Effects of industrial storage on the bioreduction capacity of brewer’s yeast

The effects of industrial storage on the changes of the cell viability and the activities of intracellular alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) in brewer’s yeast, and the corresponding capacity for the bioconversion of ethyl-3-oxobutanoate (EOB) to ethyl (S)-3-hydroxybutanoate ((S)-EHB), were investigated. The viability of fresh brewer’s yeast cells stored in industrial circulating cooling water at 1–2°C showed 4 and 15% drop after the storage of 7 and 15 days, respectively, after which cells died rapidly. The pretreatment of the stored brewer’s yeast cells by washing and screening significantly enhanced cell viability during industrial storage. The intracellular levels of ADH and G6PDH after permeabilization of these stored cells with cetyltrimetylammonium bromide (CTAB) were much higher, which showed only slight decrease within 2 weeks during the industrial storage. When the stored cells after the permeabilization treatment was used as the biocatalyst at 90–120 g/L, EOB was converted almost completely into enantiopure (S)-EHB with an enantiomeric excess (ee) more than 99% and a yield of over 96%, by fed-batch bioconversion of 560 mM EOB within 6 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lipid analysis of the plasma membrane and mitochondria of brewer’s yeast

The plasma membrane and mitochondria of bottom fermenting brewer's yeast obtained as a by-product of industrial beer production were isolated and the lipid fraction was analyzed. The phospholipid content accounted for 78 mg/g protein in the plasma membrane and 59 mg/g protein in the mitochondria. Major phospholipids in both preparations were phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine but their proportions differed significantly. In the plasma membrane phosphatidylinositol, and in the mitochondria phosphatidylcholine were present in the highest concentration (37 and 30%, respectively). The main classes of neutral lipids (triacylglycerols, ergosterol, squalene and steryl esters) were twice more abundant in the plasma membrane than in the mitochondria (61 and 33 mg/g protein, respectively). A characteristic of the neutral lipid composition of both organelles was the low content of ergosterol (12 and 7 mg/g protein, respectively) and a high content of squalene (25 and 22 mg/g protein). The main feature of the fatty acid composition of both organelles was the preponderance of saturated fatty acids (78 and 79%, respectively), among which palmitic acid was the principal one. The most expressed characteristics of lipid fractions of the analyzed plasma membranes and mitochondria, high concentration of squalene and preponderance of saturated fatty acids are the consequences of anaerobic growth conditions. The lack of oxygen had possibly the strongest effect on the lipid composition of the plasma membranes and mitochondria of bottom fermenting brewer's yeast.     

Cell wall polysaccharides: before and after autolysis of brewer’s yeast

Brewer’s yeast is used in production of beer since millennia, and it is receiving increased attention because of its distinct fermentation ability and other biological properties. During fermentation, autolysis occurs naturally at the end of growth cycle of yeast. Yeast cell wall provides yeast with osmotic integrity and holds the cell shape upon the cell wall stresses. The cell wall of yeast consists of β-glucans, chitin, mannoproteins, and proteins that cross linked with glycans and a glycolipid anchor. The variation in composition and amount of cell wall polysaccharides during autolysis in response to cell wall stress, laying significant impacts on the autolysis ability of yeast, either benefiting or destroying the flavor of final products. On the other hand, polysaccharides from yeast cell wall show outstanding health effects and are recommended to be used in functional foods. This article reviews the influence of cell wall polysaccharides on yeast autolysis, covering cell wall structure changings during autolysis, and functions and possible applications of cell wall components derived from yeast autolysis.     

