发酵法生产新型营养强化剂--花生四烯酸

本文研究了温度、PH、培养时间以及碳源、氮源等因素对高山被孢霉菌I49-N18的细胞生长、产油脂和花生四烯酸的影响,确定了该菌株发酵生产花生四烯酸的最佳培养基和最佳培养条件,使得其干菌体重（DW）可达25.9g/l,总脂（TL/DW）含量达36.6％,花生四烯酸（AA/TL）含量达71.0％。     

发酵性丝孢酵母发酵产油脂的碳源和氮源选择

以发酵性丝孢酵母(Trichosporon fermentans)为实验菌株,通过液体摇瓶发酵研究了碳源和氮源种类对其生长及细胞积累油脂的影响。结果表明:发酵性丝孢酵母以葡萄糖、乳糖、蔗糖及可溶性淀粉作为碳源,以牛肉膏、蛋白胨作为氮源发酵时,菌体生物量和细胞油脂含量均在发酵72 h时达到最大值,继续延长发酵时间不利于油脂积累。以硫酸铵为氮源,木糖为碳源时,菌体最大生长和细胞油脂最大积累不同步。发酵性丝孢酵母发酵产油脂宜选择硫酸铵作为氮源,葡萄糖作为碳源。     

不同前体物质的添加对高山被孢霉生长及脂肪酸组分的影响

通过添加植物油及吐温作为高山被孢霉合成油脂的前体物质,分别考察了不同前体物质的添加对菌体的生物量、油脂含量、油脂产量及所得油脂中各脂肪酸组分的影响。实验结果显示:添加植物油作为前体不但可以提高菌体中油脂含量,而且会使油脂中花生四烯酸的产量得到大幅度提高,其中加入菜籽油、亚麻籽油及大豆油为前体时,花生四烯酸的产量分别达到了2 063.2、2 062.8 mg/L及2 192.9 mg/L,比对照组提高了112.72%、112.68%及126.10%;而以吐温80为前体时,虽然菌体的油脂产量有所降低,但油脂中花生四烯酸的产量比对照组提高了79.91%,达到了1 744.9 mg/L。     

真菌发酵法产油脂的研究

本文以深黄被孢霉(Mortierellaisabellina)为试验菌株,采用发酵法生产油脂。采用单因素试验设计,研究了产脂培养基中碳源、氮源种类及其浓度对菌体生长和油脂积累的影响。试验确定酶法水解的玉米淀粉水解糖为最佳碳源,其最适添加浓度为100g/L;酵母膏为最适氮源,其最适添加浓度为3g/L。优化培养基条件下获得菌体生物量为12.350g/L,油脂含量为52.19%,油脂产量为6.4376g/L。但还有待于进一步优化培养基中的其它成分,以有效提高菌体油脂产量。     

利用被孢霉发酵生产花生四烯酸工艺配方优化研究

利用高山被孢霉Mortierella alpina I49-N18发酵生产花生四烯酸。优化筛选出最适于高山被孢霉斜面培养、种子摇瓶培养以及摇瓶发酵培养的配方,稳定的培养基配方为后续发酵生产提供保障。通过培养基配方单因子试验分别对碳源、氮源、无机盐、氨基酸、植物油等原料的合适添加量进行初步确定,然后设计正交实验进行验证。考察初始葡萄糖浓度在高山被孢霉发酵生产花生四烯酸过程中对脂肪酸组分的影响,分析脂肪酸主要组分在发酵过程中的变化趋势。考察结果显示发酵培养基中补充添加800 1000mg/kg磷酸盐,添加600 1000mg/kg谷氨酸钠盐、添加0.04%0.12%植物油可以有效提高发酵生产花生四烯酸的产量。通过发酵工艺放大,在200m3发酵罐规模的发酵生产中得到应用,发酵产量得到大幅度提升,培养7d发酵生产花生四烯酸(ARA)的产量达到10g/L以上。     

