs in acidic foods such as fruit juices and dehydrated fruits. In this type of browning, ascorbic acid (i.e., vitamin C) is lost from lowering its nutritional value. The reaction proceeds with the conversion of ascorbic acid to dehydroascorbic acid that will further oxidise to 2,3 biketogulonic acid (Fig. 5.6). The latter component is also prone to oxidation that results in the formation of furfural and ethyl glyoxal. These compounds may polymerise or react with amino acids through Strecker degradation (see Sect. 5.3.3) to form brown pigments. Decomposition of vitamin C takes place when in contact with oxygen. The brown colour only starts appearing once all vitamin C has been consumed in the reaction. It may occur at relatively low temperatures and pH, and the presence of amino acids or proteins may facilitate colour production.

A flowchart with the molecular structures presents the reaction pathway. It starts with ascorbic acid followed by dehydroascorbic acid, 2, 3 biketogulonic acid, ethyl glyoxal plus furfural plus reductone that result in amino acid and brown pigments.

Fig. 5.6

Reaction pathway in ascorbic acid browning

5.3.3 Maillard Reaction

Maillard reaction is the reaction of a reducing sugar with an amino acid or protein. It is of great importance in food manufacturing, as it may be desirable (e.g., in bread, soy sauce, coffee, chocolate) or undesirable (e.g., in dehydrated foods such as potato flakes, egg and milk powders, and corn starch). Maillard reaction occurs every time a food is heated, and a reducing sugar and amino acid are present in the formulation. As heat treatment is one of the most common food preservation techniques, it essentially signifies that the Maillard reaction takes place nearly always and is the most critical taste and flavour-generating reaction in foods. Some flavour changes are necessary in certain products (e.g., roasting coffee or cocoa beans). Occasionally, loss of essential amino acids may occur (e.g., lysine) in products already low in these amino acids, but this is not a significant problem when viewed from the perspective of a balanced diet. The reaction is particularly complex, and different pathways are still being explored. Nevertheless, it can be divided into three stages depending on the colour and odour formation.

5.3.3.1 Early Maillard and Amadori Rearrangement

Stage 1 is the colourless and odourless stage of the reaction that is reversible. The Maillard reaction starts with the reaction between the carbonyl group of a reducing sugar and the amino group of an amino acid yielding N-glycosylamine via Schiff-base formation (Fig. 5.7). As it is a condensation reaction, i.e., water is produced, it is favoured at low water contents. Acidic environments protonate amines and cannot react, thus making it difficult for the Maillard reaction to proceed. Generally, pentoses (e.g., arabinose) are more reactive than hexoses (e.g., glucose), while aldoses (e.g., glucose) are more reactive than ketoses (e.g., fructose). Monosaccharides are more reactive than reducing disaccharides (e.g., lactose, maltose), whereas non-reducing disaccharides (e.g., sucrose) do not react. Finally, strongly basic amino acids (e.g., lysine) are more reactive than other amino acids. Amadori rearrangement follows the first reaction, where N-glycosylamine undergoes isomerisation to form ketosamines (Amadori compounds).

Maillard reactions. a. Glucose reacts with amino acid to yield N glycosylamine and H 2 O. b. N glycosylamine interconverts to Amadori compound.

Fig. 5.7

Maillard reaction, (a) reaction of reducing sugar with amino acid, and (b) Amadori rearrangement forms Amadori compounds (ketosamines)

5.3.3.2 Advanced Maillard and Strecker Degradation

Stage 2 is where colour and odour develop and is the most diverse set of reactions that most frequently coincide. Amadori products are eventually dehydrated in a series of reactions that are not reversible. Deamination (loss of amine) and further isomerisation leads to several dicarbonyl compounds that are very reactive. Only at this point does colour start to develop, partly due to the appearance of HMF. Dicarbonyl compounds will react with α-amino acids in a reaction known as Strecker degradation to produce amino-carbonyl compounds and Strecker aldehydes (Fig. 5.8). Strecker aldehydes have an intense smell and are important flavour compounds in many foods (Table 5.2).

A chemical reaction of the Strecker degradation. Alpha di carbonyl compound plus amino acid results in amino carbonyl compound plus Strecker aldehyde.

Fig. 5.8

General scheme of the Strecker degradation

Table 5.2

Strecker aldehydes from specific amino acids and their odour characteristics

Amino acid

Strecker aldehyde

Odour at 100 °C

Odour at 180 °C

Cysteine

Acetaldehyde, propanal

Sulfide, cooked meat

Sulfide, smell of H₂S

Alanine

Acetaldehyde

Caramel, fruity

Burnt sugar

Threonine

Hydroxy propanal

Chocolate

Burnt

Valine

2-Methyl propanal

Straw, green, rye bread

Chocolate

Leucine

3-Methyl butanal

Bread, malty, green

Cream cheese

Isoleucine

2-Methyl butanal

Fruity, ester, green

Cream cheese

Glutamine

Pyrrolidone

Chocolate

Hard caramel

Strecker degradation has also been implicated in acrylamide formation, which is a carcinogen, and its consumption results in a low increased risk of developing cancer over a lifetime. Acrylamide formation requires asparagine in the first reaction and the subsequent Strecker degradation reactions. Cooking methods such as frying, baking, and roasting are likely to produce acrylamide, whereas boiling and steaming do not. Acrylamide is most commonly found in potato chips, fries, roasted coffee, cereal products, bread and bakery products, dried foods, chocolate products, and biscuits. Acrylamide formation cannot be avoided but can be controlled with good manufacturing practices. Cyclisation reactions resulting in ring formation may also occur during Stage 2, contributing substantially to flavour generation. Heterocyclic compounds are ring-like structures which contain atoms other than carbon in the ring (Fig. 5.9). Oxygen, nitrogen, and sulphur are primarily present in such molecules and play a key role in food flavour. These components may be present in minimal amounts yet, with very low detection thresholds, can significantly impact flavour profiles. Chocolate, caramel, and nutty flavours frequently occur because of heterocyclic compounds.

Chemical structures of furanone, pyrrole, thiophene, imidazole, pyrazine, acylpyridine, furan, pyranone, oxazole, and alkylpyridine.

Fig. 5.9

Examples of heterocyclic compounds formed during Maillard reaction

Acrylamide

The presence of acrylamide in foods is a very recent discovery (in the 2000s). Acrylamide in food is perhaps as old as the first-ever bread loaf that has ever been baked thousands of years ago! Acrylamide, in high doses, much higher than those encountered in food, is a carcinogen and a neurotoxin for humans. Acrylamide is not present in raw ingredients but is formed due to the high-heat processing of carbohydrate-rich foods, especially during frying, baking, and roasting. French fries, bakery products, and coffee have the highest amounts of acrylamide because of the presence of reducing sugars and asparagine, whereas meat products contain very little acrylamide.

A reaction mechanism. Asparagine reacts with dicarbonyl compound to obtain Schiff base followed by 3 amino propionamide and acrylamide.

The formation of acrylamide is quite complex and involves several different mechanisms. The presence of 3-aminopropionamide (3-APA) is a common characteristic in most of the reaction pathways. 3-APA may be produced in the reaction of asparagine with dicarbonyl compounds after decarboxylation of the formed Schiff base (imine). Subsequent hydrolysis and deamination result in the formation of acrylamide.

5.3.3.3 Late Maillard and Melanoidin Formation

Stage 3 of the reaction involves polymerisation reactions that occur randomly between carbonyls that often become attached to partly degraded proteins in the mixture. Although the polymerised pigments are initially water-soluble, as their size increases, their solubility decreases, and they precipitate. The final reaction products are called melanoidins with a particularly dark brown colour. An overview of all the stages involved in the Maillard reaction is presented in Fig. 5.10.

A flowchart of the Maillard reaction. It presents 3 stages, colorless and odorless, color and odor development, and brown pigments. Stage 1 includes an early Maillard reaction with reducing sugar plus an amino acid with Amadori rearrangement. Stage 2 includes advanced Maillard reaction with fission, dehydration, and Strecker degradation.

Fig. 5.10

Overview of the Maillard reaction

It should be noted that this idealised reaction scheme may be easily conceived when model reactions under laboratory conditions are discussed, e.g., the reaction of glucose with alanine in a test tube. However, in a food undergoing heat treatment, the complexity and diversity of the participating compounds make distinguishing, understanding, and controlling each step virtually impossible. As a result, foods whose flavour profile is based on Maillard reactions (e.g., potato crisps or coffee) are susceptible to the quality and reproducibility of raw materials (e.g., the genetic makeup of coffee beans) and processing conditions (e.g., time and temperature). For instance, reducing sugar content in potatoes varies with season, variety, or storage time. As a result, understanding the exact chemistry of this reaction is relatively futile for technological purposes. In contrast, understanding the biology of raw materials, processing conditions, and quality control is paramount to ensure the reproducibility of the quality characteristics of these foods.

5.3.3.4 Control of Maillard Browning

Although precise control of the reaction at the molecular level may be ineffective on the industrial scale, the general factors influencing the Maillard reaction may be utilised to control browning. As with all other chemical reactions, an increase in temperature tends to speed up browning. The effect of pH is not as simple as that for temperature, as different reactions in the sequence are affected differently by changes in pH. Generally, acidic environments protonate amino acids that cannot react anymore with the reducing sugars. Browning occurs faster at aw between 0.6 and 0.7 with moisture contents ~30%. This is normal for most chemical reactions, i.e., the higher the reactant concentrations, the faster the reaction rate. In addition, as the first step in Maillard browning is condensation, lower moisture content facilitates the reaction. Below 30% moisture, there is a decreased rate of browning since solutes (reactants) become trapped and cannot diffuse freely. If the reactants cannot meet each other, then the reaction will not occur. As oxygen is not involved in the Maillard reaction, it does not influence its rate. Different compounds, such as phosphates or various carboxylic acids and their salts, may act as accelerators or inhibitors, which may accelerate browning or result in more intense colour development. Tin (Sn) ions may slow browning to a certain extent because they cause strong reducing conditions. This is important when heating in tin cans, during the pasteurisation or sterilisation of various foods. Sulfur dioxide (SO₂) is frequently used as a preservative in several products, including beverages. It can block carbonyl groups as it reacts with reducing sugars and prevents the condensation step of the reaction. In addition, it may also react with products (e.g., HMF) and prevent further colour reactions from occurring.

5.4 Review Questions

5.4.1 Multiple Choice Questions

1.

Polyphenol oxidase catalyses:

(a)

Enzymatic browning in fruits and vegetables

(b)

Non-enzymatic browning in fruits and vegetables

(c)

Enzymatic browning in fruits and vegetables that contain ascorbic acid (Vit C)

(d)

Enzymatic browning in fruits and vegetables that lack ascorbic acid (Vit C)

2.

Substrates of polyphenol oxidase are:

(a)

Ascorbic acid and dehydroascorbic acid

(b)

Phenolics

(c)

Reducing sugars

(d)

Reducing sugars and amino acids

3.

We can inhibit the action of polyphenol oxidase by

(a)

Blanching, elimination of N₂, copper removal

(b)

Blanching, elimination of O₂, zinc removal

(c)

Blanching, elimination of O₂, copper removal

(d)

Blanching, increase of O₂, zinc removal

4.

Caramelisation requires:

(a)

reaction of non-reducing sugars with amino acids

(b)

acid or base, high temperatures and 3 < pH < 9

(c)

low temperatures, free amino acids and reducing sugars

(d)

high temperatures, low pH (<3) and essential amino acids

5.

Major products of caramelisation are

(a)

glucose derivatives and high and fructose derivatives at low temperatures

(b)

furanose and pyranose derivatives

(c)

reducing and non-reducing sugar derivatives

(d)

furfural and maltol derivatives

6.

Maillard reaction is the reaction between

(a)

a reducing sugar and amino acid, peptide or protein at high temperatures

(b)

a non-reducing sugar and amino acid, peptide or protein at high temperatures

(c)

a reducing sugar and amino acid, peptide or protein at low temperatures

(d)

a non-reducing sugar and amino acid, peptide or protein at low temperatures

7.

The Amadori rearrangement

(a)

Is the conversion of N-glycosylamines to 1-amino-1-deoxy-ketose in the Maillard reaction

(b)

Is the conversion of N-glycosylamines to 1-amino-1-deoxy-ketose in ascorbic acid browning

(c)

Is the conversion of N-glycosylamines to 1-amino-1-deoxy-ketose in enzymatic browning

(d)

Is the conversion of N-glycosylamines to 1-amino-1-deoxy-ketose in Strecker degradation

8.

Strecker degradation is the reaction between

(a)

dicarbonyl and ascorbic acid

(b)

dicarbonyl and non-reducing sugar

(c)

dicarbonyl and amino acid

(d)

dicarbonyl and reducing sugar

9.

Strecker aldehydes are important because:

(a)

are responsible for important flavours in the Maillard reaction

(b)

are responsible for off-flavours in the Maillard reaction

(c)

are responsible for the creation of toxic compounds in the Maillard reaction

(d)

are responsible for the creation of brown pigments in the Maillard reaction

10.

Acrylamide is formed during the Maillard reaction and requires the presence of

(a)

Asparagine and a non-reducing sugar.

(b)

Asparagine and reducing sugar.

(c)

Arginine and reducing sugar.

(d)

Arginine and non-reducing sugar.

5.4.2 Short Answer Questions

1.

Draw the reactions of monophenol and diphenol oxidation reactions of PPO.

2.

Draw the structures of three benzoic and three cinnamic acid derivatives.

3.

List and discuss the factors that affect enzymatic browning.

4.

Describe the steps of colour formation in the caramelisation of sucrose.

5.

List the different caramel colours and identify their charge.

6.

A company makes a tea beverage containing tea extract. However, the colour between batches is inconsistent. Which caramel will you recommend and why?

7.

Describe the three stages of the Maillard reaction.

8.

A company manufactures potato crisps. The optimum level of reducing sugars in potatoes is 0.5 g/100 g of potatoes. During quality control, the team detected unusually high levels of reducing sugars (1.5 g/100 g). What is the effect of higher-than-normal reducing sugars on the colour, flavour, and acrylamide concentration of potato crisps?

9.

List and discuss the factors that affect the Maillard reaction.

10.

What is Strecker degradation, and why is it important in foods?

11.

Online activity: Find online the terms “climacteric fruits” and “non-climacteric fruits”. What is the difference between them? Which type is most likely to suffer from enzymatic browning?

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024

V. KontogiorgosIntroduction to Food Chemistryhttps://doi.org/10.1007/978-3-031-53558-1_6

6. Vitamins-Minerals-Additives

Vassilis Kontogiorgos1  

(1)

Associate Professor in Food Chemistry, University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC, Canada

Vassilis Kontogiorgos

Email: v.kontogiorgos@ubc.ca

Learning Objectives

After studying this chapter, you will be able to:

Recognise the structures of vitamins found in food.

Discuss vitamin stability during food processing and storage.

Recognise the main minerals found in food.

Discuss mineral bioavailability during food processing and storage.

Discuss the classification and the role of food additives.

6.1 Introduction

Vitamins and minerals may be essential nutrients but have only an ancillary role in creating and stabilising food structure. However, they may play a central role in organoleptic properties. For example, carotenoids may provide colour and pro-vitamin activity or sodium a salty taste and the physiologically essential sodium cation. Nevertheless, vitamin degradation and mineral losses (e.g., Fe²⁺) during food processing and storage are of concern. Vitamins are classified into water-soluble (B-complex and Vitamin C) or fat-soluble (A, D, E, and K) vitamins. The vitamin and mineral losses in food are very complex and, in most cases, poorly understood. Irrespective of how carefully we process food, these nutrients will be lost to a certain extent. Food processing aims to maximise vitamin and mineral retention by optimising processes that may degrade them. There are several causes of vitamin and mineral losses, starting with the inherent variation in their content in raw ingredients. For example, genetic characteristics of plants, stage of maturity, site of growth, soil, climate, agricultural practices, and animal diet may cause substantial differences in the production of raw food ingredients. Post-harvest changes resulting from the residual enzymatic activity may reduce vitamin content (e.g., lipoxygenases or ascorbic acid oxidase). Furthermore, continued plant metabolism (e.g., respiration) results in losses during storage. Food preparation steps such as trimming, washing, and milling also result in vitamin and mineral losses during processing. Blanching may result in thermal oxidation and losses due to leaching in the water and is important for water-soluble vitamins and minerals. Canning leads to variable losses depending on the food. Post-processing storage may also lead to vitamin deterioration, depending on the food and usually, the longer the storage, the greater the losses. Finally, additives (e.g., chlorine, sulfites, or nitrites) and food composition (e.g., oxidative food environment) affect vitamin losses. The present chapter outlines the essential chemical characteristics of vitamins and minerals and how they may behave in different processing conditions.

6.2 Fat-Soluble Vitamins

Vitamin A

may be preformed vitamin A, including retinol, retinaldehyde and retinoic acid, found in animal-derived foods. It can also be pro-vitamin A, which includes carotenoids (mainly β-carotene), which can be converted to retinal in the body (Fig. 6.1) and is needed for good vision, skin and the immune system.

Chemical structures of retinol, retinaldehyde, retinoic acid and beta carotene. Each is composed of trimethylcyclohexene and a linear chain with branches.

