dairy ingredients for food processing (chandan/dairy ingredients for food processing) ||...

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Chapter 12

Enzyme - modifi ed Dairy Ingredients

Arun Kilara and Ramesh C. Chandan

Introduction

Enzymes are biocatalysts. Catalysts speed up reaction rates without undergoing permanent changes, and biological catalysts are impor-tant in all life processes. It is an axiom that life itself would not exist if not for enzymes. The operating characteristics of enzymes include substrate specifi city, optimum tem-perature, optimum pH, and other environ-mental factors. Almost all known enzymes are proteins and range in size from 13,000 to several million Da. Only a few residues within a structure of the enzyme are impor-tant for the catalytic activity.

Commercial enzymes are derived from animal, plant, and microbial sources. Although the International Union of Bio-chemists recommends scientifi c classi fi cation of enzymes, colloquial names predominate in the food industry. Most enzyme names end in “ – ase ” and are derived from the source of the enzyme or the substrates which they act on. Hence, enzymes acting on proteins are called proteases, those acting on lipids, lipases; and carbohydrates, carbohydrases. Among proteases, enzymes derived from plants such as those from papaya ( Carica papaya ) are called papain; from fi g latex, fi cin; or from pineapples, bromelain. Clearly, these proteases do not end in “ ase, ” refl ecting the vagaries of colloquial nomenclature of

enzymes. Animal - derived enzymes come from the organs of domesticated farm animals; trypsin, chymotrypsin, pre - gastric lipase, pancreatin, and the like are commonly isolated from porcine or caprine sources. Both animal and plant sources of enzymes impose severe constraints on the production of large quantities of enzymes. Therefore, with the advent of biotechnology, microbial enzymes prevail.

Microbial enzymes for commercial use are extracted from yeasts, molds, or bacteria. They are contained within the cells or secreted into the growth medium. Enzymes contained within the cells are termed endocellular, whereas those secreted into the growth medium are extracellular. Extracellular enzymes are generally low - molecular - weight proteins (30 to 50 kilodaltons) and contain disulfi de bridges. Endocellular enzymes reg-ulate the metabolism of organisms (regula-tory enzymes) and are of higher molecular weights, and often their structures involve subunits.

Materials on which enzymes act are called substrates. Those of interest in the dairy industry are proteins, lipids, and lactose. As detailed in Chapter 2 , milk proteins are clas-sifi ed as caseins and serum proteins. Milk fat consists of triglycerides and a number of other lipid substances. Lactose is the main carbohydrate in milk.

Enzymes transform fl uid milk to products. For example, treating milk with the enzyme lactase yields low - lactose milk suitable for consumption by people unable to digest

Dairy Ingredients for Food ProcessingEdited by Ramesh C. Chandan and Arun Kilara© 2011 Blackwell Publishing Ltd. ISBN: 978-0-813-81746-0

318 Chapter 12

of strategies to achieve them is critical for developing and using modifi ed protein ingre-dients. Numerous reports on enhancing the functionality of food proteins by limited pro-teolysis have been published. Functionality imparts a valuable dimension to food pro-teins, complementing their established nutri-tional value.

The functional properties of proteins are those physicochemical properties that govern their performance and behavior in food systems during their preparation, processing, storage, and consumption. These properties are infl uenced by the nature and extent of interaction of the proteins with themselves, other components, and water in the food system. The factors that affect the functional properties of food proteins have been clas-sifi ed as intrinsic factors, extrinsic factors, and process treatments or conditions. The food system and the processing dictate most of the extrinsic factors and process treat-ment/conditions and storage conditions, and hence are amenable to a limited range of maneuverability.

Endopeptidases cleave the peptide linkage between two adjacent amino acid residues in the primary sequence of a protein, yielding two peptides. Factors affecting the enzymatic hydrolysis of proteins include enzyme speci-fi city, extent of protein denaturation, sub-strate and enzyme concentrations, pH, ionic strength, temperature, and absence or pres-ence of inhibitory substances. The specifi city of an enzyme is a key factor, infl uencing both the number and location of the peptide link-ages that are hydrolyzed. Proteolysis can proceed either sequentially, releasing one peptide at a time, or through the formation of intermediates that are further hydrolyzed to smaller peptides as proteolysis progresses, which is often termed the zipper mechanism.

The extent of proteolysis is quantifi ed as the degree of hydrolysis (DH), which refers to the percentage of peptide bonds cleaved. The DH is commonly measured and monitored by the amount of base that is

lactose. The enzyme chymosin, or rennet, is a key to the manufacture of cheeses and gelled dairy products. Microbes that contain enzymes transform milk to yogurt, and cream to sour cream and cream cheese. In these microbial transformations, the lactose content of products is reduced and desirable textures are created. Various chapters in this book discuss microbially transformed dairy products.

Enzyme - modifi ed proteins and lipids are another category of dairy ingredients. Although dairy ingredients are relatively expensive to use in cost - sensitive formula-tions, in some instances, modifi cation of functionalities of ingredients is desirable and enzymes are useful for this purpose. For example, enzymes help to obtain the nutri-tionally desirable fractions of macromole-cules that have recently become popular additions to foods.

This chapter discusses protein and lipid modifi cations in dairy ingredients. Lactose modifi cation is not discussed because it is used only in fl uid milk products (beverage milk).

Protein Modifi cation

Proteins are increasingly being used to perform functional roles in food formula-tions. Common food proteins have good functional properties including solubility, gelation, emulsifi cation, and foaming. The functional properties of proteins are impaired near their isoelectric point (pI), as is the case in most acidic foods. Enzymatic modi-fi cation of food proteins by controlled prote-olysis can enhance their functional properties over a wide pH range and other processing conditions.

Choosing the right proteolytic enzyme, environmental conditions for hydrolysis, and degree of hydrolysis is crucial for enhancing the functional properties of proteins. Under-standing the molecular properties required for protein functionality and the development

Enzyme-modifi ed Dairy Ingredients 319

• A decrease in molecular weight

• An increase in the number of ionizable groups,

• The exposure of hydrophobic groups hith-erto concealed

The changes in the functional properties of a protein are a direct result of these effects.

Food - grade proteolytic enzymes have dif-ferent pH and temperature optima, and they hydrolyze a variety of peptide bonds. Depending on the specifi city of the enzyme, environmental conditions, and extent of hydrolysis, a wide variety of peptides are generated. The resultant protein hydrolyzate contains low - molecular - weight peptides as well as higher molecular - weight peptides and unhydrolyzed proteins. Membrane fi ltration can be used to fractionate the hydrolyzate into a low - molecular - weight permeate and a high - molecular - weight retentate; the func-tional properties of the two fractions may be vastly different. In the case of whey proteins, permeates have been found to have better foaming and interfacial properties than reten-tates. However, this additional fi ltration step decreases yields and increases the production costs, and should therefore be avoided if possible.

