| Lactic acid bacteria are defined as bacteria that can metabolize carbohydrates to produce lactic acid.Lactic acid bacteria are widespread in nature and the human body,with rich species diversity.They have promising application prospects due to some important capabilities including antagonizing pathogenic bacteria,enhancing immune function,strengthening the intestinal barrier,and balancing intestinal microbiota,and are expected to become safe,stable and economical biological antibacterial agents.In this study,509 strains of lactic acid bacteria were isolated from fermented food and human fecal samples collected from China,Egypt,and Sri Lanka.Pediococcus pentosaceus IM96 with excellent antibacterial activity,growth characteristics,probiotic characteristics,and safety characteristics was obtained based on screening 509 strains of lactic acid bacteria.Furthermore,using the Enterohemorrhagic Escherichia coli O157:H7-infected mice model to explore the protective effect of strain IM96 on infected mice and its anti-infection mechanism from the aspects of intestinal immunity,intestinal barrier,and intestinal microbiota in vivo.The main results were as follows:(1)Food samples and human fecal samples were collected from China,Egypt,and Sri Lanka,and the composition of microbial community structure in fermented dairy products was analyzed by 16 S r RNA gene amplicon sequencing method.Based on the genomic information of the samples,lactic acid bacteria were selectively isolated from samples by pure culture.After enrichment,separation,purification,and identification of the samples,509 strains of lactic acid bacteria were finally obtained,including 16 genera and 41 species.(2)Strains with broad-spectrum antibacterial activity were screened from 509 strains of lactic acid bacteria via antibacterial experiments,and the factors affecting their antibacterial activity and antibacterial mechanisms were explored in vitro.Antibacterial experiments were carried out with 9 common food-borne pathogens as indicator bacteria,and 3 strains of lactic acid bacteria with broad-spectrum antibacterial activity were screened and obtained: P.pentosaceus IM96,Lactiplantibacillus plantarum EG3 and Ligilactobacillus salivarius MZ84.Enterohemorrhagic Escherichia coli O157:H7 ATCC 43895(EHEC O157:H7)and Methicillin-resistant Staphylococcus aureus Sta396(MRSA Sta396)were used as indicators to investigate the influencing factors of antibacterial activity of lactic acid bacteria.The results showed that fermentation temperature,fermentation time,and carbon source type could affect the antibacterial activity of the strain and had obvious strain specificity.In the study of antibacterial mechanism in vitro,it was found that the fermentation supernatants of the 3 strains of lactic acid bacteria could inhibit the growth and reproduction of EHEC O157:H7 and MRSA Sta396.Scanning electron microscope showed that the cell integrity and morphology of EHEC O157:H7 were changed,and the cell wall was ruptured to form pores after EHEC O157:H7 was subjected to the fermentation supernatant of the 3 strains.All 3strains could inhibit the adhesion of EHEC O157:H7 to Caco-2 cells through competition,displacement and exclusion,and strain IM96 had the best inhibitory activity,with the inhibition rates of 33.90±8.50%,45.55±9.15%,and 27.70±7.46%,respectively.(3)The growth characteristics and optimal fermentation process of 3 strains lactic acid bacteria with antibacterial activity were explored,and their probiotic and safety characteristics were evaluated to lay a foundation for further research and application.The results showed that the 3 strains had the characteristics of a fast growth rate and a large number of fermentative cells,and could grow stably and rapidly under the conditions of a pH range of 5-8 and temperature range of 37-42℃.In terms of acid metabolism and production,all 3strains could metabolize to produce malic acid,lactic acid,acetic acid,succinic acid,and propionic acid.In addition,all 3 strains had good tolerance to gastrointestinal fluid,and the survival rate was above 75%.Moreover,strain IM96 had the highest adhesion ability to Caco-2 cells,and the adhesion rate was 13.21±2.14%.The results of genomic information and drug resistance analysis showed that strains IM96 and MZ84 had no virulence genes and drug resistance genes,and were sensitive to antibiotics and non-hemolytic.Based on the antibacterial activity,growth characteristics,probiotics characteristics,and safety characteristics of the 3 strains,strain IM96 was selected for the subsequent antibacterial mechanism study in vivo.(4)The protective effects and anti-infection mechanism of P.pentosaceus IM96 on infected mice were explored in vivo,using the EHEC O157:H7-infected mice model.The results showed that strain IM96 could enhance the anti-infection ability of mice in various ways.Strain IM96 could down-regulate the levels of pro-inflammatory factors IL-1β,IL-6,and TNF-α in the jejunum,and up-regulate the level of the anti-inflammatory factor IL-10 to reduce intestinal inflammation.The intestinal epithelial barrier function was enhanced by increasing the concentration of Mucin-2(MUC-2),the number of goblet cells,and the expression of tight junction proteins Occludin and Zonula occluden-1(ZO-1)in the jejunum.Additionally,the strain IM96 could regulate the intestinal microbiota,maintain the balance of the community structure,and increase the concentration of short-chain fatty acids in the intestine to resist EHEC O157:H7 invasion.In conclusion,P.pentosaceus IM96 had excellent antibacterial activity,growth characteristics,probiotic and safety properties.Further studies showed that P.pentosaceus IM96 could antagonize EHEC O157:H7 in vitro and vivo by inhibiting the growth,reproduction,and adhesion of EHEC O157:H7,reducing intestinal inflammation,enhancing intestinal barrier function,and regulating intestinal microbiota. |
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