代谢木糖的重组工业酿酒酵母构建及其乙醇发酵

为了使工业酿酒酵母(Saccharomyces cerevisiae)能直接利用木糖发酵乙醇,本研究设计带有 kanMX和ura两种不同筛选标记的强启动子TPI的载体,并将启动子插入木酮糖激酶(xylulokinase gene,XKS1)及非氧化磷酸戊糖途径(pentose phosphate pathway, PPP)关键基因之前,并构建木糖异构酶基因(xylose isomerase gene,XylA)表达载体pYES2-FBA-xylA,将该载体转入XKS1及PPP关键基因增强表达的重组菌株,XylA在工业酿酒酵母里成功表达,其活性为0.39 U/mg蛋白.qRT-PCR结果显示,XKS1及PPP四个关键基因TAL、RPE、RKI和TKL分别比出发菌株表达量提高4.08、1.62、3.98、17.36和4.17倍.重组菌株葡萄糖和木糖共发酵,重组菌株木糖代谢比原始菌株提高17.64％;研究结果表明,通过增强木糖代谢流关键基因的表达以及XI的表达,使工业酿酒酵母获得直接代谢木糖能力,这为工业酿酒酵母生产纤维素乙醇提供参考.     

Production of Pearl Millet Irrigated with Different Levels of Brackish Water and Organic Matter

ABSTRACT

The objective is to determine the growth, yield and chemical characteristics of pearl millet irrigated with different levels of brackish water and organic matter in two cultivation cycles. The experimental design was randomized blocks in a 4 × 4 factorial arrangement, composed of 4 levels of brackish irrigation (25, 50, 75 and 100% evapotranspiration), 4 levels of organic matter (0, 15, 30 and 45 t ha^?1) with 3 repetitions. Along two cultivation cycles, pearl millet crop was analyzed for: plant growth variables, fresh and dry mass production, water-use efficiency, and chemical composition. There was significant interaction between applied water levels and organic matter for lignin in the 2nd cut (P < .05). The number of leaves, number of dead leaves and fresh mass production were influenced by the isolated effect of brackish water levels in cut 1 (P < .05). In turn, fresh mass production, dry mass production, plant height, leaves length, panicle length, fresh mass production, crude protein and ether extract were influenced by the isolated effect of saline water levels in the 2nd cut (P < .05). Growth, biomass production and chemical composition variables in second cut are positively influenced by different brackish irrigation levels under low rainfall conditions.     

Biocatalytic production of dihydrocoumarin from coumarin by Saccharomyces cerevisiae

Natural dihydrocoumarin, which is of great interest in the flavor industry, was biotechnologically produced from pure coumarin or tonka bean meal with Pseudomonas orientalis, Bacillus cereus, and various Saccharomyces cerevisiae strains. Coumarin was shown to be converted to melilotic acid, which yielded dihydrocoumarin upon distillation during purification. About 1.0 g/L product was obtained from 25 g/L tonka beans with S. cerevisiae within 147 h. This dihydrocoumarin thus fulfills all of the criteria of a natural raw material and can be used as a natural flavoring in accordance with U.S. and European Union regulations.     

Ethanol Production from Modified and Conventional Dry‐Grind Processes Using Different Corn Types

Different corn types were used to compare ethanol production from the conventional dry‐grind process to wet or dry fractionation processes. High oil, dent corn with high starch extractability, dent corn with low starch extractability and waxy corn were selected. In the conventional process, corn was ground using a hammer mill; water was added to produce slurry which was fermented. In the wet fractionation process, corn was soaked in water; germ and pericarp fiber were removed before fermentation. In the dry fractionation process, corn was tempered, degerminated, and passed through a roller mill. Germ and pericarp fiber were separated from the endosperm. Due to removal of germ and pericarp fiber in the fractionation methods, more corn was used in the wet (10%) and dry (15%) fractionation processes than in the conventional process. Water was added to endosperm and the resulting slurry was fermented. Oil, protein, and residual starch in germ were analyzed. Pericarp fiber was analyzed for residual starch and neutral detergent fiber (NDF) content. Analysis of variance and Fisher's least significant difference test were used to compare means of final ethanol concentrations as well as germ and pericarp fiber yields. The wet fractionation process had the highest final ethanol concentrations (15.7% v/v) compared with dry fractionation (15.0% v/v) and conventional process (14.1% v/v). Higher ethanol concentrations were observed in fractionation processes compared to the conventional process due to higher fermentable substrate per batch available as a result of germ and pericarp fiber removal. Germ and pericarp yields were 7.47 and 6.03% for the wet fractionation process and 7.19 and 6.22% for the dry fractionation process, respectively. Germ obtained from the wet fractionation process had higher oil content (34% db) compared with the dry fractionation method (11% db). Residual starch content in the germ fraction was 16% for wet fractionation and 44% for dry fractionation. Residual starch in the pericarp fiber fraction was lower for the wet fractionation process (19.9%) compared with dry fractionation (23.7%).     