Proteases, or proteinases, are the most important class of enzymes from an eco-nomic viewpoint. Mucor protease coagulates milk for cheese manufacture. Production of protein hydrolyzates and fl avoring materials is discussed below.

Improvement in Functional Properties

There is room for improvement in the func-tionalities of commonly used food proteins, and physical, chemical, and biological methods of altering proteins have been attempted. Among the most interesting and important is the hydrolysis of proteins by enzymes, which has been covered exten-sively. Changes in functionality of proteins upon hydrolysis result from the reduced

consumed to maintain the pH during hydro-lysis (the pH stat method); by the depression of the freezing point, which indicates the increasing osmolarity (osmometry); or by the increase in solubility in trichloroacetic acid. DH values determined by different methods are often not directly comparable. Good correlation between base consumption and soluble - nitrogen content was established in the case of tryptic hydrolysis of whey protein. The base consumption and osmom-etry methods are easy to perform, allow-ing continuous monitoring of the hydrolysis process, whereas the estimation of soluble - nitrogen content using the Kjeldahl method is time - consuming and cannot be used as an on - line process control tool. The effects of enzymatic hydrolysis, using pepsin, Prolase, or Pronase, on some of the functional prop-erties of whey protein were reported as early as 1974.

Infant food formulations are hydrolyzed to a greater extent, and are classifi ed as slightly, moderately, or extensively hydro-lyzed, depending on the molecular weight distribution of the resultant hydrolyzate. Extensive hydrolysis is normally used to produce hypoallergenic hydrolyzates, with no peptides greater than 5,000 Da and almost 90% of them approximately 500 Da, whereas hydrolyzates used as protein supplements may undergo less - extensive hydrolysis.

A commonly encountered problem with extensively hydrolyzed proteins is bitterness due to the accumulation of low - molecular - weight peptides containing hydrophobic amino acids. This problem can be solved either by selecting proteases that are not bitter or by adding specifi c peptidases to de - bitter the hydrolyzate.

Globular proteins are characterized by specifi c secondary and tertiary structures involving disulfi de linkages and hydrophobic interactions between amino acid residues within the same molecule or between mole-cules. Three distinct effects accompany enzy-matic hydrolysis of proteins:

320 Chapter 12

studies showed that the observed increases in soluble nitrogen were due to the production of proteose peptones, rather than the break-down of the components of the micellar casein. It has been speculated that micellar size might be affected by such hydrolysis. The hydrolyzed micellar casein lowered interfacial tension more than the unhydro-lyzed controls, leading researchers to con-clude that the peptides generated by such hydrolysis had good surface activity.

Almost all dairy proteins could be improved by treatment with proteases. The increased solubility of enzyme - modifi ed pro-teins is advantageous in the manufacture of whey - protein - enriched nutritional supple-ments that are otherwise vulnerable to coagu-lation and fouling during thermal processing.

Emulsifi cation is facilitated by surfac-tants, and proteins are considered surface active. Stable coexistence of an oil phase and an aqueous phase in a food system is neces-sary for good emulsifi cation properties. Dispersion of one phase into another with which it is normally immiscible requires input of mechanical energy, and results in the increase of the interfacial area by several orders of magnitude. Thermodynamic insta-bility leads to the rapid separation of the two phases unless stabilized by surfactants capable of lowering the interfacial tension between the phases. Proteins, by virtue of their amphipathic nature, exhibit good emul-sifying properties. Two aspects of the behav-ior of proteins at the oil - water interface in forming stable emulsions are generally studied. The fi rst is emulsifying capacity, which is a quantitative measure of the amount of oil emulsifi ed by unit weight of the protein. The second is emulsion stability, which mea-sures the ability of the formed emulsion to remain unchanged over time.

Many proteins are surface active and are widely used to control or infl uence interfacial behavior. The primary and secondary struc-tures of proteins have been thought to be important in the observed emulsifying func-

molecular weight of the peptides, loss or alteration of native structure, and enhanced interaction of peptides with themselves and the environment. The functional properties of milk proteins are well characterized, understood, and used in food applications, but the properties of peptides generated by enzymatic modifi cation are still relatively enigmatic.

Casein, which is used in a variety of foods and whose amino acid composition and sequences are known, has been widely studied for its functional properties. Limited proteolysis improves such surface properties as emulsifi cation and foaming. The foaming property of caseins is important in whipped cream, ice cream, whipped toppings, and mousses.

Controlled enzyme modifi cation of casein and whey proteins by proteolysis generates a total hydrolyzate, comprised of unhydro-lyzed substrates, hydrolyzed substrate, and the low - molecular - weight materials. The total hydrolyzate is further fractionated into low - molecular - weight peptides and high - molecular - weight materials. Because the pro-teolytic reactions are terminated with thermal destruction of the enzyme, sometimes in con-junction with an adjustment of pH, the state of the products in the reaction milieu is usually different from that of the unreacted substrates. A majority of the published research pertains to the total hydrolyzate, while recent research has investigated the nature of the other two fractions from enzyme - modifi ed proteins.

Solubility is considered an essential pre-requisite for the manifestation of many func-tional properties, and enzyme hydrolysis has been directed toward improving solubility of milk protein substrates. Hydrolysis of micel-lar casein dispersion by alkaline milk pro-teinase (native to milk) increased the rennet coagulation time after extensive hydrolysis but had no effect on its ethanol stability. Such hydrolysis resulted in a decrease in viscosity of the micellar casein suspension. Further


activity. The amino acid composition and sequence of this peptide may also be an important consideration. Such evidence is being provided by studies in which peptides with good surface activity have been isolated and characterized with respect to their chain length and sequence. The environmental con-ditions of the test also play an important role in the functional properties of the peptides. Additionally, mixtures of peptides may have synergistic or antagonistic effects on surface activity.

Similar to emulsifi cation, foaming requires a protein to be surface active. Dispersion of air in a continuous liquid phase generates a large interfacial area. Stabilization of this air - water interface by surfactants such as pro-teins or peptides results in the formation of foam. The role and behavior of proteins at the interface is governed by the primary and sec-ondary structures of the peptide chain. A diverse array of products ranging from ice cream to cappuccino gain their identity and structure from the foaming properties of milk proteins. Enzymatic modifi cation of proteins generates peptides with altered foaming properties. In contrast to the emulsifi cation properties of enzyme - modifi ed dairy pro-teins, relatively little work has been done in the area of foaming properties. It will be interesting to understand the structural and sequential properties necessary to form good foams from these substrates. With advances in genomics, it may be possible to screen for proteases that can specifi cally hydrolyze the bonds desired, leading to a target production of enzyme – modifi ed ingredients.

