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     

Nitrogen starvation induces expression of Lg‐FLO1 and flocculation in bottom‐fermenting yeast

When exponentially growing cells of bottom‐fermenting yeast were starved for nitrogen or were grown on proline (a non‐preferred nitrogen source), flocculation was induced. This flocculation was not induced by starvation for either carbon or amino acids. Expression of Lg‐FLO1, which is required for flocculation of bottom‐fermenting yeast, was also found to be induced by starvation for nitrogen. This suggests that the flocculation of bottom‐fermenting yeast is under the control of a nitrogen catabolite repression (NCR)‐like mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Simultaneous utilization of ammonia,free amino acids and peptides during fermentative growth of Saccharomyces cerevisiae

The efficiency of nitrogen use by yeast is one of the key determinants of the successful completion of alcoholic fermentations. In this work the growth of Saccharomyces cerevisiae S288c in a synthetic medium containing ammonia and free amino acids, supplemented with yeast hydrolysate, was studied. Experiments with ¹⁵NH₄Cl and ¹⁵N‐labelled yeast hydrolysate were carried out to gain insight into which of these three classes of assimilable nitrogen sources yeast cells prefer. Co‐consumption of all three sources was observed; approximately 40% of the total nitrogen in the yeast protein fraction originated from yeast hydrolysate, while free amino acids and ammonia contributed 40 and 20%, respectively. The results indicate that several amino acids are more readily obtained from peptides, most likely when the uptake of their free forms is competitively inhibited and/or repressed. During the second half of each fermentation, a decrease in the incorporation of yeast hydrolysate‐derived nitrogen was observed. These results highlight the nutritional role of peptides in various yeast fermentations. Copyright © 2016 The Institute of Brewing & Distilling     

The Branched-Chain Amino Acid Permease Gene of Saccharomyces cerevisiae,BAP2, Encodes the High-Affinity Leucine Permease (S1)

The amino acid leucine has been shown previously to be transported into a yeast cell by at least three permeases: the general amino acid permease, a high-affinity permease (S1) and a low-affinity permease (S2). We isolated the gene BAP2 as a multicopy suppressor of the YPD⁻ phenotype of aat1leu2 yeast. BAP2 has been identified previously as encoding an amino acid permease which transports branched-chain amino acids. In order to align the genetic and biochemical studies of leucine uptake we completed a detailed kinetic analysis of yeast strains in which the BAP2 gene was disrupted and compared this to the kinetics of uptake of the parental strain. We demonstrate that BAP2 encodes the high-affinity leucine permease previously called S1. © 1997 John Wiley & Sons, Ltd.     

Effect of Fermentation on Soluble Proteins and In Vitro Protein Digestibility of Sorghum, Green Gram and Sorghum-Green Gram Blends

Changes in pH, titratable acidity and proteins of meals of sorghum, green gram and sorghum-green gram blend (70:30, w/w) during fermentation at 25°C up to 5 days were monitored. The pH of fermenting meals decreased sharply with concomitant increase in the titratable acidity within 24 hr. The proteins, free amino acids, soluble proteins and in vitro protein digestibility markedly increased within 24 hr fermentation. Supplementation of green gram with sorghum decreased the acidity and improved the contents of crude protein, soluble proteins and free amino acids of fermented sorghum meal.     

New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations

The budding yeast Saccharomyces cerevisiae is able to take up large quantities of amino acids in the form of di‐ and tripeptides via a short peptide transporter, Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this upregulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids: the first is upregulation of PTR2 in a population of cells, caused by amino acid secretion that accompanies peptide uptake; the second is loss of Ptr2p activity, due to transporter internalization following peptide uptake. Our findings emphasize the importance of proper amino acid balance in the cell and extend understanding of peptide import regulation in yeast. Copyright © 2015 John Wiley & Sons, Ltd.     

