Improvement of maltotriose fermentation by Saccharomyces cerevisiae

AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts.     

Nucleotide sequence and expression of the Enterobacter aerogenes alpha-acetolactate decarboxylase gene in brewer's yeast.

The nucleotide sequence of a 1.4-kilobase DNA fragment containing the alpha-acetolactate decarboxylase gene of Enterobacter aerogenes was determined. The sequence contains an entire protein-coding region of 780 nucleotides which encodes an alpha-acetolactate decarboxylase of 260 amino acids. The DNA sequence coding for alpha-acetolactate decarboxylase was placed under the control of the alcohol dehydrogenase I promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both S. cerevisiae and Escherichia coli. Brewer's yeast cells transformed by this plasmid showed alpha-acetolactate decarboxylase activity and were used in laboratory-scale fermentation experiments. These experiments revealed that the diacetyl concentration in wort fermented by the plasmid-containing yeast strain was significantly lower than that in wort fermented by the parental strain. These results indicated that the alpha-acetolactate decarboxylase activity produced by brewer's yeast cells degraded alpha-acetolactate and that this degradation caused a decrease in diacetyl production.     

A kinetic study of Saccharomyces strains: Performance at high sugar concentrations

Alcoholic fermentation represents a significant example of production of compounds utilizable as alternative energy sources. High ethanol concentration in the fermented wort is needed in order to reduce the energy consumption in the process of alcohol recovery. A particular Saccharomyces strain, of the oviformis species, obtained from fermented worts exhibiting high ethanol concentrations is studied and compared with a common S. cerevisiae strain in order to show its skill in fermenting very concentrated sugar solutions with an energy saving of ca. 10%.     

Nucleotide sequence and expression of the Enterobacter aerogenes alpha-acetolactate decarboxylase gene in brewer's yeast

The nucleotide sequence of a 1.4-kilobase DNA fragment containing the alpha-acetolactate decarboxylase gene of Enterobacter aerogenes was determined. The sequence contains an entire protein-coding region of 780 nucleotides which encodes an alpha-acetolactate decarboxylase of 260 amino acids. The DNA sequence coding for alpha-acetolactate decarboxylase was placed under the control of the alcohol dehydrogenase I promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both S. cerevisiae and Escherichia coli. Brewer's yeast cells transformed by this plasmid showed alpha-acetolactate decarboxylase activity and were used in laboratory-scale fermentation experiments. These experiments revealed that the diacetyl concentration in wort fermented by the plasmid-containing yeast strain was significantly lower than that in wort fermented by the parental strain. These results indicated that the alpha-acetolactate decarboxylase activity produced by brewer's yeast cells degraded alpha-acetolactate and that this degradation caused a decrease in diacetyl production.     

Isolation and Characterization of Brewer's Yeast Variants with Improved Fermentation Performance under High-Gravity Conditions

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22° Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11°C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.     

Physico-chemical and microbiological analyses of fermented corn cob, rice bran and cowpea husk for use in composite rabbit feed

An experiment was conducted to evaluate the effect of fermentation on the proximate composition of corn cob, rice bran and cowpea husk for use in composite rabbit feed formulations. The test ingredients were moistened with tap water and allowed to ferment naturally at room temperature. During fermentation, samples of the fermenting materials were extracted at zero, 24 and 48 h for physico-chemical and microbiological analyses using standard procedures. The microorganisms associated with the fermenting materials were identified as Rhizopus oligosporus, Aspergillus oryzae, Aspergillus niger, Rhodotorula, Geotrichum candidum, Candida albicans, and Saccharomyces cerevisiae. Two (R. oligosporus and S. cerevisiae) out of microorganisms present were used as starter cultures to ferment the test ingredients and the fermented products were then analyzed. From the results obtained S. cerevisiae enhanced the protein and fat contents while R. oligosporus was able to degrade the fiber significantly.     

