Antioxidative effect of yak milk caseinates hydrolyzed with three different proteases

Aim: Yak milk is a type of milk that people are less familiar with due to its remote geographical location which may have significant effects on composition, microbiota and hydrolytic outcome. Present work was designed with the aim to evaluate the antioxidative effect of peptides derived from yak milk caseinate on hydrolysis with three different proteases. Materials and Methods: In this investigation Yak milk casein was hydrolyzed by three commercially available proteases (Trypsin, Pepsin and chymotrypsin). These hydrolysates collected at different hydrolysis times (30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min, 270 min, 300 min, 330 min and 360 min) were assayed for their antioxidant activity with respect to the effect of incubation period. Results: Among all the enzyme hydrolysates, the tryptic hydrolysates showed highest antioxidant activity followed by chymotryptic hydrolysates. Further, the peptide samples showing highest activity were subjected to RP-HPLC for their partial characterization. Tryptic and peptic hydrolysates produced peaks mainly in the region of hydrophillic solvent indicating the presence of hydrophillic peptides/peptides. Conclusion: The results indicated that yak milk casein could be a resource to generate antioxidative peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.


Introduction
and an increasing number of such molecules have been identified in milk protein hydrolysates and also Food proteins, besides their nutritional roles, fermented dairy products [6,7].Investigations usually contain peptide sequences encrypted in their primary report the production of biologically active peptides structures that are capable of modulating specific through the hydrolysis of caseins isolated from bovine physiological functions.These protein fragments, termed milk.However, milk caseins from different species bioactive peptides, are inactive within the sequence of (yak, for instance), and the high genetic variability of the precursor protein, and could be released through milk proteins, might originate different bioactive enzymatic hydrolysis in vivo or in vitro.After release, peptides [8,9].The most common way to generate they might exert opioid, antihyper-tensive, immunobioactive peptides is through enzymatic hydrolysis of modulatory, antibacterial, and antioxidant activities, whole protein molecules, and the bioactivity of such among others, with potential applications in food hydrolysis-generated peptides appears to be inherent to science, technology, and nutrition [1][2][3][4].
size and specific amino acid sequences [2,5].Milk proteins are considered important sources of Proteolytic enzymes from various sources (animal, bioactive peptides that could be released through plant, microbial) have been successfully employed in enzymatic hydrolysis by digestive (gastrointestinal), the production of these molecules [6,7].fermentation, and proteolysis employing enzymes In the current study, three different commercially derived from microorganisms or plants [4,5].These available proteases were employed in the hydrolysis of proteins, mainly caseins, are commercially available in sodium caseinate obtained from yak milk, under large amounts at a high degree of purity and at low price conditions simulating human digestive tract.The which, from a technological aspect, make them biological activities (antioxidant) of the protein hydroattractive in the search for bioactive peptides [2].
lysates were then assessed, particularly considering the Therefore, the properties of milk protein-derived effect of incubation period, with the aim of evaluateing bioactive peptides have been thoroughly investigated, its potential for food applications.

Materials and Methods
Isolation of casein: Raw milk samples were collected from yak in the higher regions of Himalaya in All reagents for HPLC were prepared using HPLC defatted by centrifuging twice at 5000 g for 20 min at grade water and solvents.These including acetonitrile 4°C in a refrigerated centrifuge.The milk was filtered used for preparation of solvents were membrane (0.22 via four layers of cheese cloth and fat separated was µ PVDF, Millipore, SA, France) filtered and degassed discarded.The filtrate was diluted with equal volume using Millipore filtration assembly. of double distilled water (DDW); pH adjusted to 4.6

