impact of processing on bioactive peptides

Proteins are fundamental and integral food components,both nutritionally and functionally. Nutritionally, they are asource of energy and amino acids, which are essential forgrowth and maintenance. Functionally, they aect thephysicochemical and sensory properties of various protei-naceous foods. In addition, many dietary proteins possessspecific biological properties which make these compo-nents potential ingredients of functional or health-promot-ing foods. These proteins may also aect the technologicalfunctionality of the intended end-products. On the otherhand, it is essential to apply or develop technologies whichretain or even enhance the activity of bioactive compo-nents in food systems. This review article focuses on theeects of processing on the properties of bioactive proteinsderived from various sources. A special emphasis is given tomilk proteins as their physiological and technological func-tionality has been studied extensively. # 1998 ElsevierScience Ltd. All rights reserved

Technological processes used in food manufacture aectthe functional, nutritional and biological properties offood proteins. On the other hand, proteins may beadded as functional ingredients to foods to emulsify,bind water or fat, form foams or gels, and alter flavor,appearance, and texture [1]. In recent years, the role ofproteins in the diet as physiologically active componentshas been increasingly acknowledged. Such proteins ortheir precursors may occur naturally in raw food mate-rials exerting their physiological action direct or uponenzymatic hydrolysis in vitro or in vivo. For example, ithas become clear that dietary proteins are a source ofbiologically active peptides. These peptides are inactivewithin the sequence of parent protein and can bereleased during gastrointestinal digestion or food pro-cessing. Once bioactive peptides are liberated, they mayact as regulatory compounds with hormone-like activ-ity. Milk proteins are the most important source ofbioactive peptides, though other animal as well as plantproteins, especially soybean, also contain potentialbioactive sequences [2, 3]. In addition, it is well docu-mented that a number of amino acids possess specificphysiological properties, both beneficial and detrimentale.g. they participate in many biochemical pathways andare precursors of active metabolites. The amino acidswhich are considered to be physiologically beneficialare, for example, arginine, glutamine, histidine, lysine,taurine, tyrosine and tryptophan [2]. The best sources ofthese amino acids are meat, eggs and dairy products.On the other hand, a few amino acid derivatives, whichare formed during food processing, such as lysinoala-nine, d-amino acids and biogenic amines, may causeundesirable metabolic or even toxic events in the body[4, 5]. Many dietary proteins may naturally pose aspotential allergens and protein-derived allergenic prop-erties have been mentioned as possible side-eects ofgenetically engineered foodstus [6]. In this respect,development and application of novel processing andisolation techniques which aim at minimizing suchhealth risks will play a crucial role.

Sources of bioactive proteinsMilkMilk contains two major protein groups, caseins and

whey proteins, which dier greatly with regard to theirphysicochemical and biological properties. Caseins,

0924-2244/98/$see front matter Copyright # 1998 Elsevier Science Ltd. All rights reservedPI I : S0924-2244 (98 )00054-5

Trends in Food Science & Technology 9 (1998) 307319

Impact ofprocessing on

bioactive proteinsand peptides

Hannu Korhonen*Anne Pihlanto-Leppala,

Pirjo Rantamakiand Tuomo Tupasela

Agricultural Research Centre of Finland, FoodResearch Institute, 31600 Jokioinen, Finland

(tel.: 358-3-418-8271; fax: 358-3-418-8444;e-mail: [email protected])

Review

*Corresponding author.

which account for 80% of the total protein in bovinemilk, exist primarily in large complexes termed micelles[7]. The multiple functional properties of caseinatederivatives allow them to be used in several food pro-ducts, e.g. bakery and meat products, soups and toppings[8, 9]. The caseins are known to exhibit biologicalactivity, such as carrying of calcium, zinc, copper, ironand phosphate ions in the body (Table 1). Also, thecaseins act as precursors of a number of dierentbioactive peptides [10].The whey proteins, which account for 20% of total

milk protein, represent an excellent source of bothfunctional and nutritious proteins. The main whey pro-tein constituents are -lactoglobulin and -lactalbumin,two small globular proteins that account for some7080% of total whey protein. Minor whey proteincomponents include the immunoglobulins (Igs), glyco-macropeptide, serum albumin, lactoferrin, proteose-peptones and numerous enzymes [11, 12]. The knownbiological properties of these proteins are highlighted inTable 1. The functional versatility of whey proteins iswell known and reviewed in many articles [1317].Native -lactalbumin has good emulsifying properties,but its gelation ability is poor. By contrast, native-lactoglobulin has excellent gelling and foaming prop-erties. Whey protein concentrates (WPC) and isolates(WPI) are nowadays increasingly used by variousindustries, though their physicochemical functionality isquite limited [1820]. There seems to be considerablescope for expanded utilization of specific whey-derivedproteins, not only in the food industry, but also in thepharmaceutical, health-related and diagnostic industries[21, 22].As shown in Table 2, bioactive peptides are widely

