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.     

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 BY VARIOUS YEASTS

The formation of higher alcohols by various yeasts has been studied as a function of the nitrogen level of the medium. Two types of substrate were used. In one ammonium sulphate was the sole nitrogen source and in the other a complex amino acid mixture simulating that present in brewery wort was used. In the former medium, the patterns exhibited by the yeasts were all very much alike, indicating that the formation of higher alcohols through the biosynthetic activities of yeasts is always basically similar. In the medium containing a complex mixture of amino acids, where the higher alcohols can be formed partly or entirely from the exogenous amino acids through Ehrlich's mechanism, widely different patterns were observed, indicating that the utilization of exogenous amino acids as a source of nitrogen and the extent of regulation of biosynthetic pathways vary with species and strain. All the brewery strains seem to be rather similar and no distinct differences between top and bottom fermentation strains could be observed.     

AMINO ACID ANALYSIS OF BEER POLYPEPTIDES

The principles of amino acid analysis of proteins and polypeptides are reviewed. Analysis of the amino acid composition of dialysed beer material prepared from a wide variety of commercial and pilot brewery beers showed that the principal amino acids comprised glutamic acid/glutamine, proline, glycine and aspartic acid/asparagine. The results from the analysis of a series of pilot brewery beers brewed under standardised conditions showed that the composition of the grist may influence the amino acid composition of beer polypeptide fractions. Dialysed beer material prepared from beer brewed from grists containing torrified wheat, wheat flour and malted wheat contained greater proportions of glutamic acid/glutamine compared to material prepared from all malt beers. Further fractionation and analysis of dialysed beer material prepared from pilot brewery beers suggested that fractions MW>60000 contained polypeptide material derived from yeast mannan-protein. In addition fractions MW>60000 prepared from beer brewed from grists containing torrified wheat, wheat flour or all malted wheat may contain high molecular weight polypeptide material derived from wheat proteins. The results from the analysis of fraction MW 40,000–60000 prepared from beers brewed from grists containing all malt, 80% malt and 20% torrified wheat and 50% malt and 50% malted wheat are consistent with the presence of polypeptide material derived from cereal albumins and globulins whereas fractions MW 40,000–60000 prepared from beers brewed from 80% malt and 20% wheat flour and 100% malted wheat may contain polypeptide material derived from wheat prolamins and glutelins. The amino acid composition of fraction MW 20,000–40,000 from all pilot brewery beers investigated is consistent with the presence of polypeptide material derived from cereal prolamins and glutelins. The amino acid composition of beer polypeptide fractions may be used to detect the use of wheat adjuncts in beer brewing.     

SIMULTANEOUS ASSESSMENT OF CHANGES OCCURRING DURING BATCH FERMENTATION

Detailed study of the fermentation of wort by Saccheromyces cerevisiae on a pilot scale showed that all the oxygen and considerable quantities of certain amino acids present in the wort were absorbed by the yeast before active fermentation commenced. The pH fell rapidly after pitching and reached a constant level after 32 hr. The α acids and iso α acids in the wort diminished in parallel with the decrease in pH and the increase in concentration of suspended yeast, respectively. Esters and higher alcohols appeared as the carbohydrate content of the wort diminished. Their production did not parallel the uptake of amino acids or changes in the amount of suspended yeast. The level of diacetyl reached a maximum when valine was fully taken up from the wort. Distillation of fermenting wort caused production of diacetyl.     

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     

THE FATE OF AMINO ACIDS DURING MASHING AND HOP BOILING

In an infusion mash system the concentration of all α amino acids in sweet wort reaches a maximum value when approximately one-third of the total volume extracted has been run from the mashing vessel. The relative concentrations of each α amino acid in wort vary throughout the mash tun extraction owing to differences in rate of extraction of the amino acids and to the quantities produced by peptidase activity in the mash tun. The relative proportions of proline, alanine, glutamine and asparagine in the total α amino nitrogen content of wort decrease appreciably as the mashing process proceeds. Copper boiling results in the partial destruction of methionine, glutamic acid, lysine, arginine and valine present in sweet wort together with an increase in the concentration of ammonium ion in the wort.     