Use of Mefluidide to Alter Growth and Nutritive Value of Pearl Millet

Abstract

Pearl millet (Pennisetum americanum L. Leeke) has an inherent rapid growth rate that often presents management problems to achieve optimum utilization by livestock throughout the growing season. Both the rapid spring–summer growth rate and photoperiodism that diminishes growth in late summer—early fall creates the need to use both grazing and mechanical defoliation to have efficient use of forage production. The objectives of this 2‐year field study were to evaluate the effects of a growth regulator, mefluidide (N‐[2, 4‐dimethyl‐5‐([(trifluoromethyl) sulfonyl] amino) phenyl] acetamide), on growth characteristics and nutritive parameters of “Millex 24” pearl millet. Mefluidide was broadcast applied at different rates to determine the effect on dry matter (DM) yield and nutritive value of leaves and stems of pearl millet. Mefluidide reduced DM biomass at each weekly harvest, and at three weeks post‐treatment DM was reduced 3‐fold in Year 1 and 2‐fold in Year 2. Percent leaf, however, was nearly doubled by mefluidide application. Crude protein (CP) of leaves was not affected by mefluidide, however, CP of treated stems was significantly higher (P < 0.05) than untreated pearl millet. Fiber components were lowered (P < 0.05) in treated leaves and stems compared to untreated pearl millet. The 2‐year study showed that mefluidide enhanced nutritive value of pearl millet, but at the expense of DM.     

Factors Impacting Ethanol Production from Grain Sorghum in the Dry-Grind Process

The goal of this research is to understand the key factors affecting ethanol production from grain sorghum. Seventy genotypes and elite hybrids with a range of chemical compositions and physical properties selected from ≈1,200 sorghum lines were evaluated for ethanol production and were used to study the relationships of composition, grain structure, and physical features that affect ethanol yield and fermentation efficiency. Variations of 22% in ethanol yield and 9% in fermentation efficiency were observed among the 70 sorghum samples. Genotypes with high and low conversion efficiencies were associated with attributes that may be manipulated to improve fermentation efficiency. Major characteristics of the elite sorghum genotypes for ethanol production by the dry-grind method include high starch content, rapid liquefaction, low viscosity during liquefaction, high fermentation speed, and high fermentation efficiency. Major factors adversely affecting the bioconversion process are tannin content, low protein digestibility, high mash viscosity, and an elevated concentration of amylose-lipid complex in the mash.     

Assessing Fermentation Quality of Grain Sorghum for Fuel Ethanol Production Using Rapid Visco‐Analyzer

The Rapid Visco‐Analyzer (RVA) was used to characterize the pasting properties of 68 sorghum grains with a standard 23‐min temperature profile. The results showed a strong linear relationship between ethanol yield and final viscosity as well as setback. Ethanol yield increased as final viscosity decreased. A modified RVA procedure (10 min) with an application of α‐amylase was developed to simulate the liquefaction step in dry‐grind ethanol production. There was a remarkable difference in mashing properties among the sorghum samples with the normal dosage of α‐amylase. The sorghum samples which were difficult to liquefy in the mashing step had much higher peak viscosities than the samples that were easily liquefied. The results also showed that the relationship between conversion efficiency and mashing property was significant. Tannins cause high mash viscosities. There was a strong linear relationship between tannin content and final viscosity as well as peak viscosity. The modified RVA procedure is applicable not only for characterization of mashing properties but also for optimization of α‐amylase doses for starch liquefaction.     

