Selection Of The Bovine Blood Proteolytic Bacteria And Study Of Its Enzyme-producing Characteristics

With the continuous development of society and the growth of people's needs,slaughtered blood waste is increasing day by day,but it still has not been effectively treated and fully recycled.Improper handling of slaughtered blood may cause not only resources waste,but also increase the risk of epidemicsang and environmental pollution.This study aims to explore the feasibility of using slaughter blood protein as fertilizer through the screening of high-efficiency blood protein-degrading strains and the study of enzyme-producing properties.In this study,Use vulture feces,sludge from slaughterhouse sewers or other related substrates as separation sources,and use blood agar plates and casein plates to screen strains that degrade blood proteins identify isolated strains,study their growth characteristics and drug resistance,optimize protein production conditions,and analyze the properties of protease and the evaluation of the degradation effect of bovine blood protein,the results of the study are as follows:1.Screening,identification and growth characteristics of the isolated strain for separating bovine blood protein:Ten strains with strong enzyme-producing ability were isolated from different matrix,and a strain Nw MCC01910137 with the optimal enzyme-producing ability was screened out,this isolated strain was identified Bacillus subtilis,and it was named Bacillus subtilis strain Nw MCC01910137.Through morphology,physiology and biochemistry and molecular biological identification.The optimal carbon source for the growth of this strain is lactose,the optimal nitrogen source is yeast powder,the suitable growth temperature is 35?,and the suitable growth p H is 7.5.This strain showed low sensitivity to ceftazidime and amphotericin B,and high sensitivity to other tested antibiotics.2.Optimization of the enzyme production conditions of Bacillus subtilis strain Nw MCC01910137 by response surface methodology:the optimal enzyme production conditions are:fermentation temperature 37.4?,initial p H 7.7,soybean meal addition 3.00%(w/v),glucose 3.8%(w/v),Na Cl 0.3 g·L^-1,phosphate concentration2.5 g·L^-1,inoculation amount 4%,fermentation time 48 h.Under these conditions,the enzyme activity is 166.83 U·m L^-1.3.The purification of protease produced and enzymatic properties of protease produced:The specific activity of the protease after preliminary purification is670.23 U·mg^-1,the purification multiple reached 3.82,and the recovery rate is25.92%;the optimal reaction p H of the enzyme is 7.The optimal reaction temperature is 40?.This protease is a mesophilic protease,neutral protease;The metal ion of K⁺,Na⁺,Mg²⁺,Zn²⁺,Ba²⁺have obvious promotion effect on protease activity,Ca²⁺inhibits protease activity;The inhibitor of EDTA,PMSF,IAM can inhibit protease activity;The Michaelis constant(Km)of the protease is 10.25 mg·m L^-1,and the Vmax is 27.93?g·m L^-1·min^-1.4.Evaluation of the degradation effect of bovine blood protein:when 90%of blood added in the fermentation system,the degradation rate of blood protein by the strain Bacillus subtilis strain Nw MCC01910137 is 17.61%,the peptide content in the degradation solution is 15.16 mg·m L^-1,and the free amino acid content 0.85%(w/v),the macromolecular peptides in the blood are all degraded into small molecule peptides.In summary,this study has isolated a Bacillus subtilis strain capable of degrading bovine blood proteins,which has the potential to be a candidate functional strain for the production of organic water-soluble fertilizer by decomposing waste blood into amino acids.