Structural Complexity of the Nitrogen Source and Influence on Yeast Growth and Fermentation

The structural complexity of the nitrogen source strongly affects both biomass and ethanol production by industrial strains of Saccharomyces cerevisiae, during fermentation in media containing glucose or maltose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low glucose and maltose concentrations independent of nitrogen supplementation. At high sugar concentrations diauxie was not easily observed, and growth and ethanol production depended on the nature of the nitrogen source. This was different for baking and brewing ale and lager yeast strains. Sugar concentration had a strong effect on the shift from oxido‐fermentative to oxidative metabolism. At low sugar concentrations, biomass production was similar under both peptone and casamino acid supplementation. Under casamino acid supplementation, the time for metabolic shift increased with the glucose concentration, together with a decrease in the biomass production. This drastic effect on glucose fermentation resulted in the extinction of the second growth phase, probably due to the loss of cell viability. Ammonium salts always induced poor yeast performance. In general, supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, inducing higher biomass and ethanol production, and preserving yeast viability, in both glucose and maltose media, for baking and brewing ale and lager yeast strains. Determination of amino acid utilization showed that most free and peptide amino acids present, in peptone and casamino acids, were utilized by the yeast, suggesting that the results described in this work were not due to a nutritional status induced by nitrogen limitation.     

Effect of Sugar Catabolite Repression in Correlation with the Structural Complexity of the Nitrogen Source on Yeast Growth and Fermentation

Biomass and ethanol production by industrial Saccharomyces cerevisiae strains were strongly affected by the structural complexity of the nitrogen source during fermentation in media containing galactose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low galactose concentrations independent of nitrogen supplementation. At high sugar concentrations altered patterns of galactose utilisation were observed. Biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for baking and brewing ale and lager strains. Baking yeast showed improved galactose fermentation performance in the medium supplemented with casamino acids. High biomass production was observed with peptone and casamino acids for the ale brewing strain, however high ethanol production was observed only in the presence of casamino acids. Conversely, peptone was the nitrogen supplement that induced higher biomass and ethanol production for the lager brewing strain. Ammonium salts always induced poor yeast performance. The results with galactose differed from those obtained with glucose and maltose which indicated that supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, suggesting that sugar catabolite repression has a central role in yeast performance in a medium containing nitrogen sources with differing levels of structural complexity.     

Glucose and Fructose Fermentation by Wine Yeasts in Media Containing Structurally Complex Nitrogen Sources

Glucose and fructose fermentations by industrial yeasts strains are strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. In this study two Saccharomyces cerevisiae industrial wine strains were grown, under shaken and static conditions, in a media containing either a) 20% (w/v) glucose, or b) 10% (w/v) fructose and 10% (w/v) glucose or c) 20% (w/v) fructose, all supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Data suggest that a complex structured nitrogen source is not submitted to the same control mechanisms as those involved in the utilization of simpler structured nitrogen sources, and mutual interaction between carbon and nitrogen sources, including the mechanisms involved in the regulation of aerobic/anaerobic metabolism, may play an important role in defining yeast fermentation performance and the differing response to the structural complexity of the nitrogen source, with a strong impact on fermentation performance.     

Sucrose Fermentation by Brazilian Ethanol Production Yeasts in Media Containing Structurally Complex Nitrogen Sources

Four Saccharomyces cerevisiae Brazilian industrial ethanol production strains were grown, under shaken and static conditions, in media containing 22% (w/v) sucrose supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Sucrose fermentations by Brazilian industrial ethanol production yeasts strains were strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. Data suggest that yeast strains vary in their response to the nitrogen source's complex structure and to oxygen availability. In addition, the amount of trehalose produced could be correlated with the fermentation performance of the different yeasts, suggesting that efficient fuel ethanol production depends on finding conditions which are appropriate for a particular strain, considering demand and dependence on available nitrogen sources in the fermentation medium.     

