FORMATION OF HIGHER ALCOHOLS FROM AMINO ACIDS DERIVED FROM YEAST PROTEINS

When yeast, which had previously been grown in an amino acid rich medium and labelled with ¹⁴C-leucine, was transferred to a strongly nitrogen-limiting medium containing glucose and ammonium sulphate, the 3-methylbutanol obtained after completed fermentation was radioactive, indicating that about 13% of the leucine of yeast was transformed to this alcohol, and that at least 10% of 3-methylbutanol formed was derived from the protein of yeast. This in turn suggests that protein turnover in yeast partly occurs in such a way that carbon skeletons of amino acids are rejected and only the amino group is re-utilized.     

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.     

The influence of precultivation parameters on the catabolism of branched-chain amino acids by Staphylococcus xylosus and Staphylococcus carnosus

The influence of precultivation parameters on the ability of Staphylococcus xylosus and Staphylococcus carnosus to convert branched-chain amino acids—leucine, isoleucine and valine—into volatile flavour compounds was investigated using resting cells in a defined reaction medium. The studied precultivation parameters were: growth phase, temperature, NaCl concentration and the concentration of leucine, isoleucine and valine (only for S. xylosus). Flavour compounds were sampled by automatic static headspace collection and separated/quantified using gas chromatography/flame ionization detection (GC/FID).Main catabolic products from degradation of leucine, isoleucine and valine were the flavour intensive branched-chain acids: 2- and 3-methylbutanoic and 2-methylpropanoic acids. The precultivation parameters altered the production of the branched-chain acids significantly, but to various degrees for S. xylosus and S. carnosus.Production of branched-chain acids by S. carnosus was only influenced slightly by the growth phase and not by changing the NaCl concentration between 4.0% and 10.0% (w/w). Lowering the temperature from 28°C to 18°C significantly decreased S. carnosus’ generation of branched-chain acids. In contrast, S. xylosus was significantly influenced by all precultivation parameters, in particular by the growth phase. Cells taken from growing cultures had a much higher production of branched-chain acids compared to cells taken from stationary cultures. Addition of leucine and valine to the precultivation medium enhanced the production of branched-chain acids whereas addition of isoleucine had the opposite effect.     

Plasma concentrations and dietary requirements of leucine, isoleucine and valine: studies with the young chick

The minimal requirements of the chick for leucine, isoleucine and valine have been assessed using diets based on wheat, groundnut meal and crystalline amino acids. The results of these studies clearly indicate that a relatively low isoleucine concentration of 0.52% allows satisfactory growth rate and efficiency of food conversion with the dietary levels of leucine and valine maintained at 0.98 and 0.63%, respectively. No beneficial response occurs to further supplementation with isoleucine. However, a higher isoleucine level (0.76 %) augments the requirements of leucine and valine to 1.10 and 0.75%, respectively, as judged by growth and efficiency of food conversion responses. These patterns of interactions are reflected in the plasma concentrations of leucine, isoleucine and valine.     

Transaminase activity in extracts of cod muscle studied by the use of a 14C-labelled amino acid mixture

Changes in the amino acid fraction of buffered extracts of cod muscle during incubation in the presence of α-ketoglutarate have been studied using a ¹⁴C-labelled amino acid mixture. The patterns of radioactive amino acids pre and post incubation suggest that transamination reactions are responsible for marked changes in the levels of aspartic acid, glutamic acid, alanine, valine, leucine and iso-leucine. No changes were noted in the levels of threonine, serine, proline, glycine, lysine and arginine. Slight falls in the concentration of tyrosine and phenylalanine were seen after extended incubation times.     

