Preparation Of Antibacterial Peptides By Enzymatic Hydrolysis Of Tartary Buckwheat Protein And Its Research On Beef Preservation

Antibacterial peptides are usually short peptides which are specifically immunized against microbial invasion produced by the innate immune system of many different species,such as animals,plants or fungi.Antibacterial peptides have a small molecular weight,good thermal stability,with a broad spectrum of anti-Gram-positive bacteria and anti-Gram-negative bacteria activity.The biologically active peptides are inactive within the sequence of the parent protein.They are released by enzymatic hydrolysis,and then they can exert a variety of physiological functions.Antibacterial peptides can be used for medicine,food and feed processing,with a wide range of applications.Antimicrobial peptides,as a natural preservative,have high safety and have aroused the researchers wide interest in it.In this paper,the protease screening and enzymatic hydrolysis conditions of antimicrobial peptides prepared by enzymatic hydrolysis of buckwheat protein were optimized,and the preservation effect of tartary buckwheat antimicrobial peptides on beef was studied.The method of enzymatic hydrolysis was used to broaden the application range of tartary buckwheat protein.On the one hand,the functional properties of the hydrolyzate were improved by enzymolysis.On the other hand,the activity of the hydrolyzate was analyzed and the biologically active peptide was screened.So as to provide the theoretical basis and effective way to promote the deep development and utilization of tartary buckwheat protein,and provide some reference for the application of antimicrobial peptides in the preservation of meat products,and provide a new direction for the application of buckwheat,which has certain economic significance and social significance.The main contents and results are as follows:1.Screening of enzymatic hydrolysis of tartary buckwheat proteaseThe alkaline protease,trypsin,flavor protease,papain,neutral protease,acid protease and pepsin were used to hydrolyze the tartary buckwheat protein,and the peptide concentration and bacteriostasis were screened.Neutral protease was thought to be the best protease to prepare antimicrobial peptides by enzymatic hydrolysis of buckwheat protein.The peptides concentration was 29.25 ± 0.45 mg/mL,the inhibitory rate of E.coli and Staphylococcus aureus was 22.48 ±1.73%and 92.82 ± 3.05%at 4 h after enzymatic hydrolysis,respectively.2.Optimization of the preparation of tartary buck wheat antimicrobial peptides by enzymatic hydrolysis of tartary buckwheat protein by neutral proteasePlackett-Burman test was used to select the most significant factors based on the results of single factor test of substrate concentration,the amount of enzyme,pH,hydrolysis temperature and hydrolysis time.The preparation of antimicrobial peptides process was optimized by Box-Behnken experiment.The optimum parameters of enzymatic hydrolysis were:substrate concentration 3.22%,the amount of enzyme 4000 U/g and pH 6.86.Under the optimum conditions,the peptides concentration of was 32.41 mg/mL(the predicted value was 32.12 mg/mL).The inhibition rate of antimicrobial peptides against E.coli was 27.88±2.78%,and against S.aureus was 94.56±0.74%.3.Enzymatic Hydrolysis of Tartary Buckwheat Protein by Double Enzyme and effect of tartary buckwheat antimicrobial peptides on beef preservationIn order to obtain a small molecular weight peptide,the neutral protease was digested with tartary buckwheat protein on the basis of double enzyme enzymatic hydrolysis,through the antibacterial effect and Tricine-SDS-PAGE gel electrophoresis results,to determine the use of neutral protease and pepsin.Digestion and enzymolysis were as follows:the first step of neutral protease digestion,the substrate concentration of 3.22%,the amount of enzyme 4000 U/g,pH 6.86,temperature 50 ?,time 2.5 h;the second step by adding pepsin,The amount of enzyme added 1000 U/g,pH 2.0,temperature 37?,enzymolysis time 40 min.The antibacterial rate of the hydrolyzate was 11.78±0.88%,the inhibition rate of S aureus was 82.06± 1.37%,and the peptide concentration was 30.50±0.55%.The ultrafiltration of the enzymolysis solution was carried out by ultrafiltration.using more than 5 kDa and less than 5 kDa different concentrations of tartary buckwheat antimicrobial peptides on the preservation of beef.pH value,volatile base nitrogen(TVB-N),thiobarbituric acid(TBA),high iron myoglobin content,color difference,texture and the total number of bacteria were used to study the beef changes of the freshness at different concentrations of more than 5 kDa and less than 5 kDa during storage of 4?.30 mg/mL tartary buckwheat antibacterial peptide of Less than 5 kDa treated beef preservation period extended for 6 days,freshness was significantly better than nisin.