Implementation of Enzymatic Processes and Lactic Bacteria in the Food Industries E-Book

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Veröffentlichungsjahr: 2024

Beschreibung

Consumers are demanding healthy, natural food products with no environmental impacts. The use of ingredients of plant origin and the implementation of bioprocesses using enzymes and micro-organisms as biocatalysts represent a promising alternative to satisfy this demand.

Implementation of Enzymatic Processes and Lactic Bacteria in the Food Industries focuses on describing the latest developments in the use of enzymatic biocatalysts and lactic acid bacteria in the food industry.

The first part of the book is devoted to the presentation of different classes of enzymes, production and application processes, ways of improving enzymes and the main industrial applications using biocatalysts.

The second part of the book describes a family of micro-organisms widely used in health food processing and formulation: lactic acid bacteria and bifidobacteria. Then, the most commonly used encapsulation matrices, encapsulation processes and the process of bacterial adhesion to these matrices are discussed. Finally, the best-known fermented foods and new approaches developed in this field are presented.

Leseprobe

SCIENCES

Agronomy and Food Science,Field Directors – Jack Legrand and Gilles Trystram

Food Bioprocesses, Subject Head – Mohamed Ghoul

Implementation of Enzymatic Processes and Lactic Bacteria in the Food Industries

Coordinated by

Mohamed Ghoul

First published 2024 in Great Britain and the United States by ISTE Ltd and John Wiley & Sons, Inc.

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ERC code:LS9 Applied Life Sciences, Biotechnology, and Molecular and Biosystems Engineering LS9_5 Food sciences (including food technology, food safety, nutrition)

Preface

Mohamed GHOUL

LRGP, CNRS, Université de Lorraine, Nancy, France

To meet growing consumer demand for healthy, natural, additive-free products with low environmental impact, plant-sourced raw materials have emerged as a relevant alternative to derivatives from the petrochemical industry. However, most of these raw materials are complex and difficult to digest as they are. They therefore require numerous physicochemical and mechanical processing, as well as the implementation of numerous processing reactions.

The use of enzymes and micro-organisms as catalysts to hydrolyze, condense and/or fractionate these raw materials is well adapted. Enzymatic processes enable processes to be implemented under mild conditions and targeted molecules to be produced. Similarly, given the selectivity and specificity of these catalysts, separation and/or purification operations downstream of the production stages are simpler and less costly.

This book describes the latest developments in the use of biocatalysts in the food industry.

Part 1 is dedicated to a presentation of current enzyme processes in the food industry (general characteristics of enzymes, classifications and operating modes of the main enzymes used in the food industry, enzyme production for food applications), ways of improving enzyme processes (enzyme engineering, process engineering) and the main enzyme processes in the food industry (bakery and confectionery, malting and brewing, starch and its derivatives, milk, fats, meat and seafood products, flavors and additives, fruit juices and wines).

Part 2 begins by describing a family of micro-organisms that are widely used in food processing and in the formulation of healthy foods. These are lactic acid bacteria and bifidobacteria (taxonomic concepts and definition of “probiotic”, probiotic market and health claims, production of probiotics and metabiotics, industrial applications, development and innovation prospects, impact of lactobacilli on digestive health, etc.). Secondly, we describe the processes used to encapsulate lactic acid bacteria (atomization, extrusion, emulsification, coating), the most commonly used encapsulation matrices and the process by which bacteria adhere to these matrices. Finally, we present the best-known fermented foods and the new approaches under development in this field.

January 2024

Introduction

Mohamed GHOUL

LRGP, CNRS, Université de Lorraine, Nancy, France

The raw materials used in the various production processes for foodstuffs and non-food active ingredients have a wide variety of structures (cellulose, hemicellulose, lignin, proteins, phenolic compounds, fats, etc.). This structural complexity means that some of these materials cannot be directly assimilated by the consumer, and do not have the desired biological activities and functionalities (nutrition, health, nutraceuticals, etc.).

Digestion and transfer of the active ingredients of these raw materials through cell walls, and/or their functionalization, therefore require one or more hydrolysis and rearrangement stages, which involve several types of reactions (synthesis, hydrolysis) that are catalyzed in vitro or in vivo by complex enzyme systems. These enzyme systems can either be made up of enzymes or enzyme cocktails extracted from plant, bacterial or animal matrices through the application of appropriate unit operations, or through the use of whole cells (bacteria, yeast, fungi, etc.).

To ensure that these catalytic systems (extracted enzymes or whole cells) operate under optimum conditions, a number of factors (pH, temperature, concentration, redox potential, oxygen level, etc.) need to be carefully adjusted. Similarly, in order to preserve and maintain the activity of certain catalytic systems over a long period of time, it is necessary to carry out a protection step, such as encapsulation or immobilization. These protective operations are carried out by applying the appropriate matrices, enabling adequate transfer of substrates between the macro- and micro-environment of the catalytic system, thus maintaining the required catalytic activities and functionalities.