Evaluating biochemical methane production from brewer’s spent yeast

Anaerobic digestion treatment of brewer’s spent yeast (SY) is a viable option for bioenergy capture. The biochemical methane potential (BMP) assay was performed with three different samples (SY1, SY2, and SY3) and SY1 dilutions (75, 50, and 25 % on a v/v basis). Gompertz-equation parameters denoted slow degradability of SY1 with methane production rates of 14.59–4.63 mL/day and lag phases of 10.72–19.7 days. Performance and kinetic parameters were obtained with the Gompertz equation and the first-order hydrolysis model with SY2 and SY3 diluted 25 % and SY1 50 %. A SY2 25 % gave a 17 % of TCOD conversion to methane as well as shorter lag phase (<1 day). Average estimated hydrolysis constant for SY was 0.0141 (±0.003) day^?1, and SY2 25 % was more appropriate for faster methane production. Methane capture and biogas composition were dependent upon the SY source, and co-digestion (or dilution) can be advantageous.     

Flocculation gene variability in industrial brewer’s yeast strains

The brewer’s yeast genome encodes a ‘Flo’ flocculin family responsible for flocculation. Controlled floc formation or flocculation at the end of fermentation is of great importance in the brewing industry since it is a cost-effective and environmental-friendly technique to separate yeast cells from the final beer. FLO genes have the notable capacity to evolve and diverge many times faster than other genes. In actual practice, this genetic variability may directly alter the flocculin structure, which in turn may affect the flocculation onset and/or strength in an uncontrolled manner. Here, 16 ale and lager yeast strains from different breweries, one laboratory Saccharomyces cerevisiae and one reference Saccharomyces pastorianus strain, with divergent flocculation strengths, were selected and screened for characteristic FLO gene sequences. Most of the strains could be distinguished by a typical pattern of these FLO gene markers. The FLO1 and FLO10 markers were only present in five out of the 18 yeast strains, while the FLO9 marker was ubiquitous in all the tested strains. Surprisingly, three strongly flocculating ale yeast strains in this screening also share a typical ‘lager’ yeast FLO gene marker. Further analysis revealed that a complete Lg-FLO1 allele was present in these ale yeasts. Taken together, this explicit genetic variation between flocculation genes hampers attempts to understand and control the flocculation behavior in industrial brewer’s yeasts.     

Ochratoxin A in brewer’s yeast used as food supplement

Brewer’s yeasts are rich in vitamins of the B-group and contain other nutritive factors; therefore, they are recommended as valuable food supplements for people with special dietary requirements like pregnant women, children, and adolescents, or for people with high physical activity. Additionally, certain strains of brewer’s yeast are known to be capable of adsorbing xenobiotics such as mycotoxins. Because of that, these yeasts are regarded as having positive effects in food, beverage, and feed technology. Their potential to bind mycotoxins such as ochratoxin A (OTA), however, can subsequently lead to a contamination of such brewer’s yeasts used as food supplements. In the present study, we analyzed 46 samples of brewer’s yeasts for the occurrence of OTA by HPLC with fluorescence detector (HPLC-FLD) and for confirmatory measurements by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nearly 90 % of the samples were contaminated with OTA, the levels ranging from the limit of detection (LOD, 0.01 μg/kg) to 4.2 μg/kg. The mean and median levels of contamination were 0.49 and 0.27 μg/kg, respectively. Based on these results, the additional weekly OTA exposure by regularly consuming such supplements was assessed. Depending on different subpopulations (adults, children) and levels of contamination used for calculation, the additional OTA intake via brewer’s yeast products ranged from 9.3 % (mean case) to 114 % (worst case) of the published mean weekly OTA intake in Germany (adults 279.3 ng, children 195.3 ng). At present, maximum levels for OTA in nutritional supplements like brewer’s yeast do not exist. Based on our results, however, it is recommended that producers of these dietary supplements should include mycotoxin analyses in ongoing and future self-monitoring programs and in product quality checks.     

