| Bacterial spores were the most difficult microorganisms to kill.High-pressure thermal sterilization(HPTS)had little effect on food quality and could effectively kill spores.However,research on the killing of spores by HPTS had not been a major breakthrough for a long time.The hydrolysis of spore cortex peptidoglycan under high-pressure thermal sterilization(HPTS)was one of the important reasons for the death of bacterial spores.The spore cortex-lytic enzyme was the only enzyme that hydrolyzes the peptidoglycan of the spore and plays an important role in spore gennination.However,it wasn't known whether spore death related to the activity of spore cortex-lytic enzyme in HPTS or not.Therefore,the following studies had been conducted in this article:The cortex-lytic enzyme was extracted by using 2,6-pyridinedicarboxylic acid(DPA),L-alanine,inosine and L-alanine combined with inosine as germinating agents to induce spore germination.Colorimetric method and reversed-phase high performance liquid chromatography(RP-HPLC)were used to detect whether DPA in the enzyme solution was completely removed after the dialysis treatment.The results showed that four kinds of inducers could induce spores germination,and the effect of DPA on spore germination was significantly higher than the other three(P<0.05).The method of colorimetric method and RP-HPLC detection showed that dialysis could effectively remove DPA from the enzyme solution.Spore cortex-lytic enzyme was desalted and concentrated using a 10 kDa ultrafiltration centrifuge tube and analyzed by SDS-PAGE gel electrophoresis.Select the appropriate separation gel concentration and injection volume.The cortex-lytic enzyme was purified by dialysis,ammonium sulfate fractionation,SP-sephadex C-25 ion exchange chromatography and Superdex 75 gel filtration chromatography,the protein content and enzyme activity during each purification process were determined.It was found that when the injection volume was 15 ?L,and the separation gel concentration was 10%the SDS-PAGE separation efficiency was good.After the ammonium sulfate fractionation,the total enzyme activity was 292.71 U/L,the recovery rate was 84.17%,the total enzyme activity after SP-sephadex C-25 ion exchange chromatography was 237.96 U/L and the recovery rate was 68.43%.Finally,Superdex 75 gel filtration chromatography was used to purify the enzyme and the enzyme activity was 67.63 U/L,and the recovery rate is 19.45%.The molecular weight of the purified lyase was 61.10 kDa.Scanning electron microscopy(SEM)was used to observe the morphological changes of the spores before and after germination,and the morphological changes of coat-stripped spores before and after treatment with cortex-lytic enzyme.The effects of sodium phosphate concentration,pH,temperature,pressure and HPTS on the activity of the enzyme were studied.It could be seen by scanning electron microscopy that the spore surface after DPA-induced germination was damaged.The cortex-lytic enzyme hydrolyzes the spore cortex peptidoglycan and the surface of the coat-stripped spores collapsed.The optimum pH value of the spore cortex-lytic enzyme was 7 and the optimum sodium phosphate concentration was 0.05 mol/L,the single high pressure(150?530 MPa)treatment and the single temperature treatment(below 44 C)had no significant effect on the activity of the cortex-lytic enzyme.The HPTS conditions were 530 MPa,68?,15 min and the enzyme was inactivated.The Fourier transform infrared spectroscopy(FTIR)information of cortex-lytic enzyme before and after HPTS was collected,and it was found that the intramolecular hydrogen bonds of spore cortex-lytic enzyme was broken after HPTS. |
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