富含谷氨酸酿酒酵母的筛选及发酵培养基优化

从农家自酿葡萄酒中筛选出一株富含谷氨酸酿酒酵母菌（Saccharomyces cerevisiae）F-5,其26S rDNA核苷酸序列与S. cerevisiae TY12的26S rDNA核苷酸序列同源性为100%。以胞内谷氨酸含量为目标,采用响应面法对其发酵培养基进行了优化,建立糖蜜、工业蛋白胨和KH2PO4的二次回归模型,确定培养基最佳配方为：糖蜜（含30%蔗糖）100 mL/L、酵母浸粉10 g/L、工业蛋白胨20 g/L、MgSO4·7H2O 1 g/L、KH2PO4 0.5 g/L、FeSO4·7H2O 2 g/L。在此优化培养基中发酵培养24 h,胞内游离谷氨酸达到了3.29%,比优化前提高了87.8%。     

响应面法优化固定化酿酒酵母细胞制备工艺研究

酿酒酵母的固定化细胞技术能够实现可连续与重复再利用性强、并且能够实现酿酒酵母细胞的高密度培养.本研究利用海藻酸锰代替海藻酸钙作为固定化酿酒酵母的载体,采用响应面方法优化固定化工艺,为了降低海藻酸锰凝胶球的传质阻力,在凝胶球中添加适量的经过酶解的秸秆纤维素.结果表明,最佳工艺条件为海藻酸钠(SA)初始浓度为2.18％,秸秆纤维素(CS)添加量为0.21％,CaC12初始浓度为5.21％,在此BBD响应面模型优化条件下,制备固定化酿酒酵母细胞的包埋率理论值可达到88.02％.包埋率从48％提高为88.02％.表明了BBD响应面模型对于制备固定化细胞的工艺流程具有很高的的应用价值.     

响应曲面法优化猕猴桃酒增殖培养基配方

利用JMP和Box-Behnken响应曲面设计(RSM)研究了增殖因子对猕猴桃酒酵母WF25增殖作用的影响。研究结果表明:葡萄糖、(NH4)2HPO4、VB1、VB2和泛酸对猕猴桃酒酵母WF25细胞的增殖均有极显著的影响,MgSO4有显著的影响。当葡萄糖、(NH4)2HPO4、MgSO4、VB1、VB2和泛酸添加量分别为8.39、0.53、0.07、1.75、0.60mg/L和0.57mL/L,生物量的预测值可达3.01785×108cfu/mL。     

响应面法优化生香酵母C42增殖培养基

在摇瓶水平上对生香酵母C42培养基进行响应面法优化。首先用单因子法筛选最优碳源、氮源及无机盐。采用 Plackett- Burman法筛选影响生香酵母C42生长的主要因素、再利用最陡爬坡试验进行Box-Behnken设计,得到生香酵母C42的优化培养基配比。优化后的培养基组成为葡萄糖7.12%、酵母浸粉3.28%、KH2PO4 0.2%、(NH)2SO4 0.6%、NaCl 0.5%、MgSO4 0.03%。优化后的活菌数量可达到1.26×109 CFU/mL,比优化前的活菌数7.65×108 CFU/mL提高了64.7%。     

Selective retention of secretory proteins in the yeast endoplasmic reticulum by treatment of cells with a reducing agent

We have used four glycoproteins as markers to study how disulfide bond formation and protein folding effect the intracellular transport of proteins in yeast. Under normal conditions, the vacuolar enzyme carboxypeptidase Y (CPY) and the secretory stress-protein hsp150 acquired disulfide bonds in the endoplasmic reticulum (ER). Treatment of living cells with the reducing agent dithiothreitol (DTT) prevented disulfide formation of newly synthesized CPY and hsp150, resulting in retention of the proteins in the ER. When DTT was removed, the sulfhydryls were reoxidized, and the transport of the proteins to their correct destinations was resumed. Even mature CPY, located in the vacuole, could be reduced with DTT, and reoxidized after removal of the drug. DTT treatment blocked intracellular transport of hsp150 only when present during the synthesis and translocation of the protein. Reduction of folded hsp150, accumulated in the ER due to a sec block prior to DTT treatment, did not inhibit its secretion. The Kar2p/BiP protein, a component of the ER lumen, was found to be associated with fully translocated reduced hsp150, but not with native hsp150, suggesting that Kar2p/BiP may be involved in the putative retention mechanism. The cysteine-free pro-α-factor, and invertase which was shown to have free sulfhydryls, were secreted and modified similarly in the presence and absence of DTT, showing that the secretory pathway of yeast functioned under reducing conditions.     