咖啡渣酿酒酵母的筛选及氮源优化营养条件的研究

该文首先对比不同酵母菌酿造咖啡渣酒的产气、产酒精和产香能力,而后探究氮源的种类和浓度对酵母菌酿造咖啡渣酒的影响。结果表明,在备筛的4株酵母菌中,Saccharomyces cerevisiae D254产气能力、产酒精能力和发酵液的感官评分都较佳,适用于酿造咖啡渣酒;氮源的加入能有效解决咖啡渣酒发酵迟缓的问题,提高酵母菌生物量、酒体酒精度,降低残糖含量;其中添加0.20%磷酸氢二铵作氮源时效果最好,生物量由1.21 g/L增加至1.73 g/L,酒精度由7.3%vol增加至9.6%vol,残糖由19.52 g/L降低至5.73 g/L,且所得咖啡渣酒具有咖啡特有的芳香,酒体柔和协调。     

Efficient very‐high‐gravity fermentation of sugarcane molasses by industrial yeast strains

In this study industrial strains were inoculated, in successive cycles, at high cell density into a sugarcane‐based juice containing normal (22%, w/v) and very high sucrose (30%, w/v) levels and supplemented with peptone as a nitrogen source. At 30 °C, in shaken cultures, in the normal gravity situation, efficient sucrose utilization occurred in both the supplemented and unsupplemented medium. At higher sugar concentrations, supplementation with peptone induced a more efficient fermentation compared with the unsupplemented medium, with higher biomass accumulation and maintenance of cell viability, and ethanol levels as high as 16% (v/v). Trehalose was also high during the cycles, probably as a necessary response of the yeast to the high stress fermentation conditions. This suggests that it is possible to increase ethanol production by using very‐high‐fermentation technology and that nutritional supplementation would have a positive effect on the fermentation, allowing for efficient sugar consumption and cell viability maintenance. Copyright © 2016 The Institute of Brewing & Distilling     

Evaluation of Brazilian ethanol production yeasts for maltose fermentation in media containing structurally complex nitrogen sources

Maltose and glucose fermentations are strongly affected by the structural complexity of the nitrogen source and by the presence of oxygen. In this study five industrial Saccharomyces cerevisiae strains were grown in synthetic medium, containing maltose or glucose, supplemented with different nitrogen sources, with or without agitation. All strains were able to grow and efficiently ferment glucose, but not all strains were able to grow and ferment maltose well. Peptone and ammonium sulfate induced improved fermentation for all strains and conditions. Under agitation, as expected, higher biomass accumulation was detected. Casamino acids supplementation induced efficient maltose fermentation for all of the strains under aerated conditions, but differing maltose utilization patterns were observed for the static cultures. Copyright © 2012 The Institute of Brewing & Distilling     

Very high gravity sucrose fermentation by Brazilian industrial yeast strains: effect of nitrogen supplementation

In this study three industrial strains were inoculated into natural sugar cane juice containing 22, 30 and 35% (w/v) sucrose supplemented with nitrogen sources with differing structural complexities, which varied from a single ammonium salt (ammonium sulphate) to peptides (peptone), under aerobic and anaerobic conditions. At 30 °C, in shaken cultures, it was found that efficient sucrose utilization occurred only in media supplemented with a nitrogen source. In general, under agitation, supplementation with peptone led to more efficient fermentation compared with ammonium sulphate supplementation, with higher biomass accumulation and maintenance of cell viability. In a 35% (w/v) sucrose fermentation, under conditions with an inoculation of low cell density, nitrogen supplementation was required to obtain complete sucrose utilization, suggesting the possibility of producing wines with higher amounts of ethanol under working conditions that approach the limit of yeast alcohol tolerance. The results in this study have industrial relevance and they indicate that, under appropriate environmental and nutritional conditions, the commercial Brazilian yeast strains studied can efficiently use sugar, with high cell viability, even during very high gravity sucrose fermentation conditions. Copyright © 2012 The Institute of Brewing & Distilling     