Various sources and classes of enzymes and bacteria exist naturally, and are already used effectively in many food and non-food industries. However, to enhance the catalytic activities of these enzymes and micro-organisms, a great deal of work is regularly devoted to discovering new bacteria and enzymes, as well as improving their catalytic activities. This work either uses classical selection, cloning and mutation tools, or tools involving rational design through the application of various molecular modeling approaches.

Part 1 of this book is devoted to a presentation of (i) the different classes of industrial enzymes; (ii) enzyme production techniques; (iii) current and future avenues for enzyme improvement; (iv) techniques for intensifying enzymatic processes; and (v) a description of the main industries using industrial enzymes.

Part 2 of this book is dedicated to the description of a group of bacteria that are widely used in food processing, namely, lactic acid bacteria. A general presentation of these bacteria is given, followed by a more detailed discussion of the bifido-bacteria subgroup.

In order to maintain and prolong their activity, these bacteria can be encapsulated using a variety of techniques. The encapsulation techniques best suited to lactic acid bacteria that are used on an industrial scale are described, as well as bacteria–matrix interactions and their role in retaining bacteria in microparticles, during storage and gastrointestinal transit. Lastly, the most common industrial applications involving lactic acid bacteria are presented.

PART 1Enzymatic Processes in the Food Industry

Introduction to Part 1

Isabelle CHEVALOT

LRGP, CNRS, Université de Lorraine, Nancy, France

Enzymes are biological catalysts that regulate the metabolic pathways of all living systems. They are globular proteins that act as catalysts, accelerating the speed of a reaction by lowering the energy of activation. Unlike chemical catalysts, enzymes are of natural origin, operate under mild conditions of temperature and pressure, exhibit high specificity and are biodegradable (Subin et al. 2015). Due to these main characteristics, enzymes have commercial applications in a variety of industries. They have been used for millennia in food processing, such as bread making, brewing, cheese and wine making. However, the large-scale introduction of enzymes into manufacturing processes in the 1970s paved the way for the development of numerous food processing and additive productions (Mishra et al. 2017). The industrial enzyme market was estimated at 5.9 billion USD in 2020, and is expected to reach 8.7 billion USD by 2026, with a growth rate of 6.5% in terms of value1. Growing environmental concerns and advances in enzyme engineering and green chemistry have boosted their industrial use. Technological innovations that reduce the consumption of chemical products are contributing to market growth. Industrial enzymes are widely used in the food and beverage industry, but also in pharmaceuticals, textiles, animal feed, biofuels, cosmetics, detergents and paper. Overall, human and animal nutrition applications account for between 55% and 60% of the global enzyme market, with an estimated annual growth between 6% and 8%. Half of the enzyme market (around 50%) concerns the category of enzymes catalyzing carbohydrate hydrolysis (mainly amylases and cellulases/hemicellulases). Proteolytic enzymes account for 25–30% of the global enzyme market, while lipases represent 10% of the global enzyme market (Guerrand 2017).

In the food industry, enzymes are mainly used as additives in foods, or as processing aids in the production of foods and beverages, to improve texture, flavor and quality, as well as to extend shelf life. Enzymes also have applications as analytical tools in the food industry. However, enzyme activity and stability are influenced by operating conditions, such as pH, temperature, substrate concentration and the presence of metal ions. Enzymes require pH and temperature conditions to maintain optimum activity and stability, which may not entirely correspond to industrial process conditions. Furthermore, the reactions and substrates of interest to industry may differ from the enzyme’s natural reactions and substrates. In order to overcome these difficulties and offer high-performance catalysts on a large scale, several strategies are being considered, based either on the process itself or on the biocatalyst.

Recent developments in enzyme biotechnology, nanobiotechnology, metagenomics and proteomics have contributed to the design and development of tailor-made enzymes, with new properties and catalytic activities. There have also been numerous innovations in enzyme immobilization media. Enzymes can be used in free or immobilized form, for example, bound to an inert support, so as to enable repeated and/or continuous use of the enzyme, and also to increase their stability. Implementing a process based on immobilized enzymes on an industrial scale requires careful assessment; indeed, in addition to potential technical problems, such as loss of activity during immobilization and mass transfer limitations, it is necessary to consider the economic impact of using immobilized enzymes, including the cost of immobilization, the cost of the immobilization media and the chemical compounds used for the immobilization procedure (Sheldon and van Pelt 2013). More recently, processes have been developed, involving microwave and/or ultrasonic technologies, to increase catalytic efficiency. This intensification of biocatalytic processes is also conceived through the design of new micro-structured reactors.

Recent advances in biocatalysts and processes have played an important role in the discovery and development of new enzymes, as well as in the implementation of new processes for food and beverage processing, and co-product valuation.

