响应面法优化谷胱甘肽发酵生产培养基的研究

本研究以酿酒酵母变异株为出发菌株,利用Box—Benhnken中心组合设计和响应面法对该菌株产谷胱甘肽的培养基组成进行优化。实验结果表明：在10°Be’麦芽汁中添加38g／L葡萄糖、6．3g／L（NH4）2SO4及1．9g／L L-半胱氨酸盐酸盐时,谷胱甘肽产量达到120mg／L,生物量达到11．02g／L,比优化前分别提高了71．36％、39．67％。     

Enzymatic Production of Ethanol from Cassava Starch Using Two Strains of Saccharomyces cerevisiae

Cassava starch from TMS 30572 and Idileru were hydrolyzed with α-amylase and amylo-glucosidase before fermentation using two strains of Saccharomyces cerevisiae from palm wine and bakers’ yeast. The per cent yield of sugars and total dissolved solids were 66 % and 26% respectively while pH was 7. Spectrophotometric measurement of the cell growth revealed steady but insignificant (p ≤ 0.05) increase in cell concentrations up to 48 h fermentation time with a gradual decline by 72 h. Saccharomyces cerevisiae strain from palm wine grew best on TMS 30572 hydrolysate at 20% sugar concentration (optical density 0.663; fermentation time 48 h) while on Idileru hydrolysate it grew best at 25% (optical density 0.698; fermentation time 60 h). The pH values obtained from the fermenting hydrolysates for both yeast strains declined gradually as the fermentation progressed with the lowest pH values (3.01 for S. cerevisiae from palm wine; 3.06 for S. cerevisiae from bakers’ yeast) obtained for TMS 30572 cassava variety at 25% sugar concentration. Changes in pH were significant (p ≤ 0.05). The Saccharomyces cerevisiae strain from palm-wine had a higher conversion of available sugar into ethanol. The yield of ethanol was found to vary but the highest ethanol concentration obtained was 5.3% at 10% initial sugar concentration, which gave a sugar conversion efficiency of 37.3%. The results obtained suggest that Saccharomyces cerevisiae strains from sources other than those used conventionally can serve as good substitutes for bio-conversion processes in the industrial production of ethanol.     

Production of Table Wine from Processed Tea Bags Using Different Strains of Saccharomyces cerevisiae

Wine samples were produced from locally available tea infusions (Lipton tea, Top tea and Highland tea) using baker’s yeast (Saccharomyces cerevisiae By1) and yeast cultures from pineapple (S. cerevisiae Py6) and cocoa (S. cerevisiae CY43). Physicochemical analyses and microbial evaluation were undertaken during fermentation. Lipton tea wine produced, using baker’s yeast, pineapple yeast and cocoa yeast had highest alcoholic contents of 7.88%, 6.25% and 7.20%, respectively. Top tea wine produced using the same set of yeasts had highest alcoholic contents of 9.78%, 5.43 and 8.15% respectively, while Highland tea wine produced highest alcoholic contents of 9.78%, 7.07% and 7.61% respectively. Physicochemical analyses for all the wines produced showed that the specific gravity, total solids and pH of the must decreased as fermentation progressed while the titratable acidity remained constant throughout the must fermentation. Colony counts showed a high biomass of yeast cells that decreased as it tended towards the end of fermentation. Sensory analysis of the wines showed that Lipton and Top tea are the most acceptable organoleptically when compared with the commercial wine used as control although the three tested teas were not significantly different statistically. Baker’s yeast was rated as the best yeast for wine fermentation irrespective of the type of tea used. All the wines produced were generally accepted as they were scored above average. This study highlights the potential of using different tea infusions as alternatives to grape and other fruit musts in wine making. It also confirms that commercial yeasts such as baker’s yeast can be used in homemade wine production.     

Effect of Medium Composition on Glucoamylase Production During Batch Fermentation of Recombinant Saccharomyces cerevisiae

Plasmid stability of the recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) strain harboring a pGAC9 plasmid with glucoamylase genes has been investigated in shake flasks and in a bioreactor system using various compositions of media containing glucose or starch as the main carbon and energy source. The medium composition affected both the growth characteristics of S. cerevisiae and stability of the plasmid. Superior plasmid stability was obtained in yeast minimal medium and in complex medium with 0.5 to 2% D‐glucose. Plasmid stability of 92% was obtained in complex medium with 2% D‐glucose yielding 48 units of glucoamylase/g of cells compared to 54% plasmid stability achieved with 2% soluble starch, which yielded 23 units of glucoamylase/g of cells. The plasmid stability increased at high growth rates and decreased with increasing starch concentration in the complex media as compared to glucose medium. The kinetic characteristics of biomass and glucoamylase production were investigated, and a growth kinetic model was used to interpret the experimental results.     

