Role of the ubiquitin‐proteasome pathway on proteolytic activity in postmortem proteolysis and tenderisation of sheep skeletal muscle

The objective of this study was to investigate the effects of ubiquitin‐proteasome pathway on meat tenderisation. The sheep muscle longissimus lumborum was injected with or without PYR‐41 (inhibitor of ubiquitination) or MG‐132 (inhibitor of proteasome). Muscle samples were collected at 6, 15, 24 and 48 h after injection. Myofibrillar protein degradation, muscle ultrastructure and sarcomere length were determined. Results showed that inhibition of proteasome or ubiquitination affected sarcomere length at 48 h after treatments. Destruction of muscle ultrastructure in both treatments was reduced when compared to control. Inhibition of proteasome produced different fragments of myofibrillar proteins in comparison with control at 48 h. In conclusion, ubiquitin‐proteasome plays a role in postmortem proteolysis and might contribute to meat tenderisation.     

The proteasomal inhibitor MG132 increases the efficiency of mouse embryo production after cloning by electrofusion

In this study, we cloned mice from ES cells by a post-electrofusion MG132 treatment and improved development of cloned embryos with a sequential cultivation protocol. When 5 microM MG132, a proteasome inhibitor, were used to treat the reconstructed embryos, the capacity of in vitro development, implantation and full-term development were significantly improved. Blastocyst formation rates of the reconstructed embryos from X4 ES cells (F1 strain derived from C57BL/6 x 129sv) and J1 ES cells obtained with or without MG132 treatment were 66.9% and 26.6%, and 66.1% and 34.5% respectively (P < 0.05). A total of 146 two-cell embryos cloned from X4 ES cells with MG132 treatment were transferred to recipients, and five cloned pups (3.4%) were born, of which four survived. When the same numbers of two-cell embryos cloned from X4 ES cells without MG132 treatment were transferred, however, no live-born mice were obtained. When embryos cloned from J1 ES cells without MG132 treatment were cultured in KSOM medium for 54 h followed by culture in CZB medium containing 5.6 mM glucose for 42 h, the blastocyst rate was significantly higher than when they were cultured in KSOM continuously for 96 h (34.5% vs 17.1%). However, sequential cultivation did not improve the development of embryos cloned with MG132 treatment and that of parthenotes. In conclusion, MG132 treatment increased the developmental potential of reconstructed mouse embryos, and sequential cultivation improved development of the embryos cloned by electrofusion without MG132 treatment.     

Effect of proteasome inhibitor on sarcoplasmic protein of bovine skeletal muscle during storage

Bovine skeletal muscle, immediately after slaughter (1.5 h; 0 d), was buffered, homogenized treated with proteasome inhibitor, and stored (2 d) for SDS-PAGE and Western blotting analysis. Ubiquitin antiserum (Sigma, St Louis) reacted with bands corresponding to purified ubiquitin and small amounts of other higher-molecular-mass proteins (about 30 and 40 kDa) which were considered to be ubiquitin-protein conjugates. These bands were faint in the control 2 d sample, suggesting that they had degraded. However, these tendencies of the ubiquitin positive bands to decrease were not clearly observed in the sample treated with proteasome inhibitors (MG132 and Lactacystin). These results suggest that both ubiquitin and the ubiquitin-protein conjugates were present in the skeletal muscle immediately after slaughter and they were then degraded during storage. This degradation was partially due to the action of proteasome.     

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.     

Possible involvement of GDI1 protein, a GDP dissociation inhibitor related to vesicle transport, in an amelioration of zinc toxicity in Saccharomyces cerevisiae

The GDI1 protein related vesicle transport system was studied to investigate the possibility that an exclusion of toxic zinc (Zn) from the cytoplasm ameliorates Zn toxicity in Saccharomyces cerevisiae (yeast). A temperature‐sensitive gdi1 mutant (originally called sec19), in which the GDP dissociation inhibitor becomes inactive at the non‐permissive temperature (37 °C), was more sensitive to Zn than its parental GDI1 strain at 32 °C (a moderately non‐permissive temperature). The relative efflux of cytoplasmic Zn in the gdi1 mutant was lower than that in the control strain. Treatment with a vesicle transport‐specific inhibitor, Brefeldin A, caused an increase of Zn sensitivity and a decrease of Zn efflux in these strains. It is therefore suggested that the GDI1‐related vesicle transport system contributes to Zn tolerance in yeast. Furthermore, changes in the number of Zn‐specific fluorescent granules (zincosomes) were observed by zinquin staining in the mutant cells under Zn treatment at 32 °C and 37 °C. We concluded that the GDI1 protein is implicated in control of vesicle numbers. Collectively, the results suggest that the GDI1protein is involved in Zn efflux via small vesicle trafficking and contributes to the control of cytoplasmic Zn content, allowing yeast to survive in the presence of toxic Zn. Copyright © 2011 John Wiley & Sons, Ltd.     

