Synergistic Inactivation And Mechanism Of Thermal And Ultrasound Treatments Against Bacillus Subtilis Spores

Bacterial spore is one of the main sources of microbial contamination in the food industry.Recently,many researchers have tended to increase the effectiveness of spore inactivation by combining ultrasound and thermal treatment.However,most of these studies focused on the sporicidal effects of different combinations of ultrasound in series with thermal processing(after or simultaneous),even lack of comprehensive and systematic research on their inactivation mechanisms.These lacks will limit its development in food industry.The present study selected Bacillus subtilis spore as a model microbe to investigate the inactivation of spore by the simultaneous treatment of ultrasound and thermal processing(thermonsonication,TS).Microstructural analysis,molecular biology and proteomics were employed to clarify the morphological structure and resistant characteristics at cellular level,as well as to elucidate the targets of TS treatment on spores and the relationship between cell biological characteristics and differentially expressed proteins at molecular level.This work lays the theoretical foundation for the further optimization of TS sterilization processing.The main contents and results are summarized as follows:(1)The inactivation kinetics of B.subtilis spores by TS treatmentThe inactivation kinetics of spores by TS treatment were studied under different ultrasonic power density and temperature conditions.The resistant factors to TS treatment was also investigated by analyzing the sonochemical effect caused by TS treatment,the complicated structure of spores and the aggregated behaviors.Results showed that temperature played a critical role in killing spores by TS treatment,a slight spore reduction was observed during the initial treatment and reached nearly maximum reduction of 0.61 log at temperatures of 60 and 70 �C after 40 min.However,a significant spore inactivation was achieved as the temperature for TS was elevated to80 ?,with a maximum reduction of 2.23 log.Ultrasound had a synergetic effect with thermal treatment on spore inactivation.The production of hydrogen peroxide during TS treatment at 80 ? was lower than that at 60 ? and 70 ?,and the inactivation of spores obtained by the combination of hydrogen peroxide(0.1m M)with 80 ? was significantly lower than that caused by TS(P<0.01),indicating that the inactivation of spores by TS mainly attributed to the synergetic effect between sonomechanical force and thermal treatment.The survival curves of spore inactivation by TS treatment at 80 ?fit well with log-Logistic model,and the resistance to TS was promoted via spore coat and ?/? SASPs instead of spores aggregation.(2)Morphological mechanism of spores inactivation by TS treatmentThe effects of TS treatment at 6.7 W/m L and 80 ? on spore morphological structure,surface characteristics,stress resistance and density were investigated via electron microscopical observation and biological characteristic analysis.The results showed that the spore morphological structure significantly changed after TS treatment,and the coat,cortex,inner membrane and nuclear materials of the spore were damaged to different degrees.Further analysis showed that the destruction of structural proteins of spore outer layers led to the decrease of the hydrophobicity,absolute value of Zeta potential and adhesion of the spores.The number of TS-treated spores reduced more than that of the control ones after being exposed to subsequent thermal treatment at 85 ?and 90 ?,indicating that the thermal resistance of TS-treated spores was decreased.The release amount of pyridine-2,6-dicarboxylic acid(DPA)in the TS-treated spores was significantly higher than that in the untreated spores,indicating that the inner membrane of spore was damaged during TS treatment,thus accelerating the release of DPA in the subsequent thermal treatment,resulting in reduced thermal resistance of spore.Three layers were obtained in the spore suspension of TS-treated spores in the equilibrium density gradient.The spores in the lower separation layer were inactivated but still retained DPA,indicating that inner membrane collapses after spore inactivation.These results demonstrated that TS inactivated spores by damaging the key proteins,rather than by destroying the inner membrane.(3)Multi-target effect of TS treatment on sporesThe characteristics of spore germination and outgrowth after TS treatment were investigated,and the present work analyzed whether the damage of key enzymes and receptor proteins in the germination stage would lead to the inactivation of spore.Meanwhile,the key substances such as DNA and ATP synthetase for the outgrowth stage were also explored.The ability of TS-treated spores to germinate was directly measured by the response of the spores in fractions from equilibrium density gradients of TS-treated spores to the nutrient germinant L-alanine.Consequently,the spores in the denser band from the TS-killed spore population that retained their DPA could well germinate,although these spores were largely dead.However,the spores from the lower band germinated slower than the untreated spores,indicating that one or more proteins that are essential for spore germination were damaged by TS treatment.The decrease in AGFK germination of the TS-treated spores was more than L-valine germination,indicating that TS treatment could act on some GRs to slow spore germination to some extent.The germination of fraction from treated spores with GR-independent germinant dodecylamine was not significantly decreased,which suggested that TS did not damaged Spo VA protein.The damage to these proteins would not result in apparently spore dead,since those three artificial germination treatments mentioned above did not contribute to a better recovery of TS-treated spores.According to flow cytometry analysis,germinated TS-treated spores died in outgrowth phase.TS treatment did not destroy DNA,but ATP synthesis was intercepted at the outgrowth stage.The outgrowth of spore was blocked,causing the inactivation of spore.(4)Proteomic responses of spores to TS treatment involve large-scale alterations in metabolic pathwaysThe significant differential expression levels of proteins(DEP)in several key metabolic pathways in outgrowth stage of TS-treated spores were investigated,and the relationship between the biological function changes of spores and the DEP after TS treatment was analyzed at molecular level.A total of 167 DEPs were obtained after TS treatment at 6.7 W/m L and 80 �C.Enrichment analysis of the proteome revealed that the major categories were associated with metabolic functions,including energy metabolic processes,amino acids biosynthesis and metabolism,translation and ribosomal protein.Under the stress of TS treatment,Fructose-bisphosphate aldolase(FBPA),oxoglutarate dehydrogenase complex(OGDC),malate dehydrogenase(MD),together with cytochrome c(Cyt)oxidase were down-regulated during TS treatment,which obstructed the energy supply/metabolism in B.subtilis spores.Moreover,the down-regulation of Ado Met synthetase and Type I glutamate-ammonia ligase inhibited amino acids biosynthesis and metabolism.Meanwhile,due to the down regulation of RNA-binding protein Hfq,IF-3,and Elongation factor P(EF-P),signal transduction was blocked and molecular chaperone Dna K,and heat shock protein Grp E were damaged,making the spores more susceptible.To survive,spores call up defense mechanism-related proteins,for example,peptidylprolyl cis-trans isomerase(PPI),to protect cells against TS stress and reestablish homeostasis.Nevertheless,The germinated TS-killed spores were inhibited to pass the outgrowth stage due to the insufficient energy supply and limited ability of core protein synthesis.