啤酒高浓酿造中氨基酸对酵母发酵性能和啤酒色值的影响

探讨在24?°P高浓啤酒发酵过程中8?种氨基酸（Met、Phe、Trp、Arg、His、Ile、Leu、Lys）的不同添加量（分别为原麦汁中相应氨基酸含量的0.5、1?倍和2?倍）对酵母生理特性、发酵性能和啤酒色值的影响。结果表明：8?种氨基酸的补充可显著提高麦汁发酵度、乙醇产量,促进酵母生长,提高酵母活细胞率,改善啤酒色值。其中,补充1?倍氨基酸的高浓麦汁发酵性能较好,与对照组相比,发酵度、乙醇产量、最大悬浮酵母细胞数和发酵结束时的酵母活细胞率分别提高了6%、17%、11%和10%。添加氨基酸的高浓酿造啤酒经稀释后,啤酒色泽依然鲜亮,且添加1?倍氨基酸酿造而成的啤酒经稀释后色差（ΔE）最小,色泽最接近青岛纯生啤酒。     

SOYBEAN WHEY PROTEINS-RECOVERY AND AMINO ACID ANALYSIS

SUMMARY– About 14 million Ib of soybean whey protein of high biological value is disposed of as waste based on estimated production figures for soybean concentrates and isolates. An estimated 5–7% annual increase in consumption poses serious waste disposal problems and alternates should be sought. By simulated commercial procedures, yield and protein content of soybean whey were determined. Whey solids account for 2–28% of original nitrogen in dehulled, defatted flakes. Whole whey protein was prepared by dialysis, and whey was also fractionated by heating into heat-coagulable and supernatant proteins. Whole whey protein has a good balance of essential amino acids when compared with a system followed by the Food and Agricultural Organization of United Nations based on hen's egg protein. Heat-coagulable and supernatant proteins varied greatly: heat-coagulable fractions had 42% of the sulfur amino acid content but 181% of the tryptophan content of hen's egg protein; supernatant protein had 142% of the sulfur amino acid content but only about 60% of the isolevcine, valine, leucine and tryptophan content of hen's egg protein. All three whey protein fractions would be suitable for addition to feeds.     

INFLUENCE OF THE CONCENTRATION OF BRANCHED CHAIN AMINO ACIDS ON THE FORMATION OF FUSEL ALCOHOLS

The influence of concentrations of branched-chain amino acids on the total, anabolic and catabolic production of isobutyl, active amyl, and isoamyl alcohol by Saccharomyces cerevisiae X 2180-1A has been examined in media with high nitrogen levels. It has been found that the total and catabolic production of higher alcohols increased with increasing concentrations of the corresponding amino acids. The anabolic pathway can be suppressed by high levels of the corresponding amino acid. Lower concentrations up to 1 mm for isoleucine and 10mm for leucine stimulate the anabolic formation of active amyl alcohol and isoamyl alcohol respectively. From the data obtained, it is shown that, under the conditions used, 50% or more of the fusel alcohols may arise in a beer wort fermentation from carbohydrates.     

INHIBITION OF BEER-SPOILAGE LACTIC ACID BACTERIA BY NISIN

149 strains of bacteria, mostly brewery contaminants able to spoil wort or beer, and 12 brewing strains of yeast (8 ale and 4 lager strains) have been screened using a well-test assay for sensitivity to the food preservative, Nisin (E234), Nisin inhibited growth of 92% of the gram-positive strains, predominantly lactic acid bacteria of the genera Lactobacillus and Pediococcus. In contrast, all 32 gram-negative strains tested, except 3 Flavobacter strains, were Nisin-resistant; in addition none of the brewing yeasts showed Nisin-sensitivity. Therefore. Nisin has potential applications in preventing spoilage of worts or beers by lactic acid bacteria.     

