Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid

AIMS: To understand the impact of assimilable nitrogen and pantothenic acid on fermentation rate and synthesis of volatile compounds by Saccharomyces under fermentative conditions. METHODS AND RESULTS: A 2 x 3 factorial experimental design was employed with the concentrations of yeast assimilable nitrogen (YAN) (60 and 250 mg l(-1)) and pantothenic acid (10, 50 and 250 microg l(-1)) as variables. In media containing 250 microg l(-1) pantothenic acid, H2S production by two different species of Saccharomyces decreased when YAN was increased from 60 to 250 mg l(-1). Conversely, H2S production was significantly higher when the concentration of assimilable nitrogen was increased if pantothenic acid was deficient (10 or 50 microg l(-1)). Yeast synthesis of other volatile compounds were impacted by both assimilable nitrogen and pantothenic acid. CONCLUSIONS: While growth and fermentative rate of Saccharomyces was more influenced by nitrogen than by pantothenic acid, complicated interactions exist between these nutrients that affect the synthesis of volatile compounds including H2S. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has important implications for the winemaking industry where a better understanding of the nutritional requirements of Saccharomyces is necessary to reduce fermentation problems and to improve final product quality.     

A comparative study of the wine fermentation performance of Saccharomyces paradoxus under different nitrogen concentrations and glucose/fructose ratios

Aims:  The main goal of the present study is to determine the effects of different nitrogen concentrations and glucose/fructose ratios on the fermentation performance of Saccharomyces paradoxus , a nonconventional species used for winemaking.
Methods and Results:  Ethanol yield, residual sugar concentration, as well as glycerol and acetic acid production were determined for diverse wine fermentations conducted by S. paradoxus . Experiments were also carried out with a commercial Saccharomyces cerevisiae wine strain used as control. The values obtained were compared to test significant differences by means of a factorial anova and the Scheffé test. Our results show that S. paradoxus strain was able to complete the fermentation even in the nonoptimal conditions of low nitrogen content and high fructose concentration. In addition, the S. paradoxus strain showed significant higher glycerol synthesis and lower acetic acid production than S. cerevisiae in media enriched with nitrogen, as well as a lower, but not significant, ethanol yield.
Conclusions:  The response of S. paradoxus was different with respect to the commercial S. cerevisiae strain, especially to glycerol and acetic acid synthesis.
Significance and Impact of the Study:  The present study has an important implication for the implementation of S. paradoxus strains as new wine yeast starters exhibiting interesting enological properties.     

Natural hybrids from Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations

Several wine isolates of Saccharomyces were analysed for six molecular markers, five nuclear and one mitochondrial, and new natural interspecific hybrids were identified. The molecular characterization of these Saccharomyces hybrids was performed based on the restriction analysis of five nuclear genes (CAT8, CYR1, GSY1, MET6 and OPY1, located in different chromosomes), the ribosomal region encompassing the 5.8S rRNA gene and the two internal transcribed spacers, and sequence analysis of the mitochondrial gene COX2. This method allowed us to identify and characterize new hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii, between S. cerevisiae and Saccharomyces bayanus, as well as a triple hybrid S. bayanusxS. cerevisiaexS. kudriavzevii. This is the first time that S. cerevisiaexS. kudriavzevii hybrids have been described which have been involved in wine fermentation.     

Effect of must characteristics on the diversity of Saccharomyces strains and their prevalence in spontaneous fermentations

Aims: The aim of this study was to investigate whether grapevine variety and must characteristics influence the diversity of Saccharomyces strains and their prevalence during spontaneous fermentations. Methods and results: Musts from different grapevine varieties, all of them autochthonous from Galicia, were used to perform spontaneous fermentations. Yeasts were isolated from the must and at the beginning, in the middle and at the end of fermentations. Those yeasts identified as Saccharomyces were characterized at the strain level by analysis of mtDNA‐RFLP. The results showed a low diversity of Saccharomyces strains, which was related to must sugar content and total acidity. Moreover, from a total of 44 different Saccharomyces strains, only eleven of them appeared at frequencies higher than 20% and were able to lead fermentations. A significant correlation between yeast strains and must acidity was observed, with the predominance of certain strains at high acidity values. Conclusions: Must characteristics, such as sugar content and acidity, influence the Saccharomyces strains diversity and the leader strains during fermentation. Significance and Impact of the Study: These results showed the adaptation of certain Saccharomyces strains to must with specific characteristics; this may be considered by winemakers for yeast inocula selection. Our findings have special relevance because this is the first study carried out in Galicia dealing with the influence of must properties on yeast strains that control fermentations.     