Ethanol production from fodder beets

Various yeasts such as two strains of Saccharomyces cerevisiae, Saccharomyces diastaticus, and Kluyveromyces marxianus were investigated for their ability to ferment fodder beet juice to alcohol. Juice extracted from fodder beet roots without any additives was used as a fermentation substrate. The fermentation kinetic parameters were determined and compared for each species of yeast tested. The best species for fodder beet juice fermentation was chosen and products obtained by fermentation of one hectare of fodder beet plants are given.     

Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard

AIMS: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia. METHODS AND RESULTS: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production. CONCLUSIONS: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.     

中国酱香型白酒酿造酿酒酵母的独特生理代谢特征

【目的】酿酒酵母是白酒发酵的主要微生物,对白酒的产量及质量都有重要影响。而酱香酒酿造环境具有高温、酸性、高乙醇等胁迫因素,研究其中酿酒酵母的生理代谢特征并有目的地应用于白酒实际生产中的意义。【方法】从酱香酒酿造环境中筛选一株性能优良的酿酒酵母菌株,比较其与酿酒酵母模式菌株S288c和商业酵母的生理代谢特征。【结果】从酱香型酒醅中筛选得到性能优良的Saccharomyces cerevisiae MT1,该菌株可耐受高温42 °C,高浓度乙醇(16%,体积比),低pH (2.0),其最大比生长速率和最大比产乙醇速率分别达到了S288c的125%和114%,其乙醇转化率也要高于其他菌株。一些挥发性物质只有在MT1的发酵液中可检测到,包括苯并噻唑、2,3-二氢苯并呋喃、4-乙烯基愈创木酚以及丁羟甲苯等,MT1的苯乙醇、法呢醇、橙花叔醇、乙偶姻和大马酮量也要高于S288c。另外,MT1可以利用多种碳源发酵产乙醇,如半乳糖、麦芽糖、蜜二糖、松二糖、海藻糖和棉子糖等。【结论】来源于酱香酒酿造的S. cerevisiae MT1具有高耐受力、高效的发酵性能及更广泛的碳源利用图谱,并能生成多种挥发性物质。     

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae

Laboratory strains of yeast ( Saccharomyces cerevisiae ) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys⁺ strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.     

Interactions between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni

AIMS: To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions. METHODS AND RESULTS: Interactions between S. cerevisiae and O. oeni were investigated by double-layer and well-plate assays showing the occurrence of specific interactions for each yeast-malolactic bacteria (MLB) coupling. Heat and protease treatments of synthetic grape juice fermented by the S. cerevisiae strain F63 indicated that the inhibitory activity exerted by this yeast on O. oeni is due to a proteinaceous factor(s) which exerts either bacteriostatic or bactericidal effect depending on concentration and affects malolactic fermentation in natural grape juice and wine. CONCLUSIONS: A proteinaceous factor(s) produced by a S. cerevisiae wine strain able to inhibit O. oeni growth and malic acid fermentation was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The individuation, characterization and exploitation of yeast proteinaceous factor(s) exerting inhibitory activity on MLB may offer new opportunities for the management of malolactic fermentation.     

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.     

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

AIMS: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. METHODS AND RESULTS: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l(-1)) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. CONCLUSIONS: The yeast strain required a minimum of 267 mg N l(-1) to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality.     

Use of a PGU1 recombinant Saccharomyces cerevisiae strain in oenological fermentations

AIM: The aim of this work was the construction of an oenological Saccharomyces cerevisiae strain able to overexpress the PGU1 gene in order to be used in trial fermentations. METHODS AND RESULTS: The recombinant strain is able to secrete an active endopolygalacturonase into the medium leaving its fermentation ability essentially unchanged. Wines obtained with the recombinant strain and the untransformed counterpart did not differ in their physicochemical parameters or major sensory characteristics. The time needed for wine filtration was dramatically reduced in wines elaborated with the PGU1 recombinant strain, and was comparable to the filtration time shown by wines elaborated from must supplemented with fungal pectolytic enzymes. CONCLUSIONS: The oenological strain constructed in this work secretes an endopolygalacturonase into the wine in an efficient manner, resulting in an improvement in wine filtration but preserving wine typicality and keeping the methanol levels unchanged. SIGNIFICANCE AND IMPACT OF THE STUDY: The PGU1 recombinant strains could be used in oenological fermentations as an alternative to commercial pectolytic enzymes of fungal origin.     

