上海海洋大学学报  2020, Vol. 29 Issue (4): 578-584    PDF    
应用酸性电解水联合超声波杀灭副溶血性弧菌
许愈1, 张昭寰1, 赵莉1, 刘海泉1,2,3,4, 潘迎捷1,2,3, 赵勇1,2,3     
1. 上海海洋大学 食品学院, 上海 201306;
2. 上海水产品加工及贮藏工程技术研究中心, 上海 201306;
3. 农业农村部水产品贮藏保鲜质量安全风险评估实验室, 上海 201306;
4. 上海海洋大学 食品热加工工程技术研究中心, 上海 201306
摘要:将酸性电解水与超声波技术相结合,探究其对副溶血性弧菌的杀灭情况,并与其他杀菌措施进行对比。采用平板计数法进行菌落计数,扫描电镜观察细菌的形态变化,蛋白质泄漏揭示细胞膜通透性差异,并结合流式细胞仪分析生物学特征的改变,分别比较了酸性电解水、超声波以及联合处理对副溶血性弧菌的杀菌效果。结果显示:酸性电解水联合超声波处理可使副溶血性弧菌的数量减少2.09 log CFU/mL,亚致死菌数量为1.80 log CFU/mL,而仅用超声波处理,细菌只减少了0.63 log CFU/mL,亚致死菌数量为0.05 log CFU/mL(P < 0.05)。扫描电镜结果表明,电解水联合超声波处理对副溶血性弧菌的细胞结构有明显的破坏作用,结合二喹啉甲酸法(BCA)显示其细胞内蛋白质泄漏226.596 μg/mL(P < 0.05)。进一步的流式细胞仪分析结果显示,经联合处理后细菌细胞明显缩小,颗粒度变化增大。综上所述,相较于酸性电解水或超声波单一处理,通过菌落计数,细菌的形态变化,蛋白质泄漏与细胞生物学特征的改变可知,酸性电解水联合超声波的处理方式具有更强的杀菌效果,可作为一种新型的技术应用于水产品中副溶血性弧菌的杀灭。
关键词副溶血性弧菌    酸性电解水    超声波    杀菌效果    

副溶血性弧菌(Vibrio parahaemolyticus, VP)是一种重要的食源性致病菌,广泛存在于海洋环境中,常从鱼、虾、蟹等海产品中分离出来[1-3]。在夏秋季节中,因误食致病性副溶血性弧菌污染的水产品,可引发严重的急性肠胃炎、败血症等疾病,对人类健康造成了巨大威胁[4-6]。因此,寻找合适环保的杀菌方法,以控制副溶血性弧菌的生长和生存,具有十分重要的科研价值和实际意义。

酸性电解水(acidic electrolyzed water,AEW)是一种新型杀菌剂[7],具有低pH、高氧化还原电位和含有效氯等特性[8],能迅速并广泛地杀死细菌。相较于其他化学杀菌剂,对食品的品质影响较小[3, 9-10]。研究[11-12]发现,在液体环境中,由于超声波产生的压力波动,整个液体中将产生空化气泡。这些空化气泡爆裂将产生可破坏微生物的机械效应(冲击波、剪切力和微喷射)[13],导致细胞膜变薄,局部加热和自由基产生,可用于超声波灭活食源性病原体[14-16]。研究表明,更换超声波的液体介质,将酸性电解水作为一种新的介质,与超声波相结合,可提升超声波的杀菌和保鲜作用[17-18],但目前较少有研究应用酸性电解水结合超声波杀菌技术来提升致病性副溶血性弧菌的杀菌效率。

因此,本研究以副溶血性弧菌临床菌株作为研究对象,分别比较了酸性电解水、超声波、酸性电解水结合超声波技术对副溶血性弧菌的杀灭作用,以期探究一种更为有效的新型杀菌技术,用于水产品中常见食源性致病菌的清除。

1 材料与方法 1.1 实验材料

供试菌株:副溶血性弧菌临床菌株VPC1(Vibrio parahaemolyticus clinical1[19])由课题组从上海市人民医院临床样本中分离得到。主要试剂:胰酶大豆肉汤培养基(Tryptone Soy Broth,TSB)、胰蛋白胨大豆琼脂培养基(Ttytic Soy Agar, TSA)、硫代硫酸盐柠檬酸盐胆盐蔗糖琼脂培养基(Thiosulfate citrate bile salts sucrose agar culture medium,TCBS)、NaCl等均购自北京陆桥技术有限责任公司;实验所用试剂均为分析纯。实验用水为去离子水。

