Functional properties of sodium caseinate hydrolysates - Journal of ...

Available online at http://journal-of-agroalimentary.roJournal of Agroalimentary Processes andTechnologies 2011, 17(3), 308-314Journal ofAgroalimentary Processes andTechnologiesFunctional properties of sodium caseinate hydrolysates asaffected by the extent of chymotrypsinolysisDaniela Pralea, Loredana Dumitrascu, Daniela Borda, Nicoleta Stănciuc *“ Dunărea de Jos” University of Galaţi, Faculty of Food Science and Engineering, 800201, Domneasca Street, No.111, Galati, RomaniaReceived: 28 May 2011; Accepted: 28 July 2011______________________________________________________________________________________AbstractCasein is the main proteinaceous component of milk, where it accounts for ca. 80% of the total proteininventory. Biologically active peptides can be produced from precursor milk proteins. The most commonway to produce bioactive peptides is through enzymatic hydrolysis of whole proteins.The objectives of present study were to study the effect of enzymatic hydrolysis on the functionalproperties of sodium caseinate. Gastrointestinal enzymes, such as chymotrypsin, have been utilized togenerate hydrolysates. Functional properties were limited to solubility, foaming capacity, and 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity. These properties were evaluated and compared as afunction of hydrolysis degree.Keywords: casein, enzymatic hydrolysis, functional properties______________________________________________________________________________________1. IntroductionA major role of milk proteins is to supply amineacids and nitrogen to the young mammal. Milkproteins also constitute an important part of dietaryproteins for the adult. However, these proteinsshould not only be considered as nutrients for theyprobably have a physiological role as well.Casein is the main proteinaceous component ofmilk, where it accounts for ca. 80% of the totalprotein inventory. Until recently, the mainphysiological role of casein in the milk system waswidely accepted to be a source of amino acidsrequired by growth of the neonate. However, thedominant physiological feature of the caseinmicelle system has more recently been proven tobe the prevention of pathological calcification ofthe mammary gland [11]. Caseins can be animportant source of biologically active peptides[15].While no specific physiological property has beenproposed for the whole casein system (or itsindividual fractions, for that matter), various peptideshidden (or inactive) in the amino-acid sequence havebeen the subject of increasingly intense studies. Thepeptides are not active within the parent protein butcan be released and activated following enzymatichydrolysis [6, 8].Much work regarding those peptides, which areknown to possess bioactivities, is currently underwayregarding their release via selective enzymatichydrolysis.The beneficial health effects may be attributed tonumerous known peptide sequences exhibitingantimicrobial, antioxidative, antithrombotic,antihypertensive, immunomodulatory and opioidactivities, among others [6].___________________________________________Corresponding author: e-mail: Nicoleta.Sava@ugal.ro

Daniela Pralea et. al. / Journal of Agroalimentary Processes and Technologies 2011, 17(3)The activity of these peptides is based on theirinherent amino acid composition and sequence.The size of active sequences may vary from 2 to20 amino acid residues, and many peptides areknown to reveal multi-functional properties. Forabove reasons, milk-derived bioactive peptides areconsidered as prominent candidates for varioushealth-promoting functional foods targeted atheart, bone and digestive system health as well asimproving immune defense, mood and stresscontrol. Recent studies suggest that bioactive milkpeptides may also be beneficial in reducing the riskof obesity and development of type two diabetes[31].The most common way to produce bioactivepeptides is through enzymatic hydrolysis of wholeprotein molecules. A large number of studies havedemonstrated that hydrolysis of milk proteins bydigestive enzymes can produce biologically activepeptides [16]. The most prominent enzymes arepepsin, trypsin and chymotrypsin that have beenshown to release a number of antihypertensivepeptides, calcium-binding phosphopeptides (CPP),antibacterial, immunomodulatory and opioidpeptides both from different casein (α-, β- and κ-casein) and whey proteins, e.g., α-lactalbumin, β-lactoglobulin and glycomacropeptide [7,9,10,19].Proteins are used in all kinds of food products toprofit from their nutritional