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     

Effects of branched‐chain amino acid addition on chemical constituents in lychee wine fermented with Saccharomyces cerevisiae

This study investigated the impact of single branched‐chain amino acid addition (L‐valine, L‐leucine, L‐isoleucine) on nonvolatile and volatile constituents in lychee wine fermented with Saccharomyces cerevisiae MERIT.ferm. The individual addition of branched‐chain amino acids decreased the consumption of proline with significantly different rates. The lychee wine with added L‐leucine and L‐isoleucine had higher odour activity values (OAVs) of isoamyl alcohol, active amyl alcohol and isoamyl acetate. The lychee wine with added L‐valine had higher OAVs of isobutyric acid and isobutyl alcohol (5.55 and 2.98, respectively), which were lower in the wine with added L‐leucine (2.91 and 1.02, respectively) or L‐isoleucine (3.43 and 1.73, respectively) relative to the control wine (4.18 and 1.77, respectively), suggesting interactive effects among the amino acids. These findings demonstrated that lychee wine aroma can potentially be manipulated via the addition of selected branched‐chain amino acids.     

Chemical and taste characteristics of threadfin bream (Nemipterus japonicus) hydrolysate

The chemical and taste characteristics of threadfin bream (Nemipterus japonicus) hydrolysate produced by Alcalase hydrolysis of the fish muscle (pH 8.5, 60 °C, 120 min, enzyme/substrate ratio 20 g kg^?1) were studied. Umami, bitter and fishy were the dominant tastes. The bitter amino acids methionine, valine, isoleucine, phenylalanine, leucine and tyrosine comprised 317 mg g^?1 of the total amino acids present. The umaminess could be due to the presence of high contents of glutamic acid and inosine 5′‐monophosphate. SDS‐PAGE analysis showed that the peptides in the hydrolysate had molecular weights ranging between 7.2 and 64 kDa. Copyright © 2004 Society of Chemical Industry     

PRODUCTION OF BEER USING IMMOBILIZED YEAST ENCODING α-ACETOLACTATE DECARBOXYLASE

Genetically modified brewer's yeast encoding α-acetolactate decarboxylase (α-ALDC) was tested in immobilized yeast bioreactors for main fermentation of beer. The α-ALDC enzyme produced by the transformant catalyzes the direct conversion of α-acetolactate to acetoin without formation of diacetyl. The long lagering period required for beer maturation in conventional brewing can thus be shortened or even omitted. Three different packed bed bioreactors were employed, with volumes of 1.6 dm³, 5 dm³ and 25 dm³. The 5 dm³ column had a slightly conical geometry in contrast to the others which had cylindrical shapes. Sintered glass beads were chosen as the carrier material on the basis of experiments with the parent strain. The brewing performance of the transformant compared well with that of the parent strain in the immobilized system. Fermentation, utilization of amino acids (including isoleucine, valine and leucine) and flavour formation were practically identical with both strains, the only difference being a marked decrease in the formation of diacetyl by the transformant. Small differences were, however, observed in the long-term biochemical stability. By using yeast encoding α-ALDC in the immobilized yeast system the total (primary and secondary) fermentation time could be reduced to approximately 2–6 days, compared with 3–6 weeks in a conventional batch process.     

Mutations in five loci affecting GAP1-independent uptake of neutral amino acids in yeast

In order to identify genes involved in uptake of isoleucine, leucine and valine in Saccharomyces cerevisiae we isolated mutants that, on a complex medium, were sensitive to an inhibitor of the biosynthesis of the branched-chain amino acids. Mutants that in a secondary screen showed reduced uptake of isoleucine, leucine and valine when growing in synthetic complete medium were further characterized. Genetic analysis identified five loci, named ssy1 through ssy5. ssy2 corresponds to the previously characterized bap1 mutation, which we recently have found to be allelic to stp1. ssy1, ssy3 and ssy5 exhibit a reduced uptake of phenylalanine, methionine and threonine, as well. Furthermore, they are resistant to several neutral amino acid analogs. ssy4 only affects uptake of few neutral amino acids and is as sensitive as the wild type to the amino acid analogs tested. It was previously found that a C-terminal truncation of 29 codons of BAP2, which encodes a branched-chain amino acid permease, results in increased uptake of the branched-chain amino acids. We find epistasis of the C-terminally truncated BAP2 gene over the ssy4 mutation, while the other ssy mutations are epistatic over the truncated BAP2 gene. SSY1, SSY3 and SSY5 were cloned from a low-copy genomic library by complementation of the mutants. The SSY3 gene and the SSY5 gene show no significant homology to any sequence in the databases. SSY1 is a member of the major family of genes encoding amino acid permeases in yeast. We discuss possible roles of Ssy1p in amino acid uptake. © 1998 John Wiley & Sons, Ltd.     