Fig. 6.1

Structures with vitamin A activity

β-Carotene has one-sixth of the vitamin activity of retinol. The total vitamin A content of the diet (from both animal and plant sources) is expressed as retinol activity equivalents (RAE or RE). One IU (International Units) of retinol equals 0.3 μg RAE, one IU beta-carotene from dietary supplements equals 0.15 μg RAE, whereas one IU beta-carotene from food equals 0.05 μg RAE. Sweet potato, liver, spinach, and carrots are some sources that are rich in vitamin A, or pro-vitamin A. Vitamin A degrades similarly to oxidative degradation of unsaturated fatty acids. It is unstable to heat, light, acid, and oxygen, with up to 40% losses during thermal processing. Vitamin A degradation may be accelerated in dehydrated foods where oxidation proceeds faster.

The two most important forms of vitamin D are vitamin D₂ (ergocalciferol) and vitamin D₃ (cholecalciferol) (Fig. 6.2). Vitamin D promotes intestinal absorption of calcium and is involved in many other metabolic reactions. Vitamin D₂ is formed by the UV irradiation of the plant sterol ergosterol found in plants and fungi. Vitamin D₃ is synthesised in animals and humans after exposure to sunlight. Dietary vitamin D sources are practically insignificant compared to the amount prepared via the biosynthetic pathway (sunlight irradiation). Liver, beef, egg yolk, dairy products, some fish (e.g., salmon, tuna, and sardine) and fortified foods (e.g., milk, yoghurt, or margarine) are good sources of vitamin D. It is a stable vitamin during both heat processing and storage but is sensitive to light and oxygen. As a result, packaging materials of foods rich in vitamin D need low permeability to oxygen and light. Like vitamin A, vitamin D was measured in international units of biological activity before the pure compound was isolated. As a result, one IU equals to 0.025 μg of cholecalciferol or 1 μg of cholecalciferol equals to 40 IU.

Chemical structures of egrocalciferol and cholecalciferol. Both are composed of methylidenecyclohexanol, cyclohexane, cyclopentane and a linear chain.

Fig. 6.2

Structures with vitamin D activity

Vitamin E

is a group of fat-soluble compounds consisting of four tocopherols and four tocotrienols (Fig. 6.3) and protects cell membranes from oxidation. It is found in plant oils and processed food rich in plant oils (e.g., salad dressings), fortified cereals, oatmeal, wheat germ, asparagus, tomatoes, and green leafy vegetables. It is sensitive to light, oxygen, and heat, especially in dehydrated or deep-fried foods. Processing and storage of foods may result in substantial tocopherol losses. However, anaerobic processing (e.g., canning) has little effect. Vitamin E frequently acts as a natural antioxidant by quenching singlet oxygen.

2 chemical structures. Top. (2 R), 2, 5, 7, 8 tetramethyl 2 [(4 R, 8 R) 4, 8, 12, trimethyltridecyl], 3, 4 dihydrochromen 6 ol. Bottom. 2 methyl 2 (4, 8, 12 trimethyltrideca 3, 7, 11 trienyl), 3, 4 dihydrochromen 6 ol.

Fig. 6.3

Structures with vitamin E activity

Vitamin K

is a family of naphthoquinones with or without terpenoid side chain (Fig. 6.4) and is needed for blood coagulation. It is found in plants, plant oils, fish oils, and meats. In humans, it is synthesised by the bacteria in the colon, where it is also absorbed (10% of needs). Good sources of vitamin K are dark green vegetables (e.g., spinach and cabbage leaves), cauliflower, peas, and cereals. It is very stable to heat and oxygen but not to light.

3 chemical structures. a. 2 methyl 3 [(E, 7 R,11 R), 3, 7, 11, 15 tetramethylhexadec 2 enyl, naphthalene 1, 4 dione. b. 2 methylnaphthalene 1, 4 dione. c. 2, [(2 E, 6 E, 10 E, 14 E, 18 E, 22 E), 3, 7, 11, 15, 19, 23, 27 heptamethyloctacosa 2, 6, 10, 14, 18, 22, 26 heptaenyl], 3 methylnaphthalene 1, 4 dione.

Fig. 6.4

Structures with vitamin K activity

6.3 Water-Soluble Vitamins

Ascorbic acid (vitamin C)

occurs in fruits (especially citrus fruits) and vegetables but not in animal tissues (Fig. 6.5). It is highly unstable to oxygen, light and heat and easily leaches out from tissues during processing. Ascorbic acid is oxidised easily to dehydro-l-ascorbic acid, which retains vitamin C activity. However, this compound can be further oxidised to 2,3 biketogulonic acid, which does not have biological activity (see Sect. 5.​2.​2). It is one of the most unstable vitamins, and there are large losses during processing, storage and freezing (e.g., frozen fruits and vegetables), but it can be easily supplemented in fruit juices. Enzymes naturally found in tissues can also catalyse vitamin C decomposition, such as ascorbic acid oxidase or peroxidase. Deactivation of these enzymes is most frequently accomplished with blanching. Ascorbic acid can be used as a dough conditioner, antioxidant, an inhibitor of browning in fruits and vegetables, or colour stabiliser in meat products.

Chemical structure of (2 R), 2, [(1 S), 1, 2 dihydroxyethyl], 3, 4 dihydroxy 2 H furan 5 one.

Fig. 6.5

Structure of ascorbic acid

Vitamin B₁ (thiamine)

is involved in the metabolism of carbohydrates, and food sources of thiamine include whole grains, legumes, and some meats, fish, and breakfast cereals (Fig. 6.6). Grain processing removes much of the thiamine content, and many countries enrich cereals and flours with thiamine. Thiamine is stable to light but among the least stable vitamins at neutral or alkaline pH, oxygen, and heat. This is important, as even simple food processing steps such as heating in water may result in significant losses by decomposition or leaching. Vitamin B₁ has excellent stability in low aw foods (e.g., breakfast cereals, bread), whereas tannins may interact and inactivate it. Thiamine is inactivated in the presence of nitrites found in processed meat products. Losses in food may range from as little as ~10% in canned fruits and vegetables to up to ~60% in home-cooked meat. Regardless of the processing, around a fifth of thiamine (~20%) is generally lost.

3 chemical structures. a. Vitamin B 1, 2, [3, [(4 amino 2 methylpyrimidin 5 yl)methyl], 4, methyl 1, 3 thiazol 3 ium 5 yl] ethanol. b. Vitamin B 2, 7, 8 dimethyl 10 [(2 S, 3 S, 4 R), 2, 3, 4, 5 tetrahydroxypentyl] benzo, g, pteridine 2, 4 dione. c. pyridine 3 carboxylic acid.

Fig. 6.6

Structures with vitamin B₁, B₂ and B₃ activity

Vitamin B₂ (riboflavin)

is a vitamin involved in energy production (Fig. 6.6). Food sources include eggs, green vegetables, milk and other dairy products, meat, mushrooms, and almonds while some countries require its addition to flour. It is stable at acidic conditions but not neutral or alkaline and slight changes in its chemical structure result in loss of vitamin activity. It has good stability to conventional thermal processing, storage and oxygen but is light-sensitive. Vitamin B₃ (niacin) is the generic term for nicotinic acid and its derivatives involved in multiple metabolic activities (Fig. 6.6). Niacin is obtained from various whole and processed foods, with the highest contents found in fortified packaged foods, liver, tuna, salmon, and leafy vegetables. Some countries require its addition to flours similar to vitamin B₂. Niacin is the most stable vitamin, as it is not affected by light and thermal losses do not occur under conditions relevant to food processing. Losses of ~15% may occur during blanching (leaching) or dripping on frozen meat thawing.

Vitamin B₅ (pantothenic acid)

is found in both free and bound forms, with 85% occurring bound as a part of coenzyme A (Co-A) (Fig. 6.7). It is involved in fat metabolism and other important metabolic activities. It is widely available in meats, whole grains, potatoes, egg yolks, broccoli, mushrooms, and avocados. It is stable during food storage, especially in low aw foods, but is sensitive to heat and leaching during processing. Vitamin B₆ refers to chemically similar compounds (pyridoxine, pyridoxal, pyridoxamine), which can be interconverted in biological systems (Fig. 6.7). Its active form is pyridoxal 5΄-phosphate and it plays an important role in the metabolism of amino acids. Foods that contain large amounts of vitamin B₆ include fortified breakfast cereals, pork, turkey, beef, bananas, chickpeas, and potatoes. Pyridoxine has good stability to heat and low pH but is light-sensitive. Pyridoxamine is very sensitive to air, heat, or light and is practically destroyed during food processing. Pyridoxal is relatively stable and is the form that is used in food fortification. Fruit and vegetable canning results in ~25% losses and wheat milling of up to 90% due to the separation of the bran.

Chemical structures of pantothenic acid, pyridoxine, pyridoxal, and pyridoxamine.

Fig. 6.7

Structures with vitamin B₅ and B₆ activity

Importance of Vitamins for the Food Technologist

Many consumers worry about the adequate consumption of vitamins from their food. It is also a popular misconception that processed food lacks or has insufficient vitamins. Modern food processing has not only minimised the losses but, in some instances, has enhanced the concentration of vitamins in the food, with notable examples of the addition of vitamin B complex in breakfast cereals or vitamin D in fortified milk. In some instances, technologies such as encapsulation minimise vitamin degradation during processing and storage and improve their absorption once in the gastrointestinal tract. Because of uncontrolled temperatures and inaccurate cooking methods, loss of vitamins during home cooking is more likely to occur than in industrial food preparation. Regardless of their nutritional importance, food scientists rarely focus on vitamins during food formulation design. Vitamins do not constitute elements of food structuring and rarely influence sensory properties, unlike minerals that may contribute to both structure creation (e.g., through calcium cross-linking) and taste (e.g., NaCl). Vitamins rarely take part in relevant reactions. Overall, they have negligible technological significance except for ascorbic acid when used as a processing aid (e.g., gluten cross-linking in flours or colour stabiliser meat products). It is not an exaggeration to state that vitamins are technologically the least important of all molecules presented in this book.

Vitamin B₁₂ (cobalamin)

has a complex structure with cobalt (Co) chelated in the molecule (Fig. 6.8). It plays metabolic roles in the function of the nervous system and red blood cell formation. Various forms of cobalamin exist in which different groups are attached to the central Co atom, such as cyanocobalamin, hydroxy-cobalamin, adenosyl-cobalamin (major storage form in the liver) and methyl-cobalamin (mostly found in blood circulation). Meat, fish, milk, cheese, eggs, yeast extract, and fortified breakfast cereals are good sources of B₁₂. It is a stable vitamin, and negligible losses occur under most food processing conditions.

Chemical structure of cyanocobalamin.

Fig. 6.8

Structure of cyanocobalamin (B₁₂)

Folate (folic acid)

is made up of three distinct parts that all must be present for vitamin activity: pteridine (or pterin), p-aminobenzoic acid (PABA) and l-glutamic acid to form folate (Fig. 6.9). Folate is necessary for cell division and is required in amino acid metabolism. Although humans can synthesise all the vitamin parts, they do not have the enzyme needed for coupling the pterin molecule to PABA to form pteroic acid. Green vegetables, legumes and fortified grain products are good sources of folate. Folic acid is the synthetic form of folate used in supplements and food fortification. There are significant differences in stability depending on the exact structure of folate. Naturally occurring folates are unstable and sensitive to oxygen, oxidative agents, heat, and light. However, folic acid is very stable in acidic environments and high-temperature processing.

2 chemical structures. Top. (2 S), 2, [[4, [(2 amino 4 oxo 3 H pteridin 6 yl) methylamino] benzoyl] amino] pentanedioic acid. Bottom. 5, [(3 a S, 4 S, 6 a R), 2 oxo 1, 3, 3 a, 4, 6, 6 a, hexahydrothieno [3, 4, d] imidazol, 4, yl] pentanoic acid.

Fig. 6.9

Structures of folates (top) and biotin (bottom)

Biotin

is involved in many metabolic processes related to all macronutrients (i.e., fats, carbohydrates, and amino acids), and it may be synthesised by intestinal bacteria (Fig. 6.9). Liver, soybeans, egg yolk, cereals, legumes, and nuts are good sources of biotin. It is found to be combined with lysine (biocytin) or proteins, and proteolysis is required before its absorption. Avidin, a protein in egg white, binds biotin, making it unavailable, but denaturation of egg white during cooking improves availability. It is very stable to heat, light and oxygen and is quite well retained in foods.

Depending on the purpose, vitamins may be added to food formulations with different associated terms. Restoration refers to vitamin additions to restore their original concentration (e.g., vitamin C in orange juice). Fortification is the addition of nutrients to make food an excellent source of vitamins (e.g., breakfast cereals). Enrichment is the addition of specific amounts of vitamins lost during processing (e.g., flour enrichment with niacin, thiamine, riboflavin, and folic acid). Finally, nutrification is a generic term that encompasses the addition of nutrients to food (e.g., vitamin D addition in milk). Some strategies may be followed to optimise vitamin retention in processed foods. The most important is optimising thermal processing, where most losses generally occur, with lower-intensity thermal processes being preferable when possible (e.g., HTST). Blanching should be carried out at lower temperatures with shorter exposure to hot water and a minimum water volume to minimise leaching. Prediction of losses with the determination of the rate of vitamin degradation under the processing conditions could be a valuable tool to optimise processing parameters. Finally, packaging using correct geometry in canning and materials with minimum oxygen and light permeability should be preferred. Table 6.1 summarises vitamin stability under typical processing and storage conditions. It should be emphasised that this table should be used as a guideline, as stability highly depends on food formulation and storage conditions. For instance, the same vitamin may have additional oxidative stability in foods rich in antioxidants even if processed under the same conditions.

Table 6.1

Generalised stability of vitamins during processing and storage. U: unstable, S: stable. Stability may vary depending on food formulation, processing conditions and specific types of vitamins

Vitamin

Neutral pH

Acidic pH

Air or O₂

Light

Heat

A

S

U

U

U

U

D

S

S

U

U

U

E

S

S

U

U

U

K

S

U

S

U

S

B1

U

S

U

S

U

B2

S

S

S

U

U

B3

S

S

S

S

S

B5

S

U

S

S

U

B6

S

S

S

U

U

B12

S

S

U

U

S

Folate

U

U

U

U

U

C

U

S

U

U

U

Biotin

U

U

S

S

S

6.4 Minerals

A mineral

is a chemical element that is an essential nutrient, as it is needed to sustain life and promote growth. Minerals cannot be synthesised and must be obtained from the diet, but only small amounts are needed. The inorganic residue from food incineration (burning) is termed ash and contains minerals. Minerals are classified into macro-minerals (Ca, P, Na, K, Mg, Cl, S) and trace elements (Cu, I, Co, Fe, Mn, Se, Zn, Mo, F). Na, K, F, and Cl are present in foods as free ions with high bioavailability and rare nutritional deficiencies. The most common dietary deficiencies occur with Fe, Zn, Ca, and I. Mg, P, and Se are present in foods but bound as complexes, while Cu and Mn have low bioavailability. Other elements may be found in foods without nutritional value classed as toxic metals, posing a significant health risk, such as Pb, Hg, As, Cd, or Cr. For example, lead and mercury may be found in high levels in seafood (e.g., mussels) and arsenic in rice.

The availability and reactivity of minerals depend on their water solubility. If the mineral is insoluble in food, it will not be absorbed. Bioavailability ranges from 1% for some forms of iron to 90% for sodium and potassium. Minerals may form complexes with compounds termed ligands. In foods, ligands include proteins, carbohydrates, phospholipids, and organic acids. Ligands and metals form complexes termed chelates (Fig. 6.10). Chelate formation stabilises the metal and may either improve or reduce its adsorption.

Chemical structures of calcium oxalate, calcium ethylenediamine tetra acetic acid and ferrous bisglycinate.

Fig. 6.10

Common chelates found in food

Organic acids generally enhance bioavailability (e.g., ascorbic, citric, and lactic acids). Phytic acid is the principal storage form of phosphorus in bran and seeds and is present in many legumes, cereals and grains. Phytic acid and phytate have a strong binding affinity to dietary minerals, calcium, iron, and zinc, inhibiting their absorption and possibly leading to mineral deficiencies (e.g., in vegan diets or developing countries) (Fig. 6.11). Phytic acid, primarily as phytate in the form of phytin, is found within the hulls of seeds, including nuts, grains, and pulses.

Chemical structure of (2, 3, 4, 5, 6 pentaphosphonooxycyclohexyl) dihydrogen phosphate.

Fig. 6.11

Structure of phytic acid

Phytochemicals

like polyphenols and tannins also influence the binding of minerals. They form insoluble precipitates and are far less absorbable in the intestines. Typical examples restricting mineral absorption are polyphenolic compounds from tea, coffee, raisins, or sorghum. For example, a common practice of tea or coffee consumption immediately after a meal interferes with mineral absorption, including iron, zinc, and calcium, which may lead to mineral deficiencies in the long term.

Mineral and Vitamin Analysis

Ash content is measured after incineration of the sample at a high temperature (525 °C) in a muffle furnace. All the organic material is burnt at such high temperatures, and the inorganic residue contains the minerals. The ash may be further analysed to identify and quantify specific minerals of interest using atomic absorption spectroscopy (AAS) or inductively coupled plasma mass spectrometry (ICP-MS). Vitamin analysis in foods is quite complex. Methods usually have several steps and use specialised chromatographic protocols that must be tailored for the specific vitamin and food under consideration.