Differentiation between fermenting and spoilage yeasts in wine by total free fatty acid analysis

Differentiation between fermenting and spoilage yeasts in wine was estimated by cellular fatty acid profiles. Forty-two strains of yeasts representing 17 genera were grown on a defined liquid medium for 48 h at 25°C in a rotary shaker. After saponification of yeast cells, free fatty acid extracts were analysed by gas chromatography. Multivariate analysis was performed by Principal Components Analysis (PCA) to define clusters of fatty acids and yeasts. Strains were characterised especially by long-chain fatty acids, palmitic (C16) to linolenic (C18:3) acid under aerobic culture. Nevertheless, most of Saccharomyces cerevisiae and also Dekkera bruxellensis (former names D intermedia and Brettanomyces lumbicus) synthesized medium-chain fatty acids, octanoic (C8) acid to dodecanoic (C12) acid. With this method it was possible to differentiate fermenting grape yeasts such as S cerevisiae from spoilage yeasts on the basis of the absence of linoleic (C18:2) and linolenic (C18:3) acids. However, the method seemed unreliable for the identification of strains, more particularly those of species of S cerevisiae.     

THE INFLUENCE OF TEMPERATURE ON SOME PROPERTIES OF YEAST

The effects of temperature on the growth of yeast and on its metabolic activity in distiller's malt wort have been studied. In un-aerated fermentations, maximum yeast production takes place at about 30° C. whereas the growth rate in aerated cultures is highest at 35° C. The lag phase of the yeast studied fell from 6 hr. at 20° C. to 2·8 hr. at 25° C. and was not thereafter greatly affected by increases of temperature until 42° C. was reached, at which point growth ceased. Maltase activity was maximal at 25° C. when considered in terms of unit quantities of either yeast or fermenting wort, but the optimum temperature for initial fermentation velocity varied according to the time over which the measurement was made, being maximal at 40° C. for 0·5 hr., and at 35° C. for 2 hr. Alcohol production was highest at 25° C. whereas glycerol and higher alcohol formation took place optimally at 30° C.     

Amino Acid Profiles of Fermenting Wheat Sour Doughs

The free amino acid patterns of unfermented and fermented wheat sour doughs started with Lactobacillus plantarum were related by factor analysis to dough yield, ash content of flour, fermentation temperature and presence/absence of yeast, from data corresponding to a central composite design of samples. Individual levels of hydrophobic, acid and basic amino acids as well as total amino acid content positively correlated with extraction rate of flours. The presence of yeast promoted metabolism of histidine, aspartic acid and asparagine, particularly in samples made with whole and wholemeal flours. Proliferation of predominant amino acids by enzymatic release was more notable at higher fermentation temperatures and wholemeal samples. Sour doughs with maximum levels of hydrophobic and basic amino acids were started with no yeast bacterial cultures, made with whole and wholemeal flours and fermented at 35°C.     

Impact of the Long‐Term Maintenance Method of Brewer's Yeast on Fermentation Course,Yeast Vitality and Beer Characteristics

The effect of the long‐term maintenance method used with a brewer's yeast on its technological properties was determined in laboratory fermentation trials with a 12°P all‐malt wort. The trials were performed at a constant temperature and under conditions of constant substrate concentration. Two cultures of a bottom fermenting yeast, Saccharomyces pastorianus RIBM 95, were tested — one culture was maintained by subculturing on wort agar slopes at 4°C and the other culture underwent a three year storage in liquid nitrogen at minus 196°C. Parameters under investigation included yeast vitality measured as acidification power (AP), fermentation time needed to reach an alcohol level of 4%, the yeast cell count, sedimentation of the yeast during the fermentation, and the production of beer flavour compounds in green beer. The yeast culture stored for three years in liquid nitrogen displayed a higher count of suspended cells, required a shorter time to attenuate the wort to produce 4% alcohol and produced a 1.5 to 2.5‐fold higher concentration of a number of flavour compounds. The long‐term storage method did not affect the sedimentation ability and vitality of the yeast strain tested.     

Effect of the respiro-fermentative balance during yeast propagation on fermentation and wort attenuation

