胞内红曲色素渗透萃取发酵的机理探索

Submerged cultivation of Monascus in a nonionic surfactant micelle aqueous solution for production of Monascus pigments is exploited, in which the active growth of fungus Monascus and the "milking" intracellular pigments into the extracellular broth occur simultaneously. This "milking processing" of intracellular pigment consists of permeabilization of intracellular pigments across the cell membrane and extraction of the pigments into nonionic surfactant micelles, which is a perstraction process. The advantage of perstractive fermentation of intracellular product includes the application of conventional submerged cultivation by secretion of the intracellular pigments to its extracellular broth and the benefit of extractive microbial fermentation by solubilizating the pigments into nonionic surfactant micelles. The effect of nonionic surfactant on the permeability of cell membrane and the biocompatibility of microorganism in the nonionic surfactant micelle aqueous solution are the keys of perstractive fermentation. The solubilization of pigments into the nonionic surfactant micelles also intensifies this perstractive fermentation. Both of them should be studied extensively in the present work. The intracellular pigments of Monascus fermentation includes red pigmens, organe pigemnts, and yellow pigments.Simultaneously,the utilization of perstractive fermentation for regulation of the secondary metabolism should also be studied based on the selectvity of perstraction process.

在非离子表面活性剂胶束溶液中液态发酵红曲色素的过程中同时实现了维持微生物生长和将胞内红曲色素像"挤奶"一样挤到胞外发酵液。"挤奶"加工包括了胞内色素渗透通过细胞膜和渗透到胞外的色素被表面活性剂胶束萃取这两个连续的过程，这一发酵技术本质上是渗透萃取发酵。胞内产物的渗透萃取发酵可以采用常规胞外产物的液态发酵方式进行且同时拥有萃取微生物发酵的优点。微生物在非离子表面活性剂胶束溶液中的生物相容性和非离子表面活性剂改变细胞膜的渗透性是实现渗透萃取发酵的前提条件。本立题将重点研究非离子表面活性剂对细胞膜渗透性的影响和微生物在表面活性剂胶束溶液中生物相容性的基本规律，探索表面活性剂胶束溶液对渗透到细胞外产物的增溶作用对渗透萃取发酵的强化作用。红曲霉菌发酵的胞内产物包括红色素、橙色素和黄色素等组分。基于渗透萃取过程的选择性，进一步探索应用渗透萃取发酵技术调控复杂真菌次级产物代谢的基本策略。

微生物发酵产生胞内产物常受到产物抑制/降解的限制。以红曲霉菌发酵胞内红曲橙色素为例，研究发现胞内红曲橙色素的积累与微生物的油滴含量成正相关。油滴也许充当着红曲橙色素的储存库，起到解除产物抑制和排除产物进一步降解的作用。在非离子表面活性剂胶束溶液中，红曲霉菌能维持较高的生物活性，如较高消耗葡萄糖的速率、生物合成红曲橙色素等。同时，胞内红曲橙色素在发酵过程中被选择性地转移到细胞外环境。因此，在表面活性剂胶束溶液中实现了红曲橙色素的萃取发酵。. 萃取发酵将胞内红曲橙色素成功地转移到细胞外环境。细胞外环境中的表面活性剂胶束结构模拟了胞内油滴，具有解除红曲橙色素的抑制与降解的功能。因此，应用萃取发酵可以成功实现：1）静息细胞培养红曲橙色素，提高生物合成的速率；2）操控微生物的次级代谢，如改变了红曲橙色素与红曲黄色素的比例；3）将胞内红曲橙色素转移到细胞外环境，使红曲橙色素在细胞外环境中进一步化学修饰成为可能。特别地，化学反应对接微生物次级代谢产物能高选择性地解除产物抑制和排除产物降解。同时，该方法还提供了合成“非天然”产物的新途径。. 回收表面活性剂胶束溶液中的产物是萃取发酵生产红曲色素不可缺少的步骤。水不溶性离子液体能替代有机溶剂在表面活性剂胶束溶液中形成Winsor I微乳液。应用这一新型微乳液成功地应用于从非离子表面活性剂胶束溶液中回收红曲色素，如获得了红曲黄色素的晶体。同时，测定水溶性离子液体-非离子表面活性剂-水三元系统的相图，发现该三元系统中存在一个不溶性的“小岛”。在该小岛区域形成了离子液体-非离子表面活性剂双水相系统。这一新型双水相系统也具有从非离子表面活性剂胶束溶液中回收有机化合物的应用潜力。