Research On Effectiveness And Mechanism Of Spore Inactivation By High Hydrostatic Pressure Combined With Heat

To inactivate the bacterial spores while preserve the quality of low acid canned food(pH>4.6,Aw>0.85),Bacillus subtilis 168 and Clostridium sporogenes PA3679 spores were selected in the study.The ultra-high pressure and ultra-high temperature sterilization technique with the fluid medium of edible soybean oil,moderate pressure and heat combined with GM-TriDAP inducing spore germination homogenously before killing them technique were developed.To predict the spore germination and inactivation curve,the mathematical model were established.At last,the mechanism of moderate pressure and heat combined with GM-TriDAP induced spores germination and inactivation were revealed through the study of the features of spore inner membrane.The main results and conclusions were summarized as follows:The ultra-high pressure and ultra-high temperature spores inactivation technique was developed.Using soybean oil as the adiabatic medium,the uniform ultra-high temperature can be achieved by the adiabatic compression property of soybean oil,and the temperature was not siginificantly decreased under the pressure-holding time.The compression heat law were fitted by binary quadric equation.The fitted model indicated that 130? could be achieved at 700 MPa when the initial temperature of soybean oil was only 90 ? or above.Combination treatments with ultra-high pressure and ultra-high temperature applied simultaneously resulted in 5-log C.sporogenes PA3679 sprore reduction,and achieved the requirements of commercial sterilization of low acid canned food.The moderate pressure and heat induced spores germination and inactivation technique was developed.2-log B.subtilis spores were induced to germinate by pressures between 100 and 200 MPa,the C.sporogenes spores were not affected under the same conditions.Moreover,4-log B.subtilis spores were induced to germination and inactivation by moderate pressure at 150 MPa combined with moderate heat between 60 and 90 ?.However,less than 1-log C.sporogenes spores were germinated and inactivated.Weibull and Biphasic models were fitted to predict the B.subtilis spores germination and inactivation curves.Regression coefficients(R2),root mean square(RMSE)values and residual plot suggested that the two models produced a good fit to the data.It was concluded that two modes were assumed of spore germination and inactivation affected by heat.On the one hand,the group number of sensitive spores was increased by temperature from Weibull model.On the other hand,the inactivation rate of sesitive spores was increased by temperature from Biphasic model.The moderate pressure and heat combined with GM-TriDAP technique based on "inducing spore germination homogenously before killing them" was developed.The peptidoglycan fragments of Bacillus subtilis 168 were separated and purificated by HPLC,and the peptidoglycan fragments(PGF)was demonstrated to be GM-TriDAP by means of HPLC-MS,FTIR,1H NMR.These results indicated that the B.subtilis and C.sporogenes spores could be induced to germination by both PGF and GM-triDAP.PGF had the better impact on the spore germination than GM-triDAP,and a very low concentration of PGF with ppb level was needed to induce spores DPA releasing.Moreover,the B.subtilis and C.sporogenes spores were entirely germinated and inactivated by the treatment of PGF or GM-triDAP combined with moderate pressure and heat.For instance,7-8 log reduction of B.subtilis and C.sporogenes spores treated by PGF or GM-triDAP could be obtained at 150 MPa and 80? for 20 min.The inactivation of B.subtilis and C.sporogenes spores by moderate pressure and heat treatment combined with PGF was fitted well by first-order kinetic model.The related mechanism of spore germination induced by moderate pressure and heat combined with GM-triDAP was studied.B.subtilis and C.sporogenes spores' inner membrane was stained with Laurdan fluorescent dye.The results showed that Laurdan emission spectra exhibited very difference between dormant spores and vegetative cells.The GP values of B.subtilis and C.sporogenes spores were amounted to 0.7.However,the GP values of vegetative cells of B.subtilis and C.sporogenes were about 0.3.Moderate pressure and heat combined with GM-triDAP had no effect on the GP value of spores,the gel phase of spores inner membrane was unchanged before and after treatment.In addition,there was no change in Flow distribution of fluorescent under moderate pressure and heat treatment combined with GM-triDAP.PI could not penetrate the spore core due to the intact inner membrane of spores.Therefore,the mechanism of spore inactivation by moderate pressure and heat combined with GM-triDAP was the selective open of DPA channel in spore inner membrance.