Breweries use different yeast strains to create beers with different flavours and aromas. Yeast propagation must produce yeast that performs consistently from the first fermentation to harvesting and re-pitching in subsequent fermentations. Breweries propagate yeast in wort leading to low efficiency fermentative growth in Crabtree-positive yeast. There is limited knowledge on the impact on beer production when fermenting with yeast propagated in sugar limited and nutrient supplemented wort. It was hypothesised that propagating yeast in this way would have a positive impact on subsequent fermentation performance. Saccharomyces cerevisiae was propagated at the laboratory scale in standard wort with a high carbon to nitrogen (C:N) ratio (850) or in modified wort supplemented with yeast extract to achieve a low C:N ratio (100) and at varying sugar concentrations. Propagation in low C:N wort with 2°P sugar yielded a 27% decrease in fermentation efficiency and a 46% increase in cell production compared to 2°P high C:N wort. This suggests nitrogen is critical to the respiro-fermentative balance during growth. Yeast propagated in standard wort resulted in slower fermentations and significant under-attenuation compared to yeast grown in the modified wort with low sugar and high nitrogen. The results of this study suggest the nitrogen and sugar content drive the respiro-fermentative balance during yeast propagation. The metabolism of yeast during propagation induces significant downstream impacts on the subsequent fermentation performance and wort attenuation. © 2020 The Institute of Brewing & Distilling     

Direct Evidence That Maltose Transport Activity Is Affected by the Lipid Composition of Brewer's Yeast

A brewer's yeast strain was grown with maltose as sole carbon source under strictly anaerobic conditions with and without ergosterol and/or unsaturated fatty acid (Tween 80) supplements. Under all these conditions the MALx1 genes for maltose transporters were strongly expressed during growth. The fatty acid unsaturation indices of growing and stationary phase yeast were increased from about 20% to 56–69% by supplementation with Tween 80. Ergosterol contents were increased up to at least 4‐fold by supplementation with ergosterol and Tween 80. Maltose transport activity measured at 20°C was not increased by supplementation with Tween 80 alone, but was increased 2‐fold and 3‐fold, respectively, in growing and stationary phase yeast by supplementation with ergosterol together with Tween 80. The stimulation of maltose transport by ergosterol was greater when the transport was measured at temperatures (10°C and 0°C) lower than 20°C. The results show that proper function of maltose transporters requires adequate amounts of ergosterol in the yeast. This effect may partly explain the low maltose (and maltotriose) uptake rates both in the second half of brewery fermentations, when the sterol content of yeast has fallen, and when fresh wort is pitched with sterol‐deficient cropped yeast.     

THE INFLUENCE OF 2-ACETOHYDROXY ACIDS ON THE DETERMINATION OF VICINAL DIKETONES IN BEER AND DURING FERMENTATION

During sampling and determination of diacetyl, 2-acetohydroxy acids are easily converted to vicinal diketones. A simple procedure for gas chromatographic determination of diacetyl, 2-acetolactate, acetoin and the homologous compounds is given. By careful sampling, less than 0·01 ppm of diacetyl was detected during the main fermentation in one brewery, whereas another strain of brewer's yeast yielded a maximum of 1·7 ppm of diacetyl. When samples of fermenting liquids are exposed to air at 60°C, complete conversion of 2-acetohydroxy acids takes place in less than one hour. The possibility that part of 2-acetolactate may be converted to acetoin, however, cannot be excluded. In finished beer 2-acetolactate levels of 0·2–0·5 ppm were observed. During the main fermentation the values ranged from 0·5–2·5 ppm.     

Regulation of the amino acid permeases in nitrogen-limited continuous cultures of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, there is a general amino acid permease, regulated by nitrogen catabolite repression, and several specific permeases whose nitrogen regulation is not well understood. In this study, we used continuous cultures to analyse the effect of nitrogen limitation and pH on the activity of general and several specific amino acid permeases. General permease activity was maximal in severe nitrogen limitation and diminished 400-fold in cells grown under nitrogen excess. For the specific permeases, the maximal uptake activity was found between mild limitation and nitrogen excess, while very small activity was detected under strict limitation. These results indicate that the nitrogen regulation of the general and the specific amino acid carriers is coordinated in such a way that no redundancy exists in amino acid transport. The regulation of the specific permeases was similar to that found for a system with anabolic function in nitrogen metabolism. All of these permeases are supposed to work through a proton symport mechanism, and thus rely on pH gradients to carry out their function. We studied the effect of pH on the kinetic constants of the general permease. Our results show that the effect of pH on the K_m was different for acidic, neutral and basic amino acids, while the effect on V_max was independent of the electrical charge of the amino acids.     

