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The quality of wine, its structure, and its chemical composition are dependent on the grapes’ characteristics as raw material, alcoholic fermentation particularities, and the applied oenological practices. Awareness of the significant role that enzymes play in winemaking contributes to the development of different new strategies for optimizing the production process. Numerous studies confirmed the positive impact of using enzymes in food and beverage industries, in improving the quality of final products, and optimization of applied production technologies. This chapter aims to present the link between biochemical processes that involve enzymes and the quality of wine as a final food product.
Enzymes are functional units of cellular metabolism that catalyze biological reactions. In other words, they lend protein compounds a catalytic role in accelerating the transformation of chemicals into living organisms without being expended during the reaction. The converted substance is the substrate of action, while the compound resulting from the enzymatic activity is called the reaction product [1, 2, 3, 4]. Enzymes can speed up the reaction in cells up to 1016 times. As such, the presence of a relatively small amount of enzymes can catalyze the bioconversion of a large amount of substrate [3]. Even though enzymes are formed inside living cells, they can have in vitro activity (e.g., various enzymes in blood plasma), and they are also present in industrial processes. Enzymatic processes have been known since antiquity, with enzymes being initially used under the name of ferments in correlation with their role in the fermentation of sugars and subsequent transformation into alcohol. The earliest reference to the commercial use of enzymes is found in a description of wine in Hammurabi’s Code (ancient Babylon, about 2100 BC). Ancient texts of the early civilizations of Rome, Greece, Egypt, and China also contain a number of references to the technological process of vinegar, which is based on the enzymatic conversion of alcohol to acetic acid. Today, these compounds continue to play a key role in many food and beverage manufacturing processes, as well as in non-food products (e.g., laundry detergents that dissolve stains using proteolytic enzymes). The analysis and action of enzymes have caught the attention of scientific researchers not only as a focus of scholarly interest, but also because of their many practical needs for society [4]. Much of the research in biochemistry is devoted to analyzing the activity of enzymes. The first theory of chemical catalysis put forth by Berzelius, referred to the hydrolysis of starch, a reaction catalyzed rather by diastase (amylase) than by mineral acids. Thus, the presence of enzymes as biological catalysts specific to living organisms can explain many biological processes, such as fermentation or digestion. In a follow-up to Réanmur’s studies, Spallanzani demonstrated the role of the enzymes found in gastric juice in the process of digestion. In 1836, Schwann coined the name of the gastric juice enzyme known as pepsin, while the name trypsin, an enzyme present in gastric juice, was coined by Kühne. In 1897, Eduard Buchner extracted from yeast cells the enzymes involved in the catalysis of alcoholic fermentation, which function independently of cellular structure [5]. In 1870, the Danish chemist Hansen managed to extract renin from the stomach of calves, which significantly improved both the quality and the quantity of cheese production. In 1921, Fleming discovered lysozyme, a component of tears, saliva, leukocytes, skin, nails, and human milk, which is widely spread in both animals and plants. He published the first articles on the subject between 1922 and 1927. In 1926, James Sumner managed to isolate urease from the jack bean (Canavalia ensiformis), the first pure crystalline enzyme. His observations were of particular importance for the development of enzymology, confirming the protein structure of enzymes. The name enzyme was given by Kühne (1867), while Stern was the first to observe the first enzyme-substrate complex in 1935 [5, 6]. Steady advances over time had a major impact on the enzyme industry, such as the production and marketing of glucoamylase, which catalyzes the production of glucose from starch with superior efficiency compared to the chemical procedure of acid hydrolysis. Consequently, the launch of enzyme-based detergents was made possible [3].
As to the beverage industry, enzymes were first used in the 1930s to make wine and fruit juices, with Boidin and Effront discovering bacterial amylase. The fruit drinks and juices industry began using pectinase in the late 1940s to improve clarification and filtration. These types of enzymatic preparations also began to be tested in the oenological sector. After 1974, they were officially authorized in the oenological industry by the Ciba-Geigy Company, with Ultrazym 100 as the first enzymatic preparation proposed. It was only in the 1980s that β-glucanases were authorized, which helped to solve the problems of clarifying and filtering wines obtained from botrytized grapes. In the late 1980s, β-glycosidase-enriched pectolytic enzymatic preparations began to appear on the market following a close collaboration between French (Montpellier) and Australian (Australian Wine Research Institute) researchers, and Gist Brocades [6]. Enzymes offer flexible, high-performance solutions that ensure high-quality products boasting a higher nutritional intake, cost-effectiveness, and guaranteed consumer satisfaction. Food enzymes are cost-effective and provide reliable food security, which explains why they are increasingly in demand in the food industry. Understanding the crucial role that these oenological products have in winemaking contributes to the development of optimizing strategies in improving the structure, chemical composition of the final product, and implicitly their sensory profiles [1, 3].
Grape pomace, the main by-product of the wine industry, has been shown to be an important source of nutrients. Botella et al. [7] studied the production of hydrolytic enzymes from grape remnants (cellulases, xylanases, and pectinases) under the influence of Aspergillus awamori. The volume of industrial use of enzymes has gone up recently due to their many advantages. Being of natural origin, enzymes have no toxicity and show a negligible impact on the environment. These catalysts present the specificity of action insofar as they are selective with regard to both the substrate they act upon and the catalyzed reaction. Enzymes act effectively in moderate temperature conditions, show quick action at relatively low concentrations and the reaction rate can be easily controlled by adjusting temperature, pH, and quantity. Moreover, enzymes can be easily inactivated once the reactions have produced the desired result. These preparations are considered technological aids and are not found in the final product [2].
To date, more than 6000 different types of enzymes are known. The classification and naming of enzymes are generally based on the type and mechanism of the reaction they catalyze. The classification of enzymes is based on the principles established by the Enzyme Commission of the International Union of Biochemistry. The following criteria are laid out:
Enzymes and the reactions they catalyze are divided into six different classes, each in turn divided into subclasses.
The name of the enzyme provides information on the name of the substrate and the type of catalyzed reaction, followed by the suffix -ase, except for proteolytic enzymes, where the suffix is -in (trypsin). For example, protein hydrolysis is catalyzed by proteases.
For a correct and positive identification, each enzyme is assigned a 4-digit code number, as follows: the first digit refers to the reaction class it belongs to; the second and third digits indicate the subclass and sub-subclass; the fourth is the serial number of the enzyme in the subclass [2, 4, 6, 8].
Enzymes are classified as follows:
Oxidoreductases (EC 1). Enzymes that catalyze redox reactions belong to this class, with the oxidized substrate as hydrogen-donor. Put differently, oxidoreductases catalyze the transfer of hydrogen, oxygen, or electron atoms from one substrate to another [4, 6, 9]. The systematic name is based on the donor-acceptor oxidoreductase: dehydrogenase or reductase. The recommended name is made up of the donor’s name and the endings: dehydrogenase, oxidase, reductase, oxygenase, and peroxidase. The name oxidase is used only in cases where O2 is the acceptor, while that of oxygenase is used when part of the O2 molecule is framed in the corresponding substrate. Oxidoreductases make up about 25% of all enzymes [10].
Transferases (EC 2) are enzymes that transfer a group, for example, a methyl group or a hydroxyl glycosidic group, from one compound (generally considered a donor) to another (acceptor). The systematic names are made up according to the donor of the scheme: donor-acceptor or group transferase. Recommended common names are group transferase donor or group transferase acceptor, but the name group kinase acceptor is also accepted for some phosphotransferases (e.g., hexokinase and glucokinase). This class makes up about 30% of all known enzymes. Of these, of enological interest are reactions involving the transfer of phosphoric acid residue (H3PO4), carbonate ion (-(CO3)−2), carbon dioxide (CO2), water (H2O), ammonia (NH3), and amino groups (-NH2) [9, 10].
Hydrolases (EC 3) amount to 24% of all known enzymes to date and catalyze hydrolytic reactions. These are group transfer reactions where the acceptor is always water, the systematic name is the hydrolase substrate (e.g., peptidyl-peptide hydrolase), and the common name -ase substrate (e.g., methylesterase and o-glycosidase). Of interest in the food industry are α-amylases (EC. 3.2.1.1), β-amylases (EC. 3.2.1.2), lactase (EC. 3.2.1.23), lipase (EC. 3.1.1.3), proteases - amino peptidases (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). The hydrolases commonly found in must and wine are of fungal origin and come either from the plant’s microflora or from an external source following administration of treatments with enzymatic preparations [4, 9, 10].