Dairy proteins play an important role in gelation, another desirable property in foods. A gel is easier to recognize than defi ne. Qualitative descriptions of gels can help with this recognition. Gels are solids that are able to store the work expended in their defor-mation and recover to their original shape. Often, gels are soft solids that can be deformed and contain a large proportion of low - molecular - weight liquid. Thus, gels

tionality. However, parts of a protein mole-cule may consist of amphipathic α - helices and β - sheets with high surface activity, so that a peptide designed to have a maximally stabilized structure should have a higher potential specifi c surface activity.

As noted previously, hydrolysis of dairy proteins improves emulsifi cation properties. In order to maintain good emulsifying prop-erties the apparent molecular weights of the peptides should not be less than 5,000 Da. In addition, the important role played by enzyme specifi city in generating desirable peptides with amphiphilic properties has been demonstrated.

Commercial hydrolyzates of whey pro-teins varying between 8% and 45% degree of hydrolysis were used to make emulsions with soybean oil. The concentration of hydroly-zates was varied between 0.02% and 5% (wt/wt). The stability of these emulsions was measured by determining the average size of the emulsion droplets and their size distribu-tion both immediately after formation and after storage. The effects of heating on the stability of the emulsions were also deter-mined. As estimated by the particle sizes, the maximum emulsion capacity was observed with a 10% or 20% degree of hydrolysis. Higher degrees of hydrolysis resulted in pep-tides that were reported to be too small to act as emulsifi ers. These researchers reported that at lower than 10% degrees of hydrolysis a lowered solubility diminished the ability of hydrolyzates to emulsify oil. All of the emul-sions prepared were unstable to heat at tem-peratures of 122 ° C (251.6 ° F) for 15 minutes, but emulsions prepared with less hydrolysis were stable to heat at 90 ° C (194 ° F)for 30 minutes. To a limited extent, mixing different peptides post emulsifi cation could alter emul-sion stability.

It has generally been reported in the litera-ture that surface activity is positively corre-lated with peptide chain length. A minimum chain length of greater that 20 residues has been suggested as necessary for good surface

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two main substrates for the manufacture of cheese fl avors. The use of enzyme - modifi ed cheese (EMC)and lipid fl avors is discussed later in this chapter.

A variety of proteases is commercially available. The operational characteristics (pH, temperature of stability, specifi city, etc.) and amount and type of products vary, and the variations result in different fl avor pro-fi les in EMC. Twenty - three commercial microbial proteinase preparations derived from various Bacillus or Aspergillus spp. or from Rhizomucor niveus were assessed for proteolytic activity on azocasein at pH 5.5 or 7.0 or specifi city on sodium caseinate at pH 5.5. They were semi - quantitatively assessed for esterase, lipase, trypsin, chymotrypsin, general aminopeptidase, phosphatase, and glycosidase activities using the API - ZYM system. Selected preparations were further assayed for peptidase, esterase, and lipase activities at pH 7.0. The proteolytic activity of the Bacillus preparations was greater at pH 7.0, while that of the Aspergillus and Rhizomucor preparations was greater at pH 5.5. All of the Bacillus preparations con-tained one of two main proteolytic activities, thought to be either bacillolysin or subtilisin. Most of the Aspergillus preparations con-tained the same proteinase, thought to be aspergillopepsin I, but two preparations appeared to contain a different unidentifi ed proteinase. The proteolytic specifi city of the Rhizomucor preparation was different from that of the Bacillus or Aspergillus prepara-tions; this difference is thought to be due to an enzyme called rhizopuspepsin.

According to the results of the API - ZYM system, all preparations contained enzyme activities in addition to their main proteolytic activity, with the Aspergillus and Rhizomucor preparations containing the highest levels and widest range of activities. Generally, preparations derived from Aspergillus con-tained the highest level of general, proline, and endopeptidase activities, with the Bacillus preparations conspicuous by the

exhibit both the elastic and viscous proper-ties described by the term viscoelasticity. The gelation process can immobilize a large volume of liquid.

A classic example of gelation of milk occurs in the cheese - making process wherein the enzyme chymosin causes the formation of a soft gel. This gelation occurs due to the hydrolysis one specifi c peptide bond (Phe105 - Met106) in κ - casein by chymosin. The scis-sion of the glycomacropeptide causes the casein micelle to become sensitive to ionic calcium in the environment, leading to coag-ulation or gel formation.

Any alteration in the native conformation of a protein has the potential to induce gela-tion. Thus, enzymes modify the conforma-tion of proteins and the altered conformation increases the propensity of the protein to form gels under appropriate conditions. Sometimes other enzymes, e.g., transgluta-minase, are employed to increase the strength of the formed gels. The exact nature of the peptides that result in gelation is not easy to understand. The processing pre - treatments accorded to proteins pre - or post - modifi cation confound the understanding of the gelation process. In general, enzymatic modifi cation results in a conformation of the protein that can form aggregates, which have a propen-sity to form gels under a variety of condi-tions. Altering the environment by changing pH, ionic strength, and type of ions, or by physical treatments such as heat causes the aggregates to form gels. Structural informa-tion in this context is of limited use in delin-eating the mechanism of gelation.

Flavor Production Resulting from Hydrolysis

Commonly used hydrolyzed proteins for fl a-vorings are derived from plant materials, and milk proteins are not an economically viable source for manufacturing such fl avorings. However, enzymes are used to manufacture cheese fl avors. Lipids and proteins are the


the other hand, lipolytic rancidity has been observed in foods stored at - 29 ° C ( - 20 ° F). Some lipases, referred to as non - specifi c, hydrolyze fatty acids at any position in the triglyceride. Non - specifi c lipases break down triglycerides to free fatty acids and glycerol. Other lipases, referred to as specifi c, hydro-lyze fatty acids esterifi ed at one or three posi-tion of glycerol. The products of specifi c lipase reactions are free fatty acids and di - and mono - glycerides. Lipases, in addition to positional specifi city, also exhibit specifi city towards the type of fatty acid. Pancreatic lipase is specifi c for shorter chain fatty acids esterifi ed to glycerol. Milk fat contains short chain fatty acids.

