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.     

金属离子和氧载体对红酵母NR06胡萝卜素发酵的影响

在单因素实验的基础上,选定对β-胡萝卜素合成促进作用较明显的2种金属离子K 、Fe2 和2种氧载体正十二烷、正己烷等共4种添加物,利用二次回归正交设计实验进行优化,得到每种添加物的最佳添加量依次为0.63%、0.0094%、2.11%和1.52%,此最优组合下β-胡萝卜素的产量为7.34 mg/L,比对照组的5.99 mg/L提高了22.54%.     

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。     

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。      

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.     

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。     

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。     

Effects of dissolved oxygen level on cell growth and total lipid accumulation in the cultivation of Rhodotorula glutinis

The total amount of lipids produced in Rhodotorula glutinis is a subject which has attracted increasing attention due to the potential biodiesel conversion from these microbial oils. The effects of the dissolved oxygen (DO) level in lipid accumulation were examined in this study. Variations of different medium volumes (30, 40 and 50ml) and shaking speed (60, 150 and 210rpm) in the flask trials were adopted to explore the DO effects on lipid production. All of the results revealed that a low DO could retard cell growth, while enhancing lipid accumulation. The 5l-fermentor results also confirm that a low DO (25 ± 10%) batch could have higher lipid content than that of high DO batch (60 ± 10%). Nevertheless, the DO level would not obviously affect the lipid composition profile. Oleic acid (C18:1) was the primary fatty acid in both batches. Due to the slow biomass growth rate resulting from the low DO, a two-stage DO controlled strategy (consisting of a high DO stage and following a low DO stage) was performed to improve the cell growth and lipid accumulation simultaneously. However, the strategy was not successful on the enhancement of total lipid production as compared to other batches. Conclusively, even a low DO could retard cell growth; the total production of lipids in the batch with low DO was higher that of the high DO batch due to the enhancement of lipid accumulation. Therefore, the batch operation of R. glutinis at the low DO was suggested for the purpose of lipid production.     

高静水压对产β-胡萝卜素粘红酵母的诱变效应

利用100～500 MPa的静水高压和10～30 min的保压时间对粘红酵母（Rh. glutinis RG5）进行处理,发现处理10 min时的高压存活曲线呈现马鞍形,说明高静水压处理的最终效应可能是多种生物学效应的累积.150 MPa保压20 min时的突变株RG5-3的生物量虽然比出发菌株RG5下降了9.25%,但目的产物β-胡萝卜素的质量分数却比RG5提高了68.60%.传代实验表明,该诱变株遗传稳定性良好,没有发生性状退化及回复突变.利用限制性片段长度多态性分析（RFLP）比较发现,限制性内切酶HindⅢ和BamHⅠ切割的RG5和RG5-3的DNA片段有明显不同,证明高静水压使粘红酵母RG5在表面性状和DNA水平上都发生了改变.     

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%。     

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.     

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。     

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。     

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。     

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.     

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。     

Optimization of beta-carotene production by Rhodotorula glutinis using high hydrostatic pressure and response surface methodology

ABSTRACT:  Rhodotorula glutinis RG6 was treated by high hydrostatic pressure (HHP) of 300 MPa for 15 min for improving its ability of β-carotene production. After the treatments of 5 repeated cycles, the mutant strain RG6p was obtained, β-carotene production of which reached 10.01 mg/L, increased by 57.89% compared with 6.34 mg/L from parent strain RG6. To optimize the medium for β-carotene fermentation by mutant RG6p, a response surface methodology (RSM) approach was used in conjunction with a factorial design and a central composite design, and the maximum yield of β-carotene (13.43 mg/L), an increase of 34.17% compared to the control, was obtained at a pH 6.7 with an optimum medium (40 mL/250 mL) of yeast extract (4.23 g/L), glucose (12.11 g/L), inoculum (30 mL/L), tomato extract (2.5 mL/L), peanut oil (0.5 mL/L), and (NH₄)₂SO₄ (5 g/L).     

金属离子对红酵母菌产类胡萝卜素影响的研究

主要以红酵母AS2.2241液态发酵培养基为原料,添加不同的金属离子(Na+、Mg2+、K+、Zn2+、Fe2+、Cu2+等),在相同的培养条件下进行培养。采用酸热破壁法破坏红酵母的细胞壁,同时使用丙酮浸提色素,提取类胡萝卜素,通过检测475nm下的吸光度,筛选出对提高类胡萝卜素产量最有利的不同金属离子的添加量。     

Control of postharvest pear diseases using Rhodotorula glutinis and its effects on postharvest quality parameters

Rhodotorula glutinis was evaluated for its activity in reducing postharvest gray mold decay and blue mold decay of pear caused by Botrytis cinerea and Penicillium expansum respectively, and in reducing natural decay development of pear fruits, as well as its effects on postharvest quality of fruits. There was a significant negative correlation between concentrations of the yeast cells and infectivity of the pathogens. At concentrations of R. glutinis at 5x108 CFU/ml, the gray mold decay was completely inhibited after 7 days incubation at 20 degrees C, while the control fruit had 100% disease incidence and 2.15 cm lesion diameter respectively, at challenged with B. cinerea spores suspension of 1x105 spores/ml; No completely control was got to blue mold, when pear fruits stored at 20 degrees C for 7 d (challenged with P. expansum spores suspension of 5x104 spores/ml), but the decay was distinctly prevented with 20% and 0.60 cm of disease incidence and lesion diameter respectively, while the control fruits were 100% and 2.74 cm, respectively. Rapid colonization of the yeast in wounds was observed during the first 1 d at 20 degrees C, and then the populations stabilized for the remaining storage period. On pear wounds kept at 4 degrees C, rapid colonization of the yeast in wounds was observed during the first 3 d, and then the increase in population density of R. glutinis turned slow, which continued over 6 d after application of the antagonist until it reached a high level. Then, the populations stabilized for the remaining storage period. In the test on PDA plates, R. glutinis significantly inhibit the growth of B. cinerea and P. expansum. Spore germination of pathogens in PDB was greatly controlled in the present of living yeast cell suspensions. R. glutinis significantly reduced the natural development of decay of pear following storage at 20 degrees C for 7 days or at 4 degrees C for 30 days followed by 20 degrees C for 5 days, and did not impair quality parameters, including mass loss, firmness, TSS, ascorbic acid or titratable acidity. Thus, the application of R. glutinis can be an alternative to chemicals for control of postharvest diseases on pear fruits.