超临界CO2与姜精油协同对副溶血弧菌杀菌效果的影响与机制研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-1207

摘要/Abstract

摘要：

优化超临界CO₂低压、低温杀菌技术，降低细菌群体感应对杀菌效果的影响。采用液质联用法和苯酚硫酸法分别测定副溶血弧菌群体感应信号分子和胞外多糖的含量，采用微量扩散法测定所选群体感应抑制剂姜精油的群体感应抑制活性，优化联合超临界CO₂杀菌工艺。随着菌落总数的增加，群体感应信号分子和胞外多糖在菌悬液中积累，激活相关基因的表达从而增强菌体感应导致超临界CO₂杀菌效果下降。姜精油可作为群体感应抑制剂，其最小抑制浓度为200 μg/mL，且在亚抑菌浓度下可以显著抑制副溶血弧菌的泳动性、生物膜形成（抑制率为64.20%）及菌悬液中的胞外多糖产生（抑制率为45.1%）。联合杀菌的最优工艺为群体感应抑制剂浓度75 μg/mL，杀菌条件为15 MPa、40℃、20 min。姜精油可作为群体感应抑制剂应用于超临界CO₂杀菌可以大幅度提升其杀菌效果，为群体感应抑制剂的应用提供理论基础。

关键词: 副溶血弧菌, 群体感应, 超临界CO₂杀菌, 姜精油

Abstract:

The objective is to optimize the low pressure and low temperature sterilization technology of supercritical CO₂ and to reduce the influence of bacterial quorum sensing on the sterilization effect. The LC-MS and phenol sulfuric acid were used to determine the contents of quorum sensing signal molecules and exopolysaccharides of Vibrio parahaemolyticus, respectively. The micro diffusion method was applied to determine the quorum sensing inhibitory activity of selected quorum sensing inhibitor ginger essential oil, and the combined supercritical CO₂ sterilization process was optimized. The quorum sensing signal molecules and exopolysaccharides accumulated in the bacterial suspension with the increase of the total number of colonies, activating the expression of related genes, thereby enhancing cell sensing, leading to the decline of the bactericidal effect of supercritical CO₂. Ginger essential oil can be used as quorum sensing inhibitor, and the minimum inhibitory concentration was 200 μg/mL, and it significantly inhibited the mobility of Vibrio parahaemolyticus, biofilm formation（64.20% of inhibition rate）and the production of exopolysaccharides in bacterial suspension（45.1% of inhibition rate）at the sub inhibitory concentration. The optimal process of quorum sensing inhibitor combined with supercritical CO₂ sterilization is quorum sensing inhibitor concentration 75 μg/mL, and the sterilization conditions are 15 MPa, 40℃, and 20 min. In conclusion, ginger essential oil can be used as a quorum sensing inhibitor to enhance the effect of supercritical CO₂ sterilization, providing a theoretical basis for the application of quorum sensing inhibitors.

Key words: Vibrio parahaemolyticus, quorum sensing, supercritical CO₂ sterilization, ginger essential oil

黄亚宁, 张海娇, 韩玉谦, 刘尊英. 超临界CO₂与姜精油协同对副溶血弧菌杀菌效果的影响与机制研究[J]. 生物技术通报, 2023, 39(5): 297-305.

HUANG Ya-ning, ZHANG Hai-jiao, HAN Yu-qian, LIU Zun-ying. Effects of Supercritical CO₂ Combined with Ginger Essential Oil on the Sterilization of Vibrio parahaemolyticus and Its Mechanism[J]. Biotechnology Bulletin, 2023, 39(5): 297-305.

图/表 10

表1 四因素三水平正交优化表

Table 1 Four-factor three-level orthogonal optimization table

水平 Level	因素Factor
水平 Level	压力 Pressure/MPa	温度 Temperature/℃	时间 Time/min	QSI浓度 QSI concentration/ （μg·mL^-1）
1	10	35	10	25
2	15	40	20	50
3	20	45	30	75

图1 菌液生长曲线及信号分子含量的变化相同字母表示处理之间在统计学上无显著性差异（P>0.05），反之则说明有显著性差异（P<0.05）。下同

Fig. 1 Growth curve of bacterial solution and content variation of signal molecules The same letter indicates no statistically significant difference between the treatments（P> 0.05）, or otherwise, there is a significant difference（P <0.05）. The same below

图2 信号分子与胞外多糖含量与初始菌落浓度的关系

Fig. 2 Relationship between signal molecules, extracellular polysaccharide content and initial colony concentra-tion

图3 超临界CO2杀菌效果与初始菌落密度的关系

Fig. 3 Relationship between sterilization effect of superc-ritical CO2 and initial colony density

图4 姜精油对报告菌株紫色杆菌的群体感应抑制活性对照：不添加姜精油。A：25 μg/mL；B：50 μg/mL；C：75 μg/mL；D：100 μg/mL

Fig. 4 Quorum sensing inhibitory activity of ginger esse-ntial oil against C. violaceum Control: No ginger essential oil. A: 25 μg/mL; B: 50 μg/mL; C: 75 μg/mL; D: 100 μg/mL

图5 姜精油对副溶血弧菌泳动性的影响 A：对照；B：50 μg/mL；C：100 μg/mL

Fig. 5 Effect of ginger essential oil on the mobility of V. parahaemolyticus A: Control; B: 50 μg/mL; C: 100 μg/mL

图6 姜精油对副溶血弧菌生物膜形成量的影响

Fig. 6 Effect of ginger essential oil on biofilm formation of V. parahaemolyticus

图7 姜精油对胞外多糖含量的影响

Fig. 7 Effect of ginger essential oil on extracellular polysa-ccharide content of V. parahaemolyticus suspension

表2 四因素三水平正交优化设计及结果

Table 2 Four-factor three-level orthogonal optimization design and results

实验序号 Experiment No.	因素Factor				杀菌对数值 Sterilization logarithmic value	FDA规定的最低程度 Minimum required by FDA
实验序号 Experiment No.	压力Pressure/ MPa	温度Tempe-rature/℃	时间 Time/min	QSI浓度 QSI concentration/（μg·mL^-1）	杀菌对数值 Sterilization logarithmic value	FDA规定的最低程度 Minimum required by FDA
1	1	1	1	1	4.58	-1.42
2	1	2	2	2	5.77	-0.23
3	1	3	3	3	7.40	+1.40
4	2	1	2	3	7.43	+1.43
5	2	2	3	1	5.93	-0.07
6	2	3	1	2	5.70	-0.30
7	3	1	3	2	6.63	+0.63
8	3	2	1	3	7.46	+1.46
9	3	3	2	1	5.95	-0.05
K1	5.917	5.880	5.913	5.487
K2	6.353	6.387	6.383	5.700
K3	6.347	6.350	6.320	7.430
R	0.436	0.507	0.470	1.943
主次顺序Primary and secondary order 最优组合Optimal combination			D>A≈B≈C A₂B₂C₂D₃

表3 杀菌对数值的方差分析

Table 3 Analysis of variance of sterilization logarithm

方差来源 Source of variance	偏差平方和 Sum of squares of deviations	自由度 Freedom	F值 F value	显著性 Significance
压力Pressure	0.373	2	0.834
温度Temperature	0.479 0.390	2 2	1.061 0.865
时间Time	6.815	2	15.084	*
QSI浓度QSI concentration	8.06	8
误差Error

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超临界CO₂与姜精油协同对副溶血弧菌杀菌效果的影响与机制研究

Effects of Supercritical CO₂ Combined with Ginger Essential Oil on the Sterilization of Vibrio parahaemolyticus and Its Mechanism

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