Pre - gastric esterases are useful in generat-ing specifi c fl avors reminiscent of Italian cheeses. Esterases act on soluble substrates as opposed to lipases, which require insolu-ble substrates. Pre - gastric esterases are also known as pre - lingual lipases — a misnomer. The main sources of esterases are kid, calf, and lamb. These enzymes, used in traditional Italian piquant cheeses, are not acceptable to vegetarians and are very expensive. As judged by enzyme activity on tributyrin sub-strate, pre - gastric esterases are 1,000 times more expensive than pancreatic lipase. The cost disadvantage of pre - gastric esterases coupled with animal virus issues have led to the use of microbial esterases. Besides being less expensive, microbial esterases are protease free and useful for vegetarian products.

The free fatty acids that are generated are converted to fl avor compounds by reactions such as thiolation, oxidation, and esterifi ca-tion, yielding compounds such as thiolesters, γ - or δ - lactones, ethyl esters, and alken - 2 - ols. Thiol ester formation involves sulphur - containing amino acids such as methionine and cysteine, whereas interaction of free fatty acids with tryptophan results in indole, and tyrosine forms phenol. The amino acid leucine reacts with α - ketoglutarate to form α - isoketocaproate. Phenylalanine catabolism

absence of general and proline - specifi c pep-tidase activities. The Rhizomucor niveus preparation contained little or no general or endopeptidase activity. Esterase activity was found in all of the preparations evaluated, with only two Aspergillus preparations con-taining lipase activity.

Lipid Modifi cation

Enzymatic modifi cation of lipids is facili-tated by lipases and esterases. Lipases hydro-lyze triglycerides to free fatty acids and mono - and di - glycerides. Lipases require an insoluble substrate to be present at an inter-face; with triglycerides the interface is created by emulsifying the substrate in an aqueous medium. A potential hindrance to lipolysis is the generation of surface - active mono - and di - glycerides that block the interface. If such surfactants are not controlled, the rate of lipolysis continues to decrease. One method of controlling the release of surfactants is to include divalent cations, e.g. Ca 2 + , which can form insoluble soaps.

As noted above, plants, animals, and microorganisms produce lipases. Plant lipases derived from castor bean or wheat germ are not commercially viable sources of enzyme. Animal lipases are mainly obtained from porcine or bovine pancreas. Other enzymes derived from animals are caprine (kid and goat) pre - gastric lipases and esterases.

Microbial lipases are extracellular enzymes and in some instances inducible. The synthesis of the enzyme is under feed-back control of mono - and di - glycerides and glycerol concentrations in the growth medium. Some microbial lipases are glyco-proteins with carbohydrate moieties facilitat-ing the secretory process. Most microbial lipases have pH optima in the alkaline range (pH 8 to 9) and altering composition of the growth medium by salts and emulsifi ers can shift the optima to an acidic range. Most industrial microbial lipases are active between 30 ° C to 40 ° C (86 ° F to 104 ° F). On

324 Chapter 12

system in newborn infants. Furthermore, immunopeptides formed during milk fermen-tation may contribute to the anti - tumor effect of fermented milk.

Casein Hydrolyzates

Hydrolyzed casein is a rich source of pep-tides, many of which have physiological effects. The properties of α - casein hydroly-sates differ, depending upon the enzymes used to digest them. Trypsin hydrolysis of α - casein promotes antibody formation and phagocytosis (the engulfi ng of the cell wall of external cell material, such as bacteria) and reduces the severity of infections. Resi-dues 90 to 96 (Arg - Tyr - Leu - Gly - Tyr - Leu - Glu) and 90 to 95 (Arg - Tyr - Leu - Gly - Tyr - Leu) of α - casein have been shown to have opioid properties, increasing the body ’ s natural killer cells ’ (lymphocytes) responses and boosting the number of white blood cells.

Beta - casein is the source of numerous bio-active peptides. Residues 63 to 68 (Pro - Gly - Pro - Ile - Pr - Asn) and 191 to 193 (Leu - Leu - Tyr) of β - casein promote antibody formation and phagocytosis. Other peptides derived from β - casein have anti - hypertensive activity, pro-mote mineral absorption, or have an opiate - like effect ( β - casomorphins). Furthermore, these peptides are considered to be immune suppressive when taken orally. Because immunosuppresion prevents the body from rejecting organ and bone marrow transplants, or is used to treat auto - immune diseases such as Crohn ’ s disease, work within this fi eld could open up a new market for β - casein peptides.

Similar to the other casein peptides, the digestion of κ - casein results in several pep-tides, which adjust or change the immuno-modulatory responses of the body. The κ - casein peptide Phe - Phe - Ser - Asp - Lys pro-motes antibody formation and phagocytosis, while the κ - casein peptide Tyr - Gly increases the generation of lymphocytes in the human body.

forms phenyl lactate, benzaldehyde, phenyl-acetate, and phenylethanol. These are a few examples of fl avor - impact compounds present in cheese; they refl ect the chemical complexity of cheese fl avor.

Ingredients Involving Proteolysis and Lipolysis

Proteolysis

Proteolysis of casein and whey proteins leads to products called casein hydrolyzate and whey protein hydrolyzate. For each type of protein, various degrees of hydrolysis can be achieved. Casein hydrolyzates are often bitter due to the preponderance of hydrophobic amino acids. Whey protein hydrolyzates are non - bitter but astringent.

Milk and whey peptides are latent until released and activated, e.g., from digestion or milk fermentation. Once activated, these pep-tides have many bioactive functions, all of which relate to general health conditions or reducing the risk of certain chronic diseases. Some examples of the physiological effects of peptides are given below. These proteins are subject to considerable research world-wide. The best data so far indicate that ACE - inhibitory peptides limit the formation of angiotensin II, a potent chemical that causes the muscles surrounding the blood vessels to contract and thereby increasing blood pressure.

Recent studies have indicated that dairy peptides are opioid peptides and may play an active role in the nervous system. Once absorbed into blood, these peptides can travel to the brain and other organs and cause phar-macological properties similar to opium and morphine. This may be why newborn infants generally become calm and sleepy after drinking milk. It also has been suggested that these peptides may alleviate allergic reac-tions in humans and enhance mucosal immu-nity in the gastrointestinal tract; thus, peptides may regulate the development of the immune


increases bioavailability of calcium. CPP also maintains a state of supersaturation with respect to tooth enamel, depressing deminer-alization and enhancing remineralization and preventing tooth decay. CPP is thought to add value to a number of different oral hygiene products such as toothpaste, mouthwash, and chewing gum. CPP can be produced industri-ally from sodium caseinate using enzymes. CPP can dissolve in a wide range of pH levels, is heat stable, and can be processed at high temperatures. CPP applications include health foods, dairy products, soy products and carbonated beverages such as beer (CPP makes foam smaller and longer lasting). Some commercially available casein - derived manufacturers and products are listed in Table 12.1 and currently marketed products are listed in Table 12.2 .

