UPTAKE OF AMINO ACIDS IN BOTTOM FERMENTATIONS

The net uptake of amino acids in full-scale brewery fermentations with a strain of Saccharomyces carlsbergensis at 8–9° C. has been followed by analysing amino acids in fermenting wort and beer. The amino acids are usually taken up very incompletely and their uptake follows a certain order, not very different from that observed by British authors using top yeast fermenting at 15° C. The order is not quite constant, however; particularly the uptake of arginine and of ammonia are subject to variations. The amino acids can be divided, according to their rate of absorption and degree of utilization, into three clear-cut groups. The results suggest that it is the system allowing entry of amino acids into the cell that is of prime importance and that this is not capable of regulating the uptake to the demand of amino acids as building blocks for yeast protein. The results are considered in the light of recent investigations on amino acid permeases of yeast, and the metabolic roles of the most rapidly absorbed amino acids, serine, asparagine, glutamine, and threonine, are discussed.     

BIOCHEMICAL PHYSIOLOGY OF OBESUMBACTERIUM PROTEUS,A COMMON BREWERY CONTAMINANT

In defined media, arginine plus either glutamate or aspartate supports growth of Obesumbaeterium proteus but additional amino acids enhance yields. Glucose, fructose, mannose and galactose are rapidly utilized; maltose and starch give slower growth. Fermentation products include acetic, formic, 2-oxoglutaric, pyruvic and succinic acids. With brewer's wort as growth medium, the concentrations of arginine, lysine, NH₃, aspartate, serine, tyrosine and leucine decrease whereas several other amino acids increase in concentration. Fermentation products include acetoin, ethanol, lactic acid, fusel alcohols, some volatile acids and dimethylsulphide. Measurements of oxygen uptake by washed suspensions in the presence of a variety of substrates suggest synthesis of (i) a terminal electron transfer chain that is repressed anaerobically and (ii) a glucose oxidase complex. Radio-respirometry indicates that the EMP is not the sole pathway for glucose metabolism. Cytochrome b₁ is detected in the bacterial cells but not cytochromes a and c. Glucose represses the activities of several enzymes of the EMP and HMP, but not lactate dehydrogenase and 3 enzymes of the citric acid cycle.     

大红浙醋醋酸菌分离及其产酸关键氨基酸分析

该研究首先采用钙透明圈法从大红浙醋中分离筛选醋酸菌,并通过分子生物学技术对其进行菌种鉴定;然后研究10种氮源对巴氏醋酸杆菌的生长及产酸量的影响,并对10种氮源组分进行定性和定量分析,得出产酸代谢的关键氨基酸;最后挑出10种氨基酸进行验证。结果表明,分离筛选得到一株产酸菌株,编号为SHQ-A,经鉴定为巴氏醋酸杆菌（Acetobacter pasteurianus）。该菌株利用10种氮源发酵的产酸量在8.8～34.5 g/L范围内,且以酵母抽提物为氮源的产酸量明显高于蛋白胨。当缺少丙氨酸和精氨酸时,产酸量仅为18.0 g/L和19.2 g/L,分别低于空白组62.5%和60%,表明丙氨酸和精氨酸是产酸代谢的关键积极氨基酸;当缺少脯氨酸时,产酸量为61.2 g/L,高于空白组27.1%,表明脯氨酸是产酸代谢的关键消极氨基酸。     

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     

Berry nitrogen status altered by cover cropping: Effects on berry hormone dynamics,growth and amino acid composition of Pinot Noir

The influence of altered nitrogen (N) content, caused by cover crop, on Pinot Noir berry quality was studied. Treatments included two levels of cover cropping and clean-cultivation. Berry N-content was reduced by grass cover crop competition, with the highest reductions of N at the early stage of berry formation and at the commercial harvest stage. Growth regulators, trans-zeatin and indole-3-acetic acid, and berry growth were influenced by the changes in N-status at different stages of berry formation. The total amino acid content of berries at harvest decreased with cover cropping, while the proline to arginine ratio and assimilable amino acids content improved compared to the berries from clean-cultivated vines. Our data indicate that early stage berry development is influenced by N-status but finally it results in an improved amino acid composition for yeast fermentation in mature berries. At the same time, sugar and pigment contents were not negatively influenced by cover crop-induced changes in berry N-status.     