The Saccharomyces cerevisiae enolase‐related regions encode proteins that are active enolases

In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase‐related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2‐phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double‐null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase‐related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes. Copyright © 2012 John Wiley & Sons, Ltd.     

The Effect of Proanthocyanidins on Growth and Alcoholic Fermentation of Wine Yeast under Copper Stress

Proanthocyanidins (PAs) derived from the grape skin, as well as from grape seeds, grape stems, are an important group of polyphenols in wine. The aim of this study was to understand the effect of PAs (0.1, 1.0 g/L) on growth and alcoholic fermentation of 2 strains of Saccharomyces cerevisiae (commercial strain FREDDO and newly selected strain BH8) during copper‐stress fermentation, using a simple model fermentation system. Our results showed that both PAs and Cu²⁺ could pose significant inhibition effects on the growth of yeast cells, CO₂ release, sugar consumption, and ethanol production during the initial phase of the fermentation. Compared to PAs, Cu²⁺ performed more obvious inhibition on the yeast growth and fermentation. However, adding 1.0 g/L PAs increased in the vitality and metabolism activity of yeast cells at the mid‐exponential phase of fermentation in the mediums with no copper and 0.1 mM Cu²⁺ added, shortened the period of wine fermentation, and decreased the copper residues. It indicated that PAs could improve the ability of wine yeast to resist detrimental effects under copper‐stress fermentation condition, maintaining cells metabolic activity, and fermentation could be controlled by manipulating PAs supplementation.     

Investigating flavour characteristics of British ale yeasts: techniques,resources and opportunities for innovation

Five British ale yeast strains were subjected to flavour profiling under brewery fermentation conditions in which all other brewing parameters were kept constant. Significant variation was observed in the timing and quantity of flavour‐related chemicals produced. Genetic tests showed no evidence of hybrid origins in any of the strains, including one strain previously reported as a possible hybrid of Saccharomyces cerevisiae and S. bayanus. Variation maintained in historical S. cerevisiae ale yeast collections is highlighted as a potential source of novelty in innovative strain improvement for bioflavour production. Copyright © 2014 John Wiley & Sons, Ltd.     

Oxylipin Associated Co‐Flocculation in Yeasts

According to the lectin‐theory, the yeast Schizosaccharomyces pombe lacks the specific receptors (α‐mannans) necessary to facilitate co‐flocculation with Saccharomyces cerevisiae species. In this study we demonstrate oxylipin associated co‐flocculation between Sacch. cerevisiae and S. pombe strains using differential cell staining, immunofluoresence and ultrastructural studies. Using a 3‐hydroxy (OH) oxylipin specific antibody coupled to a fluorescing compound, 3‐OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3‐OH oxylipins was confirmed using gas chromatography‐mass spectrometry. Strikingly, when acetylsalicylic acid (aspirin), a 3‐OH oxylipins inhibitor, was added to Sacch. cerevisiae which was then mixed with S. pombe strains grown in complex media, co‐flocculation was significantly inhibited. We conclude that aspirin‐sensitive 3‐OH 8:0 is probably involved in co‐flocculation.     

Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.     