Whey - protein - derived Peptides

Whey represents a rich and heterogeneous mixture of secreted proteins with wide rang-ing nutritional, biological, and food func-tional attributes. The main constituents are β - lactoglobulin ( β - lg) and α - lactalbumin ( α - la), two small globular proteins that account for approximately 70% to 80% of total whey protein. Historically, whey has either been considered a waste product and disposed of in the most cost - effective manner, or pro-cessed into relatively low value commodities

Glycomacropeptide (GMP) is a carbohydrate - rich peptide present in liquid whey. It is derived from the splitting of κ - casein by chymosin, or rennet, during cheese or casein production. The peptide residues of this process are soluble and therefore become part of the whey. There are two dif-ferent variants of GMP, A and variant B, which differ in two amino acids. Different abbreviations are often used for GMP, such as CGMP or CMP, but these all refer to the same molecule.

GMP is rich in sialic acid. Studies indicate that sialic acid contained in GMP promotes the growth of bifi dobacteria and is involved in the brain development of newborns. It is also believed to play an anti - infective role in the small intestine. The peptides in GMP have a high content of branched chain amino acids (BCAA) and contain no aromatic amino acids, which makes them ideal ingredients in nutritional formulations for people suffering from hepatic diseases. GMP is also an ideal nitrogen source for people suffering from phenyl - ketonuria (PKU), due to the lack of phenylalanine. A person suffering from PKU is not able to metabolize phenylalanine, which causes it to accumulate and damage the central nervous system and possibly the brain. Furthermore, studies over the past thirty years have shown that GMP may infl u-ence satiety in humans, inducing the secre-tion of cholecystokinin (CKK), a group of neuropeptides known to regulate short - term control of food intake. However, this effect seems to depend on the actual structure and composition of GMP. GMP has also demon-strated a protective effect on teeth by inhibit-ing cariogenic bacteria.

Caseinphosphopeptides (CPP) are a type of phosphorylated casein - derived peptides. Interest in this ingredient is increasing due to its purported ability to bind and solubilize minerals such as calcium, zinc, iron, and magnesium. CPP forms a stable complex with calcium phosphate, which hinders calcium phosphate precipitation and thus

Table 12.1. Hydrolyzed casein (milk protein hydrolyzate) manufacturers.

Producer MPH product name

American Casein Co. HLA - 198 Arla Food Ingredients LACPRODAN and

PEPTIGEN Armor Prot é nes Vitalarmor 950 Ingredia NA Lactalis NA Kerry Ingredients NA Morinaqa NA Fonterra NA FrieslandCampina NA Glanbia BarPro

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protein, generated using pepsin, trypsin, chy-motrypsin, or other commercially available proteases, resulted in high ACE inhibition indices (i.e. 73% to 90%). Furthermore, the active peptides were usually short (3 to 8 amino acids) and could be enriched from a mixture of protein and other peptides using ultrafi ltration with low - molecular - mass cut - off membranes.

A tryptic peptide of β - lg (f142 to 148) was further characterized following reversed - phase chromatographic isolation and shown to have an ACE IC50 value of 42.6 nM. Similarly, several researchers have demon-strated that a number of β - lg - derived pep-tides have impressive ACE inhibitory activity using a variety of in vitro assay techniques. In a study in which whey proteins were treated with different lactic acid starters and digestive enzymes, it was reported that two peptides from β - lg (f9 to 14 and f15 to 20), following hydrolysis with trypsin or pepsin and characterization by amino acid and MS - analysis, had ACE inhibitory activity (Meisel, 1998 ). Four novel ACE inhibitory peptides have been reported from caprine β - lg follow-ing hydrolytic treatment with thermolysin and purifi cation. It has been demonstrated that a tetrapeptide isolated from β - lg (f142 to 145; Ala - Leu - Pro - Met), termed β - lactosin B, had signifi cant anti - hypertensive activity when administered orally to spontaneously hypertensive rats (SHR) and therefore has

such as whey powder and various grades of whey protein concentrate (WPC) and isolate (WPI).

Isolation of whey proteins as spray - dried whey powder and, in more limited quantities, as whey protein concentrate/isolate, has realized only a small portion of the commercial potential of these proteins. Indeed, whey protein concentrate, once her-alded as a value - added outlet for whey solids, is now considered a commodity item. In addition, whey - protein - based products have an unfortunate record of inconsistent and unreliable performance in food systems. Thus, expanded use of whey proteins relies on exploitation of individual whey pro-teins and their derivatives as products with increased nutritional, functional, and/or bio-logical value and increased commercial value to the dairy industry. The emergence of new technologies and methods provides fresh insight into the bioactivity of these proteins and produces new and sometimes surprising results.

β - lg is the predominant protein in cheese whey. Various peptides derived from proteo-lytic digestion of β - lg have been shown to have an inhibitory activity against angiotensin - converting enzyme (ACE), which plays a major role in the regulation of blood pressure and thereby hypertension. It has been shown that unhydrolyzed β - lg had very poor ACE inhibitory activity, but that digests of the

Table 12.2. Currently marketed dairy products containing functional peptides.

Sample Peptide Product Function Producer

Calpis VPP/IPP Sour milk Hypotensive Calpis Co (JP) Evolus VPP/IPP Fermented milk Hypotensive Valio (FI) Capolac CPP Ingredient Mineral absorption AFI (DK) PeptoPro NA Ingredient Recovery DSM Foods (NL) tensVida IPP Ingredient Hypotensive DSM Foods (NL) Cysteine Peptide Milk - protein -

derived peptide Ingredient Raise energy and

sleep FrieslandCampina (NL)

C12 FFVAPFPEVFGK Ingredient Hypotensive FrieslandCampina (NL) PRODIET F200/

Lactium TLGTLGGLLA Ingredient Anxiety reduction Ingredia (FR)

NOP - 47 NA Ingredient Vascular function Glanbia Nutritionals


(f61 to 68) disulfi de bound to f75 to 80 (Chatterton et al., 2006 ). These peptides were mostly active against Gram - positive bacteria; however, weaker effects were observed with Gram - negative bacteria. Although pepsin did not release any antibacterial peptides in one study, a different study indicated that both pepsin and trypsin released peptides from α - la, which inhibited the growth of E. coli JM103. The peptide concentration was 25 mgmL _ 1, whereas unhydrolyzed α - la did not inhibit the growth at a concentration of 100 mgmL _ 1. Peptides released from whey proteins and their bioactivities are shown in Table 12.3 and commercially available whey peptide products are shown in Table 12.4 .

