Functional properties of sodium caseinate hydrolysates - Journal of ...
Functional properties of sodium caseinate hydrolysates - Journal of ...
Functional properties of sodium caseinate hydrolysates - Journal of ...
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Available online at http://journal-<strong>of</strong>-agroalimentary.ro<strong>Journal</strong> <strong>of</strong> Agroalimentary Processes andTechnologies 2011, 17(3), 308-314<strong>Journal</strong> <strong>of</strong>Agroalimentary Processes andTechnologies<strong>Functional</strong> <strong>properties</strong> <strong>of</strong> <strong>sodium</strong> <strong>caseinate</strong> <strong>hydrolysates</strong> asaffected by the extent <strong>of</strong> chymotrypsinolysisDaniela Pralea, Loredana Dumitrascu, Daniela Borda, Nicoleta Stănciuc *“ Dunărea de Jos” University <strong>of</strong> Galaţi, Faculty <strong>of</strong> Food Science and Engineering, 800201, Domneasca Street, No.111, Galati, RomaniaReceived: 28 May 2011; Accepted: 28 July 2011______________________________________________________________________________________AbstractCasein is the main proteinaceous component <strong>of</strong> milk, where it accounts for ca. 80% <strong>of</strong> the total proteininventory. Biologically active peptides can be produced from precursor milk proteins. The most commonway to produce bioactive peptides is through enzymatic hydrolysis <strong>of</strong> whole proteins.The objectives <strong>of</strong> present study were to study the effect <strong>of</strong> enzymatic hydrolysis on the functional<strong>properties</strong> <strong>of</strong> <strong>sodium</strong> <strong>caseinate</strong>. Gastrointestinal enzymes, such as chymotrypsin, have been utilized togenerate <strong>hydrolysates</strong>. <strong>Functional</strong> <strong>properties</strong> were limited to solubility, foaming capacity, and 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity. These <strong>properties</strong> were evaluated and compared as afunction <strong>of</strong> hydrolysis degree.Keywords: casein, enzymatic hydrolysis, functional <strong>properties</strong>______________________________________________________________________________________1. IntroductionA major role <strong>of</strong> milk proteins is to supply amineacids and nitrogen to the young mammal. Milkproteins also constitute an important part <strong>of</strong> 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 <strong>of</strong>milk, where it accounts for ca. 80% <strong>of</strong> the totalprotein inventory. Until recently, the mainphysiological role <strong>of</strong> casein in the milk system waswidely accepted to be a source <strong>of</strong> amino acidsrequired by growth <strong>of</strong> the neonate. However, thedominant physiological feature <strong>of</strong> the caseinmicelle system has more recently been proven tobe the prevention <strong>of</strong> pathological calcification <strong>of</strong>the mammary gland [11]. Caseins can be animportant source <strong>of</strong> 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 <strong>of</strong> 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. / <strong>Journal</strong> <strong>of</strong> Agroalimentary Processes and Technologies 2011, 17(3)The activity <strong>of</strong> these peptides is based on theirinherent amino acid composition and sequence.The size <strong>of</strong> active sequences may vary from 2 to20 amino acid residues, and many peptides areknown to reveal multi-functional <strong>properties</strong>. 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 risk<strong>of</strong> obesity and development <strong>of</strong> type two diabetes[31].The most common way to produce bioactivepeptides is through enzymatic hydrolysis <strong>of</strong> wholeprotein molecules. A large number <strong>of</strong> studies havedemonstrated that hydrolysis <strong>of</strong> milk proteins bydigestive enzymes can produce biologically activepeptides [16]. The most prominent enzymes arepepsin, trypsin and chymotrypsin that have beenshown to release a number <strong>of</strong> 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 <strong>of</strong> food products topr<strong>of</strong>it from their nutritional value or from specificfunctional <strong>properties</strong>. Their physicochemicalbehaviour might, however, also impede the use <strong>of</strong>proteins 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 <strong>properties</strong>.Choice <strong>of</strong> enzyme and process conditionsinfluence hydrolysate composition and thereby thefunctional <strong>properties</strong>.The objectives <strong>of</strong> present study were to study theeffect <strong>of</strong> enzymatic hydrolysis on the functional<strong>properties</strong> <strong>of</strong> <strong>sodium</strong> <strong>caseinate</strong>. Thegastrointestinal enzyme chymotrypsin has beenutilized to generate <strong>hydrolysates</strong>. <strong>Functional</strong><strong>properties</strong> were limited to solubility, foamingcapacity, and 2,2-diphenyl-1-picryhydrazyl(DPPH) radical scavenging activity. These<strong>properties</strong> were evaluated and compared as afunction <strong>of</strong> hydrolysis degree.2. Materials and MethodsCasein <strong>sodium</strong> 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 <strong>of</strong> analyticalgrade.Enzymatic hydrolysis.A 5% (w/v) aqueous solution<strong>of</strong> casein <strong>sodium</strong> salt was adjusted to pH 8.0 withHCl 1 mol.L -1 and digested with 0.25% (w/w <strong>of</strong>substrate) 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 <strong>of</strong>HCl 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 <strong>of</strong> 50 mmOPA (in methanol) and 10 mL <strong>of</strong> 50 mm NAC, 5 mL<strong>of</strong> 20% (w/v) <strong>sodium</strong> dodecyl sulphate (SDS), and 75mL <strong>of</strong> 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 <strong>of</strong> 10µL <strong>of</strong> sample during hydrolysis to 2.5 mL <strong>of</strong>OPA/NAC reagent. The absorbance <strong>of</strong> the solutionwas measured at 340 nm with an UV–VIS GBCCintra 202 spectrophotometer. The absorbance valuesfor the interaction <strong>of</strong> 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 <strong>of</strong> test sample at 340nm -Abs un-hydrolyzed sample at 340 nm, M themolecular mass <strong>of</strong> 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 <strong>of</strong> peptide bonds perprotein molecule.309
Daniela Pralea et. al. / <strong>Journal</strong> <strong>of</strong> Agroalimentary Processes and Technologies 2011, 17(3)2,2-Diphenyl-1-picryhydrazyl (DPPH) radicalscavenging activity.DPPH radical-scavengingactivity was measured, using the method <strong>of</strong> Yenand Wu [29]. Sodium casein <strong>hydrolysates</strong> withdifferent DHs were dissolved in distilled water toobtain a concentration <strong>of</strong> 40 mg protein/ml. To 4ml <strong>of</strong> sample solutions, 1.0 ml <strong>of</strong> 0.2 mM DPPHwas added and mixed vigorously. After incubatingfor 30 min, the absorbance <strong>of</strong> 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 <strong>of</strong>sample. 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 <strong>of</strong>protein hydrolysate sample was dispersed in 100ml <strong>of</strong> distillated water and pH <strong>of</strong> 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 <strong>properties</strong>.Foaming capacity and stability<strong>of</strong> protein <strong>hydrolysates</strong> were determined accordingto the method <strong>of</strong> Sathe and Salunkhe [23]. Onehundred milliliters <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> casein <strong>sodium</strong> saltusing α-chymotrypsin was carried out by theOPA/NAC method. Rapid hydrolysis was observedwithin the first 30 min <strong>of</strong> reaction. Thereafter, aslower rate <strong>of</strong> hydrolysis was recorded. Themaximum extent <strong>of</strong> hydrolysis (DH 8.7±0.7%) wasobserved after 60 min <strong>of</strong> hydrolysis (Figure 1). Itseems that casein <strong>sodium</strong> salt is relative resistance toproteolysis, which is possible due to the compacttertiary structure <strong>of</strong> the protein that protects most <strong>of</strong>the enzyme susceptible peptide bonds.Figure 1. Hydrolysis curve <strong>of</strong> casein <strong>sodium</strong> salt by α-chymotrypsinFrom the degree <strong>of</strong> hydrolysis the average peptidechain length (PCL) <strong>of</strong> <strong>hydrolysates</strong> 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 <strong>of</strong> peptides in <strong>hydrolysates</strong>.However, <strong>hydrolysates</strong> 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 <strong>of</strong> exopeptidases will yield <strong>hydrolysates</strong>with mostly free amino acids combined withremaining large peptides.
