【作者】 王博；

【导师】 吴振强； 王志龙；

【作者基本信息】 华南理工大学 ， 发酵工程， 2016， 硕士

【摘要】 红曲色素作为食用色素在中国已有上千年使用历史,红曲霉菌发酵产红曲色素也被广泛地研究,但是关于红曲霉菌油脂的报道却并不多见。现有的研究指出红曲霉菌所产油脂不仅可作为生物柴油的油源,同时也具有降血脂等生理功能。因此对红曲油脂的研究具有重要意义。红曲霉中红曲色素与油脂的合成均以乙酰CoA为前体物质,分别在聚酮酶、脂肪酸合成酶的作用下生成。目前对于红曲霉菌油脂含量,及油脂与色素合成的关联性知之甚少。本研究设计在一系列pH梯度、谷氨酸钠(MSG)浓度梯度、Triton X-100浓度梯度下进行红曲霉菌常规液态发酵及萃取发酵实验,并考察红曲色素与油脂的积累情况,进而探究二者生物合成的关联性。结果表明红曲霉菌胞内色素与油脂的积累具有同步性,低pH、低氮的培养条件有利于二者同时积累,油脂含量最高可达菌体干重的50%。与常规发酵相比,高Triton X-100浓度下的萃取发酵获得的红曲色素浓度更高且主要集中在胞外(约为40 AU),而油脂含量却有所降低;并且在萃取发酵过程中,非离子表面活性剂Triton X-100有选择性地将红曲霉菌胞内色素萃取到胞外基质中,而油脂依然留在胞内。综上表明萃取发酵在一定程度上使代谢流从油脂合成方向转向色素的合成。生长细胞的液态深层培养是获得红曲橙色素的常规方式,在该过程中色素的积累与菌体的生长表现出一定的关联性;在本研究中采用萃取发酵的方式得到一定量的低胞内色素含量的菌丝体,并以该菌丝体作为静息培养的生物催化剂,成功地实现了红曲霉菌静息培养产红曲橙色素。在此过程中红曲霉菌保持一定的代谢活力但菌体并无生长,同时红曲色素、油脂都明显增加,符合静息细胞的特征。因此借助于非离子表面活性剂胶束溶液中的萃取发酵,可有效地建立起红曲霉菌静息细胞培养系统。红曲霉菌的静息细胞培养与常规方式相比具有如下优势:大大简化了培养操作(无需无菌),易实现高密度培养(24 g/L DCW),提高了红曲色素的产率(14 AU/d)及对葡萄糖转化率(1 AU/g),而且红曲霉菌静息细胞可被循环利用,循环三次后其色素合成能力依然保持稳定。更多还原

【Abstract】 Few studies of the FA profile of the genus Monscus have been published.C18:1,C18:2,C16:0,C18:0 and C16:1 were identified as the major acids.And some polyunsaturated fatty acids,such as arachidonic acid(ARA),eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA),were also found among the fatty acids.The same precursor acetyl CoA is utilized for biosynthesis of both Monascus pigments(catalyzed by polyketide)and fatty acids(catalyzed by fatty acid synthase).However,the relationship of Monascus pigments and lipid accumulation have never been discussed,as well as the lipid content of Monascus.In this work,a series of submerged fermentation of Monascus were conducted in the medium of different concentration of MSG,Triton X-100 and different pH.The results indicted that the limited nitrogen concentration and/or low initial pH 2.5 conditions favored the production of intracellular orange pigments and accumulation of microbial lipid(high content reaching to approximately 50% in dry cell weight)by Monascus anka.Interestingly,the content of pigments and the weight of lipid exhibited a positive correlated relationship.Extractive fermentation in nonionic surfactant micelle aqueous solution selectively exported the intracellular Monascus pigments into its extracellular broth,in which the concentration of intracellular pigments was negligible while the extracellular one was enhanced.The extractive fermentation provides a novel strategy for shifting of the metabolic channeling from intracellular lipid accumulation to Monascus pigment production.High pigment concentration,i.e.,approximately 40 AU of extracellular Monascus pigments,was achieved by extractive fermentation at relatively high nonionic surfactant concentration 10 g/l.This phenomenon might be attributed to the nonionic surfactant micelles acting as pigment reservoir by biomimetic of intracellular lipids.Growing cell submerged culture is usually applied for fermentation of intracellular orange Monascus pigments,in which accumulation of Monascus pigments is at least partially associated to cell growth.In the present work,extractive fermentation in a nonionic surfactant micelle aqueous solution was utilized as a strategy for releasing of intracellular Monascus pigments.Those mycelia with low content of intracellular Monascus pigments were utilized as biocatalyst in resting cell submerged culture.By this means,resting cell submerged culture for production of orange Monascus pigments was carried out successfully in the nonionic surfactant micelle aqueous solution,which exhibited some advantages comparing with the corresponding conventional growing cell submerged culture,such as non-sterilization operation,high cell density(24 g/l DCW)leading to high productivity(14 AU of orange Monascus pigments at 470 nm per day),and recycling of cells as biocatalyst leading to high product yield(approximately 1 AU of orange Monascus pigments at 470 nm per gram of glucose)based on energy metabolism.更多还原