唐泽群,姜紫薇,邱亮,王露,周婷,廖涛

• 1:武汉工程大学化学与环境工程学院
• 2:湖北省农业科学院农产品加工与核农技术研究所/农业农村部农产品冷链物流技术重点实验室

摘要(Abstract)：

【目的】研究水产品中常见的腐败希瓦氏菌（Shewanella putrefaciens）在热超声处理下进入可活不可培养（VBNC）状态的生理特性以及复苏状况。【方法】以腐败希瓦氏菌为研究对象，首先采用平板计数法测定腐败希瓦氏菌在不同温度（35、40、45、50、55、60、65、70℃）和不同功率（100、150、200、250、300 W）热超声处理下的细菌。进而在70℃、不同功率（100、150、200、250、300 W）TS条件下处理腐败希瓦氏菌，利用流式细胞学技术对不同状态的细菌群体进行计数，通过台盼蓝染色验证其存活状态，结合透射电子显微镜观察其形态变化，并使用DNA体外扩增技术检测其VBNC状态特征。最后，将经过70℃、300 W条件TS处理的菌株进行复苏实验。【结果】腐败希瓦氏菌经不同条件的热超声处理后，其细菌死亡率同温度和超声功率成正相关。流式细胞学技术和台盼蓝染色结果表明，在高热超声处理（50 kHz、300 W、超声处理30 min以及70℃加热）下的细菌会大量死亡，但仍有5.87%菌体存活，结合平板计数结果，表明这部分细菌已进入VBNC状态。透射电镜显微镜观察发现，VBNC菌体存在细胞壁和细胞膜变厚，细胞质密度增加，核区模糊的情况。体外扩增结果表明，热超声处理会降解细菌DNA，且复苏实验证明热超声处理后VBNC状态的腐败希瓦氏菌仍存在复苏能力。【结论】在300 W以及70℃加热的热超声处理下会诱导腐败希瓦氏菌进入VBNC状态，处于该状态下的菌体会对TS处理产生一系列生理特性的改变来抵抗外界环境，且当处于适宜环境下时，VBNC状态下的腐败希瓦氏菌会复苏并继续生长增殖。即使经过严格处理过程，仍需对食品安全中的VBNC细菌进行全面监控。

关键词(KeyWords)： 腐败希瓦氏菌;热超声处理;可活不可培养状态;生理特性;细菌复苏

Abstract:

Keywords:

基金项目(Foundation): 国家重点研发计划（2019YFD0902000）;; 湖北省重点研发计划（2023BCB122）;; 荆州市2023科技计划（2023FC46）

