Preparation Of Bioactive Peptides From Silkworm Pupa Protein By Simulated Gastrointestinal Digestion And Bioactivities Study On The Prepared Peptides

Silkworm pupa is the main by-product of silk reeling industry. China is a big country insericulture. The annual output of silkworm (dry base) is about500,000tons. Silkworm is rich innutrients, dried silkworm pupa protein content of about60%, fat content of25%to30%, inaddition to a variety of vitamins and iron, zinc, selenium and other trace elements. Pupa proteinis a resource of high quality protein which contains18kinds of amino acids needed by the body,including8kinds of essential amino acids, and is well-balanced. The content of essential aminoacids is up to40%, in line with the pattern recommended by FAO/WHO Food Standards (36%).Silkworm pupa protein polypeptides from simulated gastrointestinal digestion were studiedon the following aspects:Three different kinds of organic solvents were used to study the degrease performanc and oilquality, n-hexane, cyclohexane, petroleum ether which were commonly used in the oilextraction. The results showed that cyclohexane was the best solvent for oil in pupa powderwith the degreasing rate92.10%; n-hexane was best for oil defatting in entire pupa and thedegreasing ratio is up to86.15%. The pupa oil extracted by n-hexane was clear, light-color,light-smell and high-quality. Hexane was selected as the dissolvent to study the effects ofextraction temperature, extraction time and liquid-to-solid ratio on the oil extraction efficiencyby single factor experiment. The leaching rate was up to98.3%after three times whenextraction temperature was40℃, extraction time4h, liquid-to-solid ratio1:8. In this thesis,silkworm pupa protein (SPP) was used as the material to investigate the effects of spray dryingand vacuum freeze drying on the properties. For this purpose, SPP powders were made by twodifferent drying methods. Results indicated that spray dried products exhibited a little lightcolor and odor. Vacuum freeze drying needed longer time to prepare, cost more and was easy tocause browning reaction. However, vacuum freeze dried products possessed better solubility and higher powder yield. Water-holding capacity of samples through two methods did notexhibit obvious difference. But the oil absorption of vacuum freeze dried products was higherthan that of spray dried products.Bioactive peptides were prepared by simulated gastrointestinal digestion in vitro. SPP andpeptides were evaluated on their nutritional quality on the amino acid score and chemical score.Results showed that two samples both contained18kinds of amino acids which were necessaryfor the body, their compositions in line with the FAO/WHO standard patterns. The dataindicated pupa proteins and peptides might be a high quality supplement for human nutritionalsupport. Pupa proteins had an efficient digestion in vitro, the nitrogen release rate up to92.72%.Peptides prepared by simulated digestion possessed excellent antioxidant activity on reducingpower, DPPH· and OH· scavenging capacities.The hydrolysates fractions of different molecular weight (Mr)>50kDa,10~50kDa,5~10kDa, Mr<5kDa were obtained by ultrafiltration. Fractions of Mr<5kDa accounted for64.13%of the total polypeptides. Fractions of Mr<5kDa were separated by gel permeationchromatography to test the molecule mass distribution, the results showed that Mr<1kDafractions were the main activity fractions，accounting for97.54%. Polypeptide of differentmolecular weights all possessed high antioxidant activity. Among the four fractions, peptides ofMr<5kDa showed the highest reducing power, DPPH radical scavenging and·OH inhibitionrate.The last chapter studied the anti-tumor activity of gastrointestinal hydrolysates of silkwormpupa protein. MTT method was applied to determine the anti-tumor activity of gastrointestinalhydrolysates of different molecular weights on tumor cell proliferation. Results showed thatsilkwormpupa peptides could inhibit the viability of HepG2, PC-3and MDA-MB-231. Thehydrolysates fractions of10kDa<Mr<50kDa exhibited best inhibitory effect on HepG2cells,its half inhibitory concentration (IC50) of1.89mg/mL. Fractions of Mr<5kDa showed betterinhibitory activity on PC-3and MDA-MB-231respectively. Silkwormpupa proteinhydrolysates from simulated digestion showed good anti-tumor activity value, but itsmechanism needed further study.