Ingredients Derived from Lipolysis

A majority of the fl avor compounds are derived from lipids or are a result of interac-tions of lipolysis and proteolysis products. Most fl avor - impact compounds are also lipid soluble. Recent trends in healthy foods have created markets for low - and nonfat products in the United States. Nonfat products do not have enough lipids to act as effective fl avor carriers. The time of onset of fl avor release and the duration of fl avor perception are

potential as a natural antihypertensive agent for inclusion in foods.

Peptide fragments of β - lg, generated through the action of alcalase, pepsin, or trypsin, have been shown to be bacteriostatic against E. coli and against pathogenic strains of E. coli, Bacillus subtilis , and Staphylococcus aureus (Meisel, 1998 ).

Alphalactalbumin ( α - la) is the next most predominant protein in whey. The peptide with the amino acids sequence Tyr - Gly - Leu - Thr (f50 to 53), released from α - la by pepsin treatment, was shown to inhibit ACE; the accuracy of the last amino acid (Thr) is uncertain. This peptide has been termed α - lactophorin. Interestingly, proteolytic frag-ments of this peptide, i.e., the dipeptides Tyr - Gly (f18 to 19 and f50 to 51) and Leu - Phe (f52 to 53), were also observed to have an inhibitory effect. Trypsin treatment of α - la has been shown to release two antibacterial peptides, Glu - Gln - Leu - Thr - Lys (f1 to 5) and Gly - Tyr - Gly - Gly - Val - Ser - Leu - Pro - Glu - Trp - Val - Cys - Thr - Thr - Phe (f17 to 31) disulphide - bonded to Ala - Leu - Cys - Ser - Glu - Lys (f109 to 114). Treatment using another intestinal enzyme, chymotrypsin, resulted in one anti-bacterial peptide, namely, Cys - Lys - Asp - Asp - Gln - Asn - Pro - His - Ile - Ser - Cys - Asp - Lys - Phe

Table 12.3. Peptides derived from whey proteins and their bioactivity.

Precursor protein Fragment Peptide sequence Name Function

α - lactalbumin 50 – 53 Tyr - Gly - Leu - Phe α - lactorphin Opioid agonist, ACE inhibition

β - lactoglobulin 102 – 105 Tyr - Leu - Leu - Phe β - lactorphin Non - opioid stimulatory effect on ileum, ACE inhibition

142 – 148 Ala - Leu - Pro - Met - His - Ile - Arg ACE inhibition 146 – 149 His - Ile - Arg - Leu β - lactotensin Ileum contraction

Bovine serum albumin

399 – 404 Tyr - Gly - Phe - Gln - Asn - Ala Serorphin Opioid

208 – 216 Ala - Leu - Lys - Ala - Trp - Ser - Val - Ala - Arg

Albutensin A Ileum contraction, ACE inhibition

Lactoferrin 17 – 41 Lys - Cys - Arg - Arg - Trp - Glu - Trp - Arg - Met - Lys - Lys - Leu - Gly - Ala - Pro - Ser - Ile - Pro - Ser - Ile - Thr - Cys - Val - Arg - Arg - Ala - Phe

Lactoferricin Antimicrobial

328 Chapter 12

enzyme - modifi ed cream and enzyme - modifi ed butter oil. Bread formulations have been evaluated for the effects of enzyme - modifi ed butter oil on the attributes of the baked product. Commercial shortening served as the control and another control used 3% butter oil, while the experimental samples contained 2% butter oil plus 1% enzyme - modifi ed butter oil, 1% butter oil plus 1% enzyme - modifi ed butter oil, and 2% commercial shortening plus 1% enzyme - modifi ed butter oil (Arnold et al. 1975 ). The breads were judged by a trained sensory panel for fl avor, color, softness, appearance, and internal structure. The experimental breads were judged to be slightly softer, and samples with enzyme - modifi ed butter oil were superior in fl avor to the two controls. After 24 hours of storage, the control bread was stale and the experimental bread was fresh. Enzyme - modifi ed butter oil can be substituted for 35% to 40% of the shortening. Butter oil modifi ed with enzymes derived from Achromobacter lipolyticum , Penicillium roquefortii, or Geotrichum candidum is not recommended for baked goods because they produce musty and soapy fl avored bread.

Symrise Company markets Dariteen L - 11 and L - 40 lipolyzed creams; Dariteen L - 22, a lipolyzed cultured cream product; and Dariteen L - 60 and L - 95, natural dairy fl avors with a strong butter fl avor. Lipolyzed cream products are natural dairy fl avors produced by treating fresh cream with lipase. Hydrolysis of butterfat in cream liberates the fl avorful free fatty acids butyric, caproic, caprylic, and capric. To control fl avor development in the fi nal product, the lipolyzed cream is heat treated to denature lipases. Lipolyzed cul-tured cream products are inoculated with Lactobacillus delbrueckii subsp. bulgaricus to develop acidity in cream prior to lipolysis. After lipolysis, a heat treatment is used to destroy both the enzyme and the culture bacteria.

Pure butter oil is also lipolyzed, and the modifi ed product is solid at room tempera-

related to the amount of fat in the product. The partition coeffi cients of fl avor com-pounds in an oil - in - water system infl uence vapor pressure and, therefore, volatility of these molecules. In a fat - free system only a single aqueous phase exists, and release and duration of fl avor compounds is drastically different than in oil - in - water systems.

Cost reduction of food formulations is important, especially in cost - sensitive (eco-nomic) products. Costs can be lowered by careful control of the types and quantities of ingredients used in formulations. Milk fat and milk protein are expensive ingredients. Therefore, alternative, less expensive ingre-dients are desirable in the manufacture of certain products (e.g., cream in desserts, cheese in dips, or blue cheese in salad dress-ings). Local availability and cost of dairy ingredients also factor in the selection of ingredients in food formulations.

Enzyme - modifi ed Butterfat

Many different processes for modifying but-terfat are available to fl avor manufacturers. Milk - fat emulsions are modifi ed to make

Table 12.4. Commercial whey protein hydroly-zate (WPH) ingredients in the marketplace.