METHIONINE AND SULPHATE AS COMPETING AND COMPLEMENTARY SOURCES OF SULPHUR FOR YEAST DURING FERMENTATION

Study of the uptake of sulphate and methionine by an ale yeast from a range of media showed that utilisation of sulphate was fairly strictly controlled but assimilation of methionine was not. Cells never took up more than about 0.3 mMol sulphate per litre whilst methionine, up to an initial concentration of 10 mMol per litre, was completely absorbed. Sulphate-grown cells had low intracellular pools of amino acids and methinonine was never detected. Methionine-grown cells contained methionine in both cytosol and vacuole and the concentration of several other amino acids also increased in such a way to suggest that methionine catabolism was occurring. With mixed sulphur sources methionine prevented uptake of sulphate when the concentration of sulphate was high but not when it was low suggesting the presence of two sulphate transporters with different control properties. Sulphate did not influence uptake of methionine. Addition of other amino acids to the medium did reduce the rate and extent of methionine uptake but not the intracellular pool sizes. Pilot plant studies suggested that SO₂ production in a brewery is more likely to be a reflection of the overall nutritive status of the wort rather than be connected to the initial methionine concentration .     

Elucidation of the Role of Nitrogenous Wort Components in Yeast Fermentation

The Free Amino Nitrogen (FAN) content of wort prescribes efficient yeast cell growth and fermentation performance. FAN consists of the individual amino acids, small peptides and ammonia ions formed during malting, the relative amounts of which vary. In this paper, the individual constituents of FAN were dissected and their effect on both ale and lager fermentations determined. The patterns of amino acid and small peptide uptake and the changes in extracellular protease activity revealed the dynamic environment that develops during fermentation. Lysine and methionine, previously identified as key amino acids in wort fermentation, were investigated further.     

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.     

AMINO ACID COMPOSITION AND NUTRITIONAL IMPLICATIONS OF MILK FERMENTED BY VARIOUS LACTIC CULTURES

A total of nine milk products were analyzed for total and free amino acids and total and nonprotein nitrogen. The products were: (1) whole milk (WM), (2) skim milk (SM), (3) WM fermented by Streptococcus thermophilus, (4) SM fermented by S. thermophilus, (5) WM fermented by Lactobacillus bulgaricus, (6) SM fermented by L. bulgaricus, (7) WM fermented by L. acidophilus, (8) SM fermented by L. acidophilus and (9) commercially prepared buttermilk. Essential amino acid content of the fermented milk was lower than that of the whole or skim milk. Fermentation of milk by lactic cultures decreased the total lysine (except butter milk) and methionine contents of all the samples. Since these two amino acids are limiting amino acids in protein malnourished areas, lactic fermentation of milk may be undesirable based on the chemical scores of the protein. However, the free amino acid content, including that of lysine and methionine, increases significantly upon lactic fermentation of milk. This predigestion of protein might result in an increased availability of the amino acids to offset the reduction in amino acids during fermentation and may even result in a better quality protein. Biological evaluation of the fermented milk proteins is necessary to evaluate their nutritional quality. L. bulgaricus was the most proteolytic of all the organisms used.     