小麦品种对库德里阿兹威氏毕赤酵母风味代谢特性影响的研究

研究不同小麦品种对库德里阿兹威氏毕赤酵母（Pichia kudriavzevii）MT-Y01风味代谢特性的影响,并对小麦中的游离氨基酸与酵母代谢产生的挥发性风味物质进行典型关联分析（CCA）。结果表明,8种小麦的基本理化指标存在差异,游离氨基酸组成相似,但含量差异较大。小麦品种对酵母MT-Y01代谢产生挥发性风味物质有重要的影响,主要为高级醇和酯类物质（占比80%以上）。酵母MT-Y01以小麦品种鄂麦596为基质时代谢产生的挥发性风味物质总含量最高（568.5 mg/L）;以郑麦1769为基质时代谢产生的挥发性风味物质总含量最低（96.8 mg/L）;以百农201、淮麦32、徐麦35为基质时代谢产生的最高含量风味物质分别为3-甲硫基丙醇、己酸乙酯及苯甲醛。小麦品种对酵母发酵风味的影响与氨基酸关联,3-甲基丁醇、苯乙醇分别与缬氨酸、苯丙氨酸相关性强,3-甲基丁酸与天冬氨酸和天冬酰胺相关性强,苯乙醛受精氨酸影响大,丙酸乙酯、乙酸异戊酯、乙酸-2-苯乙酯受亮氨酸、半胱氨酸和赖氨酸影响大。     

DETERMINATION OF SOME LIPID CONSTITUENTS OF BAKER'S YEAST

Lipid was extracted from whole yeast cells with difficulty because of the limited porosity of the cell wall and its sensitivity to dehydrating solvents. Extraction from mechanically broken cells was rapid and complete. Metabolic alteration of lipid when cells were disintegrated was minimized by first heating a yeast suspension briefly at 90°C. Details are given for the routine analysis of yeast phospholipids by quantitative paper chromatography and of neutral lipids by column chromatography. Lysophosphatides were found in some types of yeast after drying, but not in pressed commercial yeast or in yeast grown on synthetic media. They were formed when yeast was suspended in methanol-water, as yeast phospholipase A was activated and split palmitoleic acid from lecithin and phosphatidyl ethanolamine. Phosphatidyl inositol in baker's yeast contained a higher proportion of saturated fatty acids than other phospholipids. Sterols were present free and as esters with palmitoleic and oleic acids. Phospholipid content was affected by growth conditions. A high level of inositol in the growth medium reduced the formation of neutral lipid but did not affect the content of yeast phospholipid.     

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.     

BASIS OF STABILITY OF AMINE SALTS OF LINOLEIC ACID

SUMMARY— The mechanism and generality of the known stabilization against autoxidation conferred on linoleic acid by certain basic amino acids, such as lysine and arginine, was investigated. Basic amino acids were the only class of compounds found to confer the effect. However, the smallest basic amino acid, 2,3-diaminopropionic acid, was not effective, nor was αβω-diaminc acid, 3,6-diaminohexanoic acid, although a simple isomer of lysine. The stabilization was observed only in the solid phase. Inclusion of sodium chloride in the solid matrix was deleterious to the effect. A large number of physical and chemical observations were made and correlated but it has not been possible to draw detailed conclusions about the mechanism of stabilization, nor can a detailed structure of the stabilized complex be suggested. The cause of the phenomenon appears to be closely associated with the physical arrangement of the ions in the crystal lattice.     

蛹虫草菌丝体发酵培养过程中氨基酸添加技术研究

组氨酸、精氨酸、赖氨酸是对蛹虫草生长影响较大的3 种碱性氨基酸,本实验研究在蛹虫草液体发酵过程中添加上述氨基酸的不同条件对蛹虫草菌丝体生物量和其中主要抗癌功能成分虫草素的影响。首先进行添加氨基酸种类、氨基酸的添加量、初始pH 值、培养温度对蛹虫草菌丝体生物量和虫草素含量的单因素试验,在此基础上进行四因素三水平的正交试验。结果表明：对蛹虫草菌丝体生物量影响从大到小的顺序依次是：氨基酸种类＞培养温度＞初始pH 值＞氨基酸添加量。增加蛹虫草菌丝体生物量的最佳培养条件为：添加氨基酸种类为精氨酸,培养温度为26℃,培养初始pH 值为6,100mL PDA 发酵培养液添加精氨酸0.3g。对蛹虫草菌丝体中虫草素含量影响从大到小的顺序依次是：氨基酸种类＞初始pH 值＞培养温度＞氨基酸添加量。增加菌丝体中虫草素含量的最佳培养条件是添加氨基酸种类为精氨酸,培养温度为24℃,100mL PDA 发酵培养液添加0.3g,培养的初始pH 值为6 。     