Producer WPH product name

American Casein Co. HCA - 411 Arla Food

Ingredients LACPRODAN and

PEPTIGEN Armor Prot é nes Vitalarmor 855LB Carbery Optipep, Optipep DH5A Davisco NA Fonterra NA FrieslandCampina NA Glanbia Barfl ex Hilmar Ingredients Hilmar 8360 to Hilmar

8390 Ingredia NA Lactalis NA Kerry Ingredients NA Morinaqa NA Murray Goulburn NA Milk Specialties

Global 9003, 9004, 9005, 8010,

8020, 9010, 9015, 9020 Protient 3020, 4003, 4020 Tatua NA


Cheese Flavors

Enzyme - modifi ed cheese fl avors are used in a variety of foods. Proteases along with esterases are used in a ratio of 1 : 2 to 1 : 3 to treat cheese curds over a certain period at 10 ° C to 25 ° C (50 ° F to 70 ° F). As depicted in Figure 12.1 , the use of discreet proteases helps in generating desirable fl avors.

ture. Lipolyzed cream and cultured cream enhance dairy fl avors in candies, cheese-cakes, sauces, dips, salad dressings, sweet doughs, soups, and baked goods. For subtle fl avor, the modifi ed creams are used at the 0.05% to 0.1% level, and for a more pro-nounced effect, levels of 0.1% to 0.5% are customary. Partially lipolyzed butter oil is useful in fl avoring oils, fats, cereals, snacks, and baked goods. For example, the oil used to cover popped corn results in buttered popcorn. In this application, the typical use level of the modifi ed butter oil is 0.05% to 1% in the fi nished food; these fl avors are heat stable and are suitable where high tempera-tures are used during preparation or process-ing of the food.

Caramel candy manufacture consists of mixing dry cream with water followed by the addition of corn syrup, salt, and vegetable oil. This mixture is cooked to 110 ° C (230 ° F) and evaporated milk is added under continuous agitation. Vanilla is then added and the candy cooled, cut, and wrapped. Dariteen L - 22 is used in caramel manufacture and a typical formulation is shown in Table 12.5 . In another candy formulation, for butterscotch (Table 12.6 ), Dariteen L - 95 is used. Butterscotch hard candy is made by combining sugar, corn syrup, salt, water and vegetable oil and cooking the mixture to 136 ° C (277 ° F) prior to adding Dariteen L - 95, coloring the mixture, and raising the temperature to 138 ° C (280 ° F). The candy is cooled, cut, and wrapped.

Table 12.5. Formulation for caramel candy using lipolyzed cultured cream.

Ingredient Quantity (wt.%)

Corn syrup (42 DE) 30.92 Evaporated milk 31.32 Sugar 19.32 Water 10.83 Dry cream (72% butterfat) 4.64 Hydrogenated vegetable oil 1.55 Dariteen L - 22 1.00 Salt 0.20 Vanilla extract 0.20 Lecithin 0.20 TOTAL 100

Table 12.6. Formulation for butterscotch hard candy using lipolyzed butter oil.

Ingredient Quantity (wt.%)

Sugar 51.88 Corn syrup (43 DE) 31.12 Water 14.52 Hydrogenated vegetable oil 1.03 Dairyteen L - 95 1.00 Salt 0.45 Color As desired Total 100

Figure 12.1. Contribution of proteolysis to fl avor compounds.

Protein Peptides Free amino acids

Flavor

IntermediatesFlavor

compounds

Flavor

precu

rssors

330 Chapter 12

quality of these products can be signifi cantly enhanced by the use of EMC without increas-ing the quantity of cheese in a product.

EMC also can replace cheese in food for-mulations; the levels used depend on the amount of cheese being replaced and the fl avor intensity of the EMC. For example, if a current formulation has 50% cheese and a 50% substitution is desired with an EMC that is 20 times more potent than the cheese fl avor, only 1.25% of the EMC is required to reach the substitution target without affect-ing fl avor intensity. However, this reduces the weight of the formulation, and other ingredients such as starch and oil may have

In some cases, cheese cultures are also added. The resulting material has fi ve - to 15 - fold strength of a regular cheese fl avor. The exact reaction conditions and particulars are closely held trade secrets. Figure 12.2 shows the principles involved in EMC production. After incubation to maximize the cheese fl avor, the blend is heat treated to inactivate the enzymes and cultures and spray dried. EMC paste is also available.

A variety of fl avor profi les can be devel-oped of varying compositions and product forms. Typical applications include pro-cessed cheese, cheese spreads, cheese dips, cheese analogs, and cheese sauces. The fl avor

Figure 12.2. Principles of enzyme - modifi ed cheese production. Adapted from Wilkinson and Kilcawley (2003) .

ComminutedCheese curd- Cream

Cone-bottomTank

Cone-bottomTank

Water,Emulsifying Salts

Blend,Pasteurize,

Cool

Incubate Mix-Optimum Temp.,

pH, Time

Proteasesand

Peptidases

Heat treat to inactivate culture

and enzymes

Blend,Pasteurize,

Cool

Lipases/Esterases

IncubateMix-

OptimumTemp., pH,

Time

Heat treat to inactivate culture

and enzymes

Enzyme Modified Cheese Concentrate

Blend

Cheese culture


fatty acids are liberated from milk fat by lipases. The released fatty acids are oxidized to β - keto acids, which undergo decarboxyl-ation to generate methyl ketones, which in turn are reduced to yield secondary alcohol. Blue cheese fl avors can be produced by sub-merged mycellial fermentation involving sterile milk or milk fat. The fl avors are used in salad dressings, dips, products that can be extended, and baked snack foods. The levels used in food products vary, depending on the process used to obtain the fl avor. For example, a fl avor obtained by continuous fermentation and containing 24 times the concentration of C 5, 7, 9, or 11 methyl ketones than in blue cheese can replace 100% blue cheese in a chip dip.

Compared to Italian and blue cheese, cheddar, Swiss and Dutch cheeses undergo low levels of lipolysis. Addition of rennet paste, pre - gastric esterase, or gastric lipase improves the fl avor of cheddar cheese and several patents have been granted for such an application. Enzyme - modifi ed cheddar cheese fl avor comes in various intensities, such as mild, sharp, and extra sharp. Symrise company has a fi ve - times paste and an eight - times cheese powder. All of these fl avors they are blended with other natural fl avors (WONF). Typical applications of enzyme - modifi ed cheddar cheese include cheese

to be added to restore the weight of the origi-nal formulation. Various EMCs are available to boost or replace cheddar, Swiss, blue, Romano, and other cheese varieties.

Generally, in the manufacture of enzyme - modifi ed dairy ingredients, the substrate is prepared and then the enzyme solution is pre-pared and standardized. The enzyme solution is added to the substrate and the mixture is homogenized or thoroughly mixed. The mixture of enzyme and substrate is incubated at an appropriate temperature to achieve the desired conversion of the substrate to the desired product. This is followed by enzyme inactivation, spray drying, and packaging.

Differing amounts of products are yielded when using the same substrata but different lipolytic enzymes. The addition of pre - gastric esterase in the manufacture of the Italian Romano and provolone cheeses results in the much desired piquant fl avors. Lamb gastric esterase in combination with lamb pre - gastric esterase results in provolone - like fl avor. Mucor miehei esterase is used to develop fl avor in Romano and fontina cheeses. Inclusion of pre - gastric esterase and fungal esterase (on an equal activity basis) results in good fontina fl avor but fi ve times more fungal esterase than the pre - gastric kid ester-ase that is required to develop good Romano cheese fl avor.