高山被孢霉发酵生产多不饱和脂肪酸的初步研究

高山被孢霉具有很强的脂肪合成能力,并可积累多种多不饱和脂肪酸,为进一步提高高山被孢霉多不饱和脂肪酸产量,对碳源、氮源等对高山被孢霉菌体生长和多不饱和脂肪酸（PUFA）生产的影响进行分析。研究结果表明：甘油可以作为葡萄糖的替代物用于高山被孢霉工业化发酵生产多不饱和脂肪酸;当脱脂豆粉与硝酸钾的比例为2∶1时,此时菌体形态最利于脂肪酸生产,脂肪酸和花生四烯酸的产量分别为11.20g/L和5.89g/L,花生四烯酸的产量提高了4.0倍。     

响应面法优化深黄被孢霉发酵生产花生四烯酸的培养基条件

为了降低深黄被孢霉YZ-124生产花生四烯酸的成本,研究了不同添加量的玉米黄浆水对发酵的影响,与葡萄糖培养基相比,在发酵培养基中添加一定量的玉米黄浆水对发酵产量无显著影响.在单因素实验的基础上,利用Design Expert设计了响应面实验,研究了葡萄糖浓度、不同添加量的玉米黄浆水和初始pH对花生四烯酸产量的影响.结果表明,最佳的培养基条件是葡萄糖浓度为90g/L、添加体积分数为25％的玉米黄浆水、初始pH6时,花生四烯酸(ARA)产量达到最大,为3.11g/L.     

主成分分析法在花生四烯酸高产菌株代谢组学研究中的应用

利用主成分分析法对于三株花生四烯酸高产高山被孢霉菌株的GC-MS谱图信息进行了分析,以菌株间通含的18种代谢组分为原始指标,提取出两个主成分变量,在二元主成分平面图上实现了对三个具有不同性状菌株的良好区分,同时发现主成分1可能为指向花生四烯酸积累的主成分指标,主成分2可能为指向菌体生长的主成分指标.     

Yarrowia lipolytica H222（pox1-6缺陷）利用苹果渣酶解液与葡萄糖发酵合成微生物油脂的对比研究

分别以苹果渣酶解液和葡萄糖作为发酵培养基的碳源,以Yarrowia lipolytica H222(pox1-6缺陷)为实验菌株合成微生物油脂。分析比较发酵液中细胞干重、总油脂含量、总油脂产量及脂肪酸组成。结果表明:以苹果渣酶解液为碳源的发酵液中细胞干重、总油脂含量、总油脂产量、脂滴大小均较以葡萄糖为碳源的发酵液有显著增高;当发酵至3 d时,80 g/L总糖含量的苹果渣酶解液为碳源的发酵液中总油脂产量达到最高(0.088 mg/mL),明显高于以葡萄糖为碳源的发酵液中总油脂产量,并且不同碳源的发酵液中主要脂肪酸组成和含量也存在明显差异。因此,苹果渣酶解液更有利于Yarrowia lipolitica H222(pox1-6缺陷)产生油脂。     

深黄被孢霉产脂的研究

深黄被孢霉在限制氮源的情况下培养,其生物量得到了显著提高,达到35.9 g/L,葡萄糖利用率也大大增加,甚至培养基中葡萄糖起始浓度可以达到100 g/L。氮源耗尽后,菌体中重要的脂肪酸得到积累,其油脂含量为50%~55%,微生物油脂产量为18.0 g/L产脂培养基。每消耗1 g葡萄糖,总的生物量增加0.34 g,干菌体中的油脂含量增加0.17 g。油脂中的γ-亚麻酸含量为3.5%左右,即16~19 mg/g干菌体,最多的甚至可以达到0.801 g/L发酵培养基。     

Effect of Culture Variables on Mycelial Arachidonic acid Production by Mortierella alpina