Antioxidant protection of resveratrol and catechin in Saccharomyces cerevisiae

Moderate consumption of red wine reduces the risk of heart disease and extends lifespan, but the relative contribution of wine polyphenols to these effects is unclear. In this work, the capacity of resveratrol and catechin to protect the eukaryotic microorganism Saccharomyces cerevisiae against oxidative stress caused by different agents, hydrogen peroxide, carbon tetrachloride, and cadmium, was evaluated. Under all stress conditions, both polyphenols increased tolerance, although their protection was more evident under peroxide exposure. By using mutant strains deficient in specific antioxidant defense systems (superoxide dismutases, catalase, or glutathione), it was observed that increased H2O2 tolerance produced by both polyphenols was associated with catalase, as well as the rise in survival rates caused by resveratrol under CCl4. The acquisition of tolerance was correlated with a reduction in lipid peroxidation, indicating that the antioxidant property of resveratrol and catechin involves protection against membrane oxidation.     

高压脉冲电场对酿酒酵母钝化机理研究

为探究高压脉冲电场（PEF）对微生物的钝化机理,该试验主要采用Lowry法、SDS-PAGE电泳、APIZYM试剂盒、流式细胞仪等方法测定PEF处理对酿酒酵母蛋白质含量、胞内酶活性和DNA等的影响。结果显示：PEF处理后,酿酒酵母胞内蛋白质含量显著降低（p<0.05）,SDS-PAGE 电泳图分析发现酿酒酵母胞内蛋白谱图不清晰,且有小分子和 60 KDa 以上大分子蛋白谱带缺失,酿酒酵母细胞内13种酶的活性均有不同程度的下降,细胞DNA含量降低,部分DNA变性。PEF钝化微生物与其胞内酶失活、蛋白质和DNA发生外泄或变性有关。     

Hydrogen peroxide removal with magnetically responsive Saccharomyces cerevisiae cells

Hydrogen peroxide (HP) is a promising chemical sanitizer for use in the food industry. Its residues have to be decomposed, usually using an enzyme process employing catalase. In order to offer an inexpensive biocatalyst and to simplify subsequent manipulation, we have prepared magnetically responsive alginate beads containing entrapped Saccharomyces cerevisiae cells and magnetite microparticles. Larger beads (2-3 mm in diameter) were prepared by dropping the mixture into calcium chloride solution, while microbeads (the diameter of majority of particles ranged between 50 and 100 microm) were prepared using the water in oil emulsification process. In general, microbeads enabled more efficient HP decomposition. The prepared microparticulate biocatalyst caused efficient decomposition of HP in water solutions (up to 2% concentration), leaving very low residual HP concentration after treatment (below 0.001% under appropriate conditions). The biocatalyst was stable; the same catalytic activity was observed after one month storage at 4 degrees C, and the microbeads could be used at least five times.     

Biodegradation of 3-chloro-1,2-propanediol with Saccharomyces cerevisiae

A novel enzymatic dehalogenating activity of 3-chloro-1,2-propanediol (3-MCPD) with Saccharomyces cerevisiae (baker's yeast) is reported. All bioconversion assays were carried out under aerobic conditions, at 28 degrees C, and the kinetics were monitored. The biodegradation was performed at different pH values (6.2, 7.0, and 8.2), in the presence and absence of glucose, using racemic 3-MCPD at two different concentrations (7.3 micromol/L and 27 mmol/L). Optimal conversion (68%) of racemic (R,S)-3-MCPD at a concentration of 27 mmol/L was achieved after 48 h of reaction time, at pH 8.2, and in the presence of glucose. At a concentration of 7.3 micromol/L, 73% degradation was observed after 72 h, at pH 8.2 and in the absence of glucose. Under the same experimental conditions, the conversion of pure (S)-3-MCPD (85%) was higher than that of the (R)-enantiomer (60%).     

瘤胃真菌与酿酒酵母仿生共培养提升秸秆发酵产乙醇量

玉米秸秆中具有较高的纤维素、半纤维素含量,是一种具有稳定产率、可集中处理、可代替木材作制浆原料的生物质材料。为了研究厌氧微生物与酵母共培养预处理玉米秸秆的产物,该研究模拟反刍动物消化玉米秸秆的过程,从羊瘤胃液中分离出厌氧真菌（Pecoramyces sp.）。以玉米秸秆茎皮碎为底物,与厌氧真菌、酿酒酵母菌S1145在39℃进行共培养72 h,分析发酵对秸秆茎皮降解及其代谢产物的影响。结果表明,在瘤胃真菌作用下,添加不同量的酿酒酵母可对代谢产物中乙醇含量产生影响,其中添加5 mL酿酒酵母时产生的乙醇含量最高,占总代谢产物比例为32.09%,相对于未添加酿酒酵母的对照组,乙醇含量提高了23.04百分点。研究表明,在厌氧真菌与酿酒酵母共培养预处理玉米秸秆茎皮的过程中,添加酿酒酵母可提高乙醇产量,为玉米秸秆高效资源化处理和生物质燃料生产提供了一种可靠的方法。     

