Study On The Inactivation Effects Of HPTS Combined With NaCl On Bacillus Subtilis Spores

It has been a difficult problem in the field of food industry of inactivation spores effectively for a long period of time.Using HPTS(High Pressure Thermal sterilization)technique to inactivate spore has made great progress in recent years.NaCl can produce high osmotic pressure outside the microbial membrane structure,so it has a certain bactericidal effect,but NaCl can not inactivate spores,and the inactivation effects of HPTS combined with NaCl on spores is rarely studied.Based on HPTS techno logy,the inactivation effects of HPTS combined with NaCl on Bacillus subtilis spore was studied by studying the survival of Bacillus subtilis spores,the changes of spore structure and morphology,and the changes of the composition and content of key substances related to spore survival before and after treatment.The optimal concentration of NaCl was deter mined to produce synergetic spore-inactivation effects with HPTS,and it was confirmed that high concentration of NaCl had a certain protective effect on spores when treated with HPTS.In order to lay the foundation for further clarifying the mechanism of sporeinactivation by HPTS combined with NaCl,the following studies were mainly conducted:(1)Through plate count,turbidity change of spore suspension,DPA leakage in spore,the spore inactivation effects under different concentrations of NaCl,different treatment temperatures and treatment pressures was analyzed.The results showed that HPTS combined with 15%NaCl treatment had a strong synergic effect,and improve the spore inactivation effects significantly(P<0.05).Under the condition of 600MPa-75?,the optimal treatment was combined with 15%NaCl,which could inactivate about 5.13 log CFU/mL spores.At 200MPa-65? and 75?,15%NaCl could increase the spore inactivation capacity by 2.35 and 2.26 log CFU/mL,respectively.The saturated NaCl had protective effect on spores,which was enhanced with the increase of temperature and pressure.Compared with the 15%NaCl treatment,the spores inactivation amount under the saturated NaCl treatment at 200 MPa-75? showed no significant difference(P>0.05),while the spores inactivation amount under the saturated NaCl treatment at 600MPa-65? and 75? decreased by 1.21 and 2.46log CFU/mL,respectively.With the improvement of the inactivation effects,the spore content leakage accelerated,the OD600 of the spore suspension decreased and the leakage rate of DPA increased basically in accordance with the increasing trend of the inactivation effects.(2)HPTS combined with NaCl processing had a certain influence on spore morpho logy and structure,in 600MPa-75?,15%NaCl can significantly increase the fluidity and permeability of Bacillus subtilis spore inner membrane,and the protein secondary structure of spore have also changed dramatically.The ?-helix structure decreased,and the ratio of ?-pleated sheet structure increased.In the morphology of spores,the particle size decreased significantly.The integrity of spores was destroyed,and surface collapse and fragmentation appeared.Compared with 15%NaCl,the inner membrane fluidity and permeability of the spores treated with saturated NaCl decreased significantly.The ?-helix ratio in the protein structure of the spores increased significantly,and the particle size index significantly larger too.The scanning electron microscope(SEM)images showed that the degree of spore fragmentation was weakened.With the combination of 15%NaCl and different HPTS parameters,the results showed that the elevated temperature could significantly increase the fluidity and permeability of the spore's inner membrane,and the damage degree of the secondary structure of the spore protein was more severe,the average particle size continued to decrease,and the damage degree of the individual morpho logy of the spores increased too.The fluidity of the spore inner membrane is mainly related to temperature,and the increase of temperature can significantly increase the fluidity of spore inner membrane,but the change of pressure is not obvious.(3)The components and contents of key substances of spore survival changed greatly after NaCl combined with HPTS treatment.At 600MPa-75?,the leakage of nucleic acid and protein caused by 15%NaCl was the highest,the total protein content in spore suspension was also the highest,and the activities of Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase were the lowest,and the contents of C14:1,C15:1 and C16:0 in 6 phospholipid fatty acids were significantly reduced(P<0.05).Compared with 15%NaCl treatment,saturated NaCl treatment reduced the leakage level of nucleic acid and protein,delayed the decline of Na⁺/K⁺-ATPase activity,and the contents of C13:0,C14:1and C16:0 fatty acid significantly increased(P<0.05).In 15%NaCl environment,with the increase of temperature and pressure,the nucleic acid and protein leakage in spore and total protein content increased,ATPase activity decreased.The principal component analysis and cluster analysis showed that the concentration of NaCl,temperature and pressure all had a great influence on the content of phospholipid fatty acids in spore.In conclusion,under certain HPTS conditions combined with different concentrations of NaCl treatment can promote the inactivation or protection of Bacillus subtilis spores.In this process,the fluidity,permeability of spore inner membrane,and protein secondary structure of spores have different degrees of changes.The micromorphology and macro particle size of spores as well as some main substances related to spores such as ATPase and phospholipid fatty acid also changed.