Lactobacilli Stress Response Mechanism And Polymicrobial Interaction With Yeast In Food System

Food system is a complicated system with various chemicals and microbes.Foodborne microbes exist as complex polymicrobial community instead of single species.Based on the different roles played by microbes,mutualism,synergism,and antagonism are main polymicrobial interaction patterns.Thus,according to the chemical and microbe component in food system,understanding microbial behavior and polymicrobial interaction are of importance.Microbes in food systems exist as polymicrobial community and influence each other.Also,different microbes have various growth patterns.VBNC state Lactobacilli cells have been detected in food products passed routine microbial culturing detection in a real food spoilage accident.This study simulates the food processing and storage process to study the VBNC state formation and characteristics of Lactobacilli,as well as the molecular mechanism.The polymicrobial interaction between bacteria(Lactobacilli)and fungi(yeast)is also investigated.Concerning the hyphae formation ability of Candida,the inhibition of Lactobacilli on Candida hyphae formation through the regulation of glucose signaling pathway on BLP1 gene expression is elucidated.The primary contents and results are as follows:(1)Simulating the food storage process,low temperature storage and subculturing are applied to induce the VBNC state of 6 food spoilage Lactobacilli.Over 10~6 cells/mL viable cells enter into the VBNC state in the first generation,and all viable cells enter into the VBNC state after 105-196 days or 17-32 subcultures.VBNC cells shrink and remain similar organic acids,and diacetyl production levels and food spoilage capability.Thus,Lactobacilli are able to escape from sterilization and routine microbe culturing detection by entry into the VBNC state and cause food spoilage within shelf-life.Based on the VBNC state formation phenotype,genomics study is performed on typical Lactobacillus strains,as well as transcriptomics study on normal,stressed,and VBNC state.The Lactobacilli VBNC state formation mechanism at gene function and transcription level is demonstrated.The activation of 7 stress response factors to withstand environmental stress,and the repression of 18 transport,7 metabolism,28 enzyme activity,4 biosynthesis related genes to reduce requirement to adapt to the environmental stress are the key points.The VBNC state formation mechanism varies from species to strains.(2)Given the special growth pattern and the difference in Lactobacillus-yeast ratio,initial cell concentration and yeast with different growth patterns are selected to study the polymicrobial interaction between Lactobacillus and yeast.RNA-seq is performed to determine the mechanism.The polymicrobial interactions of Lactobacillus with Saccharomyces or Candida are similar,Lactobacillus inhibits yeast growth with its own growth unaffected.Lactobacillus activates lamBDCA QS system to enhance adhesion and adhere to yeast.Signal molecules are secreted to stimulate yeast activating stress response genes.However,the down-regulating of basic DNA-RNA-protein process,cell cycle,meiosis,and mating pathways lead to the lowered growth rate.The down-regulation of starvation adjustment,high-osmolality survival,cell wall remodeling pathways and cell integrity genes WSC1/2/3 lead to the death of some cells.The lowered growth rate and cell death cause the inhibited growth of yeast.(3)The effect of Lactobacillus on hyphae formation is studied phenotypically and genetically.The activation lamBDCA QS system enhances the adhesive ability of Lactobacillus.The signal molecules secreted by Lactobacillus stimulate Candida glucose repression pathway.The up-regulation of HGT19 transporter results in the import of more glucose,and the up-regulation of HXK2 gene increases the production of Hxk2 kinase,phosphorylating glucose to glucose-6-phosphate.The Glc7/Reg1 phosphorylase complex produced by the up-regulation of GLC7 and REG1 genes,recognizes glucose-6-phosphate and inhibit the expression of SNF1gene.The lower production rate of Snf1 kinase results in the activation of MIG1 and MIG2transcriptional repressors.Meanwhile,the HCM1 transcriptional activator is down-regulated.Thus,BLP1 gene expression is inhibited and hyphae formation is repressed.(4)Due to the difference in the growth pattern and genome of Saccharomyces and Candida,the polymicrobial interaction differ,including the inhibition rate,signal molecules,stress response genes,and metabolism pathways,as well as the regulation on Candida hyphae formation.Lactobacilli activate 3-5 stress response genes and down-regulate amino acid and fatty acid metabolisms,which are the partial cause of VBNC state formation.The study on microbial behavior and polymicrobial interaction lay the foundation on the control of microbes in different food systems.