作者(Author): 唐泽群,姜紫薇,邱亮,王露,周婷,廖涛

参考文献(References)：

• [1] CHEN Y, MIAO W H, LI X X, et al. The structure, properties,synthesis method and antimicrobial mechanism of ε-polylysine with the preservative effects for aquatic products[J]. Trends in Food Science&Technology, 2023, 139:104131.
• [2] ZHUANG S, LIU Y Y, GAO S, et al. Mechanisms of fish protein degradation caused by grass carp spoilage bacteria:a bottom-up exploration from the molecular level, muscle microstructure level, to related quality changes[J]. Food Chemistry, 2023, 403:134309.
• [3] MüLLER S, VON BONIN S, SCHNEIDER R, et al.Shewanella putrefaciens, a rare human pathogen:a review from a clinical perspective[J]. Frontiers in Cellular and Infection Microbiology, 2023, 12:1033639.
• [4] GU Y J, LAN W Q, XIE J. Antibacterial mechanism of ultrasound combined with thymol against Shewanella putrefaciens:implications for cell integrity and biofilm control[J].International Journal of Food Science&Technology, 2024,59(5):3045-3055.
• [5] KIRBY R, DA SILVA M, CAPELL C, et al. Note. reaction of bacteria associated with fish spoilage to chemical and physical stress[J]. Food Science and Technology International,2001, 7(5):405-409.
• [6] REYES J E, TABILO-MUNIZAGA G, PéREZ-WON M,et al. Effect of high hydrostatic pressure(HHP)treatments on microbiological shelf-life of chilled Chilean jack mackerel(Trachurus murphyi)[J]. Innovative Food Science&Emerging Technologies, 2015, 29:107-112.
• [7] SCUDINO H, GUIMAR?ES J T, SILVA MOURA R, et al.Thermosonication as a pretreatment of raw milk for Minas frescal cheese production[J]. Ultrasonics Sonochemistry, 2023,92:106260.
• [8] URANGO A C M, STRIEDER M M, SILVA E K, et al.Impact of thermosonication processing on food quality and safety:a review[J]. Food and Bioprocess Technology, 2022,15(8):1700-1728.
• [9] QI Z, LIU C G. Ultrasound treatment reducing the production of VBNC bacteria in the process of chlorine disinfection:efficiency and mechanisms[J]. Chemical Research in Chinese Universities, 2023, 39(3):425-433.
• [10] WEI C J, ZHAO X H. Induction of viable but nonculturable Escherichia coli O157:H7 by low temperature and its resuscitation[J]. Frontiers in Microbiology, 2018, 9:2728.
• [11] ZHANG S H, YE C S, LIN W F, et al. Response to comment on “uv disinfection induces a VBNC state in Escherichia coli and Pseudomonas aeruginosa”[J]. Environmental Science&Technology, 2015, 49(17):10752-10753.
• [12] BOLLEN C, DEWACHTER L, MICHIELS J. Protein aggregation as a bacterial strategy to survive antibiotic treatment[J]. Frontiers in Molecular Biosciences, 2021, 8:669664.
• [13] LIU X, TANG R, LI H Q, et al. The physiological and ecological properties of bacterial persisters discovered from municipal sewage sludge and the potential risk[J].Environmental Research, 2022, 205:112481.
• [14] WANG L, GAO Y X, JIANG W, et al. Microplastics with cadmium inhibit the growth of Vallisneria natans(Lour.)Hara rather than reduce cadmium toxicity[J]. Chemosphere,2021, 266:128979.
• [15] AYRAPETYAN M, WILLIAMS T, OLIVER J D.Relationship between the viable but nonculturable state and antibiotic persister cells[J]. Journal of Bacteriology, 2018,200(20):e00249-18.
• [16] LIU J Y, YANG L, KJELLERUP B V, et al. Viable but nonculturable(VBNC)state, an underestimated and controversial microbial survival strategy[J]. Trends in Microbiology, 2023, 31(10):1013-1023.
• [17] ARVANITI M, OROLOGAS-STAVROU N, TSITSILONIS O E, et al. Induction into viable but non culturable state and outgrowth heterogeneity of Listeria monocytogenes is affected by stress history and type of growth[J]. International Journal of Food Microbiology, 2024, 421:110786.
• [18] DI FERMO P, DI LODOVICO S, DI CAMPLI E, et al.Helicobacter pylori dormant states are affected by vitamin C[J]. International Journal of Molecular Sciences, 2023,24(6):5776.
• [19] PAN H X, REN Q. Wake up! Resuscitation of viable but nonculturable bacteria:mechanism and potential application[J]. Foods, 2022, 12(1):82. https://doi.org/10.3390/foods12010082
• [20] FAN Z H, ZHU H C, TAO C, et al. Quantitative detection of VBNC state Pseudomonas aeruginosa contributing to accurate assessment of microbial inactivation in drinking water disinfection[J]. Water, 2024, 16(2):236.
• [21] AYRAPETYAN M, OLIVER J D. The viable but non-culturable state and its relevance in food safety[J].Current Opinion in Food Science, 2016, 8:127-133.
• [22] YOON J H, LEE S Y. Characteristics of viable-butnonculturable Vibrio parahaemolyticus induced by nutrientdeficiency at cold temperature[J]. Critical Reviews in Food Science and Nutrition, 2020, 60(8):1302-1320.
• [23] LI J H, ZHAO X H. Effects of quorum sensing on the biofilm formation and viable but non-culturable state[J].Food Research International, 2020, 137:109742.
• [24] ZHANG L N, JIANG L F, NIE X Y, et al. Factors affecting the occurrence of viable but non-culturable state in Salmonella,Escherichia coli and Staphylococcus aureus during thermosonication and prevent it with sodium pyruvate[J].International Journal of Food Science&Technology, 2021,56(5):2244-2254.
• [25] HU Z Y, BAI X H. Self-repair and resuscitation of viable injured bacteria in chlorinated drinking water:Achromobacter as an example[J]. Water Research, 2023, 245:120585.
• [26] YANG D, WANG W X, ZHAO L, et al. Resuscitation of viable but nonculturable bacteria promoted by ATP-mediated NAD+synthesis[J]. Journal of Advanced Research, 2024,60:27-39.
• [27] ZHAO F, BI X F, HAO Y L, et al. Induction of viable but nonculturable Escherichia coli O157:H7 by high pressure CO2 and its characteristics[J]. PLoS One, 2013, 8(4):e62388.
• [28] CAI Y J, CHEN X, QI H H, et al. Genome analysis of Shewanella putrefaciens 4H revealing the potential mechanisms for the chromium remediation[J]. BMC Genomics, 2024, 25(1):136.
• [29] LIAO H M, ZHANG R R, ZHONG K, et al. Induction of a viable but non-culturable state in Salmonella Typhimurium is correlated with free radicals generated by thermosonication[J]. International Journal of Food Microbiology, 2018, 286:90-97.
• [30] BAM Chapter 3:Aerobic Plate Count[S]. Food and Drug Administration, 2001.
• [31] BAI H, ZHAO F, LI M, et al. Citric acid can force Staphylococcus aureus into viable but nonculturable state and its characteristics[J]. International Journal of Food Microbiology, 2019, 305:108254.
• [32] LUO W, WANG J Q, WANG Y, et al. Bacteriostatic effects of high-intensity ultrasonic treatment on Bacillus subtilis vegetative cells[J]. Ultrasonics Sonochemistry, 2021,81:105862.
• [33] GAVAHIAN M, MANYATSI T S, MORATA A, et al.Ultrasound-assisted production of alcoholic beverages:From fermentation and sterilization to extraction and aging[J]. Comprehensive Reviews in Food Science and Food Safety, 2022, 21(6):5243-5271.
• [34] LIANG N J, KOH J, KIM B J, et al. Structural and functional changes of bioactive proteins in donor human milk treated by vat-pasteurization, retort sterilization,ultra-high-temperature sterilization, freeze-thawing and homogenization[J]. Frontiers in Nutrition, 2022, 9:926814.
• [35] CHEN S, LI X, WANG Y H, et al. Induction of Escherichia coli into a VBNC state through chlorination/chloramination and differences in characteristics of the bacterium between states[J]. Water Research, 2018, 142:279-288.
• [36] JIA Y Y, YU C G, FAN J H, et al. Alterations in the cell wall of Rhodococcus biphenylivorans under norfloxacin stress[J]. Frontiers in Microbiology, 2020, 11:554957.
• [37] LIAO X Y, LI J, SUO Y J, et al. Multiple action sites of ultrasound on Escherichia coli and Staphylococcus aureus[J].Food Science and Human Wellness, 2018, 7(1):102-109.
• [38] MA Y, ISHIHARA K, YOSHIDA K, et al. Double-strand breaks in genome-sized DNA caused by megahertz ultrasound[J]. The Journal of the Acoustical Society of America, 2021, 150(1):241-247.