6.4.1 Effect of Processing on Mineral Bioavailability

In minerals, bioavailability is more important than content, as high content does not guarantee absorption. Processing methods should influence solubility or destroy the inhibitory effect of phytic acid or tannins. Minerals, unlike vitamins, are not destroyed by heat, light, oxidising agents, or pH, but they may change their form. For example, iron changes from Fe²⁺ (ferrous) to Fe³⁺ (ferric). Iron bioavailability depends on the type of iron added and the chemical environment of food. Ascorbic acid’s effectiveness in increasing iron absorption depends on the type of food and the number of inhibiting factors present. Minerals may be removed from foods during processing (e.g., through leaching or physical separation) or added by the instruments used to process foods (e.g., iron from steel mixers or pipes). Grains generally have a high concentration of minerals in the bran and germ, and their removal by milling leaves behind a pure endosperm with low mineral content. Conversion of whole wheat to white flour results in substantial losses in iron, zinc, copper, magnesium, and selenium. Although losses of minerals are substantial during milling, mineral bioavailability may improve due to the reduction in the phytic acid content of the grain. For instance, the pearling of sorghum increases iron absorption due to the reduction in polyphenol and phytate content. Dehulling of soybeans reduces iron absorption since iron in soy hulls is similar to the highly available form of FeSO₄. Leaching out of minerals into the cooking water is lower when foods are steamed rather than boiled. Although significant losses occur by cooking, mineral bioavailability generally increases with cooking by increasing solubility due to cell wall disruption, protein denaturation and release of organic acids. Freezing and drying have a negligible influence on mineral content and absorption. Mineral availability from cereal grains can be improved by hydrolysing phytates during soaking and germination. Fermentation enhances the bioavailability of minerals by reducing the phytate content and producing lactic acid, which improves mineral solubility. Storage of food has negligible effects on the mineral content and bioavailability. Heat processing may affect bioavailability by changing mineral solubility and destroying food constituents that increase or decrease availability. Cooking and baking may eliminate ascorbic acid and its effect on iron availability. Maillard reaction products produced during browning may bind with zinc, making it unavailable for absorption. Extrusion processing of cereals can cause an increase in iron content due to contamination from the extruder and an increase in mineral bioavailability by reducing the phytate content of food, although the relationship is quite complex depending on the food formulation. The following table (Table 6.2) summarises the influence of various food processes on the mineral content of food.

Table 6.2

Influence of food processing on minerals

Processing

Effects

Milling

Minerals lost from bran removal

Heat treatments

Blanching

Losses due to leaching into the water

Pasteurisation

Few losses

Sterilisation

Losses into brine/syrup

Baking

Phytate destruction may increase absorption

Frying

Iodine loses

Drying

Negligible influence

Freezing

Losses due to blanching before freezing losses in the drip after thawing

Fermentation

Reduction of phytate content

Extrusion

Bioavailability may increase or decrease

Packaging

Tin-cans may cause strong reducing conditions

6.5 Additives

Additives

are substances used for various technological reasons, for example, preservation, colouring or flavouring. Additives are usually substances not consumed as food. For example, tomato paste frequently has citric acid as an additive. Tomato paste is the food, while citric acid is not meant to be eaten alone but is used for a specific technological reason. A crucial distinction should be made between the terms additive and processing aid. Once added, additives become a component of the food and are ingested during consumption. In contrast, processing aids are also added for technological reasons, but they are removed after processing has finished and do not consist of part of the final product. For example, hexane is used in soybean processing to extract the oil, but it is completely removed at the end of the process, and no hexane remains in the finished product. Additives have four key roles: (a) to maintain product consistency, (b) to improve or maintain food’s nutritional quality, (c) to maintain or enhance sensory properties of food and (d) to make food safe. Frequently, they may have more than one role at the same time. For instance, nitrates are used in meat products to safeguard from Clostridium botulinum growth and improve colour (i.e., c and d). In other cases, they are added for only one purpose; for instance, an emulsifier would be necessary to keep the oil dispersed in a salad dressing (i.e., a), whereas flour can be fortified with vitamin B complex (i.e., b) to improve its nutritional value. It should be noted that because of the inherent complexity of food composition, a compound that works in one product may not be suitable in another. For example, while ascorbic acid (vitamin C) can be added in large amounts in orange juice to supplement its vitamin C content (40–60 mg/100 mL), this amount would destroy dough where it may be used as dough-improver at concentrations of ~10 ppm. In this example, ascorbic acid fulfils the role (b), while in dough, it fulfils the role (a). In another example, citric acid may be used as an acidifier in a lemon-flavoured soft drink. While acetic acid also acidifies, it would not be suitable for such a drink as it would taste like vinegar and compromise the sensory properties of the beverage. Consequently, having a limited list of additives that work in different foods is not possible. As a result, there are several thousands of additives, and depending on the country, the exact number may differ. For instance, the list can be found online on the EFSA websites for the European Union and the FDA website for the USA.

While it is impossible and futile to remember all the chemicals, it is feasible to group them into classes depending on their functionality, even though frequently, there is overlap because some additives exert more than one effect (Table 6.3). It becomes evident from this discussion that the focus should be on understanding their functionality and interactions with food components.

Table 6.3

Examples of additives used in the food industry. The list is not exhaustive but contains all the major additive groups used in the food industry

Additive agent

Function

Examples

Antifoaming agents

Prevent foaming that may interfere with processing

Dimethylpolysiloxane, Propylene glycol

Acidity regulators

Maintain the pH of food

Citric acid, acetic acid

Anticaking agents

Prevents powder agglomeration, ensuring the product remains free-flowing

Sodium aluminium silicate, Sodium ferrocyanide decahydrate

Antimicrobial agents

(preservatives)

Prevent the growth of microorganisms

Sodium nitrate, Potassium sorbate

Antioxidants

Prevents lipid oxidation

BHT, TBHQ

Colourants

Adding colour

β-Carotene

Flour treatment agents

Bleaching of flour or dough to improve its baking quality

Benzoyl peroxide, Ascorbic acid, DATEM

Emulsifiers/Surfactants

Dispersing oil in water

Lecithin, Sorbitan esters

Enzymes

Various roles depending on the enzyme

Amylases

Flavourings

Adding flavour

Limonene

Humectants

Attracts and retains the moisture in the product

Glycerol, sorbitol

Sequestrants

Chelates with polyvalent metal ions to prevent oxidation

Potassium gluconate, EDTA

Stabilisers/thickeners

Improve viscosity, gelation

Guar gum

Leavening agents

Releasing CO₂ in dough products

Sodium bicarbonate

Non-nutritive sweeteners

Add sweet taste

Aspartame

Additives

Consumers are concerned about the presence of additives in their food. This concern is justified to a certain extent. For example, an additive that was deemed safe in the past turns out to be unsafe after re-evaluation of research data or new findings that were not available in the past. A recent example is titanium dioxide, which was banned in the EU in 2022 but was allowed until then. Also, additives banned in Europe are allowed in the USA (or other areas). For instance, azodicarbonamide, a bleaching agent, is prohibited in the EU but permitted in the USA. Despite the different approaches jurisdictions may take to interpret scientific data, food additives are regulated, and their safety is reassessed frequently. In addition, additives are only used when the natural alternative fails to preserve or produce a uniform product, as often, the cost increases when the formulation’s complexity increases. It is true that without additives, mass food production would not be possible, and the food industry would not exist. The dilemma is not between forbidding or allowing additives but between throwing food away or accepting a carefully assessed risk of consuming a small amount of them.

6.5.1 Safety of Food Additives

Food additives must be functional and possess a low or insignificant risk to human health. Their safety is based on risk assessment and are only used if they are deemed safe, regardless of whether they come from a natural or synthetic source. The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) formed the Joint Expert Committee on Food Additives (JECFA) in 1956 to evaluate the safety of food additives. Since then, the committee has evaluated more than 2500 food additives. The starting point for determining whether an additive can be used without harmful effects is establishing their acceptable daily intake (ADI). The ADI is an estimate of the amount of an additive in food that can be safely consumed daily over a lifetime without adverse health effects. It is measured in mg/kg of body weight. ADIs serve to protect the health of consumers and to make international trade easier. For example, according to the FDA, the ADI of aspartame is 50 mg/kg per day (50 mg/kg/day), whereas EFSA has set it to 40 mg/kg/day. Consequently, an individual weighing 70 kg can safely consume 3500 mg (or 2800 mg) of aspartame every day until the natural end of life without any health problems arising from its consumption. A can (~330 mL) of a zero-calorie soft drink contains ~200 mg of aspartame. Consumption of 3500 mg of aspartame would require drinking more than 4.5 L (>14 cans) of a soft drink daily, which is unrealistic for most people. It should also be noted that the additive is often safer than the food itself. For example, a drop of artificial blue colour in an ice cream formulation is much safer than the ice cream itself from a nutritional point of view.

The International Numbering System for Food Additives (INS) is a naming system that provides an alternative to the chemical or complex names of the compounds. The identification number usually consists of three or four digits, such as 330 for citric acid or 1100 for amylases. Other combinations are possible depending on the case (e.g., 150a is plain caramel). The E numbers are the codes used in the EU for the food additives. They represent the same compounds as the INS but with an E before the number and refer to approved additives within the EU. For example, additive amaranth has an INS code of 123; in the EU, it would be E123. Interestingly, as of 2023, this additive is banned in Australia and the USA but approved in the EU!

6.6 Review Questions

6.6.1 Multiple Choice Questions

1.

The lipid-soluble vitamins are:

(a)

A, D, E and K

(b)

A, D, E and L

(c)

A, D, E and B

(d)

A, D, E and C

2.

Vitamin A degrades with a similar mechanism as the:

(a)

Oxidative degradation of fatty acids

(b)

Hydrolysis of proteins

(c)

Enzymatic demethylesterification of pectin

(d)

Saturation of double bonds in fatty acids

3.

Pantothenic acid has two parts in its structure that are:

(a)

Pantoic acid and β-alanine

(b)

Pentoic acid and β-alanine

(c)

Pantinoic acid and α-alanine

(d)

Pentoic acid and α-alanine

4.

Which three structural parts are needed to form folate?

(a)

Pteridine, o-aminobenzoic acid and l-glutamic acid

(b)

Pteridine, p-aminobenzoic acid, and l-glutamic acid

(c)

Pteridine, m-aminobenzoic acid and l-glutamic acid

(d)

Pteridine, m-aminobenzoic acid and d-glutamic acid

5.

Biotin in food is protein-bound and is called:

(a)

Biocysteine

(b)

Biocystine

(c)

Biocyteine

(d)

Biocytin

6.

Ash, the inorganic residue from the incineration of food, contains:

(a)

All macrominerals

(b)

All trace elements

(c)

All macro minerals and trace elements

(d)

All macro minerals, trace elements and vitamins

7.

Phytic acid has a very strong binding affinity for:

(a)

Ca, Fe and Zn

(b)

Ca, Cu and Zn

(c)

Fe, Na, and Cu

(d)

Cu, Zn and Mg

8.

“The availability and reactivity of minerals depend on their water solubility”. This statement is:

(a)

True

(b)

False

9.

Ligands and minerals form complexes termed:

(a)

Chelaoates

(b)

Cheloates

(c)

Cheliceral

(d)

Chelates

10.

In minerals, bioavailability is more important than content, as high content does not guarantee absorption. This statement is:

(a)

True

(b)

False

6.6.2 Short Answer Questions

1.

Look online for the terms Daily Reference Intake (DRI), Recommended Dietary Allowance (RDA), and Daily Value (DV) and find the values for Vitamin A.

2.

Write the structures of α-, β-, γ- and δ- tocopherols and tocotrienols and identify the differences in their structures.

3.

List and discuss the causes of vitamin loss during food processing.

4.

Online activity: Search online for the terms “coordination number”, “monodentate”, “bidentate”, and “polydentate”.

5.

List and discuss the causes of mineral losses during food processing.

6.

Discuss the role of additives in foods.

7.

What is the acceptable daily intake (ADI) of additives?

8.

What are the E-numbers?

9.

Discuss why some countries allow some additives while they are banned in others.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024

V. KontogiorgosIntroduction to Food Chemistryhttps://doi.org/10.1007/978-3-031-53558-1_7

7. Colour Chemistry

Vassilis Kontogiorgos1  

(1)

Associate Professor in Food Chemistry, University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC, Canada

Vassilis Kontogiorgos

Email: v.kontogiorgos@ubc.ca

Learning Objectives

After studying this chapter, you will be able to:

Describe the interaction of light with the food surface

Describe how the colour is generated and measured

Discuss the chemistry of all classes of natural pigments

Describe colour changes during processing

7.1 Introduction

Taste, flavour, texture, and colour are foremost for food quality and sensory acceptability, with freshness, taste, and colour being the top three criteria that define food quality in consumer studies. Colour may indicate chemical changes in food, flavour, and texture (e.g., ripening stage or crust colour of bakery products) and is the first assessment of satisfaction before consuming the product. Control of colour intensity and understanding pigment interactions with other food components and their behaviour during processing and storage is essential to ensure high food quality and prolonged shelf life. Adding pigments is done due to variations in the colour of food ingredients (e.g., seasonal variations), changes occurring during processing (e.g., due to heat degradation), or to compensate for colour losses due to exposure to light, oxygen, or moisture during storage. In addition, colours that occur naturally (e.g., red in a strawberry-containing drink) may need to be enhanced or provide colour to colourless foods and create a unique sensory experience (e.g., green in mint-flavoured ice cream).

Colour is the sensory quality of an object perceived by the eyes from the light reflected by the object. Colour perception results from photoreceptor cells (cones and rods) stimulation by electromagnetic radiation with wavelengths between 380 and 780 nm. Rods are sensitive to lightness and darkness and cones to red, green, and blue, with the human eye being most sensitive at 555 nm (green). Radiation within this range is called the visible region of electromagnetic radiation. Radiations outside this range (e.g., ultraviolet or microwave) are not detectable by the human eye. The visible region of the electromagnetic spectrum is the stimulus for colour perception. However, the light source, object properties, and the anatomy of the eye and the brain result in a unique and individual sensory experience. The human eye is estimated to distinguish about ten million different colours. Light source (e.g., sunlight, fluorescent, tungsten, neon, or blacklight) is an important parameter that dramatically influences colour perception. For example, rice that appears white under sunlight (consisting of ultraviolet, visible, and infrared radiations) will appear purple when it is illuminated with blacklight (consisting of long-wave ultraviolet light (UV-A), very little visible light and no infrared radiation). Background differences also play an important role in colour perception, as colours may appear different depending on their surrounding colours and shapes (e.g., bright vs. dark background or the Chubb illusion). Other parameters, such as sample size (i.e., the same colour looks different in objects of varying size), angle of observation (i.e., colours may be different from different angles of observation), or observer differences (i.e., the sensitivity of an individual’s eyes and brain) may also influence colour perception.

7.2 Interactions of Light with Food

Objects are characterised by how much light they absorb, reflect or transmit at each wavelength of interest. When light is incident on an object, a part of it is absorbed (i.e., “stays inside the object”), reflected (i.e., bounces back to the observer) or transmitted (i.e., goes through the object) (Fig. 7.1). A pigment derives its colour from the selective absorption of specific wavelengths of visible light. Sometimes, a pigment found in food may also emit light, as in fluorescence pigments. For example, when tonic water is illuminated with black light, it emits bright blue light because of the quinine found in the formulation. Reflected light may present different forms of reflectance depending on the surface properties of food. In specular reflection (i.e., mirror-like), the angle of incidence on the surface is equal to the angle of reflection. Ideal specular reflection is never observed in foods. In diffuse reflection, the light beam is scattered at many angles rather than just one. Combining both modes of reflection (specular and diffuse) results in gloss reflectance (Fig. 7.1b).

A, A diagram of reflected, transmitted, and absorbed light on the food surface. B, A diagram of specular, diffused, gloss reflections on food surface. Diffused light rays are scattered. On a glossy surface, light rays are more scattered. C, A photo of blueberries. D, A photo of cherries.

Fig. 7.1

(a) Modes of light interaction with the food surface and (b) different types of reflection, (c) blueberries appear matte because of diffuse reflection of light, and (d) cherries appear glossy because of the gloss reflectance of light on their surface

In practical terms, a food that strongly scatters light in all directions appears matte (i.e., not shiny) (Fig. 7.1c), whereas when specular reflection is dominant, it appears glossy (i.e., shiny) (Fig. 7.1d). For instance, the external cell layer of fruits (i.e., cuticle) consists of cutin, a waxy material imparting fruits with a glossy appearance (e.g., apples, cherries, etc.). Degradation of the cutin layer during storage because of biochemical changes or abrasion during transportation may result in loss of gloss. Cloudiness and translucency are two terms that are related to transmitted light. Cloudy liquids contain particles or droplets that scatter light in all directions (e.g., cloudy lemonade, milk, or cloudy apple juice). Transparent liquids do not contain particles, or particles are very small in the range of nanometres, unable to scatter light. As a result, the fluid appears clear (e.g., clear apple juice or cranberry juice).

A method to describe colour that relates the colour of a pigment to its UV/Vis spectrum in solution is to define the colour with the three attributes: hue, intensity and brightness. The hue of a pigment can be characterised by the wavelength of maximum absorbance (λmax) from the UV/Vis spectrum. A shift of the absorption band towards longer wavelengths (e.g., from yellow to orange) is called a bathochromic shift (or red shift) (Fig. 7.2). A shift in the opposite direction(e.g., from yellow to blue) is known as a hypsochromic shift (or blue shit). Most frequently, shifts in the absorption spectra occur because of a structural change in the molecule. The most common causes that induce colour changes include high-temperature treatments, pH changes, light, and oxygen.

A multiline graph of absorbance versus wavelength. It plots 3 bell curves in the visible spectrum. The highest point of the middle bell curve is lambda max. The curve on the right side indicates a bathochromic shift. The curve on the left side indicates a hypsochromic shift.

