Studies On Preparation And Antioxidant Activity Of Bioactive Peptides From Yeast

Beer is produced and consumed a lot in China each year, which leads to an abundant byproduct-spent brewer yeast. But at present, it is mostly used to produce the feed for animals and is not really effectively utilized. In order to make full use of protein component in yeast. we meant to hydralyze the spent brewer yeast to produce peptide product with definate bioacitivity and high adding values. The feasible industrial processing of peptides with high yield and high bioactivity was proposed in this study; modern technologys of puritification and analyse were also used to information about the relationship between the activity and structure.The hydrolyzing ability of commercial enzyme(sAlcalase,Neutrase,Protamex,N120,Papin,Flavorzyme) to yeast cell was firstly studied. Alcalase was selected for its most significant effect in producing yeast bioactive peptides, and its enzymatic factors were studied, including protease dosage, pH value, temperature and enzymatic time. The regression models were established between the impact factors and two indexes-yield of Total Nitrogen(TN) and Amino Nitrogen(AN). The results indicated that the optimal processing conditions for preparing protein peptides from the yeast with Alcalase were as follows: pH values 8. 7, temperature 50.8℃, A lcalase dosage 361.7U/g (as dry yeast basis), time 24h. Under these conditions, the yields of TN were 50.17% and AN 32.80%.Several methods of cell disruption were used in order to increase the dissolved rate of proteins in yeast. In this study, biochemical, ultrasonic, and homogenizing cell disruption were tried to disrupte the yeast cell wall.The experiment data showed that the homogenizing cell disruption was the most suitable method of cell disruption in the industry: the yield of products increased to 65% under the pressure of 50MPa. The bioactive peptides were then separated through a series of ultrafiltration membranes with molecular weight cutoffs of 10K, 3K, and 1KDa; and three types of permeates including 3K ~10KDa,1 K ~3KDa, and <1 KDa were obtained. The antioxidative efficacy of each fraction was determinted.The peptide fractions of molecular weight<1 KDa showed the strongest antioxidative activity :the ratio of DPPH scavening was 45.40%(1mg/ml).The peptide fractions of molecular weight<1 K were further separated by consecutive chromatographic methods including ion exchange chromatography on a SP Sephadex Fast Flow column and gel filtration on a Sephadex G-15 column. The loading parameters were: gradient elution seperation, starting buffer of 1M HAC and elution buffer of 1M NaCl. After seperation on ion exchange chromatography ,fraction 33 showed the strongest antioxidative activity with :the ratio of DPPH scavening 89.38%(1mg/ml).The O2-·and·OH radical scavenging activitity and anti-linoleic acid peroxidation capacity of the four fractions were investigated and compared . The data showed that all of the four fractions had certain antioxidative activity in vitro,while the Fraction 33 showed the strongest radical scavenging activitity : O2- radical scavenging ratio66.24%(10mg/ml),·OH radical scavenging ratio 58.11%(25mg/ml),anti-linoleic acid peroxidation ratio 46.27%(50mg/ml).The Fraction 33 was identified with LC-ESI-MS. The results indicated that the isolated peptides were composed of three peptides, which were composed of 4 or 7 amino acid residues each, the MW were 538.28,778.22,570.47,respectively; and the most probable structures were as Asn-Tyr- Leu-Glu, Asp-Val-Thr- Glu-Thr- Leu- Thr and Gln-Val-Pro-Asn-Leu.