Daniela Pralea et. al. / <strong>Journal</strong> <strong>of</strong> Agroalimentary Processes and Technologies 2011, 17(3)Moreover, proteases acting according to a one-byonemechanism will yield <strong>hydrolysates</strong> with otherpeptide length distributions than proteases thathydrolyse proteins according to a zipper typereaction.Given the relative higher resistance to proteolysis<strong>of</strong> casein <strong>sodium</strong> salt, it was decided to test theinfluence <strong>of</strong> thermal treatment on the proteinsusceptibility to enzymatic hydrolysis at neutralpH. Thus, protein solutions were subjected to heattreatment in the temperature range <strong>of</strong> 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 minutes<strong>of</strong> hydrolysis in the case <strong>of</strong> thermal pretreatment <strong>of</strong>the protein solution at 90°C.At 95°C, the increase in DH was significant in thefirst 10 minutes <strong>of</strong> hydrolysis, followed by asequential decrease in the rate <strong>of</strong> 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 <strong>of</strong> <strong>hydrolysates</strong> with differentDHs are depicted in Figure 3.Bars represent standard deviations from duplicatedeterminations. It can be seen that <strong>hydrolysates</strong>with 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 <strong>of</strong> hydrolysis.Antioxidative activity <strong>of</strong> protein <strong>hydrolysates</strong>depends on the proteases [14] and hydrolysisconditions employed [20].Figure 3. DPPH radical-scavenging activity <strong>of</strong> <strong>sodium</strong>casein <strong>hydrolysates</strong> as a function <strong>of</strong> hydrolysis timeWu et al. [28] suggested that during hydrolysis, awide variety <strong>of</strong> smaller peptides and free amino acidsis generated, depending on enzyme specificity. Itseems that changes in size, level and composition <strong>of</strong>free amino acids and small peptides affect theantioxidative activity.Research on various casein <strong>hydrolysates</strong> has shownthat they possess radical scavenging activities [4,5].Suetsuna et al. [25,26] found that casein <strong>hydrolysates</strong>exerted 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 <strong>of</strong> 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 <strong>hydrolysates</strong> affected bothcellular catalase activity and GSH content in Jurkatcells. They found that casein <strong>hydrolysates</strong> containeda certain degree <strong>of</strong> electron donating capacity asdetermined by the ferric reducing antioxidant power(FRAP) assay (17–32 mmol L -1 ) [22].Solubility.The solubilities <strong>of</strong> <strong>sodium</strong> casein<strong>hydrolysates</strong> with different DH in the pH range <strong>of</strong> 2to 8 are given in Figure 4. All <strong>hydrolysates</strong> werehighly soluble over a wide pH range with more than90% solubility.In general, the degradation <strong>of</strong> proteins to smallerpeptides leads to more soluble products.311
Daniela Pralea et. al. / <strong>Journal</strong> <strong>of</strong> Agroalimentary Processes and Technologies 2011, 17(3)Enzymatic hydrolysis potentially affects themolecular size and hydrophobicity, as well as polarand ionizable groups <strong>of</strong> protein <strong>hydrolysates</strong>. As itcan be seen from Figure 2, the <strong>hydrolysates</strong>solubility varies slightly as a function <strong>of</strong> DH andpH. It should be mentioned that the solubilityvalues were quite high at pH 4.Figure 4. Solubility <strong>of</strong> protein <strong>hydrolysates</strong> 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 <strong>hydrolysates</strong> generallyshow low solubility at their isoelectric points.Solubility variations could be attributed to both netcharge <strong>of</strong> peptides that increase as pH moves awayfrom pI and surface hydrophobicity that promotesthe aggregation via hydrophobic interaction. Dueto the high solubility <strong>of</strong> the <strong>sodium</strong> casein<strong>hydrolysates</strong> over a wide pH range, it waspresumed that products had a low molecularweight and were hydrophilic in nature.Foaming <strong>properties</strong>.Enzymatic hydrolysis <strong>of</strong><strong>sodium</strong> <strong>caseinate</strong> 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 <strong>of</strong> hydrolysis,with a slightly decrease after 40 min (Figure 5).Figure 5. Foaming capacity and foam stability <strong>of</strong><strong>sodium</strong> casein <strong>hydrolysates</strong>(Bars represent standard deviations from two repetitions).312The foam stability <strong>of</strong> the control was greater than that<strong>of</strong> the treated samples. Samples treated with α-chymotrypsin showed gradual decrease in foamstability with an increase in chymotrypsinolysis time.The lowest value <strong>of</strong> foam stability was measuredafter 80 min <strong>of</strong> hydrolysis.Hydrolysis <strong>of</strong> casein generally resulted in increasedfoam-forming ability <strong>of</strong> the <strong>hydrolysates</strong> compared tothe parental proteins [2,3,18]. The effect <strong>of</strong>hydrolysis on foam stability seems to depend onenzyme specificity and degree <strong>of</strong> hydrolysis [18,24].The above results show that a limited amount <strong>of</strong>hydrolysis is desirable to increase foaming but foamstability is greatly decreased as a result <strong>of</strong> 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 <strong>of</strong> reaction. Further hydrolysis islikely to result in peptides, which lack the ability tostabilize the air cells <strong>of</strong> the foam.Hydrolysis <strong>of</strong> casein results in a reduction <strong>of</strong>molecular weight, which might promote foamformation due to the faster diffusion <strong>of</strong> molecules tothe interface [27]. On the other hand, peptides formedduring hydrolysis might destabilize protein foams bydisplacement <strong>of</strong> 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 <strong>of</strong> proteins is minimal [12].4. ConclusionsSome functional <strong>properties</strong> <strong>of</strong> <strong>sodium</strong> <strong>caseinate</strong><strong>hydrolysates</strong> were tested in this study. DPPH radicalscavengingactivity <strong>of</strong> protein <strong>hydrolysates</strong> variedslightly with the DH. The antioxidant activitydecreases an advanced hydrolysis. All <strong>hydrolysates</strong>were highly soluble over a wide pH range with morethan 90% solubility. The foaming <strong>properties</strong> <strong>of</strong> theprotein <strong>hydrolysates</strong> were a function <strong>of</strong> hydrolysisdegree. Further studies are needed to identifypeptides from <strong>hydrolysates</strong> 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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