高山被孢霉原生质体融合及全合成培养基研究

发布时间：2018-07-01 12:59

本文选题：高山被孢霉 + 花生四烯酸　；参考：《中国科学技术大学》2017年硕士论文

【摘要】：花生四烯酸(arachidonic acid,简称AA或ARA)在人体许多生理过程中起重要作用。作为一种重要的功能性油脂,在食品、药品、保健品和化妆品等领域的需求正逐步增长,市场前景继续呈扩大趋势。利用高山被孢霉(Mortierella alpina)发酵生产ARA因其ARA含量高、油脂组成合理等特点一直作为工业生产的主要方式。M.alpina是《食品安全国家标准食品营养强化剂使用标准》(GB 14880—2012)中生产ARA的指定菌种,对其菌种进行选育是提高ARA产量与品质的关键因素之一。Malpi.的油脂组成直接影响到产品质量,在实际生产过程中发现当二十四碳烷酸(24C,本蜡酸)在总油脂中含量超过8%时,终产品油脂在低温冷藏时析出白色沉淀物。因此,本课题利用自然杂交的育种方法对低24C菌种进行了筛选。将24C及ARA含量均较低的一株野生菌YS-88447与生产用菌株G5进行自然杂交育种,筛选获得一株ZJ-3菌株,其24C含量为3.39%,对比生产菌株(24C含量为9.16%)降低了 170%;其ARA产量为5.21 g/L,对比生产菌株(ARA含量为4.86 g/L)提高了 6.72%。本课题探索并建立了 Fe2+与磁场共同作用诱变M alpina的方法,对G5进行诱变后筛选得到一株油脂含量达44.85%的高产菌株C16,其ARA产量为6.43g/L,24C含量为8.98%。为快速获得高油脂含量、低24C的ARA高产菌株,加速育种过程,本课题利用原生质体融合技术定向筛选优良性状相结合的菌株。将上述获得的优良性状菌株ZJ-3和C16进行原生质体融合,对原生质体制备及再生过程中相关因素进行研究,确定了最佳条件。为提高融合子筛选效率和准确率,本课题首次尝试使用流式细胞分选法并结合双灭活两种方法取得了良好的效果。经过对融合子再生及发酵培养,筛选获得菌株YR-2,其油脂含量达43.18%,24C含量为3.26%,ARA产量达6.32 g/L,并具有良好的遗传稳定性,达到预期目标。本研究为高山被孢霉菌种选育提供了新思路,可促进ARA生产的产能升级与产品品质提升。一直以来,M alpina研究及发酵生产所用氮源原料均为天然培养基(含酵母粉),其成分复杂、不同批次易存在较大差异,不利于精细实验分析以及工业生产的稳定。因此,使用无机氮源对其进行替换实现全合成培养基发酵具有现实意义。本课题通过分类及单因素缺失实验确定了氨基酸混合物、无机盐以及生长因子的添加量,初步确定了M.apina全合成培养基成分。虽目前获得的ARA产量为天然培养基发酵的70%,但为研究高山被孢霉生长、代谢、遗传育种等提供了有力的支持。将得到的最适氨基酸组合、维生素浓度和无机盐组分补充进天然发酵培养基,实验组较对照组ARA产量提高14.7%,说明氨基酸、维生素和无机盐对M.alpina发酵具有促进作用,为工业生产培养基的优化提供了数据支撑。
[Abstract]:Arachidonic acid (AA) or arachidonic acid (AA) plays an important role in many physiological processes. As an important functional oil, the demand for food, medicine, health products and cosmetics is increasing gradually, and the market prospect continues to expand. The fermentation of Mortierella alpina was used to produce ARA because of its high content. The rational composition of oils and fats has been the main way of industrial production. M.alpina is the designated strain of ARA production in the National Food Safety Standard for the use of Food Nutrition fortifiers (GB 14880-2012). The breeding of its strain is one of the key factors to improve the yield and quality of ARA. It is found that when the content of 24 carboalkanoic acid (24C) in the total oil exceeds 8%, the final product oil precipitates white precipitate at low temperature and cold storage. Therefore, the breeding method of natural hybridization was used to screen the low 24 C strain. A wild strain YS-88447 with low content of 24C and ARA was bred with production strain G5, and a strain of ZJ-3 was screened out. The yield of ARA was 5.21 g / L and that of producing strain (ARA was 4.86 g / L) was increased by 6.72%, compared with that of producing strain (4.86 g / L), the yield of ARA was 5.21 g 路L ~ (-1) and the content of ARA was 4.86 g / L, compared with that of producing strain (9.16%). In this paper, the method of mutagenic M alpina by the interaction of Fe 2 and magnetic field was explored and established. After mutagenesis, a high yield strain C16 with oil content of 44.85% was obtained, and the yield of C16 was 6.43 g / L ~ (24) C = 8.98%. In order to obtain high-yield ARA strains with high oil content and low 24C, and accelerate breeding process, protoplast fusion technique was used to screen the strains with high oil content and low 24C. The protoplasts of ZJ-3 and C16 were fused and the relative factors during protoplast preparation and regeneration were studied and the optimum conditions were determined. In order to improve the efficiency and accuracy of fusion screening, flow cytometric sorting method and dual inactivation method were used to obtain good results. After regeneration and fermentation of the fusion, the strain YR-2 was obtained. The oil content of YR-2 was 43.18 and the content of 24C was 3.26. The yield of ARA was 6.32 g / L, and the strain had good genetic stability and reached the expected goal. This study provides a new idea for the breeding of P. alpine, which can promote the production capacity upgrade and product quality improvement of ARA production. The nitrogen sources used in the study and fermentation of M alpina have always been natural medium (containing yeast powder), which is complex in composition and easy to differ in different batches, which is not conducive to fine experimental analysis and stability of industrial production. Therefore, it is of practical significance to use inorganic nitrogen sources to replace them to realize the fermentation of full synthetic medium. The contents of amino acid mixture, inorganic salt and growth factor were determined by classification and single factor deletion experiment. The composition of M. apina total synthetic medium was preliminarily determined. Although the yield of ARA obtained at present is 70% of that of natural medium, it provides a powerful support for the study of the growth, metabolism, genetics and breeding of P. alpine. The optimum combination of amino acids, vitamin concentration and inorganic salt components were added into natural fermentation medium, and the production of ARA in the experimental group was increased by 14.7than that in the control group, indicating that amino acids, vitamins and inorganic salts could promote the fermentation of M.alpina. It provides data support for the optimization of industrial production medium.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q93

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