食源性致病菌大肠杆菌O157:H7乳酸亚致死性损伤与复活研究
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摘要
亚致死性损伤菌(SI菌)难以彻底消除,造成极大的食品安全隐患,已引起高度重视,成为近年来的研究热点。乳酸存在于很多酸性食品或者发酵食品中,可能导致SI菌的产生。本研究以具有强耐酸性的食源性致病菌大肠杆菌O157:H7为研究对象,通过TSA/VRBA双培养基计数方法得到亚致死性损伤率及复活率,结果显示:乳酸会导致SI菌产生,损伤率随时间延长而增大,37℃下pH 4.0乳酸处理90 min时损伤率达到100%。损伤菌在37℃下minA培养基(加入酪蛋白水解物)中孵育80 min可完全复活。minA培养基中去除PO43-或酪蛋白水解物均使复活时间延长到120 min,去除葡萄糖复活时间延长到100 min。Mg2+缺失在复活中无显著作用。通过荧光素二乙酸酯/碘化丙啶双染色法以及A260和A280检测,观察到损伤菌的细胞膜通透性增强,代谢活性降低,外渗核酸及蛋白质增多,复活后恢复到正常菌状态。这些结果说明在复活过程中,细胞膜修复,代谢活性增强,氨基酸摄取或氨基酸依赖型耐酸系统的激活,ATP合成可能是复活的关键因素。
Sublethally injured(SI) bacteria are difficult to be sterilized thoroughly, causing great potential risk of food safety. It has caused great concern and become research focus in recent years. Lactic acid exists in a variety of fermented food and can induce SI bacteria. Acid-resistant foodborne pathogen Escherichia coli O157:H7 was studied in this study, and ratios of sublethal injury and resuscitation were obtained by TSA/VRBA counting method. It showed SI cells can be induced by lactic acid. The ratio of injury increased with exposure time increasing, and reached 100% after exposure by lactic acid at pH 4.0 for 90 min(37 ℃). SI cells can completely resuscitate in minA(with casamino acid)for 80 min at 37 ℃. It can completely resuscitate in minA lacking of PO43-or lacking of casamino acid for 120 min, and can resuscitate in minA lacking of glucose for 100 min. However, no significant effect of Mg2+was observed in recovery.Through FDA/PI dying and A260/A280 detecting, it was observed that membrane permeability increased and metabolic activity decreased of SI cells. In addition, nucleic acid and protein increased outside SI cells. Repaired cells regained activity. These results indicated that membrane was recovered and metabolic activity increased during resuscitation. In addition,uptake of amino acids or activation of amino acid-dependent acid resistance system, and ATP synthesis might be key factors in resuscitation.
Sublethally injured(SI) bacteria are difficult to be sterilized thoroughly, causing great potential risk of food safety. It has caused great concern and become research focus in recent years. Lactic acid exists in a variety of fermented food and can induce SI bacteria. Acid-resistant foodborne pathogen Escherichia coli O157:H7 was studied in this study, and ratios of sublethal injury and resuscitation were obtained by TSA/VRBA counting method. It showed SI cells can be induced by lactic acid. The ratio of injury increased with exposure time increasing, and reached 100% after exposure by lactic acid at pH 4.0 for 90 min(37 ℃). SI cells can completely resuscitate in minA(with casamino acid)for 80 min at 37 ℃. It can completely resuscitate in minA lacking of PO43-or lacking of casamino acid for 120 min, and can resuscitate in minA lacking of glucose for 100 min. However, no significant effect of Mg2+was observed in recovery.Through FDA/PI dying and A260/A280 detecting, it was observed that membrane permeability increased and metabolic activity decreased of SI cells. In addition, nucleic acid and protein increased outside SI cells. Repaired cells regained activity. These results indicated that membrane was recovered and metabolic activity increased during resuscitation. In addition,uptake of amino acids or activation of amino acid-dependent acid resistance system, and ATP synthesis might be key factors in resuscitation.
引文
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[2]石慧,陈卓逐,阚建全.大肠杆菌在食品加工贮藏中胁迫响应机制的研究进展[J].食品科学, 2016, 37(9):250-257.
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[16] FOSTER J W. Escherichia coli acid resistance:tales of an amateur acidophile[J]. Nature Reviews Microbiology,2004, 2(11):898-907.
[17] LU P, MA D, CHEN Y, et al. L-glutamine provides acid resistance for Escherichia coli through enzymatic release of ammonia[J]. Cell Research, 2013, 23(5):635-644.
[18] MILLER J H. A short course in bacterial genetics:A laboratory manual and handbook for Escherichia coli and related bacteria[M]. Plainview, NY:Cold Spring Harbor Lab Press, 1992:45.
[19]柴阿丽,韩云,武军,等.基于FDA-PI双荧光复染法的茄病镰刀菌孢子活性检测[J].中国农业科学, 2015, 48(14):2757-2766.
[20]谌丽斌,梁文艳,曲久辉,等. FDA-PI双色荧光法检测蓝藻细胞活性的研究[J].环境化学, 2005, 24(5):554-557.
[21] CHANG C H, CHIANG M L, CHOU C C. The effect of temperature and length of heat shock treatment on the thermal tolerance and cell leakage of Cronobacter sakazakii BCRC 13988[J]. International Journal of Food Microbiology, 2009, 134(3):184-9.