Study of natural mango juice spoilage and microbial contamination with Penicillium expansum by high resolution H NMR spectroscopy

High resolution ¹H NMR has been applied to monitor the changes in the composition of natural mango juice subjected to spoilage and to microbial contamination with Penicillium expansum. A vast number of compounds undergoing changes upon these processes have been identified and their variations followed throughout time (132 h). Besides the formation of typical fermentation products (e.g. acetate, lactic acid, acetoin and isopropanol/2,3-butanediol) and the utilization of the major sugars (sucrose, glucose and fructose), there were changes in organic acids (e.g. decreases of quinic and shikimic acids with formation of 3,4,5-trihydroxycyclohexane acid in spoiled juice, and decreases of citric and malic acids in contaminated juice), amino acids (decreases of alanine, leucine, isoleucine and valine), and less abundant components such as oligosaccharides and aromatic compounds. The possibility of using these changes as early indicators of natural spoilage or P. expansum contamination is discussed.     

Production of volatile compounds by Lactobacillus sakei from branched chain α-keto acids

Lactobacillus sakei belongs to the main flora of raw fermented sausages and is used as starter culture. Bacterial starter cultures can convert amino acids to α-keto acids by aminotransferases. These α-keto acids are the precursors of aroma active aldehydes, alcohols and carboxylic acids. In this study the formation of aldehydes, alcohols and carboxylic acids from leucine, isoleucine, valine and the corresponding α-keto acids are analysed in model fermentations with two different strains of L sakei. In the absence and upon addition of leucine, isoleucine and valine they produced 1 μg/ml 3-methylbutanoic, 0.2 μg/ml 2-methylbutanoic acid and 3 μg/ml 2-methylpropanoic acid, respectively. Upon addition of α-ketoisocaproic acid, α-keto-3-methyl-pentanoic acid or α-ketoisovaleric acid the amount of the corresponding carboxylic acid was increased to 40 μg/ml 3-methylbutanoic acid, 20 μg/ml 2-methylbutanoic acid and 35 μg/ml 2-methylpropanoic acid. The response patterns of the strains and amounts of carboxylic acids produced were similar. This behaviour was typical when compared with other strains of L. sakei and suggests general lack of transaminase activity and a limit in the transport of branched chain amino acids and their conversion to volatiles, some of which can contribute to the aroma of fermented sausages.     

Amino acid catabolism by Lactococcus lactis during milk fermentation

Two wild-type Lactococcus lactis strains isolated from naturally Tunisian fermented milk (Leben), and one laboratory strain, were used to investigate the ability of L. lactis to transform amino acids into aroma compounds during milk fermentation. The α-ketoacid acceptor used for leucine transamination, the first step of catabolism, was identified by gas chromatography/mass spectrometry analysis of the ¹⁵N-labelled amino acids that formed from ¹⁵N-labelled leucine in fermented milk. Furthermore, the amino acids produced or catabolized by the laboratory strain via transamination were identified by comparing the free amino acids in milk fermented with the wild-type strain and the double mutant for aromatic and branched-chain aminotransferases, which cannot transaminate amino acids. The three L. lactis strains strongly catabolized leucine and valine during milk fermentation. The principal amino acid formed via leucine and valine transamination was glutamate indicating that α-ketoglutarate was the principal α-ketoacid acceptor and was generated during milk fermentation.     

