利用粘红酵母生产微生物油脂研究进展

粘红酵母是一种良好的产油微生物,其油脂可用来生产生物柴油。本文介绍了粘红酵母生产油脂的情况,包括其常用基本培养基,廉价工农业废弃物培养基,以及促进粘红酵母生长和产油的方法和培养条件,并对粘红酵母生产微生物油脂方面存在的问题和未来的发展方向进行了概述。      

响应面优化粘红酵母油脂提取工艺的研究

采用酸热耦合超声波法提取粘红酵母油脂,在单因素实验的基础上,通过响应面建立了1g湿菌体所得油脂质量的二次多项数学模型,分析了模型的有效性和各因素的交互作用,并得出最佳工艺条件为:酸处理时间34min,超声处理时间7.5min,提取液(氯仿-甲醇)体积比1.9∶1。在此优化条件下,1g湿菌体所得油脂质量的理论值为60.47mg,实验值为57.33mg,相对误差为5.48%。     

黏红酵母产油脂培养基的响应面优化

利用单因素试验和响应面设计相结合,对黏红酵母产油脂培养基进行了优化。单因素试验得到初步发酵培养基成分为葡萄糖、蛋白胨、KH2PO4。经响应面优化发现,当发酵培养基中葡萄糖含量为73.40g/L,蛋白胨含量为1.06 g/L,KH2PO4含量为3.56 g/L时,油脂产量的理论预测值可达到3.49 g/L,比优化前提高了13%。气相分析其油脂组成,多不饱和脂肪酸质量分数为26.97%。然后又对高产菌株的发酵特性进行研究,在10 d时,生物量和油脂产量达到最高,此时达到发酵终点,生物量为47.98 g/L(菌体湿重),油脂产量达到7.81 g/L。     

黏红酵母Rhodotorula glutinis微生物油脂提取工艺研究

采用酸热耦合超声波法对黏红酵母Rhodotorula glutinis微生物油脂提取工艺进行研究。选取酸处理时间、沸水浴时间、超声处理时间、超声功率、提取液配比、提取时间、NaCl浓度等7个微生物油脂提取相关因素,首先通过Plackett-Burmen试验设计筛选出3个显著因子:提取液配比(P=0.010 5)、超声处理时间(P=0.010 5)、酸处理时间(P=0.036 4)。然后通过Box-Behnken试验设计,对3个显著因素进行优化组合,得到最佳工艺条件,即酸处理时间34 min,超声处理时间7.5 min,提取液配比1.9∶1。与原始工艺条件相比,1 g湿菌所得油脂重量从42 mg增加到57.33 mg,提高了36.5%。     

固定化酵母细胞转化肉桂酸生产L-苯丙氨酸

建立了一套较之传统方法更为准确的测定固定化细胞生产L-苯丙氨酸(L-phe)的新方法,采用高效液相色谱法(HPLC)测定反复冲洗凝胶所得冲洗液可以更准确地测定L-phe的生成,并采用新方法研究了固定化黏红酵母细胞的活力与稳定性。复合凝胶聚乙烯醇(PVA)+琼脂糖为最适载体,最佳质量浓度为PVA 120 g/L、琼脂糖10 g/L,反复冻融4次。此法得到的固定化细胞颗粒,机械强度良好,酶活保持时间长达200 h,L-苯丙氨酸产量高。通过流化床反应器测定其半衰期为100 h,200 h后总产量为44.58 mg/10 mL,固定化细胞无崩解现象。     