Genetic improvement of brewer’s yeast: current state, perspectives and limits

Brewer’s yeast strain optimisation may lead to a more efficient beer production process, better final quality or healthier beer. However, brewer’s yeast genetic improvement is very challenging, especially true when it comes to lager brewer’s yeast (Saccharomyces pastorianus) which contributes to 90% of the total beer market. This yeast is a genetic hybrid and allopolyploid. While early studies applying traditional genetic approaches encountered many problems, the development of rational metabolic engineering strategies successfully introduced many desired properties into brewer’s yeast. Recently, the first genome sequence of a lager brewer’s strain became available. This has opened the door for applying advanced omics technologies and facilitating inverse metabolic engineering strategies. The latter approach takes advantage of natural diversity and aims at identifying and transferring the crucial genetic information for an interesting phenotype. In this way, strains can be optimised by introducing “natural” mutations. However, even when it comes to self-cloned strains, severe concerns about genetically modified organisms used in the food and beverage industry are still a major hurdle for any commercialisation. Therefore, research efforts will aim at developing new sophisticated screening methods for the isolation of natural mutants with the desired properties which are based on the knowledge of genotype–phenotype linkage.     

Bioconversion of ethyl 4-chloro-3-oxobutanoate by permeabilized fresh brewer’s yeast cells in the presence of allyl bromide

Ethyl(R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) are obtained by cetyltrimetylammonium bromide (CTAB) permeabilized fresh brewer’s yeast whole cells bioconversion of ethyl 4-chloro-3-oxobutanoate (COBE ) in the presence of allyl bromide. The results showed that the activities of alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) in CTAB permeabilized brewer’s yeast cells increased 525 and 7.9-fold, respectively, compared with that in the nonpermeabilized cells and had high enantioselectivity to convert COBE to (R)-CHBE. As one of co-substrates, glucose-6-phosphate was preprepared using glucose phosphorylation by hexokinase-catalyzed of CTAB permeabilized brewer’s yeast cells. In a two phase reaction system with n-butyl acetate as organic solvent and with 2-propanol and glucose-6-phosphate as co-substrates, the highest (R)-CHBE concentration of 447 mM was obtained with 110–130 g/l of the CTAB permeabilized cells at optimized pH, temperature, feeding rate and the shake speed of 125 r/min. The yield and enantiomeric excess (ee) of (R)-CHBE reached 99.5 and 99%, respectively, within 6 h.     

The influence of fermentation conditions and recycling on the phospholipid and fatty acid composition of the brewer’s yeast plasma membranes

Phospholipid (PL) and fatty acid (FA) compositions of the plasma membrane (PM), as well as the FA composition of the PM phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) in the pure culture (zero generation) and the first three recycled generations of the bottom-fermenting brewer’s yeast, have been determined. The PL composition differed markedly among the generations; in the zero generation, phosphatidylinositol (PtdIns) was the main PL, accounting for 27% of total PLs, followed by phosphatidic acid and PtdCho. In all recycled generations, the main PL was PtdCho with a marked increase in the first generation compared with the zero (32% and 20%, respectively), followed by PtdIns in the first and second generations. In the FA composition of the PM, 22 FAs were identified, ranging from C₁₀ to C₂₆. The compositions of the PM FAs, as well as those of PtdCho and PtdEtn, were characterised by a high preponderance of C₁₆ acids. Saturated FAs prevailed in the zero generation, whilst unsaturated prevailed in the first and second generation. Although the profiles of FAs in PtdCho and PtdEtn were similar, some marked differences were observed, pointing out to their specific functions in the regulation of membrane properties.     

Effect of inhibitors on acid production by baker’s yeast

Glucose-induced acid extrusion, respiration and anaerobic fermentation in baker’s yeast was studied with the aid of sixteen inhibitors. Uranyl(2+) nitrate affected the acid extrusion more anaerobically than aerobically; the complexing of Mg²⁺ and Ca²⁺ by EDTA at the membrane had no effect. Inhibitors of glycolysis (iodoacetamide, N-ethylmaleimide, fluoride) suppressed acid production markedly, and so did the phosphorylation-blocking arsenate. Fluoroacetate, inhibiting the citric-acid cycle, had no effect. Inhibition by uncouplers depended on their pK_a values: 2,4,6-trinitrophenol (pK_a 0.4) < 2,4-dinitrophenol (4.1) < azide (4.7) < 3-chlorophenylhydrazonomalononitrile (6.0). Inhibition by trinitrophenol was only slightly increased by its acetylation. Cyanide and nonpermeant oligomycin showed practically no effect; inhibition by dicyclohexylcarbodiimide was delayed but potent. The concentration profiles of inhibition of acid production differed from those of respiration and fermentation. Thus, though the acid production is a metabolically dependent process, it does not reflect the intensity of metabolism, except partly in the first half of glycolysis.     