Fate of the worty flavours in a cold contact fermentation

3-Methylbutanal, 2-methylbutanal and 3-methylthiopropionaldehyde are described in the literature as preponderantly responsible for the worty taste of alcohol-free beers. Even in a cold contact process, such aldehydes are reduced through fermentation. The chemical removal of aldehydes by amino acids or proteins does not exceed 20% of the initial concentration, although such mechanisms appear much more effective at 28°C. The role of Saccharomyces cerevisiae brewer's yeast, in the reduction of wort aldehydes, is confirmed here. Only viable yeasts allow a significant decrease in carbonyl level. Unfortunately, residual concentrations are higher for Strecker aldehydes among which 3-methylthiopropionaldehyde is characterized by a very low flavour threshold. Effects of yeast strain, pitching rate and inhibitors have been assessed.     

BROWNING BEHAVIOR OF TAURINE AND PROLINE IN MODEL AND DRIED SQUID SYSTEMS

Model systems containing taurine or proline mixed with glucose at pH 6.4 were adjusted for water activity (Aw) to 0.99, 0.90, 0.84, 0.75 and 0.60 with celite. The browning rates measured during 96 h at temperatures between 5° and 45°C increased with increased temperature and reduced water activity. The model system containing taurine had a higher browning rate than that with proline at the same Aw and temperature. The browning rate with taurine at Aw = 0.60 increased 34 times at 45°C and 7 times at 35°C compared to that at 25°C. Decreases in Aw from 0.90 to 0.60 resulted in increased browning rate, 4.1 times at 45°C and 1.3 times at 35°C. The browning rate in model systems with proline followed the same pattern. Temperature had a more pronounced effect on browning rate than Aw. Browning was very slow in model systems at temperatures below 25°C. Evidence is provided that the Maillard reaction occurred in the mantle of squid (Illex argentinus) during air drying at 35°C. Dehydration resulted in loss of 50% of the taurine (from 22.3 to 11.6 mmoles/100g dry mass) and more than 60% of the proline (from 23.4 to 8.9 mmoles/100g dry mass). Total free amino acids remained unchanged when the moisture level was above 50% or Aw = 0.95. The browning intensity of the squid mantle increased 5 fold during drying. Taurine and proline were digested most at water activity above 0.95, while other amino acids were lost at a greater rate in the intermediate water activity range.     

Characterization of Common Squid Using Several Freshness Indicators

Changes in several components and properties were examined to find appropriate indicators of freshness of common squid during storage at 0°C, 5°C and 10°C. Total viable counts of bacteria, pH and volatile basic nitrogen did not change significantly during 2 wk storage at 0°C. Unlike usual fish meat, K value in squid muscle changed very quickly, but Hx/AMP ratio was a better indicator. Agmatine increased before initial decomposition started but was not suitable as an early freshness indicator. Free basic amino acids, arginine and ornithine also changed rapidly. Basic amino acids such as arginine and ornithine appeared to be appropriate indicators of freshness of common squid.     

The effect of long‐term storage on amino acid content of ready‐to‐eat entrées

The effect of 24‐month storage at three different temperatures (6 ± 2 °C, 26 ± 2 °C, 37 ± 2 °C) on quality of three types of ready‐to‐eat entrées (MRE) was evaluated by means of amino acid, ammonia, moisture and crude protein contents. Spicy risotto, Pork meat with carrots and potatoes and Pork goulash with pasta, that are included into the Czech combat rations, were selected for the experiment. During storage, all the explored samples did not significantly differ in moisture and crude protein content. Higher losses of amino acids (e.g. sulphuric amino acids, lysine, leucine) were detected with the increasing storage time and temperature. Growing losses of amino acids resulted in rising ammonia content as a product of amino acid degradation process. The biological value expressed by essential amino acid index declined with higher temperature and longer time of storage. The dependence of amino acid losses on moisture content was observed, too.     

Effect of Natural Fermentation of Yams (Discorea rotundata) on Characteristics of Processed Flour

The effects of natural fermentation on chemical and physical composition of tubers (Dioscorea rotundata) for flour production were studied. A fission yeast isolated from palmwine was evaluated as inoculum for fermenting yam samples. Tubers were blanched (10 and 20 min at 70°C) before fermentation for 96 hr at 30°C. Acid production and pH changes varied with time and were influenced by availability of fermentable sugars. Fermented products, including samples inoculated with yeasts, had higher protein values. Blanching and fermentation improved color and texture, producing flours with acceptable sensory qualities after 48 hr. Fermentation offers potential for preservation and improvement in quality of yam flour.