distributed among milk proteins. Many studies haveshown in vitro formation of bioactive peptides frommilk proteins and in some studies in vivo formation hasalso been found [3, 23]. In addition to liberation duringdigestion in vivo, bioactive peptides may be liberatedduring the manufacture of milk products. For example,hydrolysed milk proteins used for hypoallergenic infantformulas, clinical diets and as food ingredients, consist

exclusively of peptides. Proteolysis during milk fermen-tation and cheese ripening leads to the formation ofvarious peptides. Indeed, casomorphins, ACE-inhibi-tory peptides and phosphopeptides have been found infermented milk products [2426].

ColostrumIn bovine colostrum, the protein content is 34 times

higher (up to 150 g/l vs 3040 g/l) than in normal milk.This is primarily attributed to a high concentration ofwhey proteins. Among colostral whey proteins, the Igsrepresent up to 75% of total protein in the first milkingcompared with 10% in normal milk [27, 28]. Ig(immunoglobulin) enriched preparations have beenintroduced to the market in many countries as calf milkreplacers [29]. It has been suggested that infant formulascould be fortified with colostral Igs and lactoferrin (LF)[30, 31]. Also, preparations containing specific colostralIgs (antibodies) produced in colostrum by hyper-immunization of pregnant cows may, in the future, findapplications in the prevention and treatment of humanmicrobial diseases [21, 32]. A preventive or therapeuticecacy of such products has been demonstrated againstdierent gastrointestinal infections [33]. A few so-calledimmune milk products are already on the market in theUSA and Australia. Since bovine colostrum also con-tains other biologically active compounds such as

Table 1. Biological activity of major milk proteins

Protein Concentrationg/l

Function

Caseins (; and ) 28 Ion carrier (Ca, PO4, Fe, Zn, Cu), precursors of bioactive peptides-lactoglobulin 1.3 Retinol carrier, fatty acids binding, possible antioxidant-Lactalbumin 1.2 Lactose synthesis in mammary gland, Ca carrier, immunomodulation, anticarcinogenicImmunoglobulins A, M and G 0.7 Immune protectionGlycomacropeptide 1.2 Antiviral, bifidogenicLactoferrin (LF) 0.1 Antimicrobial, antioxidative, immunomodulation, iron absorption, anticarcinogenicLactoperoxidase 0.03 AntimicrobialLysozyme 0.0004 Antimicrobial, synergistic eect with immunoglobulins and LF

Data from Korhonen [21]

Table 2. Bioactive peptides derived from milk proteins

Bioactive peptides Protein precursor Bioactivity

Casomorphins - and -Casein Opioid agonists-Lactorphin -Lactalbumin Opioid agonist-Lactorphin -Lactoglobulin Opioid agonistLactoferroxins Lactoferrin Opioid antagonistsCasoxins -Casein Opioid antagonistsCasokinins - and -Casein AntihypertensiveCasoplatelins -Casein, Transferrin AntithromboticImmunopeptides - and -Casein ImmunostimulantsPhosphopeptides - and -Casein Mineral carriersLactoferricin Lactoferrin Antimicrobial

Data from Meisel and Schlimme [3].

308 H. Korhonen et al./Trends in Food Science & Technology 9 (1998) 307319

growth-promoting factors and essential nutrients [12,3436], research in this field seems highly promising.

Transgenic milk proteinsTransgenic animals have been employed as in vivo

experimental models for assessing the ability andimpact of foreign gene expression in a biological sys-tem. Transgenic mice are most commonly used, whiletransgenic sheep, goats, pigs and cows have also beendeveloped for specific, applied purposes. Transgenictechnology has been applied in these animals with thepurpose of altering the properties of milk by adding anew protein or recovering the protein for other uses,such as pharmaceuticals [37, 38]. Studies carried out intransgenic mice suggest that human lysozyme and -casein are good candidates for beneficially alteringcheese manufacturing properties of milk [39]. Also, thereis a growing interest in employing genetic engineeringfor production of human milk proteins, peptides,growth factors and other bioactive substances [40, 41].Human milk proteins that have been cloned from amammary gland library are -lactalbumin, lactoferrin,lysozyme, collagen, -casein and -casein [40, 42].Human lactoferrin [43] and human lysozyme [44] havebeen expressed in transgenic mice or cattle with reason-able expression in the milk but, so far, no in vivo studieson the functional or physiological properties of suchmilks have been reported. On the other hand, humanantithrombin III and 1-antitrypsin from the milk oftransgenic livestock are currently in clinical trials [45].