The yeast Saccharomyces cerevisiae Pdr16p restricts changes in ergosterol biosynthesis caused by the presence of azole antifungals

Pdr16p belongs to the family of phosphatidylinositol transfer proteins in yeast. The absence of Pdr16p results in enhanced susceptibility to azole antifungals in Saccharomyces cerevisiae. In the major fungal human pathogen Candida albicans, CaPDR16 is a contributing factor to clinical azole resistance. The current study was aimed at better understanding the function of Pdr16p, especially in relation to azole resistance in S. cerevisiae. We show that deletion of the PDR16 gene increased susceptibility of S. cerevisiae to azole antifungals that are used in clinical medicine and agriculture. Significant differences in the inhibition of the sterol biosynthetic pathway were observed between the pdr16Δ strain and its corresponding wild‐type (wt) strain when yeast cells were challenged by sub‐inhibitory concentrations of the azoles miconazole or fluconazole. The increased susceptibility to azoles, and enhanced changes in sterol biosynthesis upon exposure to azoles of the pdr16Δ strain compared to wt strain, are not the results of increased intracellular concentration of azoles in the pdr16Δ cells. We also show that overexpression of PDR17 complemented the azole susceptible phenotype of the pdr16Δ strain and corrected the enhanced sterol alterations in pdr16Δ cells in the presence of azoles. Pdr17p was found previously to be an essential part of a complex required for intermembrane transport of phosphatidylserine at regions of membrane apposition. Based on these observations, we propose a hypothesis that Pdr16p assists in shuttling sterols or their intermediates between membranes or, alternatively, between sterol biosynthetic enzymes or complexes. Copyright © 2013 John Wiley & Sons, Ltd.     

Disc Stack Centrifuge Operating Parameters and Their Impact on Yeast Physiology

Hydrodynamic stresses imposed on brewing cells of Saccharomyces cerevisiae during beer processing can have a detrimental impact on beer quality. The use of centrifuges has become an efficient way to increase brewery throughput as they decrease clarification times and improve fermenter and tank conditioning efficiency. The effect of a disc stack centrifuge on yeast and beer physical stability has been investigated. In this study, a commercial ale yeast strain has been subjected to different operating conditions during centrifugation. Cell viability and intracellular pH decreased due to processing conditions encountered during yeast cropping with a centrifuge. A relationship has been established that yeast cell wall mannan, an unfilterable haze constituent, as a function of G‐force and centrifugation cycles, is released from the cell wall while concurrently, particle sizes between 0.5‐2.8 μm and beer haze increased. Furthermore, yeast intracellular glycogen and trehalose levels were depleted as a result of centrifugation.     

Evaluating potential applications of indigenous yeasts and their β‐glucosidases

The potential applications of wild yeast strains with β‐glucosidase activity were investigated by assaying their enzymatic production under simulated oenological conditions, coupled with the exploration of the potential applications of the β‐glucosidases by studying the enzymatic activity and stability under similar oenological conditions. The assay of enzymatic locations revealed that the β‐glucosidase activities from these wild strains occurred in the extracellular fraction, and in whole and permeabilized cells. The effects of different oenological factors on β‐glucosidase production indicated that the F6 Trichosporon asahii strain had higher β‐glucosidase production than the other strains under low pH conditions. However, the F35 Hanseniaspora uvarum strain and the F30 Saccharomyces cerevisiae strain showed higher β‐glucosidase production under high‐sugar conditions. Furthermore, the influence of oenological factors on the activity and stability of the β‐glucosidases revealed that the enzyme from the F6 T. asahii strain had a stronger low‐pH‐value resistance than the other yeast β‐glucosidases. These results suggest that the F35 H. uvarum, F30 S. cerevisiae and the F6 T. asahii β‐glucosidases may have some potentially applicable values in the fermentation industry. Copyright © 2015 The Institute of Brewing & Distilling     

Characterization of the respiration‐induced yeast mitochondrial permeability transition pore

When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration‐induced mannitol permeability in mitochondria from this strain. Potassium (K⁺) had varied effects on the respiration‐induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K⁺ inhibited respiration‐induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K⁺ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration‐induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration‐induced yPTP was also inhibited by NAD, Mg²⁺, NH₄⁺ or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration‐induced yPTP as those previously described for the ATP‐induced yPTP and reconcile previous strain‐dependent differences in yPTP solute selectivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Oxidized proteins and their biological impact