CAMBRIDGE PRIZE LECTURE IMPROVING YEAST FERMENTATION PERFORMANCE

A number of factors affecting yeast fermentation performance have been investigated. These include the mode of substrate feeding, nutrient supplementation, temperature, osmotic pressure, oxygen, intracellular ethanol accumulation, and yeast ethanol tolerance. Nutrient supplementation was observed to play a key role in yeast fermentations employing high gravity media and at high temperatures. Furthermore, complete attenuation of high gravity wort (25°P) could be achieved by optimizing the yeast pitching rate, fermentation temperature, and level of wort oxygenation. Genetic manipulation techniques, such as spheroplast fusion, were successfully employed to obtain ethanol and osmotolerant yeast strains. In addition, a number of stable 2-deoxy-D-glucose resistant mutants, isolated from brewing and non-brewing yeast strains, were observed to rapidly utilize maltose and maltotriose in the presence of high concentrations of glucose. Fermentation and ethanol production rates were increased in these strains. Therefore, employing strategies of optimized fermentation conditions and strain development have resulted in improvements in yeast fermentation performance.     

玫瑰香干红葡萄酒自然发酵过程中优势酵母分离鉴定及其应用潜力分析

为酿造特色玫瑰香干红葡萄酒,开发本土发酵剂是目前重要发展方向。从昌黎产区玫瑰香干红葡萄酒自然发酵醪中共分离得到337株酵母,通过WL(Wallerstein laboratory)鉴别培养基对其进行分类鉴定、计数,将其鉴别为7大类,根据计数结果选取优势酵母接种至BIGGY(bismuth sulphite glucose glycine yeast)固体培养基,从中进一步筛选不产H2S酵母,进行分子鉴定,结果显示HBKS-Y1、HBKS-Y3为酿酒酵母,HBKS-Y2为葡萄汁有孢汉逊酵母。考察3株本土酵母酿造因子耐性,发现3株酵母对乙醇、SO2、糖度均具有较高耐性,满足葡萄酒酿造要求;以商用活性干酵母FX10葡萄汁发酵为对照,考察3株酵母对玫瑰香葡萄汁单菌、混菌发酵理化指标和香气成分的影响,3株本土酵母单菌和混菌发酵各指标(例如:还原糖、酒精度、总酸、挥发酸、甘油和乙醛含量)均符合干红葡萄酒酿造标准。同时发现,与HBKS-Y2单菌发酵相比,3株酵母混菌发酵总酸和挥发酸含量降低,对葡萄酒有益,且混菌发酵对葡萄酒其他理化指标没有不利影响。本土酵母发酵尤其是混菌发酵,表征玫瑰香品种香气的萜烯醇类物质含量明显高于活性干酵母发酵。以上结果表明,3株本土酵母可以应用于玫瑰香干红葡萄酒的酿造,且3株酵母混菌发酵对突出玫瑰香干红葡萄酒品种香气具有一定的应用潜力。     

红提葡萄酒发酵酵母菌的分离及耐受性研究

该实验从红提葡萄发酵过程中分离出酵母菌株,并对其耐受性进行研究,旨在提高红提葡萄酒酿造品质。通过培养不同发酵时期菌株,并用肉眼和显微镜分别观察其菌落和细胞形态,再测定其发酵能力、耐糖性、耐酸性、耐酒精度。结果表明,共分离出酵母菌140株,经菌落形状和细胞形态鉴定后,获得典型的三类共6株酵母菌;在发酵过程中,加入分离菌株比自然发酵可多发酵1.6%葡萄糖,多产生0.78% CO2和0.82%vol的酒精,且发酵趋势和安琪酵母、自然发酵过程一致;以安琪酵母为对照,对6株菌进行耐糖性、耐酸性、耐酒精度试验,得出综合耐受性较好的酵母菌株为A3、B7和C1。     

不同有机氮源对葡萄酒发酵的影响

分别使用酵母浸粉和混合氨基酸作为模拟葡萄汁（36 °Bx）的有机氮源发酵葡萄酒,以保证葡萄酒的正常发酵和最终产品品 质。 通过测定发酵过程中的二氧化碳生成量、还原糖、可同化氮、甘油和挥发性化合物含量变化,比较酵母浸粉和混合氨基酸对葡萄酒 品质的影响。 结果表明,使用酵母浸粉耗还原糖量为295.7 g/L,生成乙醇97.20 g/L、甘油26.50 g/L、乙酸1.08 g/L和乙酸乙酯46.05 mg/L, 与使用混合氨基酸相比,多消耗还原糖130.47 g/L,多生成乙醇46.14 g/L、甘油7.95 g/L和乙酸0.54 g/L,增幅分别为78.95%、90.38%、 42.84%和99.35%。 使用酵母浸粉比混合氨基酸的发酵程度大,速度快。 因此,可用适量酵母浸粉替代混合氨基酸作为葡萄酒发酵的 氮源补充。     