EggA hens egg consists of 13% protein (shell, 3%; egg

white, 11% and yolk, 17%). Eggs are a rich source ofproteins with dierent physicochemical and biologicalcharacteristics as shown in Table 3 [46]. Egg white pos-sesses multiple functionalities such as gelation, emulsifi-cation, foaming, water binding and heat coagulation,

which makes it a highly desirable protein in many foods.These properties of egg white can be attributed to com-plex interactions among its protein constituents, namelyovalbumin, conalbumin, lysozyme, ovomucin, globulinsand other minor proteins [47]. Whole egg or egg whitepowders are commercially manufactured and used inmany food products and also non-food applications.Among specific egg proteins, lysozyme can easily beseparated from egg white using crystallization or ion-exchange resins. Purified lysozyme has shown promise asa food preservative, e.g. in prevention of late fermentationof hard cheese and in reduction of pathogenic bacteriaon meat surfaces [46]. Egg immunoglobulins (IgY) canbe enriched and isolated in a highly purified form usinga serial filtration system or ultracentrifugation com-bined with a chromatographic purification process [48,49]. IgYs have already found use in immunoassay tech-niques and may, in the future, find applications asingredients of functional foods and feeds aimed at pre-venting or curing gastrointestinal infections [50].

Other sourcesIn addition to milk and egg, bioactive proteins are

found in many other biological materials. Among them,there are animal proteins such as gelatin and fish muscleproteins and also plant proteins such as corn -zein, riceglutelin and prolamin, wheat gluten and soya protein[51, 52]. The biological activity of most of these proteinsis attributed to specific peptide sequences, which arefreed by enzymatic hydrolysis. In the following, exam-ples of such bioactive proteins are described. Oshima etal. [53] isolated nine angiotensin-converting enzyme(ACE)-inhibitory peptides from bacterial collagenasedigests of gelatin. Also, digestion with mammaliantrypsin and -chymotrypsin produced these peptides. Inthe hydrolysates of fish meat obtained by pepsin, trypsin,chymotrypsin, thermolysin or denazyme AP (a proteasefrom Aspergillus oryzae), potent ACE-inhibitory peptides

Table 3. Proteins in egg albumen

Protein Amount ofalbumen (%)

Isoelectric point Molecularweight (Da)

Characteristics

Ovalbumin 45 4.5 45,000 PhosphoglycoproteinOvotransferrin(conalbumin)

12 6.1 76,000 Binds metal ions

Ovomucoid 11 4.1 28,000 Inhibits trypsinOvomucin 3.5 5.55.0 5.58.3106 Sialoprotein, viscousLysozyme 3.4 10.7 14,300 Lyses some bacteriaG2-Globulin 4.0 5.5 3.04.5104 G3-Globulin 4.0 4.0 Ovoinhibitor 1.5 5.1 49,000 Inhibits serine proteasesFicin (cystatin) inhibitor 0.05 5.1 12,700 Inhibits thioproteasesOvoglycoprotein 1.0 3.9 24,400 SialoproteinOvomacroglobulin 0.5 4.5 7.69.0105 Strongly antigenicAvidin 0.05 10 68,300 Binds biotin

Data from Froning [46]

H. Korhonen et al./Trends in Food Science & Technology 9 (1998) 307319 309

have been detected [51]. Among corn proteins, threedistinct zein classes are distinguished, ; - and -zein.-zein is a major component of maize endosperm pro-tein and consists of 7585% of the total zein. It containspolypeptides of molecular weight of 21,00025,000 and10,000Da. When -zein was hydrolyzed with thermo-lysin, ACE-inhibitory peptides were obtained [54]. Inrice proteins, antihypertensive activity has been demon-strated in peptides originating from glutelin and prola-min [55, 56]. Gliadin and glutenin are the main wheatendosperm storage proteins and form 85% of thewheat flour protein content. When hydrated, gliadinand glutenin form a colloidal complex known as wheatgluten [57, 58]. Zioudrou et al. [59] discovered opioidactivity in hydrolysates of wheat gluten. Of all oilseeds,soyabeans are commercially the most important sourceof protein. The protein content (mainly glycinins) ofsoyabeans is much higher than that of cereal grains. Byenzymatic hydrolysis of soya proteins, functional ingre-dients suitable, for example, for whipping or foaminghave been developed [60, 61]. Soyabeans have beenshown to possess anti-carcinogenic properties [62] andboth animal and human studies have demonstrated thata soya protein diet reduces high plasma cholesterollevels [2]. The actual mechanism by which soya proteinsmight lower blood lipid concentrations in humansremains, however, to be elucidated.

