Inactivation Of Alicyclobacillus Spp.in Apple Juice By Pulsed Light And The Analysis Of Its Mechanisms

Alicyclobacillus spp.is a common spoilage bacterium used in apple juice production,with acidophilic and heat-resistant properties and budding spores that can withstand pasteur sterilization.Spore germination can cause a large number of bacteria to reproduce,produce metabolites such as guaiacol,2,6-dibromophenol and 2,6-dichlorophenol,cause corruption and deterioration of juice,which can have a serious impact on juice processing and export trade.Therefore,this study was carried out to investigate the effect of pulsed intense light treatment on the production of metabolites of Alicyclobacillus spp.in apple juice and to systematically evaluate the effect of sterilization treatment on juice quality.The main research contents and results are as follows:（1）The common guaiacol-producing bacteria A.acidoterrestris DSM-3923,A.acidiphilus DSM-14558,A.cycloheptanicus DSM-4006,A.acidocaldarius SD-4 and SD-19 were used as test strains to evaluate the killing effect of pulsed light treatment on five strains and elucidate its mechanism.The results showed that after 24-36 pulsed light treatments（corresponding to a fluence of 23.52-35.28 J/cm²）,Alicyclobacillus spp.（10⁴,10⁵ and 10⁶ CFU/m L）could be completely inactivated in 8-20°Brix juice samples with a total processing time of less than 8min.Of these,strains SD-4,SD-19 and DSM-4006 were more sensitive to pulsed light treatment than DSM-3923 and DSM-14558.The effect of pulsed light sterilization on the metabolism of guaiacol,the product of five strains of Alicyclobacillus spp.was analyzed.After three flashes of pulsed light treatment,only 10⁶ CFU/m L of Alicyclobacillus spp.were metabolized to produce guaiacol,and guaiacol was not detected in the other treatment groups,indicating that the cells that reached the inactivation point after pulsed light treatment did not germinate under appropriate conditions,and the bacterial cells were completely inactivated.Compared with the control group,the protein content in the supernatant of the bacterial suspension increased by 32.96%～661.98%,the intracellular ROS release increased by0.21～0.47 times,the intracellular ATP content decreased by 89.89%～98.67%after 18 flashes.The pulsed light treatment increased the permeability of the cell membrane to different degrees,and the cell appearance and morphology were all impaired.In addition,pulsed light treatment alters the fluidity of the cell membrane of bacterial cells by increasing the content of saturated andω-cyclic fatty acids,thus adapting to the unfavorable external environment.Changes in the characteristic peaks of the infrared spectra of macromolecules such as proteins,carbohydrates and nucleic acids in Alicyclobacillus spp.are mainly associated with C-H stretching vibrations,O-H stretching vibrations,decarboxylation behaviour,carbonyl CO and double bond stretching vibrations.This supports the bactericidal mechanism of the photochemical effect of pulsed light,whereby bacterial DNA absorbs UV light from the pulsed light spectrum to form thymine dimers,blocking the transcription and replication of microbial DNA,thus preventing the synthesis of proteins essential for microbial life activity and ultimately leading to bacterial inactivation.（2）A.acidoterrestris DSM-3923,the most tolerant strain to pulsed light,was used as a representative strain to evaluate the effect of pulsed light treatment on the inactivation of DSM-3923 spores,to establish the corresponding bactericidal kinetic model and to elucidate the mechanism.The results showed that 40 flashes（corresponding to a fluence of 39.2 J/cm2）could completely inactivate DSM 3923 spores in apple juice samples,and the log-linear+shoulder+tail model could simulate the bactericidal process.The R² of the fitted curves was all above 0.90.The pulsed light treatment increased the permeability of the spore cell membrane and caused a significant increase in intracellular protein leakage,and the protein content of the supernatant increased by 26.70%after 40 flashes.At the same time,the intracellular ATP content decreased to 0.96 times that of the control group after 40 flashes,suggesting that the pulsed light treatment may further affect intracellular ATP synthesis or ATP synthase activity,thereby limiting normal cellular physiological processes and damaging the bacteria.In addition,the intracellular ROS release was reduced to 0.84 times that of the control group by 40 flashes,suggesting that the pulsed light treatment may cause oxidative damage by stimulating the release of large amounts of ROS from the spores,ultimately leading to the death of the spores.DSM-3923 spores had their cell membrane integrity disrupted by the pulsed light treatment,and the morphology of the budding spores was damaged and deformed,losing their barrier protection to the cells.（3）The effects of pulsed intense light treatment on the conventional physicochemical parameters and soluble sugars,organic acids,monomeric phenols and volatile flavor substances of apple juice were systematically evaluated.The results showed that the pulsed intense light treatment increased the malic acid content of apple juice by 26.62%and decreased the oxalic acid and citric acid content by 66.67%and 76.99%,respectively,but did not significantly change the total sugar,total acid,total phenol and soluble sugar contents.In addition,there was some loss in the content of esters and acids and a significant increase in the content of aldehydes,alcohols and ketones among the volatile components;there was a decreasing trend in the content of DPPH,ABTS radical scavenging and T-AOC in apple juice,but no significant change in the-O^2-radical reducing capacity.