Today, the integration of enzymes in food processing is well known, and research dedicated to improving enzymes and optimizing the processes that use them is still in progress. The aim of Part 1 of this book is to provide an up-to-date review of the use of enzymes in food processes, their production, enzyme engineering methods for their improvement and biocatalytic process intensification technologies.

Note

1

MarketsandMarkets.com

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1General Characteristics of Enzymes

Isabelle CHEVALOT1, Mohamed GHOUL1 and Seraphim PAPANIKOLAOU2

1 LRGP, CNRS, Université de Lorraine, Nancy, France

2 LFMB, Agricultural University of Athens, Greece

Enzymes are proteins that speed up chemical reactions. This acceleration process is called catalysis, and enzymes act as catalysts for chemical reactions. In enzymatic reactions, the molecules present at the start of the reaction are called “substrates”. Enzymes transform the substrates into different molecules called “products”. As with all catalysts, enzymes are not consumed by the reactions they catalyze and do not alter the equilibrium of these reactions. However, enzymes differ from most other catalysts by being much more specific. Numerous works detail the structure, functions, mechanisms, kinetics and technologies of enzyme catalysts (Pelmont 1997; Buchholz et al. 2005; Cornish-Bowden et al. 2005; Illanes 2008).

1.1. Notion of catalysis

Two factors are specifically involved in the rate laws for a chemical transformation: the pre-exponential factor (A) and the activation energy (Ea). The classical expression for the rate constant is:

[1.1]

This constant is only valid for a given system. From a set of reagents, through an appropriate reaction pathway, the system evolves toward the formation of products. Ea corresponds to the height of the potential barrier (Figure 1.1).

Unlike thermodynamics, which does not consider the path followed (the principle of initial and final states), chemical kinetics is largely dependent on it. By modifying this path, the potential barrier is altered. If Ea increases, the reaction-rate constant decreases and vice versa.

The principle of enzymatic catalysis is related to a reduction in the energy required for the reaction (Figure 1.1). The activation energy corresponds to the amount of energy absorbed by the substrate molecules required to move to an unstable transition or activated state, in which bonds are more fragile and easier to break. The transition state is located at the top of the energy barrier.

The enzyme reduces the activation energy by creating an environment in which the transition state is stabilized.

Figure 1.1.Schematic representation of the role of enzymes as biological catalysts, which act by lowering the activation energy of reactions, enabling the formation of reaction intermediates with lower activation energy.

1.2. Notion of specificity

One of the most remarkable properties of enzymes is their specificity. Some enzymes have a very strict specificity, only catalyzing one particular reaction. Other enzymes will be specific to a particular type of chemical bond or functional group.

This specificity is directly related to the structure of enzymatic proteins. Indeed, native proteins fold into a unique functional tertiary conformation that confers their biological activity, which may be that of a catalyst. This overall structure of the macromolecule enables a particular region to adopt a spatial structure, recognized by the protein’s specific ligand. In the case of enzymes, this particular region is known as the active site. Within the active site, a distinction is made between amino acids that constitute the binding site (these amino acids have no chemical functions involved in the reaction) and amino acids that constitute the catalytic site. The active site is made up of a small number of amino acids which, in most cases, are not contiguous in the polypeptide chain. These amino acids are characterized by side chains, whose chemical nature (ionizable groups) and structure (steric hindrance) are specifically adapted to substrate recognition. Enzyme specificity is determined by the complementary form, charge, hydrophilic/hydrophobic nature of the substrates and their three-dimensional structure.

Three types of specificity can be distinguished as follows:

Chemo-specificity

: it includes enzymes that are specific to certain functional groups, such as the amino, phosphate or methyl groups.

Regio-specificity

: it includes enzymes that are specific to a functional group, depending on its position within the structure of the molecule.

Stereo-specificity

: it includes stereospecific enzymes that only act on a steric or optical isomer and not on their homologous isomers.

1.3. Nomenclature

The International Commission on Enzymes, founded in 1955, has established an enzyme nomenclature system based on the type of reaction each enzyme catalyzes. Under this system, enzymes and coenzymes, their units of activity, standard assay methods and the symbols used to designate reaction kinetics are all grouped together in a single system. All enzymes are assigned a four-digit number according to the class, subclass and sub-subclass in which they have been classified. Each enzyme has been assigned a code number, consisting of four digits separated by dots. The first digit indicates the main class to which the enzymes belong:

Oxidoreductases

catalyze redox reactions in which hydrogen or oxygen atoms, or electrons are transferred between molecules. This extended class includes dehydrogenases (hydride transfer), oxidases (electron transfer to molecular oxygen), oxygenases (oxygen transfer from molecular oxygen) and peroxidases (electron transfer to peroxide). The second digit of the code indicates the reducing equivalent donor involved in the reaction. For example, glucose oxidase (EC 1.1.3.4 or β-D-glucose: oxygen 1-oxydoreductase), laccase (EC 1.14.18.1) and lipoxygenase (EC 1.13.11.12) are major enzymes of this class used in the food industry.

