Effect And Mechanism Of Ultrasound In The Inactivation Of Bacillus Cereus Spores

As a green non-thermal physical sterilization technology,ultrasound treatment can destroy microorganisms in a short period time.At the same time,it maintains food quality and reduces the damage to functional components in food,which has a broad application prospect in the food industry.Spore is a dormant state of vegetative cells with dense structure.Thus,spore has strong resistance to various processing techniques.It is urgent to find an effective way to inactive spores.Although,the inactivation effect of ultrasound to spore is minor,it can reduce the resistance of spore.Moreover,it used to have synergistic effect with other technologies.In this thesis,the inactivation effect and mechanism of Bacillus cereus spores were studied using ultrasonic combined with heat,pressure,and electrolyzed water treatments.The changes of protein expression after ultrasonic treatment were monitored using DIA Proteomics,the key proteins of spores were found and the molecular mechanism of spore stress after ultrasonic treatment was clarified.The main research contents and results are as follows:(1)The inactivation effect of ultrasound to sporeThe inactivation effect of ultrasound with different power on Bacillus cereus spores was studied.The results showed that the inactivation effect increased significantly with the increase of ultrasound power,but the reduction was less than 1log CFU/m L after ultrasound treatment for 30 min.The ultrastructure of spores was observed by electron microscope.It was found that ultrasound treatment could promote the detachment of the exosporium and greatly reduce the particle size of spores.But the inner structure maintained intact.The detachment of exosporium resulted in significant change in the surface properties.For example,the absolute value of zeta potential decreased which leading to the decrease in stability of the spore suspension.Moreover,the hydrophobicity of spore decreased significantly,thus the adhesion to hydrophobic surface(such as the processing of pipes)of spores decreased.Flow cytometry combined with SYTO 16/PI was used to monitor the physiological heterogeneity of spores after ultrasound treatment.The results indicated that ultrasound caused the decrease of permeability of cortex.At the same time,the leakage of DPA,nucleic acid and protein was monitored.Ultrasonic treatment induced part release of DPA and nucleic acid.The leakage of protein was significant,a large number of proteins leaked out of the cell,and the thermal resistance of spores was reduced consequently.However,the release of DPA protein and other substances was a “all-or-nothing” process without a gradual accumulation.(2)The response mechanism to ultrasound using proteomicsDIA proteomics was used to analyze the changes of protein expression level after ultrasound treatment.The results showed that 340 proteins changed the expression level significantly after ultrasound treatment(fold change?2.0 and p < 0.05),among which207 proteins decreased significantly.The differential expression proteins were mainly distributed in metabolism,cell process,and genetic information processing pathways.The expression of many cytochrome c oxidase polypeptides and ATP synthase in the oxidative phosphorylation of spores decreased significantly,which reduced the ability to pump protons across the membrane and transfer electrons through the respiratory chain,the ATP synthesis decreased subsequently.Moreover,most key proteins in amino acid synthesis pathway decreased significantly,especially in histidine synthesis pathway.The expression of 6-phosphogluconate dehydrogenase and gluconokinase in pentose phosphate pathway was significantly up-regulated,and the metabolism of glucose was significantly enhanced.The expression of key proteins related to tricarboxylic acid cycle(TCA)was decreased,which led to the decrease of metabolism of three major nutrients.What's more,ultrasound not only induced down-regulation of fatty acid synthesis enzymes,but also promoted fatty acid metabolism.Then,the intracellular fatty acid content decreased.In addition,the expression of ATP binding protein in carbohydrate transport was significantly up-regulated after ultrasound treatment,and the ability of spores to transport carbohydrate was enhanced;while the expression of ATP binding protein in phosphate transport was down regulated,and the ability of phosphate transport was decreased.(3)The inactivation effect of Manothermosonication to sporeUltrasound treatment had minor inactivation effect on spores,so it was further combined with other technologies.The results showed that the combination of ultrasound and heat treatment(TS)had synergistic inactivation effect.The permeability of the inner membrane and cortex of spores changed using flow cytometry combined SYTO 16/PI.The DPA leakage of the treated spores was significantly higher than that with ultrasound treatment singly,while the spore structure was not significantly changed.Although the inactivation effect was not increased by ultrasound combined with pressure(MS),the release of DPA was close to the total amount of spores.Thus,the resistance of spores decreased significantly.At the same time,combined heat treatment with MS(MTS)resulted in high inactivation of spores.The permeability of cortex and inner membrane changed,and the structure of spores destoryed significantly.In addition to exosporium,the inner cortex,coat and other inner structures of spores were damaged.Although MTS had a great inactivation effect,the equipment was damaged seriously,the energy consumption was high.What's more,there were a lot of probe debris in the samples after MTS treatment.There are still some defects in the actual production,so the equipment design still needs to be further improved.(4)The synergistic inactivation effect of ultrasound and electrolyzed waterThe results showed that the ultrasound treatment had no significant effect on the characteristics of electrolyzed water(EW)with low available chlorine concentration(ACC).However,the main indicators of EW with high ACC were significantly reduced,such as ACC especially the concentration of HCl O,and the bactericidal effect reduced significantly.EW was a short-term processing technology,and the reduction of spores did not increase significantly with the increase of processing time.In addition,the inactivation effect did not have significant difference between EW with different ACC.Interestingly,the inactivation of spores treated by ultrasound and EW in different orders was similar to EW alone,which might due to the "cross protection".The inactivation amount of ultrasound combined with EW treatment was higher than the sum of ech treatment,which was synergistic effect.Flow cytometry combined with SYTO 16/PI dye was used to evaluate the physiological heterogeneity of spores.It was found that the permeability of inner membrane and cortex of spores was significantly changed by ultrasound combined with EW treatment.The results of electron microscope showed that the spore structure was relatively complete after the EW treatment.However,the exosporium of spore detached after simultaneous ultrasound and EW treatment.Besides,the coat and cortex were seriously damaged.(5)The molecular mechanism of spores in response to stressThe protein expression level of spores treated by ultrasound combined with EW treatment was analyzed by DIA proteomics.There were 197 differentially expressed proteins after simultaneous treatment.The differentially expressed proteins were mainly distributed in the metabolic pathways,indicating that spores responded to stress by regulating intracellular metabolism.Spores enhanced resistance by regulating protein expression under stress,such as significantly up-regulated SASP,which led to "cross protection".The expression of several key proteins in oxidative phosphorylation pathway decreased significantly.Thus,the ability of ATP production decreased significantly.After the simultaneous treatment,the key proteins of stress response had the superposition effect,which led to the synergistic inactivation of spores.Through the analysis of different protein expression under stresses,the key proteins of spore to deal with external stresses were mainly involved in energy metabolism and substance transport.