Impact of processing on bioactive proteinsIn the conventional industrial manufacture of various

foodstus, the proteins contained in raw materials arereadily subjected to alterations with regard to theirfunctional or biological properties. pH changes and

certain chemical treatments aect functional propertiesby modifying specifically one or more amino acids. Forexample, acidic treatments destroy glutamine andasparagine, whereas alkaline treatments destroy cystine,serine and threonine, and produce lysinoalanine and d-amino acids (Table 4) [1, 5]. Other chemical treatments,such as acylation, glycosylation, phosphorylation,reductive alkylation, succinylation or lipophilizationmay improve functionality of the proteins but theyentail also negative eects due to possible residual che-micals and modification of amino acids. Chemicaltreatments of dietary proteins are, therefore, practisedwith caution in the food industry [63, 64].The most common treatments applied to proteinac-

eous raw materials are dierent heat treatments, fer-mentation processes and extrusion technology. A noveltechnology being introduced currently in the Europeanfood industry is high pressure treatment which isexpected to replace in the future heat treatment prac-tises in the manufacture of a variety of foodstus. Thesemethods are discussed hereunder in more detail.

Heat treatmentHeating is one of the oldest, most common, and most

widely used methods of modifying proteins, for exam-ple, to make food proteins more edible. It is also used inmany dierent food products to form protein gels or setstructure. Such food products include yogurt, sausages,and bread. Depending on the intensity of heat treat-ment, the nutritive value of proteins may be aectedeither in a positive or negative way. Heat is also used tomodify the functional properties of protein ingredients.Heat denatures proteins and may be used, for example,

Table 4. Physicochemical changes and positive (+) or negative () nutritional eects of process treatment and storage on proteins andamino acids

Treatment/Condition Physicochemical changes Nutritional eects

PROCESSINGHeat treatment Protein denaturation Improvement of intrinsic digestibility (+)

Reduction of trypsin inhibitor activity (+)Destruction of heat-sensitive amino acids ()

Intramolecular reactions Cross-linkages ()Reaction with sugars Destruction of lysine ()

pH modification Solubility Risk of oxidation ()Acid or alkaline hydrolysis Improvement of digestibility (+)

Unspecific peptide bond breakage ()Destruction of pH-sensitive amino acids ()Cross-linkages ()Isomerization (racemization) ()

Enzymatic hydrolysis Reaction with proteases Peptides (+/)Reaction with oxygenases Oxidation of amino acids through lipid or polyphenol oxidation ()

Membrane separation Protein fractionation Protein/peptide enrichment (+)Change in amino acid composition (+/)

STORAGE Reaction with sugars Destruction of lysine ()Presence of oxygen Oxidation ()Reaction with polyphenols Oxidation ()

Data modified from Finot [5]


to improve water-binding ability and emulsification. Onthe other hand, heating usually decreases the solubilityof proteins due to their aggregation or coagulation.Heat denaturation temperatures of dierent dietaryproteins vary from 60 to 90 C [65]. Accordingly, theactivity of bioactive proteins is reduced by dierent heattreatments. At a standard pasteurization temperature of72 C for 15 s, the bioactive whey proteins retain most oftheir activity [5, 12]. Although most thermal denatura-tion is irreversible in nature, certain proteins mayundergo reversible denaturation when the thermalinfluence is removed. For example, the thermal dena-turation of -lactalbumin is primarily a reversibleprocess with 8090% renaturation at pH values above3.3. Below pH 3.3, the ability of the protein to return tothe native conformation is reduced. The reversibility of-lactalbumin is calcium dependent, the regenerationbeing reduced when a chelator that binds endogenousCa2+ is added [65].During heat treatment, the lysine residues of proteins

can react with reducing carbohydrates of the same foodsystem resulting in the so-called Maillard or non-enzymatic browning reaction [5]. Depending on theintensity of heat treatment, this reaction aects the sen-sory properties (aroma, flavour and appearance) of theproduct and reduces its nutritional value as the bioa-vailability of lysine is reduced. Ingestion of Maillardreaction products may also induce detrimental eects atthe cellular level in the body [5]. Milk is highly sensitiveto the Maillard reaction because of its high levels oflactose and lysine-rich proteins. Standard pasteurizationof milk does, however, not cause any destruction oflysine and the UHT treatment destroys less than 2% oflysine. In can sterilization of milk destroys 1015% ofavailable lysine. During the storage of milk powders,the evolution of lysine blockage depends on wateractivity and temperature. In model studies, it has beenshown that up to 5060% of lysine can be blocked dur-ing long-term storage with no browning development[5]. In milk and infant formulas, the lysine loss has alimited nutritional eect, as they contain much morelysine than the recommended level for adults andinfants. A number of compounds have been demon-strated to inhibit the Maillard reaction. In milk pro-ducts, active sulfhydryl groups of whey proteins inhibitheat-induced browning. Also, high pressure treatmentcan inhibit the Maillard reaction in milk [66].