餐厨垃圾水解液培养酿酒酵母发酵产油脂的影响因素

为提高餐厨垃圾水解液发酵产油脂量,探究其发酵生产特征和影响因素,以Saccharomyces cerevisiae As2.516为供试菌株,以初始餐厨垃圾水解液加入量90%(V/V)、搅拌速率180 r/min、通气量2.5 L/min、发酵周期10 d为基础发酵条件进行1 L发酵罐发酵。实验结果表明发酵过程受初始p H值、温度、接种生物量、还原糖含量及无机盐、金属离子浓度影响。其中最适宜初始培养条件为:p H值6、温度30℃、接种量10%,该条件下生物量、油脂产量及产物转化率最大。以实验最适宜培养条件为基础发酵条件时,S.cerevisiae发酵第7d油脂产量最高,为4.26 g/L,生物量12.95 g/L,油脂产率32.9%。经气相色谱分析发酵所得脂肪酸主要由C16及C18脂肪酸组成,其中不饱和脂肪酸占72.09%。餐厨垃圾水解液中无机盐离子含量丰富,外加钾、镁、锰等金属离子抑制菌体生长并影响油脂合成,除微量Cu SO4·5H2O(1×10-4 g/L)外,无需添加其它无机盐。S.cerevisiae利用餐厨垃圾水解底物具有谱宽和抗逆性。     

不同酿酒酵母对柑橘果酒香气成分的影响

研究不同来源酿酒酵母对柑橘果酒香气成分的影响,以锦橙表皮和柑橘园土壤为酵母的来源,经培养、分离、纯化、初筛和复筛得到适于柑橘果酒酿造的酵母菌株。确定其最适生长温度、pH 值及对乙醇和SO2 的耐受性,并用GC-MS 分析其对柑橘果酒香气的影响。结果显示：筛选得到3 株酵母菌L3、L5、L9,其最适生长温度为28～30℃,最适pH 值在3.5 左右,其中L3 耐低温和高温的能力最强,L9 乙醇耐受力最强,L5 对SO2 的耐受能力最强。3 株酵母所酿柑橘果酒的香气成分有特异性差异,庚酸乙酯、萜品油烯等13 种物质为L3 酵母菌所酿柑橘果酒独有;月桂醇、乙酸异丁酯等8 种物质仅见于L5 酵母菌所酿柑橘果酒中;正辛醇、5- 甲基呋喃醛等4 种物质只在L9 酵母菌所酿柑橘果酒中存在。     

The Production of Isoamyl Acetate from Amyl Alcohol by Saccharomyces cerevisiae

Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as “lees oil” or “fusel oil”, of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L‐leucine and amyl alcohol in fermentations carried out by a high ester‐producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro‐DL‐leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.     

响应面法优化酿酒酵母谷胱甘肽的提取条件

利用Box-Benhnken 中心组合设计和响应面法优化乙醇提取酿酒酵母胞内谷胱甘肽的条件。结果表明,谷胱甘肽最佳提取条件为乙醇体积分数43.7%、乙醇添加量7.03mL/0.1g 干酵母粉、抽提时间80min、谷胱甘肽提取率达最大为9.54mg/g。     

不同发酵条件对酿酒酵母甘油产量的影响

为提高酿酒酵母的甘油产量,分别考察不同初始葡萄糖和果糖质量浓度、pH值、发酵温度及SO2添加量对酿酒酵母D254甘油产量的影响。对酿酒酵母D254不同发酵初始条件进行单因素试验,其他因素固定条件下,葡萄糖质量浓度180g/L时酵母菌体生长平稳、生长量最高;果糖质量浓度108g/L时酵母甘油产量最高;pH值为3.5更适宜酵母菌体生长和合成甘油;在发酵温度和SO2添加量的单因素试验中也分别得出适宜发酵温度为28℃和适宜SO2添加量为 20mg/L。通过单因素试验,筛选出最利于酿酒酵母D254生长和产甘油的各因素的最佳质量浓度,进行Plackett-Burman发酵条件组合试验,得到发酵条件最佳组合为：初始葡萄糖质量浓度216g/L、果糖质量浓度144g/L、发酵温度32℃、pH 3.0、SO2添加量40mg/L,此条件下,酿酒酵母D254获得最高甘油产量达655.64μmol/L。     

High Pressure Inactivation Kinetics of Saccharomyces cerevisiae Ascospores in Orange and Apple Juices

High pressure inactivation kinetics (D and z values) of Saccharomyces cerevisiae ascospores were determined in fruit juices and a model juice buffer at pH 3.5 to 5.0. Approximately 0.5 to 1.0 × 10⁶ ascospores/mL were pressurized at 300 to 500 MPa in juice or buffer. D-values ranged from 8 sec to 10.8 min at 500 and 300 MPa, respectively. The range for z-values was 115 to 121 MPa. No differences (P≥0.05) in D (at constant pressure) or z-values among buffers or juices at any pH were determined, indicating little influence of pH in this range and absence of protective or detrimental effects of juice constituents.     