Aging is characterized by an increased accumulation of damaged macromolecules and oxidized protein build up is considered to be a hallmark of cellular aging. Advanced glycation end products (AGE) have been analyzed in aging human peripheral blood lymphocytes since such glycoxidative modifications have been reported to increase with age in a variety of cellular and tissular systems and are believed to contribute to the intracellular age‐related accumulation of damaged proteins, a process that has been associated with the cellular functional deficits that occur with age. The pattern of glycated protein has been studied using two dimensional gel electrophoresis followed by Western blotting with an anti‐AGE antibody. The protein silver stain and the immunoblot patterns were not superimposable indicating that glycoxidative modifications are targeting only a restricted set of proteins. Among these preferential protein targets, seven of them exhibited a significant age‐related increased immunoreactivity with the anti‐AGE antibody suggesting that the corresponding modified proteins might serve as biomarkers of aging lymphocytes [ 1 ]. Other age‐related protein modifications such as carbonyl formation and conjugation with the lipid peroxidation product 4‐hydroxy‐2‐nonenal are also currently studied. The age‐related accumulation of altered protein raises the problem of the efficacy of intracellular protein maintenance, in particular the protein degradation and the protein repair systems. Indeed, if these systems that take care of the removal or repair of damaged proteins are affected with aging, they would therefore directly contribute to the increased intracellular load of functionally impaired protein. Since cytosolic oxidized protein degradation and basal protein turnover have been shown to be mostly carried out by the proteasomal system, the fate of proteasome in aging has been addressed and a decline of proteasomal proteolytic activity has been reported [ 2 ]. The impact of aging on human lymphocyte 26S proteasome has been recently investigated and age‐related alterations of proteasome structure and function have been evidenced. Indeed, we observed a decline of 26S proteasome specific activity which is correlated to an increasing yield of post‐translational modifications of proteasome subunits [ 3 ]. In fact, some proteasome subunits and particularly assembly and catalytic subunits are specifically modified with age. According to bidimensional westernblotting, some subunits were found either glycated, conjugated with the lipid peroxidation product 4‐hydroxy‐2‐nonenal or even ubiquitinated. In vitro treatment of the proteasome by glyoxal, that promotes the formation of N?‐carboxymethyllysine adducts, or by the lipid peroxidation product 4‐hydroxy‐2‐nonenal was found to inactivate its peptidase activities although to different extent. In other studies aimed at monitoring the effect of oxidative stress on proteasome structure and function, we have shown on an in vivo rat model that coronary occlusion/reperfusion resulted in inactivation of the proteasome [ 4 ]. This inactivation is associated with selective modification by the lipid peroxidation product 4‐hydroxy‐2‐nonenal of three 20S proteasome α‐subunits. In contrast, the observed inhibition of proteasome upon exposure of human keratinocytes to UV stress was mainly due to the stress‐induced formation of endogeneous inhibitors, including certain oxidatively modified proteins [ 5 ]. In addition, the role of the peptide methionine sulfoxide reductase, one of the very few protein repair enzyme described, and its possible implication in the age‐related decline of protein maintenance has been investigated. The peptide methionine sulfoxide reductase system (Msr A and Msr B) catalyzes the reduction of methionine sulfoxide to methionine within proteins and its activity and the expression of Msr A have been shown to decline in different organs of aged rats [ 6 ] and more recently in senescent human fibroblasts. Moreover, we have recently shown that the peptide methionine sulfoxide reductase Msr A is present both in the cytosol and in the mitochondrial matrix, although under different isoforms [ 7 ]. In conclusion, during aging or in certain oxidative stress situations, impairment of both peptide methionine sulfoxide reductases and proteasome activity appears as a critical factor in the decreased efficacy of intracellular protein maintenance, contributing to the increased intracellular load of modified and functionally impaired proteins that may ultimately lead to a global deterioration of cellular homeostasis.     

Breeding of high malate‐producing diploid sake yeast with a homozygous mutation in the VID24 gene

Malate is an important taste component of sake (a Japanese alcoholic beverage) that is produced by the yeast Saccharomyces cerevisiae during alcoholic fermentation. A variety of methods for generating high malate‐producing yeast strains have been developed to date. We recently reported that a high malate‐producing strain was isolated as a mutant sensitive to dimethyl succinate (DMS), and that a mutation in the vacuolar import and degradation protein (VID) 24 gene was responsible for high malate productivity and DMS sensitivity. In this work, the relationships between heterozygous and homozygous mutants of VID24 and malate productivity in diploid sake yeast were examined and a method was developed for breeding a higher malate‐producing strain. First a diploid yeast was generated with a homozygous VID24 mutation by genetic engineering. The homozygous integrants produced more malate during sake brewing and grew more slowly in DMS medium than wild‐type and heterozygous integrants. Thus, the genotype of the VID24 mutation influenced the level of malate production and sensitivity to DMS in diploid yeast. Then a homozygous mutant from a heterozygous mutant was obtained without genetic engineering by ultraviolet irradiation and culturing in DMS with nystatin enrichment. The non‐genetically modified sake yeast with a homozygous VID24 mutation exhibited a higher level of malate productivity than the parent heterozygous mutant strain. These findings provide a basis for controlling malate production in yeast, and thereby regulating malate levels in sake. Copyright © 2016 The Institute of Brewing & Distilling     