1.2 主要仪器与设备

发射扫描电子显微镜(Sirion 200),美国FEI公司;OptiMair®垂直流超净工作台、Airstream®A2型二级生物安全柜,Esco China公司;pH/ORP测量仪表(model pH 430),美国Corning Life Sciences公司;高浓度有效氯测定仪(RC-2Z),日本Kasahara Chemical Instruments公司;超声波细胞粉碎机(Scientz-IID),宁波新芝生物科技公司;电解水制备仪(FW-200),日本AMANO公司;流式细胞仪(BD FACSVerse),美国BD公司;高速台式离心机(5804R),德国Eppendorf公司。

1.3 实验方法 1.3.1 酸性电解水的制备

参考文献[10]的方法,将0.10%的NaCl溶液,在电解水仪器中电离5 min获得酸性电解水,分别用pH/ORP测定仪和有效氯测定仪,测定酸性电解水pH、氧化还原电位(oxidation reduction potential, ORP)和有效氯成分(available chlorine concentration, ACC)。见表 1

表 1 酸性电解水物理化学性质 Tab.1 Physicochemical properties of AEW
1.3.2 菌株准备

从-80 ℃冰箱内取出副溶血性弧菌临床菌株VPC1,用平板划线法将副溶血性弧菌接种至TCBS琼脂培养基中,37 ℃过夜培养,挑取单菌落于9 mL胰酶大豆肉汤培养基(TSB,3% NaCl)试管中,在37 ℃、180 r/min的摇床中培养10 h,连续活化2次后备用。

1.3.3 悬液定量杀菌实验

将活化后的副溶血性弧菌悬液置于离心管中离心10 min (25 ℃,3 000 g),收集菌体细胞用生理盐水调整菌体浓度为9 log CFU/mL。

悬液定量杀菌实验一共分为4组,分别为空白对照组、酸性电解水组、超声波组和电解水结合超声组。空白对照组即生理盐水组,将菌悬液与0.85%NaCl溶液各2 mL混合均匀。AEW组中,将菌悬液与AEW各2 mL混合均匀,处理2 min。超声波组中,在25 ℃、参考王顺民等[20]的方法进行超声波杀菌,取菌悬液与0.85%NaCl溶液各2 mL混合均匀,50 W超声功率超声2 min。电解水结合超声组(A/U)中,菌悬液与AEW各2 mL混匀,50 W超声处理2 min。

将以上处理过的样品[7, 21],分别取1 mL混合液加入到9 mL终止液(0.5%Na2S2O3和0.85%NaCl)中终止酸性电解水杀菌过程[7]。利用0.85%NaCl溶液进行10倍梯度稀释,取100 μL样品稀释液进行菌落涂布。副溶血性弧菌经过杀菌处理后,有3种不同的生理状态,分别是活菌、死菌以及亚致死损伤状态[22]。当原料充足、条件适宜时亚致死细菌具备自我修复能力[22-23]。TOMLINS等[24]指出选择性培养中无法生长的亚致死细菌,在非选择性培养基中可形成菌落。其中TSA固体培养基(非选择性培养基)进行活菌数量计数,TCBS固体培养基(具选择性培养基)进行亚致死副溶血性弧菌计数。

1.3.4 扫描电镜观察细菌形态变化

用扫描电子显微镜观察空白对照组、酸性电解水组、超声波组和电解水结合超声组的副溶血性弧菌微观结构。

将以上实验组用4%戊二醛固定,8 h后,PBS缓冲液清洗。分别用30%、50%、70%、90%和100%的梯度乙醇进行脱水处理(每个梯度洗脱两次,每次洗脱10 min, 8 000 r/min离心5 min),梯度洗脱完全后,8 000 r/min离心5 min后弃上清,用无水乙醇悬浮菌液,悬浮均匀后取1滴菌液滴于盖玻片上, 放置数分钟,待稍微干燥后,置于-80 ℃冰箱中冷冻4 h,冷冻干燥后,离子喷金,电镜观察细菌微观结构[7]

1.3.5 BCA法进行细菌蛋白泄漏测定

配制一系列浓度梯度为0、25、50、100、250、500、750和1 000 μg/mL的牛血清蛋白(BSA)标准工作液,在酶标仪562 nm波长下测吸光度,绘制蛋白定量标准曲线。根据所得线性回归方程Y=9.999 7X+0.082 5(R2=0.999),对AEW组、超声波组及联合组处理的细菌蛋白泄漏量进行定量测定。