Romano cheese fl avor from Symrise Company contains Romano cheese, water, salt, and potassium sorbate. The product is available in 25 - kg (approximately 50 lbs) polyethylene - lined pails. Romano cheese is ground through a 0.63 - cm (1/4 - inch) plate and mixed with warm water at 60 ° C to 70 ° C (140 ° F to 158 ° F) to obtain a lump - free slurry. The remaining ingredients are added and the temperature is raised to 82 ° C (180 ° F) and held for 15 minutes prior to fi lling in contain-ers, which are then thermally processed. A formulation for a tomato sauce with Romano cheese is given in Table 12.7 .

Blue cheese is another popular cheese fl avor; it involves four enzymatic steps. First,

Table 12.7. Tomato sauce with Romano cheese made with and without Dariteen - 310.

Ingredients Content (wt.%)

Without Dariteen

With Dariteen

Tomato puree 44.02 44.02 Water 26.83 28.83 Tomato paste 17.77 17.77 Romano cheese 6.00 3.00 Dariteen - 310 0 1.00 Salt 0.62 0.62 Dried onion 0.53 0.53 Sugar 0.49 0.49 Oregano 0.18 0.18 Garlic powder 0.01 0.01 Total 100 100

332 Chapter 12

analogs, spaghetti sauces, and cheese sauces. Typical application of Dariteen - 245 (a 20 - times sharp cheddar fl avor) in a soup formu-lation is provided (Table 12.8 ).

The process steps for the manufacture of this product are: First grind the cheese through a 0.31 cm (0.25 ” ) plate. Combine non fat dry milk, whey, fl our, corn starch, salt, monosodium glutamate and Tureen AYP 65 yeast extract with water and oil. Add the grated cheese, Dariteen 245 and Dariteen L - 22, paprika and capsicum oleoresins, mix and heat to 88 ° C (190 ° F) and hold for 10 minutes., fi ll cans and thermally process the containers. This is a condensed soup and can be hydrated with an equal part of water prior to consumption.

Swiss cheese fl avor for use in fondues, quiches, crackers, and sauces also is com-mercially available. A typical formulation (Table 12.9 ) is processed by grinding cheese through a 0.31 - cm (0.25 - inch) plate. Nonfat dry milk, buttermilk solids, starch, salt, Tween AYP 65, and xanthan gum are mixed thoroughly in a kettle. This mixture is com-bined with water and grated Swiss cheese

Table 12.8. Ingredient formulation for cheddar cheese soup with and without Dariteen enzyme - modifi ed fl avor.

Ingredient Content (wt.%)

Without Dariteen

With Dariteen

Water 52.59 53.83 Sharp cheddar cheese 20.00 16.00 Nonfat dry milk 12.00 1.23 Whey powder 5.00 4.00 Flour 4.00 4.40 Oil 4.00 5.28 Dariteen - 245 Cheddar

fl avor 0 0.75

Dariteen L - 22 lipolyzed cream fl avor

0.60 0.60

Corn starch 0.50 0.50 Salt 0.40 0.50 Tureen AYP 65 yeast

extract 0.20 0.20

Oleoresin capsicum 0.10 0.10 Oleoresin paprika 0.01 0.01 Total 100 100

Table 12.9. Formulation for a frozen Swiss cheese sauce with and without Dariteen - 410 Swiss cheese fl avor.

Ingredients Quantity (wt. %)

Without Dariteen

With Dariteen

Water 71.7 74.4 Swiss cheese (aged

more than 60 days) 15.0 11.3

Nonfat dry milk 6.0 6.0 Buttermilk solids 3.0 3.0 Oil 2.0 2.0 Starch 1.5 1.5 Dariteen - 410 Swiss

cheese fl avor 0 1.0

Salt 0.4 0.4 Tureen AYP 65 yeast

extract 0.3 0.3

Xanthan gum 0.1 0.1 Annatto extract As desired As desired Total 100 100

and oil are added, along with Dariteen 410 and annatto extract. The mixture is heated to 88 ° C (190 ° F) for fi ve minutes and the product is packaged and frozen.

Strongly fl avored cheese is produced by adding protease and lipase mixtures to cheese curd and then curing at 10 ° C to 25 ° C (50 ° F to 77 ° F) 1 to 2 months. The ratio of esterase to lipase activity should be 2 or 3 : 1. Over treatment results in the methyl ketones 2 - heptanone and 2 - nonanone. Concentrated cheese fl avorings are produced by the rapid modifi cation of slurries of milk solids or casein, various fats, and emulsifi ers.

Suggested Readings

Arnold , R.G. , Shahani , K.M. and Dwivedi , B.K. 1975 . “ Enzyme modifi ed fl avors . ” J. Dairy Sci. 58 : 1127 – 1132 .

Chatterton , D.E.W. , Smithers , G. , Roupas , P. and Brodkorb , A. 2006 . “ Bioactivity of b - lactoglobulin and a - lactalbumin — Technological implications for processing . ” Int. Dairy J . 16 : 1229 – 1240 .

Godfrey , T. and West , S. 1996 . Industrial Enzymology . 2nd ed. Stockton Press , New York, NY .

Hannon J.A. , Kilcawley K.N. , Wilkinson M.G. , Delahunty C.M. and Beresford T.P. 2006 . “ Production of ingredient - type cheddar cheese with accelerated fl avor development by addition of enzyme - modifi ed cheese powder . ” J. Dairy Sci. 89 : 3749 – 3762 .


Kilara , A. 1985 . “ Enzyme modifi ed lipid food ingredi-ents . ” Process Biochem. 20 ( 3 ): 35 – 45 .

Kilara , A. 1985 . “ Enzyme modifi ed protein food ingre-dients . ” Process Biochem , 20 ( 5 ): 149 – 158 .

Meisel , H. 1998 . “ Overview of milk protein derived peptides . ” Int. Dairy J . 8 : 363 – 373 .

Wilkinson , M. and Kilcawley , K. 1999 . “ Enzyme modi-fi ed cheese fl avor ingredients. ” DPRC 10. Dairy Products Research Institute, Moorepak, Ireland.

Wilkinson , M.G. and Kilcawly , K.N. 2003 . “ Enzyme - modifi ed Cheeses . ” Encyclopedia of Dairy Sciences , Vol. 1. Roginski , H. , Fuquay , J.W. and Fox , P.F. (eds). Academic Press , New York, NY . 434 – 438 .

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