冰酒中的毕赤属和汉逊属酵母菌的筛选及其在葡萄酒酿造中的应用

从冰葡萄酒自然发酵过程中分离、鉴定出10株毕赤属(Pichia)和汉逊属(Hanseniaspora)酵母,对其耐受性(酒精、糖、酸、SO2)进行研究,筛选得到4株耐受性能优良的酵母菌株,编号为"HO"和"HU"(Hanseniaspora属)、"PO"和"PK"(Pichia属).再将此4个酵母菌株与商业酿酒酵母(S...     

甘蔗酒酿造酵母的筛选

选择常用商业果酒酵母菌株KD、DV10、Q23、EC1118、安琪和H7Y7作为供试酵母菌株,以川蔗17为原料发酵生产甘蔗酒,通过分析发酵液中糖含量变化情况及酒精度、澄清度,比较不同菌株发酵特性,结果发现EC1118菌株发酵彻底,产酒能力最强,发酵后甘蔗酒的高级醇含量最低;H7Y7菌株发酵的甘蔗酒中酯类物质最高,为其他菌株的3～6倍;安琪酵母发酵液最易澄清,但其发酵后的甘蔗酒中甲醇含量为其他菌株的5倍以上。感官评分结果显示,不同酵母菌株发酵的甘蔗酒感官评分依次为H7Y7EC1118DV10安琪酵母Q23KD。研究结果表明,供试酵母菌株中EC1118和H7Y7更适宜用于甘蔗酒酿造。     

SOME EFFECTS OF WORT COMPOSITION ON THE RATE AND EXTENT OF FERMENTATION BY BREWING YEASTS

The time required to ferment worts of varied composition to a given extent is dependent upon the extent of exponential growth in the early stages of fermentation; in the worts studied this is determined by the concentration of assimilable nitrogen. When the concentration of all the non-carbohydrate nutrients in malt wort is halved by dilution with carbohydrate, the addition of appropriate quantities of serine or arginine restores the rate of fermentation to that of the malt wort. Minor nutrients, other than amino acids specifically required by the yeasts used, are thus present in at least two-fold excess in the malt wort. The yeast produced during exponential growth in malt wort (sp.gr. 1·040) is able to ferment rapidly much greater quantities of fermentable carbohydrate than are present in that wort. The majority of the strains of yeast examined ferment equally well when either glucose or maltose is added to malt wort and do so whether the sugar is added prior to fermentation or towards the end; however, one strain fails to ferment satisfactorily if a substantial quantity of glucose is added to wort prior to fermentation, because of the subsequent failure of the yeast to adapt to ferment maltose. It is suggested that most brewing strains do not require to adapt to maltose utilization during the fermentation of wort.     

西藏传统青稞酒酿造用藏曲中主要酵母菌的分离及酿造特性研究

采用WL酵母形态鉴别培养基从不同地域来源藏曲中分离筛选出48株典型酵母形态菌株,进一步通过分子鉴定为酿酒酵母(Saccharomyces cerevisiae)31株,扣囊复膜孢酵母(Saccharomycopsis fibuliger)9株,Saccharomycopsis malanga 3株。通过青稞汁培养基发酵研究了不同种类酵母的酿造性能,发现酿酒酵母是青稞酒发酵主要产酒微生物,S.malanga是主要产酸微生物。通过26种香气代谢物的分析比较3种酵母产香性能发现酿酒酵母是青稞酒发酵主要产香微生物,能够产丰富的酯类、醇类和有机酸类香气物质,扣囊复膜孢酵母也对青稞酒中微量香气物质具有重要贡献,而S.malanga产香能力较弱。该文系统研究了西藏传统酿造青稞酒酿造用藏曲中主要酵母菌的酿造性能和风味功能,对青稞酒酿造风味品质的科学控制提供了依据。     

