高压热杀菌(high-pressure thermal sterilization, HPTS)能够有效的杀灭食品中最难杀死的芽孢,研究表明,HPTS结合酸处理能取得更好的杀灭效果。为明确HPTS结合酸杀灭芽孢的机理,该研究调整芽孢悬浮液的pH值为1、4、7,于550 MPa结合25、65、75 ℃处理20 min。采用平板计数法、紫外分光光度法、流式细胞术和傅里叶红外变换光谱对HPTS结合酸处理后枯草杆菌芽孢的杀灭率、紫外吸收泄漏量、内膜通透性及芽孢成分变化进行研究。结果表明,随着pH值降低,HPTS结合酸对芽孢的杀灭作用明显增强,芽孢最多被杀灭了6.25 lgCFU/mL;紫外吸收泄漏量及内膜通透性都显著增加;同时芽孢内膜磷脂相态发生转变,膜脂流动性增加;蛋白质二级结构变化,导致蛋白变性;核酸物质变性,皮层肽聚糖和细胞壁结构改变。综上,酸处理是辅助HPTS杀灭芽孢的有效手段,两者具有良好的协同杀菌作用。
High-pressure thermal sterilization (HPTS) is effective in inactivating the most difficult-to-inactivate bacterial spores in foods, and studies have shown that HPTS combined with acid treatment can achieve better inactivating effect.To clarify the mechanism of inactivating bacterial spores by HPTS combined with acid, the pH of the bacterium suspension was adjusted to 1, 4 and 7, and the suspension was treated at 550 MPa combined with 25, 65 and 75 ℃ for 20 min in this study.The killing effect, UV absorption leakage, inner membrane permeability and changes in spore composition of Bacillus subtilis after HPTS combined with acid treatment were investigated by plate counting, UV spectrophotometry, flow cytometry and Fourier infrared transform spectroscopy.It was found that:(1) Under normal temperature and pressure, the pH value alone could not cause the inactivation of bacterial spores.The inactivating effect of HPTS combining with acid on bacterial spores increased effectively with the decreasing of pH, and bacterial spores were inactivated by up to 6.25 lgCFU/mL.(2) The UV absorption leakage reflects the changes in the permeability of the inner membrane of bacterial spores.OD260 and OD280 values increased significantly after HPTS combined with different pH treatments, the UV absorption leakage of bacterial spores increased, indicating that HPTS combined with different pH caused some degree of damage to the permeability barrier of bacterial spores.As the pH decreased, the amount of UV-absorbing material leaked from bacterial spores after 550 MPa combined with 25 ℃/65 ℃/75 ℃ treatment increased significantly, indicating that acid would further damage the permeation barrier of bacterial spores, allowing more nucleic acid and protein-based UV absorbers to leak out.(3) Flow cytometry is commonly used to detect damage to bacterial cell membranes.The bacterial spores were stained with the fluorescent dye PI, which releases red fluorescence after embedding double-stranded DNA.Compared to the action of HPTS alone, the proportion of positive areas after treatment with HPTS combined with pH 4 and pH 7 was essentially similar, whereas the proportion of positive areas after treatment with HPTS combined with pH 1 was significantly greater, indicating that HPTS combined with pH 1 had a stronger damaging effect on the inner membrane of bacterial spores than HPTS alone.It can be assumed that acid increased the permeability of the inner membrane of bacterial spores and improved the inactivating effect.(4) Fourier transform infrared spectroscopy is now widely used for the comprehensive detection of the physiological state of microorganisms.By applying the second derivative to the original infrared spectrum, changes in the lipid phase state of bacterial cell membranes and substances such as proteins and nucleic acids can be specifically analyzed.The wavelength range for infrared detection in this experiment was 4 000-500 cm-1.The 3 000-2 800 cm-1 band reflects the phase changes of the membrane lipids.The infrared spectroscopy(IR) absorption peaks at the original 2 960 cm-1 and 2 919 cm-1 of bacterial spores treated with HPTS combined with acid shifted and the IR absorption peaks changed more strongly, indicating that the phospholipid phase state of the inner membrane of bacterial spores shifted, the phospholipid molecules of its inner membrane changed from a gel state to a liquid crystal state, and the membrane lipid mobility increased.The 1 700-1 600 cm-1 band reflects changes in protein secondary structure in the protein amide I band.The IR absorption peak of 1 674 cm-1 representing the β-folded structure of the protein shifted to 1 676 cm-1 for bacterial spores treated with HPTS combined with acid, meanwhile, the IR absorption peaks of 1 652 cm-1 and 1 644 cm-1 representing the α-helical structure of the protein shifted to 1 650 cm-1 and 1 646 cm-1, indicating that changes in the secondary structure of proteins resulted in protein denaturation.The 1 300-900 cm-1 band reflects changes in nucleic acid backbone vibrations.The bacterial spores treated with HPTS combined with acid showed a shift in the position of the IR absorption peaks at 1 080 cm-1 and 1 220 cm-1 towards the lower wave numbers and a weakening of the intensity of the absorption peaks.This indicates that the nucleic acid material within bacterial spores is denatured and the peptidoglycan layer and cell wall structure are significantly altered, possibly causing bacterial spores' cortex to rupture and thus leading to bacterial spores' death.The experimental results show that acid treatment is an effective means to assist HPTS in inactivating bacterial spores, with good synergistic bactericidal effect.
[1] WELLS-BENNIK M H J, EIJLANDER R T, DEN BESTEN H M W, et al.Bacterial spores in food:Survival, emergence, and outgrowth [J].Annual Review of Food Science and Technology, 2016, 7(1):457-482.
