Study On The The Bactericidal Mechanism Of HPTS Combined With ?-polylysine On Bacillus Subtilis Spores And Its Inactivation In Different Media

High-pressure thermal sterilization(HPTS)has been recognized for its good bactericidal effect on bacterial spores.This experiment takes Bacillus subtilis spores as the research object to explore the mechanism of HPTS combined with ?-polylysine on the inactivation of Bacillus subtilis spores before and after the inactivation,and the inactivation effect in food media,and promote the application of HPTS technology in the food industry.The following experimental results are obtained:(1)In this chapter,HPTS(200 MPa combined with 25?,65?,75?;550 MPa combined with 25?,65?,75?;pressure holding time:20 min)combined with ?-polylysine(0.1%?Polylysine,0.3%s-lysine)were used to treat Bacillus subtilis spores.The change in the size of the spore size after the treatment was measured with a laser particle size analyzer.The external morphology of the spores before and after the treatment was observed by scanning electron microscope.With the increase of spore size,the spore size gradually becomes smaller,and the surface of the spores gradually appears sunken,wrinkled,and even collapsed.With the increase of the concentration of ?-polylysine,the spore morphology changes more significantly.200 MPa-75? combined with 0.3%?-polylysine.After 550 MPa-75? combined with 0.3%?-polylysine treatment,the inner membrane structure of the spore was severely damaged,the nucleus volume increased,and the nuclear material was lost.The OD600 value of the spore suspension after treatment was measured by spectrophotometry.The OD600 value of the spore suspension was the lowest after 550 MPa-75? combined with 0.3%?-polylysine,and the release of the spore content was the greatest.The results of plate count showed that different treatments had a certain effect on the survival concentration of spores,and the change of the survival concentration of spores was consistent with the changes in morphology and OD600 value.(2)This chapter studies the effect of HPTS combined with ?-polylysine(0.1%?-polylysine,0.3%?-lysine)treatment on the fluidity and permeability of the spore inner membrane.Flow cytometry was used to detect the changes in the permeability of the spore inner membrane,550 MPa-75? combined with 0.3%?-polylysine,the permeability of the inner membrane of the spore changed the most significantly(P<0.05),and the percentage of positive areas reached 83.2%.Combining different concentrations of ?-polylysine has a certain effect on the permeability of the spore inner membrane.The absorption intensity of OD260 and OD280 was measured by ultraviolet spectrophotometry to determine the leakage of nucleic acid protein material.It was found that the leakage of UV absorption increased under this condition of 200 MPa-75? combined with 0.3%?-polylysine,and the leakage of nucleic acid and protein increased significantly(P<0.05).200 MPa-25? combined with ?-polylysine UV absorption leakage is not significant(P>0.05),but the PI fluorescence staining method shows that the cell membrane permeability of the sample under this pressure treatment has a small deviation,which may be due to The cell membrane permeability has changed,but the degree of permeability change is not enough to make the intracellular UV-absorbing substances seep out of the cell,so the experimental results obtained by the two detection methods are inconsistent.Changes in the fluidity and permeability of the inner membrane of the spore may cause water molecules to enter the spore more easily,and the resistance of the spore to the external environment is reduced,thereby producing a killing effect on the spore.Fourier infrared spectroscopy(FT-IR)was used to collect the infrared spectra of Bacillus subtilis spores before and after HPTS treatment.The original spectra were debaselined and normalized,and the infrared spectra and second derivative spectra of Bacillus subtilis spores under different treatment conditions were analyzed.It was found that the lipid,protein,nucleic acid,and polysaccharide of the spore membrane changed after treatment.(3)In this chapter,550 MPa-65/75?,0.1%and 0.3%?-polylysine concentration were selected,the inactivation rule under different treatment conditions was studied,and the inactivation effect under different treatment conditions was analyzed 550 MPa-65?/75?combined ?-polylysine,the leakage of nucleic acid and protein UV absorbing substances increased significantly(P<0.05),but for 550 MPa-65?,the treatment time seemed to have no significant effect on the leakage of nucleic acid and protein UV absorbing substances.The leakage of UV absorbing material increased significantly after 550 MPa-75?-20 min treatment.The sterilization effect of the linear model and Weibull model under the different treatment conditions is used to fit,and the fitting degree of the model is quantitatively and qualitatively evaluated.From the model test factors,Weibull model has lower residual square sum(RSS)and higher regression coefficient(R2),because the Weibull model has a better fitting effect.According to the change of Weibull parameters n(shape factor)and b(scale factor).Weibull model can better predict the death of microorganisms under specific conditions than linear model.