Antibacterial Effect And Mechanism Of Enterocin TJUQ1

Bacteriocins are antibacterial peptides or proteins synthesized by ribosome.Due to their broad antibacterial spectrum,high safety and low resistance,they have attracted wide attention in biomedical and food fields.Lactic acid bacteria are microorganisms isolated from food,and the bacteriocins produced by them have an attractive prospect as food biological preservatives.Taking fermented food as a sample,this paper screened lactic acid bacteria producing bacteriocin,optimized the fermentation conditions for producing bacteriocin,purified bacteriocin,the properties were analyzed,and studied the antibacterial effect and mechanism of bacteriocin.In this study,Listeria monocytogenes CMCC 1595 was used as the indicator bacteria to screen lactic acid bacteria producing bacteriocin from celery fermentation broth by agar well difusion assay.Through morphological,physiobiochemical and 16S r DNA analysis,the strain was identified as Enterococcus faecium and named E.faecium TJUQ1.The biological characteristics of growth curve,optimal growth temperature,salt resistance,acid resistance and bile salt resistance of E.faecium TJUQ1were studied.Single factor and response surface method were used to optimize the fermentation conditions of E.faecium TJUQ1,the optimal medium were sucrose 30 g/L,tryptone 15g/L,yeast extract 6 g/L,beef extract 15.20 g/L,K₂HPO₄1.93 g/L,anhydrous sodium acetate 4 g/L,ammonium citrate 1.5 g/L,2 mmol/L Zn Cl₂,1.0 m L/L Tween 80.The optimal culture conditions were temperature 30°C,initial p H 7.19,inoculation quantity5%(V/V),loading volumes 120 m L/250 m L,fermentation under static condition for 18h.The maximum bacteriocin activity of TJUQ1 was 816.87±5.21 AU/m L under optimal fermentation conditions,which was 1.78 times higher than non-optimized condition.E.faecium TJUQ1 was cultured under optimized fermentation conditions,the bacteriocin was purified by ammonium sulfate precipitation,reversed phase chromatography and cation exchange chromatography.After purification,the specific activity was 44,566.41±874.69 AU/m L,and the purification fold was 35.89±2.34.The molecular weight of bacteriocin TJUQ1 was determined to be 5520 Da by Tris-Tricine SDS-PAGE and MALDI-TOF MS.Primers were designed with the known gene enterocin B,the genomic DNA of E.faecium TJUQ1 was used as the template,and the PCR test result was positive,but the translated amino acid sequence had only 58%homology with enterocin B,indicating that bacteriocin TJUQ1 was a novel bacteriocin and was named enterocin TJUQ1.The secondary structure of enterocin TJUQ1 was measured by circular dichroism as 32.6%helix,19.5%beta,12.9%turn and 35.0%random.In addition,the physical and chemical properties of enterocin TJUQ1 were studied.It can be degraded by a variety of proteases,but not affected by amylase and lipase.It has significant acid and alkaline resistance and thermal stability.Enterocin TJUQ1 has a broad antibacterial spectrum and can inhibit a variety of gram-positive and gram-negative bacteria,such as Staphylococcus aureus,Bacillus,L.monocytogenes,Escherichia coli and Salmonella enterica.Enterocin TJUQ1 was applied to L.monocytogenes CMCC 1595,minimum inhibitory concentration,time-inhibition curve,and the changes of its extracellular conductivity,nucleic acid,protein,ATP and lactate dehydrogenase were measured.The changes of bacterial cell structure were observed by SEM and TEM.The results showed that the antibacterial mechanism of enterocin TJUQ1 on L.monocytogenes CMCC 1595 was to form pores in the cell membrane,increase its permeability,and destroy its integrity,leading to the leakage of ions,nucleic acids,proteins,ATP and lactate dehydrogenase intracellular components,and finally leading to death.In conclusion,the enterocin TJUQ1 produced by E.faecium was a novel bacteriocin,and its molecular weight,physical and chemical properties,antibacterial spectrum and antibacterial mechanism were determined,which provided a basis for the development and utilization of bacteriocin.