Lyases (EC 4) represent 13% of the overall number of enzymes known. They catalyze the addition or removal of non-hydrolytic groups from the structure of the substrate, with the formation of double bonds or acyclic structures of the type C–C, C–O, and C–N. The systematic name consists of substrate group—lyases. The (common) historical names are created according to the group removed, namely: decarboxylase, when carbon dioxide is removed; dehydratase, when water is removed; aldolase, when the deleted group is of the aldehyde type. Out of the total lyases carbon-carbon lyases, carbon-nitrogen lyases, and carbon-oxygenates are most significant for the wine industry [4, 9, 10].
Isomerases (EC 5) catalyze isomerization reactions, as a result of which a molecule is converted from one isomer to another. They amount to 3% of all known enzymes. In general, the systematic name corresponds to the traditional one and is formed by: substrate isomerization type class name. Depending on the type of isomerism, the enzymes in this class can be divided into subclasses, namely: isomerases, racemases, cis- and trans-isomerases, epimerases, mutases, tautomerases, or cycloisomerases. Of these, the following are important in the enology field: lactate racemase, glucose isomerase, glucose phosphate isomerase, carotenoid isomerase, and triosephosphate isomerase [10].
Ligases (EC 6) are enzymes that catalyze the binding of two molecules coupled with the hydrolysis of a pyrophosphate bond to ATP or a similar triphosphate. Enzymes in this class are involved in condensation reactions. The systematic name is A: B ligase form (ADP-forming), where A and B are the two substrates, followed by the class name and the product resulting from hydrolysis. Ligases represent 5% of all known enzymes. Also called synthetases, they have the property of catalyzing bimolecular reactions to form new carbon-carbon or carbon-heteroatom bonds. Of these, those of interest in the enology industry are asparagine synthetase, acetyl-coA synthetase, succinyl-coA synthetase, glutamine synthetase, glutathione synthetase, and pyruvate carboxylase [6, 9].
Enzymes are macromolecular organic substances of protein origin that are spherical in shape and have primary, secondary, tertiary, and quaternary-type structures. They can be classified into holoproteins and heteroproteins. The catalytic properties of enzymes are generated by the spatial structural configuration of the molecule that participates in the development of enzymatic activity and by the existence of catalytic sites in their molecule to which the substrate, activator, or inhibitor, will bind, as appropriate [2]. There are also exceptions, such as the ribonuclease P, defined as a protein-ribonucleic acid complex whose enzymatic activity is not due to the protein, but to the ribonucleic acid [11]. In terms of chemical structure, enzymes are simple proteins (mono-components), such as trypsin, pepsin, lipase, sucrose, amylase, or conjugates (bicomponents), that is, apoenzymes and coenzymes. Some coenzymes are derivatives of vitamins (NAD, TDP, coenzyme A, etc.) or metal ions (Zn, Mn, Ca, etc.). Apoenzymes and coenzymes show a synergistic action by only acting in tandem. The apoenzyme is the protein macromolecule responsible for the specificity of the reaction, while the coenzyme triggers the reaction, both forming a fermentative complex [10]. The composition of the apoenzyme includes the catalytic site (a distinct area framed by a group of amino acids, which is set apart from the rest of the amino acids by their function, in which the specific reaction substrates bind) and the allosteric site (a distinct area for the activator or inhibitor to bind). Enzymes with both catalytic and regulatory functions are called allosteric enzymes [6]. The mechanism of enzymatic action has been explained by many researchers. Thus, Ogston [12] reported that at a wavelength of 280 nm, three points of interaction between enzyme and substrate were identified, which explains the phenomenon of stereospecificity of enzymes. These interactions have either a binding or a catalytic function. Binding sites (active sites) connect to specific groups in the substrate to ensure a stable orientation of the enzyme and substrate molecules with the reaction group in the vicinity of the catalytic sites. The three-point interaction theory cannot explain thoroughly the action and specificity of the enzyme, and there are other hypotheses in this regard. The action of enzymes is considered to occur in two stages, as shown in Figure 1: the active site of the enzyme initially combines with the substrate to form an enzyme-substrate complex (ES); the latter then decomposes to form the products (P) and the free enzyme (E), which can react yet again [9].
Figure 1.
Schematic representation of the enzymes’ action (left) and illustration of the lock-and-key and induced fit model (right) [13].
For the reaction to take place, the reacting molecules (substrate) require a certain amount of energy (activation) to traverse the transition state of the reaction and turn into reaction products. In 1888, the catalytic action of enzymes was explained by the Swedish chemist Svante Arrhenius, who proposed that the substrate and enzyme are combined to form an intermediate compound known as the enzyme-substrate complex (ES). This complex decomposes into a reaction product (P) and an active enzyme (E). The total enzyme-catalyzed reaction can be represented as: S + E → ES → P + E.
In general, enzyme-catalyzed reactions cover the following stages:
The substrate molecule comes into contact with the active site of the enzyme through non-covalent bonds. The active site is the area of the enzyme that combines with the substrate.
The substrate and the enzyme form an enzyme-substrate complex.
The substrate molecule is transformed into a reaction product either by rearranging the atoms or by decomposing the substrate or combining it with another molecule.
Dissolution of the ES complex leads to the formation of the reaction product, which is released by the active site of the enzyme.
The nature of the enzyme is unchanged and can catalyze a new reaction [6].
The mechanisms of enzymatic action are commonly explained by two proposed models:
The lock-and-key hypothesis. In 1894, Fischer put forth his theory by suggesting that both substrate and enzyme have specific geometric shapes that match. The hypothesis specifies that the active site of an enzyme has a unique configuration that is complementary to the substrate structure (key), and therefore allows the two molecules to match [6]. According to this model, the structures show rigidity by remaining fixed throughout the binding process [2, 9].
The induced fit hypothesis. In 1958, Koshland proposed some changes to the lock-and-key hypothesis detailed above by positing that the essential functional groups on the active site of the free enzyme are not in their optimal positions for catalysis. Because enzymes are so flexible, when the substrate molecule binds to them, the active site of the enzymes takes on a favorable geometric shape to reach the transition state. As per Koshland’s suggestion, the substrate induces a configuration change in the enzymes that aligns the amino acid residues or other groups so as to bind and catalyze the substrate [2, 6].
The specificity of the enzymes can be exhibited either on the substrate or on the reaction. In other words, an enzyme has an affinity for the substrate it acts on and for the reaction it catalyzes [11]. Enzymes have varying degrees of specificity. For example, the enzyme alcohol dehydrogenase catalyzes the dehydrogenation of high-efficiency ethanol and low-efficiency methanol. Such an enzyme is seen as specific to a compound, and not to a class of substances. Moreover, with regard to the reaction specifics, an enzyme can only catalyze a transformation of the substrate. For example, L-amino acid oxidase catalyzes the oxidation of L-amino acids to produce the corresponding keto-acids, ammonia, and hydrogen peroxide. However, the racemization of L-amino acids into D-amino acids is catalyzed by an enzyme other than L-amino acid oxidase, that is, amino acid racemase [11]. To act as a catalyst, most enzymes need a molecule known as a cofactor, which is a non-protein chemical compound bound to an inactive protein part of the enzyme (apoenzyme) to increase its biological activity [4, 6]. The active complex of the apoenzyme (protein part) together with the cofactor (coenzyme/prosthetic group) constitutes the holoenzyme. Two categories of cofactors are known, namely: coenzymes and prosthetic groups. The cofactor may be a metal ion and/or an organic molecule. As a specific type of cofactor, coenzymes are organic molecules that bind to enzymes to ensure their functioning. Many coenzymes are derived from vitamins. Prosthetic groups are also cofactors that often bind closely to proteins or enzymes through a covalent bond [6]. Enzymes have the ability to catalyze biochemical reactions in cells, something specific to catabolic and anabolic processes. For example, the enzymes involved in photosynthesis are located in the chloroplasts, those of the glycolytic cycle are found in the cytoplasm, the enzymes of the Krebs cycle are present in the mitochondria, etc. Moreover, the intensity of the physiological process depends on the activity of the involved enzymes. For example, phosphofructokinase is involved in the phosphorylation reaction of fructose in plants, causing biodegradation of hexoses in the glycolytic cycle; phenylalanine ammonia lyase plays a role in the biosynthesis of phenols, anthocyanins, lignins; chlorophylls are responsible for catalyzing the chlorophyll decomposition reaction; polyphenol oxidases are involved in the catalysis of oxidation reactions of polyphenols, etc. [11].
