AMINO ACID BALANCE IN YEAST FERMENTATION WITH A SYNTHETIC AMINO ACID MIXTURE AS NITROGEN SOURCE

Two brewery yeasts, one bottom- and one top-fermenting strain, were allowed to ferment an 8% glucose solution containing as nitrogen source an amino acid mixture simulating that obtained when yeast was autolysed. The amounts given were approximately twice as high as the expected requirements. After completion of fermentation the total amounts of each amino acid in the whole system, i. e., in medium and yeast, were determined. The results show that the yeast had not taken up amino acids according to its own composition. The amino acids previously found to be rapidly absorbed from brewery wort were present in the whole system in considerably smaller amounts than in the original medium, indicating that these acids had been utilized as a nitrogen souce or for other purposes. The acids which are taken up slowly from brewery wort were present in larger amounts than in the original medium, indicating that they had been synthesized despite the excess in the medium. The two strains showed relatively similar behaviour.     

Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose 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 sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

Concomitant extracellular accumulation of alpha-keto acids and higher alcohols by Zygosaccharomyces rouxii

Alpha-keto acids are key intermediates in the formation of higher alcohols, important flavor components in soy sauce, and produced by the salt-tolerant yeast Zygosaccharomyces rouxii. Unlike most of the higher alcohols, the alpha-keto acids are usually not extracellularly accumulated by Z. rouxii when it is cultivated with ammonium as the sole nitrogen source. To facilitate extracellular accumulation of the alpha-keto acids from aspartate-derived amino acid metabolism, the amino acids valine, leucine, threonine and methionine were exogenously supplied during batch and A-star cultivations of (routants of) Z. rouxii. It was shown that all alpha-keto acids from the aspartate-derived amino acid metabolism, except alpha-ketobutyrate, could be extracellularly accumulated. In addition, it appeared from the concomitant extracellular accumulation of alpha-keto acids and higher alcohols that in Z. rouxii, valine, leucine and methionine were converted via Ehrlich pathways similar to those in Saccharomyces cerevisiae. Unlike these amino acids, threonine was converted via both the Ehrlich and amino acid biosynthetic pathways in Z. rouxii.     

ABSORPTION OF AMINO ACIDS BY YEASTS FROM A SEMI-DEFINED MEDIUM SIMULATING WORT

The biosynthetic pathways involved when α-amino acids are absorbed by yeasts from a semi-defined medium simulating wort have been measured using ¹⁵N and ¹⁴C isotopes. When grown in this medium under brewery conditions, a complex transaminase equilibrium system operates within the cell, showing that the theory of intact assimilation of amino acids into yeast protein is invalid. A relatively high level of specificity was found in respect of the transfer of carbon skeletons of amino acids in the medium to carbon skeletons of yeast protein amino acids and the contributions in respect of each amino acid have been measured throughout growth. Simple sugars have been shown to contribute significantly to the carbon skeletons of most amino acids found in yeast and the extent of these syntheses has been quantitatively assessed.     

FORMATION OF HIGHER ALCOHOLS FROM 14C-LABELLED VALINE AND LEUCINE

The formation of isobutanol and 3-methylbutanol during alcoholic fermentation by brewer's yeast has been studied by adding uniformly ¹⁴C-labelled valine and leucine to the complex amino acid mixture which is used as nitrogen source. In the presence of ¹⁴C-leucine, only 3-methylbutanol becomes radioactive whereas, in the presence of ¹⁴C-valine, both isobutanol and 3-methylbutanol acquire label. The specific radioactivity of these compounds is, in all cases, inferior to that of the parent amino acids showing that a part of these higher alcohols is always formed by the synthetic functions of the yeast. The results suggest that the regulating effect of the valine and leucine level of the medium on the synthesis of these amino acids in yeast cells is rather limited. They furthermore indicate that intact assimilation of amino acids is not the only mode of their utilization, even in media with excessive levels of amino acids. An attempt is made to explain the dependence of the anabolic and catabolic modes of higher alcohol formation upon the nitrogenous nutrient level of the medium. The presence of radioactive isoleucine as impurity in the ¹⁴C-leucine used has allowed an approximative calculation of the formation of 2-methylbutanol from isoleucine which is very similar to the formation of isobutanol from valine.     