value or from specificfunctional properties. Their physicochemicalbehaviour might, however, also impede the use ofproteins in products, for example, in high-energydrinks, where high viscosity or limited solubilityrestricts the protein concentration. Proteinfunctionality can be modified by enzymatichydrolysis, which alters, for instance, solubility,viscosity, and emulsion and foam properties.Choice of enzyme and process conditionsinfluence hydrolysate composition and thereby thefunctional properties.The objectives of present study were to study theeffect of enzymatic hydrolysis on the functionalproperties of sodium caseinate. Thegastrointestinal enzyme chymotrypsin has beenutilized to generate hydrolysates. Functionalproperties were limited to solubility, foamingcapacity, and 2,2-diphenyl-1-picryhydrazyl(DPPH) radical scavenging activity. Theseproperties were evaluated and compared as afunction of hydrolysis degree.2. Materials and MethodsCasein sodium salt from bovine milk and α-chymotrypsin (EC 3.4.21.1, type II from bovinepancreas, 40 units/mg protein) were purchased fromSigma-Aldrich, Inc. (St. Louis, MO, USA). Allsolvents and chemical reagents were of analyticalgrade.Enzymatic hydrolysis.A 5% (w/v) aqueous solutionof casein sodium salt was adjusted to pH 8.0 withHCl 1 mol.L -1 and digested with 0.25% (w/w ofsubstrate) α-chymotrypsin for 10 to 80 min at 37°C.The reaction was terminated by heating at 70°C for15 min and the pH was adjusted to 4.6 by addition ofHCl 1 mol.L -1 . The digest was centrifuged at 6000xgfor 20 min and the supernatant was freeze dried untiluse.Hydrolysis degree.The DH% values were determinedby the modified o-phtaldialdehyde (OPA) method.This involved using N-acetyl cysteine (NAC) as thethiol reagent [25]. The OPA/NAC reagent (100 mL)was prepared daily by combining 10 mL of 50 mmOPA (in methanol) and 10 mL of 50 mm NAC, 5 mLof 20% (w/v) sodium dodecyl sulphate (SDS), and 75mL of borate buffer (0.1 M, pH 9.5). The reagent wascovered with aluminum foil to protect from light andallowed to stir for at least 1 h before use.The OPA assay was carried out by the addition of 10µL of sample during hydrolysis to 2.5 mL ofOPA/NAC reagent. The absorbance of the solutionwas measured at 340 nm with an UV–VIS GBCCintra 202 spectrophotometer. The absorbance valuesfor the interaction of amino groups with OPA weretaken after 2 min standing for un-hydrolyzed sampleand after 10 min standing for hydrolyzed samples. Astandard curve was prepared using L-leucine (0-1.5mM).The DH value was calculated based on equation 1:DH (%) =∆A×M × d 100×ε × c N(1)where ∆ Abs is the Abs of test sample at 340nm -Abs un-hydrolyzed sample at 340 nm, M themolecular mass of the test protein (Da), d the dilutionfactor, e the molar extinction coefficient at 340 nm(6000 L mol -1 cm -1 ), c the protein concentration(g.L 1 ) and N the total number of peptide bonds perprotein molecule.309

Daniela Pralea et. al. / Journal of Agroalimentary Processes and Technologies 2011, 17(3)2,2-Diphenyl-1-picryhydrazyl (DPPH) radicalscavenging activity.DPPH radical-scavengingactivity was measured, using the method of Yenand Wu [29]. Sodium casein hydrolysates withdifferent DHs were dissolved in distilled water toobtain a concentration of 40 mg protein/ml. To 4ml of sample solutions, 1.0 ml of 0.2 mM DPPHwas added and mixed vigorously. After incubatingfor 30 min, the absorbance of the resultingsolutions was measured at 517 nm using aspectrophotometer (UV-VIS Cintra 202, GBCScientific Equipment, Australia).The control was conducted in the same manner,except that distilled water was used instead ofsample. DPPH radical scavenging activity wascalculated according to the equation 2 [29,30]:DPPH radical-scavenging activity (%)=(1 − ( A517sample − A517control))× 100 (2)A control517Solubility.To determine protein solubility 1 g ofprotein hydrolysate sample was dispersed in 100ml of distillated water and pH of the mixture wasadjusted to 2, 4, 6 and 8 with HCl 1 N and NaOH 1N. The mixture was stirred at room temperature for15 min and centrifuged at 6000xg for 15 min.Protein contents in the supernatant weredetermined using the Lowry method. Proteinsolubility was calculated according to the equation3:SPSolubility (%) = x100TP(3)where SP is the protein content in supernatant andTP is the total protein content in sample.Foaming properties.Foaming capacity and stabilityof protein hydrolysates were determined accordingto the method of Sathe and Salunkhe [23]. Onehundred milliliters of 1% sample solution werewhipped to incorporate the air for 3 min at roomtemperature. The whipped sample