利用响应面方法优化产L-乳酸的合成培养基

采用响应面方法对拟干酪乳杆菌Lactobacillus paracasei (L.paracasei)的一种产乳酸的合成培养基进行了优化.该文将培养基中的混合氨基酸、核苷类物质和混合维生素看作3大类营养成分.首先利用Plackett-Burman实验设计考察了培养基中包括该3类营养物质在内的8种营养成分对菌体生长和乳酸合成的影响,筛选出了影响菌体生长和乳酸合成的3个主要因素,即混合氨基酸、KH2PO4和CaCO3;在此基础上利用最陡爬坡实验逼近最大响应区域;然后利用Box-Behnken实验设计及响应面分析法确定了最佳培养条件.经过3次实验验证,乳酸实际浓度与预测浓度十分接近,已经超过了在复合培养基中培养的水平.这些结果为L.paracasei的代谢流定量研究奠定了基础.     

响应面法优化酿酒酵母工程菌产甜蛋白monellin发酵培养基

对酿酒酵母工程菌WHF9/monellin在液体培养基中产甜蛋白monellin的条件进行了优化.采用单因子试验筛选出细胞生长培养基的最适碳源为蔗糖,氮源为玉米浆干粉,且应添加微量元素溶液.在此基础上,利用Plackett-Burman试验设计筛选出影响菌体生物量的3个显著因素:蔗糖、玉米浆干粉、微量元素溶液.用最陡爬坡路径逼近最大响应区域后,利用Box-Behnken设计和响应面分析法对显著因素进行优化,得出蔗糖、玉米浆干粉、微量元素溶液的最佳浓度分别为102.2g/L,33.9g/L,4.5mL/L.菌株在优化后的生长培养基中的生物量比在YPD培养基中提高了81.38％.在诱导剂半乳糖最适浓度102.2g/L条件下,菌株在优化后的培养基中monellin的表达量达到226.7mg/L,比在YPG中表达的monellin提高了4.2倍,且表达的monellin具有生物活性.     

Enhancement of Protein Secretion in Saccharomyces cerevisiae by Overproduction of Sso Protein,a Late-acting Component of the Secretory Machinery

Increased production of secreted proteins in Saccharomyces cerevisiae was achieved by overexpressing the yeast syntaxins, Sso1 or Sso2 protein, the t-SNAREs functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane. Up to four- or six-fold yields of a heterologous secreted protein, Bacillus α-amylase, or an endogenous secreted protein, invertase, were obtained respectively when expressing either one of the SSO genes, SSO1 or SSO2, from the ADH1 promoter on a multicopy plasmid. Direct correlation between the Sso protein level and the amount of secreted α-amylase was demonstrated by modulating the expression level of the SSO2 gene. Quantitation of the α-amylase activity in the culture medium, periplasmic space and cytoplasm suggests that secretion into the periplasmic space is the primary stage at which the SSO genes exert the secretion-enhancing function. Pulse-chase data also support enhanced secretion efficiency obtained by SSO overexpression. Our data suggest that the Sso proteins may be rate-limiting components of the protein secretion machinery at the exocytosis step in yeast. © 1997 John Wiley & Sons, Ltd.     

Optimisation of the medium composition for production of protease and soybean peptides by Bacillus subtilis SHZ using response surface methodology

Responses surface methodology was employed to enhance the production of protease and soybean peptides by Bacillus subtilis SHZ. For screening of medium composition significantly influencing protease and soybean peptides yield, the two-level Plackett–Burman design was used. Among thirteen variables tested; KH₂PO₄, glucose and defatted soybean flour (DSF) were selected based on their high significant effect on both protease activity and soybean peptides yield. Then, a three-level Box–Behnken design was employed to optimise the medium composition for the production of the protease and soybean peptides in submerged fermentation. Mathematical models were then developed to show the effect of each medium composition and their interactions on the production of protease and soybean peptides. The model estimated that, the maximal protease activity (320 ± 1 U mL⁻¹) could be obtained when the concentrations of glucose, KH₂PO₄, DSF were set at 8–9 g L⁻¹, 2–3 g L⁻¹, 55–65 g L⁻¹, respectively; while a maximal yield of soybean peptides (8.5 ± 0.1 g L⁻¹) could be achieved when the concentrations of glucose, KH₂PO₄, DSF were set at 7–9 g L⁻¹, 3–4 g L⁻¹ and 55–58 g L⁻¹, respectively. These predicted values were also verified by validation experiments.     