Fig. 7.2

Bathochromic and hypsochromic shifts relative to λmax of a pigment

Intensity is quantified by the molar extinction coefficient (ε) at its λmax value and measures how strongly a molecule absorbs light at a specific wavelength. For example, from Beer-Lambert’s law (A = εcl, where A is the absorbance, c is the concentration, and l is the path length that light travels), we see that for the same concentration, the pigment with higher ε will have greater absorption. The molar extinction coefficient depends on the molecule’s structure, and modifying even one functional group may result in colour changes. Finally, the brightness of a colour refers to the shape of its absorption band (Fig. 7.3). Pigments with bright colours have narrow absorption bands, whereas broad absorption bands produce dull colours. It should be emphasised that very rarely, the colour of a food is due to the presence of a single molecule. Usually, there are hundreds of molecules responsible. Consequently, a strategy to achieve bright food colours would be to limit the presence of multiple compounds. This is not easy to achieve with natural pigments and is usually a characteristic of artificial colours in very simple formulations. For instance, the colour of a sports drink (very bright reds, greens or blues) is usually a simple aqueous solution with only one artificial pigment present. In contrast, giving a blue colour to a yoghurt using an anthocyanin extract from blueberries may contain hundreds of molecules with colour, making it challenging to control brightness.

A graph of absorbance versus wavelength. It plots two bell curves with different amplitudes. The bell curve with smaller width indicates narrow absorption band with a bright color. The bell curve with large width indicates broad absorption band with a dull color.

Fig. 7.3

Colour brightness refers to the shape of the absorption band of the pigment

The above descriptions refer to the analytical technique known as absorbance spectroscopy, usually when the sample is a simple homogeneous solution. This is rarely the case with foods, and the technique of visible reflectance spectroscopy is used for colour measurement. A suitable mixture of red, green and blue lights can match any coloured light. This observation forms the basis of colour measurement with visible reflectance spectroscopy and the colour classification systems (or colour spaces). Various colour spaces are available depending on the underlying fundamental principles used to describe them and the desired application (e.g., CIE 1931 XYZ, Munsell system, Adobe RGB and many others). CIELAB colour space, abbreviated as L*a*b*, is a system of colour classification that approximates human vision and is most frequently used in industry and research for colour measurements of food. Colour is expressed as three values: L* for the lightness that ranges from 0 (black) to 100 (white), a* ranging from green (−128) to red (+127), and b* fluctuating from blue (−128) to yellow (+127) (Fig. 7.4).

A representation of three bands labeled L asterisk, a asterisk, b asterisk. L asterisk ranges between 0 and 100. a asterisk ranges between negative 128 and positive 127. b asterisk ranges between negative 128 and positive 127.

Fig. 7.4

The L*a*b* colour space

In the L*a*b* system, numerical changes in these values correspond to approximately the same amount of visually perceived differences. L*a*b* values can be converted to a single colour function called colour difference (∆E*) using the following equation:

with ΔL* = L*sample − L*standard, Δa* = a*sample − a*standard, and Δb* = b*sample − b*standard. Other equations that take more parameters into account are also available, but the one presented above provides useful information about the differences between two colours. For example, quite frequently, the difference in colour during processing (e.g., frying or baking) needs to be closely monitored and controlled. Two colours are different when ΔE* ≥ 1. When ΔE* > 3, this is perceived as a significant colour deviation. However, depending on the food, occasional batch-to-batch colour variations may be tolerated with ΔE* > 4 (e.g., baked or fried foods) in contrast to others where slight deviations may signify product rejection (e.g., meat products). Colourimetry is used extensively in the food industry in quality control to ensure consistent colour, product development (e.g., new processes or ingredient replacement), and shelf-life determination to monitor colour changes during storage and in consumer studies to identify which colour is preferred.

Absorbance and Reflectance Spectroscopy

Absorbance spectroscopy, or spectrophotometry, is the analytical technique that measures the amount of light absorbed by a sample at a specific wavelength. Spectrophotometry is commonly used to measure the concentration of compounds in solutions as the intensity of colour is proportional to the concentration and follows Beer-Lambert’s law. The main regions of the electromagnetic spectrum used are the ultraviolet (UV) and the visible (Vis). Because of this, the technique is also called “UV-Vis spectroscopy”. While this technique is a staple in every analytical laboratory worldwide, it is not used to measure the colour of objects.

Colourimetry uses the technique of reflectance spectroscopy. This technique measures the amount of light reflected from the food’s surface. Two models can be used to describe the reflected light intensity: the diffuse reflection model (Lambertian reflectance) and the specular reflection model. The diffuse reflection model predicts that light scatters in all directions after hitting the surface. The specular reflection model supports the idea that the light beam hits the surface and is reflected at only one angle. Real objects are considered to reflect light with both mechanisms and ultimately, the reflected light depends on its colour and surface characteristics.

The reflectance spectra of an object picked up by the instrument are related to colour using established mathematical relationships. The first system was designed in 1931 by the “Commission Internationale de l’Éclairage” (CIE), known as the “CIE 1931 XYZ” colour space. The “CIELAB” colour space, described in the main text, was designed in 1976 and is useful for detecting small colour differences. The Munsell colour system is sometimes also used (e.g., in the beer industry), but the CIELAB has now replaced it.

7.3 Pigment Chemistry

There are various physical and chemical processes responsible for colour. For instance, colour may be generated from gas excitations (e.g., neon lights) or vibrations and rotations of specific chemical groups (e.g., halogens). In organic compounds responsible for giving colour to foods, their colour is usually explained with the molecular orbital theory, which is a theory that describes the electronic structure of molecules with quantum mechanics. Organic molecules with double bonds can form molecular orbitals known as π orbitals. Electrons may absorb energy from light and move from a ground state to an excited state, known as electronic transition. The most common electronic transition relevant to colour is known as π → π*, with π being the ground and π* the excited state. Once the molecules have absorbed energy, the colour observed is the complementary colour of the absorbed one. The pairs of colours which, when combined, cancel each other out and produce a grey are called complementary. These colours are found opposite to each other on a colour wheel (Fig. 7.5). For example, the complementary colour of violet is light green, and as a result, when the organic pigment absorbs violet at ~400 nm, the colour we see is light green at ~550 nm.

A pie chart. It presents the ranges of different absorbed and observed colors. Red between 750 and 650 nanometers. Orange between 650 and 580 nanometers. Yellow between 580 and 560 nanometers. Dark green between 525 and 490 nanometers. Light blue between 490 and 460 nanometers and others.

Fig. 7.5

The colour wheel showing the approximate wavelength of each colour. The observed colour is the complementary colour of the absorbed colour. For example, if the pigment absorbs red colour, we see dark green because it is located opposite red on the colour wheel

Molecules with only one double bond absorb energy in the UV region, which is not visible to humans, so they do not produce any visible colour. As a result, more double bonds are needed to create a molecule capable of absorbing in the visible spectrum. A chromophore is the part of the molecule responsible for its colour and usually involves a conjugated system. As we saw in the chapter on lipids, a conjugated system is when a molecule has alternating single and double bonds. Molecules with large conjugated systems can absorb from the visible spectrum and produce colours. Usually, five or more conjugated double bonds are needed to absorb in the visible range. For example, retinol (Fig. 7.6a) has a conjugated system of five double bonds, absorbs the dark blue part of the spectrum and appears yellow (the complementary colour of dark blue). A bathochromic shift (to higher wavelengths) is observed as double bonds increase in a conjugated system. For example, the energy that β-carotene absorbs with 11 double bonds shifts to higher wavelengths (Fig. 7.6b). In that case, it absorbs in the light blue region of the spectrum (~450 nm), and the colour we observe is orange (as in carrots).

4 chemical structures. A, (2 E, 4 E, 6 E, 8 E), 3, 7 dimethyl 9, (2, 6, 6 trimethylcyclohexen 1 yl), nona, 2, 4, 6, 8 tetraen 1 ol. B, beta carotene. C, (6 E, 8 E, 10 E, 12 E, 14 E, 16 E, 18 E, 20 E, 22 E, 24 E, 26 E), 2, 6, 10, 14, 19, 23, 27, 31 octamethyldotriaconta, 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 30 tridecaene. D, beta cryptoxanthin.

Fig. 7.6

Conjugated systems and colour variations. (a) Retinol has a conjugated system that spreads over five carbon-to-carbon double bonds (red double bonds), giving it a yellow colour, (b) β-carotene has a conjugated system of eleven carbon-to-carbon double bonds, giving an orange colour, (c) lycopene also has a conjugated system with eleven carbon-to-carbon double bonds. Because of structural differences compared to carotene (blue squares), its colour is red, and (d) β-cryptoxanthin has a –OH group (in blue) compared to β-carotene. This induces a bathochromic shift, giving red colour instead of orange

The exact amount of the shift depends on the specific molecular structure and not only the number of double bonds. For instance, lycopene found in tomatoes, also with 11 double bonds in its conjugated system (Fig. 7.6c), absorbs at even higher wavelengths in the dark green region of the spectrum (~500 nm), and we observe red. Generally, adding a double bond creates a bathochromic shift of ~30 nm. Oxygen may also induce a bathochromic shift, but generalising it is difficult. For example, the difference between β-cryptoxanthin and β-carotene is one hydroxyl group. Adding a hydroxyl group to the β-carotene’s structure induces a bathochromic shift, and β-cryptoxanthin has a strong red colour instead of orange (Fig. 7.6d).

Food colour results from the presence of pigments falling into two classes: natural and artificial, both with advantages and disadvantages. L*a*b* values can be related to pigment concentration, and relationships may be drawn between concentration vs. intensity of the colour. The five groups of natural pigments are classified most frequently based on their aqueous or lipid solubility or chemical structure (Table 7.1). Anthocyanins and betalains are water-soluble, whereas carotenoids and chlorophylls are lipid-soluble and are of plant origin. However, some carotenoids may also be found in non-plant foods. For example, the pigmentation of shrimps is due to the presence of various carotenoids, most frequently astaxanthin and β-carotene, whereas the colour of egg yolk is due to carotenoids lutein and zeaxanthin. Myoglobin is the only natural pigment derived from animal sources and is water-soluble. In terms of chemical structure, natural dyes are classified into four major groups: isoprenoid derivatives (carotenoids and xanthophylls), tetrapyrrole derivatives (chlorophylls, heme), flavylium cation derivatives (anthocyanins, flavonoids, tannins) and betalamic acid derivatives (betalains) (see below for structures).

Table 7.1

Classification of the main groups of natural food pigments

Group

Source

Solubility

Structure

Anthocyanins

Plants

Water soluble

Flavylium cation derivatives

Betalains

Plants

Water soluble

Betalamic acid derivatives

Carotenoids

Plants

Lipid-soluble

Isoprenoid derivatives

Chlorophylls

Plants

Lipid-soluble

Tetrapyrrole derivatives

Myoglobin

Animals

Water soluble

Tetrapyrrole derivative

7.3.1 Myoglobin

Myoglobin (Mb) is a protein found in muscle cells responsible for meat colour (Fig 7.7a). This protein has a prosthetic group called haem containing iron and can react with oxygen (Fig 7.7b).

A, A diagram of the structure of myoglobin. The haem is present at the center. It presents a ribbon-shaped structure. B, Chemical structure of 3, [18, (2 carboxyethyl), 8, 13, bis (ethenyl), 3, 7, 12, 17 tetramethylporphyrin 21, 24 diid, 2, yl], propanoic acid, iron 2+.

Fig. 7.7

(a) Structure of myoglobin. Haem is located within the protein (in purple), and (b) the structure of haem. The structure of myoglobin was drawn based on PDB code 1WLA

In contrast to haemoglobin, found in red blood cells and is essentially found in the entire body, Mb is only found in muscle tissues of animals. It should be noted that since blood is drained during slaughtering, the colour of meat is almost exclusively due to the presence of Mb, and any residual haemoglobin plays only a minor role. The state of iron oxidation in haem (i.e., ferrous (Fe²⁺) or ferric (Fe³⁺)) determines the colour of meat. The colour of Mb in fresh meat and under the complete absence of oxygen is purple and is termed deoxy-Mb (Fe²⁺) (Fig. 7.8). When Mb interacts reversibly with oxygen but without changes in the state of Fe oxidation, it forms oxymyoglobin (oxy-Mb (Fe²⁺)) with a bright red colour, the typical appealing colour of red meat. When iron is oxidised to Fe³⁺, metmyoglobin (met-Mb) is formed, which is brown. Reducing Fe³⁺ to Fe²⁺ will revert colour to red, but this process is not used in practice. The addition of nitrates and nitrites used in cured meat products creates nitrous oxide (NO) that interacts with Mb, yielding NO-Mb (nitroso myoglobin, Fe²⁺) with a pink colour after the application of heat (e.g., cooked hams). Carbon monoxide (CO) may also be applied to improve colour that proceeds with the formation of CO-Mb (Fe²⁺) (carboxy myoglobin, bright red).

A schematic process diagram. Fresh meat myoglobin is converted into oxymyoglobin by oxygenation. Oxymyoglobin is converted into metmyoglobin by oxidation. It is converted into denatured metmyoglobin. Nitrosyl hemochrome is converted into denatured metmyoglobin by oxidation.

Fig. 7.8

Colour of myoglobin with changes in iron oxidation

Green colours may also be formed due to the presence of sulfmyoglobin (H₂S) or chole globin (ascorbate, reducing agents), oxidised porphyrins or excess of NO (“nitrite burn”). Colour intensity depends on Mb concentration, and it varies between different species in the following order: chicken (0.02 mg/g) < pork (2 mg/g) < lamb (6 mg/g) < beef (8 mg/g). Additionally, older animals have a higher concentration of myoglobin than young, while muscles that are used for motion have a higher concentration of Mb than those that are used for support.

7.3.2 Chlorophyll

Chlorophyll is the green pigment in plant tissues (e.g., leafy vegetables), allowing plants to absorb light energy through photosynthesis. Minor differences in the structure and light absorption properties of chlorophylls (e.g., chlorophyll a, chlorophyll b, etc.) result in various shades of green. Chlorophyll has two main components: a porphyrin ring and a phytol tail. The porphyrin ring is similar to haem but with bound Mg²⁺ instead of Fe²⁺ (Fig. 7.9).

Chemical structure of chlorophyll a. M g 2 plus ion is at the center. It is surrounded by a porphyrin ring. The phytol tail is connected to the porphyrin ring.

Fig. 7.9

Structure of chlorophyll a

Phytol tail is the hydrophobic part of the molecule and gives chlorophyll its lipophilic characteristics. Food processing always causes some changes to chlorophyll pigments. Food composition, processing conditions (e.g., NaCl, pH, temperature), and heating duration influence colour (Fig. 7.10). Chlorophyllase, an enzyme naturally found in the chloroplasts and other locations of plant cells, degrades chlorophylls by catalysing the cleavage of the phytol tail from the porphyrin ring to produce chlorophyllide, with a dark green colour. The enzyme is inactivated at 100 °C, but vegetable blanching is usually carried out at temperatures between 70 and 100 °C. Consequently, chlorophyllase is active, giving blanched vegetables a dark green colour. However, with extensive heating or low pH, Mg²⁺ is lost, resulting in pheophorbide with an olive green colour. Another route of chlorophyll degradation is during processing at low pH, where Mg²⁺ is lost, but the phytol chain remains to produce pheophytin with olive-green colour. Further heating at higher temperatures (e.g., sterilisation) gives pyropheophytin, also olive-green. In reality, all these reactions occur simultaneously during processing, and depending on the nature of the product and the purpose of processing, one of these reactions will have a controlling influence on the final colour. Frequently, thermal processing may result in the complete conversion of chlorophyll to pheophytin, and additives may be needed to stabilise the colour (Fig. 7.10).

A reaction pathway. Chlorophyll by blanching induced chlorophyllase activity, is converted into chlorophyllide. It is then converted into pheophorbide when heat and acid are applied. The pheophytin is changed when chlorophyll is treated with heat and acid. It is then converted into Pyro pheophytin.

Fig. 7.10

Colour changes in chlorophyll with processing conditions

7.3.3 Carotenoids

Carotenoids are fat-soluble compounds with yellow, orange, and red shades and are the most widespread pigments. There are two carotenoid classes, hydrocarbon carotenes and oxygenated xanthophylls, with over 700 different compounds in these two classes. Carotenes are hydrocarbon chains, whereas xanthophylls have oxygen atoms in their structure and are both insoluble in water (Table 7.2).

Table 7.2

Examples of carotenoids their sources and colour

Carotenoid

Class

Examples of sources

Colour

α-Carotene

Carotene

Carrots, pumpkin

Yellow-orange

β-Carotene

Carotene

Carrots, appricot, mango

Orange

Lycopene

Carotene

Tomatoes, watermelon

Red

Zeaxanthin

Xanthophyll

Corn

Yellow

Astaxanthin

Xanthophyll

Crustaceans

Red

Lutein

Xanthophyll

Egg yolks

Yellow

Crocetin

Xanthophyll

Saffron

Orange-yellow

Bixin

Xanthophyll

Annatto

Red

Carotenoids are formed with head-to-tail polymerisation of isoprene units that result in the presence of conjugated double bonds (Fig. 7.11). Head-to-tail polymerisation proceeds with the linkage of C1 of the first isoprene to the C4 of the second isoprene, also known as 1–4 linked terpenes. Potentially, the links could also be 1–1 (tail-tail) and 4–4 (head-head). However, in most naturally occurring terpenes, the isoprene rule states that there are no 1–1 or 4–4 links. A terpene that does not obey the isoprene rule is called an irregular terpene, with the most common example being β-carotene (Fig. 7.12).