Isolation and characterization of brewer's yeast variants with improved fermentation performance under high-gravity conditions

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22 degrees Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11 degrees C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.     

The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations

AIM: To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. METHODS AND RESULTS: Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l(-1)) and biotin (0, 1 or 10 microg l(-1)) as variables. All media contained 240 g l(-1) glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 microg l(-1) had no effect. Nitrogen x biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H(2)S). Maximum H(2)S production occurred in the medium containing 60 mg l(-1) YAN and 1 microg l(-1) biotin. CONCLUSIONS: Nitrogen x biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.     

Fermentation of corn starch to ethanol with genetically engineered yeast

Expression of the glucoamylase gene from Aspergillus awamori by laboratory and distiller's strains of Saccharomyces cerevisiae allowed them to ferment soluble starch. Approximately 95% of the carbohydrates in the starch were utilized. Glycerol production was significantly decreased when soluble starch was used instead of glucose. Ethanol yield on soluble starch was higher than that on glucose. The rate of starch fermentation was directly related to the level of glucoamylase activity. Strains with higher levels of glucoamylase expression fermented starch faster. The decline in starch fermentation rates toward the end of the fermentation was associated with accumulation of disaccharides and limit dextrins, poor substrates for glucoamylase. The buildup of these products in continuous fermentations inhibited glucoamylase activity and complete utilization of the starch. Under these conditions maltose-fermenting strains had a significant advantage over nonfermenting strains. The synthesis and secretion of glucoamylase showed no deleterious effects on cell growth rates, fermetation rates, and fermentation products.     

Studies on Osmophilic Yeasts

Assimilation of galactose and maltose by Saccharomyces rouxii, which is a typical salt-tolerant yeast playing an important role in soy-brewing, was negligible or extremely poor in the medium containing 18% NaCl, although the assimilation in the ordinary medium was vigorous. The yeasts which were able to assimilate and ferment galactose, maltose and/or saccharose in the high-saline medium were limited to a few strains. From the studies on balances of fermentation products, it was revealed that ethanol yield based on sugar consumed in the 18% NaCl-medium was lower than that in the ordinary medium, suggesting that other fermentation products than ethanol should be accumulated. Analytical results of the fermented broths showed much polyalcohol production in the high-saline medium.     

Occurrence and taxonomic characteristics of strains of Saccharomyces cerevisiae predominant in African indigenous fermented foods and beverages

Indigenous fermented foods and beverages play a major role in the diet of African people. The predominant yeast species seen is Saccharomyces cerevisiae, involved in basically three groups of indigenous fermented products: non-alcoholic starchy foods, alcoholic beverages and fermented milk. These products are to a great extent made by spontaneous fermentation and consequently S. cerevisiae often coexists with other microorganisms even though a microbiological succession usually takes place both between and within species. The functions of S. cerevisiae are mainly related to formation of alcohols and other aroma compounds, but stimulation of e.g. lactic acid bacteria, improvement of nutritional value, probiotic effects, inhibition of undesired microorganisms and production of tissue-degrading enzymes may also be observed. Several different isolates of S. cerevisiae have been shown to be involved in the fermentations and some of the isolates show pheno- and genotypic characteristics that deviate from those normally recognised for S. cerevisiae.     

Maltotriose fermentation by Saccharomyces cerevisiae

Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin A. This strain produced equivalent amounts of ethanol from 20 g l⁻¹ glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific α-glucosidase. However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose by these Saccharomyces cerevisiae cells. Journal of Industrial Microbiology & Biotechnology (2001) 27, 34–38. Received 13 January 2001/ Accepted in revised form 29 May 2001