EXTRUDED SORGHUM AS A BREWING RAW MATERIAL

Small scale mashes (50 g total grist) with grists containing up to 50% by weight of extruded whole sorghum produced worts of high extract yield and low viscosity. Increasing the proportion of extruded sorghum in the grist resulted in decreasing wort filtration volume, total nitrogen and free amino nitrogen content. The wort filtration behaviour of mashes containing sorghum extruded at 175°C was superior to that of mashes containing sorghum extruded at 165°C or 185°C. The results from such small scale mashing experiments suggested that extruded sorghum compared favourably to extruded barley and extruded wheat as a brewing adjunct. Worts and beers were produced on a pilot brewery scale (100 1) from grists comprising 70% malt + 30% extruded sorghum and 100% malt under isothermal infusion mashing conditions. Mashes containing sorghum extruded at 175°C showed comparable wort filtration behaviour to that of the all malt control mash whereas mashes containing sorghum extruded at 165°C or 185°C showed poor wort filtration behaviour. Worts produced from grists containing extruded sorghum fermented more quickly than the control wort and attained lower values of final gravity. The resulting beers were filtered without difficulty. Beers produced from grists containing extruded sorghum contained lower levels of total nitrogen and free amino nitrogen compared to the control beer consistent with extruded sorghum contributing little or no nitrogenous material to the wort and beer. Beers brewed from grists containing extruded sorghum were of sound flavour and showed reasonable foam stability behaviour.     

Studies on Wheat Steep Liquor Produced During Starch Production

Chemical analysis of wheat steeping liquor, produced from soaking of wheat grains revealed the presence of 1% total soluble solids with 7,15% proteins. A method was devised to obtain large amounts of precipitates with protein content up to 9,84%. Glutamic acid and lysine constitute the largest proportions of the amino acids. Nine essential amino acids were found. The raw wheat steeping liquor and the supernatant resulting after separation of the protein were used as a growth medium for the production of fodder yeast Candida tropicalis.     

QUANTITATIVE GAS CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS IN BEER AND WORT

Amino acids present in beer and wort samples were isolated using Dowex 50W-X8 ion exchange resin. These acids were converted to N-trifluoroacetyl methyl esters and were separated on a 10 ft. x 1/4 in. copper column packed with 3/4% Carbowax 1540 and 1/4% NPGS on Chromosorb W. Complete separation and quantitative estimation was obtained for eleven amino acids: valine, alanine, isoleucine, leucine, glycine, proline, aspartic acid, threonine, methionine, glutamic acid, and phenylalanine. Standards of the acids showed a relative standard deviation of 4% and less for nine acids while threonine and methionine gave deviations of 9% and 15% respectively. The other naturally-occurring amino acids did not interfere with the separation of the above eleven. Amino acid assimilation data is presented along with other beer and wort analyses.     

ESTIMATION OF ALPHA AMINO NITROGEN

The free alpha amino nitrogen in wort and beer has been estimated by reaction with trinitrobenzene sulphonic acid (TNBS) either at 40°C for 2 h or at 60°C for 30 min. Statistical analysis shows that for worts there is no significant difference between results obtained under the two sets of conditions, but for beers the values are higher when determined at 60°C by amounts which vary from beer to beer. However, there is no loss in precision (± 2 SD) so that the more rapid method can be used with advantage.     

RATE-LIMITING ENZYMES IN THE LIBERATION OF AMINO ACIDS IN MASHING

When 29 malts representing a wide variation in free amino nitrogen of wort were mashed for 3 h at 45°C, the increases in TCA-soluble amino nitrogen varied from 47 to 116 μmoles per gramme of malt. The carboxypeptidase activities of the malts showed only small positive correlations with the increase of amino nitrogen during mashing. Inactivation of the different malt carboxypeptidases by 70 to 90% with diisopropylfluorophosphate decreased the liberation of amino nitrogen by only 5 to 25%. Moreover, addition of purified malt carboxypeptidase I to the mashing suspensions had no effect on the liberation of amino nitrogen. It is therefore evident that the carboxypeptidases do not have a major rate-limiting role in the liberation of amino acids in mashing, although it has earlier been shown that they are essential enzymes in this process. The proteinase activities of the malts, determined using two methods, correlated strongly with the liberation of amino nitrogen in mashing. Addition of crude malt proteinase to the mash increased, and addition of purified barley proteinase inhibitor decreased the measured proteinase activity and the liberation of amino acids roughly to the same extent. These results indicate that the most important rate-limiting enzyme activity in the liberation of amino acids in mashing is the proteinase activity of the malt.