wasimmediately transferred into a 500 ml cylinder andthe total volume was read after 30 s. The foamingcapacity was calculated according to the equation4:A − BFoaming capacity (%) = x100B(4)where A is the volume after whipping (ml) and Bis the volume before whipping (ml).310The whipped sample was allowed to stand at roomtemperature for 3 min and the volume of whippedsample was then recorded. Foam stability wascalculated according to the equation 5:A − BFoam stability (%) = = x100(5)Bwhere A is the volume after standing (ml) and B isthe volume before standing (ml).3. Results and discussionsHydrolysis degree.Hydrolysis of casein sodium saltusing α-chymotrypsin was carried out by theOPA/NAC method. Rapid hydrolysis was observedwithin the first 30 min of reaction. Thereafter, aslower rate of hydrolysis was recorded. Themaximum extent of hydrolysis (DH 8.7±0.7%) wasobserved after 60 min of hydrolysis (Figure 1). Itseems that casein sodium salt is relative resistance toproteolysis, which is possible due to the compacttertiary structure of the protein that protects most ofthe enzyme susceptible peptide bonds.Figure 1. Hydrolysis curve of casein sodium salt by α-chymotrypsinFrom the degree of hydrolysis the average peptidechain length (PCL) of hydrolysates can be estimatedaccording to equation 6, assuming that the entirehydrolysate s are soluble [1].100PCL =(6)DH %The peptide chain length is related to the averagemolecular weight of peptides in hydrolysates.However, hydrolysates with similar PCL may havesubstantially different peptide molecular weightdistributions. Enzyme specificity, for example,influences the peptide size distribution: a pureendoprotease will yield peptides with varyinglengths, whereas an enzyme mixture that is mainlycomposed of exopeptidases will yield hydrolysateswith mostly free amino acids combined withremaining large peptides.

Daniela Pralea et. al. / Journal of Agroalimentary Processes and Technologies 2011, 17(3)Moreover, proteases acting according to a one-byonemechanism will yield hydrolysates with otherpeptide length distributions than proteases thathydrolyse proteins according to a zipper typereaction.Given the relative higher resistance to proteolysisof casein sodium salt, it was decided to test theinfluence of thermal treatment on the proteinsusceptibility to enzymatic hydrolysis at neutralpH. Thus, protein solutions were subjected to heattreatment in the temperature range of 85 to 95°Cfor 15 minutes, followed by cooling in ice water (2min) and hydrolysis at 37°C for one hour. In theseexperiments, a higher enzyme substrate ratio(1:50) was used. The results are given in Figure 2.It can be seen a significant increase in the DH afterheat pretreatment at 85°C and 90°C. For example,DH was about four times greater after 60 minutesof hydrolysis in the case of thermal pretreatment ofthe protein solution at 90°C.At 95°C, the increase in DH was significant in thefirst 10 minutes of hydrolysis, followed by asequential decrease in the rate of hydrolysis. Evenin these cases, DH values were higher whencompared with the un-treated solutions.Figure 2. The heat-induced changes in DH ( nothermal treatment, 85°C, 90°C, 95°C)DPPH radical-scavenging activity.DPPH radicalscavengingactivities of hydrolysates with differentDHs are depicted in Figure 3.Bars represent standard deviations from duplicatedeterminations. It can be seen that hydrolysateswith DH between 5 and 8.7% exhibited the highestDPPH radical-scavenging activity. As thehydrolysis time increased to 80 min, DPPHradical-scavenging activity decreased.Nevertheless, no significant differences wereobserved for the first 60 min of hydrolysis.Antioxidative activity of protein hydrolysatesdepends on the proteases [14] and hydrolysisconditions employed [20].Figure 3. DPPH radical-scavenging activity of sodiumcasein hydrolysates as a function of hydrolysis timeWu et al. [28] suggested that during hydrolysis, awide variety of smaller peptides and free amino acidsis generated, depending on enzyme specificity. Itseems that changes in size, level and composition offree amino acids and small peptides affect theantioxidative activity.Research on various casein hydrolysates has shownthat they possess radical scavenging activities [4,5].Suetsuna et al. [25,26] found that casein hydrolysatesexerted scavenging activity towards the superoxideanion, hydroxyl radical, and DPPH radical. It issuggested that the casein-derived Glu-Leu sequenceis important for this radical scavenging action, thusthe primary structure of the protein plays a role indetermining the antioxidant activity.Although