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.     

SYNTHESIS OF HIGHER ALCOHOLS IN THE GENUS ZYMOMONAS

Several strains of bacteria of the genus Zymomonas were examined with respect to their potential for higher alcohol synthesis. All strains studied were able to produce higher alcohols during growth in a simple medium containing glucose and yeast extract. The higher alcohols produced were mainly n-propanol and iso-amyl alcohol. In contrast to Saccharomyces cerevisiae, only trace amounts of higher alcohols were produced from glucose by resting cells. When amino acids or other precursors were added to the fermentation medium, the resting cells formed higher alcohols. The stimulation of n-propanol synthesis by precursors was the most pronounced. The results obtained indicate that, with minor differences, the mechanisms of higher alcohol synthesis are comparable to these used by yeasts.     

THE GROWTH OF TOP-FERMENTATION YEASTS IN BINARY MIXTURES OF VARIOUS NITROGEN NUTRIENTS

The growth of English top-fermentation yeasts in various nitrogen sources, used singly—including ammonium phosphate and a number of amino acids—has previously been investigated and reported upon. But malt wort, the natural medium for yeast growth, contains, not a single nitrogen source, but a mixture of many derived from the breakdown of barley proteins. It is therefore important to know whether growth in such mixtures is simply the average of growth in the individual nitrogen sources or whether the different nitrogen sources interact with each other in such a way as to produce enhanced or diminished growth. The object of the work described in this paper was to ascertain whether the admixture of two different nitrogen sources introduces any new effects unpredictable from the known behaviour of the same nitrogen sources used singly. Nutrient media were prepared containing graded proportions of various selected pairs of nitrogen sources. Four different top-fermentation brewery yeasts were cultured in these media, measurements being made of fermentation, nitrogen assimilation and rate and extent of yeast growth. It was thus possible to observe the influence of the percentage composition of any particular binary mixture of nitrogen nutrients upon the growth and fermentation of a variety of yeasts. Among the mixtures investigated there was generally found a slight enhancement of growth and fermentation over what would be expected from the individual behaviour of the nitrogen sources. In a few cases, however, the observed enhancement was very large and was practically independent of the particular variety of yeast used. No important depressive effects were found in any of the mixtures. It may therefore be concluded that the mere admixture of different nitrogen sources can make a special contribution to yeast growth and fermentation; this contribution is generally relatively small but may in some circumstances becomes very significant. A chemical interpretation of some of the phenomena of yeast growth in mixed nutrients has been put forward.     

Biosynthesis of higher alcohol flavour compounds by the yeast Saccharomyces cerevisiae: impact of oxygen availability and responses to glucose pulse in minimal growth medium with leucine as sole nitrogen source

Higher alcohol formation by yeast is of great interest in the field of fermented beverages. Among them, medium‐chain alcohols impact greatly the final flavour profile of alcoholic beverages, even at low concentrations. It is widely accepted that amino acid metabolism in yeasts directly influences higher alcohol formation, especially the catabolism of aromatic and branched‐chain amino acids. However, it is not clear how the availability of oxygen and glucose metabolism influence the final higher alcohol levels in fermented beverages. Here, using an industrial Brazilian cachaça strain of Saccharomyces cerevisiae, we investigated the effect of oxygen limitation and glucose pulse on the accumulation of higher alcohol compounds in batch cultures, with glucose (20 g/l) and leucine (9.8 g/l) as the carbon and nitrogen sources, respectively. Fermentative metabolites and CO₂/O₂ balance were analysed in order to correlate the results with physiological data. Our results show that the accumulation of isoamyl alcohol by yeast is independent of oxygen availability in the medium, depending mainly on leucine, α‐keto‐acids and/or NADH pools. High‐availability leucine experiments showed a novel and unexpected accumulation of isobutanol, active amyl alcohol and 2‐phenylethanol, which could be attributed to de novo biosynthesis of valine, isoleucine and phenylalanine and subsequent outflow of these pathways. In carbon‐exhausted conditions, our results also describe, for the first time, the metabolization of isoamyl alcohol, isobutanol, active amyl alcohol but not of 2‐phenylethanol, by yeast strains in stationary phase, suggesting a role for these higher alcohols as carbon source for cell maintenance and/or redox homeostasis during this physiological phase. Copyright © 2014 John Wiley & Sons, Ltd.     

