Comparative Assessment Of Bioactive Peptides Obtained From Buffalo And Bovine Casein Hydrolysate

Buffalo milk is consumed in many parts of the world and ranked at the second position for worldwide milk production.In addition,it has higher protein content,especially of caseins,than that of bovine milk.However,there is a high degree of homology(>92%)between each of the caseins in buffalo milk and the corresponding casein in bovine milk,which emphasises their potential as substrates for release of bioactive peptides.Recently,several studies have described the generation of antioxidant peptides from different milk proteins by in vitro enzymatic hydrolysis and these have been isolated and identified bioactive peptides.The objectives of this study were to;first generate antioxidant peptides from buffalo casein and compared antioxidant activities of bovine casein and buffalo casein after hydrolysis by alcalase,trypsin,pepsin,and papain enzymes to identify treatments of protein hydrolysates that produce the highest antioxidant activity,second to achieve fractionation,purification and identification of the antioxidant peptides.In the first study,we carried out to evaluate and camper the physiochemical and characteristics of buffalo casein with bovine casein.The casein powder was characterized in terms of particle size distribution,zeta-potential,solubility,and scanning electron microscopy.The bovine casein contained highest protein content(93.96%).Buffalo casein had the largest particle size and more white color than bovine casein(173.7 and 94.7 nm)respectively.Zeta-potential for buffalo and bovine casein showed negative charge and mobility suitable to solubility index(99 and 95)in aqueous.Scanning electron microscopy images of buffalo casein was showed large size,as well as surface of smooth of these structural characteristics which was similar bovine casein.Hence,buffalo casein could be considered as a natural source for coffee whiteners and ingredient food in supplement food products to use in functional foods and pharmaceutical applications.In the second study,Buffalo casein(CB)and bovine casein(CN)were hydrolyzed using alcalase,trypsin,pepsin and papain to produce peptide hydrolysates.A comparative assessment of the degree of hydrolysis(DH),SDS-PAGE,amino acid composition and reverse-phase HPLC(RP-HPLC)of both bovine and buffalo casein hydrolysates(CNH and CBH)was carried out.Antioxidant activities of CNH and CBH were evaluated using various free radicals in-vitro antioxidant assays.The highest DH was observed in CN(87.28%)and CB(83.9%)with alcalase,while hydrolysis of the same substrates with trypsin gave a DH of 65.84% and 63.42%,respectively.SDS-PAGE confirmed molecular weight of peptidehydrolysates from alcalase and trypsin were smaller than 3.5 KDa.RP-HPLC analysis of CNH and CBH exhibited considerable variation in peptide hydrolysate composition.Amino acid composition showed high hydrophobic amino acids like tyrosine,valine,lysine,histidine and methionine in both CBH and CNH treated with alcalase and trypsin.All CNH and CBH showed varying degree of antioxidant activity.Altogether,alcalase-CBH and trypsin-CNH had the highest free radical scavenging potential and protection against hydroxyl radical induced by DNA nicking.Therefore,the utilization of alcalase-CBH and trypsin-CNH could provide the beneficial antioxidant properties in fractionation,purification and identification of the antioxidant peptides.The third study was carried out to be hydrolysis of buffalo and bovine caseins by alcalase and trypsin to produce novel antioxidant peptides.The casein hydrolysates were purified using ultrafiltration(UF)and further characterized by RP-HPLC.The fractions produced higher antioxidant activities were identified for their peptides using LC MS/MS.All UF-VI(MW < 1 k Da)fractions showed higher antioxidant activity.Hydrolysate produced by alcalase for buffalo casein(UF-VI with 54.84 fold purification)showed higher antioxidant activity than that obtained by trypsin.Trypsin hydrolysate contained high amount of hydrophobic amino acids while alcalase hydrolysate consisted mainly of Ser,Arg,Ala and Leu.The antioxidant peptides identified by LC MS/MS were RELEE,MEDNKQ and TVA,EQL in buffalo casein produced by both trypsin and alcalase treated hydrolysates,respectively.Mechanism and reaction pathways of selected antioxidant peptides with ABTS were proposed.Conclusively,buffalo casein provided novel antioxidant peptides differ to bovine casein,suggesting that buffalo casein is a novel source of antioxidant.