Cationic amino acid transport by bovine mammary tissue

Cationic amino acid transport (arginine and lysine) into bovine mammary tissue occurs by a sodium independent and saturable mediated system. Concentrative uptake ratios (cell concentration/media concentration) for both arginine and lysine varied between 6 and 22. High concentrations of specific inhibitors of the neutral amino acid transport systems had no effect upon arginine or lysine uptake. Both arginine and lysine were strong inhibitors of each others uptake, whereas ornithine showed less specificity for inhibition of arginine and lysine uptake. In the presence of all amino acids, cationic amino acid uptake occurred at a rate equivalent to that with cationic substrates alone. The presence of the independent cationic amino acid transport system in bovine mammary tissue accounts for the high cationic amino acid uptake by measuring arteriovenous differences of plasma across the bovine mammary gland. This transport system is responsible for excess uptake (beyond milk protein synthesis requirements) of cationic amino acids for catabolism to other amino acids or potential oxidation.     

BIOCHEMISTRY OF TEA FERMENTATION: CONVERSION OF AMINO ACIDS TO BLACK TEA AROMA CONSTITUENTS

SUMMARY: ¹⁴C-amino acids were added to fresh tea-leaf homogenate undergoing conversion to black tea. After conversion (30 min, 25°C), the volatile compounds present in the headspace over the reaction mixture were collected and analyzed by gas chromatography. Results showed that leucine, isoleucine, valine and phenylalanine were partially converted to the aldehydes expected from a Strecker degradation. These aldehydes are constituents of black tea aroma. Further, drying of the fermented mixture caused an additional amount of the aldehydes to be formed. In contrast, no detectable volatile compounds were formed from aspartic acid, glutamic acid, glutamine, arginine, threonine. serine or theanine under the same conditions. Production of aldehydes from amino acids was shown to be dependent on the enzymic conversion process: Tea leaf which had been inactivated by steam treatment was not effective in causing formation of volatile aldehydes from the amino acids. Identical results were obtained in a model tea fermentation system composed of a crude soluble enzymes extract from tea leaves, purified epigallocatechin gallate and ¹⁴C-amino acids. Ascorbic acid was found to inhibit formation of aldehydes from amino acids in this model tea fermentation system; dehydroascorbic acid by itself was found to be effective in causing formation of volatile aldehydes from amino acids.     

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.     

The potential of dairy lactic acid bacteria to metabolise amino acids via non-transaminating reactions and endogenous transamination

The metabolism of amino acids by 22 starter and 49 non-starter lactic acid bacteria (LAB) was studied in a system consisting of amino acids and non-growing cells without added amino acceptors such as alpha-ketoglutarate. There were significant inter- and intra-species differences in the metabolism of amino acids. Some amino acids such as alanine, arginine, aspartate, serine and branched-chain amino acids (leucine, isoleucine and valine) were utilised, whereas other amino acids such as glycine, ornithine and citrulline were produced. Alanine and aspartate were utilised by some LAB and accumulated during the incubation of other LAB. Arginine was degraded not only by Lactococcus lactis subsp. lactis (the lactococcal subspecies known to catabolise arginine), but also by pediococci, heterofermentative lactobacilli (Lactobacillus brevis and Lb. fermentum) and some unidentified homofermentative lactobacilli. Serine was utilised predominantly by homofermentative Lb. paracasei subsp. paracasei, Lb. rhamnosus and Lb. plantarum. Of the LAB studied, Lb. brevis and Lb. fermentum were the most metabolically active, utilising alanine, arginine, aspartate, glutamate and branched-chain amino acids. Leuconostocs were the least metabolically active, showing little potential to metabolise amino acids. The formation of ammonia and acetate from amino acid metabolism varied both between species and between strains within species. These findings suggest that the potential of LAB for amino acid metabolism via non-transaminating reactions and endogenous transamination will impact both on the physiology of LAB and on cheese ripening, especially when transamination is rate-limiting in the absence of an exogenous amino acceptor such as alpha-ketoglutarate.     