1.3.6 流式细胞仪数据分析

将制备好的空白对照组、酸性电解水组、超声波组和电解水结合超声组菌悬液稀释至1×105个/mL,混匀,取500 μL至流式管中,排除杂质,圈出细胞群,调节电压参数,使副溶血性弧菌细胞群位于坐标系中央,通过使用前向光散射(FSC)表示细胞的相对大小,使用侧光散射(SSC)表示细胞的粒度,测量该参数间接测定杀菌作用。采用BD FACSVerse流式细胞仪进行分析,以前向角散射(FSC)为横坐标,侧向角散射(SSC)为纵坐标,作图进行分析[25]

1.4 数据处理与统计分析

实验结果为3次重复的平均值,用SPSS 19.0进行数据统计和分析检验水准为0.05,运用LSD法比较,以P < 0.05为差异显著。

2 结果与分析 2.1 酸性电解水结合超声波联合处理副溶血性弧菌的杀菌效果

通过TSA和TCBS培养基进行菌落计数,得出亚致死状态细菌的数量。如表 2所示,在酸性电解水处理组中,副溶血性弧菌的活菌数量减少了1.37 log CFU/mL,亚致死损伤菌落数为0.61 log CFU/mL。超声功率处理组中(50 W),活菌数量减少了0.63 log CFU/mL,亚致死损伤量为0.05 log CFU/mL。使用超声波和低浓度酸性电解水(AEW)进行处理时,活菌数量减少了2.9 log CFU/mL,亚致死损伤的菌落数量为1.80 log CFU/mL(P < 0.05)。结果显示,相较于酸性电解水或超声波单独处理,酸性电解水结合超声波联合处理具有更好的杀菌效果。

表 2 不同条件处理2 min后副溶血性弧菌的死亡和亚致死数量 Tab.2 Surviving and sublethally injured V.parahaemolyticus populations after various treatments for 2 minutes
2.2 扫描电镜分析细菌细胞结构的变化

图 1可知,与对照组比,酸性电解水处理可使副溶血性弧菌的细胞开始回缩,细胞间间隙减小,并产生粘连[26-28]。超声处理仅能使副溶血性弧菌回缩变小,但是形态变化不明显。而在电解水联合超声波处理组中,副溶血性弧菌细胞结构破坏最为明显, 细胞严重皱缩,并从中间发生断裂。通过扫描电镜观察到细胞的细胞壁和细胞膜受到严重破坏,推测是超声波的损伤作用[29-30]。超声波杀菌机理主要是超声产生的空穴气泡瞬间爆裂产生冲击波和高速剪切力,分散细菌,使细菌更易受到外界压力,同时,细胞结构受到不同程度的损伤,释放胞内物质[29]。细胞壁损伤表明酸性电解水可以最终渗透细菌细胞壁,很可能在超声处理中,酸性电解水更容易透过细胞壁[21, 28]

图 1 不同条件处理后副溶血性弧菌的扫描电镜图片 Fig. 1 SEM photographs of V. parahaemolyticus strains after various treatments
2.3 细胞膜完整性的影响

表 3可知,酸性电解水处理后,蛋白质泄漏量随之增加(P < 0.05),超声处理后蛋白质泄漏量几乎无变化(P>0.05),该结果与SEM形态变化和细胞结果一致。相较于未处理组,联合处理后的蛋白质泄漏量虽有所增加,但与酸性电解水处理后的蛋白泄漏量大致相同(P>0.05)。这一结果表明,酸性电解水结合超声处理可使细菌细胞内蛋白质大量流出,而酸性电解水处理是导致这一现象的主要原因。

表 3 不同处理组副溶血性弧菌蛋白泄漏情况 Tab.3 The leakage of proteins from V. parahaemolyticus after various treatments
2.4 细菌生物学特征的影响

采用流式细胞仪观察细菌生物学特性的变化。根据流式细胞仪的特点,无需免疫荧光染色,通过物理参数FSC和SSC,可以测出处理组前后细菌的形态、大小变化。由图 2可见,副溶血性弧菌主要分布在坐标系中央,细菌超声杀菌前后的物理参数变化可全面地体现出来。副溶血性弧菌细胞大小没有明显改变。与未处理组相比,电解水组细胞内的颗粒度变化较大。联合处理组中,细胞变小,颗粒度变化增大,这表明了联合处理对副溶血性弧菌细胞的损伤更为严重,各个副溶血性弧菌的物理参数FSC与SSC差异增大。

