该文探究了姜黄素介导的光动力技术(photodynamic technology, PDT)协同柠檬酸处理对副溶血性弧菌的灭活效果和机理。通过平板菌落计数法测定光动力技术对副溶血性弧菌的灭活效果、扫描电镜观察细菌形态变化,酶标仪检测PDT处理后菌体活性氧(reactive oxygen species,ROS)水平以及过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、腺苷三磷酸酶(adenosine triphosphatase,ATPase)、碱性磷酸酶(alkaline phosphatase,AKP)的活性变化,琼脂糖凝胶电泳分析基因损伤程度。结果表明,PDT杀菌效果随姜黄素浓度增加、光照时间延长和柠檬酸(0.5 mg/mL)的添加而显著增强(在最高浓度姜黄素条件下P<0.05)。当姜黄素浓度为2 μmol/L,柠檬酸质量浓度为0.5 mg/mL,光照2 min后,PBS中的副溶血性弧菌菌落总数从7.43 lg CFU/mL降至0。光敏剂姜黄素被蓝光激活后通过电子转移和能量转移产生具有强氧化性的ROS,菌体内ROS水平不断升高,胞内抗氧化酶(CAT、SOD)活性下降,ATPase活性下降,上清液中AKP活性上升,细菌发生严重形变甚至趋向于扁平,DNA条带变暗甚至消失,最终导致细胞死亡。综上,姜黄素介导的PDT对副溶血性弧菌有较强的杀灭作用,添加柠檬酸显著加强杀菌效果,为PDT在水产食品安全防控领域的推广应用提供了参考。
In this paper, the inactivation effect and mechanism of curcumin-mediated photodynamic technology (PDT) combined with citric acid treatment on Vibrio parahaemolyticus were studied. The inactivation effect of PDT on V. parahaemolyticus was determined by flat colony counting method, and the morphological changes of bacteria were observed by scanning electron microscope. The levels of reactive oxygen species (ROS) and the activities of catalase (CAT), superoxide dismutase (SOD), adenosine triphosphatase (ATPase), alkaline phosphatase (AKP) was detected by microplate reader after PDT. The degree of gene damage was analyzed by agarose gel electrophoresis. The results showed that the germicidal effect of PDT was significantly enhanced with the increase of curcumin concentration, light time and the addition of 0.5 mg/mL citric acid (at the highest concentration of curcumin conditions P<0.05). When the concentration of curcumin was 2 μmol/L, the concentration of citric acid was 0.5 mg/mL and light exposure time was 2 min, the total colony number of V. parahemolyticus in PBS decreased from 7.43 lg CFU/mL to 0 CFU/mL. As a photosensitizer, curcumin was activated by blue light to produce strong oxidizing ROS through electron and energy transfer. With the increasing level of ROS in the bacteria, the activities of CAT, SOD, and ATPase decreased, the activity of AKP in the supernatant increased, the bacteria seriously deformed and even tended to be flat, and the band of genomic DNA became darken or even disappear, eventually leading to cell death. In conclusion, curcumin-mediated PDT can effectively inactivate V. parahemolyticus, and the addition of citric acid can significantly enhance the germicidal efficacy, which provided a reference for the popularization and application of PDT in the field of aquatic food safety prevention and control.
[1] WANG D, FLINT S H, PALMER J S, et al.Global expansion of Vibrio parahaemolyticus threatens the seafood industry:Perspective on controlling its biofilm formation[J].LWT, 2022, 158:113182.
[2] FANG M M, WANG R F, AGYEKUMWAA A K, et al.Antibacterial effect of phenyllactic acid against Vibrio parahaemolyticus and its application on raw salmon fillets[J].LWT, 2022, 154:112586.
[3] ASHRAFUDOULLA M, NA K W, HOSSAIN M I, et al.Molecular and pathogenic characterization of Vibrio parahaemolyticus isolated from seafood[J].Marine Pollution Bulletin, 2021, 172:112927.
[4] 孟媛媛, 刘海泉, 潘迎捷, 等.光动力杀菌机制及在食品应用中的优势与不足[J].食品工业科技, 2022, 43(22):414-421.
MENG Y Y, LIU H Q, PAN Y J, et al.Mechanism of photodynamic inactivation and its advantages and disadvantages in food applications[J].Science and Technology of Food Industry, 2022, 43(22):414-421.
[5] GHATE V S, ZHOU W B, YUK H G.Perspectives and trends in the application of photodynamic inactivation for microbiological food safety[J].Comprehensive Reviews in Food Science and Food Safety, 2019, 18(2):402-424.
[6] YANG Q Q, FARHA A K, KIM G, et al.Antimicrobial and anticancer applications and related mechanisms of curcumin-mediated photodynamic treatments[J].Trends in Food Science & Technology, 2020, 97:341-354.
[7] LIANG Z X, LIU X L, QIN Z R, et al.Photodynamic inactivation of Shigella flexneri by curcumin[J].LWT, 2022, 153:112491.
[8] DIAS L D, BLANCO K C, MFOUO-TYNGA I S, et al.Curcumin as a photosensitizer:From molecular structure to recent advances in antimicrobial photodynamic therapy[J].Journal of Photochemistry and Photobiology C:Photochemistry Reviews, 2020, 45:100384.
[9] HUANG J M, CHEN B W, LI H H, et al.Enhanced antibacterial and antibiofilm functions of the curcumin-mediated photodynamic inactivation against Listeria monocytogenes[J].Food Control, 2020, 108:106886.
[10] TAN L J, LI H H, CHEN B W, et al.Dual-species biofilms formation of Vibrio parahaemolyticus and Shewanella putrefaciens and their tolerance to photodynamic inactivation[J].Food Control, 2021, 125:107983.