Characterization of a Caffeine-Resistant Mutant of Aspergillus parasiticus: Role of Amino Acid Metabolism

Aspergillus parasiticus BCRI, a caffeine-resistant mutant of A. parasiticus NRRL 2999, produced abundant amounts of aflatoxins (AF) in yeast extract-sucrose broth only when the medium was supplemented with caffeine. However, little AF production occurred in glucose-mineral salts medium (GMS) regardless of the level of caffeine supplementation. Caffeine-dependent AF production was restored if GMS were fortified with peptone or some other source of amino acids. Subsequent studies indicated that this effect could be achieved by supplementing GMS with specific amino acids, particularly proline, alanine, methionine, arginine or asparagine. Restoration of caffeine-dependent AF synthesis did not occur when GMS was supplemented with purine bases or nucleotides. The results indicated that caffeine-dependent AF production in BCRI was dependent on amino acid catabolism.     

Influence of addition of ammonium and different amino acid concentrations on nitrogen metabolism in spontaneous must fermentation

The effect of the addition of different amino acid concentrations in must on yeast nitrogen metabolism during alcoholic fermentation was studied. To do this, fermentations of Mazuelo must, poor in nitrogen compounds, were carried out. Ammonium and different concentrations of amino acids (0, 45, 120, 250 and 450 mg/l) were added to the must. Addition of 45, 120 and 250 mg/l of proteic amino acids to the must increased the rate of fermentation. Proline was mainly consumed in fermentations with smaller amounts of amino nitrogen and, at the same time, this amino acid showed the highest residual concentration in the final wines. The consumption of other proteic amino acids was directly proportional to their concentration in the musts, with the exception of leucine and isoleucine that were synthesized. However, a difference in the percentages of the amino acids consumed by the yeasts was observed. The percentages of aspartic acid, alanine and arginine consumed were higher in the fermentations supplemented with amino acids than in the fermentation where only ammonium was added. The percentages of tyrosine and phenylalanine consumed gradually increased with increase of their initial concentration.     

Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.     

Effects of nitrogen catabolite repression‐related amino acids on the flavour of rice wine

The quality of rice wine is highly dependent on the content of the flavour compounds produced by the budding yeast Saccharomyces cerevisiae. In this study, the effects of three amino acids (arginine, glutamate and glutamine) related to nitrogen catabolite repression on the formation of flavour compounds were investigated. Each of these amino acids could promote the growth of S. cerevisiae, and a total of 83 flavour compounds were found in a model system of rice wine production. The effects of arginine, glutamate and glutamine on the content of the higher alcohols, amino acids and esters were significant, whereas the effects on the aldehydes and organic acids were slight. The results of this study could facilitate the development of new strategies to control the flavour pattern and improve the quality of rice wine. Copyright © 2015 The Institute of Brewing & Distilling     

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

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

Application of Plackett–Burman experimental design for investigating the effect of wort amino acids on flavour‐active compounds production during lager yeast fermentation

Aroma‐active higher alcohols and esters are produced intracellularly in the cytosol by fermenting lager yeast cells, which are of major industrial interest because they determine aroma and taste characteristics of the fermented beer. Wort amino acid composition and their utilization by yeast during brewer's wort fermentation influence both the yeast fermentation performance and the flavour profile of the finished product. To better understand the relationship between the yeast cell and wort amino acid composition, Plackett–Burman screening design was applied to measure the changes in nitrogen composition associated with yeast amino acids uptake and flavour formation during fermentation. Here, using an industrial lager brewing strain of Saccharomyces pastorianus , we investigated the effect of amino acid composition on the accumulation of higher alcohols and volatile esters. The objective of this study was to identify the significant amino acids involved in the flavour production during beer fermentation. Our results showed that even though different flavour substances were produced with different amino acid composition in the fermentation experiments, the discrepancies were not related to the total amount of amino acids in the synthetic medium. The most significant effect on higher alcohol production was exercised by the content of glutamic acid, aromatic amino acids and branch chain amino acids. Leucine, valine, glutamic acid, phenylalanine, serine and lysine were identified as important determinants for the formation of esters. The future applications of this information could drastically improve the current regime of selecting malt and adjunct or their formula with desired amino acids in wort. Copyright © 2017 The Institute of Brewing & Distilling