Bioconversion of heptanal to heptanol by Saccharomyces cerevisiae

Saccharomyces cerevisiae is widely known for its catalytic activity on substrates such as aldehyde and ketone. Interestingly, the activity of S. cerevisiae on heptanal (C₆H₁₃CHO), in spite of its being a very common aldehyde, has not been explored. The main objective of this study was therefore to investigate the bioconversion of heptanal, using a strain of the yeast S. cerevisiae. Bioconversion parameters such as incubation period, pH, concentration of substrate, yeast and maltose were also optimized. The study revealed heptanol as the major product. The optimum conditions for biotransformation were found to be: 3 days incubation; pH 7.0; heptanal concentration 0.15 ml/100 ml medium; and S. cerevisiae concentration of 0.15 g/100 ml medium. Reduction in maltose content (to 0.3 g maltose/100 ml medium) showed increased conversion of heptanal. Heptanoic acid and 2‐hydroxyheptanoic acid were obtained as two minor co‐products. The overall study showed that S. cerevisiae converted heptanal to heptanol by a yield of 68.9 ± 1.1% w/w under optimum conditions. Copyright © 2010 John Wiley & Sons, Ltd.     

Isolation of glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae and their use in baker's yeast production

 Glutathione biosynthesis-deficient mutants of Saccharomyces cerevisiae 0511 were obtained by mutation under specific conditions. A total of 3388 strains were isolated and among them were found 46 mutants sensitive to methylglyoxal. The intracellular glutathione concentration of mutant strain S. cerevisiae 3033 was 0.0172 g/g dry weight, which was a decrease of >76% compared to that of the parent. The growth of mutant strains S. cerevisiae 3033 and S. cerevisiae 1116 in the medium with glutathione present and absent was compared to that of the parent strain. The sensibility of the baker's yeast strains studied to antifoaming agents, butanol and acetic acid was also investigated. The relationship between glutathione presence in the cell and the sensitivity of strain S. cerevisiae 3033 to antifoaming agents and butanol was ascertained, while such a connection with the presence of acetic acid in the molasses medium used for baker's yeast cultivation was not observed. The higher sensitivity of strain S. cerevisiae 3033 to some chemical compounds in the molasses nutrition medium was shown. Received: 2 November 1999 / Revised version: 15 February 2000     

Impact of dried,creamed and cake supply formats on the genetic variation and ethanol tolerance of three Saccharomyces cerevisiae distilling strains

Scotch whisky fermentations typically employ high‐gravity fermentation practices to maximize product formation and to minimize both energy and water inputs. This approach increases ethanol concentrations at the end of fermentation, creating stressful conditions for the yeast. In this work we examined the relative tolerance of four Saccharomyces cerevisiae distilling yeast strains, supplied in dried, creamed, cake or slurry format, to ethanol under CO₂‐induced anaerobic conditions. The cells were assessed for their capacity to recover and grow on inhibition spot plates and to maintain cell viability in ethanol‐dosed suspensions. Variations in ethanol tolerance were observed between strains and between the same strain supplied in different formats. The creamed yeast format typically exhibited a higher tolerance to ethanol. One possible explanation for this observation is that cells surviving the dehydration and rehydration process might incur sub‐lethal genome damage. Thus the genetic integrity of the most ethanol‐tolerant strain was assessed as a function of supply format (two dried and one creamed). The mitochondrial DNA was examined using mitochondrial restriction fragment length polymorphism and the chromosomal DNA using pulsed field gel electrophoresis and polymerase chain reaction with both ITS and delta‐specific primers. In one dried yeast sample, genetic integrity was compromised, highlighting the requirement for yeast intake quality assurance programmes. Copyright © 2012 The Institute of Brewing & Distilling