Jing and Kitts [13] showed that casein doesnot affect cellular enzymes, Laparra et al. [17]reported that CPP reduced glutathione (GSH)concentration and increased GSH-reductase activityin Caco-2 cells. In addition, Phelan et al. [21]reported that casein hydrolysates affected bothcellular catalase activity and GSH content in Jurkatcells. They found that casein hydrolysates containeda certain degree of electron donating capacity asdetermined by the ferric reducing antioxidant power(FRAP) assay (17–32 mmol L -1 ) [22].Solubility.The solubilities of sodium caseinhydrolysates with different DH in the pH range of 2to 8 are given in Figure 4. All hydrolysates werehighly soluble over a wide pH range with more than90% solubility.In general, the degradation of proteins to smallerpeptides leads to more soluble products.311

Daniela Pralea et. al. / Journal of Agroalimentary Processes and Technologies 2011, 17(3)Enzymatic hydrolysis potentially affects themolecular size and hydrophobicity, as well as polarand ionizable groups of protein hydrolysates. As itcan be seen from Figure 2, the hydrolysatessolubility varies slightly as a function of DH andpH. It should be mentioned that the solubilityvalues were quite high at pH 4.Figure 4. Solubility of protein hydrolysates asinfluenced by pH values(Bars represent standard deviations from two repetitions)The pH affects the charge on the weakly acidic andbasic side chain groups and hydrolysates generallyshow low solubility at their isoelectric points.Solubility variations could be attributed to both netcharge of peptides that increase as pH moves awayfrom pI and surface hydrophobicity that promotesthe aggregation via hydrophobic interaction. Dueto the high solubility of the sodium caseinhydrolysates over a wide pH range, it waspresumed that products had a low molecularweight and were hydrophilic in nature.Foaming properties.Enzymatic hydrolysis ofsodium caseinate caused an increase in the foamcapacity initially and then a decrease withhydrolysis time. The foam volume was found to besimilar at both control and 10 min of hydrolysis,with a slightly decrease after 40 min (Figure 5).Figure 5. Foaming capacity and foam stability ofsodium casein hydrolysates(Bars represent standard deviations from two repetitions).312The foam stability of the control was greater than thatof the treated samples. Samples treated with α-chymotrypsin showed gradual decrease in foamstability with an increase in chymotrypsinolysis time.The lowest value of foam stability was measuredafter 80 min of hydrolysis.Hydrolysis of casein generally resulted in increasedfoam-forming ability of the hydrolysates compared tothe parental proteins [2,3,18]. The effect ofhydrolysis on foam stability seems to depend onenzyme specificity and degree of hydrolysis [18,24].The above results show that a limited amount ofhydrolysis is desirable to increase foaming but foamstability is greatly decreased as a result of suchhydrolysis. This is probably due to an initial increasein the polypeptide content, which allows more air tobe incorporated. However, the polypeptides do nothave the strength required to give stable foam. Thedecrease in foam stability manifests itself primarilyin the initial 60 min of reaction. Further hydrolysis islikely to result in peptides, which lack the ability tostabilize the air cells of the foam.Hydrolysis of casein results in a reduction ofmolecular weight, which might promote foamformation due to the faster diffusion of molecules tothe interface [27]. On the other hand, peptides formedduring hydrolysis might destabilize protein foams bydisplacement of proteins or by disturbingprotein/protein interactions [27, 30]. Furthermore,hydrolysis leads to increased charge density, whichnegatively influence foam stability, because foamstability was shown to improve when electrostaticrepulsion of proteins is minimal [12].4. ConclusionsSome functional properties of sodium caseinatehydrolysates were tested in this study. DPPH radicalscavengingactivity of protein hydrolysates variedslightly with the DH. The antioxidant activitydecreases an advanced hydrolysis. All hydrolysateswere highly soluble over a wide pH range with morethan 90% solubility. The foaming properties of theprotein hydrolysates were a function of hydrolysisdegree. Further studies are needed to identifypeptides from hydrolysates and their completecharacterization.AcknowledgementThe authors acknowledge financial support from theNational University Research Council (NURC, PN-II-ID-PCE-2008-2, Idea, ID 517) Romania.

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