Research On Structure And Application In Milk Antiseptic Of Brevilaterin From Brevibacillus Laterosporus

Our group previously screened for a Brevibacillus laterosporus which can highly produce antibacterial peptides named brevilaterin,the researches showed that brevilaterin has highly efficient and broad-spectrum antibacterial ability and good stability,and it was expected to be developed as a food preservative,however,the structure of brevilaterin is unclear.Clarifying the structure was the key to rational use of brevilaterin,So this study established a high performance liquid chromatography method to purify brevilaterin from fermentation broth of Brevibacillus laterosporus,meanwhile,the structure identification of brevilaterin was done by mass-spectrography and NMR,at last,the brevilaterin as food preservatives in milk was investigated to provide theoretical guidance for the application of milk.Brevilaterin was separated and purified by low pressure rapid purification chromatography system to obtain a mixture,then using a semi-preparative HPLC for single component separation acquired six brevilaterin(brevilaterin X、A、B、C、D、E)that purity was great than or equal to 98%.The MALDI-TOF MS uncovered the molecular weight of brevilaterin X、A、B、C、D、E were 1568.9753,1555.0334,1601.0044,1569.0189,1583.0344,1583.0946 Da respectively,and their structures were all determined by MALDI-TOF/TOF MS bonding with NMR including 1H-1H TOCSY,1H-1H NOESY,13C-1H HSQC spectra and formed by 13 amino acids including three modified amino acids which were Aba(2-amino-2-butenoic acid),ornithine,valinol(2-amino-3-methyl-1-butanol)at C terminus,respectively,and an N-terminal C6 fatty acid chain:Hmp(2-hydroxy-3-methylpentanoic acid).The primary structures were Hmp-Aba-V-Orn-I-V-V-K-V-L-K-Y-L-valinol(brevilaterin X),Hmp-Aba-V-Orn-V-V-VK-V-L-K-Y-L-valinol(brevilaterin A),Hmp-Aba-M-Orn-I-V-V-K-V-L-K-Y-L-valinol(brevilaterin B),Hmp-Aba-V-Orn-I-V-V-K-V-L-K-Y-L-valinol(brevilaterin C),Hmp-Aba-V-Orn-I-V-I-K-V-L-K-Y-L-valinol(brevilaterin D),Hmp-Aba-L-Orn-I-V-V-K-V-L-K-Y-L-valinol(brevilaterin E).By investigating brevilaterin preservation effect in row milk,on the sixth day,blank control group showed that the number of bacteria was 7Lg CFU/mL,mold and yeast was 6.5Lg CFU/mL,the acidity was 45 °T;while 1 mg/mL brevilaterin can reduce Aerobic thermophilic bacteria by 2Lg CFU/mL,Coliform bacteria by 2Lg CFU/mL,mold and yeast by 1.5Lg CFU/mL,Lactobacillus by 1.5Lg CFU/mL,the acidity by 29 ° T.Brevilaterin can extend the shelf life of row milk to sixth day.In addition,this study investigated the inhibition effect of brevilaterin on B.subtilis,L.monocytogenes,S.aureus by inoculating them 1 × 105 CFU/mL to contaminate UHT whole fat milk,respectively.The results showed that 0.4 mg/mL brevilaterin achieved bactericidal effect on B.subtilis within 24 hours,0.5 mg/mL brevilaterin made L.monocytogenes sterilization within 3 days,and 1 mg/mL brevilaterin has a bactericidal effect on S.aureus within 8 hours,all processing groups can be maintained to the fourteenth day,and the state of milk was in good condition within 14 days.