Formation And Further Inactivation Of Sublethally Injured Saccharomyces Cerevisiae Induced By PEF

Pulsed electric field （PEF） is a kind of non-thermal processing technology which can beapplied to the microbe inactivation of liquid foods. Current researchs at home and abroadmostly focus on its influence on microbial sterilization, enzyme passivation, and nutrientcomponents of fruit and vegetable juices. Limited reports are found about the formation anddetection of sublethally injured microorganisms caused by PEF recently. In this study, thedetection methods of sublethally injured Saccharomyces cerevisiae cells and its formationmechanism were investigated. The further inactivation methods of sublethally injuredS.cerevisiae cells were also discussed.The sublethally injured S.cerevisiae induced by PEF was detected through the count ofNon-selective medium and Selective medium which containing of4%NaCl. PEF treatmentunder the electric field intensity of20kV/cm for different time（from100μs to500μs） wouldproduce large quantities of sublethally injured S.cerevisiae cells with the range of58.11%to89.54%, the quantity and PEF processing time were positively correlated.Fluorescent techniques in combination with flow cytometry（FCM） and related kits wereused for damage detection of sublethally injured S.cerevisiae cells on membrane, intracellularenzyme activity and nucleic acids （including DNA and RNA） caused by PEF. The resultsshowed that PEF treatment increased cell membrane permeability, decreased membranefluidity and reduced the total lipid relative content from94.74%to90.83%comparing withthe untreated group; caused the degradation of RNA, but almost had no impact on the DNA ofS. cerevisiae cells. PEF treatment enabled the vitality of Ca²⁺-ATPase increased from0.23mgprot/ml to28.57mgprot/ml; while the activity of alkaline phosphatase and glucosidaseincreased significantly at the same time; the activity of esterase and aromatic amine enzymesdecreased slightly; the activity of lipase did not change essentially.The repair of sublethally injured S.cerevisiae cells in yeast extract buffer, peptone bufferand glucose buffer systems and in the presence of Ca²⁺, K⁺and Mg²⁺inorganic ions wereinvestigate. Sublethally injured S.cerevisiae cells induced by PEF treatment could self-repairwithin30to70minutes under different buffer systems; The presence of Ca²⁺, K⁺and Mg²⁺played a protective role to S.cerevisiae cells and effectively promoted the repair of sublethallyinjured S.cerevisiae cells in the buffer system and freshly squeezed strawberry juice, withpromoting effect of K⁺> Ca²⁺> Mg²⁺. The knock out of Heat shock protein104,Trehalose-6-Phosphate Synthase1and Temperature Shock-Inducible Protein1didn’t changedthe sensitivity of S.cerevisiae cells to PEF processing, while PEF treatment increased thesensitivity of sublethally injured S.cerevisiae cells to temperature.Take wild-type yeast strain and the three stress protein gene-deficient yeast strains asobjects, the further inactivation of sublethally injured S.cerevisiae cells were explored underPEF treatment in combination with cold shock and different water bath temperature as4oC,15oC,35oC and55oC. PEF treatment was more conducive to inactivate S. cerevisiae cells under4degrees temperature of water bath, what’s more, cold shock under4degreestemperature for one hour following PEF treatment were found to be effective in killing thesublethally injured S.cerevisiae cells for more than1.70log10CFU/mL comparing withuntreated one.