Studies On Enzymatic Hydrolysis Of Poultry Blood Corpuscle And Isolation,identification,and Characterization Of The Antioxidant Peptide

To find new and effective approaches for utilizing poultry blood corpuscle,we hydrolyzed the duck blood corpuscle with proteases and decolored its hydrolysate.The changes of organoleptic qualities and nutritional properties were investigated for blood corpuscle after decoloration.Meanwhile,we evaluated the nutritional and functional properties of these hydrolysates.Furthermore,the antioxidant peptides were prepared by means of enzymatic hydrolysis or fermentation with Bacillus natto to explore the antioxidant potential of blood corpuscle.The antioxidant stabilities of blood corpuscle peptides were also evaluated in this study.The main results were summarized as follows:1.The results showed that acidic protease was the best enzyme to hydrolyze duck blood corpuscle and the optimal hydrolysis conditions were temperature 50?,pH 3.5,E/S 6000 U/g and time 7 h.Under these conditions,the degree of hydrolysis(DH)and hydrolysis yield were 25.10±0.65%and 60.09±1.77%,respectively.When hydrolyzing blood corpuscle with neutrase(3000 U/g)and flavourzyme G(1000 U/g),the optimal hydrolysis parameters to get the highest DH(32.06±0.59%)and TCA-index(56.25±0.32%)were substrate concentration 14%,temperature 51?,pH 7.0 and time 7.5 h.Another hydrolysate,obtained through hydrolysis by alacase for 3 h and followed by papain for 6 h,was optimized by Box-Behnken design to get the highest DH(26.31±2.59%)and TCA-index(51.95±0.26%),and the optimal conditions were temperature 55?,pH 9.0 and substrate concentration 12%.2.The physical and chemical methods were good ways to decolor the hydrolysate to prepare blood corpuscle peptide protein powder with light color and pleasure taste,which enriched in protein and essential amino acids.Moreover,the results of nutritional,functional and antioxidant properties indicated that the three kinds of blood corpuscle peptides enriched small peptides and amino acids,possessing excellent solubility(>60%)and antioxidant activity.3.The results of screening proteases displayed the pepsin hydrolysate had the highest DPPH radical scavenging activity.Under the optimal conditions(E/S 2.55%,pH 2.0 and time 3.0 h),the DPPH radical scavenging activity,superoxide ion scavenging activity and reducing power of pepsin hydrolysate were 93.01±2.51%,73.29±3.64%and 1.96±0.09,respectively.After purification and separation,the peptide was identified as MGQKDSYVGDEAQSKRGILT(2182.1 Da)and the DPPH radical scavenging activity of this purified peptide(100 ?g/ml)was 13.72-fold of its hydrolysate.Interestingly,the antioxidant peptide prepared by acidic protease was similar to that of pepsin.At the same time,we also adopted response surface method to optimize the enzymatic hydrolysis of blood corpuscle using papain and flavourzyme.The hydrolysate under the optimal parameters(E/S 2.0%,temperature 50? and time 6.0 h)had the highest antioxidant activity with 94.99±0.31%of DPPH radical scavenging activity,57.39±2.82%of superoxide ion scavenging activity and 1.83±0.06 of reducing power.The purified peptide was identified as AEDKKLIQ(943.5 Da),and at the concentration of 100 ?g/ml,it showed good DPPH radical scavenging activity(42.45±1.32%),superoxide jon scavenging activity(49.40±0.56%),hydroxyl radical scavenging activity(56.29±2.04%)and reducing power(0.14±0.00).4.In this study,the chicken blood corpuscle was also fermented by Bacillus natto to prepare antioxidant peptide.Under the optimal conditions(3%inoculum,4%blood corpuscle,3%glucose,and time 36 h),the DPPH radical scavenging activity,hydroxyl radical scavenging activity and reducing power of fermented product were 80.63±0.34%,76.84±7.58%and 3.84±0.05,respectively.The purified peptide was identified as TSFGDAVKNLDNIK(1521.7 Da),and at the concentration of 100 ?g/ml,its DPPH radical scavenging activity,hydroxyl radical scavenging activity and reducing power were 58.98±4.72%,49.64±0.86%and 0.67±0.01,respectively.5.The antioxidant activity of blood corpuscle peptides(PH?PH-I?AH?AH-I?PFH?PFH-I and BNH)obtained by enzymatic hydrolysis or microbial fermentation were affected by different factors.In summary,PH-1,AH,AH-1,PFH-I and BNH had excellent thermal stability,and the peptides except PH-I(good pH stability)lost their antioxidant activities under alkaline conditions.The increasing concentrations of Cu2+resulted in declining the antioxidant activities of all peptides,while combining PFH-I with Zn2+showed good synergetic antioxidant activity.After treated by two-stage in vitro digestion model system,the blood corpuscle peptides(PH,PH-I,AH,PFH and PFH-I)had increased antioxidant activities during pepsin digestion but decreased antioxidant activities during intestinal digestion,while both AH-I and BNH possessed good resistance against gastrointestinal enzymes.