酸性电解水对肉源性荧光假单胞菌的致死效应

doi:10.3864/j.issn.0578-1752.2019.09.012

摘要/Abstract

摘要：

【目的】研究酸性电解水（AEW）能否有效控制肉源性荧光假单胞菌,探究处理后菌体变化,为新型杀菌剂的开发提供思路。【方法】使用不同有效氯浓度酸性电解水（20、40和60 mg?L ^-1）将肉源性荧光假单胞菌分别处理5、10、15、20、25和30 min后,通过平板计数检测酸性电解水对肉源性荧光假单胞菌的致死效果;收集未处理及20、40和60 mg?L ^-1酸性电解水各处理5 min后菌液,利用扫描电子显微镜观测酸性电解水对肉源性荧光假单胞菌细胞形态的影响;利用多种荧光探针检测酸性电解水对肉源性荧光假单胞菌细胞膜完整性、胞内pH、胞内ATP浓度、膜电位的影响;提取胞外聚合物（EPS）探究酸性电解水与EPS的相互作用。【结果】20、40和60 mg?L ^-1的酸性电解水均可在5 min内杀死10 ⁷CFU/mL肉源性荧光假单胞菌,酸性电解水浓度大于40 mg·L ^-1时对肉源性荧光假单胞菌的杀菌效果没有显著影响（P>0.05）,10 min处理后均已无活菌检出。扫描电子显微镜结果表明3种浓度酸性电解水的处理均可使肉源性荧光假单胞菌菌体干瘪皱缩,失去原本饱满的细胞形态,60 mg·L ^-1电解水处理下破损程度最大。酸性电解水可显著降低肉源性荧光假单胞菌的膜完整性、胞内pH、膜电位、胞内ATP浓度和EPS含量（P<0.05）。20、40和60 mg·L ^-1酸性电解水处理后菌体膜完整性分别降低到2.26%、1.87%和1.20%,膜电位分别消散40%、50%和50%,胞内pH由7.50分别降低到6.53、5.90和5.83,3种浓度处理后胞内ATP浓度已降至最低。杀菌效果显著依赖于酸性电解水浓度的升高,当处理浓度高于40 mg?L ^-1时,胞内pH、膜电位和胞内ATP浓度的降低无显著差异。酸性电解水处理下EPS中蛋白、总糖含量显著降低,松散胞外聚合物（L-EPS）中蛋白和总糖分别降低71.08%和62.23%,而紧密胞外聚合物（B-EPS）中蛋白和总糖分别降低99.32%和40.62%。当EPS反向添加到酸性电解水中时则可显著降低酸性电解水氧化还原电位（ORP）（P<0.05）,但对有效氯浓度（ACC）和pH没有显著影响（P>0.05）。【结论】酸性电解水对肉源性荧光假单胞菌具有良好的杀菌效果,可破坏肉源性荧光假单胞菌细胞膜,同时EPS对酸性电解水杀菌效果具有一定的阻碍作用。

关键词: 肉源性荧光假单胞菌, 酸性电解水, 杀菌, 致死效应

Abstract:

【Objective】The objective of this study was to investigate whether the acidic electrolyzed water (AEW) could effectively control meat-borne Pseudomonas fluorescens and to investigate the changes induced in bacteria, which was to provide a reference for the development of novel disinfection approaches.【Method】In this paper, the meat-borne P. fluorescens was treated by AEW with available chlorine concentrations of 20, 40, and 60 mg?L ^-1 for 5, 10, 15, 20, 25 and 30 minutes. The lethal effect of AEW on meat-borne P. fluorescens was determined by plate counting, and the characteristic morphology of meat-borne P. fluorescens after 5 min treatment of 20, 40, and 60 mg?L ^-1 AEW was observed under scanning electronic microscopy (SEM) images. In addition, multiple fluorescent probes were used to measure the effects of AEW on cell membrane integrity, intracellular pH, intracellular ATP concentration and membrane potential. The extracellular polymeric substances (EPS) was also extracted to preliminarily investigate the interaction between EPS and AEW. 【Result】 All treatments of AEW with researched concentrations of 20, 40, and 60 mg?L ^-1could reduce the viable cell number of meat-borne P. fluorescens by 7 log CFU/mL in 5 min, and the increasing concentration of AEW above 40 mg?L ^-1 played no significant role in the survival of bacterial cell (P>0.05). No viable cells were detected after 10 min treatment of AEW. SEM observations showed that meat-borne P. fluorescens cells exposed to all concentrations of AEW treatment exhibited surface indentations and cell distortions, which indicated the loss of typical structure. AEW significantly decreased the bacterial membrane integrity, membrane potential, intracellular ATP concentrations, intracellular pH and EPS content (P<0.05). Under treatments of 20, 40 and 60 mg?L ^-1 AEW, the membrane integrity decreased to 2.26%, 1.87% and 1.20%, respectively; the membrane potential declined by 40%, 50% and 50%, respectively; the intracellular pH dropped from 7.50 to 6.53, 5.90 and 5.83, respectively. The intracellular ATP concentration was decreased nearly under detection. The decrease significantly depended on the increasing concentration of AEW, however, when the concentration of AEW was above 40 mg?L ^-1, there were no significant changes in membrane potential, intracellular ATP concentrations and intracellular pH. After treatment with AEW, the contents of protein and carbohydrate in EPS were significantly decreased (P<0.05). The protein and carbohydrate of L-EPS were decreased by 71.08% and 62.23%, respectively, which those of B-EPS were decreased by 99.32% and 40.62%, respectively. When added to AEW, EPS decreased the ORP of AEW significantly (P<0.05), but played no significant role in AEW’s pH and ACC (P>0.05).【Conclusion】AEW had good bactericidal effect against meat-borne P. fluorescens and it impaired the cell membrane, destroying cell homeostasis. EPS played an important role in hindering the lethal effect of AEW on bacteria.

Key words: meat-borne Pseudomonas fluorescens, acidic electrolyzed water, disinfection, lethal effect

蔡林林, 胡海静, 衣晓坤, 王虎虎, 徐幸莲, 彭斌. 酸性电解水对肉源性荧光假单胞菌的致死效应[J]. 中国农业科学, 2019, 52(9): 1614-1623.

CAI LinLin, HU HaiJing, YI XiaoKun, WANG HuHu, XU XingLian, PENG Bin. Lethal Effect of Meat-Borne Pseudomonas fluorescens to Acidic Electrolyzed Water[J]. Scientia Agricultura Sinica, 2019, 52(9): 1614-1623.

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NaCl浓度 NaCl concentration (%)	电流Electricity (A)	有效氯浓度 ACC (mg?L^-1)	氧化还原电位 ORP (mV)	pH
2.0	16	20.33±0.47c	1116.00±2.45c	2.91±0.01a
3.5	18	40.33±0.47b	1152.67±1.70b	2.78±0.00b
4.6	19	61.33±0.47a	1167.67±0.94a	2.69±0.00c

类型 Type	处理 Treatment	蛋白 Protein (μg?mg^-1)	总糖 Carbohydrate (μg?mg^-1)	蛋白/总糖 Protein/Carbohydrate ratio
松散胞外聚合物L-EPS	0	583.68±72.05a	112.33±0.73a	5.20±0.18a
松散胞外聚合物L-EPS	40 mg?L^-1 AEW	168.78±2.96b	42.36±0.14b	3.98±0.08b
紧密胞外聚合物B-EPS	0	59.23±1.28a	19.72±0.66a	3.01±0.15a
紧密胞外聚合物B-EPS	40 mg?L^-1 AEW	0.40±0.28b	11.71±1.23b	0.03±0.02b