FermentationNatural or controlled fermentation has been exploi-

ted by mankind for thousands of years to preserve dif-ferent foodstus and to retain or alter their nutritive orsensory properties. Typical examples of fermented pro-ducts are ripened cheese varieties, fermented dry sau-sages, and fermented soya bean (tofu), cereal (bread)and vegetable (sauerkraut) products. The fermentation

process involves usually natural or added microorgan-isms (starter cultures), e.g. lactic acid bacteria, whichduring their growth hydrolyse sugars and proteinsavailable in their surrounding medium. As a result,peptides with dierent amino acid sequencies and singleamino acids are formed. The degree of proteolysis ishighly dependent on the bacterial species involved andphysical conditions of fermentation [67]. The peptidesand amino acids derived from proteins during fermen-tation often change the functional, rheological, sensoryand biological properties of the fermented product.Recently, it has been established that during milk

fermentation, bioactive peptides are formed from milkproteins. Nakamura et al. [25] isolated two ACE-inhi-bitory peptides Val-Pro-Pro and Ile-Pro-Pro, from sourmilk. ACE- inhibitory activity was also found in ripenedcheese types. This activity increases during cheesematuration, but decreases when the proteolysis exceedsa certain level [26]. Caseinophosphopeptides can beformed during cheese ripening due to plasmin andmicrobial protease activity [68, 69]. Laeneur et al. [70]showed that -casein hydrolysed by lactic acid bacteriahas immunomodulatory activity which could be relatedto interaction with monocyte-macrophage and T-helpercells. Sutas et al. [71] showed that caseins hydrolysedwith a probiotic Lactobacillus GG strain and digestiveenzymes generate compounds with specific suppressiveor stimulatory eects on human lymphocyte prolifera-tion in vitro. Further, Rokka et al. [72] identified severalknown bioactive peptides with ACE-inhibitory, opioidor immunomodulatory activities from the casein hydro-lysates used in the above study. These results suggestthat during fermentation of milk with probiotic bac-teria, peptides with distinct bioactivities can be formed.Such peptides may contribute to the well-documentedhealth-promoting properties of fermented dairy pro-ducts and probiotic lactic acid bacteria [7376]. Somepossible actions of fermented milks in vivo are describedin Fig. 1.

Ultra high pressureUltra high pressure (UHP) processing is a non-thermal

process in which foods are subjected to high isostaticpressures of 1001000MPa at room temperature.UHP processing can aect protein conformation andlead to protein denaturation, aggregation or gelation,depending on the protein system, the applied pressure,the temperature and the duration of the pressuretreatment. Low pressures usually induce reversiblechanges such as dissociation of protein-protein com-plexes, the binding of ligands and conformational changes[66]. Pressures higher than 500MPa induce, in mostcases, irreversible denaturation. The UHP process alsoinactivates microorganisms and, therefore, this techni-que provides an alternative to heat treatments. On theother hand, there are, considerable dierences in protein


denaturation and aggregation induced by high pressurecompared with heat. The use of high pressure to modifythe functionality of food proteins was recently reviewedby Heremans et al. [66] and Messens et al. [77]. Refer-ence is made hereunder to these reviews. Althoughblood plasma and egg white proteins are known to besensitive to heat and readily form gel networks at mod-erate operating temperatures, at 80 C (30min), nogelation occurs if these proteins are pressurized for30min at a pressure of 400MPa. This stability may bepositively correlated with the high amount of disulfidebonds stabilizing the three-dimensional structure ofboth proteins. Again, -lactoglobulin appears far moresensitive towards pressure than ovalbumin and bovineserum albumin (BSA). UHP process has been shown todestabilize casein micelles in reconstituted skim milk.The size distribution of the spherical casein micelleschanged from &200 nm to 120 nm after pressurization.Subsequent heating of skim milk at 30 C and atatmospheric pressure restored the original size distribution.In another study [78], the antigenicity of whey proteinhydrolysates treated with high pressure was found to belower than that of heat-treated hydrolysates. Mussa andRamaswamy [79] studied the kinetics of microbialdestruction and changes in physico-chemical character-istics of fresh raw milk caused by UHP treatment whichwas conducted at 200400MPa for various holdingtimes (5120min). The treatment led to an ecientdestruction of microorganisms and a prolonged shelf-life of milk up to 18 days at 5 C and 12 days at 10 C. It

was concluded that UHP processing of milk may be auseful alternative for extending the shelf-life with qual-ity advantages. Other potential applications of UHPtreatment on milk include low-temperature inactivationof enzymes and stabilization of fermented dairy pro-ducts, improved coagulation of milk, and the manu-facture of dairy gels and emulsions with novel textures[77]. Furthermore, studies have been undertaken on theeects of UHP treatment on meat proteins myosin andmetmyoglobin, egg white, ovalbumin and soya proteins.Additional experimental research on protein model sys-tems and real food products is required to understandthe potential of this technology in the restructuring offood proteins and stabilizing their biological activities.