酿酒酵母Y3401产己酸乙酯发酵条件的优化

以酿酒酵母Y3401为研究对象,对该菌株发酵产己酸乙酯条件进行优化。首先,通过单因素对其培养基条件(pH值和糖度)及诱导条件(温度、转速、接种量、乙醇添加量、己酸添加量、前体添加时机和诱导时间)进行优化,然后通过Plackett-Burman试验,筛选出5个显著影响酿酒酵母Y3401产己酸乙酯的因素,再通过最陡爬坡试验确定最大响应区域,最后采用Box-Behnken试验设计及响应面分析,确定其最优产己酸乙酯条件为:糖度14 Brix、初始pH 7、温度25℃、转速180 r/min、接种量3.5%、乙醇添加量8%、己酸添加量0.059%、前体添加时机30 h、诱导时间31 h。在此培养条件下己酸乙酯产量达10.1 mg/L。本研究结果有助于酿酒酵母Y3401更好地应用于白酒发酵中。     

代谢工程改造酿酒酵母生产L-苹果酸

为了研究胞质还原路径对酿酒酵母积累L-苹果酸的影响,通过在酿酒酵母中过量表达源于黄曲霉的丙酮酸羧化酶(Afpyc)、苹果酸脱氢酶(Afmdh)及C4-二羧酸转运蛋白(Afmae),成功构建了L-苹果酸合成的胞质还原路径。结果表明:(1)当低水平表达Afpyc时,其丙酮酸浓度降低了42%,但不能积累L-苹果酸;(2)当共表达Afpyc和Afmdh时,菌株W005积累了1.93 g/L的L-苹果酸,与对照菌株W004相比细胞干重提高了350%,丙酮酸降低了65.9%;(3)当共表达Afpyc、Afmdh和Afmae时,菌株W006的L-苹果酸产量提高了21.2%,达到2.34 g/L;4)通过提高接种量至初始OD_(600)=2,L-苹果酸的产量提高到3.28 g/L。通过在酿酒酵母中过量表达黄曲霉胞质还原路径的关键基因,使得工程菌能够积累L-苹果酸,为目标产物的高效积累提供了一种可借鉴的思路。     

酿酒酵母生产谷胱甘肽分批发酵动力学研究

以麦芽汁为发酵培养基,对7.5L 自动发酵罐中酿酒酵母Y518 分批发酵生产谷胱甘肽的实验数据进行分析,建立谷胱甘肽分批发酵动力学模型。通过对符合菌体生长的Logistic 方程、产物生成的Luedeking-Piret 方程和基质消耗的物料衡算方程进行最优参数估计和非线性拟合,分别得到了相应的动力学模型和最佳模型参数值。对动力学模型的拟合曲线进行分析,发现模型的计算值与实验值能较好地拟合,表明本实验建立的分批发酵动力学模型能较好地反映谷胱甘肽分批发酵过程。     

Sweet Wine Production by Two Osmotolerant Saccharomyces cerevisiae Strains

The use of Saccharomyces cerevisiae to produce sweet wine is difficult because yeast is affected by a hyperosmotic stress due to the high sugar concentrations in the fermenting must. One possible alternative could be the coimmobilization of the osmotolerant yeast strains S. cerevisiae X4 and X5 on Penicillium chrysogenum strain H3 (GRAS) for the partial fermentation of raisin musts. This immobilized has been, namely, as yeast biocapsules. Traditional sweet wine (that is, without fermentation of the must) and must partially fermented by free yeast cells were also used for comparison. Partially fermented sweet wines showed higher concentration of the volatile compounds than traditionally produced wines. The wines obtained by immobilized yeast cells reached minor concentrations of major alcohols than wines by free cells. The consumption of specific nitrogen compounds was dependent on yeast strain and the cellular immobilization. A principal component analysis shows that the compounds related to the response to osmotic stress (glycerol, acetaldehyde, acetoin, and butanediol) clearly differentiate the wines obtained with free yeasts but not the wines obtained with immobilized yeasts.     

