Study On The Combined Antibacterial Effect Of Antimicrobial Peptides From Brevibacillus Laterosporus With Food Additives

With the development of food industry and the improvement of requirements for food safety,natural food additives are becoming increasingly popular.Brevilaterin is an antimicrobial peptide from Brevilbacillus laterosporus discovered earlier in the laboratory.It has the advantages of high acid-base stability,broad antimicrobial spectrum and good thermal stability.In order to further broaden the antibacterial spectrum of Brevilaterin and reduce its usage in food preservation,the combined antimicrobial effects of Brevilaterin with food additives were researched in this study.Screened the food additives that had synergistic effect with Brevilaterin and formed the additive compound product with broad-spectrum,with a view to provide theoretical guidance for the application of Brevilaterin in food preservation.Minimum inhibitory concentrations（MICs）of Brevilaterin and food antibacterial agents against Staphylococcus aureus、Listeria monocytogenes、Bacillus cereus and Bacillus subtilis were determined by broth micro-dilution method.The results of broth micro-dilution method showed:nisin,polylysine,cinnamaldehyde and protamine had better bacteriostatic effect;the MICs of these food additives were less than 0.5 mg/m L.Combined effects of Brevilaterin with food additives were determined by micro-dilution checkerboard test.The results of checkerboard test showed:using S.aureus as indicator bacteria,the fractional inhibitory concentration index（FIC_I）values of Brevilaterin with Polylysine was 0.5,suggested that Brevilaterin showed synergic effect with Polylysine;the FIC_I values of Brevilaterin with nisin,protamine were 0.75 and 0.625,suggested that Brevilaterin showed partial synergic effect with nisin,protamine,respectively;using L.monocytogenes as indicator bacteria,the FIC_I values of Brevilaterin with Polylysin,eprotamine,eugenol,sodium benzoate,Na₃PO₄ and EDTA-Na₂were 0.625,0.625,0.625,0.532,0.75 and 0.75,suggested that Brevilaterin showed partial synergic effect with these six food additives,respectively;using B.cereus as indicator bacteria,the FIC_I values of Brevilaterin with tea polyphenol was 0.5,suggested that Brevilaterin showed synergic effect with tea polyphenol;using B.subtilis as indicator bacteria,the FIC_I values of Brevilaterin with tea polyphenol,cinnamaldehyde,Na₃PO₄ and potassium sorbate were 1,suggested that Brevilaterin showed additive effect with these four food additives,respectively.It can be seen from the above results that Brevilaterin showed combined antimicrobial effects with Polylysine and cinnamaldehyde respectively against gram-positive.MICs and combined effects of Brevilaterin and food additives against Escherichia coli、Salmonella typhimurium、Enterobacter sakazakii and Pseudomonas aeruginosa were determined by broth micro-dilution method and micro-dilution checkerboard test.The results of broth micro-dilution method showed:polylysine,cinnamaldehyde,eugenol and tea polyphenol had better antibacterial effect against gram-negative bacteria;the MICs of these food additives were less than 1 mg/m L.The results of checkerboard test showed:using E.coli as indicator bacteria,the FIC_I values of Brevilaterin with citric acid and EDTA-Na₂ were 0.5 and 0.141,suggested that Brevilaterin showed synergic effect with these two food additives agents,respectively;using E.sakazakii,as indicator bacteria,the FIC_I values of Brevilaterin with Na₃PO₄ and EDTA-Na₂ were 0.5,suggested that Brevilaterin showed synergic effect with these two food additives,the FIC_I value of Brevilaterin with kojic acid was 0.75,suggested that Brevilaterin showed partial synergic effect with kojic acid;using Salmonella as indicator bacteria,the FIC_I values of Brevilaterin with Na₃PO₄ and EDTA-Na₂ were 0.5 and 0.1875,suggested that Brevilaterin showed synergic effect with these two food additives,the FIC_Ivalues of Brevilaterin with citric acid,kojic acid and sodium benzoate were 0.75,suggested that Brevilaterin showed partial synergic effect with these food additives;using P.aeruginosa as indicator bacteria,the FIC_I values of Brevilaterin with EDTA-Na₂ was0.1875,suggested that Brevilaterin showed synergic effect with EDTA-Na₂,the FIC_Ivalues of Brevilaterin with citric acid,kojic acid and Na3PO₄ were 0.75,suggested that Brevilaterin showed partial synergic effect with these food additives.It can be seen from the above results that Brevilaterin showed synergic effect with EDTA-Na₂,citric acid and Na₃PO₄respectively against gram-negative.According to the synergic effect results,colony counting method was used to study the combined effect of Brevilaterin、nisin and citric acid.The results showed that Brevilaterin combined with nisin and citric acid had good antibacterial effect against gram-positive and gram-negative bacteria when they were at a concentration of 20 AU/m L,0.3125 mg/m L and 0.625 mg/m L.Brevilaterin combined with nisin and citric acid had a good antibacterial effect against S.aureus in milk when they were at a concentration of200 AU/m L,1.25 mg/m L and 3.125 mg/m L,the colony number was 4Lg CFU/m L within8 d,and decrease by 3.81Lg CFU/m L compared with using Brevilaterin（200 AU/m L）,nisin（1.25 mg/m L）,citric acid（3.125 mg/m L）only.Brevilaterin combined with nisin and citric acid had a good antibacterial effect against E.coli in milk when they were at a concentration of 800 AU/m L,3.125 mg/m L and 6.25 mg/m L.The colony number was5Lg CFU/m L within 8 d,and decrease by 2.82Lg CFU/m L compared with using Brevilaterin（800 AU/m L）,nisin（3.125 mg/m L）,citric acid（6.25 mg/m L）only.