昌黎产区产酶酵母多样性及其应用潜力分析

利用优选酵母混菌发酵,是改善葡萄酒品质及增香的有效途径。以赖氨酸培养基、WL培养基和产酶筛选培养基为基础,分析了昌黎4个葡萄园土样中产酶酵母的多样性,结果表明,该地区产酶酵母丰富,50%的产酶酵母能同时产2种或2种以上的酶。对产酶酵母酿造因子耐性分析表明,菌株GY1、M9和J24具有高糖耐性,适合于高糖低醇果酒酿造;菌株GY13和M9具有高酒精耐性,酒精含量为15.000%时活性较强;菌株J24和GY1具有高酸耐性,pH 2.5时具有较高活性;7株菌15℃时具有较高活性。综合分析发现,菌株M9(Torulaspora delbrueckii)同时产β-葡萄糖苷酶、果胶酶和纤维素酶,除酸耐性较差外,具有高糖、高酒精度和低温耐性,在高糖低醇果酒酿造中具有很大应用潜力。     

产香酵母的筛选及其苹果酒发酵特性

从分离自赤霞珠葡萄酿酒不同时期发酵液中的酵母菌中筛选产香菌株,并对其苹果酒发酵特性进行研究。结果表明,经感官评定初筛,获得7株产香较优的酵母菌,并对其进行耐酒精、耐SO2和耐盐性能分析,其中菌株B2-13具有较好的发酵性能,经分子生物学鉴定菌株B2-13为葡萄酒有孢汉逊酵母（Hanseniaspora uvarum）。菌株B2-13发酵苹果酒酒精度为4.2%vol～5.5%vol,糖度为2.6～44 g/L,共鉴定出32种香气成分,主要有酯类（6种）、醛酮类（4种）、醇类（6种）、酸类（8种）等,其中异戊醇、苯乙醇含量最高,分别为0.36 mg/mL和0.16 mg/mL,另外还有赤霞珠干红葡萄酒的特有香气物质3-羟基-2-丁酮（0.16 mg/mL）。菌株B2-13是一株具有较好的苹果酒发酵和产香性能的酵母菌株,有良好的工业化生产应用前景。     

烟台贵人香葡萄自然发酵醪中本土酿酒酵母的选育及酿造特性研究

自葡萄原料生产地选育优良酿造特性的本土酵母是开发地方特色葡萄酒品种的重要措施。本研究以烟台地区贵人香葡萄自然发酵醪为材料,筛选酿酒酵母并对其发酵特点和耐受能力进行测定,进而选育本土酿酒酵母用于霞多丽干白和赤霞珠干红葡萄酒的酿造试验。采用孟加拉红选择性培养基筛选本土酵母菌,并经WL鉴定培养基和5.8S ITS序列鉴定获得12株酿酒酵母。通过测定发酵后残糖量和酒精度及耐受能力(酒精度、SO2、酸、高糖),选育4株酵母用于酿造霞多丽干白葡萄酒,对酒样指标、主要挥发物和香气组分分析,显示菌株YGF2、YGF5和YGF10的酿造特性和发酵指标较好,并用于酿造赤霞珠干红葡萄酒。结果表明菌株YGF2酿造的葡萄酒质量较好,香气浓郁,部分指标优于商品酿酒酵母,具备生产区域特色葡萄酒的潜力。     

优良野生葡萄酒酵母的筛选及性能评价

以河北昌黎葡萄酒产区4个不同品种的葡萄为野生酵母分离源,分离到了58株酵母。以此作为出发菌株,通过酒精耐受性试验、SO2耐受性试验、模拟发酵试验,以发酵力、发酵速率、糖利用率等为标准初步筛得4株具有应用价值的野生酵母。用这4株酵母在酒厂进行了小型的葡萄酒发酵试验,将所得酒样进行理化分析及感官品评,并采用气-质联用仪(GC-MS)分析其香气成分,最终获得了对这4株酵母发酵性能的综合评价,认为其中2株具有应用于生产的潜能。