ExtrusionAt present, the major technique for texturization of

plant proteins is thermoplastic extrusion. In the extru-sion process, the raw material is exposed to high pres-sure (10,00020,000 kPa), high temperature and shearforces. Extrusion is applied in the manufacture ofinstant and snack foods, in particular. Starting materi-als are usually protein isolates, concentrates and flours.During extrusion, disulphide bonds play an importantrole in protein interactions. Mei and Tung [80] observedthat the solubility of wheat proteins decreased and bothaggregation and fragmentation occurred during theextrusion process. Proteins aggregated mostly throughnon-specific hydrophobic interaction and inter-molecular disulphide bond formation. Glutenins and

Fig. 1. Possible in vivo functions of probiotic fermented milks.


gliadins were mainly responsible for the aggregation.Tae [81] studied eects of extrusion on the functionalproperties of co-precipitated proteins originating fromsoy flour and wheat gluten. As compared to native pro-teins, extruded proteins had significantly improvedviscosity, gelation and foaming stability. Hu et al. [82]observed that among the extrusion parameters, thetemperature used had the greatest impact on the nutri-tional quality of wheat, rice and soyabean proteins.Addition of soyabean improved significantly proteinquality and stability of rice and wheat extrudates. Inanimal studies, it has been shown that extruded grainslower serum and liver cholesterol levels as compared toraw grain diet or control diet based on casein [83]. Fur-ther research is required to verify these findings and toestablish the eects of extrusion on the bioactive prop-erties of dietary proteins.

Processing options modulating functionalityStructurefunction relationshipsThe functional properties of proteins in a food matrix

are highly influenced by their molecular structure, pro-tein interactions with other components, e.g. water,other proteins, carbohydrates, lipids and ions, as well asthe conditions of processing. Apart from sensory prop-erties, typical functions of proteins in food systemsinclude adhesion and cohesion, emulsification, gelation,foaming, texturization, water, lipid and flavor bindingand retention. These functionalities are described indetail in recently published books and review articlesand will not be discussed further in this review [64, 8488].

Enzymatic modification of proteinsProteins can be hydrolysed with acid, alkali or

enzymes to yield peptides or, eventually, amino acids.For example, acid hydrolysis is being used to producehydrolysed vegetable proteins, which have meaty flavorprofiles. Alkali treatments are used in the production ofgelatin. Various enzymatic hydrolytic treatments, how-ever, have become the most important tools formodifying the functionality of dietary proteins [60, 84,89]. Enzymatically modified proteins have long beenavailable in many conventional foods such as ripenedcheese and fermented soya protein products. Moreover,pure protein hydrolysates have been shown to havevaluable dietetic properties and high nutritional value[90]. Modification of milk proteins by enzymatic treat-ments is described in more detail below.

Whey protein hydrolysatesThe most commonly used enzymes in the production

of whey protein hydrolysates are pepsin, trypsin andchymotrypsin. Also, plant-originated papain and somebacterial and fungal proteases have been used in studiesreviewed by Lahl and Braun [91], Panyam and Kilara

[92] and Nielsen [60]. The ability of enzymes to hydro-lyse whey proteins is highly variable. Pepsin digests-lactalbumin and denatured, but not native -lacto-globulin [93]. Trypsin hydrolyses -lactalbumin slowlybut -lactoglobulin remains almost undegraded [94].Chymotrypsin hydrolyses readily -lactalbumin but -lactoglobulin is degraded slowly. Liske and Konrad [95]demonstrated that BSA and -lactoglobulin werehydrolysed by papain but -lactalbumin was resistant.However, -lactalbumin was hydrolysed completely atacidic pH when calcium binding was absent [97]. Enzy-matic modification of milk proteins by controlled pro-teolysis can alter their functional properties over a widepH range and other processing conditions [63, 91]. Thehydrolysis of peptide bonds can increase the number ofcharged groups and hydrophobicity, decrease molecularweight, and modify molecular configuration [97]. Chan-ges in functional properties are greatly dependent on thedegree of hydrolysis. The most common changes infunctionality of whey proteins are an increase in solubi-lity and a decrease in viscosity. When the degree ofhydrolysis is high, hydrolysates often tolerate strongheating without precipitating, and solubility is high evenat pH 3.54.0. Hydrolysates also have far lower viscos-ity than intact proteins. The dierence is especiallystriking in solutions with a high protein concentration.Other eects are altered gelation properties, enhancedthermal stability, increased emulsifying and foamingabilities and decreased emulsion and foam stabilities[95, 98, 99].