Arginase activity is a useful marker of nitrogen limitation during alcoholic fermentations

Nitrogen deficiency in musts is one of the causes of sluggish or stuck fermentations. In this work we propose that arginase activity determination can be useful for detecting nitrogen starvation early in vinification. CAR1 and YGP1 genes are not specifically induced under conditions of nitrogen starvation. However, a significant increase in the enzymatic activity of arginase, the product of the CAR1 gene, is detected in vinifications carried out with musts containing limiting amounts of nitrogen. Moreover, on adding ammonia to a nitrogen-deficient vinification, even at late stages, this enzymatic activity is repressed, and growth rate is restored simultaneously. We also investigate the role of ethanol toxicity in nitrogen starvation. The results suggest that ethanol produced during vinification or exogenously added up to 8% (v/v) concentration does not cause nitrogen starvation under the conditions tested because arginase activity is not increased.     

Effect of feed zone in fed-batch fermentations of Saccharomyces cerevisiae

In production-scale, fed-batch fermentations, feed is often added to a single point at the top of the fermentor, which, combined with poor mixing, results in formation of a "feed zone" rich in nutrients. Frequent exposure of the culture to high concentrations of nutrients in the feed zone for sufficient duration can produce unexpected effects on its performance. The effect of the feed zone was evaluated by conducting aerobic fed-batch fermentations of Saccharomyces cerevisiae with both complex and defined media. The broth was recirculated between a recycle loop and a bench-scale fermentor, and feed was intermittently added into the recycle loop to simulate the circulation of cells through the feed zone. Experiments were carried out for a range of residence times in the recycle loop from 0.5 to 12 min. Biomass yields from the complex-media fermentations were not affected by exposure to high nutrient levels in the recycle loop for residence times up to 12 min. Ethanol consumption was reduced by as much as 50% for residence time in the loop up to 3 min. Very long exposure of yeast cells to excess nutrient levels (12 min) gave acetic acid formation. In a defined medium, the simulated feed zone effect increased biomass yield by up to 10%, but had no effect on ethanol levels. This study indicates that the feed zone effect on biomass yield in yeast fermentation, using complex substrates, will be negligible under fully aerobic conditions.     

Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR

Real-time, or quantitative, PCR (QPCR) was developed for the rapid quantification of two of the most important yeast groups in alcoholic fermentation (Saccharomyces spp. and Hanseniaspora spp.). Specific primers were designed from the region spanning the internal transcribed spacer 2 (ITS2) and the 5.8S rRNA gene. To confirm the specificity of these primers, they were tested with different yeast species, acetic acid bacteria and lactic acid bacteria. The designed primers only amplified for the intended group of species and none of the PCR assays was positive for any other wine microorganisms. This technique was performed on reference yeast strains from pure cultures and validated with both artificially contaminated wines and real wine fermentation samples. To determine the effectiveness of the technique, the QPCR results were compared with those obtained by plating. The design of new primers for other important wine yeast species will enable to monitor yeast diversity during industrial wine fermentation and to detect the main spoilage yeasts in wine.     

Nitrogen demand of different yeast strains during alcoholic fermentation. Importance of the stationary phase

The nitrogen demand of industrial yeast strains were compared. Substantial differences were found between strains. These did not change regardless of the initial medium composition and added nitrogen source. To separately study growth and stationary phases, we ran fermentations with different nitrogen feeding profiles: a) exponentially fed fermentations with a long growth phase, and b) constant rate fermentations with nitrogen addition during the stationary phase. Differences between stains mostly appeared during the second phase. Measuring nitrogen requirements under such conditions would thus be an interesting complementary test when selecting new strains especially for enological purposes since most fermentation kinetics are nitrogen limited.     

Saccharomyces cerevisiae biodiversity in spontaneous commercial fermentations of grape musts with 'adequate' and 'inadequate' assimilable-nitrogen content

AIM: To evaluate whether intraspecific diversity of Saccharomyces cerevisiae in wine fermentations is affected by initial assimilable-nitrogen content. METHODS AND RESULTS: Saccharomyces cerevisiae isolates from two spontaneous commercial wine fermentations started with adequate and inadequate nitrogen amounts were characterized by mitochondrial DNA restriction analysis. Several strains occurred in each fermentation, two strains, but not the same ones, being predominant at frequencies of about 30%. No significant differences were detected by comparing the biodiversity indices of the two fermentations. Cluster analysis demonstrated that the strain distribution was independent of nitrogen content, the two pairs of closely related dominant strains grouping into clusters at low similarity. CONCLUSIONS: The genetic variability of S. cerevisiae in wine fermentations seemed not to depend on the nitrogen availability in must. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrogen content did not affect the genetic diversity but may have induced a 'selection effect' on S. cerevisiae strains dominating wine fermentations, with possible consequences on wine properties.     