粘红酵母酪氨酸解氨酶在大肠杆菌中的异源表达

酪氨酸解氨酶是苯丙氨酸代谢途径的关键酶之一。酪氨酸经其催化生成对香豆酸,可进一步生成白藜芦醇、柚皮素等具有抗氧化、抗衰老作用的苯丙素类天然产物。作者选取来源于粘红酵母(Rhodotorula glutinis)的酪氨酸解氨酶,对其基因进行大肠杆菌(Escherichia coli)密码子偏好性优化,合成基因后于E.coli BL21(DE3)中成功表达。经过硫酸铵分级沉淀、QFF阴离子交换层析、分子筛纯化后得到了纯化的酪氨酸解氨酶,比酶活达到1.78 U/mg。在相同的培养条件下,以不同的质粒作为表达载体,获得了不同的对香豆酸产量。其中以酪氨酸为底物发酵24 h,当以p ET-32a(+)作为表达载体时,获得最高的对香豆酸产量196.3 mg/L。经密码子优化后的酪氨酸解氨酶基因可更好地应用于苯丙素类物质的合成途径构建,不同载体的应用也为生物法生产对香豆酸提供了更多的选择依据。     

索氏提取法测定黏红酵母含油量的改进

研究分别用索氏提取的传统法和改进法测定了黏红酵母菌体含油量,并利用SPSS统计软件比较分析了两种测定方法的准确性。结果表明,按照传统增重法测定废水培养基和基础培养基发酵黏红酵母菌体的含油量分别为16.2%和9.3%,而用改进后减重法测得其含油量分别为20.6%和15.3%;SPSS分析表明,传统索氏增重法计算平行试验的标准误差分别为0.002 9和0.008 1,改进减重法计算平行试验的标准误差分别为0.000 8和0.004 6。可见,传统增重法测定黏红酵母菌体含油量数据偏小,而利用改进后的减重法测定准确度较高,餐厨固体垃圾含油量的测定结果进一步验证了改进后的减重法优于传统增重法。     

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.     

黏红酵母离子注入诱变及其发酵产生β- 胡萝卜素条件的研究

利用低能N+ 注入黏红酵母高压突变株G-39,经筛选获得高产β- 胡萝卜素的离子诱变株GL-5,其β- 胡萝卜素产量由出发菌株的9.64mg/L 提高到17.36mg/L,增加了80.08%。通过均匀设计试验法,初步确定了诱变株GL-5 的β- 胡萝卜素发酵最适条件(葡萄糖40g/L、蛋白胨30g/L、酵母膏10g/L、番茄汁3ml/L、核黄素0.5mg/L、初始pH6.0、摇瓶装量50ml/250ml、接种量50ml/L),使其β- 胡萝卜素产量进一步由17.36mg/L 提高到34.21mg/L,比对照增加了98.09%。这表明,离子诱变株GL-5 是一株优良的高产β- 胡萝卜素突变株,离子注入对该菌种具有良好的诱变效果。     

Production of carotenoids by Rhodotorula glutinis MT-5 in submerged fermentation using the extract from waste loquat kernels as substrate

BACKGROUND: The aim of present study was to investigate the feasibility of the hydrolysate extracts from waste loquat kernels as substrate in submerged culture of yeast Rhodotorula glutinis MT‐5 for carotenoid production. RESULTS: Loquat kernel was found to have high protein (22.5%) and total carbohydrate (71.2%) contents. Dried and powdered loquat kernels were subjected to acid hydrolysis with 2 mol L^?1 HCl. The hydrolysate obtained was used for the preparation of loquat kernel extract and detoxified loquat kernel extract. The detoxification of hydrolysate was performed with Ca(OH)₂. Among the 10 R. glutinis isolates, the MT‐5 was found to be best in order to produce carotenoid using the extract as substrate. Production media prepared with detoxified loquat kernel extract or loquat kernel extract gave maximum biomass concentrations of 12.64 and 11.37 g L^?1, and maximum carotenoid concentrations of 72.36 and 62.73 mg L^?1, respectively. CONCLUSION: This study has provided effective processes for the conversion of waste material of plant origin to the extracts which are very rich in term of total fermentable sugar. The practicability of the extracts as fermentation substrate was proven in carotenoid production. To the best of our knowledge, this is the first report on use of this waste material as a substrate in yeast fermentations. Copyright © 2011 Society of Chemical Industry     