酿酒酵母CWY132以糖蜜为碳源生产2-苯乙醇的培养条件

对酿酒酵母CWY132利用糖蜜为碳源,生物转化L-苯丙氨酸(L-Phe)生成2-苯乙醇的液-液两相分批补料培养工艺进行了研究。发现在以聚丙二醇(PPG1500)作为抽提剂的液-液两相培养中,采用分批补料方式添加糖蜜和L-Phe使2-苯乙醇产量明显提高。在0、4、8、12、24 h分别添加40 g/L糖蜜,4、8 h分别添加12 g/L、3g/L L-Phe的液-液两相分批补料培养中,2-苯乙醇产量最高达到9.03 g/L,其中抽提相中2-苯乙醇浓度22.5 g/L,比优化前的液-液两相单批培养中的产量4.82 g/L提高了87%。底物L-Phe的摩尔转化率达到0.82 mol/mol。     

Rapid protein extraction from Saccharomyces cerevisiae

We have developed and evaluated an easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate (SDS)–gel electrophoresis and Western blotting. The procedure comprises a centrifugation step to harvest the cells, addition of a sample buffer and heating, then another centrifugation step before applying the extracted proteins found in the supernatant to an SDS gel. It is applicable to the study of large numbers of samples in 1 day. This procedure is easier, quicker, and as efficient as procedures using base and 2-mercaptoethanol, but somewhat less efficient than lysis with glass beads under certain conditions.     

基于CRISPR-Cas9系统的Saccharomyces cerevisiae基因删除

建立CRISPR-Cas9介导的在Saccharomyces cerevisiae双倍体细胞中进行基因敲除的方法。以can1基因敲除后的表型验证该CRISPR-Cas9系统的有效性,can1基因的失活效率达到4%。利用该系统又分别敲除了pdc、adh3、adh2、adh1、 pdh等基因,单基因编辑效率分别为4/48、3/48、1/48、3/28、1/16。确定了基因连续敲除的方法流程,pdc、adh3、adh2三个基因全部敲除,整个过程用时17 d。探索了双基因一次转化同时敲除的方法,将adh5、lip两个基因同时敲除用时6 d,基因编辑效率分别为9/32和10/32。     

Formation of Volatile Sulfur-Containing Compounds by Saccharomyces cerevisiae in Soymilk Supplemented with L-methionine

The formation of volatile sulfur-containing flavor compounds from L-methionine catabolism by Saccharomyces cerevisiae EC-1118 was investigated in soymilk supplemented with this sulfur-containing amino acid with a focus on methionol (3-methylthio-1-propanol). Methionol produced from L-methionine metabolism of yeast in soymilk was extracted by solid-phase microextraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). The effects of time (0 – 72 h), L-methionine concentration (0.05–0.20%), yeast extract concentration (0.00–0.30%), initial medium pH (4.5–6.9), and temperature (25–34°C) on the production of methionol were investigated. One-liter scale-up fermentation of soymilk by Saccharomyces cerevisiae EC-1118 was conducted for 48 h under static conditions with 0.15% of L-methionine, 0.10% of yeast extract, initial medium pH of 5.5, and temperature of 25°C, yielding 190 ppm of methionol. GC-olfactometry dilution analysis was conducted on extracts of the fermented soymilk, and no single compound was found to account for the overall odor perception. Methional (3-methylthio-1-propanal) and methionol were determined to be most potent odor-active compounds in the fermented soymilk. The fermented soy milk may serve as a complex sulfur flavor concentrate for applications in foods such as fermented soy products, enzyme-modified, and imitation cheeses.     

Production of senescent cells of Saccharomyces cerevisiae by centrifugal elutriation

The centrifugal elutriator has been used as a baby machine by loading the chamber with a population of mixed-generation daughter cells and allowing this population to grow, divide and age under continuous washing-out of newborn daughter cells. Clear peaks in the number of elutriated cells were reproducibly obtained for at least ten generations. The parent cells growing in the chamber continued to divide at the steady-state generation time of 95–100 min, showing no change in cycle time during aging. The washed-out daughter cells increased in volume during the first five generations from their steady-state value of 17 μm³ to a maximum of 34 μm³. As to be expected, the generation times of these large daughters, determined in a synchronous batch culture, were shorter (130 min) than that of the steady-state daughters (240 min), even when derived from 15-generation parents. No indication for a volume increase of daughter cells without bud was observed when a population was allowed to grow in the chamber without washing-out the smaller daughter cells. The 15-generation parent population, recovered from the chamber, had an average volume of 80 μm³ and consisted of: (i) 71% cells with more than ten scars, (ii) 13% cells with one to nine scars, and (iii) 17% daughter cells. The production of senescent cells by undisturbed growth in the elutriator chamber has been prolonged to 29 generations. The method is therefore suitable to examine what factors determine the life span of budding yeast.