Enzymes are globular proteins soluble in aqueous solvents or dilute saline solutions. Their solubility increases through weak ionic interactions, such as hydrogen bonds. Some of the factors that influence or interfere with this process and have an effect on the solubility of enzymes are salt concentration, pH, temperature, and solvent structure. Solubility can be increased by adding neutral salt in low concentrations. When using salts with a higher solubility, such as ammonium sulfate, some proteins will precipitate only in certain concentrations. Most proteins will precipitate at more than 80% (NH4)2SO4. Cations such as Zn2+ and Pb2+ decrease the solubility of enzymes to form insoluble complexes with the enzymatic protein. Proteins are also precipitated by the addition of acids, such as trichloroacetic acid or picric acid, due to the formation of insoluble salts, a property used in analytical techniques to separate proteins from solutions. The solubility of proteins can be reduced in a narrow pH range called the isoelectric point, when they are electrically neutral. When the temperature varies between 40°C and 50°C, the solubility of the enzymes increases. At temperatures above these, the tertiary structure is disrupted, and the protein is denatured and loses its activity [9].
The stability of enzymes is a very important factor that must be taken into account when administering them in the course of the technological process. Enzymatic reactions are influenced by factors such as presence and concentration of enzymes and substrate, pH level, temperature, pressure, and presence of inhibitors and activators [2, 6, 9, 11]. The reaction rate varies in direct proportion to the concentration of the enzyme and the substrate. Thus, by increasing the concentration of the enzyme above a certain threshold, the reaction rate will remain constant. On the other hand, by keeping the enzyme within constant limits while increasing the substrate concentration, the reaction rate will vary exponentially, as shown in Figure 2.
Figure 2.
Dependence of reaction rate on concentration—C (a), substrate—S (b), pH level (c), and temperature—T (d) [14].
Consequently, the larger the available reactant surface, the higher the reaction rate, and as the particle size decreases, the total surface area increases. This allows for the participation of several reactant molecules in the chemical reaction. Most biological reactions occur in solution and their reaction rate is therefore directly proportional to the concentration of the reactant [5, 11]. Figure 3 shows how the concentration varies depending on glucose isomerization and sucrose hydrolysis. The reaction rate is directly proportional to the concentration of the reactant under constant conditions of temperature and pH. In 1867, Guldberg and Waage posited a quantitative relationship between the molar concentration of the reactant (reactions) and the reaction rate [5].
Figure 3.
Change in substrate concentration relative to reaction time. Fructose isomerization in fructose (left) and sucrose hydrolysis (right) [5].
The enzymatic reaction rate goes up with the rise in temperature to a maximum (optimal) threshold, only to go down with additional increases in temperature, which further cause denaturation of the enzyme [2, 5]. Thus, most enzymes show a maximum efficiency at temperatures between 35°C and 40°C for plant enzymes, and between 20°C and 30°C for those of animal origin. At temperatures above 60°C, enzymatic activity decreases or the protein component degrades completely. Enzymes are sensitive to the action of heat and change their properties due to temperature variation (they are thermolabile). For example, ribonuclease reduces its activity with increasing temperature but resumes it rapidly after cooling. However, some enzymes are more resistant and keep up their activity even at higher temperatures, as is the case of enzymes of various thermophilic bacteria, which remain active up to about 85°C [11]. There are also enzymes that can operate at very low temperatures, even in freezing conditions (e.g., β-galactosidase). Although the action of these enzymes is slower, it incurs lower costs both in terms of the amount of enzymes administered (smaller amounts of enzymes are required to meet the activation energy requirement) and energy consumption [6]. Endogenous enzymes of plants and fruits can be made up of isoenzymes with different thermal stabilities. The thermal stability of plant peroxidase isoenzymes has been long investigated to identify appropriate mechanisms and kinetic models for enzyme inactivation. For example, deamination of asparagine and glutamine residues, hydrolysis of peptide bonds to aspartic acid residues, oxidation of cysteine residues, thiol-disulfide exchange, destruction of disulfide bonds, and the chemical reaction between the enzyme and other compounds such as polyphenols can cause irreversible inactivation of enzymes at high temperature levels [11]. The dependence of the reaction rate on temperature can be expressed by means of the Arrhenius equation:
k=Ae−EaRT
where k is a rate constant; A is the pre-exponential factor; Ea marks the activation energy; and R is the universal constant of the ideal gas at the absolute temperature T [2].
A high pressure has a major influence on enzymatic activity. Thus, at values over 3 kilobars, enzymes will be reversibly inactivated, while at a pressure of over 7 kilobars the process will be irreversible [11]. By applying high pressure, the activity of enzymes and the development of microorganisms are significantly inhibited, which allows for the protection of nutrients and flavor compounds. Microorganisms show extra sensitivity to high pressure, with their growth being inhibited at values between 300 and 600 MPa. On the other hand, a low pH level will emphasize this effect. However, bacterial spores can withstand pressures over 1200 MPa. In general, proteins are irreversibly denatured at ambient temperature by applying pressures above 300 MPa. Below this value, reversible changes in the structure of protein compounds occur. In the case of enzymes, even slight changes in the steric arrangement and mobility of the amino acid residues involved in catalysis can lead to their diminution and loss of activity [2]. The activity of enzymes is significantly influenced by the concentration of hydrogen ions in the reaction medium. Enzymes usually have a bell-shaped activity related to the pH profile (Figure 2). Decreased enzymatic activity on either side of the optimal pH can appear due to two causes. In the first case, the pH can influence the stability of the enzyme by inactivating it irreversibly. In the second situation, the pH can influence the kinetic parameters of the enzymatic reaction, namely: stability of the ES complex, reaction rate, rate inhibition, or both. The pH dependence of enzyme-catalyzed reactions is similar to that of acid and base-catalyzed chemical reactions. In steady-state conditions, the integrated form of the Michaelis-Menten model is used:
Km′lnS0S+[S0−S=Vmaxt=kcat×ETt
where K’m is the apparent Michaelis constant; [ET] corresponds to the total enzyme concentration; [S0] and [S] refer to the substrate concentration at time zero and time t, respectively; kcat is the zero-order constant for the enzymatic reaction under conditions of substrate saturation; and t represents the reaction time [9].
The optimal pH value generates a maximum enzymatic activity and is influenced by the origin of the enzyme, type, and reaction medium. For example, pepsin has an optimal pH level between 1.4 and 2.5, while pancreatic amylase will show a maximum activity at pH 6.8. The relationship between enzymatic activity and pH level depends on the acid-base behavior of the substrate and the enzyme. Furthermore, the optimum pH for an enzymatic reaction is not the same as that of its normal intracellular environment. The activity of enzymes can also be inhibited by various chemical compounds, either endogenous (various metabolites) or exogenous (toxic agents, drugs). Ions and metal compounds, which are active as prosthetic groups or which ensure the stabilization of the configuration of the enzyme or the enzyme-substrate complex, are the activators of enzymatic reactions [2]. Enzyme inhibitors are low molecular weight chemical compounds that have the ability to completely reduce or inhibit the catalytic activity of the enzyme reversibly or permanently. That is to say, an enzyme inhibitor is a substance that slows down an enzyme-catalyzed reaction. It can alter one or more amino acids required in the enzymatic catalytic activity. Most natural inhibitors react reversibly with the enzyme and are classified into two types: specific and non-specific. The most common enzyme inhibitors with a wide range of applications in the food industry include protease inhibitors, polyphenol oxidase, and amylase or lipase inhibitors. For instance, protease inhibitors are substances that act directly on proteases to lower catalytic velocity. They usually mimic the protein substrate by binding to the active site of the enzyme and are specific for the active site of a class of proteinase. Protease inhibitors are usually classified according to the class of protease they inhibit (cysteine, serine, aspartic, and metalloprotease inhibitors). Most extracellular protein inhibitors produced by microorganisms belong to the genus Streptomyces. A number of pathogenic gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, or Erwinia chrysanthemi, appear to be able to get protection against their own proteases by producing periplasmic protease inhibitors, such as ecotin [2]. Succinate dehydrogenase, which is responsible for the catalytic reactions involving the transformation of succinic acid into fumaric acid, is inhibited by dicarboxylic acids: malonic, malic, and oxalic [11]. In addition, food may be contaminated with pesticides, metal ions, and other chemicals in a polluted environment that may become inhibitory in certain circumstances [2].