[11] TEMBA B A, FLETCHER M T, FOX G P, et al.Inactivation of Aspergillus flavus spores by curcumin-mediated photosensitization[J].Food Control, 2016, 59:708-713.
[12] GONG C, LI Y J, GAO R C, et al.Inactivation of specific spoilage organism (Pseudomonas) of sturgeon by curcumin-mediated photodynamic inactivation[J].Photodiagnosis and Photodynamic Therapy, 2020, 31:101827.
[13] RANDAZZO W, AZNAR R, SÁNCHEZ G.Curcumin-mediated photodynamic inactivation of norovirus surrogates[J].Food and Environmental Virology, 2016, 8(4):244-250.
[14] CHO G L, HA J W.Synergistic effect of citric acid and xenon light for inactivating foodborne pathogens on spinach leaves[J].Food Research International, 2021, 142:110210.
[15] GHATE V, KUMAR A, ZHOU W B, et al.Effect of organic acids on the photodynamic inactivation of selected foodborne pathogens using 461 nm LEDs[J].Food Control, 2015, 57:333-340.
[16] 檀利军, 胡钰梅, 陈博文, 等.姜黄素介导的光动力技术对副溶血性弧菌与腐败希瓦氏菌的杀灭效果[J].食品科学, 2022, 43(3):83-91.
TAN L J, HU Y M, CHEN B W, et al.Inactivation of curcumin-mediated photodynamic technology on Vibrio parahaemolyticus and Shewanella putrefaciens[J].Food Science, 2022, 43(3):83-91.
[17] SHI Y G, JIANG L, LIN S, et al.Ultra-efficient antimicrobial photodynamic inactivation system based on blue light and octyl gallate for ablation of planktonic bacteria and biofilms of Pseudomonas fluorescens[J].Food Chemistry, 2022, 374:131585.
[18] MAO G X, ZHENG L D, CAO Y B, et al.Antiaging effect of pine pollen in human diploid fibroblasts and in a mouse model induced by D-galactose[J].Oxidative Medicine and Cellular Longevity, 2012, 2012:750963.
[19] ZHAO Q F, SHAO L, HU X T, et al.Lipoxin a4 preconditioning and postconditioning protect myocardial ischemia/reperfusion injury in rats[J].Mediators of Inflammation, 2013, 2013:231351.
[20] TOMOTAKE H, KOGA T, YAMATO M, et al.Antibacterial activity of citrus fruit juices against Vibrio species[J].Journal of Nutritional Science and Vitaminology, 2006, 52(2):157-160.
[21] LUND P, TRAMONTI A, DE BIASE D.Coping with low pH:Molecular strategies in neutralophilic bacteria[J].FEMS Microbiology Reviews, 2014, 38(6):1091-1125.
[22] WIKENE K O, BRUZELL E, TØNNESEN H H.Characterization and antimicrobial phototoxicity of curcumin dissolved in natural deep eutectic solvents[J].European Journal of Pharmaceutical Sciences, 2015, 80:26-32.
[23] LI T M, ZHAO Y L, MATTHEWS K, et al.Antibacterial activity against Staphylococcus aureus of curcumin-loaded chitosan spray coupled with photodynamic treatment[J].LWT, 2020, 134:110073.
[24] DE OLIVEIRA E F, TOSATI J V, TIKEKAR R V, et al.Antimicrobial activity of curcumin in combination with light against Escherichia coli O157:H7 and Listeria innocua:Applications for fresh produce sanitation[J].Postharvest Biology and Technology, 2018, 137:86-94.
[25] DE OLIVEIRA E F, COSSU A, TIKEKAR R V, et al.Enhanced antimicrobial activity based on a synergistic combination of sublethal levels of stresses induced by UV-A light and organic acids[J].Applied & Environmental Microbiology, 2017, 83(11):e00383-e00317.
[26] FREITAS M A, PEREIRA A H, PINTO J G, et al.Bacterial viability after antimicrobial photodynamic therapy with curcumin on multiresistant Staphylococcus aureus[J].Future Microbiology, 2019, 14(9):739-748.
[27] ZHU Y L, ZHANG S.Antibacterial activity and mechanism of lacidophilin from Lactobacillus pentosus against Staphylococcus aureus and Escherichia coli[J].Frontiers in Microbiology, 2020, 11:582349.
[28] SHLAR I, DROBY S, RODOV V.Modes of antibacterial action of curcumin under dark and light conditions:A toxicoproteomics approach[J].Journal of Proteomics, 2017, 160:8-20.
[29] KHAN S, MOHAMMED RAYIS P, RIZVI A, et al.ROS mediated antibacterial activity of photoilluminated riboflavin:A photodynamic mechanism against nosocomial infections[J].Toxicology Reports, 2019, 6:136-142.
[30] 韩启明, 张芳, 孟祥红.姜黄素光动力技术对食源性致病菌抗氧化酶体系的影响研究[J].合肥工业大学学报(自然科学版), 2021, 44(12):1704-1709.
HAN Q M, ZHANG F, MENG X H.Effect of curcumin-based photodynamic technology on antioxidant enzyme of foodborne pathogenic bacteria[J].Journal of Hefei University of Technology (Natural Science), 2021, 44(12):1704-1709.
[31] PATEL A, MALINOVSKA L, SAHA S, et al.ATP as a biological hydrotrope[J].Science, 2017, 356(6339):753-756.
[32] WU J, MOU H J, XUE C H, et al.Photodynamic effect of curcumin on Vibrio parahaemolyticus[J].Photodiagnosis and Photodynamic Therapy, 2016, 15:34-39.