Impact of fluorides on the removal of heavy metals from an electroplating effluent using a flocculent brewer’s yeast strain of Saccharomyces cerevisiae

Abstract

Besides several toxic heavy metals, electroplating effluents can have in solution different cations and anions, which may influence heavy metals removal by the biomass. Among them, fluorides are commonly used in the electroplating industries and thus can be found in the respective wastewaters. In the present work, the effect of the presence of fluorides in the efficiency of chromium(III), copper(II) and nickel(II) removal, from an effluent, by heat-inactivated cells of a brewing flocculent strain of Saccharomyces cerevisiae was evaluated. The presence of fluorides severely decreased (>60%) the removal of chromium(III) by yeast biomass. This effect impaired the effective treatment of the effluent according to the US Environmental Protection Agency and the Portuguese law; conversely, a higher removal of copper(II) and nickel(II) was observed. This behaviour can be understood by metal speciation. In the presence of fluorides, chromium(III) was mainly complexed, becoming unavailable for yeast accumulation; this effect decreased the efficiency of chromium(III) removal. Thus, in the presence of fluorides, less chromium(III) is associated with biomass and consequently more yeast binding sites remain available for the uptake of other metals present in solution. This fact explains the increase of copper(II) and nickel(II) removal in the presence of fluorides.     

Intracellular pH of baker’s yeast

The distribution of bromophenol blue between the cell and the medium was used to calculate the intracellular pH of yeast. In buffered media the intracellular pH exhibited a plateau at pH _i =5.8 for low external pH values and another at pH _i =7.6 for high external pH values. The production of H ions by the yeast during utilization of glucose is not accompanied by an alkalinization of the cell interior. The pH _i even decreases somewhat in the presence of glucose and K ions.

1. (1)
   Внутриклеточный pH дрожжей вычисляли на основании распределения бромфеноловой сини между клетками и средой.     
   
   

Synthesis of hydroquinone-α-glucoside by α-glucosidasefrom baker’s yeast

Hydroquinone-α-glucoside was synthesised from hydroquinone and maltose as glucosyl donor by transglucosylation in a water system with α-glucosidase from baker’s yeast. Only one phenolic –OH group was α-anomer-selectively glucosylated. The optimum conditions for transglucosylation reaction were at 30 °C for 20 h with 50 mM hydroquinone and 1.5 M maltose in 100 mM sodium citrate/phosphate buffer at pH 5.5. The glucoside was obtained at 0.6 mg/ml with a 4.6% molar yield with respect to hydroquinone.     

Bioethanol production from the hydrolysate of Palmaria palmata using sulfuric acid and fermentation with brewer’s yeast

Seaweeds, particularly species of red macroalgae, are promising resources for bioethanol production because of their exceptionally high carbohydrate content. Of 20 seaweeds evaluated, Palmaria palmata (Rhodymenia palmata) contained the highest carbohydrate content (469.8 mg g^?1 seaweed) with a carrageenan content of 354 mg g^?1 seaweed. Such a high carrageenan content makes the high-volume production of bioethanol feasible. Acid hydrolysis of P. palmata in 0.4 M H₂SO₄ at 125 °C for 25 min released 27 mg of glucose, 218.4 mg of reducing sugars, and 127.6 mg of galactose per gram of seaweed. Ethanol fermentation of these hydrolysis products using an inoculum concentration of 1.5 mg mL^?1 at 30 °C and 72 h in a shaking incubator at 130 rpm yielded 17.3 mg of ethanol per gram of seaweed.     

