Mechanisms Of Sublethal Heat Treatment To Enhance The Heat Resistance Of Salmonella Enterica Serovar Enteritidis PT 30 And Its Application In Freeze-dried Surrogate

Microorganisms subjected to sublethal temperatures for a certain period of time exhibit increased resistance to heat,which is an important defense mechanism of microorganisms against heat stress.However,it has been less studied and the changes in gene expression are still unclear.In this study,Salmonella enterica serovar Enteritidis PT 30（S.Enteritidis）and its non-pathogenic alternative strain,Enterococcus faecium BRRL B-2354（E.Faecium）,were used to investigate the changes in gene expression of S.Enteritidis under sublethal heat stress.S.Enteritidis cells were cultured in Tryptone Soy Broth（TSB）medium to Logarithmic phase（LP）or stationary phase（SP）and subjected to sublethal heat treatment at 42℃for 20-180min（i.e.LP/SP-42℃-time）.Untreated S.Enteritidis cells（set as Sal-TSB and Sal-TSA）cultured with TSB or Tryptic Soy Agar（TSA）at 37℃were used as controls and subjected to isothermal heat lethal treatment at 65℃to characterize their resistance to heat(D_65℃).Transcriptome sequencing was performed by Tru Seq PE Cluster Kit v30c B0t0HS（Illumina）in a paired-end approach on S.Enteritidis prepared from Sal-TSB,Sal-TSA and the highest treatment group with D_65℃values,to preliminarily reveal the expression of resistance-related genes in S.Enteritidis in the context of sublethal heat.The mechanism of enhanced heat resistance of S.Enteritidis after sublethal heat stress was initially revealed from the expression of resistance-related genes.To extend the application of its heat resistance enhancement and related mechanisms in low-moisture foods,Enterococcus faecium NRRL B-2354（E.faecium）was selected as replacement for S.Enteritidis,for sublethal heat treatment（42-50℃）and selected the group with the greatest heat resistance enhancement for freeze-drying under the protection of 10%skim milk powder to produce lyophilized bacterial powder.This method counteracts the inevitable damage that the freeze-drying process can cause to bacterial cells and leads to less resistant inocula that are unlikely to qualify in microbiological studies.The bacterial powder was inoculated with three types of LMFs（peanut flour,egg flour and onion flour）as a model for LMFs to validate freeze-dried E.faecium powder,compared with S.Enteritidis（Sal-TSA）and E.faecium（EF-TSB）prepared by the conventional solution method.The specific results of the study are as follows:（1）In this study,sublethal heat treatment was applied to S.Enteritidis at logarithmic and stable phases,and its resistance to heat was enhanced after sublethal heat treatment:the initial population produced by Sal-TSA（10.02±0.11 log CFU/m L）was higher than any other group incubated with Sal-TSB（8.19±0.11 log CFU/m L）,and the D_65℃-value（1.72±0.09 min）was higher than that of Sal-TSB（1.15±0.06 min）.The D_65℃-values of S.Enteritidis cells after sublethal heat treatment were 24.5%-60.8%higher than Sal-TSB.The LP-42℃-60 min group produced the highest resistance to heat(D_65℃=1.85±0.13 min),which was 60.8%and 7.7%higher than Sal-TSB and Sal-TSA,respectively.（2）Transcriptomic analysis of the Sal-TSB,Sal-TSA and LP-42℃-60 min groups revealed that 483 up-regulated and 443 down-regulated S.Enteritidis genes were identified in the LP-42℃-60 min group compared to the Sal-TSB group（Log₂Fold Change>1,p<0.05）.Rpo regulators were fully involved in the heat acclimation process of S.Entertidis:rpo S,rpo N and rpo E were upregulated and rpo H,rpo B and rpo C were downregulated.KEGG enrichment analysis revealed that differentially expressed genes were mainly enriched in secondary metabolite biosynthesis,tricarboxylic acid cycle and ribosome metabolism pathways;in GO enrichment analysis,differentially expressed genes were mainly associated with redox processes and redox enzymes,etc.This suggests that sublethal heat treatment enhances heat resistance of S.Enteritidis may be related to changes in gene expression of energy metabolism,membrane function and proteins,which initially reveals how bacteria regulate themselves to resist the stress of lethal high temperature environment.（3）The non-pathogenic alternative to S.Enteritidis,E.faecium,was subjected to sublethal preheating treatment at（42-50℃,5-180 min）,and its resistance to heat was enhanced and peaked at 45℃-60 min and 50℃-5 min.The freeze-dried alternative to S.Enteritidis could be stored at room temperature（22℃）for 40 d after the 50℃-5 min treatment group.It showed higher potential than the wet inoculation method S.Enteritidis（Sal-TSA）in all three low-moisture foods,exhibiting good potential for freeze-drying replacement bacteria.E.faecium was subjected to sublethal preheating treatment at（42-50℃）to characterize the degree of change in its resistance to heat,and the most heat-resistant treatment group was protected by 10%skim milk powder to prepare lyophilized bacterial powder and inoculated into three types of LMFs（peanut powder,egg powder and onion powder）as a model for LMFs to validate lyophilized E.faecium（lyophilized EF-50℃-5 min）for applicability.It was found that the thermal resistance(D_65℃-value)of sublethal heat-treated E.faecium increased to varying degrees during all treatments compared to Sal-TSA and EF-TSB and peaked at 45℃-60 min and 50℃-5 min(D_65℃=9.95±0.74 min and D_65℃=9.93±0.35 min).The survival of the lyophilized EF-50℃-5 min inoculum and its resistance to heat remained good over 40 d.In the LMFs,the resistance to heat(D_85℃or D_75℃)of the lyophilized E.faecium prepared in this study was the same or higher than that of S.Enteritidis（Sal-TSA）.In conclusion,sublethal heat treatment can improve the thermal resistance of S.Enteritidis to different degrees in logarithmic and stable phases.Transcriptome sequencing analysis revealed that Rpo regulators were fully involved in the thermal adaptation process of S.Enteritidis,and the differentially expressed genes were mainly enriched in the secondary metabolite biosynthesis,tricarboxylic acid cycle and ribosomal pathways.In addition,the lyophilized E.faecium（EF-50℃-5 min）prepared using the principle of sublethal preheating treatment to enhance microbial resistance to heat was able to replace the traditional liquid inoculation of S.Enteritidis with peanut flour,egg flour,and onion powder for microbial validation.Therefore,the results of this study provide a certain theoretical basis for investigating the mechanism of enhancing the heat resistance of S.Enteritidis by sublethal heat stress in terms of gene expression,and provide a new way for the dry inoculation of S.Enteritidis with low-moisture foods for heat resistance,which can be combined with liquid culture method and sublethal preheating treatment to achieve industrial scale production of lyophilized inoculum.