Enzymatic Preparation And Physiological Activities Of Silk Fibroin Peptide

Fibroin, derived from silk cocoon, is a new source of food protein,possessing nutritionaland medicinal values. In China, a large number of cut cocoon with low economic values areproduced due to the deduction for the need of seed-making, resulting in a tremendous wasteof protein resources In this dissertation, the enzymatic hydrolysis technology was explored forproduction of bioactive silk peptides. The effects of reaction conditions on silk fibroinhydrolysis and peptide yield were investigated and a new silk pretreatment and peptideproduction systeme was established. In adiition, activities of fibroin from cocoon were testedand the effects of hydrolysis degree on the fibroin peptide activities were revealed.The sericin removation from cocoon was iniitally researched. Results showed that thesericin removing-rate reached22.08%by means of removaling twice using Na2CO3asmedium. Determination of properties of fibroin from cocoon showed that the proteincontent,oil content, moisture content, total sugar content and ash content were94.58%,0.44%,3.13%,0.66%and1.25%, respectively.Then six proteases from different sources were tested for their catalytic activity inhydrolysis of fibroin.Among them, Alcalase2.4L, an alkaline protease, showed the highestactivity and was chosen as the best catalyst for fibroin hydrolysis. By the orthogonal test, theoptimum hydrolysis conditions for Alcalase2.4L catalyzed hydrolysis of fibroin weredetermined as substrate concentration5%, enzyme dosage1000U/g, temperature60℃，pH8.5and reaction time240min, under which the highest DH value of the reaction reached18.69%.Thirdly, the in vitro ACE inhibitory activity, antioxidant activity, α-glucosidaseinhibitory activity and antibacterial activity of silk fibroin peptide were measured and theeffect of hydrolysis degree on those activities of the product were also investigated. Resultsshowed that the ACE-inhibition rate of silk fibroin peptide reached74.07%when the DH was17.12%. The silk fibroin peptide had the best antioxidant effect when the DH was14.97%.Thevalue of ORAC, DPPH· scavenging and·OH scavenging were determined as30582.6μmolTE/g,80.89%,71.23%,respectively. The α-glucosidase-inhibition rate of silk fibroin peptidereached44.90%when the DH was16.08%. The silk fibroin peptide had the best inhibitioneffect on Escherichia coli and Staphylococcus when the DH was18.69%and the diameter ofinhibition zone on Escherichia coli and Staphylococcus were27mm and21mm,respectively.The antibacterial effect intensified with the increase of the peptideconcentration within certain range.The minimum inhibitory concentrations of silk fibroin peptide on Escherichia coli and Staphylococcus were both10mg/ml.Finally, four kinds of fibroin peptited with different activities by control of the DH of thereaction(DH=14.97%,16.09%,17.12%,19.69%) were prepared for investigating teir molarweight distribution and amino acid composition. It was found that amino acid compositions ofthe four kinds of silk fibroin peptides were similar to fibroin and the contents of Ser, Ala,Glyand Tyr in silk fibroin peptide were higher than the unhydrolyzed protein. MALDI-TOF massspectrometry of the hydrolysate showed that the molecular weight of four kinds of silk fibroinpeptide were all below2.4kDa.Only a slight difference were found in the molecular weight ofthe four kinds of peptides.This research had high therotical value, which can enrich our knowleges on naturalproducts degradation and bioactive compounds production. In addition, it provides referencesfor preparation of new types of bioactive peptide and further value-added utilization of silkproteins.