阿魏蘑与胶红酵母共培养促进产漆酶的机理研究

In the food industry, processing assisted by laccase has become a mild technology due to its priority. In order to realize the universal and large-scale application of laccase in food industry, great effort has been made to reduce the cost of application by improving the expression level of laccase. In recent years, it is an effective method to enhance laccase production using co-culture technology. However, the promoting mechanism of the co-culture in laccase fermentation is still not revealed. Thus, the best laccase enhancement by the co-culture is difficult to be obtained through heavy work load and more repeated experiments. In this project, laccase production was investigated in the co-culture system of Pleurotus ferulae and Rhodotorula mucilaginosa The stimulator in the co-culture system was verified and the effects of stimulator on hyphae morph ology, cell membrane permeability and laccase gene expression of P. ferulae were also illustrated. The promoting mechanism of the co-culture system was disclosed at the cell and molecular levels by combining the proteomic technology. The obtained results provided the theoretical basis for the screening of the co-culture strain and stimulator and the strategy design of the high-effective and stable co-culture process. It would greatly promote the large-scale application of laccase in food industry with low cost. This project would provide an important theoretical supplement and a valuable theory reference for the future study on more complex co-culture systems in laccase production and the other co-culture systems in food fermentation industry.

在食品工业中，应用漆酶进行加工和处理已成为一种极具优势的温和技术。然而，要实现其在食品工业中更大规模和更广泛的应用，如何提高漆酶发酵水平从而降低应用成本已经成为亟待解决的问题。近年来，利用共培养技术可以有效提高漆酶发酵水平，但其确切的共培养促进机理尚不清楚，导致其研究工作量繁重、重复性大，无法达到最佳的效果。本项目以阿魏蘑和胶红酵母共培养体系为研究对象，在研究共培养促进因子的基础上，研究共培养促进因子对阿魏蘑菌体形态、细胞通透性和漆酶基因表达调控的影响，结合蛋白质组学技术的分析，以期从细胞和分子水平揭示共培养促进漆酶表达的机理。这将为共培养菌株或促进因子的理性筛选，高效稳定的共培养发酵过程控制策略的设计提供良好的理论依据；也为漆酶发酵中更为复杂的共培养体系及食品发酵工业其他相关共培养体系的研究提供理论参考。

在食品工业中，应用漆酶进行加工和处理已成为一种极具优势的温和技术。然而，要实现其在食品工业中更大规模和更广泛的应用，如何提高漆酶发酵水平从而降低应用成本已经成为亟待解决的问题。近年来，利用共培养技术可以有效提高漆酶发酵水平，但其确切的共培养促进机理尚不清楚。本项目以阿魏蘑和胶红酵母共培养体系为研究对象，筛选并发现了胶红酵母的代谢产物β-胡萝卜素是共培养过程中胶红酵母促进阿魏蘑产漆酶的重要促进因子。促进因子使得阿魏蘑菌体表面的粗糙度增大，细胞壁骨架物质β-1,3葡聚糖和几丁质含量减少，细胞壁厚度降低，细胞膜通透性增加。β-胡萝卜素作为异源物质引起了阿魏蘑的应激反应，异源物质降解和能量消耗相关的基因普遍性地上调，而氧化应激相关基因下调，发酵液中活性氧簇（ROS）处于较低水平。在共培养条件下，漆酶同工酶组成比例发生变化，漆酶基因呈现出一部分上调（lacc1，lacc2，lacc12）另一部分下调（lacc4，lacc6，lacc9）的现象。而β-胡萝卜素促进了lacc2及其两个小亚基（sspoxA3a和sspoxA3b）基因转录水平的同时提高，正是共培养促进阿魏蘑漆酶发酵水平提高的重要原因。共培养条件下的上述变化与四个转录因子（cluster-1041.76581、cluster-1041.20044、cluster-1041.40661、cluster-1041.39592）密切相关。本项目明确了阿魏蘑和胶红酵母共培养体系中的重要促进因子，并从细胞和分子水平揭示了共培养促进漆酶表达的机理。这为漆酶共培养菌株对的理性筛选，以及高效稳定的共培养发酵过程控制策略的设计提供了良好的理论依据；也为漆酶发酵中更为复杂的共培养体系及食品发酵工业其他相关共培养体系的研究提供理论参考。