Allergenic peptidesMilk, egg, soya and wheat proteins may provoke

allergic reactions in sensitized people [6]. Processingmay alter the content and/or properties of these aller-gens, reducing or increasing the allergenicity of thestarting material. In earlier studies, conflicting resultswere obtained with regard to the eect of heat treatmenton the allergenicity of whey proteins. Standard pasteur-ization of milk does not seem to cause any significantreduction of the antigenicity of milk proteins, while milksterilization may even exacerbate allergic reactions[100]. It has been postulated that heat treatment andhomogenization of milk, due to mechanical disintegra-tion of casein micelles and milk fat globules, increasethe ability of milk proteins to elicit allergic reactions insensitized persons [101, 102]. Further studies have,however, not been carried out to substantiate thishypothesis. In contrast, many studies have shown thatthe allergenicity of milk proteins, in particular that of -lactoglobulin, can be reduced substantially if the pro-teins are hydrolysed with pepsin or trypsin or usingcombinations of proteolytic enzymes [60, 103, 104].Heat treatment or high-pressure treatment prior tohydrolysis increases the DH (degree of hydrolysis)value, further reducing antigenicity of whey proteins


[105, 106]. The hydrophilic amino acids, such as lysine,arginine, glutamate, and aspartate residues seem to playimportant roles in allergenic peptides. The antigenicityof such peptides can probably be lowered further byusing proteases specific for these amino acid residues.On the other hand, it has been reported that peptidesconsisting of four amino acids only can cause an allergicreaction in consumers allergic to milk, even thoughthese small peptides will not be able to sensitize a person[107]. Partially or extensively hydrolysed milk proteinshave found increasing use in hypoallergenic infant for-mulas and dietetic products [6, 108110]. The problemsrelated to enzymatic hydrolysis of proteins are the pro-duction of a high amount of free amino acids, a bittertaste and an increase of the formula osmolarity [111].Current research in this field is focusing on technologi-cal reduction in whey of the amount of -lactoglobulinwhich is considered the major allergen because it isabsent in human milk. On the other hand, infant for-mulas enriched with -lactalbumin are being developedto mimic human milk as it is rich in this protein [64].

Bioactive peptidesA lot of scientific interest has focused on physiologi-

cally active peptides derived from food proteins. Thesepeptides are inactive within the sequence of the pre-cursor protein and can be released by enzymatic pro-teolysis. Milk proteins are a rich source of bioactivepeptides; both casein and whey proteins have beenfound to act as precursors of bioactive peptides [3, 64,36]. Opioid peptides (exorphins) are receptors of opioidligands with agonistic or antagonistic activities. Thesepeptides can be released by the digestion of bovinecasein and whey proteins [112, 113]. -casein opioidpeptides (-casomorphins) have been detected in theduodenal chyme of minipigs [114] and in the humansmall intestine [115] as a consequence of in vivo diges-tion. Casein of other species like ovine, bualo andhuman milk can be regarded as precursors of exorphinssince they contain the amino acid sequences character-istic for exogenous opioid peptides [116]. Exorphins canbe found also from wheat gluten proteins and their dif-ferent classes of saline-soluble gliadin and glutein. Thesequences of these peptides have not yet been deter-mined [59, 117]. Angiotensin-converting enzyme (ACE)acts on blood pressure regulation and inhibition of thisenzyme can exert an antihypertensive eect. ACE-inhi-bitory peptides have been isolated from enzymaticdigest of food proteins. Oshima et al. [53] reported thatcollagen and gelatin digests contain ACE-inhibitorypeptides. Enzymatic digestion of casein produces ACE-inhibitory peptides; also fermentation of milk producesACE-inhibitory peptides [25, 118, 119]. Some of theseidentified ACE-inhibitory peptides have also beenshown to have an antihypertensive eect in vivo [25,120]. ACE-inhibitory peptides have also been found

from whey, fish and maize protein digests [51, 121].Immunomodulating casein peptides have been found tostimulate the proliferation of human lymphocytes andthe phagocytic activities of macrophages [122]. Anti-microbial peptides from lactoferrin have been shown tokill sensitive microorganisms [123]. Casein phospho-peptides can form soluble organophosphate salts andmay function as carriers for dierent minerals, espe-cially calcium. Bovine s1; s2 and -casein containphosphorylated regions which can be released by enzy-matic hydrolysis and specific phosphopeptides havebeen identified in the intestinal contents of minipigsafter ingestion of a diet containing casein [114]. There iscommercial interest in the production of bioactive pep-tides with the purpose of using them as active ingre-dients in functional foods. The development oftechnology for industrial-scale production of such pep-tides is currently in progress [124].