BIOSYNTHETIC FORMATION OF HIGHER ALCOHOLS BY YEAST. DEPENDENCE ON THE NITROGENOUS NUTRIENT LEVEL OF THE MEDIUM

In previous investigations it has been shown that in batch fermentations the yield of higher alcohols formed biosynthetically (in the sense that yeast synthesizes the carbon skeletons needed) is mainly negatively correlated with the intial level of nitrogenous nutrients in the medium. The validity of this relationship has been established with different nitrogen sources and sugars, and at different sugar concentrations. Its causes have been studied in simple systems, using single, rapidly-absorbed nitrogen sources, usually ammonium salts. Under such conditions it is the amount, rather than the concentration, of nitrogen source that is of importance. Yields of higher alcohols per unit weight sugar consumed are practically identical at varying nitrogen levels as long as nitrogen is available in the medium, but they increase sharply after nitrogen exhaustion, by a factor of about 2–3. However, if allowance is made for the production of valine, leucine, and isoleucine during the period when nitrogen is present, there is no rise in the total production of carbon skeletons along these pathways after the nitrogen is exhausted. On the contrary, the yields of valine-isobutanol and isoleucine-2-methylbutanol skeletons per unit weight of sugar are appreciably reduced in the absence of nitrogen, whereas the yields of leucine-3-methylbutanol skeletons are nearly unchanged.     

Effect of growth phase and dry-cured sausage processing conditions on Debaryomyces spp. generation of volatile compounds from branched-chain amino acids

The generation of volatile aroma compounds by Debaryomyces spp. from branched-chain amino acids (BCAAs) was investigated. The yeast, that was a natural isolate from sausages, was grown in a meat model system and incubated with leucine, isoleucine and valine under various conditions of interest in dry-cured sausages processing. In the meat model system, Debaryomyces spp. showed ability for growing and, simultaneously, raising the pH, using lactate, generating ammonia and several volatile compounds, and altering the free amino acids content. The production of volatile compounds from BCAAs by Debaryomyces spp. was negatively affected by the presence of salt, although pH reduction to 4.5 increased the yield of alcohols and aldehydes. Cells transition from exponential to stationary growth phase diminished alcohol and aldehyde production, but increased acid generation. Thus, it can be expected that the addition of Debaryomyces spp. as a starter culture can modify the flavour pattern of dry-cured sausages.     

Properties and substrate specificities of proteolytic enzymes from the edible basidiomycete Grifola frondosa

Highly active proteolytic enzymes are found in the fruiting bodies of Grifola frondosa. The general properties and substrate specificities of these proteases from G. frondosa (ProGF) were studied. The optimal pH for ProGF activity was pH 3 or 7 using hemoglobin or Hammersten casein as a substrate, respectively. The ProGF exhibited over 70% of maximal activity within the pH range of 4.5–8.5. In terms of temperature, the ProGF were maximally active at 55 °C, while over 80% of maximal activity was observed within the range of 50–75 °C. These proteases were substrate-specific, mainly cleaving at Ala¹⁴-Leu¹⁵, Tyr¹⁶-Leu¹⁷, and Pro²⁸-Lys²⁹ bonds, with occasional cleavage of Phe²⁴-Phe²⁵ bonds in the oxidized insulin B-chain. The ProGF also liberated hydrophobic amino acids, such as valine, leucine, and phenylalanine, using the oxidized insulin B-chain as a substrate. When soy protein was used as a substrate, valine, leucine, phenylalanine, and tyrosine were selectively released from the hydrolysate. Thus, over the time course of incubation, the peptide concentration increased as the average peptide chain length decreased. These results indicate that the ProGF include both endopeptidases recognizing leucine, phenylalanine, and lysine at the P1′ position, and aminopeptidases preferentially releasing hydrophobic and aromatic amino acids such as valine, leucine, phenylalanine, and tyrosine.     

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.     

Enantiomer Separation of D-L Branched Amino Acids by Capillary Electrophoresis in Sport Nutritional Supplements

A capillary electrophoresis method was developed for the chiral separation of D‐L valine, isoleucine, and leucine. The separation of derivatized amino acids with 9‐fluorenylmethyl‐chloroformate was performed by micellar electrokinetic capillary chromatography. We optimized the method by varying β‐cyclodextrin and sodium dodecyl sulphate concentrations in the presence of 2‐propanol. The proposed method was applied to the determination of D‐forms of valine, Isoleucine, and leucine in the presence of an excess of relative L forms, in commercial supplements for sport nutrition. Results demonstrated that the separation of enantiomers was possible up to an enantiomeric ratio of 1:100. The analysis of selected products confirmed the enantiomeric purity of utilized amino acids.