A reaction pathway. One molecule of myrcene is formed from two molecules of isoprene by head to tail polymerization. The molecular formula of isoprene is C 5 H 8. The molecular formula of myrcene is C 10 H 16.

Fig. 7.11

Head-to-tail polymerisation of isoprene to form myrcene. Myrcene is the simplest monoterpene and a main component of essential oils

Chemical structure of 1, 1 prime, [(1 E, 3 E, 5 E, 7 E, 9 E, 11 E, 13 E, 15 E, 17 E), 3, 7, 12, 16, tetramethyloctadeca, 1, 3, 5, 7, 9, 11, 13, 15, 17 nonaene 1, 18 diyl], bis, (2, 6, 6 trimethylcyclohex 1 ene).

Fig. 7.12

β-carotene is a hydrocarbon carotene and an irregular terpene. The conjugated bonds of β-carotene are shown in red. The irregular double bond formed by 1–1 polymerisation (tail-tail) is shown in green. Other examples of carotenoid structures and oxygenated xanthophylls are shown in Fig. 7.6

The colour of carotenoids results from their large conjugated system described previously, and their absorption shifts to longer wavelengths as the number of conjugated bonds increases (i.e., a bathochromic shift). Carotenoids require at least seven conjugated double bonds before the appearance of yellow colour. The double bond configuration in carotenoids is trans type, although cis may also be present. Carotenoids with trans bonds have deep colour, whereas increased cis configuration results in colour fading. This configurational change may occur during processing and storage under light, oxygen, acid, and heat.

Common unit operations during food processing do not particularly influence the colour of carotenoids, although this depends on the specific food formulation. While they are relatively stable during food processing, they are susceptible to oxidation because of their highly unsaturated structures. In addition, they are sensitive to light and acid, resulting in colour fading. Blanching destroys lipoxygenase that bleaches carotenoids, thus preserving their colour in frozen foods. More severe heat treatments (e.g., sterilisation) do not generally influence colour. In contrast, dehydration degrades pigments, and dry fruits and vegetables usually have faded colour compared to their fresh counterparts.

7.3.4 Anthocyanins

Anthocyanins are found in flowers, fruits, and vegetables, with more than 700 molecules in this pigment group. The colour varies between blue, pink, or violet shades but has strong pH dependency due to the molecule being amphoteric. The building block of anthocyanins is known as a flavylium cation (Fig. 7.13), and they usually contain glucose or galactose. Hydrolysis of the monosaccharides gives anthocyanidins with a higher absorption maximum than their corresponding anthocyanin (Fig. 7.14).

Chemical structure of Flavylium cation. It has a benzene ring in conjunction with another benzene ring, from which one carbon is replaced with O plus. The adjacent carbon is bonded to another benzene ring.

Fig. 7.13

Flavylium cation is the building block of anthocyanins. The numbering of the atoms is also shown

A reaction pathway. (2 S, 3 R, 4 S, 5 S, 6 R), 2, [2, (3, 4 dihydroxyphenyl), 7 hydroxy 3, [(2 S, 3 R, 4 S, 5 S, 6 R), 3, 4, 5 trihydroxy 6, (hydroxymethyl), oxan, 2, yl], oxychromenylium, 5, yl], oxy 6, (hydroxymethyl), oxane 3, 4, 5 triol on hydrolysis yields 2, (3, 4 dihydroxyphenyl), chromenylium 3, 5, 7 triol.

Fig. 7.14

Cyanin is an anthocyanin containing two glucose molecules linked with a glycosidic bond. Hydrolysis of the glycosidic bonds and release of glucose result in cyanidin, the aglycone of cyanin

Flavylium has several hydroxy (-OH) and methoxy (-OMe) substituents, and the type and number of substitutions result in different colours. More -OH group substitutions tend to shift the colour to red, whereas adding -OMe groups tend to shift the colour to blue. A higher number of -OH groups decreases the stability of the pigment, whereas more -OMe groups increase its stability. In foods, pelargonidin, cyanidin, delphinidin, peonidin, malvidin, and petunidin are the most common anthocyanidins (Fig. 7.15).

Chemical structures. A, Pelargonidin. Molecular formula is C 15 H 11 O 5 positive. B, Cyanidin. Molecular formula is C 15 H 11 O 6 positive. C, Delphinidin. Molecular formula is C 15 H 11 O 7. D, Peonidin. Molecular formula is C 16 H 13 O 6 positive. E, Petunidin. F, Malvidin.

Fig. 7.15

Common anthocyanidins occurring in food. The structure’s colour indicates its approximate colour at the typical pH of foods

Anthocyanin colour is highly dependent on the pH. At pH < 2, the orange-red flavylium cation predominates. As the pH increases, two competing reactions may occur between the hydration reaction on position 2 of the flavylium cation and the proton transfer reactions of the acidic hydroxyl groups. The first reaction gives colourless carbinol pseudobases (~pH 5), which can undergo ring-opening to pale yellow or colourless chalcones (~pH 6). The second reaction gives rise to violet quinonoidal bases (~pH 4). Further deprotonation of the quinonoidal base can occur at pH between 6 and 7 to form more bluish quinonoid anions (Fig. 7.16). At the typical pH of foods (< 7.0), anthocyanins are present as a mixture of equilibrium forms.

Two reversible reaction pathways. A, Quinoidal base in the alkaline medium is converted into flavylium cation. p H is less than 2. B, Carbinol pseudo base is converted into chalcone pseudo base. p H is between 3 and 6.

Fig. 7.16

Colour changes of anthocyanins with changes in pH. The starting point is the flavylium cation in the middle at pH <2. (a) Violet quinoidal bases start forming at pH ~4. Further increase of pH between 6 and 8 results in blue colour, and (b) colourless carbinol pseudobases form at pH ~5, which can change to pale yellow or colourless chalcones at pH ~6. Glu is glucose

Anthocyanins are unstable and sensitive to many factors that may act in coordination depending on the food. Strategies to retain anthocyanin colour in food must consider every aspect of processing and storage. Generally, heat processing shifts the equilibrium towards chalcones with oxidation and cleavage of covalent bonds by heat, resulting in unpleasant yellow or brown colours. Oxygen degrades colour from purple to brown and must be removed from packaging (e.g., vacuum packaging). Light also accelerates degradation, and packaging of food rich in anthocyanins should be in containers impermeable to light. High concentrations of sugars (e.g., jams) stabilise anthocyanins because of the lowering of aw. However, a low concentration of sugars accelerates degradation. Anthocyanins degrade faster in the presence of ascorbic acid, which is important for food formulations made with fruits. Table 7.3 summarises the factors influencing anthocyanin colour.

Table 7.3

Summary of main parameters influencing the colour of anthocyanins

Parameter

Colour

Effect

-OH

Shifts to red

Stabilises structure

-OMe

Shifts to blue

Destabilises strucutre

Lowering pH

Shifts to red

–

Increasing pH

Shifts to blue

–

Monosaccharide addition in the structure

Shifts to red

–

Sugar addition in the formulation

–

Stabilises colour

Temperature

Yellow-brown

–

Oxygen

Brown

–

Light

Yellow-brown

Destabilises colour

Low aw

–

Stabilises colour

Ascorbic acid

–

Destabilises colour

Flavonoids are the wider class of molecules to which anthocyanidins also belong and are water-soluble compounds with a primarily yellow colour. They occur widely in plant materials, with over 6000 compounds known to date. They do not have a technological significance, but some may be antioxidants or biologically active. For instance, isoflavonoids, also known as phytoestrogens, may exert biological activity because of their structural similarity to estradiol, a major female hormone. Their structures indicate subtle differences concentrated on the presence or absence of a ketone or hydroxyl group (Fig. 7.17).

Chemical structures of 7 compounds, such as flavanones, flavones, anthocyanidins, flavonols, flavanolols, and isoflavonoids.

Fig. 7.17

Structures of flavonoids. The red colour identifies the differences between the structures

7.3.5 Betalains

Betalains are red and yellow pigments divided into betacyanins (red, violet) and betaxanthins (yellow, orange). Interestingly, they only occur in the Caryophyllales order of flowering plants such as beets, amaranth and cacti. Betacyanins have a similar colour to anthocyanins but are not affected by pH, whereas betaxanthins are less common. Betalains are nitrogen-containing compounds formed by the condensation of betalamic acid with an amine (Fig. 7.18). Substitution with different amines and monosaccharides produces distinct red shades. For example, betanin from red beets contains glucose. They are stable at pH 4.0–7.0, but heat processing in acidic conditions hydrolyses betanin, resulting in yellow colours. Oxygen and light result in bleaching, and packing should be carried out under a vacuum. Some naturally found enzymes (e.g., peroxidases) may catalyse oxidative degradation of betalains.

3 chemical structures. a, (2 S, 4 E), 4, (2 oxoethylidene), 2, 3 dihydro 1 H pyridine 2, 6 dicarboxylic acid. b, (2 S), 1, 2, [(2 S) 2, 6 dicarboxy 2, 3 dihydropyridin 4, 1 H, ylidene] ethylidene, 5, (beta d glucopyranosyloxy), 6 hydroxy 2, 3 dihydro 1 H indol 1 ium 2 carboxylate. c, (2 S), 1, [(2 E), 2, [(2 S), 2, 6 dicarboxy 2, 3 dihydro 1 H pyridin 4 ylidene], ethylidene], pyrrolidin 1 ium 2 carboxylate.

Fig. 7.18

Betalains structure, which are betalamic acid derivatives

7.3.6 Other Natural Pigments

Other pigments may be obtained from natural sources frequently without purification and have variable stability on exposure to light, heat or oxygen. They are often more expensive than the previously mentioned alternatives or may have limited shades. Table 7.4 is a list of some common natural pigments.

Table 7.4

Natural pigments from less widespread sources

Pigment

Colour

Source

Chemical structure

Spirulina

Blue

Algae

Phycocyanins

Saffron

Orange-Yellow

Crocus

Crocin

Annatto

Red

Achiote tree

Bixin-Norbixin

Cochineal

Red

Insects

Carminic acid

Turmeric

Yellow

Turmeric

Curcumin-diketone

Paprika oleoresin

Red

Peppers

Capsanthin-Capsorubin

Genipin

Blue

Genip tree

Geniposide

Elderberry

Blue-purple

Elderflower

Anthocyanins

7.3.7 Artificial Colours

Artificial colours are needed because the range of shades and quantities from natural sources are limited. In addition, synthetic dyes are made to provide an almost limitless range of alternatives for food manufacturers to improve visual attractiveness. In addition, chemically synthesised dyes have many advantages over natural colours such as a brighter and broader range of shades, greater stability during processing and storage (e.g., against heat, light, oxygen, etc.) and are generally cheaper. Caramel colours that are produced through caramelisation reactions have been described in Sect. 5.​3.​1.​2. The other major artificial pigments are the azo-dyes, followed by triaryl methane and xanthine dyes, all entirely synthetic. Azo dyes are derivatives of diazene (H-N=N-H). The N=N group in their structure is called the azo group, with some dyes having more than one azo group (i.e., di-azo dyes). They have a general structure written as R − N=N − R΄, with R and R΄ usually being aryl groups (Fig. 7.19). Dyes may also be converted into lakes, usually using aluminium hydroxide. The difference between dyes and lakes is that dyes are water-soluble, whereas lakes are not. However, lakes can be dispersed into fats, oils and other non-aqueous formulations (e.g., cake mixes). For example, a blue sports drink is coloured with a dye, whereas a blue chewing gum with a lake. A dye is converted into a lake that is insoluble in water. It should be emphasised that lakes are not oil soluble, but they may be dispersed in oil. The list of authorised artificial pigments undergoes safety assessment from relevant authorities depending on the regulations of each region. Depending on the region, around 10–15 synthetic pigments are currently authorised for food use. The complete lists are available online on the websites of the relevant institutions (e.g., EFSA, FDA, etc.). The lists are periodically re-evaluated and may change when new toxicological data on their safety come to light.

4 chemical structures. a, trisodium, 5 oxo 1, (4 sulfonatophenyl), 4, [(4 sulfonatophenyl) diazenyl], 4 H pyrazole 3 carboxylate. b, di azo dye. c, 3, 7 dihydropurine 2, 6 dione. d, triaryl methane.

Fig. 7.19

Examples of structures of an azo dye (tartrazine), di-azo dye (brilliant black), xanthine (erythrosine), and triaryl methane (brilliant blue). Azo groups are highlighted in blue for the first two structures

7.4 Review Questions

7.4.1 Multiple Choice Questions

1.

Specular and diffuse reflectance of light on fruit surface results in:

(a)

Gloss appearance

(b)

Matte appearance

(c)

Fluorescent appearance

(d)

Translucent appearance

2.

In the L*a*b* system of colour, classification b* is:

(a)

Colour variation from blue to yellow

(b)

Colour variation from red to green

(c)

Colour variation from white to black

(d)

Colour variation from blue to green

3.

Oxygenation of myoglobin results in:

(a)

Oxymyoglobin

(b)

Metmyoglobin

(c)

Denatured myoglobin

(d)

Denatured metmyoglobin

4.

Oxidation of myoglobin results in:

(a)

Oxymyoglobin

(b)

Metmyoglobin

(c)

Denatured myoglobin

(d)

Denatured metmyoglobin

5.

Chlorophylls are water-soluble components that are sensitive to pH. This statement is:

(a)

True

(b)

False

6.

The olive-green colour of green vegetables after blanching is due to the loss of:

(a)

Mg

(b)

Fe

(c)

Ca

(d)

Zn

7.

Anthocyanins have a very similar structure to carotenoids, but they give blue/purple shades. This statement is:

(a)

True

(b)

False

8.

Increasing the pH of a delphinidin solution will change its colour from:

(a)

Blue to green

(b)

Blue to red

(c)

Green to blue

(d)

Red to blue

9.

Betalains are typical derivatives of the flavylium cation. This statement is:

(a)

True

(b)

False

10.

 Porphyrin ring is present in:

(a)

Chlorophylls and heme

(b)

Flavylium cations and heme

(c)

Betalamic acid and chlorophyll

(d)

β-carotene and betacyanins

7.4.2 Short Answer Questions

1.

Discuss the purpose of colouring food and the parameters that affect colour perception.

2.

What are bathochromic and hypsochromic shifts, and why are they important for food colour?

3.

Describe why some colours are bright and others are dull.

4.

What is a chromophore, and how do the conjugated systems influence colour?

5.

A company makes biscuits for children and uses β-carotene to give the product a bright orange colour. The product is packed in transparent plastic film. A batch of the product has been mishandled during transportation, and it has been exposed repeatedly to the sun and high temperatures. The table below shows the L*a*b* values after colour measurements of the fresh and transported product.

L*

a*

b*

Fresh

65.3

52.9

57.1

Transported

67.1

41.2

58.1

(a)

Calculate ΔΕ* between the fresh product and the transported product

(b)

Is the colour difference perceivable?

6.

A food product absorbs light green. What is the colour we see?

7.

Explain why some meat products (e.g., cooked ham, sausages) are pink.

8.

Discuss the reactions in chlorophyll during blanching and heating in an acidic environment.

9.

Discuss the difference between carotenes and oxygenated xanthophylls and give two examples.

10.

What is the difference between cyanin and cyanidin? Name the major anthocyanidins found in food.

11.

Discuss the factors that influence the colour of anthocyanins.

12.

Online activity: Search for the terms “apocarotenoid”, “bixin”, and “norbixin”. In which natural food colouring do we find these compounds?

13.

Draw and discuss the reactions that occur in anthocyanins with changes in pH.

14.

Online activity: Find the pigments with E-numbers E160d, E161b and E162.

15.

Online activity: Find Ponceau S, Allura Red, and Brown HT structures and identify the azo group.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024

V. KontogiorgosIntroduction to Food Chemistryhttps://doi.org/10.1007/978-3-031-53558-1_8

8. Flavour Chemistry

Vassilis Kontogiorgos1  

(1)

Associate Professor in Food Chemistry, University of British Columbia, Faculty of Land and Food Systems, Vancouver, BC, Canada

Vassilis Kontogiorgos

Email: v.kontogiorgos@ubc.ca

Learning Objectives

After studying this chapter, you will be able to:

Recognise the chemical structures of major compounds responsible for flavour

Discuss the chemical structure of compounds with sweet and bitter tastes

Discuss how flavour is generated and its sources

Discuss how flavour is delivered and its stability in foods

8.1 Introduction

The flavour is the sensory impression of food determined mainly by the chemical senses of taste, smell and trigeminal senses. Taste is perceived with the tongue, smell with the nose, and trigeminal senses with the trigeminal nerve across our face. The trigeminal nerve detects chemical irritants, temperature changes, and texture, which are critical to the overall flavour perception. The mechanisms we perceive these three sensory impressions are biologically distinct as they have different pathways to transfer the signals to the brain. However, from a consumer perspective, they are indistinguishable, resulting in a unique sensory experience most frequently described in sensory testing as “overall satisfaction”. Taste and smell are purely chemical senses because molecules present in the food trigger them when they associate with the tongue’s receptors. For instance, when a sweet molecule interacts with sweet receptors in the tongue, it transfers information interpreted as “sweetness” from the brain. In contrast, the trigeminal nerve can detect chemical (e.g., capsaicin results in a pain sensation) and physical sensations (e.g., temperature (hot, cold) or force (soft, hard)). Chemicals that prompt a chemical sense are naturally found in the raw materials but are also frequently added to food. The main reasons are to add a compound that exists in nature but is not typical of the product (e.g., flavoured mineral water with citrus extracts), to add a flavour that has been lost or modified during processing (e.g., flavours lost from a fruit drink during heat treatments), and to create a flavour that does not exist in the raw material (e.g., in cocoa or coffee bean roasting). The present chapter explores the chemical structures of molecules responsible for inducing a sensory perception associated with taste, smell and trigeminal senses.