7. Use of commercial enzymes in food and non-food industry
People have used enzymatic systems since ancient times, albeit with scarce information about them, to preserve food or ferment food or bread [1, 15]. Numerous desired or undesirable changes in the aromatic profile and physicochemical properties of untreated fruits, vegetables, oilseeds, cereals, and food of animal origin are catalyzed by one or more enzymes. Whether activated intentionally or not, these enzymes influence the final quality of the food or drink in which they are present. Over time, major advances have been made in the field of enzyme chemistry with a focus on achieving a well-defined end product [3]. With technological progress, new enzymes have been developed that are characterized by a wide applicability and specificity [15]. Their use in industrial processes has shown increasing promise as they can eliminate the need for high temperatures, extreme pH values, and organic solvents and, at the same time, ensure high substrate specificity, low toxicity, high purity of the final product, low environmental impact, and easy inhibition of enzymatic activity [15]. Figure 4 indicates some industrial applications of enzymes in food and non-food fields.
Figure 4.
Use of enzymes at industrial scale.
In the food industry, this technology allows for diversifying assortments and obtaining new products, improving nutritional value, reducing production costs, optimizing processing, and reducing the amount of waste, plus new solutions for food and packaging safety [6]. Enzymes are commonly used in the fruit processing industry to improve the pressing yield, extract and improve color and flavor characteristics, and clarify and decompose insoluble carbohydrates (pectins, hemicelluloses, and starch).
Enzymes play a key role in the production of beer and whiskey by helping to extract the sugars needed for fermentation, viscosity control, and to increase stability under storage conditions. Moreover, in the technology of beer, the administration of various enzymatic preparations can lead to a dietary product with low-calorie intake. Enzymes contribute significantly to improving the quality and stability of wines, reducing the period of alcoholic fermentation, promoting the clarification process, and ultimately facilitating filtration. The food industry is currently experiencing a growing trend in the demand for high-quality foods and beverages with outstanding, healthy sensory characteristics at competitive costs. The global market for enzymatic preparations used in the food industry, including beverages, reached approximately $1.69 billion in 2018, with growth expected to continue in the coming years, which poses a challenge to producers aiming to obtain innovative products. Most existing biotechnological applications are of microbial origin. Microbial enzymes are superior to those from animal and plant sources due to ease of production and genetic manipulation, various catalytic activities, etc. [8, 15]. The microorganisms used to produce the enzymes include about 50 bacteria considered safe by the FDA (GRAS - generally recognized as safe) and also fungi. The bacteria are mainly Bacillus subtilis, Bacillus licheniformis, and various species of Streptomyces. Fungi are usually of the genus Aspergillus, Mucor, and Rhizopus. Microorganisms can be grown in large quantities in a relatively short period of time by means of well-established fermentation methods. Large-scale production of microbial enzymes has many economic advantages due to cheap culture media and short fermentation stages [8, 9]. Globally, enzymes such as α-amylase, glucoamylase, lipase, pectinase, chymosin, and protease are most commonly used in the food processing industry. α-Amylase contributes to the transformation of starch into dextrins and is used in the production of corn syrup for various applications, such as sweetening various foods. In the production of high-quality beer, glucoamylase (hydrolytic enzymes) transforms dextrins into glucose by converting residual dextrins into fermentable sugars [6]. Proteases are of particular interest in the food industry due to their specific properties, such as high production yield, substrate specificity, high activity, and environmentally friendly nature. Also, the activity of these enzymes occurs in a wide range of temperatures (20 °C–80°C) and pH values (pH = 3–13), which increases its scope [15]. Also known as proteolytic enzymes, proteases are the largest class of such compounds in the human genome. They have the property of selectively catalyzing the hydrolysis of peptide bonds. Proteases are available in a wide variety of microorganisms, plants, and animals. Microbial production offers many benefits in terms of technical and economic properties, such as higher yields in a shorter time and reduced costs, plus a higher overall productivity [15]. The main field of application of proteases is the dairy industry, especially cheese manufacturing. Renin was initially preferred in cheese manufacturing due to its high specificity, while microbial proteases produced by GRAS microorganisms, such as Mucor miehei, Mucor pusilis, Bacillus subtilis, and Endothia parasitica, appeared not long after. For many years, proteases have also been used to produce low-allergenic milk proteins as ingredients for baby milk formulas. Proteases can also be used for the synthesis of peptides in organic solvents. The food industry uses the invertase produced by Kluyveromyces fragilis, Saccharomyces cerevisiae, and Saccharomyces carlsbergensis to make candy and jam. β-Galactosidase (lactase), produced by Kluyveromyces lactis, Kluyveromyces fragilis, or Candida pseudotropicalis, is used to hydrolyze lactose in milk or whey, while α-galactosidase secreted by Saccharomyces carlsbergensis is used to crystallize beet sugar [8]. Aspartic proteases, which play a role in the degradation of protein materials, comprise a small group of enzymes, among which cathepsin, renin, and pepsin are predominant. Their applications are well established in food processing in the manufacture of both traditional and modern products and are now being extended to new fields. They are widely used in cheese making, wine preservation, and also for clarifying beverages [15].
Cysteine protease, also known as bromelain, is isolated from the stem, fruit, or other parts of pineapple plants. It has a wide range of uses, from industrial to pharmaceutical domains. For most industrial applications, conventional production methods, such as extraction, concentration, and drying, are used. However, state-of-the-art applications in the pharmaceutical industry involve a much higher purity of bromelain, which is obtained through chromatographic methods, such as gene filtering or affinity chromatography [15].
Asparaginases are among the most widely clinically used enzymes, particularly in treating various cancers, insofar as they convert asparagine into aspartic acid and ammonia. Similarly, there has been a steady interest in their capacity to minimize the content of acrylamide in foods containing starch, and fried or baked products. Acrylamide is generated as a by-product of Maillard reactions between asparagine and reductive sugars. Reactions usually occur at temperatures above 100°C, being intended to alter the chromatic and aromatic profile of starchy foods, whether fried or baked. In 1994, acrylamide was first classified as group B2 - a possible carcinogen by the International Agency for Research on Cancer. Extensive efforts have been made to reduce the formation of acrylamide during baking or frying by incorporating the asparaginase. When used to reduce the formation of acrylamide in food, asparaginase can be isolated from fungal species and is considered safe, as it presents high specificity and minimal activity compared to glutamine. The main disadvantage of using asparaginase comes from marketing restrictions in some countries due to the associated problems at the industrial level. The incorporation of asparaginase in the food industry requires extensive research on the enzymatic effect and pre-/post-processing conditions. Purification of the enzyme needs extensive attention, as it influences the attenuation activity of acrylamide [15].
Lipases are universal enzymes that are present in all living things (plants, animals, fungi, and bacteria). Their basic function is to catalyze the hydrolysis of lipids into free fatty acids and glycerol at the interface of aqueous and organic solvents. Lipases catalyze a wide range of reactions that are significant from an industrial point of view, and present enantio-selectivity due to which they come to be seen as indispensable in food, pharmaceuticals, biofuels, detergents, cosmetics, leather industry, biosensor production, etc. [15]. For the production of fungal lipases, hosts such as Aspergillus oryzae, Rhizomucor miehi, Thermomyces lanuginosus, and Fusarium oxysporum [8] are used. In the food industry, lipases are used to improve the aromatic profile, reduce the time required for the maturation of cheeses, and obtain special products with superior qualities [6].
Cellulose, hemicellulose, pectin, and lignin are major components of the plant’s cell wall. Hemicellulose is the second most abundant carbohydrate polymer on earth. α-L-arabinofuranosidase has a potential application in agro-industrial processes due to its synergistic effect with other hemicellulases. For example, α-L-arabinofuranoses are used in various industries: as a natural quality enhancer in bread manufacture; in the beverage industry to improve the aromatic profile of wines or to clarify fruit juices; in the production of pharmaceuticals, etc. [15].