粘红酵母苯丙氨酸解氨酶合成条件优化

首先利用Plackett-Burman设计及最陡爬坡试验对在摇瓶中对粘红酵母合成苯丙氨酸解氨酶的培养基和培养条件进行优化筛选,然后通过单因子试验确定最适诱导物。在此基础上,进行发酵罐葡萄糖浓度、产酶pH值以及诱导物添加时间的优化。结果显示优化的发酵培养条件为葡萄糖1g/L,蛋白胨35g/L,NaCl 5g/L,KH2PO4 0.25g/L,（NH4）2HPO4 1.5g/L;接种量4%;初始pH值为5;控制产酶pH值为7;诱导物为L-苯丙氨酸,分别在发酵8h和26h时添加;在上述优化条件下,最高比酶活为40.85U/g,比未优化前提高了7.3倍。     

粘红酵母发酵生产红色素及其稳定性研究

该文研究了碳源、氮源、温度、pH、无机离子等对红色素的产生量及粘红酵母生长的影响，获得较佳粘红酵母发酵生产天然红色素的工艺条件。并研究了光、热、山梨酸钾、Vc对产生的红色素稳定性的影响，获得了粘红酵母生产红色素稳定应用条件。     

Activation of torularhodin production by Rhodotorula glutinis using weak white light irradiation

The effects of the irradiation of weak white light on the growth of the red yeast Rhodotorula glutinis and its production of carotenoids were investigated. The ability of beta-carotene and torularhodin, which are final products of carotenoid biosynthesis in R. glutinis, to quench singlet oxygen has also been investigated. Weak white light irradiation that has no effect on the growth of Saccharomyces cerevisiae inhibited the growth of R. glutinis. Simultaneously, the production of torularhodin by R. glutinis markedly increased. In a mutant of R. glutinis, which exhibited increased production of torularhodin, an increase in torularhodin production was shown as a result of light irradiation during the logarithmic growth phase. An experiment using 3-(1,4-epidioxyl-4-methyl-1,4-dehydro-1-naphtyl) propionic acid clarified that torularhodin inhibited 2,5-diphenyl-3,4-benzofran decomposition by singlet oxygen quenching more strongly than did beta-carotene. This result is consistent with the report that carotenoids having a longer polyene chain may exhibit a more potent ability to quench singlet oxygen. These results suggest that the biosynthesis of carotenoids in R. glutinis may play an important role in protecting against oxidative damage caused by light irradiation, and in particular, torularhodin which has a potent singlet oxygen quenching ability may be important. We suggest that acquisition of the ability to produce torularhodin may be an important property for this yeast to promote its wider distribution in the natural world.     

Properties of a high-torularhodin-producing mutant of Rhodotorula glutinis cultivated under oxidative stress

To characterize the properties of torularhodin, which is one of the carotenoid pigments produced by the yeast Rhodotorula sp., a mutant which produces large amounts of torularhodin was constructed and its tolerance against oxidative stress was investigated. The mutant we obtained was capable of producing large amounts of torularhodin in response to irradiation with blue light. The mutant, incubated under irradiation with white light that resulted in an increased production of torularhodin, exhibited resistance to growth inhibition induced by the addition of methylene blue as the generator of singlet oxygen. Leakage of lactate dehydrogenase to the growth medium from the mutant was not increased as compared to that from a parent strain and a high-beta-carotene-producing mutant. These results suggest that an increase in the production of torularhodin reduces the susceptibility to injury induced by an active oxygen species.     