Effect of culture conditions on biomass, lipid, and arachidonic acid production was investigated in the oleaginous fungus Mortierella alpina CBS 528.72 under shake flask conditions. Several factors have been found to affect the biomass buildup and lipogenesis in this fungus, complicated by the fact that different strains demonstrate varying optimization conditions. Growth, lipid accumulation, and arachidonic acid production in the strain investigated were influenced by media, pH, temperature, carbon source, nitrogen source, etc. The results indicated that the most effective medium for growth and arachidonic acid production was glucose yeast extract medium. The optimum pH and temperature were found to be 6.5 and 28°C, respectively. On the same weight basis, glucose was the most efficient carbon source for biomass and lipid production in this fungal strain which yielded 6.8 g/L dry biomass and 40.2% (w/w) total lipid after 7 days of cultivation. Maximum arachidonic acid (ARA) production of 40.41% achieved in rhamnose-containing media was not concomitant with higher biomass and lipid yields. Efficacy of organic carbon sources, viz, yeast extract and peptone over inorganic sources like sodium nitrate, ammonium sulfate, ammonium chloride, etc, was established in the present study. M. alpina CBS 528.72 grown in peptone acquired the highest lipid content (42.0% (w/w)). However, the ARA content (28.74%) proved to be significantly less than that grown in yeast extract (35.28%). Furthermore, it was found that the biomass and ARA production declined drastically in a medium with vegetable oils as the sole carbon source but triggered the lipogenic pathway leading to higher accumulation of total lipids. Under the ideal conditions mentioned above, the maximum biomass, total lipid, and arachidonic acid production were 6.8 g/L, 41.6%, and 35.28% total fatty acid, respectively, in shake flask system.     

Using crude glycerol and thin stillage for the production of microbial lipids through the cultivation of Rhodotorula glutinis

Single cell oils (SCO) produced from oleaginous microorganisms are a potential alternative oil feedstock for biodiesel production. The worldwide production of glycerol, a 10% (w/w) byproduct produced in the transesterfication process of oils converted to biodiesel, is increasing as more biodiesel is being produced. For the purposes of cost reduction, crude glycerol was regarded as a suitable carbon source for the cultivation of Rhodotorula glutinis. In addition to using renewable crude glycerol, waste solution collected from the brewing company (called thin stillage) was adopted as a substitute to replace a costly nitrogen source used in the medium. The results of using mixture of crude glycerol and thin stillage indicated about a 27% increase in total biomass as compared to that of using crude glycerol with a standard medium. Using glycerol instead of glucose as the carbon source could also alter the lipid profile, resulting in an increase in linolenic acid (C18:2) to comprise over 20% of the total lipid. Successfully using renewable crude glycerol and thin stillage for the cultivation of oleaginous microorganisms could greatly enhance the economic competition of biodiesel produced from SCO.     

外源油脂的添加促进卷枝毛霉脂肪酸的积累

卷枝毛霉（Mucor circinelloides）可利用多种碳源实现脂质积累,不同的碳源对其脂质积累的影响不同。在不改变其它发酵培养条件和碳氮比的情况下,以100%葡萄糖为对照,添加不同比例的外源油脂（大豆油）作为另一种碳源,对其细胞生长和脂质生成的影响进行了研究。采用氯仿-甲醇法提取脂质,通过气相色谱技术（gas chromatography,GC）测定其脂肪酸组成及含量。同时,采用实时荧光定量PCR法检测了与脂肪酸合成相关的脱饱和酶基因在菌株中的表达情况。结果表明：碳氮比不变的情况下,最佳碳源混合比例为葡萄糖35 g/L,大豆油24.3 g/L,该培养基中生长的卷枝毛霉的生物量、脂质含量和脂质产量在发酵前期迅速增长,在发酵后期的增长趋势逐渐平缓,在发酵72 h之后略有下降,其最高生物量、脂质含量和产量分别为19.60 g/L、42.62%和8.35 g/L,而对照组（以葡萄糖为单一碳源）仅为14.72 g/L、34.41%和5.06 g/L。与对照组相比,经混合碳源培养的卷枝毛霉的生物量、脂质含量和产量分别提高了33.15%、23.8%和65.10%,且可以将大豆油中的α-亚麻酸...     