Transferases

catalyze the transfer of an atom, or group of atoms, between two molecules, apart from certain enzymes included in other groups (such as oxidoreductases and hydrolases). The International Commission recommends that the names of transferases end with X-transferase, where X is the transferring group. For example, glucanotransferase (EC 2.4.1.19) is used for the modification of starch into cyclodextrins. The second digit describes the transferred group.

Hydrolases

include enzymes catalyzing the hydrolytic cleavage of bonds, such as C – O, C – N, C – C, and a few others. They are classified according to the type of hydrolyzed bond. They are currently the most common class of enzymes used in enzyme technologies. In the food industry, the majority of enzymes classified as such include α-amylases (EC 3.2.1.1), β-amylases (EC 3.2.1.2), lactases (EC 3.2.1.23), lipases (EC 3.1.1.3) and proteases, including aminopeptidase (EC 3.4.11), trypsin (EC 3.4.21.4), subtilisin (EC 3.4.21.62), papain (EC 3.4.22.2), ficin (EC 3.4.22.3), pepsin (EC 3.4.23.1) and chymosin (EC 3.4.23.4).

Lyases

are involved in the non-hydrolytic elimination of certain groups by breaking various chemical bonds. This elimination often generates double bonds. The second digit in the classification indicates the broken bond. These include aldolases, decarboxylases and dehydratases. For example, acetolactate decarboxylase (EC 4.1.1.5) is used in the beer industry.

Isomerases

are enzymes that can catalyze various molecular isomerization reactions. These enzymes catalyze geometric or structural changes within a molecule. Depending on the type of isomerism involved, they may also be referred to as racemases, epimerases, cis-trans isomerases, isomerases, tautomerases, mutases or cycloisomerases, such as phosphoglucose isomerase (EC 5.3.1.9).

Ligases

, also known as synthetases, catalyze the synthesis of new bonds between two molecules. They link molecules by covalent bonds in biosynthetic reactions. These reactions require energy through hydrolysis of an ATP diphosphate bond or a similar triphosphate: this property justifies the difficulty of their application on an industrial scale. The second digit indicates the type of bond synthesized.

The second digit designates the enzyme sub-class, defined according to its mechanism of action. The third digit designates the nature of the molecule acting as acceptor in the case of electron transfer. The fourth digit is a sequence number within the group and sub-group. When an enzyme ends in 99, it is incompletely characterized.

Table 1.1 shows the six classes, the type of reaction catalyzed by the enzyme and an example for each class.

Table 1.1.Enzyme nomenclature

No.

Classes

Reaction

Examples

1

Oxidoreductases

Oxidation-reduction reactions

Glucose oxidase (EC 1.1.3.4)

2

Transferases

Functional group transfer

Hexokinase (EC 2.7.1.2)

3

Hydrolases

Hydrolysis reactions

Carboxypeptidase A (EC 3.4.17.1)

4

Lyases

Addition of double bonds

Pyruvate decarboxylase (EC 4.1.1.1)

5

Isomerases

Isomerization reactions

Malate isomerase (EC 5.2.1.1)

6

Ligases

Bond formation (C–C, C–S, C–O and C–N) with ATP cleavage

Pyruvate carboxylase (EC 6.4.1.1)

1.4. Mechanism of enzyme catalysis

Understanding the mechanism of enzyme catalysis is essentially based on the identification of the amino acids making up the enzyme structure, and their functional groups directly involved in catalysis. Methods for the determination of these active groups are based on the determination of the enzyme’s primary structure, as well as on the analysis of changes in the enzyme resulting from variations in various environmental factors, such as pH or temperature, the use of reagents specific to certain functional groups or structural analogs of the substrate.

The speed of an enzymatic reaction depends on the efficiency of the collisions between the substrate and enzyme molecules that will form the transition state. This efficiency depends on the orientation of the molecules in relation to one another and on a minimum energy required, which is the activation energy, as described above.

There are two main types of catalytic mechanisms:

General acid-base catalysis: this is the most common mechanism in enzyme catalysis. The reaction is accelerated by the transfer of a proton. For example, this proton may come from the imidazole group on the side chain of histidine.

Nucleophilic (or covalent) catalysis: this mainly concerns enzymatic reactions involving several substrates, as in the case of enzymes catalyzing a two-substrate “ping-pong” mechanism. Part of the first substrate is transferred to the enzyme via the formation of a covalent bond; this part of the substrate is then transferred to the second substrate.

Figure 1.2.Mechanism of serine protease enzymes.

This is the case for serine proteases, such as chymotrypsin, which share a cleavage mechanism based on a catalytic triad comprising serine, aspartic acid and histidine. This triad promotes polarization of the OH group on serine. The polypeptide is inserted into the serine protease in such a way that the carbonyl group is close to serine. The serine OH group attacks the carbonyl group, and the histidine nitrogen accepts the serine OH. An intermediate enzyme-substrate association is formed, and the hydrolysis product is released (Figure 1.2).