Glucose oxidases are often used to remove oxygen from food or glucose from drinks for diabetics. These enzymes play an important role in defining the color, texture, flavor, and preservation of food. Lipases are used in the food industry to hydrolyze fats, improve taste characteristics, reduce the feeling of bitterness, or enhance preservation [6]. Lacases are increasingly used in various industrial oxidative processes, such as delignification, bioremediation, modification of plant fibers, ethanol production, biosensors, biofuels, etc. Industrial uses involve an increase in enzyme immobilization, usually from a heterologous host, such as Aspergillus spp. [8].
Enzymes are also used in a wide range of agro-biotechnological processes, and their main use is in the production of supplements to improve the nutritional quality of animal feed. For example, the use of phytases in agriculture as an ingredient in animal feed aims to improve the absorption of phosphorus from plants during the digestion of monogastric animals. Thus, phytase enables the release of phosphorus from plant matter, which contains about 2/3 of phosphorus as phytate, and reduces the phosphate load that impacts on the environment [8]. Another perspective on the use of phytases refers to human nutrition. It is known that ingestion of high amounts of food phytate severely hinders the absorption of important trace elements, such as iron and zinc, in the digestive tract. Due to this anti-nutritional effect of phytate, a large part of the population shows deficiencies with regard to these nutrients. There are two ways to reduce phytate dietary intake and its negative effects. One is to develop low phytate cultures by disrupting inositol polyphosphate kinases or other mutations in phytic acid biosynthesis. Although this approach has validated its main objective, low phytate maize and soybeans have been shown to have diminished seed yield and germination. Supplementing phytases in foods for human consumption is a more effective way to reduce the negative effect of phytate. To this end, Fujita et al. [16] tested a mutant strain of Aspergillus oryzae with high phytase activity in beer production. Haros et al. [17] used exogenous microbial phytase as an additive in bread making to improve physical and baking parameters, such as dosing time (24% reduction) width/height ratio of bread slices (5% reduction), specific volume (21% increase), and crumb firmness (28.3% reduction). While commercial use is under continuous testing, the potential role of thermophilic phytases as potent additives in the cellulose and paper industry has been suggested. Furthermore, phytase could act synergistically with xylanase in the preparation of multienzymes in xylanase-producing microorganisms such as Streptomyces cupidosporus [17].
In the chemical industry, the use of enzymes sometimes involves lower energy consumption, increased catalytic efficiency, much smaller amounts of waste and by-products, and lower volumes of wastewater. Hydrolases and ketoreductases that are stable in organic solvents are usually used for this purpose. They can also be used to produce various compounds, such as L-amino acids. About 150 biocatalysts are used in the chemical industry and are developed with the broadening application of genomic and protein engineering [8].
Enzymes are equally important in the pharmaceutical industry. For instance, penicillin acylases are used in the preparation of β-lactam antibiotics, such as semisynthetic penicillins and cephalosporins. This group of antibiotics accounts for about 60-65% of the total antibiotic market. Enzymes are also involved in the preparation of chiral drugs and peptide synthesis. Furthermore, esterases, proteases, lipases, and ketoreductases are used in the preparation of chiral alcohols, carboxylic acids, amines, and epoxies [8].
The use of enzymatic preparations in winemaking is becoming more common in view of their many time-confirmed technological advantages. Endogenous enzymes play a key part in grape ripening/maturation [18]. They act by degrading the cell wall to favor the dissolution of vacuolar contents. The role of endogenous enzymes is incomplete since it is limited by winemaking conditions, such as pH of the must and insufficient activity due to the limited timespan of pre-fermentative treatments [19]. For these reasons, enzymatic preparations of an exogenous nature are often used in the technological process of wine depending on the winemaker’s purpose. A sound knowledge of the nature and structure of macromolecules in must and wine offers new perspectives for the administration of enzymes in winemaking, especially in what concerns pressing, clarifying, filtering, and extracting various constituents with a role in defining the organoleptic characteristics of wine and its stabilization processes [18]. Moreover, the administration of such oenological products ensures the optimization of the process through a rigorous control on the quality of operations by allowing for superior loading of pressing and centrifugation equipment, reducing pressing times, favoring decantation and clarification of pressed juice, reducing energy consumption, and leading to an overall increase in production efficiency. The dosage of enzymes depends on the degree of ripeness of the grapes and the target one has in mind. For red wines, a larger dose variation is possible depending on incubation time. The enzymatic activities involved in the hydrolysis of pectic substances are carried out by pectin esterase, polygalacturonase, pectin lyase, rhamnogalacturonase, rhamnogalacturonan acetylesterase, arabinase, and galactanase. Other enzymatic activities come from hemicellulose and cellulose and are usually present in different amounts in the basic preparations of pectinases. The combined action of all these enzymes leads to partial hydrolysis and solubilization of neutral acid and polysaccharides in grapes [18]. Most enzymes are present in enzymatic preparations as isoenzymes and act differently depending on pH level, optimum temperature, and degree of pectin esterification. The most commonly used commercial enzymes are pectinases, glucanases, and glycosidases and less frequently lysozymes and urease [18].
Generally, the use of enzymatic preparations that have been purified to remove cinnamyl esterase activities is recommended for the production of white and rosé wines. Enzymatic preparations used in the manufacture of white wines are not thought to have such activity since it is already generated in nature by the species Aspergillus niger and Botrytis cinerea, and it is responsible for the hydrolysis of p-coumaric and ferulic acids, which, following decarboxylation, leads to the formation of 4-vinylphenol and 4-vinylguaiacol. These compounds are responsible for the medicinal odor in white wines specifically [18]. Lao et al. [20] presented that using purified pectolytic enzymes makes it possible to reduce the concentration of 4-vinylphenol in wines obtained from Sauvignon blanc grapes by more than 50%. Enzymatic preparations can be supplied in granular, liquid, or powder form. The latter has disadvantages due to the allergenic potential of enzymatic dust. The granular form has the double benefit of lacking preservatives and having good stability during storage, while liquid enzymes generally contain preservatives [18]. The concomitant use of bentonite and enzymatic preparations is to be avoided, as the enzyme will be inhibited due to the specific adsorption of bentonite and the latter will have reduced effectiveness due to blockage of active centers by the enzyme protein. Bentonite treatment should preferably take place after enzymatic treatment. Bentonite gel will help flocculate enzymatically hydrolyzed pectins. The activity and efficiency of an enzyme vary widely, depending on temperature and pH. Accordingly, pectinases can be administered at temperatures ranging between 10°C and 55°C. At temperatures below 10°C, the dose of the preparation should be increased, while at above 55°C the enzyme will be inactivated. β-Glucanases can only be used at temperatures above 15°C, as they require a longer incubation time. Enzyme dosage should also be increased with low pH values. Enzyme activity is not inhibited by optimal doses of sulfur dioxide in wine. With red wines, to the extent that inhibition of enzymatic activity may occur under the action of phenolic compounds, this allows for an increase in the dose of product administered. Alcoholic concentrations up to a level of 14% vol. do not impact negatively the action of enzymes. On the contrary, they may have an activating role on β-glucanases used to release flavor compounds [18]. The main benefits of using enzymes in the winemaking process are to do with their specificity of action, that is, less likely to produce unwanted secondary substances; their biodegradable nature and low impact on the environment; their capacity to get activated in conditions of low temperature, neutral pH, and normal atmospheric pressure; a significant reduction in energy consumption. Besides the many benefits, some unwanted activities of commercial preparations used in winemaking have been reported. They show high sensitivity to changes in physicochemical environmental conditions and can be distorted with relative ease (temperature, pH, infestations), which leads to an increase in the concentration of methanol during alcoholic fermentation under the action of methyl ethyl esterase. The action of cinnamyl esterase, present in enzymatic preparations based on pectinases, is responsible for the formation of a larger number of volatile compounds [21]. These preparations are considered technological aids which are not found in the final product. Figure 5 illustrates the main enzymatic preparations used in the technology of winemaking and some recommendations for their administration.
Figure 5.
Enzymes preparations usually used in enology.
Comprehensive knowledge of regulations governing all treatments administered during the production stage is required, including timings, legally allowed amounts, and method of use. The use of enzymatic preparations in the beverage industry must comply with the regulations and recommendations of the International Organization of Vine and Wine, the Association of Manufacturers of Fermentation Enzyme Products, the World Health Organization, the United Nations Food and Agriculture (FAO), and the Food Chemical Codex [18].