WORT COMPOSITION AND BEER FLAVOUR. I. THE INFLUENCE OF SOME AMINO ACIDS ON THE FORMATION OF HIGHER ALIPHATIC ALCOHOLS AND ESTERS

Wort, to which was added various amounts of glucose solution containing either (NH₄)₂SO₄ or one of the amino acids alanine, leucine, isoleucine, valine or proline, was fermented, and in the resulting beers the concentrations of ethyl acetate, isoamyl acetate, n-propanol, isobutanol, and amyl alcohols were determined by gas chromatography. The nitrogen source had some effect on the formation of higher alcohols, in addition to the effect due to conversion of some of the amino acids to the corresponding alcohols via the Ehrlich pathway. Ethyl acetate formation was not significantly affected by the nitrogen source, and iso-amyl acetate formation increased when amyl alcohol formation increased.     

Apple Aminoacid Profile and Yeast Strains in the Formation of Fusel Alcohols and Esters in Cider Production

The amino acid profile in dessert apple must and its effect on the synthesis of fusel alcohols and esters in cider were established by instrumental analysis. The amino acid profile was performed in nine apple musts. Two apple musts with high (>150 mg/L) and low (<75 mg/L) nitrogen content, and four enological yeast strains, were used in cider fermentation. The aspartic acid, asparagine and glutamic acid amino acids were the majority in all the apple juices, representing 57.10% to 81.95%. These three amino acids provided a high consumption (>90%) during fermentation in all the ciders. Principal component analysis (PCA) explained 81.42% of data variability and the separation of three groups for the analyzed samples was verified. The ciders manufactured with low nitrogen content showed sluggish fermentation and around 50% less content of volatile compounds (independent of the yeast strain used), which were mainly 3‐methyl‐1‐butanol (isoamyl alcohol) and esters. However, in the presence of amino acids (asparagine, aspartic acid, glutamic acid and alanine) there was a greater differentiation between the yeasts in the production of fusel alcohols and ethyl esters. High contents of these aminoacids in dessert apple musts are essential for the production of fusel alcohols and most of esters by aromatic yeasts during cider fermentation.     

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.     

Effect of the nitrogen source on the fatty acid composition of Saccharomyces cerevisiae

The source and content of nitrogen in the medium are very important in the development of alcoholic fermentations since they both affect the growth of Saccharomyces cerevisiae. Furthermore, the composition of the growth medium and the environmental conditions are known to affect the cell membrane fatty acid composition. The aim of this work was to study how the nitrogen source affects the membrane fatty acid composition. A mixture of amino acids and ammonia delayed the yeast growth when a high content of yeast assimilable nitrogen was present in the media. Cells grown in the mixed nitrogen source had a lower content of total fatty acids with a higher unsaturation degree than cells grown on sole ammonia.     

Role of amino acids and glucose in development of burnt off-flavours in liver sausage during heat processing

The effect of amino acids and glucose on the development of burnt off-flavours was investigated in meat model systems for liver sausage. At a constant glucose concentration, the burnt flavour was intensified by addition of an amino acid mixture. In a batter without liver, the development of burnt off-flavours, similar to those in liver sausage, could be induced through addition of exogenous amino acids and glucose. The endogenous amino acids from liver reacted more intensely than the exogenous amino acids in the formation of the burnt flavour components. After heating batches with added exogenous amino acids and glucose the free amino acid contents (73–89% of initial content) were higher than in batches to which liver was added (42–53% of initial content). The differences in the free amino acid content of glycine, glutamic acid and threonine were linearly related to the intensity of burnt flavour. Results indicate that burnt flavour is due to the Maillard reaction.     