1.5. Single-substrate enzyme kinetics

The study of enzyme kinetics aims to identify and describe the mechanisms of biochemical reactions catalyzed by enzymes (enzymatic reaction), by studying their speed, that is to say, their evolution over time. Starting with isolated enzymes and moving toward organized, integrated metabolic systems, enzyme kinetics is used to quantitatively describe the catalytic properties of enzymes, as well as the mechanisms involved in regulating them. Enzymatic kinetics is an essential element in understanding how enzymes work and helps define the conditions for implementing biocatalytic processes in the food industry. The speed of an enzymatic reaction (V) is expressed by:

the amount of substrate lost per unit of time with:

[1.2]

or the quantity of product formed per unit of time with:

[1.3]

A theory to explain the catalytic action of enzymes was proposed by Arrhenius at the end of the 19th century. He proposed that the substrate and enzyme form an intermediate transition state known as the ES complex. This can be represented schematically, as shown below:

The existence of this intermediate ES complex has been demonstrated experimentally, and the reaction takes place with the formation of the product(s); the enzyme then returns to its original form, once the reaction is complete.

The determination of enzyme kinetics is a fundamental means of describing, predicting and calculating how enzymes can bind to substrates, in order to transform them into products, as well as determining the efficiency of catalysis.

For this purpose, at the beginning of the last century, a quantitative theory of enzyme kinetics was proposed, which led to the following equations being written, based on the assumption of a pseudostationary state such that:

Speed of appearance of ES = speed of disappearance of ES:

[1.4]

[1.5]

[1.6]

[1.7]

[1.8]

[1.9]

[1.10]

[1.11]

[1.12]

[1.13]

Km is also known as the Michaelis–Menten constant. It is the apparent dissociation constant of the enzyme-substrate complex. Km is the substrate concentration [S0] required to reach half the maximum velocity (in other words, when ν0 = 1/2 Vmax). In cases where k1 and k−1 are much larger than k2 (in other words, k1, k−1 ≫ k2), the Michaelis–Menten equilibrium hypothesis is valid and Km ≅ Ks is used to describe the enzyme’s affinity for the substrate.

The catalytic constant k2 or kcat is also known as the turnover number. It can be obtained from the expression Vmax = k2 [ET]. Turnover indicates the maximum number of substrate molecules that can be converted into product per enzyme molecule, per unit of time. Turnover varies from 1 to 104 per second for most enzymes.

The term kcat/Km is used to designate the catalytic efficiency of an enzyme and is used to classify them. A high value of kcat/Km (close to that of k1) indicates that the substrate is closely bound to the enzyme, and the frequency of collisions between the enzyme and the substrate is the limiting factor.

The Michaelis–Menten graph representing ν0 as a function of [S] does not allow us to accurately determine Km and Vmax, since ν0 only reaches Vmax at infinite substrate concentrations. To overcome this problem, Linweaver and Burk (1934) proposed a linearization of the Michaelis–Menten equation using a double inverse representation (Figure 1.3):

[1.14]

Figure 1.3.Graphical representation of the Michaelis–Menten equation and its linearization using the Lineweaver–Burk method

1.6. Effects of the environment on activity

Several factors can affect the speed at which enzymatic reactions take place, such as the substrate/product/cofactor concentration (which will influence kinetics and inhibition by excess substrate/product), the presence of effectors (inhibitors, activators) acting through various mechanisms and the environment (pH, temperature, solvent, oxidizing/reducing environment, etc.). Enzyme stability is an important factor to consider in the food industry.

1.6.1. Effect of pH on enzyme activity

All enzymes have a specific pH value or pH range for optimum activity. A change in pH can affect enzyme activity, with extremely low or high pH values potentially leading to changes in enzyme structure. The pH value at which an enzyme shows its maximum activity is called the optimum pH. This is an important criterion for their use in the food industry. The relationship between enzyme activity and pH is generally described by a bell-shaped curve. The rate of reaction is highest at the optimum pH and decreases on either side of this optimum pH. This effect may be linked to irreversible enzyme degradation due to extreme pH levels, or to an effect on the ionization state of the enzyme or substrate.

1.6.2. Effect of temperature on enzyme activity

Temperature is certainly the most important parameter to control in food processing. As with pH, each enzyme has its own optimum temperature range, and enzyme performance generally improves with increasing temperature, which is linked to molecular agitation. The number of collisions increases, thus raising the energy required to increase the reaction speed. Above the optimum temperature, enzymes can be denatured, moving from an active to an inactive form. In addition to the optimum temperature, denaturation kinetics over time are also crucial parameters. Depending on the process temperature, a wide range of enzymes is available for various purposes in the food industry.

