Studies On Probiotic Characteristics Of Pediococcus Pentosaceus And Its Regulation Under Heat Stress As Well As Modulatory Effect On Gut Microbiota Of Mice

Pediococcus pentosaceus is known as an important species of lactic acid bacteria for improving synthesis and metabolism of nutrient,regulating immune,reducing cholesterol and other functions of.It has been authorized as an edible species by National Health and Family Planning Commisson.In the food industry,P.pentosaceus has been widely used to improve the nutrition,flavor and security of fermented foods.Thus,screening and application of some strains of P.pentosaceus in fermented foods may obtain tremendous value.The nutrition and flavor are variable in Chinese traditional fermented foods,the sources containing large number of probiotics.In this study,we targeted 8 strains of P.pentosaceus(WPPE01,WPPE02,WPPE03,WPPE04,WPPE05,FPPE03,DC05,DC09)from Chinese traditional fermented foods(pomelo peel,dou-chi and sourdough)and investigated their biology properties,i.e.its survival capacity(acid,bile salt,salt and heat tolerance),probiotic properties(auto-aggregation properties,adhesion to intestinal cell lines,antimicrobial properties,effects on the adhesion of Escherichia coli O157: H7 on Caco-2 cells)and safety evaluation(haemolytic activity,antibiotic resistance),with the type strain P.pentosaceus CGMCC1.2695 as a control.The results showed that all 8 isolates were able to survive at acid and bile salt conditions.Auto-aggregation ability of isolates was positively related to the adhesive ability to the Caco-2 cells.Except Bacillus cereus ATCC14579,other common pathogens(Listeria monocytogenes CMCC54007,Staphylococcus aureus CMCC26003,Escherichia coli O157: H7,Salmonella Typhimurium ATCC13311,Pseudomonas aeruginosa MCC10104,Shigella song ATCC25931)were inhibited by 8 isolates of P.pentosaceus.Thereof the adhesion of E.coli O157: H7 on Caco-2 cells was strongly inhibited by P.pentosaceus through competition and displacement,with highest inhibition values of 65.70%.Moreover,all isolates showed none hemolysis and susceptibility to some antibiotics,and survived with at least 84% in 3% salt concentration of medium,their resistance index(RI)was less than 0.53 by the treatment at 55 oC for 15 min.Considering heat resistance of 8 isolates of P.pentosaceus,we further analyzed the mechanism of heat stress response of 5 srains(CGMCC1.2695,WPPE03,WPPE04,FPPE03,DC09).First the heat adaptation and lethal temperature was tested by plate counting method,then the cell morphology was checked through scanning electron microscope,finally,the mRNA level of genes relevant to heat shock protein and fatty acid synthesis was analyzed by reverse transcription-quantitative PCR(RT-qPCR).Our results showed that the adaptation and lethal condition was at 47 oC for 60 min and 62 oC for 15 min,respectively.The cell surface appeared sunken and wrinkled,and partial cells were broken with cytoplasm leaking out.However,the cell morphology was protected when there was a heat adaptation.In addition,expression of heat shock protein relevant genes was significantly up-regulated under heat stress(P<0.05),and the genes involved in fatty acid synthesis was up-regulated or down-regulated,indicating that the heat resistance was closely related to heat stress response of P.pentosaceus.To study the safety and effects on gut microbiota of P.pentosaceus,BABL/c mice were gavaged respectively with 5 strains(CGMCC1.2695,WPPE03,WPPE04,FPPE03,DC09)of P.pentosaceus(109 CFU/mL,200 ?L for every two days)for 2 weeks.The results showed that all of the strains had no significantly effect on the body weight,immune organ indices,serum biochemical indexes and histological pattern of mice liver(P<0.05).Real-time PCR test showed that P.pentosaceus WPPE03 and WPPE04 increased the populations of Bifidobacterium and Lactobacillus in faeces,whereas reduced the potential enteropathogenic bacteria e.g.Bacteroides and Clostridium perfringens.Hence,P.pentosaceus WPPE03 and WPPE04 enhanced the number of probiotics in the intestinal flora,thus regulated gut microbiota.The heat response mechanism of P.pentosaceus may provide the theory reference for studying other probiotics under the challenge of high temperature during the fermentation.In conclusion,among 8 strains of P.pentosaceus,WPPE03 and WPPE04 might be useful in food fermentation for their beneficial probiotic properties.