8.2 Sweet Molecules

From a nutritional point of view, sweet molecules used in the food industry are classified as nutritive (or caloric) and non-nutritive (or non-caloric). When nutritive sweeteners are metabolised, they release dietary calories, whereas our bodies do not metabolise non-nutritive sweeteners. This allows us to formulate foods that still taste sweet but with low calories. It should be emphasised that replacing nutritive sweeteners with non-nutritive is particularly challenging, and it highly depends on the nature of the product and its processing conditions. For example, artificial sweeteners do not caramelise during baking nor interact with the gluten network of the dough to influence its rheology and processing characteristics. As a result, it isn’t easy to replace sucrose in sweet bakery products. In another example, artificial sweeteners do not depress the freezing point in ice cream formulations and do not influence the viscosity of the mix. As a result, ice crystal formation, melting characteristics and frozen storage stability of ice cream will be challenging to control. In contrast, artificial sweeteners are easier to handle in beverages where a sweet taste is desired (e.g., soft drinks). However, it should be realised that sweeteners have different characteristics in how we perceive their sweet taste, aftertaste, persistence after swallowing or off-flavours, most notably bitter or metallic off-flavours. Consequently, mixing sweeteners in various proportions followed by sensory evaluation is the only way to evaluate whether the sweetener is appropriate for the target product.

The sweet taste of various molecules is rated on a sweetness scale relative to sucrose. Taste panellists quantify the sweetening strength of sweeteners, usually using a 10% sucrose solution as a reference (Table 8.1). Sweeteners are then tasted at a series of dilutions to determine the concentration as sweet as the 10% sucrose solution. For example, if the panellists judge a 1% sweetener solution as sweet as a 10% sucrose solution, the sweetener is ten times as potent as sucrose. The concentration of 10% sucrose has been chosen because it is usually a target concentration in soft drink manufacturing that provides the most desirable sweetness and viscosity built up, and, in most soft drinks, it usually fluctuates narrowly between 8–12%. Specifically, when the concentration falls below ~8%, it is not considered very sweet, while above ~12%, it can lead to an intensely sweet taste, which is undesirable by most consumers. For example, a typical orange juice has ~9% carbohydrates, whereas a cola drink ~10%.

Table 8.1

Examples of sweetener strength relative to a 10% sucrose solution

Sweetener

Relative strength to sucrose

Sucrose

1

Acesulfame-K

200

Cyclamates

40

Aspartame

180

Saccharin

300

Sucralose

600

Neotame

10,000

Erythritol

0.6

Glycyrrhizic acid

40

The structure-function relationship of sweet molecules is generally well-understood. It has allowed the creation of many artificial non-calorific sweeteners with structures quite different from those of sweet carbohydrates (e.g., glucose). Initially, a sweet-tasting molecule (also termed glycophore) must be water-soluble, as this will allow strong association through hydrogen interactions between the molecule and the taste receptor. In addition, a sweet molecule must have the correct molecular shape and the proper electronic distribution that allows it to interact with the taste receptor. The current theory of sweetness, known as AH-B-γ theory or the triangle theory, suggests that the spatial arrangement of atoms in interaction with tongue receptors is triangular (Fig. 8.1).

A schematic representation. It presents B minus bonded to Y and A H plus to form a triangle. B minus is bonded to A H plus via hydrogen bonds. There is a hydrophobic interaction between Y and Y.

Fig. 8.1

The spatial arrangement of the appropriate groups of a sweet molecule should be triangular to interact with the sweet-taste receptors of the tongue. Yellow triangle: sweet molecule, grey triangle: tongue receptor

The sweet molecule binds to the taste receptor site in a complementary manner through hydrogen bonding and hydrophobic interactions. The AH⁺ area contains functional groups such as hydroxyl, amine, or some other groups with hydrogens available to hydrogen bond to some partially negative atom such as oxygen. The B⁻ area contains functional groups such as hydroxyl, carboxyl, or some other group with partially negative oxygen available to hydrogen-bond to a partially positive atom, such as hydrogen, on the sweet molecule. Finally, the γ area is perpendicular to the other two regions interacting through hydrophobic interactions with the tongue receptors. Functional groups such as benzene rings and multiple –CH₂ and –CH₃ groups are usually found in the γ-area (Fig. 8.1). Two receptors responsible for detecting sweet taste have been identified, namely T1R2 and T1R3. When sweet molecules bind to these receptors, they send signals to the brain, resulting in the perception of sweetness. From a structural point of view, sweet molecules are classified into five main classes: carbohydrate-based, peptide-based, sulfonamides, glycosides, and saponins, explored in the following sections (Fig. 8.2).

Six molecular structures. A, Glucopyranosiduronic acid. B, Methyl L alpha aspartyl L phenylalaninate. C, Brazzein a protein. It has a ribbon like structure. D, 1, 1 dioxo 1, 2 benzothiazol 3 one. E, (2 S, 3 R) butane 1, 2, 3, 4 tetrol. F, Stevioside.

Fig. 8.2

Examples of structure variability in sweet molecules. Despite the extraordinary differences in structure, all these molecules taste sweet. (See the text for a description of their properties)

8.2.1 Carbohydrate-Based Sweet Molecules

Carbohydrate-based sweet molecules and ingredients are monosaccharides, disaccharides, alditols, high fructose corn syrups (HFCS), invert sugar, glucose syrups, and sucralose. The structure of the first three has been described in Chap. 2. The main alditols used as sweeteners are erythritol, xylitol, mannitol, sorbitol, isomalt, and maltitol. Sucralose is an artificial sweetener, and its structure is very similar to that of sucrose. It is produced by selective chlorination of sucrose, replacing three hydroxy groups in the C1 and C6 positions of the furanose ring and the C4 position of the pyranose ring of the molecule.

Glucose syrups are not pure glucose solutions, i.e., they do not contain only glucose molecules. They are defined as concentrated solutions of nutritive sweeteners derived from starch with a dextrose equivalent of >20%. Glucose syrups contain varying amounts of glucose, maltose and oligosaccharides, depending on the enzymatic hydrolysis used to make them. For instance, a syrup with a DE = 30 contains only about 9% glucose, whereas with DE = 95 contains 93% glucose (Table 8.2). As it becomes evident, glucose syrups are made for specific applications depending on their composition and cannot be exchanged with each other. For example, syrup for ice cream may not be suitable for bakery products. HFCS consists of fructose, glucose and oligosaccharides. The most common HFCSs are HFCS 42 and HFCS 55, meaning that 42% or 55% are fructose, and the rest are glucose and oligosaccharides. HFCS 42 is mainly used in bakery products, cereals and other processed foods, whereas HFCS 55 is used in soft drinks. High-maltose corn syrup is a syrup where ~50% of the main carbohydrate is maltose, which is less sweet than high-fructose corn syrup. Finally, invert sugar syrup is produced by the hydrolysis of sucrose, either enzymatically or with acid hydrolysis, and consists of approximately 50% glucose and 50% fructose.

Table 8.2

Typical corn syrup composition with different dextrose equivalents (DE)

Carbohydrate (%)

DE 30

DE 65

DE 95

Glucose

9

35

93

Maltose

10

35

1

Maltotriose

12

10

2

Oligosaccharides

69

20

4

Total

100

100

100

8.2.2 Sweet Peptides and Proteins

Several sweet peptides and proteins have been identified as sweet and can be either natural or artificial. The shared chemical characteristic in these molecules is the presence of peptide bonds, as they are all amino acid derivatives. The most common peptide-based artificial sweeteners are aspartame, neotame, advantame, and alitame. A common structural feature of the first three is that they are all dipeptides with l-aspartic acid and l-phenylalanine as the backbone structure. Alitame is a dipeptide derivative of l-aspartic acid and d-alanine. Sweet proteins are naturally found and extracted from plants that grow in tropical rainforests. Thaumatin is the most notable, with the sweetness developing slowly after consumption and the only one approved for food use worldwide. Other sweet proteins are monellin, brazzein and miraculin, which are not approved for food use (Fig. 8.2).

8.2.3 Sulfonamides

A sulfonamide is an organosulfur functional group with the structure R₁ − SO₂ − NR₂R₃ consisting of a sulfonyl group (O=S=O) connected to an amine group (−NH₂). The three sulfonamides used as synthetic sweeteners are acesulfame potassium (also known as acesulfame-K or ace-K), saccharin and cyclamic acid. They have different sweetness intensity, bitterness and processing characteristics. Saccharin is ~300 times, whereas cyclamic acid is ~40 times as sweet as sucrose. They are both stable in a broad range of pH and temperatures but leave a bitter aftertaste. Acesulfame-K is ~200 times sweeter than sucrose, with no aftertaste at low concentrations but a bitter metallic taste at higher levels (Fig. 8.2).

8.2.4 Glycosides

Steviol glycosides are the compounds responsible for the sweet taste of the leaves of Stevia rebaudiana. The generic name stevia refers to a sweetener made of a mixture of steviol glycosides. They are chemically glycosides with glucose as their main monosaccharide and steviol as the aglycone. There are several different steviol glycosides with slight differences in their structure, but stevioside, rebaudioside A and rebaudioside C are most commonly used commercially (Fig. 8.2).

8.2.5 Saponins

Saponins comprise amphiphilic glycosides of triterpenes or steroids and are usually found in plants. Glycyrrhizin is a pentacyclic triterpenoid glycoside obtained from the root and stolons of the plant Glycyrrhiza glabra, commonly known as liquorice. Glycyrrhizin is the chemical responsible for the sweet taste of liquorice extract. Because of its amphiphilic structure, it interacts strongly with taste buds, and the sweet taste lingers, creating a unique tasting experience (Fig. 8.2).

8.2.6 Sweetener Synergy and Suppression

Artificial sweeteners have several features to consider when designing a food formulation. A common characteristic is that the higher the concentration, the greater the sweetness of the solution. However, increasing the concentration indefinitely is impossible as bitterness or metallic tastes start appearing. Once a concentration has been selected without undesirable off-tastes, the main feature will be their maximum sweetness intensity and the time to reach this maximum, as different sweeteners have different capacities. Also, once the sweetener comes into contact with the taste buds, it takes time until it reaches maximum intensity. This is a crucial characteristic in soft drinks where fast maximum intensity is generally required as the oral residence time is limited to a few seconds. In contrast, a slow time to maximum intensity would be needed for products like chewing gums, where the oral residence time is extended. Once this has also been decided, the last characteristic is the residual sweetness (lingering) after consumption. Depending on the product, this may be designed to be long or short. Finally, the sweetener will behave differently depending on the interactions with the other ingredients. For instance, a sweetener in ice cream containing milk proteins, fat and polysaccharides will taste completely different in a soft drink that is primarily a diluted flavoured emulsion with few other ingredients.

As it is evident, no sweetener can achieve all these conditions simultaneously and, in most cases, blending is required. When two sweeteners are blended, the perceived intensity of the mixture may be equal to (additivity), greater than (synergy) or less than (suppression) the sum of the individual sweetness intensities. For example, consider solution A, a 10% sucrose solution with a sweetness score of 1. Solution B, a 4400 ppm cyclamate solution, is also scored with 1, as it gives a similar sweetness perception to solution A. Mixing solution A and B at a 1:1 ratio yields a solution containing 5% sucrose and 2200 ppm cyclamates. This is expected to have a sweetness score of 0.5, i.e., half as sweet as solutions A and B because the concentrations have been halved. However, even though the mixture contains lower concentrations of both sweet molecules, it tastes sweeter, receiving a score of 1.2 in sensory tests. The two molecules (sucrose and cyclamates) act synergistically, thus reducing sucrose concentration to 5% without affecting sweetness perception. In contrast, mixing aspartame and sucrose leads to a lower sweetness perception with a score of 0.7, suppressing sweetness. Mixing multiple sweeteners in different ratios can mask off-flavours (e.g., metallic, bitter or astringent aftertastes) and improve overall sweetness perception, i.e., maximum intensity, time to reach maximum intensity, and residual sweetness (lingering).

Sensory Evaluation

Sensory evaluation is the scientific discipline that uses statistical analysis to measure food characteristics that are perceived by our senses (i.e., taste, smell, sight, touch and hearing). It is almost impossible to perform modern food product development without some form of sensory evaluation, as it is the only way to quantitatively evaluate the intensity of sensory qualities the human senses perceive. Even though analytical methods can reveal the food composition in great detail, there is little correlation between the chemical composition and sensory perception. For example, a sweetener may be sweet at low concentrations and bitter at higher concentrations. While the chemical composition in the low- and high-concentration samples is the same, the sensory perception is different and can only be evaluated by humans. Texture, sensed via the trigeminal nerve, is another example of a food quality in which instrumental analysis rarely correlates to the texture during consumption.

While the statistical analysis involved in the sensory evaluation can be intricate, ultimately, only three questions are answered: (a) Which products are preferred? (b) Are products different in any way? and (c) How do products differ in specific sensory characteristics? The first question is answered with tests called “affective”, the second “discrimination”, and the third “descriptive”. Each of these tests has different requirements, procedures, and analytical approaches. A skilled food technologist should have a strong grasp of food chemistry and sensory evaluation to develop products based on scientific principles.

8.3 Bitter Molecules

Bitter molecules are closely related to sweet molecules from a molecular structure-receptor relationship. Sweet molecules need two polar groups and a non-polar for functionality, whereas bitter molecules need one polar and one non-polar to cause a bitter taste. Bitter compounds have a lower detection threshold and are less soluble in water than sweet molecules. In addition, very frequently, the stereochemistry of the molecule influences bitterness. For example, hesperidin, found in citrus fruits, is tasteless, but neo-hesperidin is bitter (Fig. 8.3). Bitter molecules activate the bitter taste receptors (TAS2R), initiating signal transduction. Twenty-five TAS2Rs have been found to recognise the structurally diverse bitter compounds. The interaction of the bitter compounds with TAS2Rs is related to their chemical structure. Bitter compounds are structurally diverse, but many are alkaloids. Alkaloids are organic nitrogen-containing bases and can be classified into various groups depending on the fine details of their structure, showing large structural diversity. For example, quinine, commonly used in beverages (e.g., tonic water), is a quinoline alkaloid, whereas caffeine (coffee) and theobromine (cacao) are purine alkaloids. Other bitter structures include glycosides (naringin in grapefruit), phloroglucinol derivatives (humulone in beer) or bitter peptides (Fig. 8.4). Bitter peptides may be produced in fermentations from protein hydrolysis, and they are usually detrimental to the product (e.g., leucine-leucine). Glucosinolates are bitter compounds responsible for the characteristic bitter taste of cruciferous vegetables of the family Brassicaceae. Terpenoids (see below) and saponins (e.g., solanine in potatoes and tomatoes) may also give a bitter taste to raw materials that contain such compounds.

Four molecular structures of 4 compounds. A, Hesperidin. It is tasteless. The molecular formula is C 28 H 34 O 15. B, Neo hesperidin. It has a bitter taste. C, 2 S, 2 amino 3 (1 H indol 3 yl) propanoic acid. It has a bitter taste. D, 2 R, 2 amino 3 (1 H indol 3 yl) propanoic acid. It has a sweet taste.

Fig. 8.3

Stereochemistry influences bitterness of molecules

5 chemical structures. A, R, [(2 S, 4 S, 5 R) 5 ethenyl 1 azabicyclo, 2 dot 2 dot 2, octan 2 yl, 6 methoxyquinolin 4 yl, methanol. B, 1, 3, 7 trimethylpurine 2, 6 dione. C, 3, 7 dimethylpurine 2, 6 dione. D, humulone. E, naringin.

Fig. 8.4

Structures of common bitter food molecules

8.4 Salty, Sour and Umami Molecules

Salty taste depends on the nature of the cations and anions of the salt crystal structure. Many cations induce a salty taste, and the associated anions modify its perception. However, only sodium chloride (NaCl) is used in foods. Sodium ions pass directly through the ion channels of the tongue, leading to an action potential and perception of saltiness in the brain. Other salts may occasionally used in special applications, such as KCl, which produces salty and bitter tastes, whereas HCO₃⁻ (bicarbonate), SO₄²⁻ (sulfate), Ca²⁺ and Mg²⁺ are necessary for the taste of water.

The sour taste detects acids with a mechanism similar to the salty taste. Hydrogen ion channels detect the concentration of protons (H⁺) dissociated from an acid. There is no strong correlation between pH and sour taste. However, organic acids have different taste profiles. For example, citric acid provides a “fresh taste” sensation and is the most frequently used acidifier in the food industry. In contrast, lactic has a “cheesy” taste profile. While many organic acids contribute to sour taste, those used as additives are few. The most common are citric, ascorbic, lactic, phosphoric, malic, acetic, tartaric and fumaric acids. Phosphoric acid is the only inorganic acid used in foods as an ingredient, mainly in soft drinks. Other organic acids, such as benzoic acid or sorbic acid, may also be used as preservatives at a lower concentration.

