本文選題：高山被孢霉 + 花生四烯酸��； 參考：《中國科學(xué)技術(shù)大學(xué)》2017年碩士論文

【摘要】：花生四烯酸(arachidonic acid,簡稱AA或ARA)在人體許多生理過程中起重要作用。作為一種重要的功能性油脂,在食品、藥品、保健品和化妝品等領(lǐng)域的需求正逐步增長,市場前景繼續(xù)呈擴(kuò)大趨勢(shì)。利用高山被孢霉(Mortierella alpina)發(fā)酵生產(chǎn)ARA因其ARA含量高、油脂組成合理等特點(diǎn)一直作為工業(yè)生產(chǎn)的主要方式。M.alpina是《食品安全國家標(biāo)準(zhǔn)食品營養(yǎng)強(qiáng)化劑使用標(biāo)準(zhǔn)》(GB 14880—2012)中生產(chǎn)ARA的指定菌種,對(duì)其菌種進(jìn)行選育是提高ARA產(chǎn)量與品質(zhì)的關(guān)鍵因素之一。Malpi.的油脂組成直接影響到產(chǎn)品質(zhì)量,在實(shí)際生產(chǎn)過程中發(fā)現(xiàn)當(dāng)二十四碳烷酸(24C,本蠟酸)在總油脂中含量超過8%時(shí),終產(chǎn)品油脂在低溫冷藏時(shí)析出白色沉淀物。因此,本課題利用自然雜交的育種方法對(duì)低24C菌種進(jìn)行了篩選。將24C及ARA含量均較低的一株野生菌YS-88447與生產(chǎn)用菌株G5進(jìn)行自然雜交育種,篩選獲得一株ZJ-3菌株,其24C含量為3.39%,對(duì)比生產(chǎn)菌株(24C含量為9.16%)降低了 170%;其ARA產(chǎn)量為5.21 g/L,對(duì)比生產(chǎn)菌株(ARA含量為4.86 g/L)提高了 6.72%。本課題探索并建立了 Fe2+與磁場共同作用誘變M alpina的方法,對(duì)G5進(jìn)行誘變后篩選得到一株油脂含量達(dá)44.85%的高產(chǎn)菌株C16,其ARA產(chǎn)量為6.43g/L,24C含量為8.98%。為快速獲得高油脂含量、低24C的ARA高產(chǎn)菌株,加速育種過程,本課題利用原生質(zhì)體融合技術(shù)定向篩選優(yōu)良性狀相結(jié)合的菌株。將上述獲得的優(yōu)良性狀菌株ZJ-3和C16進(jìn)行原生質(zhì)體融合,對(duì)原生質(zhì)體制備及再生過程中相關(guān)因素進(jìn)行研究,確定了最佳條件。為提高融合子篩選效率和準(zhǔn)確率,本課題首次嘗試使用流式細(xì)胞分選法并結(jié)合雙滅活兩種方法取得了良好的效果。經(jīng)過對(duì)融合子再生及發(fā)酵培養(yǎng),篩選獲得菌株YR-2,其油脂含量達(dá)43.18%,24C含量為3.26%,ARA產(chǎn)量達(dá)6.32 g/L,并具有良好的遺傳穩(wěn)定性,達(dá)到預(yù)期目標(biāo)。本研究為高山被孢霉菌種選育提供了新思路,可促進(jìn)ARA生產(chǎn)的產(chǎn)能升級(jí)與產(chǎn)品品質(zhì)提升。一直以來,M alpina研究及發(fā)酵生產(chǎn)所用氮源原料均為天然培養(yǎng)基(含酵母粉),其成分復(fù)雜、不同批次易存在較大差異,不利于精細(xì)實(shí)驗(yàn)分析以及工業(yè)生產(chǎn)的穩(wěn)定。因此,使用無機(jī)氮源對(duì)其進(jìn)行替換實(shí)現(xiàn)全合成培養(yǎng)基發(fā)酵具有現(xiàn)實(shí)意義。本課題通過分類及單因素缺失實(shí)驗(yàn)確定了氨基酸混合物、無機(jī)鹽以及生長因子的添加量,初步確定了M.apina全合成培養(yǎng)基成分。雖目前獲得的ARA產(chǎn)量為天然培養(yǎng)基發(fā)酵的70%,但為研究高山被孢霉生長、代謝、遺傳育種等提供了有力的支持。將得到的最適氨基酸組合、維生素濃度和無機(jī)鹽組分補(bǔ)充進(jìn)天然發(fā)酵培養(yǎng)基,實(shí)驗(yàn)組較對(duì)照組ARA產(chǎn)量提高14.7%,說明氨基酸、維生素和無機(jī)鹽對(duì)M.alpina發(fā)酵具有促進(jìn)作用,為工業(yè)生產(chǎn)培養(yǎng)基的優(yōu)化提供了數(shù)據(jù)支撐。
[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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q93

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