Antimicrobial Activity Of Lactobionic Acid And Its Antimicrobial Mechanism Of Yersinia Enterocolitica

Yersinia enterocolitica?Y.enterocolitica?is a zoonotic gut pathogen that began to attract widespread attention in the 1980s and it is a common foodborne pathogen in infectious diarrhea.Y.enterocolitica is widely found in the environment,animals,plants and foods.Since it can grow and reproduce at a low temperature about 4°C,it can be found in refrigerators and frozen foods.Therefore it is a hidden danger in food refrigerating and storage.Many countries have issued regulations for the detection of Y.enterocolitica.At present,the research on Y.enterocolitica at home and abroad focuses on detection methods and pathogenic mechanisms.The antibacterial mechanism of Yersinia enterocolitica was studied from the perspectives of cell growth,protein and gene after the determination of lactobionic acid bacteriostatic resistance,which provided theoretical support for the prevention of bacteria.1.Lactobionic acid was use as a bacteriostatic agent,and Escherichia coli CCTCC AB93154,Y.enterocolitica BNCC 336699/CMCC 52207,Staphylococcus aureus CCTCC AB91093,Bacillus cereus CCTCC AB 2011085,Pseudomonas fluorscens CGMCC 1.6279 and Proteus vulgaris CCTCC AB 91103 were chosen as test strain,which are susceptible to spoilage for bacterial species.The Oxford cup method was used as bacteriostasis test.The test results showed that lactobionic acid had significant inhibitory effects on gram-negative bacteria and gram-positive bacteria.The minimum inhibitory concentration?MIC?for Y.enterocolitica was 2.5 mg/mL,and the minimum inhibitory concentration against P.vulgars was 5.0 mg/mL,minimum inhibitory concentration of 12.5 mg/mL for B.cereus,15.0 mg/mL for E.coli,15.0 mg/mL for P.fluorscens and 20.0 mg/mL for S.aureus.2.Y.enterocolitica was chosen as the test strain.High-temperature heating,changing pH,adding metal ions and other processing methods of lactobionic acid were adopted to study the stability of lactobionic acid.The heat treatment which was from 60°C to 140°C had little effect on the bacteriostatic activity of lactobionic acid;and the addition of Na⁺,K⁺,Fe³⁺and Mn²⁺did not affect the bacteriostatic activity.However 0.1 mol/L Al³⁺increased the bacteriostatic activity?P<0.05?,while 0.1 mol/L Mg²⁺significantly reduced the inhibitory?P<0.05?;pH had a significant effect on the antibacterial activity?P<0.01?.The inhibitory effect of lactobionic acid at normal pH was the best.When pH was 6.0,the mean diameter of the inhibitory circle of lactobionic acid on Y.enterocolitica was the smallest,which was reduced by 3.83mm than normal.The decrease in pH had a significant increasing effect on antibacterial activity when pH<6.0?P<0.05?.3.The research on the antibacterial mechanism mainly focuses on the growth and metabolism of cell,the integrity of cell wall cell membrane,the inhibition of bacterial protein synthesis and the influence on the expression of key pathogenic genes.The growth curve of Y.enterocolitica showed that lactobionic acid delayed the arrival of logarithmic phase,and the number of bacterial cells in the stable phase of lactobionic acid treated group was significantly lower than that of the control group,indicating that lactobionic acid was against Y.enterocolitica.Increased alkaline phosphatase levels in lactobionic acid treated cultures demonstrated that the cell wall integrity was disrupted,what's more,the increased electrical conductivity and nucleic acid leakage,were also the evidence of cell membrane permeability.The phenomenons were more intuitively reflected in the ultrathin section photos of the cells observed by transmission electron microscopy.Transmission electron microscopy also observed the cytoplasmic solidification in the lactobionic acid-treated group,and even showed a cavity with low electron density,indicating that lactobionic acid exerts a great influence on the cytoplasm and organelles in the bacteria.Compared with the control,the band of the lactobionic acid treatment group disappeared in multiple regions at SDS-polyacrylamide electrophoresis,including protein tyrosine phosphatase that plays an important role in cell growth and metabolism.It was proved that lactobionic acid caused the disruption of some proteins which played crucial part of the bacteria.The real-time PCR results showed that the expression level of the important virulence gene ail?gene adhesion invasive locus gene?in the lactobionic acid treated group was down-regulated.