| The raw material was skimmed silkworm pupa powder from An Kang,Shaan Xi provice.After hydrolyzed, the hypoglycemic activity was tested. Two hypoglycemic activity characterization models was found, one of which was the influence on a-Glucosidase activity and the influence on glucose metabolism of Saccharomyces cerevisiae. According to inhibitory rate of a-Glucosidase activity, the enzymatic hydrolysis process was optimized at first and verified by the test of the influence on glucose metabolism of Saccharomyces cerevisiae The highest hypoglycemic activity polypeptide was filter out.Silkworm pupa protein's composition,secondary structure microstructure and amino acid composition was studied. Weight percent and hypoglycemic effect of different protein fractions was compared, including albumin, globulin, gliadin and glutenin. Silkworm pupa polypeptide was ultrafiltrated and the hypoglycemic effect of membrane fractions was tested. Silkworm pupa hypoglycemic polypeptide products were prepared, including Silkworm pupa hypoglycemic polypeptide lozenge and Tremella&silkworm pupa hypoglycemic polypeptide powder.1,Two hypoglycemic activity characterization models was found, one of which was the influence of a-Glucosidase activity and the influence of the glucose metabolism of Saccharomyces cerevisiae. p-Nitrophenyl-a-D-glycopyranoside(PNPG) was degraded into p-nitrophenol(PNP) by a-Glucosidase.PNP has absorption peak at400nm. The enzymatic activity of a-Glucosidase was reflected by the concentration of PNP. The changes of residual sugar content and the amount of pyruvic acid in fermentation broth was chosen to characterized the influence on the glucose metabolism of Saccharomyces cerevisiae. In a word,the influence of a-Glucosidase activity model was simple repetitive and short time-consuming, which applies to the initial activity characterization. Due to clear background and easy cultivation, Saccharomyces cerevisiae is widely used.The influence of the glucose metabolism of Saccharomyces cerevisiae model was time-consuming and was used to further activity verification.2,Prepare silkworm pupa hypoglycemic polypeptide and optimize the the enzymatic hydrolysis process. According to a-Glucosidase activity inhibitory rate, the optimized enzymatic hydrolysis process was as follows:Skimmed silkworm pupa was crushed and screened with40mesh sieve. After heat pretreatment at60℃,10min,the skimmed silkworm pupa was hydrolyzed into polypeptide for240min,with0.1%cellulose and2%"Alcalase+Protamex"(1:1).The inhibitory activity of "Alcalase+Protamex"(1:1) hydrolyzed polypeptide was98.64%at200mg/mL, while BaiTangPing's was98.39%at60mg/mL.It was showed that polypeptide from the optimized process could promote glucose metabolism and pyruvic acid formation, according to the result of glucose metabolism model of Saccharomyces cerevisiae3,Classify silkworm pupa protein and study their nature:By soxhlet extrac tion, silkworm pupa powder was skimmed. The grease content was18.49%and t he protein content was77.33%.Based on protein solubility, skimmed silkworm pu pa powder was classified into albumin(55%),globulin(6%),gliadin(20%) and gluten (19%).The total protein of silkworm pupa was prepared by "Alkali soluble and a cid precipitation" method. In190-260nm,the secondary structure of total silkwor m pupa protein was as follows:a-spiral structure accounted for40.1%,β-folding structure accounted for14.2%(including8.4%anti-parallel and5.8%parallel struc ture), β-rotation accounted for16.9%and random coil structure accounted for19.0%.The microstructure was various among total protein, albumin, globulin, gliad in and gluten. In spite of same amino acid elements, amino acid analysis show ed that each component protein owned different amino acid composition. Gluten' s essential amino acid content was39.27%,which was the highest one. The order of a-Glucosidase inhibitory activity was total protein hydrolyzate's> albumin h ydrolyzate's>globulin hydrolyzate's>glutenin hydrolyzate's>gliadin hydrolyzate's."Alcalase+Protamex"(1:1)hydrolyzed polypeptide was separated into different molecular weight fractions by ultrafiltration membrane. They were>300ku(weigh t percent:32.97%),5-300ku(weight percent:24.20%),<lku (weight percent:26.14%),1-5ku (weight percent:16.68%).Their a-Glucosidase activity inhibitor y was different. From the highest to the lowest,it was1-5ku,5-300ku,unsepera ted mix polypeptide,<1ku,>300ku.4,Two kinds fo silkworm pupa hypoglycemic polypeptide products were prepared.1) Silkworm pupa hypoglycemic polypeptide lozenge was prepared with "Alcalase+Protamex"(1：1) hydrolyzed polypeptide as raw material. Its physical and chemical indicators were determined. The optimal formula was50%"Alcalase+Protamex"(1=1) hydrolyzed polypeptide,0.3%acesulfame,0.9%citric acid,3%sodium carboxymethyl cellulose,30%maltodextrin,0.3%orange flavor,14.7%starch,0.8%magnesium stearate. The average tablet weight was0.499g/film. Polypeptide content was0.245g/film. The moisture content was1.69%and the hardness is10.84kg/mm2.2) Tremella&silkworm pupa hypoglycemic polypeptide powder was prepared. Skimmed silkworm powder was hydrolyzed for240min, by "Alcalase+Protamex"(1:1) at55℃,pH7.The hydrolysis degree was19.01%. Tremella liquid(polysaccharide content was36.53%) was prepared by Commercial dried Tremella,whose polysaccharide content was64.89%. The optimal formula was0.15%(m/V) citric acid,0.01%(m/V) acesulfame, Tremella liquid:silkworm pupa polypeptide liquid1:1(V/V). After Freezing-dring for48h, T he Tremella&silkworm pupa hypoglycemic polypeptide powder was gained. Its moisture content was less than0.05%.Ash content was17.4%.Total number of bacteria was273cfu/g and E.coil ware not detected. The protein content was57.98%,including20.34%polypeptide and essential amino acid50.57%.The final product showed dose-depent a-Glucosidase inhibitory activity. At10mg/mL,the a-Glucosidase inhibitory activity order was Bai Tang Ping> Tremella&silkworm pupa hypoglycemic polypeptide powder>"Alcalase+Protamex"(1:1)hydrolyzed polypeptide. |
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