Bitter peptidesIt has long been known that peptides and amino acids

can produce many types of taste sensation [63]. A pro-blem in the use of proteolysis for improving function-ality and nutritional value has been the formation ofbitter peptides, which are formed, for example, from -lactoglobulin [60]. Bitterness is generally related to thehydrophobicity of the amino acids in the peptides. Inearlier studies, activated charcoal, various resins, glassfibre and hexyl sepharose were applied for the elimina-tion or reduction of bitter peptides from the hydro-lysates. Also, the plastein reaction which can occurwhen a protein hydrolysate is incubated with a protease,is able to debitter protein hydrolysates [60]. Othermethods used for reducing or eliminating bitterness areselective chromatographic separation, masking andenzymatic treatment [125, 126].

Novel fractionation techniques of bioactive proteinsThe rapid development of membrane and gel filtra-

tion techniques in the 1970s provided new possibilitiesfor a large-scale concentration of whey proteins and themanufacture of whey protein concentrates and isolates[127131]. Also, manufacture of demineralized wheypowders has become possible on a large scale throughapplication of diafiltration or ion exchange chromato-graphy. Techniques for the isolation of individual wheyproteins on a laboratory scale by salting-out, ion-exchange chromatography and/or crystallization hasbeen available for a long time. Owing to the uniquefunctional and biological properties of many of thewhey proteins, a need has arisen to develop novel,gentle methods for their enrichment or isolation on alarge scale [10, 132]. To this end, pilot and industrial-scale technological methods have been developed forisolation, in a purified form, of several individualwhey proteins, such as -lactalbumin, -lactoglobulin,


lactoperoxidase, lactoferrin, glycomacropeptide andimmunoglobulins [133140]. Also, methods for fractio-nation of micellar whole casein and selective separationof -casein have been developed [124]. For isolation orenrichment of milk proteins combinations of membraneseparation and chromatographic techniques, such asmicrofiltration, ultrafiltration, reverse osmosis, nanofil-tration, gel filtration and ion-exchange chromatographyhave been applied [131, 141]. Recently, an ion-exchangemembrane technique has been employed successfullyfor separation of lactoferrin and lactoperoxidasefrom cheese whey [142]. A potential future method forlarge scale fractionation and enrichment of bioactivepeptides is a membrane bioreactor based on serialultrafiltration membranes with dierent cut-o values[124, 143]. Also, further research is needed on theextraction and purification of the three casein fractionsand other minor whey proteins, such as polypeptidegrowth factors, complement factors, enzymes andtransgenic proteins.

Safety implications of bioactive proteinsThe development of functional foods is likely to entail

the increased use of dierent protein sources known tocontain bioactive components. These protein compo-nents may be natural constituents of plant or animalorigin or genetically modified or transferred fromanother source. The introduction into the diet of func-tional foods supplemented with these compounds mayraise the issue that such food products might causeallergies. Although the bioactive proteins or peptidesdescribed in this article are not known to possess spe-cific allergic or toxic eects, their addition to any dif-ferent food system warrants careful consideration aboutpotential health risks. Specifically, their interactionsduring processing or storage with other proteins, sugarsand lipids need to be researched with a view to possibleformation of toxic, allergenic or carcinogenic sub-stances. Some concern has been raised that the transferof proteins from one food source to another by geneticmodification could lead to the appearance of a majorfood allergen in a normally allergen-free product. In thisrespect, analytical methodologies need to be developedto detect the presence of such allergens.

Further development and research needsThe occurrence of many natural bioactive proteins or

their precursors in animal and plant proteins is now wellestablished. There are, however, a great number of sci-entific and technological issues to be solved before thesesubstances can optimally be exploited for human nutri-tion and health. Below is a list of the most importantfuture research needs related to bioactive proteins:

. Basic research on potential bioactivity of minorproteins of milk, egg, vegetables, cereals, and fruits.

. Technological functionality of bioactive proteins,e.g. lactoferrin, immunoglobulins, egg proteinsand bioactive peptides.

. Interactions of bioactive proteins/peptides/aminoacids with other food components during proces-sing and eects of these interactions on bioactivity.

. Eects of conventional and novel processing tech-nologies on the bioactivity of the proteins.

. Development of novel fractionation and purificationmethods for bioactive proteins and their hydro-lysates.

. Basic research on transgenic production of bioac-tive proteins and potential side-eects, e.g. aller-genicity and toxicity of such proteins.

. Evaluation of ecacy of bioactive proteins in ani-mal model and human clinical studies per se and infood systems.

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