Use of Phytases in Ethanol Production from E‐Mill Corn Processing

Effects of phytase addition, germ, and pericarp fiber recovery were evaluated for the E‐Mill dry grind corn process. In the E‐Mill process, corn was soaked in water followed by incubation with starch hydrolyzing enzymes. For each phytase treatment, an additional phytase incubation step was performed before incubation with starch hydrolyzing enzymes. Germ and pericarp fiber were recovered after incubation with starch hydrolyzing enzymes. Preliminary studies on phytase addition resulted in germ with higher oil (40.9%), protein (20.0%), and lower residual starch (12.2%) contents compared to oil (39.1%), protein (19.2%), and starch (18.1%) in germ from the E‐Mill process without phytase addition. Phytase treatment resulted in lower residual starch contents in pericarp fiber (19.9%) compared to pericarp fiber without phytase addition (27.4%). Results obtained led to further investigation of effects of phytase on final ethanol concentrations, germ, pericarp fiber, and DDGS recovery. Final ethanol concentrations were higher in E‐Mill processing with phytase addition (17.4% v/v) than without addition of phytase (16.6% v/v). Incubation with phytases resulted in germ with 4.3% higher oil and 2.5% lower residual starch content compared to control process. Phytase treatment also resulted in lower residual starch and higher protein contents (6.58 and 36.5%, respectively) in DDGS compared to DDGS without phytase incubations (8.14 and 34.2%, respectively). Phytase incubation in E‐Mill processing may assist in increasing coproduct values as well as lead to increased ethanol concentrations.     

New Saccharomyces cerevisiae baker's yeast displaying enhanced resistance to freezing

Three procedures were used to obtain new Saccharomyces cerevisiae baker's yeasts with increased storage stability at -20, 4, 22, and 30 degrees C. The first used mitochondria from highly ethanol-tolerant wine yeast, which were transferred to baker's strains. Viability of the heteroplasmons was improved shortly after freezing. However, after prolonged storage, viability dramatically decreased and was accompanied by an increase in the frequency of respiratory-deficient (petite) mutant formation. This indicated that mitochondria were not stable and were incompatible with the nucleus. The strains tested regained their original resistance to freezing after recovering their own mitochondria. The second procedure used hybrid formation after protoplast fusion and isolation on selective media of fusants from baker's yeast meiotic products resistant to parafluorphenylalanine and cycloheximide, respectively. No hybrids were obtained when using the parentals, probably due to the high ploidy of the baker's strains. Hybrids obtained from nonisogenic strains manifested in all cases a resistance to freezing intermediate between those of their parental strains. Hybrids from crosses between meiotic products of the same strain were always more sensitive than their parentals. The third method was used to develop baker's yeast mutants resistant to 2-deoxy-d-glucose (DOG) and deregulated for maltose and sucrose metabolism. Mutant DOG21 displayed a slight increase in trehalose content and viability both in frozen doughs and during storage at 4 and 22 degrees C. This mutant also displayed a capacity to ferment, under laboratory conditions, both lean and sweet fresh and frozen doughs. For industrial uses, fermented lean and sweet bakery products, both from fresh and frozen doughs obtained with mutant DOG21, were of better quality with regard to volume, texture, and organoleptic properties than those produced by the wild type.     

Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: evaluation of extraction methods and immunolocalization

Yeast-derived haze-protective mannoprotein material (HPM) offers protection to white wines from commercially unacceptable turbidities. HPM extraction methods have been evaluated using three winemaking strains of Saccharomyces cerevisiae. Digestion with Zymolyase of cells pretreated with DTE and EDTA gave the greatest yields of active material. Heat treatment of cells with SDS also released active material but the quantities were low. Treatment of the cells in an autoclave or with a French pressure device was less effective. A detailed study was conducted on the strain Maurivin PDM. SDS was not necessary to extract HPM from PDM; boiling the cells for 5 min in Tris buffer was sufficient. HPM could also be extracted with EDTA during the pretreatment of the cells prior to Zymolyase digestion. The data suggest that HPM was noncovalently linked to other cell wall components and loosely associated with the cell wall. An immunological investigation showed that a specific mannoprotein with haze-protective activity, HPF1, was located primarily on the outermost and innermost layers of the cell wall.     