富含核糖核酸酿酒酵母的选育及其高密度发酵工艺

该研究首先以酿酒酵母（Saccharomyces cerevisiae）J-5为出发菌株进行诱变选育,筛选出一株核糖核酸（RNA）含量优于菌株J-5的诱变菌株J-5-9,其RNA含量在摇瓶中达到了13.12%,比出发菌株提高了10.62%。选取糖蜜为碳源,应用正交试验优化菌株J-5-9发酵培养基组成为：糖蜜3%,酵母浸粉2%,磷酸二氢钾0.01%,谷氨酸钠0.2%,硫酸亚铁0.1%。最后利用优化发酵培养基和碳源、氮源和磷源的流加补料工艺,在10 L发酵罐中培养诱变菌株J-5-9,RNA含量达到8.11%,比优化前提高了18.22%,同时细胞生物量达到188 g/L（湿质量）,实现了酿酒酵母高产RNA的高密度发酵。这说明诱变菌株J-5-9是一株很有潜力的工业化生产菌株。     

Near-infrared spectroscopic monitoring of biomass, glucose, ethanol and protein content in a high cell density baker's yeast fed-batch bioprocess

The use of at-line NIRS to monitor a high cell density fed-batch baker's yeast bioprocess was investigated. Quantification of the key analytes (biomass, ethanol and glucose) and the product quality indicator (percentage protein content) was studied. Biomass was quantitatively modelled using whole matrix samples (as was percentage protein content). The dominance of the whole matrix spectrum by biomass, and its associated light scattering effects, were overcome by use of filtrate samples and adapted (semi-synthetic) filtrate samples, which allowed successful ethanol and glucose modelling, respectively. Calibrations were rigorously challenged via external validation with large sample sets relative to the calibration sample size, ensuring model robustness and potential practical utility. The standard errors of calibration for biomass, glucose, ethanol and total intracellular protein were (g/l) 1.79, 0.19, 0.79 and 0.91, respectively, comparable to those of the primary assays. The calibration strategies necessary to generate quantitative models for this range of analytes in such a complex high cell density bioprocess fluid are discussed.     

Large-scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes.

Sequencing of the yeast genome has shown that about one-third of the yeast ORFs code for unknown proteins. Many other have similarity to known genes, but still the cellular functions of the gene products are unknown. The aim of the B1 Consortium of the EUROFAN project was to perform a qualitative phenotypic analysis on yeast strains deleted for functionally orphan genes. To this end we set up a simple approach to detect growth defects of a relatively large number of strains in the presence of osmolytes, ethanol, high temperature, inhibitory compounds or drugs affecting protein biosynthesis, phosphorylation level or nucleic acids biosynthesis. We have now developed this procedure to a semi-quantitative level, we have included new inhibitors, such as hygromycin B, benomyl, metals and additional drugs interfering with synthesis of nucleic acids, and we have performed phenotypic analysis on the deleted strains of 564 genes poorly characterized in respect to their cellular functions. About 30% of the deleted strains showed at least one phenotype: many of them were pleiotropic. For many gene deletions, the linkage between the deletion marker and the observed phenotype(s) was studied by tetrad analysis and their co-segregation was demonstrated. Co-segregation was found in about two-thirds of the analysed strains showing phenotype(s).     

酵母发酵条件的优化及其发酵造纸污泥产乙醇

优化了酿酒酵母Saccharomyces cerevisiae GIM-2发酵工艺条件并对其发酵造纸污泥产乙醇进行了研究。以模拟造纸污泥水解液中糖成分的混合糖为试验原料,采用响应面分析法优化酵母发酵生产乙醇工艺,得优化条件:温度33.1℃,pH值5.4,摇床转速50r/min,发酵24h糖醇转化率为38%。造纸污泥在纤维素酶作用下水解48h,纤维素转化率为58.2%,水解液在优化条件下发酵24h糖醇转化率为28.4%,产率达0.14g乙醇/g污泥,是理论值的37.3%。     

