Preparation Of Silkworm Pupa Protein Peptide And Its Anti-inflammatory Activity

Inflammation is the body’s defense response to infection,injury or chemical stimulation.However,uncontrolled excessive inflammatory responses are harmful to the body and have been implicated in the development of many diseases.At present,the commonly used anti-inflammatory drugs are gradually limited in practical application because they have a variety of side effects.Food-derived bioactive peptides have the advantages of wide sources,fast absorption and high safety.They have broad application prospects in the prevention and treatment of inflammation.Silkworm pupa has rich resources and high nutritional value,but due to the lack of deep processing technology,limited application and low resource utilization.In addition,silkworm pupa and its related products have been reported to have a variety of activities,but their anti-inflammatory activities are rarely reported,and the mechanism of action is still unclear.Many bioactive peptides have been isolated and purified from natural resources by microbial fermentation,but the research on anti-inflammatory peptides is still blank.Most of the anti-inflammatory peptides are mainly prepared by enzymatic hydrolysis.Therefore,this paper breaks through the traditional model,prepares the silkworm pupa peptide by Bacillus subtilis natto liquid fermentation,evaluates its anti-inflammatory activity based on the cell inflammation model and explores its mechanism of action,in order to provide a theoretical basis for the high-value utilization of silkworm pupa and the research and development of food-derived anti-inflammatory peptides.The main research contents and results of this paper are as follows:Three different silkworm pupa materials(silkworm pupa,defatted silkworm pupa and silkworm pupa protein)were fermented by Bacillus subtilis natto.The inflammatory model was established by LPS induced RAW264.7 macrophages,and the anti-inflammatory activity of its fermentation products was evaluated with the release of nitric oxide(NO)as the index.The results showed that among the fermentation products of three different silkworm pupa materials,the polypeptide yield of the silkworm pupa protein fermentation product was the highest.The three products fermented by Bacillus subtilis natto had no toxicity to RAW264.7 macrophages and promoted the growth of cells.Among them,the anti-inflammatory effect of the silkworm pupa protein fermentation product is the best,and the release of NO was significantly reduced in a dose-dependent manner.The preparation technology of silkworm pupa peptide was studied by a single factor experiment and response interview,and the optimum fermentation conditions were determined.The results showed that 2.6 g of silkworm pupa protein was added to every 100 m L of fermentation substrate,the inoculation amount of Bacillus subtilis natto was 5.0 m L(the concentration of seed solution was 10~7～10~8CFU/ml),the initial p H was 7.0 and fermentation temperature was37℃for 35.4 h.Under these conditions,the highest yield of the polypeptide was14.58%.In addition,the silkworm pupa peptide obtained under the optimal combination of fermentation process parameters was in LPS induced RAW264.7 macrophages showed good anti-inflammatory activity in the in vitro inflammatory model.The silkworm pupa peptides were separated and purified by ultrafiltration and semi-preparative liquid phase technology,and the components with the strongest anti-inflammatory activity were selected.Their structures were further identified by mass spectrometry,and the most promising anti-inflammatory peptides were selected by bioinformatics.The results showed that SPP-I-F4separated components were identified by mass spectrometry and 7 peptides were obtained.Among them,GYALPHAILR and YALPHAILR have the most anti-inflammatory potential,which was reported for the first time.The inhibitory effects of GYALPHAILR and YALPHAILR on inflammatory mediators(such as NO and cytokines)were investigated,and the effect of silkworm pupa anti-inflammatory active peptide on the expression of the inflammatory signal pathway was analyzed by Western blot.The results showed that the two silkworm pupa anti-inflammatory peptides had no toxicity to cells and could significantly reduce the secretion of NO,IL-1βand IL-6 in the cell inflammatory model,as well as by regulating mitogen-activated protein kinase(MAPK)and nuclear factor kappa-B(NF-κB)signaling pathway plays an anti-inflammatory role.In conclusion,the fermentation products with definite anti-inflammatory activity can be obtained from silkworm pupa protein by Bacillus subtilis natto fermentation.Two novel anti-inflammatory peptides were isolated,which played anti-inflammatory effects by inhibiting NO,inflammatory factors and regulating MAPK and NF-κB signaling pathways.The research conclusion of this paper provides theoretical and technical support for the development of silkworm pupa protein health promoting products.