Quercetin increases oxidative stress resistance and longevity in Saccharomyces cerevisiae

Quercetin, the major flavonol found in several fruits and vegetables, is a natural antioxidant with potential anticancer and antiaging activities. In this paper, the effect of quercetin in Sacharomyces cerevisiae cells submitted to oxidative stress was studied. Hydrogen peroxide resistance increased in cells pretreated with quercetin. Cellular protection was correlated with a decrease in oxidative stress markers, namely, levels of reactive oxygen species, glutathione oxidation, protein carbonylation, and lipid peroxidation. The acquisition of H2O2 resistance was not associated with the induction of antioxidant defenses or with iron chelation. Oxidative stress is a limiting factor for longevity. In agreement, quercetin also increased 60% chronological life span. These results support the utilization of yeast as a useful model to screen in vivo for natural antioxidants with putative health beneficial effects.     

利用木糖产乙醇酵母菌株的构建

以酿酒酵母缺陷型(Saccharomyces cerevisiae)与黏红酵母(Rhodotorula glutinis)为亲本菌株,分别采用聚乙二醇(PEG)和电击方法进行原生质体融合。对得到的融合细胞进行木糖发酵验证,通过检测发酵液中乙醇的含量,筛选出1株产乙醇的重组酵母菌株。利用该重组酵母进行木糖、葡萄糖共发酵,发酵条件为接种量3%,发酵温度27~30 ℃,pH 5.5~6.5,发酵组分为4%木糖、3%蛋白胨、2%酵母浸粉和12%葡萄糖,发酵周期40 h,结果重组酵母可发酵木糖,木糖消耗速率明显低于葡萄糖,乙醇浓度为72 g/L。     

Fiber Separated from Distillers Dried Grains with Solubles as a Feedstock for Ethanol Production

In the dry-grind process, corn starch is converted into sugars that are fermented into ethanol. The remaining corn components (protein, fiber, fat, and ash) form a coproduct, distillers dried grains with solubles (DDGS). In a previous study, the combination of sieving and elutriation (air classification), known as the elusieve process, was effective in separating fiber from DDGS. In this study, elusieve fiber was evaluated for ethanol production and results were compared with those reported in other studies for fiber from different corn processing techniques. Fiber samples were pretreated using acid hydrolysis followed by enzymatic treatment. The hydrolyzate was fermented using Escherichia coli FBR5 strain. Efficiency of ethanol production from elusieve fiber was 89–91%, similar to that for pericarp fiber from wet-milling and quick fiber processes (86–90%). Ethanol yields from elusieve fiber were 0.23–0.25 L/kg (0.027–0.030 gal/lb); similar to ethanol yields from wet-milling pericarp fiber and quick fiber. Fermentations were completed within 50 hr. Elusieve fiber conversion could result in 1.2–2.7% increase in ethanol production from dry-grind plants. It could be economically feasible to use elusieve fiber along with other feedstock in a plant producing ethanol from cellulosic feedstocks. Due to the small scale of operation and the stage of technology development for cellulosic conversion to ethanol, implementation of elusieve fiber conversion to ethanol within a dry-grind plant may not be currently economically feasible.     

Evaluation of Waxy Grain Sorghum for Ethanol Production

The objective of this research was to investigate the fermentation performance of waxy grain sorghum for ethanol production. Twenty‐five waxy grain sorghum varieties were evaluated with a laboratory dry‐grind procedure. Total starch and amylose contents were measured following colorimetric procedures. Total starch and amylose contents ranged from 65.4 to 76.3% and from 5.5 to 7.3%, respectively. Fermentation efficiencies were in the range of 86.0–92.2%, corresponding to ethanol yields of 2.61–3.03 gallons/bushel. The advantages of using waxy sorghums for ethanol production include easier gelatinization and low viscosity during liquefaction, higher starch and protein digestibility, higher free amino nitrogen (FAN) content, and shorter fermentation times. The results showed a strong linear relationship between FAN content and fermentation rate. Fermentation rate increased as FAN content increased, especially during the first 30 hr of fermentation (R² = 0.90). Total starch content in distillers dried grains with solubles (DDGS) was less than 1% for all waxy varieties.