8.1 Action of enzymes on the reaction yield obtained by pressing the must
The extraction yield of the juice can be significantly improved under the action of enzymes. Commercial preparations show various enzymatic activities at low pH values (pectin methyl esterases, polygalacturonases, pectin lyases, and hemicelluloses). These preparations may also contain various glycosides and proteases which are responsible for secondary transformations. Therefore, it is necessary to ensure a high degree of enzyme purity [19]. Pectinases are considered to be among the most important enzymes in the commercial sector, especially in the processing of fruit juices, that is, as adjuvants for the clarification and stabilization of juices, and to obtain a high yield as well. The degradation of cell walls under the action of pectinases allows for the wider diffusion of the constituents inside the vacuoles and facilitates a better extraction of the must during pressing [18]. The outcome depends on the amount of grape pectin, which varies according to the degree of maturation and grape variety, the enzymatic preparation administered (the type of enzymatic activity), and the conditions of administration (specific incubation time, pH of the environment, temperature, and presence of inhibitors). If pectinases are applied to grapes before pressing, they will increase the yield of juice extraction and color compounds [21]. The increase in pressing yield can reach at least 10%, in correlation with a reduction of up to 20–50% in the time needed for pressing, depending on the quality of grapes and the targeted result. When enzymes are applied without pre-fermentative maceration, their action mainly occurs at the time of pressing. At the maceration-fermentation stage, the enzymes are added immediately after the reception of the grapes. This improves the pressing efficiency and also the enzyme’s degree of action. Pre-maceration is usually performed for about 3–4 hours at temperatures around 20°C, and 6–10 hours at temperatures below 15°C [18].
8.2 Influence of enzymes on wine clarification
During the processing of white and rosé wines, and especially after pressing, the must is rich in solid particles. Negatively charged pectin molecules form a protective layer around positively charged solid particles and keep them in suspension. Excessive turbidity of the must lends an herbaceous aroma to the wine, not to mention the hydrogen sulfide odor and a high content of isoamyl alcohol [18]. Consequently, clarification of the must before the alcoholic fermentation is particularly important as it considerably reduces the formation of aromatic compounds that give the wine unpleasant spicy notes or a salty sensation. Enzymatic preparations for clarification have predominantly pectolytic activities. Hydrolysis of pectic substances (Figure 6) leads to a significant reduction in the viscosity of the must [19]. During winemaking operations, segments of grape pectic compounds are released into the must after crushing and pressing. They form colloids that reduce or prevent the sedimentation of solid particles, especially skin fragments. Removal of solid particles is an important operation in the technology of obtaining white wines. Enzymatic hydrolysis of pectic structures is considered the most efficient method of decomposing colloids as it allows for the separation of captured solid particles. The presence of polygalacturonase and pectinase activity in grapes favors the clarification of the must after crushing. However, the activity of these enzymes is often insufficient since the time needed to obtain optimal clarification under the action of grape pectinases cannot compare with the time spent in classical winemaking processes. Therefore, for improving the efficiency of the clarification process, commercial preparations based on pectinases may be added [21]. Such preparations are added before the fermentation of musts of white varieties obtained after pressing and without prior maceration to accelerate clarification. Enzyme administration is recommended as a pre-fermentative treatment because the high levels of alcohol resulting from fermentation tend to inhibit enzymatic activity.
Figure 6.
Action of enzymes on grape pectin chains.
Moreover, the use of pectolytic enzymes in wine technology is often associated with the maceration after heating the harvested grapes technique for red wines. This involves heating the must to 50°C and maintaining it at this temperature for several hours to solubilize the anthocyanins in the skin. The procedure sees the extraction of procyanidins in excess, which imprints astringency on the wine. In this way, the wines acquire an intense color but are not suitable for long-term aging. During heating, large amounts of pectin can be extracted from grapes, a phenomenon that does not occur in traditional processing. It becomes therefore necessary to administer a pectolytic preparation to reduce the viscosity of the must and remove the colloidal protective action of macromolecules with six carbon atoms (e.g., hexanol and hexanal) [19]. Following this process, the extraction of anthocyanins is intensified due to the decomposition of the cellular structure by the enzyme, which allows for easier dissolution of pigments. In traditional winemaking, the use of pectolytic enzymes triggers a significantly accelerated release of pigments, while maceration time can be shortened from 4 to 2 days. A potential disadvantage of this process is that the anthocyanins in the wines produced in this way can be unstable due to the hydrolysis of anthocyanin glycosides into their much more unstable aglycone forms. Secondary activities of enzymatic preparations are considered responsible for this glycosidic action [19]. The clarification of the must is carried out in three stages. The first stage is depectinization, characterized by the partial decomposition of pectins and the reduction of the must’s viscosity. The second stage, flocculation, is described by an increase in turbidity and the formation of insoluble complexes. The third stage, sedimentation, is mainly characterized by a strong reduction in the turbidity and precipitation of complex molecules. Enzymes improve the first stage, thereby helping to accelerate the subsequent steps [18]. Significant improvements in clarification’s degree have been reported with the use of pectolytic enzymes, β-glucanases, or proteases. Of these, proteases have been studied as an alternative to bentonite treatment, which would induce many chemical changes in the environment. Thus, Mojsov et al. [21] highlighted the degradation of enzymes that cause wine turbidity by administering enzymatic preparations based on lysozyme obtained from Botrytis cinerea.
8.3 Impact of enzymes on wine filtration
Enzymes for maturation and filtration consist mainly of pectinases and β-glucanases. Excess colloids are able to prevent filtration. Pectinases partially hydrolyze grape’s polysaccharides and release smaller polysaccharide fragments. The latter usually presents a linear molecular structure; given the fact that these fragments can obstruct the different stages of wine filtration, their elimination before filtration is necessary. β-Glucanases hydrolyze glucan-type polysaccharides from Botrytis cinerea or yeast cell walls. Such polysaccharides are characterized by a high molecular weight and prevent or even make filtration impossible. The glucans released in wines by yeast (Saccharomyces cerevisiae) depend on the media used for yeast fermentation. At the same time, β-glucans can stimulate the extraction of certain macromolecules as mannoproteins which have an important role in stabilizing proteins in wines. A reduction in the size of these components makes them more soluble, maintains the colloidal structures in the wine during filtration, and diminishes the risk of filter blockage. The administration of enzymatic treatments can result in volumes up to five times higher during a filtration cycle, which helps to increase filtration efficiency with a reduction in costs, without affecting the sensory properties of the wine. These hypotheses were also confirmed by Mojsov et al. [21]. It is recommended that these enzymatic preparations be administered at the end of the alcoholic fermentation at a temperature above 15°C [18].
8.4 Action of enzymes on lactic acid bacteria
Due to their antibacterial action, lysozymes can inhibit the growth of bacteria, lactic acid bacteria in particular. Lysozyme administration can be an alternative to reducing the dose of SO2 in low pH white wines [10]. These enzymes are able to degrade the cell walls of lactic acid bacteria - among other types, which make them an effective tool in preventing malolactic fermentation and increasing the stability of wines. The maximum regulated amount is 0.5 g/L in must or wine [22]. These oenological preparations are obtained by extraction from egg whites. For this reason, wines treated with lysozymes have to be labeled as “potentially allergenic.” As pointed out in the literature, lysozymes reduce the concentration of biogenic amines in wine. In general, wines to which lysozymes have been administered are not to be conditioned by bentonite fining [10].
8.5 Influence of enzymes on color and basic physicochemical parameters of wines
The physicochemical properties of wines depend on the characteristics of the raw material, technological specificities, and the conditions in which fermentation takes place [10]. No significant influence has been reported on the main physicochemical parameters of wines [23, 24] following administration of enzymatic treatments. The visual characteristics of a wine depend on the degree to which its chemical structure and the compound’s nature are able to absorb, transmit, and reflect light radiation from the visible spectral domain (between 380 and 750 nm). In recent years, oenological practices have considered enhancing the chromatic characteristics of wines by focusing on improving the extraction of color compounds. Although initially used to reduce turbidity and promote clarification, pectolytic enzymes have been demonstrated to be effective in intensifying color intensity and brightness, as well as the extraction of phenolic compounds [19, 21]. Similar results regarding the significant action of enzymes on the chromatic characteristics of white wines have been published by Ducasse et al. [25]. Guérin et al. [26] reported an improvement in wine brightness generated by the use of diverse enzymatic preparations. On the other hand, Bautista-Ortin et al. [27] obtained indecisive results in terms of changes in color parameters (intensity and hue), while Bozaran & Bozan [28] showed a reduction in color intensity and stability. These differences can be explained by the use of different enzymatic preparations and winemaking technology, but also by the presence of other uncontrolled factors in experimental studies. Along the same lines, the results published by Scutarașu [24] confirm the significant impact of using various enzymatic treatments on the values of the chromatic parameters of wines in the sense that a higher level of clarity is obtained (Figure 7). Bentonite treatment usually generates a significant decrease in the main chromatic parameters (clarity, chromaticity, and saturation) and increased values for tonality.
