Effect Of Lipopolysaccharide Structure On Sensitivity Of Salmonella To Blue Light And Underlying Mechanism

Blue light is a new type of non-thermal sterilization method developed in recent years.Due to the multiple advantages of wide sterilization spectrum and high safety,it has a good potential for sterilization and preservation of various foods.At present,the bactericidal mechanism underlying blue light is still not fully revealed.The action mechanism widely accepted is mediated by the highly cytotoxic reactive oxygen species and subsequent cellular damage.Previous studies in our laboratory also showed that outer membrane damage is also an important mechanism.The structure of lipopolysaccharide（LPS）on the outer membrane is likely to regulate the blue light sensitivity of bacteria;and how the diverse LPS structures affect blue light sensitivity and its mechanism need to be explored.Therefore,this study mainly focused on Salmonella enterica serovar Typhimurium.Firstly,the inactivation effect of blue light on pathogenic bacteria such as S.Typhimurium and its application in fresh beef sterilization and preservation were explored.Then,the LPS structural gene of S.Typhimurium SL1344 was knocked out without trace,and different LPS structural mutant strains were constructed to reveal the difference of blue light sensitivity between LPS structural mutant strains.Finally,the multiple influence mechanism of LPS structure on S.Typhimurium blue light sensitivity was revealed through the changes of extracellular membrane characteristics and differential transcriptomics.The research contents and results are as follows:（1）Firstly,415 nm blue light was used to study the in vitro sterilization curve of blue light on pathogenic bacteria such as S.Typhimurium and its role in the sterilization and preservation of fresh beef.The results showed that blue light had a significant inactivation effect on pathogenic bacteria such as S.Typhimurium.In PBS buffer,when the irradiation dose reached109.44 J·cm^-2,the inactivation rate of pathogenic bacteria such as S.Typhimurium exceeded90%.When S.Typhimurium and other pathogenic bacteria were inoculated on the surface of fresh beef,the inactivation rate was more than 90%under the same dose of blue light,and the inactivated bacteria did not resume growth during the cold storage period.In addition,by measuring the physical and chemical parameters of fresh beef after blue light irradiation,the results showed that 109.44 J·cm^-2 dose of blue light irradiation did not have a negative impact on the quality of fresh beef,and the color value and p H value of beef surface were better than those of non irradiated samples,indicating that 415 nm blue light had a better bactericidal and fresh-keeping effect on fresh beef.（2）Different LPS structural mutant strains of S.Typhimurium SL1344 were constructed,and their effects on bacterial blue light sensitivity were systematically revealed.Using Red recombinant knockout technology,SL1344 LPS structural mutant strain was constructed by knocking out five glycosyltransferases and O-antigen ligase related to the synthesis of LPS core polysaccharide main chain structureΔwaa C,Δwaa F,Δwaa G,Δwaa I,Δwaa J andΔwaa L.By measuring the blue light lethal curve of each mutant strain and wild-type SL1344,the results showed that compared with wild-type SL1344,all mutants were more sensitive to blue light.In addition,except for strainΔwaa L,the sensitivity of each mutant to blue light showed a certain rule:the shorter the LPS chain length,the higher the blue light sensitivity of strain.The results showed that the gradual shortening of LPS structure could gradually improve the sensitivity of bacteria to blue light.（3）Finally,the multiple mechanisms of LPS structure affecting the sensitivity of S.Typhimurium to blue light were explored.Firstly,by measuring the outer membrane permeability,the key index of outer membrane stability,the results showed that with the truncation of the LPS structure,the permeability of the outer membrane of the cells increased,and the increase of outer membrane permeability of all mutant strains except strainΔwaa L was highly consistent with the change of blue light sensitivity,indicating that the stability of outer membrane was the main factor affecting blue light sensitivity.At the same time,in order to reveal the effect of LPS structural changes on intracellular metabolism,differential transcriptome analysis was carried out for representative SL1344,Δwaa C andΔwaa L.The results showed that LPS structural truncation would not only affect the membrane components of cells,but also have a significant impact on biological processes such as membrane transport and intracellular metabolism,and the shorter the LPS chain length,the more significant the impact.This study shows that the structural change of LPS can regulate the sensitivity of bacteria to blue light by changing the stability of extracellular membrane,and may also cause the change of blue light sensitivity through the change of intracellular metabolism.This paper shows that the change of the core sugar structure of LPS significantly affects the blue light sensitivity of S.Typhimurium,which plays a role through multiple action mechanism,which needs to be further confirmed.This study enriches and deepens the theory of blue light sterilization,and will also lay a foundation for further improving the sterilization strategy based on blue light.