Study On Preparation Of Edible Bird's Nest Peptide With High Whitening Activity

Edible bird's nest(EBN)is a traditional health food in China.It is popular among consumers because of its whitening activity.However,the relationship between the whitening substance and the activity of edible bird's nest is still unclear.Swallow shred is a by-product in the process of bird's nest processing,which contains feather impurities and does not form a cup,leading to its low commercial value.However,the basic components of swallow shreds are not much different from those of bird's nest.In order to establish the relationship between the components and the whitening activity of the EBN digested products,the enzymatic hydrolysis method was used to prepare the whitening peptide to improve the utilization rate and whitening activity of the protein.Finally,the composition and whitening activity of the peptides were studied by ultrafiltration.The main research contents and results were as follows:The purpose of this study was to study the contribution of various components of the digested products to its whitening activity.Firstly,the composition and content of the digested products were studied.The results showed that the digestibility and utilization rate of swallow shreds was low,and the dissolution rate of protein and sialic acid was less than 50%after simulated digestion.In the digested products of swallow shreds,17.41%sialic acid existed in free form,11.85%combined with glycan,and 14.98%combined with water-soluble protein.Then,the whitening activity of the digested products was studied by the antioxidant experiment of HepG2 cells and the tyrosinase activity inhibition experiment of B16 cells.The results showed that the digested products had significant antioxidant activity and tyrosinase inhibition activity,with EC₅₀ values of 1.84 mg/mL and 7.22 mg/mL,respectively.The results of H₂O₂induced oxidative damage showed that the cell survival rate increased from 40%to 57.37%when the concentration of digested product was 1 mg/mL.Cell experiments showed that the whitening activity of free sialic acid was higher than that of glycan sialic acid.Finally,the contribution rate of free sialic acid,glycan sialic acid and protein in the digestive products to the whitening activity was further analyzed.The results showed that the main antioxidant component in the digestive products was protein,with a contribution rate of 85.87%;the main component inhibiting tyrosinase activity was free sialic acid,with a contribution rate of 63.43%.In order to further improve the utilization rate and whitening activity of the protein,the peptide was prepared by enzymolysis.First of all,four kinds of protease were selected to hydrolyze swallow shreds.The inhibition rate of mushroom tyrosinase and free radical scavenging ability in vitro were used as the main indexes to screen the kinds of enzymes.The results showed that flavourzyme was the best enzyme.Then,the enzymatic hydrolysis process was optimized.It was found that under the conditions of heating at 120?for 1 h,enzyme dosage of 6000 U per gram substrate and hydrolysis time of 60 min,the inhibition rate of mushroom tyrosinase and protein dissolution rate of flavourzyme hydrolysate(10 mg/mL)were78%and 83.90%,respectively.The results showed that the antioxidant activity and tyrosinase inhibitory activity of the hydrolysates were significantly increased(p<0.05),and the EC₅₀values were 1.19 mg/mL and 5.11 mg/mL,respectively.The results of H₂O₂ induced oxidative damage showed that the cell survival rate increased from 40%to 70%when the concentration of flavourzyme hydrolysate was 1 mg/mL.As the whitening activity of enzymatic hydrolysates of swallow shreds is easily affected by digestion,the cell experiment was conducted to further study the changes of whitening activity of enzymatic hydrolysates after simulated digestion.The results showed that the antioxidant activity of enzymatic hydrolysates after digestion decreased,with EC₅₀ value of 1.51 mg/mL;tyrosinase inhibitory activity increased,with EC₅₀value of 1.83 mg/mL.The results showed that the main components of antioxidation and inhibition of tyrosinase activity were protein components,and the activity contribution rates were 95.48%and 74.78%respectively.In order to further study the whitening activity of polypeptides in protein fraction of enzymatic hydrolysate of swallow shreds,three fractions with molecular weight less than 3 kDa(F3),3 kDa?10 kDa(F3-10)and more than 10 kDa(F10)were obtained by ultrafiltration with3 kDa and 10 kDa ultrafiltration membranes.The yields of the three fractions were 53.51%,12.55%and 32.12%,respectively.The results of cell experiment showed that the antioxidant activity and tyrosinase inhibitory activity of F3 were significantly higher than those of other components(p<0.05),and the EC₅₀ values were 1.26 mg/mL and 4.17 mg/mL,respectively.The results of H₂O₂ induced oxidative damage showed that the cell survival rate increased from40%to 63.17%when F3 fraction was 1 mg/mL.F3-1 and F3-2 fractions were separated from F3 by dextran gel G15,and F3-2 had stronger whitening activity.The results showed that SAPLRVY,NELQKLLTY and PADTGDLTY in F3-2 had antioxidant activity and tyrosinase inhibitory activity.