光镊拉曼光谱技术结合响应面法优化红酵母产类胡萝卜素的发酵条件

拉曼光谱具有快速、实时、无损分析的特点,在生物学领域应用广泛。作者利用光镊拉曼光谱技术(LTRS)测定红酵母细胞中类胡萝卜素。采用Plackett-Burman法对影响红酵母发酵的相关因素进行评价,发现葡萄糖、蛋白胨、pH和温度对类胡萝卜素的产量影响显著。利用BoxBehnken设计和响应面分析法对影响类胡萝卜素产量的关键因素进行优化,获得的最优发酵条件为:葡萄糖42.86 g/L,蛋白胨5 g/L,酵母提取物5 g/L,KH2PO41 g/L,MgSO4。7H2O 0.5 g/L,pH 7.0,温度28.3℃,优化后类胡萝卜素产量较优化前提高了45%。     

Biological control of postharvest green mold decay of oranges by Rhodotorula glutinis

The biocontrol activity of Rhodotorula glutinis on green mold decay of oranges caused by Penicillium digitatum was investigated in vitro and in vivo. Significant control was achieved with a washed cell suspension and an unwashed cell culture mixture of R. glutinis. Treatment of wounds with autoclaved cell cultures or cell-free culture filtrate did not prevent decay. The protection provided by the washed yeast cells was dose-dependent. The higher the concentration of R. glutinis, the better the effect of the biocontrol capacity. At concentrations of yeast of 1×10⁹ colony-forming units per milliliter or higher and pathogen spore suspensions of 5×10⁴ spores per milliliter, green mold was almost inhibited after 4-days incubation at 20 °C. The interval between the pathogen inoculation and the antagonist application significantly influenced the biocontrol ability. The biocontrol efficacy of R. glutinis applied before the pathogen was better than that of applied after the pathogen. Surprisingly, R. glutinis was also effective in controlling green mold at low temperature (4 °C). Rapid colonization of the yeast in wounds was observed during the first 3 days at 20 °C, and remained stable after 5-days incubation. On fruits stored at 4 °C, even after 21 days, the population of R. glutinis in wounded fruits was more than 1,600-fold of what it was just prior to storage. In the test on potato dextrose agar plates, agar disks of R. glutinis nutrient yeast dextrose agar cultures placed on PDA plates seeded with pathogens did not inhibit the growth of P. digitatum. Spore germination of pathogens in potato dextrose broth was greatly controlled in the presence of living cell suspensions.     

红酵母发酵废糖蜜产类胡萝卜素的研究

对红酵母利用糖蜜发酵产类胡萝卜素进行了研究,结果表明:对糖蜜的预处理的最佳酸为硫酸;利用经过酸处理后的糖蜜发酵,菌株的生物量为30.75g/L,单细胞色素含量为104μg/g,色素产量为26mg/L;最佳发酵时间为168h。使用糖蜜代替葡萄糖做廉价碳源对红酵母进行发酵,确定添加量为170g/L,色素产量最大值为葡萄糖发酵的0.97倍,生物量为1.04倍。      

Effects of high hydrostatic pressure on the growth and beta-carotene production of Rhodotorula glutinis

The effects of high hydrostatic pressure (HHP) on the biomass and beta-carotene biosynthesis of Rhodotorula glutinis R68 were studied. After treatment with five repeated cycles at 300 MPa for 15 min, the barotolerant mutant PR68 was obtained. After 72 h of culture, the biomass of mutant PR68 was 21.6 g/l, decreased by 8.5% compared to the parental strain R68, but its beta-carotene production reached 19.4 mg/l, increased by 52.8% compared to the parental strain R68. The result of restriction fragment length polymorphism (RFLP) analysis suggested that mutant strain PR68 was likely to change in nucleic acid level, and thus enhanced beta-carotene production in this strain as a result of gene mutation induced by HHP treatment.     

粘红酵母胞内L-苯丙氨酸解氨酶释放的研究

分别考察了液氮冷冻法、超声破碎法、甘氨酸与丙氨酸化学渗透法，不同化学渗透剂TritonX-100、Tween80、Span20、溴代十六烷基吡啶，有机溶剂甲苯、脲及EDTA对粘红酵母（Rhodotorula glutinis）胞内苯丙氨酸解氨酶（即PAL酶）释放及活力的影响，比较发现超声破碎与TritonX-100联用酶释放效果显著，先加入10％Triton X-100渗透6h，再在800W超声破碎30min，蛋白释放率达76．7％。总酶活70．3％，为提取胞内PAL酶提供了一种新方法。