Influence of nitrogen on the biological aging of sherry wine

Nitrogen compounds are essential to the growth and metabolism of yeasts. The uptake and metabolism of nitrogen compounds by Saccharomyces cerevisiae depend not only on the strain and its physiological condition, but also on the chemical and physical properties of its environment. The effect of the addition of different amino acids (L ‐proline, L ‐threonine, L ‐arginine, L ‐glutamic acid, L ‐leucine and L ‐valine) to nitrogen‐depleted natural or nitrogen‐free synthetic wine on the cell growth, flor velum formation and sherry wine compound production was investigated under controlled biological aging by S. cerevisiae var. capensis strain G1 a typical flor yeast. The formation of flor velum was dependent on particular amino acid, oxygen availability and the composition of wine. Consumption of glycerol was related with the cell growth; in contrast, acetaldehyde tended to be released. Amino acid supplementation resulted in the release to wine of amino acids, esters and higher alcohols. The amino acid which was released in nearly all cases was L ‐leucine. Addition of L ‐glutamic acid resulted in the release mainly of ethyl acetate, in the case of L ‐leucine isoamyl alcohols were released, and for L ‐valine isobutanol. In the three cases, 1,1‐diethoxyethane was released in large quantities. The findings might indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential. Copyright © 2006 Society of Chemical Industry     

Survey of hydrogen sulphide production by wine yeasts

Twenty-one strains of commercial wine yeasts and 17 non-Saccharomyces species of different provenance were surveyed for their ability to produce hydrogen sulphide in synthetic grape juice medium indicator agar with different nitrogen sources, as well as in natural grape juice. Bacto Biggy agar, a commercially available bismuth-containing agar, was used to compare our results with others previously reported in the literature. Under identical physiological conditions, the strains used in this study displayed similar growth patterns but varied in colony color intensity in all media, suggesting significant differences in sulphite reductase activity. Sulphite reductase activity was absent for only one strain of Saccharomyces cerevisiae. All other strains produced an off-odor to different extents, depending significantly (P <0.05) on medium composition. Within the same species of some non-Saccharomyces yeasts, strain variation existed as it did for Saccharomyces. In natural musts, strains fell into three major groups: (i) nonproducers, (ii) must-composition-dependent producers, and (iii) invariable producers. In synthetic media, the formation of sulphide by strains of S. cerevisiae results from the reduction of sulphate. Therefore, this rapid screening methodology promises to be a very useful tool for winemakers for determining the risk of hydrogen sulphide formation by a given yeast strain in a specific grape juice.     

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.     

Influence of Nutrients Addition to Nonlimited-in-Nitrogen Must on Wine Volatile Composition

ABSTRACT: Several factors can affect production of volatile compounds in wines, including nutritional differences in the must. Among the nutritive compounds of must, the amino acids are of great importance due to the fact that they can serve as precursors of esters or may play an indirect role in supporting cellular metabolic activities. The aim of this study was to examine the influence of the addition of a complex nutrient such as the yeast autolysate to a nitrogen nonlimited must of Chardonnay variety on the formation of esters, alcohols, and acids. Yeast autolysate supplied fatty acids, amino acids (above all, tyrosine, threonine, and glutamic acid), and an insoluble fraction formed by yeast cell walls. It was observed that the nutrient enrichment of the Chardonnay must did not favor the formation of either esters or alcohols. These results demonstrate that when juice is sufficient in nitrogen, the addition of amino acids does not improve the volatile composition of wine. Under these conditions, the amino acids would have probably been used for other cellular processes that do not produce volatile compounds.     

Amino acid uptake by wild and commercial yeasts in single fermentations and co-fermentations

Musts require nitrogen-containing compounds in order to ensure yeast development. This study examined the nitrogen-nutrient requirements of two commercial yeasts and three wild strains isolated from inoculated fermentations. The results showed that wild strains generally consumed lower amounts of amino acids than commercial yeasts. Most amino acids were assimilated during the exponential growth phase; only a few – including asparagine and histidine – were metabolized until the end of fermentation. The study also sought to determine whether industrial drying affected yeast nitrogen requirements.