高温高浓发酵工业啤酒酵母菌种的构建

高温高浓发酵技术作为一项新兴的啤酒生产技术,它为啤酒生产带来诸多利益的同时,也存在着发酵结束后酵母絮凝性下降、高级醇生成量过高等系列问题。为提高高温高浓发酵条件下酿酒酵母的絮凝性同时降低高级醇的合成能力,首先构建了以酿酒酵母BAT2基因为整合位点过表达FLO5基因的菌株,重组菌株S6-BF的絮凝性达到67.67%,比出发菌株S6提高了29%,而高级醇生成量仅降低5.9%;进一步构建以BAT2基因为整合位点再次过表达FLO5基因的菌株,与出发菌株S6相比,重组菌株S6-BF2的絮凝性提高了63%,达到85.44%,高级醇生成量下降至159.58 mg/L,降低了9.0%;通过弱化线粒体支链氨基酸转氨酶(BAT1)的表达,高级醇的生成量得到进一步的降低,达到142.13 mg/L,比原始菌株S6降低了18.4%,同时重组菌株S6-BF2B1的絮凝性没有受到影响;风味物质的测定结果表明啤酒中醇酯比例较为合理。研究结果对工业啤酒酵母发酵后的沉降分离和提高啤酒风味品质有着重要的意义。     

Predominance of Saccharomyces uvarum during spontaneous alcoholic fermentation, for three consecutive years, in an Alsatian winery

AIMS: The purpose of this study was to determine the origin of the yeasts involved in the spontaneous alcoholic fermentation of an Alsatian wine. METHODS AND RESULTS: During three successive years, must was collected at different stages of the winemaking process and fermented in the laboratory or in the cellar. Saccharomyces yeasts were sampled at the beginning and at the end of the fermentations. Saccharomyces cerevisiae clones were genetically characterized by inter-delta PCR. Non-S. cerevisiae clones were identified as Saccharomyces uvarum by PCR-RFLP on MET2 gene and characterized at the strain level by karyotyping. The composition of the Saccharomyces population in the vineyard, after crushing and in the vat was analyzed. This led to three main results. First, the vineyard Saccharomyces population was rather homogeneous. Second, new non-resident strains had appeared in the must during the winemaking process. Finally, the yeast population in the vat only consisted in S. uvarum strains. CONCLUSION: This 3-year study has enabled us to show the involvement of indigenous S. uvarum in the alcoholic fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a first insight into the polymorphism of S. uvarum strains involved in a spontaneous alcoholic fermentation.     

酿酒酵母类丙酮酸脱羧酶基因缺失对高级醇生成量的影响

【目的】通过构建酿酒酵母类丙酮酸脱羧酶基因(YDL080C)缺失的工程菌株,研究该基因对酿酒酵母浓醪发酵产高级醇特别是异戊醇的影响。【方法】以酿酒酵母工业菌株AY-15的单倍体a-8或α-22的基因组DNA为模板,PCR分别扩增YDL080C上下游非编码区片段YA和YB;以pUG6质粒为模板,PCR扩增KanMX抗性基因片段。分别将YA、YB和KanMX片段连入pUC19载体,构建重组质粒pUC-YABK;并以其为模板,PCR扩增YA-KanMX-YB重组盒,分别电转化单倍体a-8和α-22。将转化子和亲本分别进行酒精浓醪发酵,发酵结束后测定其发酵性能和高级醇的生成量。【结果】筛选获得了YDL080C基因缺失突变株。酒精发酵后发酵性能和高级醇测定结果显示,转化子的异戊醇及总高级醇生成量与对应的单倍体亲本相比没有明显变化,但酒精度分别比亲本提高了0.6(%,v/v)和0.4(%,v/v)。【结论】YDL080C基因缺失对降低酿酒酵母发酵产高级醇特别是异戊醇没有明显作用,但会使酒精度有所提高。     