[2] SETLOW P, JOHNSON E A.Spores and Their Significance [M].United States:Elsevier, 2019.
[3] LUONG T S V, MOIR C, CHANDRY P S, et al.Combined high pressure and heat treatment effectively disintegrates spore membranes and inactivates Alicyclobacillus acidoterrestris spores in acidic fruit juice beverage [J].Innovative Food Science & Emerging Technologies, 2020, 66(1):102523.
[4] JUNEJA V K, GOLDEN C E, MISHRA A, et al.Predictive model for growth of Bacillus cereus during cooling of cooked rice [J].International Journal of Food Microbiology, 2019, 290(2):49-58.
[5] LIANG D, ZHANG L, WANG X, et al.Building of pressure-assisted ultra-high temperature system and its inactivation of bacterial spores [J].Frontiers in Microbiology, 2019, 10:1 275.
[6] EVELYN E, SILVA F V M.Heat assisted HPP for the inactivation of bacteria, moulds and yeasts spores in foods: MLog reductions and mathematical models [J].Trends in Food Science & Technology, 2019, 88(8):143-156.
[7] SEVENICH R, MATHYS A.Continuous versus discontinuous ultra-high-pressure systems for food sterilization with focus on ultra-high-pressure homogenization and high-pressure thermal sterilization:A review [J].Comprehensive Reviews in Food Science and Food Safety, 2018, 17(3):646-662.
[8] TOLA Y B, RAMASWAMY H S.Combined effects of high pressure, moderate heat and pH on the inactivation kinetics of Bacillus licheniformis spores in carrot juice [J].Food Research International, 2014, 62:50-58.
[9] ROBERTS C M, HOOVER D G.Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin [J].Journal of Applied Bacteriology, 1996, 81(4):363-368.
[10] INOKUCHI T, ARAI N.Relationship between water permeation and flip-flop motion in a bilayer membrane [J].Physical Chemistry Chemical Physics, 2018, 20(44):28 155-28 161.
[11] CLÉRY-BARRAUD C, GAUBERT A, MASSON P, et al.Combined effects of high hydrostatic pressure and temperature for inactivation of Bacillus anthracis spores[J].Applied and Environmental Microbiology, 2004, 70(1):635-637.
[12] SEVENICH R, REINEKE K, HECHT P, et al.Impact of different water activities (Aw) adjusted by solutes on high pressure high temperature inactivation of Bacillus amyloliquefaciens spores [J].Frontiers in Microbiology, 2015, 6:689.
[13] ALDRETE-TAPIA J A, TORRES J A.Enhancing the inactivation of bacterial spores during pressure-assisted thermal processing [J].Food Engineering Reviews, 2021, 13(3):431-441.
[14] PORÉBSKA I, SOKOŁOWSKA B, SKA,PSKA S, et al.Treatment with high hydrostatic pressure and supercritical carbon dioxide to control Alicyclobacillus acidoterrestris spores in apple juice [J].Food Control, 2017, 73:24-30.
[15] WUYTACK E Y, MICHIELS C W.A study on the effects of high pressure and heat on Bacillus subtilis spores at low pH [J].International Journal of Food Microbiology, 2001, 64(3):333-341.
[16] SETLOW B, LOSHON C A, GENEST P C, et al.Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol [J].Journal of Applied Microbiology, 2002, 92(2):362-375.
[17] AL-QADIRI H M, AL-ALAMI N, AL-HOLY M, et al.Using Fourier transform infrared (FT-IR) absorbance spectroscopy and multivariate analysis to study the effect of chlorine-induced bacterial injury in water [J].Journal of Agricultural and Food Chemistry, 2008, 56(19):8 992-8 997.
[18] 章中, 孙静, 张津瑜,等.高压热杀菌处理对枯草杆菌芽孢皮层裂解酶活力的影响 [J].食品工业科技, 2018, 39(15):90-95.
ZHANG Z, SUN J, ZHANG J Y, et al.Effects of high pressure thermal sterilization on the activity of cortex-lytic enzyme extracted from Bacillus subtilis spores [J].Science and Technology of Food Industry, 2018, 39(15):90-95.
[19] AMOR K B, BREEUWER P, VERBAARSCHOT P, et al.Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress [J].Applied and Environmental Microbiology, 2002, 68(11):5 209-5 216.
[20] MOUSSA M, PERRIER-CORNET J M, GERVAIS P.Damage in Escherichia coli cells treated with a combination of high hydrostatic pressure and subzero temperature [J].Applied and Environmental Microbiology, 2007, 73(20):6 508-6 518.
[21] YU C X, IRUDAYARAJ J.Spectroscopic characterization of microorganisms by Fourier transform infrared microspectroscopy [J].Biopolymers, 2005, 77(6):368-377.
[22] 张良. 高静压与温度协同杀灭芽孢的效果与机制研究 [D].北京:中国农业大学, 2015.
ZHANG L.Research on effectiveness and mechanism of spore inactivation by high hydrostatic pressure combined with heat [D].Beijing:China Agricultural University, 2015.
[23] KILIMANN K.High pressure inactivation of bacteria:Mathematical and microbiological aspects [D].Munich:Technischen Universität München, 2005.
[24] PAUL C, FILIPPIDOU S, JAMIL I, et al.Bacterial spores, from ecology to biotechnology [J].Advances in Applied Microbiology, 2019, 106:79-111.
[25] POPHAM D L.Specialized peptidoglycan of the bacterial endospore:The inner wall of the lockbox [J].Cellular and Molecular Life Sciences, 2002, 59(3):426-433.