有机碳源对单针藻细胞生长、油脂积累和光合作用的影响

研究不同有机碳源对单针藻(Monoraphidium sp.)细胞生长,油脂积累和光合作用的影响,探讨其细胞生长和油脂积累的最佳碳源浓度。结果表明单针藻具有利用有机碳源进行混合营养生长的能力,葡萄糖、蔗糖对其细胞生长、总脂含量和光合放氧速率具有明显的促进作用,甘氨酸能够促进细胞生长,但总脂含量下降,乙酸钠则表现为抑制作用;在BG-11培养基中加入5g/L葡萄糖后,细胞生物量、总脂产量和光合放氧速率分别为7.8、3.2g/L和240.3μmolO2·(mg·chla)-1·h-1,是同等光合自养条件下的5.6、8.0和1.3倍;单针藻细胞生长、总脂积累的最佳葡萄糖添加浓度为10g/L。     

Maximizing the production of γ-linolenic acid in Mortierella ramanniana var. ramanniana as a function of pH,temperature and carbon source,nitrogen source,metal ions and oil supplementation

A locally isolated (Perryvale, Alta., Canada) fungal genus, Mortierella ramanniana var. ramanniana, was evaluated as a potential industrial producer of γ-linolenic acid (GLA). Six growth variables (pH, temperature, carbon source, nitrogen source, and metal ions and oil supplementation) were systematically manipulated. The results indicated that the GLA production for this particular strain, could be maximized by using a basal growth medium consisting of 5% dextrose and 1% yeast extract, supplemented with 5 mg/L Mn²⁺ with incubation at 20 °C. The lipid yield under optimum conditions was 54.2% of the total dry biomass and consisted of 84.3% unsaturated fatty acids. The yield of GLA per gram biomass represented 13.3% of the total lipid content, slightly higher than that of the typical evening primrose oil GLA yield. The study establishes this variety as an effective intermediate fungal source of GLA.     

不同碳、氮源质量浓度对高山被孢霉脂质积累及SNF1复合体转录调控的影响研究

高山被孢霉(M.alpina)是一株具有较强脂质合成能力的产油微生物,其脂质积累受培养基中碳、氮源调控。探究了不同葡萄糖和酒石酸铵质量浓度对M.alpina生长及脂质积累的影响,并进一步研究不同碳、氮源质量浓度下M.alpina SNF1复合体各亚基的转录水平。结果表明:初始酒石酸铵质量浓度一定时,M.alpina脂肪酸含量随碳氮比(C/N)的增加而提高,C/N为24.6时其脂肪酸含量比C/N为4.6时提高42.6%,初始葡萄糖质量浓度一定时,氮源限制下(C/N=76.7)M.alpina脂肪酸产率达到2.6 g/L,是氮源存在时的1.5~3.2倍,与葡萄糖相比氮源水平对M.alpina脂质积累影响更为显著。氮源一定时高葡萄糖质量浓度(C/N=24.6)下SNF1复合体各亚基转录水平比对照组提高13.9~20.5倍;而氮限制导致的高C/N同样可促进其转录水平的提高。高C/N和氮限制均属于营养失衡信号,说明M.alpina SNF1转录水平的变化是其对胞外营养水平的响应方式,并与脂质积累水平具有相关性。     

不同碳源对酵母代谢有机酸的影响

对啤酒酵母在以葡萄糖、果糖、麦芽糖作碳源的培养基中的代谢生长状况进行了跟踪检测,应用高效液相色谱检测各发酵液中酒石酸、苹果酸、琥珀酸和柠檬酸的含量变化,得出了碳源对啤酒酵母有机酸代谢的影响:其中酒石酸、苹果酸和柠檬酸含量差别明显,琥珀酸含量差别较小。     

基于pH值调控的高山被孢霉发酵生产花生四烯酸的补料工艺

为了提高高山被孢霉（Mortierella alpina）发酵生产花生四烯酸（arachidonic acid,ARA）的产量,研究pH值控制及不同底物流加方式对M. alpina产ARA的影响。在0～90 h菌体生长阶段pH值维持在6.0,90 h以后上调至6.5,并在此基础上考察了3 种补料方式（一次性补料、恒定葡萄糖速率补料和pH值反馈补料）对菌体生物量、油脂产量及ARA产量的影响。结果表明：发酵过程中pH 6.0时可以促进菌体的生长,而pH 6.5时有利于油脂的合成。与其他补料方式相比,pH值反馈补料不仅使发酵周期缩短至160 h,而且在该条件下得到ARA最大生产强度及产量,分别为1.32 g/（L·d）和8.82 g/L。