The role of indigenous yeasts in traditional Irish cider fermentations

AIMS: To study the role of the indigenous yeast flora in traditional Irish cider fermentations. METHODS AND RESULTS: Wallerstein laboratory nutrient agar supplemented with biotin, ferric ammonium citrate, calcium carbonate and ethanol was employed together with PCR-restriction fragment length polymorphism analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene in the identification of indigenous yeasts at the species level, from traditional Irish cider fermentations. By combining the molecular approach and the presumptive media it was possible to distinguish between a large number of yeast species, and to track them within cider fermentations. The Irish cider fermentation process can be divided into three sequential phases based on the predominant yeast type present. Kloeckera/Hanseniaspora uvarum type yeasts predominate in the initial 'fruit yeast phase'. Thereafter Saccharomyces cerevisiae type yeast dominate in the 'fermentation phase', where the alcoholic fermentation takes place. Finally the 'maturation phase' which follows, is dominated by Dekkera and Brettanomyces type yeasts. H. uvarum type yeast were found to have originated from the fruit. Brettanomyces type yeast could be traced back to the press house, and also to the fruit. The press house was identified as having high levels of S. cerevisiae type yeast. A strong link was noted between the temperature profile of the cider fermentations, which ranged from 22 to 35 degrees C and the yeast strain population dynamics. CONCLUSIONS: Many different indigenous yeast species were identified. The mycology of Irish cider fermentations appears to be very similar to that which has previously been reported in the wine industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of indigenous yeast species in 'Natural' Irish cider fermentations.     

Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malolactic wine fermentations

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10 °C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry     

Gaseous environments modify physiology in the brewing yeast Saccharomyces cerevisiae during batch alcoholic fermentation

Aims:  To investigate the impact of different gaseous atmospheres on different physiological parameters in the brewing yeast Saccharomyces cerevisiae BRAS291 during batch fermentation.
Methods and Results:  Yeasts were cultivated on a defined medium with a continuous sparging of hydrogen, helium and oxygen or without gas, permitting to obtain three values of external redox. High differences were observed concerning viable cell number, size and metabolites produced during the cultures. The ethanol yields were diminished whereas glycerol, succinate, acetoin, acetate and acetaldehyde yields were enhanced significantly. Moreover, we observed major changes in the intracellular NADH/NAD⁺ and GSH/GSSG ratio.
Conclusions:  The use of gas led to drastic changes in the cell size, primary energy metabolism and internal redox balance and E _h . These changes were different depending on the gas applied throughout the culture.
Significance and Impact of the Study:  For the first time, our study describes the influence of various gases on the physiology of the brewing yeast S. cerevisiae . These influences concern mainly yeast growth, cell structure, carbon and redox metabolisms. This work may have important implications in alcohol-related industries, where different strategies are currently developed to control better the production of metabolites with a particular attention to glycerol and ethanol.     

The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism

Many yeast species have growth rates on D-xylulose of 25-130% of those on glucose, but for Saccharomyces cerevisiae this ratio is only about 6%. The xylulokinase reaction has been proposed to be the rate-limiting step in the D-xylulose fermentation with S. cerevisiae. Over-expression of xylulokinase encoding XKS1 stimulated growth on D-xylulose in a S. cerevisiae strain to about 20% of the growth rate on glucose and deletion of the gene prevented growth on D-xylulose and D-xylulose metabolism. We have partially purified the xylulokinase and characterised its kinetic properties. It is reversible and will also accept D-ribulose as a substrate.     

The role of peroxisomes in xylose alcoholic fermentation in the engineered Saccharomyces cerevisiae

Xylose is a second‐most abounded sugar after glucose in lignocellulosic hydrolysates and should be efficiently fermented for economically viable second‐generation ethanol production. Despite significant progress in metabolic and evolutionary engineering, xylose fermentation rate of recombinant Saccharomyces cerevisiae remains lower than that for glucose. Our recent study demonstrated that peroxisome‐deficient cells of yeast Ogataea polymorpha showed a decrease in ethanol production from xylose. In this work, we have studied the role of peroxisomes in xylose alcoholic fermentation in the engineered xylose‐utilizing strain of S. cerevisiae. It was shown that peroxisome‐less pex3Δ mutant possessed 1.5‐fold decrease of ethanol production from xylose. We hypothesized that peroxisomal catalase Cta1 may have importance for hydrogen peroxide, the important component of reactive oxygen species, detoxification during xylose alcoholic fermentation. It was clearly shown that CTA1 deletion impaired ethanol production from xylose. It was found that enhancing the peroxisome population by modulation the peroxisomal biogenesis by overexpression of PEX34 activates xylose alcoholic fermentation.     

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae

Bioconversion of xylose—the second most abundant sugar in nature—into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.     

Intestinal absorption of biotin and biocytin in the rat

The uptake of biotin and biocytin was investigated in rat intestine using the everted sac technique. It has been shown that at biotin and biocytin concentrations !ess than 40 and 50 nM respectively, absorption proceeds by a saturable process, whereas at higher concentrations uptake by passive diffusion predominates. Fractionation of solublized brush border preparations indicates that biotinidase is the only protein which binds biotin in this preparation.