The umami taste is the characteristic taste of salts of glutamic acid and is perceived through taste receptors that respond to the presence of glutamates. Umami is the basic fifth taste, and its receptors have recently been identified on the human tongue (in the ‘00s). Monosodium glutamate (MSG), 5΄-ribonucleotides, especially inosine 5΄-monophosphate (IMP) and guanosine 5΄-monophosphate (GMP) may all act synergistically, resulting in a unique umami sensation depending on the formulation (Fig. 8.5). MSG is naturally found in various foods (e.g., vegetables, meats, poultry, fish, shellfish, or aged cheese) and, together with ribonucleotides, are frequently used in snacks as flavour enhancers. Protein hydrolysates are a complex mixture of peptides and amino acids produced by the partial hydrolysis of proteins that may also taste umami. The name of a protein hydrolysate is specific to the ingredient. It includes the identity of the food source from which the protein was derived, for example, “hydrolysed wheat gluten”, “hydrolysed soy protein”, or “autolysed yeast extract”. They are produced using heat and acid or enzymes and are most frequently used in snacks and savoury products to impart strong savoury sensations in combination with umami-tasting compounds.

Three chemical structures. A, sodium, (2 S), 2 amino 5 hydroxy 5 oxopentanoate. B, The molecular formula is C 5 H 8 N N a O 4. B, [(2 R, 3 S, 4 R, 5 R), 5, (2 amino 6 oxo 1 H purin 9 yl), 3, 4 dihydroxyoxolan 2 yl], methyl dihydrogen phosphate. C, The molecular formula is C 10 H 14 N 5 O 8 P. C, [(2 R,3 S, 4 R, 5 R), 3, 4 dihydroxy 5 (6 oxo 1 H purin 9 yl), oxolan 2 yl], methyl dihydrogen phosphate.

Fig. 8.5

Structures of umami-tasting molecules

8.5 Trigeminal Perception Molecules

Other flavour-inducing molecules with a broad range of chemical structures are associated with trigeminal nerve perception. These sensations include pain, cooling, warming, and tingling. Pain signals are induced, most notably from capsaicin (in chilli peppers), 6-gingerol (in ginger), and piperine (in peppercorn), with the generic term “spicy” used to describe these sensations (Fig. 8.6). Because of the commercial significance of spicy flavours, the Scoville scale has been devised to quantify spiciness intensity. This scale measures spiciness in empirical Scoville Heat Units (SHU) that measure at what dilution a chilli pepper extract must be diluted until the spiciness is no longer perceivable. The higher the capsaicin concentration, the higher the SHU rating, indicating a spicier taste. Indicatively, a bell pepper has 0 SHU, a jalapeño chilli ~5000 SHU, a Bird’s eye chilli ~70,000 SHU and a Habanero chilli >150,000 SHU. For comparison, pure capsaicin is about 16,000,000 SHU, piperine ~150,000 SHU, and gingerol ~60,000 SHU.

5 chemical structures. A, (E), N, [(4 hydroxy 3 methoxyphenyl)methyl], 8 methylnon 6 enamide. B, tannins with polyphenols. C, 5 methyl 2 propan 2 ylcyclohexan 1 ol. D, [(3 S) 3 amino 3 carboxypropyl], [[(2 S, 3 S, 4 R, 5 R), 5, (6 aminopurin 9 yl), 3, 4, dihydroxyoxolan 2 yl] methyl], methylsulfanium. D, (2 E, 4 E), 5, (1, 3 benzodioxol 5 yl), 1 piperidin 1 ylpenta 2, 4 dien 1 one.

Fig. 8.6

Flavour-inducing molecules associated with the trigeminal nerve perception

The cooling sensation is most usually attributed to the presence of menthol, which binds to the temperature and pain receptors located on the tongue. For example, menthol and capsaicin bind to protein receptors associated with temperature and pain perception. Low concentrations of menthol result only in odour. However, it induces a cooling sensation at intermediate concentrations, whereas at even higher concentrations, it causes pain. Similarly, capsaicin results in a pleasant spiciness at low concentrations, but at intermediate concentrations, pain is experienced. At even higher concentrations, heat accompanied by sweating is also experienced in addition to excruciating pain. There are many molecules with similar effects, such as eugenol (cloves), cinnamaldehyde (cinnamon), eucalyptol (eucalyptus oil) or carvacrol (oregano), just to name a few.

Tannins (polyphenols) are responsible for astringency, that is, the dry-mouth sensation important in tea, coffee, and wines. The exact mechanism of astringency perception is still debated in the literature. However, current evidence shows that it is a trigeminal sensation similar to menthol or capsaicin. Finally, the effervescence sensation from carbonated beverages is also perceived by the trigeminal nerve and contributes to the flavour profile of drinks.

8.6 Flavour Characteristics and Sources

Industrial flavours are rarely a single molecule. They consist of tens or hundreds of molecules, depending on the preparation method. Generally, flavours have four characteristics that make them unique and distinguishable: the primary and secondary characters, the complexity, and the balance. The primary character is essential to recognising the target food constituting the primary component of the flavour, and it is necessary to create realistic flavour preparations. The secondary character is not critical for recognition but contributes important descriptive characteristics. For example, pure benzaldehyde rarely invokes the flavour of cherries, even though it is the main chemical of cherry flavour. However, a cherry extract is easily distinguishable, as it contains several hundred secondary compounds, making the extract flavour characteristic of cherries. The third quality of flavours is their complexity, which varies radically depending on the purpose of use. Essentially, complexity refers to the number of different chemicals that simultaneously coexist in the flavour preparation and contribute to the recognition and quality of the flavour. The level of complexity may vary from as few as 15 components in simple fruit flavours to up to 300 in the most complex flavours of processed food (e.g., Maillard reactions). Finally, flavour balance is important, as synthetic flavours may become unbalanced when a compound overwhelms the others. Mixing artificial flavours to replicate a natural counterpart is challenging, as it may easily result in unbalanced flavours. Natural flavours are usually balanced.

Flavours can be either natural, process, or artificial. The exact definitions vary depending on the legal framework of each region (e.g., FDA, EU, etc.). Regardless of the legal definitions, natural flavours are extracted from plant or animal materials and are not chemically modified further, whereas artificial flavours are obtained by chemical synthesis. Process flavours are those created during processing and refer to caramelisation, Maillard reactions, and those produced during fermentation or through enzymatic action (e.g., protein hydrolysates). The vanilla flavour can illustrate the differences between natural and artificial flavours. The natural vanilla flavour is extracted from vanilla beans and diluted in ethanol. The five main compounds contributing to vanilla flavour are vanillin, 4-hydroxy benzaldehyde, 4-hydroxy benzoic acid, vanillic acid, and 3-methoxy benzaldehyde. In addition to these five components, the extract has over 150 compounds contributing to the natural vanilla flavour. However, we may chemically synthesise only these five main molecules and mix them in ethanol in a particular proportion. The result is an artificial vanilla flavour that smells natural but only contains these five compounds. Most commercial flavours are chemically synthesised and formulated to resemble their natural counterparts rather than extracted from natural sources.

Flavours may be generated from both natural and artificial sources (Table 8.3). Plants (e.g., elderberry flower) or animals (e.g., fish oil) can be used to extract purified flavours or make flavouring preparations.

Table 8.3

Flavour sources with some typical examples

Sources

Examples

Natural

Purified extracts (e.g., vanilla extract)

Concentrates (e.g., peach purée)

Raw ingredients (e.g., crushed garlic)

Artificial

Made by chemical synthesis of either food or non-food precursors (e.g., petrochemicals).

Process

Thermal

Maillard reaction, caramelisation, protein hydrolysates

Enzymatic

Fermentation (microbial enzymes), autolysed yeast extracts

Smoke

Liquid or aerosol (i.e., real smoke)

A flavouring preparation is a product of natural origin which is not highly purified. For example, concentrated apple juice or yeast extracts are flavouring preparations. Fermentation can also provide unique flavour characteristics to food that, in most cases, are impossible to replicate using another technology. The flavour profile of fermented foods is very complex depending on the starting raw material, cultures, and fermentation conditions. Lactic acid and ethanol fermentation produce a broad range of compounds that impart to the product unique flavour characteristics (e.g., diacetyl, acetaldehyde, or acetic acid). Fat and oil hydrolysis products also give flavour to fermented foods (e.g., fruity esters or short and medium-chain fatty acids). Process flavours are generated mainly through Maillard reactions or caramelisation, as described in the previous sections. Finally, smoke flavours can be either obtained by collecting the smoke through distillation into a fluid (liquid smoke), which can then be applied in various foods (e.g., bacon) or used directly by introducing the smoke to the product from burning appropriate woods (e.g., hickory). The composition of smoke flavour depends on wood type and moisture content. It most frequently consists of complex phenolic compounds, acids, carbonyl-containing compounds, and tars (Fig. 8.7). The smoke flavour is usually regulated separately from other flavours, as it contains unidentified substances with different safety requirements.

6 chemical structures. A, 2 methoxyphenol. B, 2, 6 dimethoxyphenol. C, 2 methylphenol. D, 3 methylphenol. E, 4 methoxyphenol. F, 5, E, 2, (4 hydroxyphenyl), ethenyl, benzene 1, 3 diol.

Fig. 8.7

Structures of molecules found in smoke flavour

Plants belonging to the genus Allium (e.g., onion, garlic, shallot, leek, and chives) play a necessary role in producing flavours of savoury food that generate organosulfur compounds with strong flavours. These flavours play a central role in savoury food manufacturing, and it is pretty challenging to create such products without including flavours from Allium species. These flavours may be present in food formulations as raw ingredients (e.g., garlic paste) or very frequently as dehydrated powders (e.g., onion powder). Intact onions have very little odour, but cutting breaks cells and mixes isoalliin (substrate) with alliinase (enzyme), producing sulfenic acids and other products (Fig. 8.8). In addition, the enzyme lachrymatory factor synthase (LFS) converts sulfenic acids into a volatile molecule called syn-propanethial S-oxide responsible for tearing and the hot, pungent sensation of raw onion. Onion odours are derived from spontaneous reactions which convert sulfenic acids into thiosulphinate and other organosulfur compounds with flavour. Similar to onion, in garlic, the product of the reaction of alliin with alliinase yields allicin. Allicin is a very reactive molecule that spontaneously breaks down into other compounds, such as diallyl sulfide and diallyl disulfide. Garlic flavours degrade rapidly with heat processing.

Two reaction pathways. A, When isoalliin is treated with alliinase, it gives allyl methyl disulfide, methyl propyl disulfide, dipropyl trisulfide, and lacrymatory factor representation. B, When alliin is treated with alliinase, it gives diallyl sulfide, allyl methyl trisulfide, and diallyl trisulfide.

Fig. 8.8

Flavour generation from plants of Allium species, (a) onion and (b) garlic

Glucosinolates are natural components of many plants belonging to the Brassicaceae family, such as mustard, cabbage, or horseradish (Fig. 8.9). Glucosinolates are glycosides where the aglycone is a sulphur-containing compound. Similarly to other biochemical reactions in plants, the substrate and the enzymes are separated in the intact plant. When the plant cells are disrupted, glucosinolates come into contact with the enzyme myrosinase that hydrolyses them to isothiocyanates with pungent flavours. For example, mustard, wasabi, and radish are hot because of isothiocyanates and not capsaicin, as in chilli peppers.

A reaction pathway. One molecule of glucosinolate is converted into one molecule of glucose, one molecule of isothiocyanate, and one molecule of sulfate. The glucosinolate has myrosinase. It has a substituted oxane moiety. The molecular formula of isothiocyanate is S C N.

Fig. 8.9

Flavours derived from members of the Brassicaceae family

Lipoxygenase-generated off-flavours are those produced by the action of lipoxygenase on free polyunsaturated fatty acids (Fig. 8.10). Linoleic acid is the most common substrate in plant-based foods. Lipoxygenase has been associated with quality deterioration because of its involvement in off-flavour and odour production, loss of pigments such as carotenes and chlorophylls, and destruction of essential fatty acids.

A reaction pathway. A long chain hydrocarbon compound with C O O H at one end with molecular formula C 18 H 30 O 2 is treated with O 2 and lipoxygenase. The products are hexenal and nonadienal.

Fig. 8.10

Lipoxygenase generated off-flavours

Terpenes are hydrocarbons made up of isoprene (Fig. 7.​11), occurring widely in plants, while terpenoids are their oxygenated derivatives (alcohols, aldehydes, and ketones) (Fig. 8.11). Terpenes and terpenoids usually have 5n carbon atoms, with n being between 2 and 6, with associated differences in their class name (Table 8.4).

Twelve chemical structures. These are L carvone, D carvone, damascenone, citral, pinene, methyl chavicol, ionone, myrcene, geranyl isobutyrate, eucalyptol, linalool and limonene.

Fig. 8.11

Structures of common terpenes and terpenoids found in food

Table 8.4

Classification of terpenes and terpenoids based on the number of isoprene units in their structure. Notice that terpenes with 5 or 7 isoprene units are not usually found in food

Classification

Isoprene units

Carbon atoms

Example

Monoterpene/oid

2

C₁₀

Myrcene/Carvone

Sesquiterpene/oid

3

C₁₅

Zingeberene/Geosmin

Diterpene/oid

4

C₂₀

Abietane/Retinol

Triterpene/oid

6

C₃₀

Squalene/Cholesterol

Tetraterpenes/oid

8

C₄₀

Carotenoids

Terpenes and terpenoids are the primary constituents of essential oils, and volatile terpenes possess extremely strong character and impact the flavour properties of food. Essential oils consist of many terpenes and terpenoids that are all highly important in the uniqueness of flavours in herbs and spices. For example, limonene is the major terpene of citrus oils, but differences in the specific type and number of other terpenes in the composition result in the characteristic aroma of the oils from different botanical sources, e.g., orange, lemon, or grapefruit. Terpenes are sensitive to heat treatments and can be easily oxidised or evaporated. As a result, processes and packaging should be designed accordingly to minimise or avoid flavour losses.

8.7 Flavour Delivery

Food composition and microstructure control flavour release and perception in foods, and both need to be considered when designing foods. There are three ways to introduce a flavour into food. The flavouring can be added as a solution, emulsified, or encapsulated. When added as a solution, the flavour is dissolved into a suitable material and mixed directly in the food formulation. For example, depending on its solubility, the flavour can be dissolved in vegetable oils or animal fats, water, ethanol or glycerin and then introduced into the food. In beverages, flavour oils (e.g., orange oils) are emulsified most frequently with gum Arabic or modified starches. In this way, flavour oils that are not miscible with the aqueous phase of the formulation stay dispersed in the drink throughout the shelf life of the product. Flavours can also be encapsulated using various polysaccharides (e.g., maltodextrins or modified starches), proteins or lipids and converted into a dry powder (e.g., with spray drying). Encapsulation is quite similar in scope to emulsification, but it results in dry powder, whereas in emulsification, the protected flavour is in the liquid phase. Consequently, flavours may be dispersed uniformly in a dry form (e.g., powdered sauces). Encapsulation can protect flavours from other food ingredients and environmental conditions like heat, moisture and acidity that cause degradation.

Flavours should also be released from the food in a controlled manner to avoid flavour saturation at the beginning of eating and flavour depletion in the subsequent stages of oral processing. This process is termed flavour release, which refers to the release of flavour components from the food structure at a specific rate. Flavour encapsulation technologies can be used to modulate perception by generating flavour impact and persistence where needed. It is essential in products that chewing is sustained for a prolonged time and can be nicely illustrated with chewing gums. Premium chewing gums allow menthol release for extended periods (even hours) after the first bite. In lower-quality products, the flavour may be depleted within the first 5 min of mastication. In liquid beverages or snacks, controlled release creates an aftertaste, i.e., a taste remaining in the mouth after swallowing. In foods, aftertaste is more important than prolonged flavour release, as the oral residence time of the bolus is less than a minute for solid foods and just a few seconds for liquid products. In these cases, the flavour is released by the degradation of the encapsulating material. However, the encapsulating material (e.g., polysaccharide) may interact strongly with the oral tissues and release flavour sustainably even after swallowing. This is typical of flavoured snacks (e.g., chicken-flavoured crisps or cheese-flavoured crackers), where the aftertaste may last for particularly long times even after consuming a single segment of the product.

Encapsulation

Encapsulation is the process that entraps active components (e.g., flavours, vitamins or drugs) within a carrier material known as the wall. The wall and the active form supramolecular complexes are stabilised by non-covalent interactions (e.g., hydrogen bonds, ionic or hydrophobic interactions or a combination). The supramolecular complexes form particles of micrometres (microencapsulation) or nanometres (nanoencapsulation) in size. Suitable wall materials for food applications are carbohydrates, proteins, or lipids. There are many encapsulation applications in food processing, resulting in food with higher nutritional value, a lower dose of synthetic preservatives, and better sensory properties of the product. For instance, it can be used to enhance the stability of sensitive compounds during production, storage, and ingestion (e.g., vitamins or drugs), decrease evaporation and degradation of volatile flavours, taste masking (e.g., astringency of polyphenols) or protect unsaturated fatty acids from oxygen, water, or light. Microscopy image reproduced with permission from Parris, Cooke, Hicks, J. Agric. Food Chem., 2005, 53, 4788–4792

A diagram of two concentric circles. The inner circle is flavor and it is surrounded by a layer of wall. A S E micrograph presents the encapsulated oregano oil in Zein particles. A cluster of spherical particles is presented.

8.8 Flavour Interactions and Stability

During processing and storage, flavours interact with other food components in many ways with frequently unpredictable consequences. While it is impossible to describe all the interactions between flavours and food components individually, some general guidelines may be drawn.