[22]陈婵娟.副溶血性弧菌亚致死损伤状态的研究[D].上海:上海海洋大学, 2013.
[23] BUSCH S V, DONNELLY C W. Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua[J]. Applied and Environmental Microbiology, 1992, 58(1):14-20.
[24] BI X, WANG Y, ZHAO F, et al. Inactivation of Escherichia coli O157:H7 by high pressure carbon dioxide combined with nisin in physiological saline, phosphate-buffered saline and carrot juice[J]. Food Control, 2014, 41(2):139-146.
[25] RAY B, SPECK M L. Metabolic process during the repair of freeze-injury in Escherichia coli[J]. Applied Microbiology, 1972, 24(4):585-590.
[2]石慧,陈卓逐,阚建全.大肠杆菌在食品加工贮藏中胁迫响应机制的研究进展[J].食品科学, 2016, 37(9):250-257.
[3] BI X, WANG Y, ZHAO F, et al. Sublethal injury and recovery of Escherichia coli O157:H7 by high pressure carbon dioxide[J]. Food Control, 2015, 50:705-713.
[4]史贤明,施春雷,索标,等.食品加工过程中致病菌控制的关键科学问题[J].中国食品学报, 2011, 11(9):194-208.
[5] WESCHE A M, GURTLER J B, MARKS B P, et al. Stress, sublethal injury, resuscitation, and virulence of bacterial foodborne pathogens[J]. Journal of Food Protection, 2009, 72(5):1121-1138.
[6] RILEY L W, REMIS R S, HELGERSON S D, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype[J]. The New England Journal of Medicine, 1983, 308(12):681-685.
[7] SUMNER J. Update:Multistate outbreak of Escherichia coli O157:H7 infections from Hamburgers-Western United States, 1992-1993[J]. Mmwr Morbidity&Mortality Weekly Report, 1993, 42(14):258-263.
[8] CDC. Multistate outbreak of Shiga toxin-producing Escherichia coli O157:H7 infections linked to ground beef(final update)[R/OL].(2014). http://www.cdc.gov/ecoli/2014/O157H7-05-14/.
[9] WU V C. A review of microbial injury and recovery methods in food[J]. Food Microbiology, 2008, 25(6):735-744.
[10] CHO K M, MATH R K, ISLAM S M A, et al. Novel multiplex PCR for the detection of lactic acid bacteria during kimchi fermentation[J]. Molecular and Cellular Probes, 2009, 23(2):90-94.
[11] FDA. CFR-Code of Federal Regulations Title 21. U.S. Food and Drug Administration, Rockville, M D[R/OL].(1984). www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfr search.cfm?fr=184.1061.
[12] EFSA. Scientific Opinion on the evaluation of the safety and efficacy of lactic acid for the removal of microbial surface contamination of beef carcasses, cuts and trimmings[J]. EFSA Journal, 2011, 9(7):2317.
[13]汪陈洁.乳酸对常见食源性致病菌的抗菌活性与作用机理[D].武汉:华中农业大学, 2014.
[14] EGYCHI Y, ITOU J, YAMANE M, et al. B1500, a small membrane protein, connects the two-component systems EvgS/E-vgA and PhoQ/PhoP in Escherichia coli[J]. Proceedings of the National Academy of Sciences of the United States of Ame-rica, 2007, 104(47):18712-18717.
[15]陈卓逐,阚建全,石慧.大肠杆菌耐酸分子机制研究进展[J].食品科学, 2015, 36(21):273-278.
[16] FOSTER J W. Escherichia coli acid resistance:tales of an amateur acidophile[J]. Nature Reviews Microbiology,2004, 2(11):898-907.
[17] LU P, MA D, CHEN Y, et al. L-glutamine provides acid resistance for Escherichia coli through enzymatic release of ammonia[J]. Cell Research, 2013, 23(5):635-644.
[18] MILLER J H. A short course in bacterial genetics:A laboratory manual and handbook for Escherichia coli and related bacteria[M]. Plainview, NY:Cold Spring Harbor Lab Press, 1992:45.
[19]柴阿丽,韩云,武军,等.基于FDA-PI双荧光复染法的茄病镰刀菌孢子活性检测[J].中国农业科学, 2015, 48(14):2757-2766.
[20]谌丽斌,梁文艳,曲久辉,等. FDA-PI双色荧光法检测蓝藻细胞活性的研究[J].环境化学, 2005, 24(5):554-557.
[21] CHANG C H, CHIANG M L, CHOU C C. The effect of temperature and length of heat shock treatment on the thermal tolerance and cell leakage of Cronobacter sakazakii BCRC 13988[J]. International Journal of Food Microbiology, 2009, 134(3):184-9.
[22]陈婵娟.副溶血性弧菌亚致死损伤状态的研究[D].上海:上海海洋大学, 2013.
[23] BUSCH S V, DONNELLY C W. Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua[J]. Applied and Environmental Microbiology, 1992, 58(1):14-20.
[24] BI X, WANG Y, ZHAO F, et al. Inactivation of Escherichia coli O157:H7 by high pressure carbon dioxide combined with nisin in physiological saline, phosphate-buffered saline and carrot juice[J]. Food Control, 2014, 41(2):139-146.
[25] RAY B, SPECK M L. Metabolic process during the repair of freeze-injury in Escherichia coli[J]. Applied Microbiology, 1972, 24(4):585-590.