Denaturation kinetics follow a first-order law. Thus:

[1.15]

[1.16]

[1.17]

E is the enzyme activity at time t, E0 is the enzyme activity at time t0 and Kd is the denaturation constant.

In practice, residual enzyme activity is monitored after different heating times. The graphical representation Ln E/E0 = f(t) is used to determine the slope –kd.

1.6.3. Effect of inhibitors on enzyme activity

Enzyme activity can be inhibited by various substances, such as small molecules or ions that can bind to the enzyme. Inhibitors are substances that reduce the speed of enzyme-catalyzed reactions. They can either act on the substrate or combine directly with the enzyme. There are two types of inhibitors that directly interact with the enzyme: reversible inhibitors, which bind to enzymes reversibly and can be removed by dialysis to restore enzyme action, and irreversible inhibitors, which cannot be removed by dialysis. In the case of reversible inhibition, dissociation of the enzyme-inhibitor (EI) complex is much faster than in the case of irreversible inhibition, as there are no covalent interactions between the inhibitor and the enzyme.

When the inhibitor binds to the enzyme, because of its structural similarity to the substrate, and competes for the same binding site as the substrate, this is known as competitive inhibition. The inhibitor prevents the substrate from binding to the active site. A competitive inhibitor competes with the substrate for the binding site, resulting in a decrease in enzymatic activity, due to the reduced binding between the substrate and the enzyme. Competitive inhibition can be overcome by increasing the substrate concentration. Competitive inhibition not only depends on the concentration of the inhibitor and the substrate, but also on their relative affinity for the binding site. Thus, the degree of inhibition is greater when the inhibitor concentration is higher than that of the substrate. Conversely, when substrate concentration is high, the inhibitor competes with the substrate for unavailable binding sites, thus reducing the degree of inhibition. When substrate concentrations are much higher, inhibitor molecules are displaced by substrate molecules, leading to non-significant inhibition. As a result, Vmax remains unchanged. However, the apparent KM clearly increases as a result of inhibition and is referred to as K’M:

In the presence of a competitive inhibitor I, the Michaelis–Menten equation becomes:

[1.18]

For example, the hydrolysis of pectin by a polygalacturonase (PG) has been described, demonstrating the presence of strong competitive inhibition of the product, with an inhibition constant KI of 3.13 g/L (Belafi-Bako et al. 2007).

In non-competitive inhibition, there is no competition for the substrate binding site; the inhibitor can bind to the enzyme regardless of substrate binding; this means that the binding sites corresponding to the substrate and inhibitor molecules are different. Non-competitive inhibitors are substances which, when added to the enzyme, can modify the enzyme so that it cannot bind to the substrate. Non-competitive inhibitors can bind to the enzyme at the same time as the substrate. A complex is formed, consisting of the enzyme, the substrate and the inhibitor (ESI). Both EI and ESI complexes are non-productive. As the inhibitor cannot be detached from the enzyme by a higher concentration of substrate (as is the case of competitive inhibition), the apparent Vmax changes. However, as the substrate can still bind to the enzyme, the Km remains the same:

In the presence of a non-competitive inhibitor I, the Michaelis–Menten equation becomes:

[1.19]

For example, a plant-based flavonoid called luteolin has been shown to exert an effective inhibitory effect on peroxidase activity in a non-competitive manner. The interaction between luteolin and peroxidase induces the formation of a complex due to hydrogen bonds and hydrophobic interactions (Li et al. 2021).

While competitive inhibitors bind to free enzymes and form the EI complex, uncompetitive inhibitors only bind to the enzyme–substrate complex. Here, the inhibitor does not compete for the substrate binding site, but binds to a completely different site. Both Vmax and Km are modified:

In the presence of an uncompetitive inhibitor I, the Michaelis–Menten equation becomes:

[1.20]

This is the case, for example, with the enzymatic browning of apples, which is linked to polyphenol oxidases, but can be limited by oligochitosans, which are inhibitors of these enzymes. It is thus shown that the type of inhibition is uncompetitive, and that these oligochitosans could therefore be considered as new types of anti-browning agent (Guo et al. 2018).

Figure 1.4.Impact of different types of inhibitors on enzyme kinetics. Direct and double inverse representation to determine the catalytic parameters Km and Vmax for competitive, non-competitive and uncompetitive inhibition

Figure 1.4 summarizes the direct and double inverse representations of enzyme rates in the presence of competitive, non-competitive and uncompetitive inhibitors.

1.7. Multi-substrate enzyme kinetics

When more than one substrate is involved, the reaction mechanism can be divided into two main categories: sequential and ping-pong.

In sequential mechanisms, all substrates combine with the enzyme before the reaction takes place. Sequential mechanisms can be divided into two subgroups (ordered or random), depending on whether or not there is a predetermined sequence of bonds between the substrates and the enzyme.