Proteins bind aldehydes and ketones through hydrophobic interactions, whereas alcohols are bound both hydrophobically and through hydrogen bonds. Most of the time, binding is reversible, so release during mastication is ensured. Noncovalent, reversible flavour binding needs time until an equilibrium is established. During food formulation, the product is frequently left stored for a few days, and the flavour is reevaluated to assess the flavour partitioning into the hydrophobic regions of the protein. Once the equilibrium between bound and free flavour has been established, reformulation may occur until the desired flavour characteristics are obtained. In the case of covalent interactions that may occur during heat processing, parts of flavour molecules may react with proteins irreversibly. Consequently, the flavour strength is diminished, and reformulation will also be needed. Occasionally, the irreversible binding of off-flavours may be advantageous as the undesired compounds can no longer be detected.

Lipids play a crucial role in the flavour and texture profile of foods and may also influence their characteristics by interacting with the other components. The interactions of flavours with lipids depend on their content, chain length, degree of saturation and the solubility of the flavour component in the lipid phase. Reducing the lipid content can significantly influence its overall flavour profile, even if the changes are minimal. This occurs because lipids modify substantially the vapour pressure of the fat-soluble flavour compounds (i.e., the way they evaporate). In lipid-rich foods, flavour compounds are distributed between the lipid and the aqueous phase, depending on their solubility. Ultimately, the solubility of flavours in water and oil depends on their hydrophobicity. As a result, hydrophobic flavours have a greater capacity to dissolve into triglycerides than hydrophilic. In addition, the flavour-binding capacity of fats is lower than that of oils as more flavour can diffuse into a liquid than solid lipid. The binding capacity of unsaturated lipids is greater than saturated. This occurs because the double bonds in unsaturated fatty acids create kinks in the carbon chain, allowing more flavour compounds to interact and bind with lipids. TAGs with long-chain fatty acids tend to bind less flavour than those with short-chain fatty acids, but as mentioned above, the chain length primarily influences flavour solubility. Longer chain triglycerides can retain hydrophobic flavour compounds, reducing their release into the mouth. Depending on the application, this can lead to delayed flavour perception and a slower release, which may be desirable or undesirable.

Polysaccharides can modify flavour release mainly due to viscosity modification and interactions of flavours with the polysaccharide chains. The interactions are especially hydrogen bonding due to the large number of -OH groups in polysaccharides. For example, maltodextrins interact with alcohols stronger than esters and ketones. In starch, volatile flavours can be entrapped in the helical structure of amylose, as the interior of the amylose helix is hydrophobic and can interact strongly with hydrophobic flavours. Starches with low amylose content (e.g., tapioca with ~17% amylose) and waxy starches consisting only of amylopectin have a weak binding capability. Those with a higher amylose content (like potato or maize) show greater interaction with flavours. Amorphous sugars (e.g., hard candy) generally retain more flavour than crystalline sugars. Finally, In some fruit-based products, sucrose can help preserve the volatile fruit flavours by reducing aw and preventing volatile loss through evaporation.

Finally, flavours have stability problems, particularly in the presence of oxygen and light, as they may be easily oxidised and lose intensity or change the product’s flavour profile. For example, certain plastic packaging materials have high permeability to O₂. In addition, flavour molecules may also diffuse in or out of the packaging, resulting in flavour changes during storage. Volatile components (e.g., aldehydes or ketones) may evaporate, or flavour oils may physically separate (e.g., oil separation). Reactions with other food ingredients may also occur, reducing the strength and may even alter the unique character of the flavour. For example, evaporation may occur during processing, such as in heat treatments or vacuum packaging.

8.9 Review Questions

8.9.1 Multiple Choice Questions

1.

A sweet-tasting molecule is required to satisfy the following conditions:

(a)

To have a weak base, an electronegative group and a hydrophobic group

(b)

To have a weak acid, an electronegative group and a hydrophobic group

(c)

To have a weak acid, a hydroxyl group and a hydrophobic group

(d)

To have a weak acid, a hydroxyl group and an amino group

2.

Bitter tasting molecules have:

(a)

One polar and one non-polar group in their structure

(b)

One polar and two non-polar groups in their structure

(c)

Two polar and two non-polar groups in their structure

(d)

Two non-polar groups in their structure

3.

Bitter-tasting molecules are structurally diverse, mostly being:

(a)

Alkaloids, glycosides, peptides

(b)

Glycosides, ketones, acids

(c)

Alkaloids, amino acids, aldehydes

(d)

Peptides, acids, xanthines

4.

Alliinase in onions catalyses the conversion of:

(a)

Isoalliin to thiosulfinate

(b)

Alliin to thiosulfinate

(c)

Isoallicin to thiosulfinate

(d)

Alicin to thiosulfonate

5.

Myrosinase is the enzyme responsible for flavour generation in:

(a)

Allium family

(b)

Brassicaceae family

(c)

In both Allium and Brassicaceae families

(d)

Nutmeg to convert myristic acid to aldehydes with odour

6.

The stereochemistry (i.e., d-, l- configurations) may change how a molecule smells. This statement is:

(a)

True

(b)

False

7.

Most essential oils consist of limonene, pinene and myrcene in different proportions, resulting in different smells. This statement is:

(a)

True

(b)

False

8.

Smoke flavours are chemically:

(a)

Phenolic compounds

(b)

Aldehydes

(c)

Terpenes

(d)

Pinenes

9.

Quinine tastes

(a)

Sweet

(b)

Salty

(c)

Bitter

(d)

Umami

10.

Guanosine 5΄-monophosphate tastes:

(a)

Bitter

(b)

Salty

(c)

Sweet

(d)

Umami

8.9.2 Short Answer Questions

1.

Describe the AH-B-γ theory of sweetness. What are the classes of sweet-tasting molecules?

2.

What is the difference between glucose syrup, HFCS, and high-maltose syrups?

3.

What is a common structural feature of sweet peptides?

4.

Discuss why we must blend sweeteners to achieve the ideal sweetness instead of using only one.

5.

What are the classes of sweet-tasting molecules? Name two molecules from each class.

6.

What are the main molecules responsible for the umami taste?

7.

What are the main molecules that induce a trigeminal perception? Identify which sensation each molecule causes.

8.

Discuss the main characteristics of flavours.

9.

Describe the main reactions during flavour generation in Allium species and the Brassicaceae family.

10.

Draw the structure of limonene, α-pinene, citral, geraniol, and linalool. What is the difference between terpenes and terpenoids? Which are terpenes or terpenoids from the structures you have just drawn? Identify the isoprene chain in citral, geraniol, and linalool. How many isoprene units can you count?

11.

Discuss the methods we use to deliver flavour in food formulations.

12.

Discuss the interactions between flavours and proteins, polysaccharides and lipids.

13.

Discuss technical problems with flavours related to their chemical and physical stability.

14.

A savoury snack contains “yeast extract” and “malt extract”. What is the role of these ingredients in the formulation? How are they produced, and what do they most likely taste?

Appendices

Chapter 1 – Water

1d, 2c, 3d, 4b, 5a, 6b, 7c, 8a, 9c,10a, 11b, 12c, 13a, 14b, 15d

Chapter 2 – Carbohydrates

Monosaccharides: 1d, 2a, 3a, 4a, 5b, 6c, 7b, 8a, 9a, 10b

Polysaccharides: 1a, 2c, 3a, 4d, 5a, 6a, 7b, 8c, 9c, 10c

Chapter 3 – Proteins

Proteins: 1a, 2b, 3b, 4b, 5c, 6a, 7a, 8c, 9b, 10a

Enzymes: 1a, 2b, 3c, 4d, 5a, 6b, 7c, 8d, 9a, 10a

Chapter 4 – Lipids

1b, 2b, 3d, 4a, 5d, 6c, 7a, 8a, 9a, 10a

Chapter 5 – Browning

1a, 2b, 3c, 4b, 5d, 6a, 7a, 8c, 9a, 10b

Chapter 6 – Vitamins-Minerals-Additives

1a, 2a, 3a, 4b, 5d, 6c, 7a, 8a, 9d, 10a

Chapter 7 – Colour chemistry

1a, 2a, 3a, 4b, 5b, 6a, 7b, 8a, 9b, 10a

Chapter 8 – Flavour chemistry

1b, 2a, 3a, 4a, 5b, 6a, 7b, 8a, 9c, 10d

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Index

A

Acceptable daily intake (ADI)127

Accretion96

Acesulfame potassium150

Acetal22

Acrylamide110

Actin65

Activation energy69

Active site69

Additives125

Aglycone27

AH-B-γ theory148

Albumen64

Albumins53

Aldaric acid25

Alditol27

Aldonic acid24–25

Aldoses19

Alginate37

Alkaloids152

Allicin157

Alliinase156

Allosteric inhibition73

α-amylase

α-helix

α-lactalbumin

α-relaxation

Amadori compounds110

Amadori rearrangement110

Amidated pectin39

Amino acid48

Aminopeptidases78

Amphoteric50

Amyloglucosidase75

Amylopectin32

Amylose32, 33

Anomeric carbon22

Anthocyanidins140

Anthocyanins140

Antioxidants92

Apo-enzyme70

Artificial colours143

Ascorbic acid119

Ascorbic acid browning109

Aspartame150

Auto-oxidation91

Axial23

Azo-dyes144

B

Bacteriocins51

Bathochromic shift133

Beer-Lambert’s law133

β-amylase

β-conglycinin

Betacyanins143

β-glucosidase

β-lactoglobulin

Betalains143

Betalamic acid143

β-pleated sheets

β-relaxation

β-strands

Betaxanthins143

Biocytin122

Biotin122

Bitter152

Blooming98

Bloom strength66

Boat22

Brightness133

C

Caffeine152

Capsaicin154

Caramel colours106

Caramel flavours106

Caramelisation106, 107

Carbinol pseudobases141

Carboxypeptidases78

Carotenes139

Carotenoids139

Carrageenan36

Casein62

Casein micelle62

Catalyst68

Cellulases76

Cellulose39

Cellulose derivatives39

Ceramides89

Cerebroside89

Chair22, 96

Chelators92

Chiral carbon20

Chitin40

Chitosan40

Chlorophyll138

Chlorophyllase138

Cholecalciferol118

Cholesterol91

Chromophore135

Chymosin78

Cis-87

Coagulation58

Cobalamin122

Co-enzyme70

Co-factor70

Collagen66

Colligative properties4

Colour131

Colour difference134

Colour spaces134

Competitive inhibition73

Complementary colour135

Conformation22

Conformers22

Conjugated double bond87

Conjugated proteins53

Conjugated system135

Crystallisation95

C-terminal51

Cyanocobalamin122

Cystine51

D

Degree of esterification39

Degree of polymerisation31

Denaturation57, 59

Dextrorotatory20

Dextrose20

Dextrose equivalents36

Diacylglycerol88

Diastereoisomerism20

Diazene144

Dietary fibre39, 41

Diffuse reflection132

Dipeptide51

Diphenol oxidase104

Disaccharide30

Disulfide bond51

Disulfide bridge51

E

EC number74

Egg-box model37

Enantiomerism20

Enantiomers20

Endoacting enzymes75

Enediol25

Enol24

E numbers128

Enzymatic browning103

Enzyme68

Enzyme affinity72

Enzyme inhibitors73

Enzyme kinetics71

Epimers20, 22

Equatorial23

Ergocalciferol118

Esterification24, 88

Exoacting enzymes75

F

Fat85

Fat-soluble vitamin117

Fatty acids85

Fibrous proteins53

Fischer projection22

Flavonoids141

Flavylium cation140

Folding54

Folic acid122

Food enzymes74

Free radical91

Freeze-concentrated matrix13

Fructans30

Furanose22

G

Galactomannan39

Ganglioside89–90

Gelatin66

Gelatinisation34, 35

Gelation58

Glass transition10

Glass transition temperature12

Glass transition zone12

Glassy12

Gliadin53, 65

Globular proteins53

Globulins53

Gloss reflectance132

Glucaric acid25

Glucoamylase75

Gluconic acid25

Glucono delta-lactone25

Glucose isomerase76

Glucose syrup36, 76, 150

Glucosinolates152, 157

Glutelins53

Gluten-free35

Glutenin53, 65

Gluten network65

Glycinin64

Glycophore148

Glycoproteins53

Glycoside27

Glycosidic bond27

Glycyrrhizin151

Gum Arabic40

H

Haem137

Haemoproteins53

Haworth projection22

Heat-stable enzymes73

Heat-stable proteins57

Hemiacetal22

Hemicelluloses41

Hexagonal ice2

High amylose starch34

High fructose corn syrup (HFCS)76, 149, 150

High methoxy pectins39

Hofmeister series60

Holoenzyme70

Homogalacturonan38

Hue133

Hydrogenation100

Hydrogen bond2

Hydrolysis60

Hydrolytic rancidity79, 94

Hydroperoxides91

Hydrophobic interactions4

Hydrophobic moieties57

Hydroxymethyl furfural107

Hypsochromic shift133

I

Imine51

Induced fit theory70

Initiation91

Interesterification101

International Numbering System for Food Additives (INS)127

Inulin30

Invertase77

Invert sugar30, 77

Iso-amylases76

Isoelectric point50

Isoenzyme69

Isoprene rule140

K

Ketosamines110

Ketoses19

L

L*a*b*134

Lactase77

Lactone25

Lactose30

Lactose free77

Lake144

Lecithin89

Legumes64

Legumin fraction64

Levorotatory20

Ligands124

Lignin41

Lipases78

Lipid oxidation91

Lipids85

Lipolysis94

Lipoproteins53

Lipovitellenin65

Lipovitellins65

Lipoxygenases79

Loop54

Low methoxy pectins39

Lyotropic series60

M

Macro-minerals123

Maillard reaction109, 110

Maltodextrin36

Maltol107

Maltose30

Maltose syrup76

Margarine99

Melanins104

Melanoidins111

Melting point97

Metal-activated enzymes70

Metalloenzyme70

Metalloproteins53

Metmyoglobin137

Michaelis-Menten constant71

Milk62

Mineral123

Modified starch24, 32, 34

Moisture migration10

Moisture sorption isotherm8

Molar extinction coefficient133

Molecular mobility8

Monoacylglycerol88

Monomolecular layer8

Monophenol oxidase104

Monosaccharide19

Monosodium glutamate154

Monounsaturated86

Motif54

Mucoproteins53

Mutarotation23

Myofibrillar proteins65

Myoglobin137

Myosin65

Myrosinase157

N

Naphthoquinones118

Naringin79

Naringinase79

Niacin119

Non-competitive inhibition73

N-terminal51

O

Oil85

Oligomer55

Oligosaccharide30

Omega fatty acids86

Optical activity20

Osborne protein classification53

Osmosis5

Ovalbumin64

Ovomucoid64

Ovotransferrin64

Oxidative rancidity91

Oxymyoglobin137

P

Pantothenic acid120

Pasting curve34

Pectic enzymes77

Pectin38

Pectinase77

Pectin methyl esterase77

Peptide bond51

Peroxidases79

Phosphocholine89

Phospholipases79

Phospholipids89

Phosphoproteins53

Phosvitin65

Photo-oxidation91

Physical ageing12

Phytic acid124

Phytochemicals124

Phytoestrogens141

Phytol138

Phytosterols91

Plasticiser14

Polarity2

Polygalacturonase77

Polymorphic transition97

Polymorphism97

Polypeptide51

Polyphenol oxidase79, 103

Polysaccharide31

Polyunsaturated86

Porphyrin ring138

Pregelatinised starch35

Primary structure54

Processing aid126

Prolamin53

Pro-oxidants91

Propagation91

Prosthetic group70

Proteases78

Protein52

Protein hydrolysates154

Pullulanase76

Pyranose22

Pyridoxine120

Q

Quaternary structure55

Quinonoidal bases141

R

Random coils55

Reaction velocity71

Recrystallisation96

Reducing sugars27

Rennin78

Resistant starch35

Retinol118

Retrogradation34

Rhamnogalacturonan-I38

Riboflavin119

S

Salting in60

Salting out60

Salty152

Schiff base51, 110

Scoville scale154

Secondary structure54

Selectivity70

Shortening99

Singlet oxygen93

Smoke flavours156

Solid fat content100

Solubility57

Sour153

Specificity70

Specular reflection132

Sphingoid bases89

Sphingolipids89

Sphingomyelin89

Sphingosine89

Stanols91

Starch32, 33

Starch granule32

Starch paste34

Stereoisomerism20

Stereospecific numbering89

Steric hindrance22

Sterols90

Stevia151

Steviol glycosides151

Storage proteins53

Strecker aldehydes110

Strecker degradation110

Substrate69

Sucrose30

Sulfonamide150

Surimi79

T

Tautomer24

Tautomerism24

Tempering97

Termination91

Terpenes158

Terpenoids158

Tertiary structure55

Thaumatin150

Theobromine152

Thiamine119

Tocopherols118

Tocotrienols118

Trace elements123

Trans-87

Transglutaminases79

Triacylglycerols (TAGs)88

Trigeminal nerve154

Triplet oxygen93

Tropomyosin66

Troponin66

Tuning fork96

Type-A gelatin66

Type-B gelatin66

U

Umami154

Unit cell95

Uronic acid26

V

Vicilin fraction64

Visible reflectance spectroscopy133–134

Vitamin A118

Vitamin B119

Vitamin C119

Vitamin D118

Vitamin E118

Vitamin K118

Vitrification12

W

Water activity6

Water-soluble vitamin117

Waxy maize34

Whey proteins62

X

Xanthan40

Xanthophylls139

Z

Zwitterion50