The Study On The High Hydrostatic Pressure With Physical And Chemical Factors To Spore's Cortical Peptidoglycan

Hydrolysis of peptidoglycans in the spore coat under ultra-high pressure combined heat treatment(HPTS)is an important cause of bacterial spore death.This article focuses on Bacillus subtilis spores,and the hydrolysis of peptidoglycan from the spore coat is the main line.Bacillus subtilis spores were first to study the effects of pressure and other auxiliary substances on the induction of spores,and to analyze and compare the effects of combination treatment with different substances on the spore and hydrolysis of cortical peptidoglycan.but it was found that it was not possible to hydrolyze peptidoglycans in the cortex.Therefore,the cortical peptidoglycan was extracted separately,and the physical and chemical indicators and its structure were studied and analyzed to determine its characteristic substances;finally,the HPLC method for the search and optimization of the characteristic substances was used to analyze the hydrolysate of HPTS-treated peptidoglycan in the lower cortex and the hydrolysis of cortex peptidoglycan was clarified.The hydrolysis of scaffolds advances the understanding of the mechanism of HPTS killing spores.The specific research content and main conclusions are as follows:(1)This article studied the pressure combined with different substances on spore induction,and the effects of combined treatment of UHP combined with acid,sugar and ethanol on Bacillus subtilis spore and cortical peptidoglycan were compared and analyzed.UHP combined with acid and sugar had a certain effect on the germination of spores.The degree of induction was acid>sugar,and the lower the pH,the lower concentration of sugar,the more extent OD600 declined.The content of DPA released by 150MPa combined with acid and sugar was significantly higher than in 450 MPa pressure.Pressure combined with acid has synergistic effect on the release of DPA,while pressure has no synergistic effect with sugar,and the release of DPA is significantly lower than the DPA content released by UHP alone.Although in 150 and 450 MPa pressure combined with acid release some substances from spore,high pressure combined with pH=1 acid can hydrolyze the spores.Under the pressure of 150MPa and 450 MPa had no significant difference in the effect of spores and cortical peptidoglycan hydrolysis.At 150 MPa and 450 MPa pressure combined with different concentrations of ethanol almost had no effect on the germination of spores,and protected the internal structure of spores more completely.(2)This article studied the structure and composition of the spore cortex peptidoglycan.The typical structure characteristics of peptidoglycan were found by infrared spectrum analysis.It was found that mutanolysin can hydrolyze peptidoglycan more completely than lysozyme.The content of peptidoglycan in the cortex and peptidoglycan was mainly determined by carbohydrate and protein.The amino acids in the peptidoglycan hydrolysates were measured by HPLC,and it was found that the contents of amino acids in the cortical peptidoglycan of bacterial spores were significantly higher than that in the cell wall peptidoglycan of bacterial vegetative cells.This difference in component contents and structure is probably the reason for the unique properties of spore's cortical peptidoglycan.At the same time,the untreated cortex peptidoglycan showed a long rod shape by scanning electron microscopy while the peptidoglycan which was enzymatically hydrolysated had obvious collapse and depression.(3)This article studied the hydrolysis Law of Peptidoglycan after HPTS treatment.MALDI-TOF MS was used firstly to analysis its hydrolysis situation.However,the non enzymatic hydrolysis of peptidoglycan was determined or not because of the absence of the corresponding mass spectrum library and the large molecular standard.Then focusing on the NAG and NAM which were the small-molecule characteristic hydrolysate of the peptidoglycan,HPLC was used to analyze the peptidoglycan sample after HPTS treatment.During optimization of analysis conditions of HPLC,it was found that Cis columm,amino column and sugar-D column couldn't separate and identify effectively NAG and NAM.Finally Aminex HPX-87H column separated and identified the standard substances NAG and NAM successfully,and using the column NAM was determined in the supernatant of cortical peptidoglycan sample treated with HPTS,which confirmed that non-enzymatic hydrolysis of the peptidoglycan took place under HPTS treatment.