In ping-pong mechanisms, the product is formed before all of the substrates have bound to the enzyme, which means that the enzyme can exist as two catalytically active species, each recognizing a substrate and transforming it into a product, with a conformational change into the other species.

Ping-pong mechanisms can also be subdivided into sequential and ordered mechanisms, but only apply to reactions with more than two substrates.

Mechanisms can be represented according to the nomenclature proposed by Cleland (1963).

1.7.1. Ordered sequential mechanism

In this case, enzyme E has no binding site for substrate B and forms the EA complex alone with A, before being able to bind B to give the EAB complex.

1.7.2. Random sequential mechanism

In this case, the enzyme can initially bind either of the substrates, so that the two secondary enzyme complexes, EA and EB, can be formed, and then bind the other substrate to generate the reactive ternary complex EAB.

1.7.3. Ping-pong mechanism

2Classification of Enzymes Used in the Food Industry

Latifa CHEBIL and Mohamed GHOUL

LRGP, CNRS, Université de Lorraine, Nancy, France

Enzymes are complex molecules produced by various microorganisms (α-amylase, β-amylase, glucose isomerase, pullulanase, cellulase, invertase, lipase, lactase, catalase, etc.), animals (trypsin, chymotrypsin, pepsin, chymosin, catalase, amylase, lipase, etc.) and plants (α-amylase, β-amylase, β-glucanase, bromelain, papain, etc.). Both in vivo and in vitro, they catalyze many reactions that are necessary for the life of biological systems. These catalytic properties have been exploited by man for centuries in order to transform raw materials into products characterized by sought-after features for nutritional, health, well-being and environmental purposes (Mantovani et al. 2005; Raveendran et al. 2018).

According to sources, between 3,500 and 5,000 enzymes have been identified and listed. To facilitate this classification, the International Union of Biochemistry and Molecular Biology (founded in 1955) has proposed a simplified nomenclature, assigning a code to each enzyme. This code is made up of the letters “CE”, followed by four numbers separated by dots, reflecting the type of activity and the position of the enzymes’ action on the various substrates. This approach has made it possible to group enzymes into six major classes, according to their activity: oxidoreductases (CE1), transferases (CE2), hydrolases (CE3), lyases (CE4), isomerases (CE5) and ligases (CE6) (Kuddus 2018).

2.1. Oxidoreductases (CE1)

This enzyme class catalyzes oxidoreduction reactions. The main sub-classes are glucose oxidases, peroxidases, polyphenol oxidases, lipoxygenases, catalases, dehydrogenases, oxygenases and hydroxylases. These enzymes are used in the food industry to modify the texture, color, nutritional value and shelf life of products. Table 2.1 illustrates the use of this class of enzymes in some food applications (Semenova 2006; Chandrasekaran 2016).

Table 2.1.Main uses of oxidoreductases in the food industry

Product

Function

Sub-class

Milk

Removes hydrogen peroxide

Catalase (CE 1.11.1.6)

Breads and pastries

Strengthens cohesion and whitens dough

Glucose oxidase (CE 1.1.3.4), Lipoxygenases (CE 1.13.11)

Wine and beer

Enhances aroma and improves must clarification

Laccases (CE 1.10.3.2)

Wine and beer

Enhances color

Polyphenol oxidases (CE 1.10.3.1)

Fruit juices

Removes oxygen

Glucose oxidase (CE 1.1.3.4)

Fruit juices

Enhances color and stabilizes juice

Laccases (CE 1.10.3.2)

The best-known reactions that are catalyzed by these enzymes are the enzymatic browning of fruit and vegetables, the oxidation of fatty acids and the formation of lactones.

2.2. Transferases (CE2)

This class of enzymes transfers radicals or atomic groups between a donor substrate and an acceptor substrate. Depending on the functional group transferred, a distinction is made between methyltransferases, glycosyltransferases and phosphotransferases. These enzymes are used in the meat tenderization process, in the gelation and polymerization of milk proteins, and in the synthesis of oligosaccharides and cyclodextrins. Table 2.2 lists some of the uses of transferases in food applications (Semenova 2006; Chandrasekaran 2016).

Table 2.2.Some transferase applications in the food industry

Product

Function

Sub-class

Milk

Gelation, polymerization

Transglutaminases (CE 2.3.2.13)

Meat

Modification of viscoelastic properties

Transglutaminases (CE 2.3.2.13)

Syrup

Synthesis of fructose oligomers

Fructosyltransferase (CE 2.4.1.9)

Starch

Cyclodextrin production

Glycosyltransferase (CE 2.4.1.19)

2.3. Hydrolases (CE3)

Table 2.3.Main hydrolase applications in the food industry

Product

Function

Sub-class

Milk

Milk coagulation-cheese production

Proteases (chymosin (CE 3.4.23.4), papain (CE 3.4.22.2))

Milk

Protein hydrolysis

Proteases (CE 3.4.21.1)