图 2 流式细胞仪检测副溶血性弧菌散点图(FSC/SSC) Fig. 2 Scatter plot of V. parahaemolyticus (VPC1)by flow cytometer (FSC/SSC)
3 讨论与结论

本研究主要通过菌落计数进行杀菌效果定量比较、结合扫描电镜对细胞结构可视化观察、蛋白质泄漏比较细胞膜通透性,流式细胞仪观测副溶血性弧菌物理参数FSC与SSC的变化,探究了酸性电解水联合超声波技术对副溶血性弧菌的杀灭作用。不同于未处理组菌落数量8.80 log CFU/mL,酸性电解水联合超声波杀菌处理后,菌落数量为6.71 log CFU/mL(P < 0.05),观察发现细菌细胞壁表面粗糙,细菌从中间发生断裂(图 2),测量蛋白质泄漏量为226.596 μg/mL,存在显著性差异(P < 0.05),另外流式图显示坐标系中央的菌落物理参数FSC与SSC发生改变,细胞变小,颗粒度变化增大。联合杀菌处理不仅可以影响副溶血性弧菌的菌落数量及质量,而且可使该菌连续暴露于超声波和电解水两种压力之中,从而提升了杀菌效率。

GARCÍA等[31]研究表明,用脉冲电场处理苹果汁中亚致死受损的大肠杆菌O157:H7,低温储存下受损细胞群也更加敏感易死亡。本研究中,不同于单一酸性电解水或超声波单独处理,酸性电解水结合超声波杀菌过程中菌落数量减少2.09 log CFU/mL(P < 0.05),造成这一现象的原因可能是超声波通过破坏副溶血性弧菌细菌细胞壁的结构,使其通透性增加,使得酸性电解水在超声过程中更容易渗透并杀死副溶血性弧菌。因此,本研究将酸性电解水与超声波技术相结合,提供了一种新型的杀菌方式,可应用于水产品中副溶血性弧菌的清除,以降低该菌的风险,从而有效提升水产品的质量安全。

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Combination of acidic electrolyzed water with ultrasonic for killing Vibrio parahaemolyticus
XU Yu1, ZHANG Zhaohuan1, ZHAO Li1, LIU Haiquan1,2,3,4, PAN Yingjie1,2,3, ZHAO Yong1,2,3     
1. College of Food Sciences and Technology, Shanghai Ocean University, Shanghai 201306, China;
2. Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China;
3. Laboratory of Quality & Safety Risk Assessment for Aquatic Product on Storage and Preservation, Ministry of Agriculture and Rural Affairs, Shanghai 201306, China;
4. Engineering Research Center of Food Thermal-Processing Technology, Shanghai Ocean University, Shanghai 201306, China
Abstract: This study evaluated the bactericidal effects were evaluated against Vibrio parahaemolyticus by acidic electrolyzed water (AEW) combined with ultrasound which is a method different from the traditional sterilization methods. The effects were compared by viable plate count analysis. The physiological response was studied by scanning electronic microscopy (SEM) and cell membrane permeability revealed by protein leakage. Flow cytometry analysis revealed changes in the physiological states of V.parahaemolyticus. The effects of the combination of SAEW and ultrasound on the reductions of V.parahaemolyticus were evaluated in comparison with acidic electrolyzed water or ultrasound. The results revealed that AEW combined with ultrasonic showed bacterial reduction in V.parahaemolyticus by 2.09 log CFU/mL and the sub-lethal bacterial reduction was 1.80 log CFU/mL, when compared to the reduction of 0.63 log CFU/mL and the sub-lethal bacterial reduction of 0.05 log CFU/mL(P < 0.05) after ultrasonic treatment only. Scanning electron microscopy (SEM) results showed that electrolyzed water combined with ultrasonic treatment had obvious destructive effect on the cell structure of V.parahaemolyticus. The protein leakage of V.parahaemolyticus detected by bicinchoninic acid (BCA) were 226.596 μg/mL, respectively, and difference was significant (P < 0.05). Flow cytometry analysis showed that the cells became smaller and the change in particle size increased. In summary, acidic electrolyzed water combined with ultrasound could be more effective in reducing V.parahaemolyticus, the physiological response changes, protein leakage and changes in the physiological states, when compared with acidic electrolyzed water or ultrasound alone. The combination of acidic electrolyzed water and ultrasound could be an new technique to reduce the risk of V.parahaemolyticus.
Key words: Vibrio parahaemolyticus     acidic electrolyzed water     ultrasound     bactericidal effect