Influence of growth phase and zeolite clinoptilolite on the concentration of sphingoid bases in Saccharomyces uvarum brewer’s yeast

Sphingolipids having a long-chain sphingoid base backbone are primarily located in the yeast’s plasma membrane. They are found in various types of foods, and although they are not essential food ingredients, they play an important role as bioactive molecules in preventing certain human diseases. Today, due to its high nutritional value, brewer’s yeast is increasingly being used in the food and pharmaceutical industry. The aim of this study was to evaluate the potential of S. uvarum, a by-product of the brewing industry, as an economically feasible source of sphingolipids. For that purpose, the growth phase dependence on sphingolipid production in S. uvarum as well as the effect of zeolite addition to the growth medium was investigated. The experiments were designed to explore the dependence of growth phase on sphingolipids metabolism, by comparing initial (starter) culture of brewer’s yeast (laboratory propagated, designated as zero yeast generation, serving here as control), and surplus brewer’s yeast (a residue produced after 5 successive beer fermentations), by-product of beer fermentation, with and without the addition of zeolite. HPLC analysis of individual molecular species of sphingoid bases obtained by acid hydrolysis of complex sphingolipids from S. uvarum yeast produced the following results: about 65% of total sphingoid bases represents C18 phytosphingosine, about 32% represents unknown long-chain base, and about 1.5–2% represents C18 DL-erythro-sphinganine. In the case of C18 phytosphingosine, production was about 11.5-fold higher during exponential phase compared with the other growth phases. For C18 DL-erythro-sphinganine, production was highest during the lag and acceleration phase of growth. In most cases, zeolite addition (1%) to the growth medium resulted in an increase up to 2.5-fold in the sphingoid bases level.     

Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts

High-gravity brewing, which can decrease production costs by increasing brewery yields, has become an attractive alternative to traditional brewing methods. However, as higher sugar concentration is required, the yeast is exposed to various stresses during fermentation. We evaluated the influence of high-gravity brewing on the fermentation performance of the brewer’s yeast under model brewing conditions. The lager brewer’s strain Weihenstephan 34/70 strain was characterized at three different gravities by adding either glucose or maltose syrups to the basic wort. We observed that increased gravity resulted in a lower specific growth rate, a longer lag phase before initiation of ethanol production, incomplete sugar utilization, and an increase in the concentrations of ethyl acetate and isoamyl acetate in the final beer. Increasing the gravity by adding maltose syrup as opposed to glucose syrup resulted in more balanced fermentation performance in terms of higher cell numbers, respectively, higher wort fermentability and a more favorable flavor profile of the final beer. Our study underlines the effects of the various stress factors on brewer’s yeast metabolism and the influence of the type of sugar syrups on the fermentation performance and the flavor profile of the final beer.     

Subcellular shifts of trimeric G-proteins following activation of Baker’s yeast by glucose

Addition of glucose to a resting cell suspension of the yeastSaccharomyces cerevisiae was accompanied by marked shifts of the Gα-protein subunits from the plasma membrane to the cell interior. This process was rapid with half-times between <10 and 20 s. The decrease of the plasma membrane pool of the G_iα/G_oα- and G_qα/G₁₁α-protein subunits correlated with an increase in acid-sensitive forms of these proteins which was recovered in the mitochondrial and/or lysosomal membrane fraction. In contrast to cells from higher organisms glucose-stimulated yeast exhibits an extremely rapid type of the redistribution (internalization). The question remains’open as to the functional significance of the internalized forms of the G-proteins as these remain sequestered from the plasma membrane. well after glucose has been consumed.     

Efficient uptake of recombinant α-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys

To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.     

Transport of ethanol in baker’s yeast

Ethanol is transported into various strains of baker's yeast by simple diffusion (no effect of inhibitors and a linear concentration dependence of the initial rate of uptake and final distribution in cells). It distributes itself in 96.6 +/- 16.2% of intracellular water.