Milk

Lactose hydrolysis

Lactases (CE 3.2.1.108)

Milk

Cheese maturation

Aminopeptidases (CE 3.4.21.4), trypsin (CE 3.4.11)

Product

Function

Sub-class

Milk

Flavor development

Lipases (triacylglycerol lipases (CE 3.1.1.3))

Bread and pastries

Improvement of yeast fermentability, color and volume by sucrose hydrolysis

α-Amylases (CE 3.2.1.1) and β-amylases (CE 3.2.1.2)

Bread and pastries

Gluten hydrolysis and improvement of texture and rheology

Proteases (CE 3.4.21.112)

Pastries and Bakery

Improvement of pasta and bread quality

Cellulases (CE 3.2.1.4), endo-1,4-β-xylanase (EC 3.2.1.8)

Malt, beer and wine

Polysaccharide hydrolysis

α-Amylases (CE 3.2.1.1) and β-amylases (CE 3.2.1.2), α-glucosidases (CE 3.2.1.20), cellulases (CE 3.2.1.4), invertases (CE 3.2.1.26)

Malt, beer and wine

Improvement of filtration and clarification

Pectinases (CE 3.2.1.15), hemicellulases (CE 3.1.1.73), cellulases (CE 3.2.1.4)

Juices and fruits

Improvement of extraction and clarification

Pectinases (CE 3.2.1.15)

Juices and fruits

Improvement of liquefaction

Cellulases (CE 3.2.1.4)

Syrup

Polysaccharide hydrolysis

α-Amylases (CE 3.2.1.1) and β-amylases (CE 3.2.1.2), β-glucosidases (CE 3.2.1.21), cellulases (CE 3.2.1.4)

This enzyme class hydrolyzes a substrate in the presence of water to form two or more products. The best-known subclasses are α-amylases, β-amylases, glucoamylases, lactases, cellulases, proteases, invertases, pectinases, phosphatases and esterases. These enzymes are widely used for the hydrolysis of starch, liquefaction, as well as the clarification of fruit juices and wine. These enzymes can be added at various stages of the raw material transformation process, or produced and activated in situ, like in the barley malting process. Table 2.3 shows some applications involving these enzymes (Chandrasekaran 2016; Raveendran et al. 2018).

2.4. Lyases (CE4)

These enzymes catalyze the removal of a group of atoms from a substrate by cleaving C–C, C–O, C–S, C–N type bonds. This mechanism is different from the hydrolytic mechanism characterizing the hydrolases presented above. The best-known sub-classes are decarboxylases, aldolases, dehydratases and some pectinases. For example, acetolactate decarboxylase is used in the beer maturation process. Aldolases are used in the removal of certain aromas, or in the formation of aromas in beer and other beverages. Table 2.4 summarizes the use of some enzymes in this class in food processing (Mantovani et al. 2005).

Table 2.4.Some examples of industrial applications for lyases

Product

Function

Sub-class

Beer

Beer maturation

Acetolactate decarboxylases (CE 4.1.1.5)

Alginate

Oligomer production

Alginate lyases (CE 4.2.2.3)

Alginate

Oligomer production

Guluronate lyases (CE 4.2.2.3)

Fruit juices

Clarification

Pectin lyases (CE 4.2.2.10)

2.5. Isomerases (CE5)

This class includes enzymes that catalyze the rearrangement of groups of atoms in a molecule by isomerization. The major sub-classes are isomerases (glucose isomerases, xylose isomerases), epimerases and racemases. The isomerization of glucose to produce fructose is a solid example of the use of this enzyme class in the food industry. Table 2.5 gives some examples of the application of this enzyme class in the functioning of food products (Chandrasekaran 2016).

Table 2.5.Industrial applications of isomerases

Product

Function

Sub-class

Glucose

Fructose production

Glucose isomerases (CE 5.3.1.5)

D-Xylose

D-Xylulose: conversion of hemicellulose into ethanol

Xylose isomerases (CE 5.3.1.5)

2.6. Ligases (CE6)

Ligases, also known as synthases, are a class of enzymes that catalyze the creation of covalent bonds between two molecules. There are many different types of ligases depending on the type of covalent bond they catalyze (carbon–carbon bond, carbon–nitrogen bond, carbon–sulfur bond, etc.). They generally use the energy of an ATP (or GTP) molecule to catalyze the reaction. Examples of ligases include aminoacyl tRNA synthetases (which bind amino acids to corresponding tRNAs) or DNA ligases (which join two pieces of DNA together) (Chandrasekaran 2016).

3Mode of Action of the Main Enzymes Used in the Food Industry

Catherine HUMEAU1, Mohamed GHOUL1 and Seraphim PAPANIKOLAOU2

1 LRGP, CNRS, Université de Lorraine, Nancy, France

2 LFMB, Agricultural University of Athens, Greece