海洋弧菌(Vibrio sp.)LA-05发酵液粗提物的群体感应抑制活性及其发酵条件优化

Quorum sensing inhibitory activity and fermentation condition optimization of crude extract of the marine bacterial strain Vibrio sp. LA-05

摘要:

【背景】海洋微生物代谢产物被认为是发掘新型天然活性物质的潜在来源，其中不可避免地包含新型群体感应抑制剂(quorum sensing inhibitors, QSIs)。响应面法是运用多元二次回归模型拟合各因素和响应值之间的函数关系，对各因素及其交互作用进行评价，可有效地从影响代谢产物发酵条件的各因素中预测出最佳条件。【目的】以紫色杆菌(Chromobacterium violaceum) ATCC 12472^T为指示菌株，研究海洋弧菌(Vibrio sp.) LA-05发酵液粗提物的群体感应抑制活性。在单因素试验基础上，应用响应面法对菌株发酵条件进行优化，以提高其发酵代谢产物中活性物质的产量。【方法】使用琼脂平板扩散法进行初筛后，通过分析发酵液粗提物对紫色杆菌生长曲线及紫色素产量的影响研究其群体感应抑制活性。在单因素试验基础上，采用Box-Behnken设计原则，以紫色素抑制率为响应值，通过响应面试验优化菌株发酵条件，从而提高其发酵液中活性物质的产量。【结果】在基本不影响紫色杆菌正常生长的浓度范围内，粗提物可不同程度地抑制紫色素的产生。模型预测结果表明，最优发酵条件为发酵时间37.59 h、发酵温度19.28 ℃、摇床转速154.21 r/min、接种量2.37%，紫色素抑制率预测值为53.38%。经试验验证，紫色素抑制率为52.85%±0.64%，与模型理论预测值相近，表明该回归模型与实际实验结果拟合程度较高，模型可信度好，具有可行性。【结论】海洋弧菌(Vibriosp.) LA-05发酵液粗提物具有群体感应抑制活性。本实验通过响应面法获得了该菌株的最优发酵条件，这为后续新型群体感应抑制剂的研究奠定了基础。

Abstract:

[Background] The metabolites of marine microorganisms are considered ideal sources for discovering natural active substances, which inevitably include novel quorum sensing inhibitors (QSIs). Response surface method uses multivariate quadratic regression model to fit the relationship between each factor and response value and evaluate the effects of factors and their interaction, which can effectively screen out the best conditions of the factors affecting the microbial fermentation for metabolite production. [Objective] The quorum sensing inhibitory activity of the crude extract of the marine bacterial strain Vibrio sp. LA-05 was investigated with Chromobacterium violaceum ATCC 12472^T as the indicator strain. To improve the production of active substances in the crude extract, we employed response surface method to optimize the fermentation conditions of the strain on the basis of single factor experiments. [Methods] After preliminarily screening by the agar plate diffusion method, the growth curves and violacein production of C. violaceum ATCC 12472^T were employed to evaluate the quorum sensing inhibitory activity of the crude extract. On the basis of single factor experiments, fermentation conditions were optimized by Box-Behnken design and response surface method with violacein inhibition rate as a response value to improve the production of active substances in the crude extract. [Results] Within the concentration range that did not affect the growth of C. violaceum ATCC 12472^T, the crude extract inhibited the production of violacein to various degrees. The fermentation conditions were optimized as fermentation with the inoculum amount of 2.37% at 19.28 ℃ and 154.21 r/min for 37.59 h, the extract obtained under which had the expected inhibition rate of 53.38%. The verification experiment showed that the inhibition rate of violacein was 52.85%±0.64%, close to the model predicted value. This result indicated that the regression model had a good fitting degree with the experimental results, being reliable and feasible. [Conclusion] The crude extract of the marine bacterial strain Vibrio sp. LA-05 possesses quorum sensing inhibitory activity. The fermentation conditions of this strain are optimized by response surface method, which will lay a foundation for the research and development of novel QSIs.