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.     

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.     

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.     

The uptake of inorganic sulphate by a brewer’s yeast

The uptake of³⁵S-labelled inorganic sulphate by a brewer’s yeast has been examined. Optimum uptake by cell suspensions required the presence in the medium of glucose, ammonium ions and citrate. The omission of phosphate produced little or no effect. Ammonium ions could be replaced almost completely byL-glutamine but not by a number of amino acids. After one hour approximately 60% of the sulphate-sulphur accumulated appeared in protein. This was comparable to the rate of entry of methionine-sulphur into yeast protein. Sulphate uptake was inhibited by azide, 2,4-dinitrophenol, iodoacetate and mercuric ions. Arsenate was inhibitory at high concentrations but stimulated uptake at low concentrations. Selenate inhibited uptake competitively and appeared to have an affinity for the sites of uptake comparable with that of sulphate. Uptake was also partly suppressed byL-methionine,L-ethionine,L-cysteine andDL-homocysteine.     

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.     

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.     

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.     

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.     

Integrated expression of the α-amylase, dextranase and glutathione gene in an industrial brewer’s yeast strain

Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase’s shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3 days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21’s better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product.     

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.     

Lipid raft–dependent plasma membrane repair interferes with the activation of B lymphocytes

Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca²⁺-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca²⁺-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR–lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte–mediated immune responses.     

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.     

Construction of dextrin and isomaltose-assimilating brewer’s yeasts for production of low-carbohydrate beer

Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer’s bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer’s yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both α-1,4- and α-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.     

NMR analysis of the CSF and plasma metabolome of rigorously matched amyotrophic lateral sclerosis,Parkinson’s disease and control subjects

Introduction

Amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are two severe neurodegenerative disorders for which the disease mechanisms are poorly understood and reliable biomarkers are absent.

Objectives

To identify metabolite biomarkers for ALS and PD, and to gain insights into which metabolic pathways are involved in disease.

Methods

Nuclear magnetic resonance (NMR) metabolomics was utilized to characterize the metabolite profiles of cerebrospinal fluid (CSF) and plasma from individuals in three age, gender, and sampling-date matched groups, comprising 22 ALS, 22 PD and 28 control subjects.

Results

Multivariate analysis of NMR data generated robust discriminatory models for separation of ALS from control subjects. ALS patients showed increased concentrations of several metabolites in both CSF and plasma, these are alanine (CSF fold change = 1.22, p = 0.005), creatine (CSF-fc = 1.17, p = 0.001), glucose (CSF-fc = 1.11, p = 0.036), isoleucine (CSF-fc = 1.24, p = 0.002), and valine (CSF-fc = 1.17, p = 0.014). Additional metabolites in CSF (creatinine, dimethylamine and lactic acid) and plasma (acetic acid, glutamic acid, histidine, leucine, pyruvate and tyrosine) were also important for this discrimination. Similarly, panels of CSF-metabolites that discriminate PD from ALS and control subjects were identified.

Conclusions

The results for the ALS patients suggest an affected creatine/creatinine pathway and an altered branched chain amino acid (BCAA) metabolism, and suggest links to glucose and energy metabolism. Putative metabolic markers specific for ALS (e.g. creatinine and lactic acid) and PD (e.g. 3-hydroxyisovaleric acid and mannose) were identified, while several (e.g. creatine and BCAAs) were shared between ALS and PD, suggesting some overlap in metabolic alterations in these disorders.

     

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.