Study On The Effect Of Glucose On The Killing Of Bacillus Subtilis Spores Treated By HPTS

As an important factor affecting the safety of food and its shelf life,spores have become a major challenge in food processing research.This thesis took the heat and pressure-resistant Bacillus subtilis spores as the research object,and explored the effects of HPTS(High pressure thermal sterilization)combined with glucose treatment on the spore killing effect.On this basis,the fluorescence polarization method and the flow cytometry were used to observe the influence on the inner membrane of the spores,and the damage of HPTS combined with glucose treatment to the spore morphology was observed by scanning electron microscopy,and the changes produced by the treatment on the key substances of spores were further investigated by Fourier infrared spectroscopy to provide some theoretical basis for the application of HPTS in food sterilization.The main research contents and results of this paper are as follows:(1)Effect of HPTS combined with glucose treatment on Bacillus subtilis sporesThe HPTS conditions were:200 MPa-25?,65?,75?;600 MPa-25?,65?,75?,and 15%,30%,60%glucose(W/V)were used to treat Bacillus subtilis for 20 min.The HPTS treatment without glucose addition was used as a comparison to investigate the inactivation effect of HPTS combined with glucose treatment on spores and the release of 2,6-pyridine-acid(Pyridine-2,6-dicarboxylic acid),and spore suspension particle size distribution.The results showed that the inactivation effect of HPTS alone was higher than that of HPTS combined with glucose treatment,and the addition of glucose helped to weaken the inactivation effect of HPTS on spores,and the higher the concentration,the stronger the protective effect on spores.The release of DPA increased with the increase of temperature and pressure.In addition,HPTS combined with glucose treatment increased the particle size of the spore suspension and formed spore aggregates.(2)Effect of HPTS combined with glucose on the spore inner membrane of Bacillus subtilisUltraviolet absorbing material method,electrical conductivity,and flow cytometry were used to observe the effect of HPTS combined with glucose on the permeability of the spore inner membrane,and the fluorescence polarization method was used to observe the changes in the inner membrane fluidity.The results showed that after HPTS combined with glucose treatment,with the increase of pressure and temperature,the inner membrane permeability of the spore increased,and the amount of released nucleic acids and proteins also increased,indicating that the combined treatment of HPTS with glucose would cause some damages to the inner membrane of the spore,the electrical conductivity results showed that as the glucose concentration increased,the spore ion release showed a downward trend,which is inconsistent with the changes in cell membrane permeability,indicating that the increase in glucose concentration could affect the solubility of metal ions.The result of fluorescence polarization showed that the treatment with the addition of glucose did have a certain effect on the fluidity of the spore inner membrane.(3)Effect of HPTS combined with glucose on the morphology of Bacillus subtilis spores and intra-spore materialScanning electron microscopy was used to observe the effect of HPTS combined with glucose treatment on the morphology of Bacillus subtilis.The results showed that the surface of the spores appeared depressed after HPTS treatment,and the surface of the spores was not significantly broken after the addition of glucose treatment,indicating that the addition of glucose would protect the morphology of Bacillus subtilis spores.The morphology of the bacillus spore played a protective role.The results of the measurement of ATP-enzyme activity within the spores showed that the addition of glucose had a weakening effect on the reduction of enzyme activity compared to the HPTS treatment alone.Through Fourier Transform Attenuated Total Reflectance Infrared Spectroscopy(ATR-FTIR)combined with the second derivative,the effect of HPTS on the structure of lipids,proteins and nucleic acids combined with glucose was explored.The results showed that the characteristic peaks of infrared spectra of Bacillus subtilis spores before and after HPTS treatment were basically similar;the second-order derivations of membrane lipids,proteins and nucleic acids were performed for 3000-2800 cm-1,1700-1600 cm-1 and 1300-900 cm-1 subgenera to investigate the changes of membrane lipids,proteins and nucleic acids of bacterial bacteriophages after HPTS combined with glucose treatment.After Gaussian fitting of the bands,it was found that the protein structure changed from order to disorder.(4)Study on the inactivation kinetics of HPTS combined with glucoseThe effect of HPTS combined with glucose on the Bacillus subtilis spores were fitted by means of the first level dynamics model and Weibull,and the model was evaluated.From the test factors of the model,it was found that the Weibull model had a lower mean square error and a higher regression coefficient.Therefore,the Weibull model has a better fitting effect.