啤酒酵母和乳酸菌发酵棕榈粕渣饲料制备工艺的响应面优化

通过单因素实验和响应面法对啤酒酵母和乳酸菌发酵棕榈粕渣饲料制备工艺进行研究。结果表明:基料制备最佳发酵条件为发酵时间5 d、棕榈仁渣目数70目、菌料比1∶103、发酵温度30℃、含水量36%,该条件下基料中总菌落数为7.20×10~(10)个/g;发酵产物最佳发酵条件为发酵温度30℃、棕榈果渣与空果串配比1∶2、棕榈果渣及空果粉碎长度4 cm、配料比1∶10(基料与棕榈果渣和空果串质量比)、含水量35%、发酵时间36 d,该条件下发酵产物营养成分OD值为0.966。对发酵饲料主要成分进行分析,其粗蛋白、粗脂肪、粗纤维、粗灰分、钙、磷、含水量分别为7.20%、7.61%、26.20%、5.20%、0.38%、0.26%、16.20%,总菌落数为9.0×10~7个/g。该发酵方法工艺简单,具有良好的工业化生产前景。     

Yeast viability during fermentation and sur lie ageing of a defined medium and subsequent growth of Oenococcus oeni

Interactions between the yeast strain used for primary oenological fermentation and the bacterium used to conduct subsequent malolactic fermentation were studied under model winemaking conditions. A commercial Saccharomyces cerevisiae wine yeast (strains, EC 1118, AWRI 835 and CY-3079) was grown in a defined medium whose composition approximated grape juice. Fermentations by all strains reached dryness, and retained a cell viability of greater than 90% upon completion of fermentation. Highest total viable cell number and percentage of viable cells were recorded for EC 1118. A sur lie ageing of the fermented medium over a 12 week period revealed a bi-phasic decay of culture viability for all strains. Thus 99% of cells had died within 2 weeks post-fermentation. Viabilities were then stable for the subsequent 4–6 week period before a second decline phase ensued and ended in either a minimal ( ca 100 CFU/mL, EC 1118) or no viable cells being detected at 12 weeks of ageing. The growth response of an Oenococcus oeni inoculum to yeast culture supernatants, previously aged for up to 12 weeks in the presence or absence of yeast lees, was evaluated in a bio-assay. In this way, yeast strains could be designated as being either inhibitory, neutral or stimulatory to the growth of O. oeni (strain Lc5p). Inhibition by supernatants of strain EC 1118 was evident, but found to be reduced by ageing the supernatant (with or without lees). Conversely, longer ageing on yeast lees increased the magnitude of the stimulatory response in O. oeni (strain Lc5p) to the supernatant from the wine yeast (strain CY-3079).     

硫酸二乙酯化学诱变选育高核糖核酸酿酒酵母及培养基组成优化

该研究以酿酒酵母（Saccharomyces cerevisiae）BY23为出发菌株,采用硫酸二乙酯（DES）对其进行化学诱变,筛选出一株生长性能好、胞内核糖核酸（RNA）含量高的突变株BY23-195,并以胞内RNA含量为评价指标,通过单因素及正交试验对其糖蜜培养基成分进行优化。结果表明：突变株BY23-195生长性能较好,在酵母浸出粉胨葡萄糖（YEPD）培养基中,RNA含量较出发菌株BY23提高了18.85%。最优糖蜜培养基组分为糖蜜（糖度调至12 °Bx）、酵母浸粉5%、硫酸铵0.05%、磷酸二氢钾0.05%、硫酸亚铁0.05%、硫酸锌0.10%。在此最优培养基组成下,突变株BY23-195胞内其RNA含量达到16.01%,较优化前（13.66%）提高了17.20%。     

响应面法优化嗜酸乳杆菌增殖培养基

为了获得低成本,易配制且增菌效果优良的嗜酸乳杆菌工业化培养基,研究采用Plackett-Burman试验设计筛选出了嗜酸乳杆菌生长的显著影响因子,通过响应面设计试验,得到了嗜酸乳杆菌的优化增殖培养基配比.结果显示:酵母浸粉,葡萄糖,硫酸锰为乳酸菌生长的显著影响因子,优化的增殖培养基配比为:酵母浸粉8.682％,葡萄糖3.842％,硫酸锰0.02％,硫酸镁0.058％,柠檬酸三铵0.2％,Tween-80 0.1％,磷酸氢二钾0.1％,乳清粉0.5％;采用优化培养基后,细胞密度可达到16.49×109cfu/mL;较经典MRS培养基,活菌数提高了20.61倍,其成本较MRS培养基降低了约30％.