Figure 7.
Effect of enzymes on wine color and clarity [24].
8.6 Impact of enzymes on wine phenolic compounds
The phenolic compounds in wine may originate in both grapes and external sources, such as the wood of the barrel in which they are stored and the cork used for bottling; alternately, they can appear after the administration of various oenological treatments. Figure 8 represents the influence of enzymatic preparations on the content of phenolic compounds in some white wines studied by Scutarașu [24].
Figure 8.
Influence of enzymes on phenolic profile of Sauvignon blanc (left) and Fetească regală (right) [24].
The level of these compounds depends on plant characteristics, analyzed variety, geographical location, specific year and harvesting procedure, and winemaking practices [10]. Phenolic compounds belonging to the group of flavones and flavonoids, especially hydroxycinnamic constituents (caffeic, p-coumaric, and ferulic acids) are mainly responsible for the color of white wines. In addition, the most common flavonoid derivatives in white wines are represented by quercetin, hesperidin, kaempferol, and rutin [29]. The proportions of the phenolic compounds are variables, participating in numerous physical, chemical, and biochemical processes. As a rule, in the first phase of the fermentation process, the oxidation of phenolic compounds that come from the raw material occurs under the action of enzymes. Some phenolic compounds may participate in the polymerization reaction with various flavor compounds. Hydroxycinnamic acids are involved in many oxidation reactions. Phenolic acids have proven to be important markers for Fetească regală and Sauvignon blanc varieties from different wine regions of Romania and France [30]. The effects of enzymatic treatments on the chemical composition of wines have been studied intensively and far-reaching research on the influence of similar oenological products [27, 31, 32] reported significant increases in the phenolic content of wine. In general, the extraction of phenolic compounds occurs with the maceration of must and during alcoholic fermentation, and it depends on the variety and quality of the grapes and on winemaking technology. The effect of fungal laccase has been studied extensively due to its capacity of reacting with a wide range of phenolic compounds. Lacasse treatment is likely to increase the effect of conventional stabilization treatments [18]. Pectinases have been shown to be effective in enriching the medium in protocatechuic, caftaric, trans- and cis-resveratrol acids with the Sauvignon blanc variety, and in p-coumaric and gentisic acids with the Fetească regală variety (both from Iași vineyard, Romania) on condition they are administered in the must at the beginning of alcoholic fermentation [24]. Fining wines (previously treated with enzymes) with bentonite leads to lower values of phenolic compound concentrations. This phenomenon is due to the indirect adsorption effect of protein-binding phenolic compounds [10].
8.7 Effect of enzymatic treatments on wines’ amino acids level
Wine amino acids can result from the degradation of grape proteins following the metabolism of yeasts and lactic acid bacteria, and from the autolysis of yeasts and bacteria. The profile and concentration of these compounds in wines can be influenced by several factors, such as grape variety, cultivation (treatments with nitrogen), and winemaking technology (e.g., maceration-fermentation process), as a result of amination and transamination of aldehydes and ketones, etc. Amino acids are particularly important for the formation and development of wine aromas (they are metabolic precursors of higher alcohols, volatile acids, and esters), and prove to be major factors in determining the authenticity and typicality of beverages. Insufficient amounts of such compounds can lead to incomplete fermentation and undesirable changes in the wine, such as hydrogen sulfide production and increased acetic acid proportions [33]. Amino acid concentration is also an important criterion for classifying wines according to their composition characteristics [34]. These compounds are highly reactive, being precursors of many flavor compounds, such as higher alcohols, esters, lactones, amines, etc. [10]. Most of the studies are focused on studying amino acids for classifying and differentiating wines according to variety, age, winemaking technologies, authentication, and typicity assessment [35]. According to the data presented by Cosme et al. [36], the synthesis of amino acids in grapes usually occurs at the end of the ripening stage, with proline and arginine being the main identified nitrogen compounds, followed by alanine, aspartic acid, and glutamic acid in smaller amounts. Numerous authors highlighted an important variation of the amino acid profile, depending on the grape variety and enzyme treatment. In this regard, Scutarașu [24] presented considerable amounts of some essential amino acids, such as histidine, isoleucine, phenylalanine, and tryptophan in wines treated with pectolytic enzymes preparations. The administration of pectolytic enzymes was more effective in the Fetească regală wines, in applied work conditions, although the β-glycosides generated the highest values of most amino acids in the Sauvignon blanc. Agustini et al.[37] obtained high proline and arginine concentrations in wine. The two compounds are not consumed during alcoholic fermentation due to anaerobic conditions and arginine metabolism. Beltran et al. [38] reported high amounts of asparagine (approximately 45 mg/L), lysine (16 mg/L) and proline (approximately 500 mg/L). The data published by Scutarașu [24] indicate a major impact of both the type of enzyme administered and the grape variety on the characteristics of the wine (Figure 9).
Figure 9.
Effect of enzymes on amino acid content in Fetească regală wines (mg/L) [24].
Considerable amounts of some essential amino acids, such as histidine, isoleucine, phenylalanine, and tryptophan, were documented in the samples of Fetească regală and Sauvignon blanc (from Iași vineyard, Romania) treated with pectinases. As concerns the increased proportions of the amino acids under research, the administration of pectolytic enzymes was more efficient for Fetească regală wines, while β-glycosides generated the highest values of most amino acids in Sauvignon blanc samples when applied before alcoholic fermentation. Burin et al. [39] demonstrated a reduction in amino acid levels following the application of various fining and stabilization treatments, including the administration of pectolytic enzymes. Pinu et al. [40] have monitored the level of nitrogen compounds and their variation during the winemaking. Some amino acids, such as tyrosine, glycine, or arginine, were not exhausted by Saccharomyces cerevisiae during Sauvignon blanc alcoholic fermentation, which confirms previous observations on white wines made by Pinu et al. [40]. According to Cotea et al. [10], bentonite treatment can reduce wine protein levels by up to 15%.
8.8 Influence of enzymes on wines’ volatile compounds
Wine’s volatile compounds may originate from the grapes, being transferred to the must during processing, or may form during alcoholic fermentation, due to the biochemical reactions that occur in the wine. The administration of various pre-fermentative treatments significantly influences the aromatic profile of wines. The action of enzymatic treatments on the cell walls of grapes’ skin is illustrated in Figure 10.
Figure 10.
The action of enzymes on the cell walls of grapes’ skin—control (A) and after the administration of maceration enzymes (B) [18].