Results
with 1N HCl and the mixture was stirred for 30 The total protein content of casein as estimated by minutes.The precipitate so formed was separated by Lowry's method was found to be 220ug/0.01ml of filtration through four layers of cheese cloth, washed, sodium caseinate [containing 3gm casein/50ml of solubilized in distilled water at pH 7.0 (equal to initial distilled water].Degree of hydrolysis (DH) was volume of milk) with 1N NaOH, re-precipitated and washed 3-4 times with distilled water.The wet casein, determined by quantification of protein in these after thorough washing with distilled water, was air-hydrolysates.The higher the DH, the higher the content dried by spreading on a sheet of filter paper at room of released amino groups.DH is reported to affect the temperature.The concentration of protein in various biological activity of protein hydrolysates.Therefore, caseins formed was estimated by Lowry's method [10].
the biological activity of peptides depends on the protein substrate, enzyme specificity, and hydrolysis Hydrolysis of casein: Casein prepared isoelectrically conditions [16][17][18].Degree of hydrolysis found was treated with three different enzymes according to maximum with pepsin treatment.This shows that the method of Abubakar et al. [11] and Pihlantopepsin utilized more protein as substrate to cause Leppala et al. [12] with some modifications (enzyme: hydrolysis as compared to chymotrypsin and trypsin.substrate ratio is taken as 1:100) (Table -1).Initially, Treatment with trypsin yields minimum DH indicating casein was incubated for 30 min, 60 min, 90 min, 120 that yak casein is most resistant to this enzyme.min, 150 min (so as to simulate human digestive tract Treatment of yak casein with pepsin for 360 min shows conditions where protein takes maximum about 120 highest hydrolysis.The same result of hydrolysis minutes to get digested) and after that for 180 min, 240 obtained when treated with chymotrypsin and trypsin.min, 300 min and 360 min to access the effect of higher It shows that degree of hydrolysis increases with the incubations.The degree of hydrolysis in various samples time of incubation.was estimated by using Hull's method [13].
The comparative inference of antioxidant status Assay of antioxidant activity: Antioxidant activity was of hydrolysates with reference to different incubation measured using 2,2-Diphenyl-1-picrylhydrazyl periods were tabulated (Table -2).Tryptic hydrolysates (DPPH) radical-scavenging assay as described by showed highest activity followed by chymotryptic and Brand-Williams et al.DPPH is a free radical that accepts an electron or a chromatograms only the major peaks showing hydrogen radical, becoming a stable molecule.For this significant rise from the baseline has been considered reason, it is employed as a substrate to evaluate the and taken into count.
antioxidant activity of peptides and protein hydrolysates.Results varied widely along with hydrolysis Discussion time, and a relationship between hydrolysis time and The degree of hydrolysis (DH) measures the DPPH activity could not be established.Proteolysis of content of peptide bonds cleaved in the substrate by a food proteins is usually reported to enhance the DPPHproteolytic agent (proteases, in the current case): the scavenging activity of hydrolysates [7].The DPPHhigher the DH, the higher the content of released amino scavenging activity of yak milk protein hydrolysates groups.DH is reported to affect the antioxidant activity obtained with Alcalase was observed to increase during of protein hydrolysates.Therefore, the biological the hydrolysis process for up to 7 h [22].Here also, the activity of peptides depends on the protein substrate, higher DPPH-scavenging activity was evidenced with enzyme specificity, and hydrolysis conditions [16-18].
higher degree of hydrolysis.Nevertheless, this is not DH found to be maximum with pepsin treatment, while always observed [23].Specifically, bovine casein in case of cow and buffalo caseins DH is usually high hydrolysates obtained with diverse proteolytic with trypsin.This difference can be attributed to enzymes were shown to possess lower DPPH activity difference in amino acid sequence of yak casein from than the whole protein [24].that of cow and buffalo casein e.g., in yak casein there Maximum percentage inhibition was shown by 1 is glycine and lysine at 192 and 59 positions in αs peptic hydroysates.Based on the result, hydrolysates casein while in cow and buffalo its glutamate and release short peptides having the antioxidant activity to glutamine respectively [19]; similarly glycine, relevant level which is directly related to incubation threonine and isoleucine at 33, 47 and 130 position in period or time of hydrolysis.In general, scavenging 2 αs casein in yak while its glutamate, alanine and activity was found to increase with the time of threonine respectively in cow and buffalo [20]; and incubation in case of all the enzymes.alanine in yak κ casein at 148 position while its Partial characterization of peptides showing aspartate in cow and buffalo casein [21].This shows maximum activity was done by RP-HPLC under two that pepsin utilized more protein as substrate to cause different wavelengths using aqueous as well as organic hydrolysis as compared to chymotrypsin and trypsin.
solvents.Peaks at 214 nm showing the presence of non- [14] with some modifications.peptic hydrolysates.The antioxidant activity goes on Each sample assay is carried out in triplicate & data are increasing with the incubation period.represented as a mean of three values along with the Hydrolysates showing maximum antioxidant activity standard deviation.were further subjected to RP-HPLC.Treatment with trypsin results in 6-8 peaks at 214 nm and 2-3 peaks at Reverse Phase-High Pressure Liquid Chromatography (RP-HPLC) of hydrolysates: The hydrolysates showing 280 nm were observed (Figure 1(a) & 1(b)).However, maximum activity were resolved on RP-HPLC for the peptic hydrolysate shows 3-4 peaks at 214 nm and

2- 3
peaks at 280 nm (Figure 2(a) & 2(b)).In case of Scavenging activities of yak caseinate chymotryptic hydrolysates, results are almost same as hydrolysates were determined using DPPH radicals.in peptic hydrolysates (Figure 3(a) & 3(b)).In all these Uttarakhand.Casein was prepared from the collected separation of different fractions of peptides following milk sample using the method of isoelectric the procedure given by Hernandez-Ledesma et al.[15].precipitation.Immediately after collection, milk was

Table - 1
. Conditions employed for hydrolysis of yak caseinate