The free forms of varietal (terpenes) and combined (terpene glycoside) aromas are subject to oxidation and hydrolysis and are influenced by numerous biochemical and technological factors [10]. Most varietal aromas develop during fermentation, which suggests that the microbial species responsible for the fermentation process play a special role in releasing them from non-aromatic precursors. Enzyme preparations based on β-glycosidases can be added during winemaking to stimulate the extraction of volatile compounds from glycosidic bonds, especially monoterpenes, norisoprenoids, and benzenoids [21]. The varietal character of white wines is mainly defined by the presence of molecules with a characteristic odor, among which monoterpenic alcohols play a prominent role. These compounds are found in grapes as free, volatile, odorous molecules, and as non-volatile glycosidic precursors, known as bound terpenes. In many grape varieties, the number of bound terpenes may be higher than the number of free terpenes. Consequently, the distinctiveness of wines could be increased by the release of terpenes with glycosidic bonds [21]. The presence of glycosylated precursors and volatile compounds in grapes was reported by Cordonnier & Bayonove [41]. In the late 1980s, enzymatic preparations containing glycosidases (β-glycosidase, α-arabinosidase, α-rhamnosidase) were developed to improve the aromatic profile of certain wines. These enzymatic preparations are usually added at the end of alcoholic fermentation and during wine transfer, in the absence of bentonite, to prevent inhibition of the enzyme. The optimum temperature for such enzyme treatments has to be in excess of 15°C, and an incubation time of a few weeks to a month is needed. The development of aromas has to be controlled by organoleptic analysis, and enzymatic action can be inhibited by the addition of bentonite. Small amounts of bentonite (20 g/hL) are usually sufficient to block the activity of enzymes completely [18]. Although much of a wine’s aroma is attributed to alcohols and esters derived from yeast’s metabolism, several grape varieties, such as Muscat, Gewürztraminer, Riesling, and Chardonnay, are characterized by specific, fragrant notes due to the presence of volatile monoterpenes such as linalool, geraniol, α-citronellol, and nerol [19]. These are released from the grapes during pressing, fermentation, and storage. Unlike many volatile fruit compounds, these compounds are glycosidically bound and are released slowly through acid hydrolysis exclusively, during wine aging. As the activity of endogenous glycosidases is very modest, there has been considerable interest in adding enzymes that promote the extraction of monoterpenes during winemaking. The secondary activities of fungal pectinases (e.g., Aspergillus niger) or extracellular glycosidases of various Candida yeasts may be used for this purpose [19]. Mateo & Stefano [42] pointed to the likely inhibition of β-glycosidase activity in the presence of ethanol and glucose. Enzymatic preparations have to be free of cinnamyl decarboxylase, which is instrumental in the formation of ethyl-phenols that give off an animal odor [19]. Numerous studies have indicated enrichment in the flavor profile of wines following administration of various enzymatic preparations. Thus, Masino et al. [31] obtained an increased level of the compound 4-vinylphenol in pectinase-treated samples. The action of pectolytic enzymatic preparations and β-glycosidases in obtaining white wines was also studied by Rusjan et al. [43] who recorded a significant increase in the concentrations of some monoterpenes (such as geraniol, nerol, linalool, or α-terpineol) compared to the control variant. Later on, Rusjan et al. [43] studied the effect of enzymatic preparations on white wines’ terpenes. In this situation, the level of linalool did not increase significantly compared to the control sample. These results are supported by the use of enzymatic preparations with reduced α-rhamnosidase, α-arabinosidase, and β-glycosidase activity. Consequently, the choice of enzyme preparations suitable for the purpose proposed is of particular importance. Armada et al. [44] studied the effect of administering pectolytic enzymes in white wines obtained from the Albariño variety on the evolution of volatile compounds. All samples exhibited different aroma profiles, compared to the untreated ones, and samples obtained following the application of maceration enzymes showed the highest level for ethyl esters or phenethyl acetate. The use of maceration enzymes in combination with fining enzymes has been proved inappropriate due to the fact that glycosidic enzymes block the formation of flavor compounds. The main monitored components revealed differences between wines treated only with maceration enzymes (glycosidases) and wines to which other types of enzyme treatments were applied. Rocha et al. [45] reported a significant increase in the concentrations of geraniol, terpenoids, phenols, alcohols, and esters for the Maria Gomez variety, while no major changes in these compounds were observed for the Bical variety. The two varieties come from the same geographical area (Bairrada), which indicates that the extraction of flavor compounds under the influence of enzymes is closely related to the aromatic potential of the analyzed variety. According to other authors, the main volatile compounds of Sauvignon blanc wines are mercaptans (4-mercapto-4-methyl-2-pentanone), while others present methoxypyrazines (represented by 3-mercaptohexyl) as the defining compounds for the mentioned variety [46]. The aromatic profile of the wine depends on many factors, including the winemaking technology and the particulars of the geographical region. With reference to the study conducted by Scutarașu [24], the fining of wines (to which enzymes were added) with bentonite triggered changes in the proportions of volatile compounds depending on the compounds’ class, grape variety, and administrated enzyme types. Regarding the level of carbonyl compounds in Sauvignon blanc wines, bentonite treatment led to increased quantities of acetoin (3-hydroxy-2-butanone) and benzaldehyde. Bentonite-treated Fetească regală samples exhibited reduced levels of acetoin. Other studies reported similar changes in these compounds [47]. Some authors have focused on the impact of bentonite on ethyl esters concentrations. For instance, Vincenzi et al. [47] reported a decreasing trend in the proportion of ethyl alcohol esters, being protein bound. Lambri et al. [48] reported a decrease in the content of ethyl butyrate and ethyl hexanoate in Chardonnay wines. Sanborn et al. [49] obtained a decrease in the level of ethyl decanoate and phenylethyl acetate in Gewürztraminer wines, while no changes were reported for Chardonnay wines. In the experimental samples obtained by Scutarașu [24], this hypothesis was confirmed by ethyl butanoate and ethyl dodecanoate in Sauvignon blanc samples and ethyl hexanoate, ethyl octanoate, ethyl 3-hydroxybutanoate, ethyl decanoate, and ethyl 4-hydroxybutanoate in most variants of Fetească regală wines, respectively. Moreover, regarding the level of fatty acids, the main precursors of aromatic esters, a decrease of butanoic, octanoic, and decanoic acids content in the Fetească regală samples was registered, correlated with an increase in the ratio of hexanoic and octadecanoic acid. In bentonite-treated Sauvignon blanc wines, 3-methylbutanoic, hexanoic, octanoic, and decanoic acids had higher values. The interaction of this treatment with fatty acids has been studied by several authors. Vincenzi et al. [47] also reported an increase in decanoic and dodecanoic acid concentrations as well as a decrease in the amount of octanoic acid in Muscat wines. McKinnon [33] showed a positive correlation between the level of decanoic acid and phenylalanine. Table 1 presents the impact of enzymes on some volatile compounds in wines.
Table 1.
Effect of enzyme treatment on wine’s volatile compounds.
8.9 Effect of enzymatic treatments on sensory properties
As far as the consumer is concerned, the organoleptic characteristics of foods are the decisive factor influencing purchase choice. The administration of enzyme treatment is mainly aimed to enrich and improve the sensory profile. In direct correlation with the data presented in this chapter, major organoleptic differences have been reported between wines treated with various enzymatic preparations. Enrique et al. [51] indicated a significant increase in the intensity of the sensory descriptors studied in samples treated with pectolytic enzymes. Scutarașu [24] confirmed that pectinases can improve the sensory characteristics of wines compared to β-glycosides (Figure 11) and that the samples are generally characterized by the lowest intensity for some negative descriptors, such as phenolic, mineral, or bitter taste. This research highlighted that β-glycosides can give effective results when administrated before alcoholic fermentation, in must.
Figure 11.
Effect of enzymes on sensory properties of wines [24, 52].
Sun et al. [52] obtained higher levels of acidic fruits, sweet fruits, and other notes in wine treated with enzymes. The application of H. uvarum extracellular enzyme enhanced fruity and floral aroma, especially the acidic fruits notes [52]. According to the data presented by Bautista-Ortin et al. [27], wines treated with pectinases had higher scores for their herbaceous, dryness, astringency, and bitterness characteristics, and showed lower equilibrium than the control sample. McKinnon [33] reported a positive correlation between leucine levels and fruity or floral notes in samples treated with pectolytic enzymes. Gonzáles-Lázaro et al. [53] indicated that pectolytic enzymes did not show effective results in sparkling wines when these preparations are administrated on unripe grapes.
Wine quality is dependent on grape characteristics and winemaking technology. Enzymes’ activity is influenced by their concentration, substrate, pH, temperature, pressure, and the presence of inhibitors and activators. Several authors confirmed the positive impact of using enzymes on wine quality. However, higher concentrations of phenolic compounds and amino acids and enriched volatile and sensory profiles can be obtained when enzyme preparation is used. Enzymes contribute to optimizing the technological process in view of improving the quality of the final product, while giving effective results when they are administrated at different moments in winemaking. Summing up all the above, enzymatic preparations will remain in focus for the near future to analyze possible new applications in the food and non-food industries.
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Written By
Elena Cristina Scutarașu, Camelia Elena Luchian, Lucia Cintia Colibaba and Valeriu Cotea
Submitted: 12 May 2022Reviewed: 19 May 2022Published: 21 June 2022
Open access peer-reviewed chapter
