Hyperosmotic stress response by strains of bakers' yeasts in high sugar concentration medium

Four strains of bakers' yeast were analysed for their hyperosmotic responses when in media that mimic conditions occurring in bread doughs. Two of the strains produced strong fermentative activity in medium with low osmotic stress, but produced considerably less ethanol in high sucrose concentration medium. Two other strains produced more similar fermentation activities across the range of media tested. The strains that were inhibited by high sucrose concentration were unable to produce significant amounts of glycerol under hyperosmotic conditions. By contrast, the yeasts that were not inhibited significantly by high sucrose produced a considerable amount of glycerol. The strains that produced significant glycerol exhibited efficient expression of the glycerol-3-phosphate dehydrogenase gene GPD1. These novel data on the molecular responses of industrially relevant strains of bakers' yeasts are prerequisite to designing strategies for improving the performance of industrial yeasts in high sugar concentration media.     

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.     

Mutants of bakers' yeasts producing a large amount of isobutyl alcohol or isoamyl alcohol,flavour components of bread

Summary Mutants resistant to 4-aza-dl-leucine were derived from strains of the bakers' yeast Saccharomyces cerevisiae and selected with respect to overproduction of isobutyl alcohol (i-BuOH) or isoamyl alcohol (i-AmOH). Many mutants that produced i-BuOH or i-AmOH more than the parent strains were obtained. In the evaluation of these mutants, bread containing more i-BuOH was evaluated as giving a favorable characteristic flavour, but bread with more i-AmOH was unfavorable. These mutants were able to ferment dough at similar rates to commercial bakers' yeasts. The mutants overproducing i-BuOH or i-AmOH were released from inhibition of the key enzymes, acetohydroxy acid synthase and -isopropylmalate synthase, respectively, in the pathway of branched-chain amino acids synthesis.Offprint requests to: M. Watanabe     

Growth Requirements of San Francisco Sour Dough Yeasts and Bakers' Yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

Growth requirements of san francisco sour dough yeasts and bakers' yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

Influence of invertase activity and glycerol synthesis and retention on fermentation of media with a high sugar concentration by Saccharomyces cerevisiae.

In the past, the fermentation activity of Saccharomyces cerevisiae in substrates with a high concentration of sucrose (HSuc), such as sweet bread doughs, has been linked inversely to invertase activity of yeast strains. The present work defines the limits of the relationship between invertase activity and fermentation in hyperosmotic HSuc medium. Fourteen polyploid, wild-type strains of S. cerevisiae with different invertase levels gave a similar ranking of fermentation activity in HSuc and in medium in which glucose and fructose replaced sucrose (HGF medium). Thus, invertase is unlikely to be the most important determinant of fermentation in sweet doughs. Yeasts produce the compatible solute-osmoprotective compound glycerol when exposed to hyperosmotic environments. Under low sugar concentrations (and nonstressing osmotic pressure), there was no correlation between glycerol and fermentation activities. However, there was a strong correlation between the ability of yeasts to ferment in HSuc or HGF medium and their capacity to produce and retain glycerol intracellularly. There was also a strong correlation between intracellular glycerol and fermentation activity of yeasts in a medium in which the nonfermentable sugar alcohol sorbitol replaced most of the sugars (HSor), but the ability to produce and retain glycerol was greater when yeasts were incubated in HGF medium under the same osmotic pressure. The difference between the amounts of glycerol produced and retained in HSor and in HGF media varied with strains. This implies that high fermentable sugar concentrations cause physiological conditions that allow for enhanced glycerol production and retention, the degree of which is strain dependent. In conclusion, one important prerequisite for yeast strains to ferment media with high concentrations of sugar is the ability to synthesize glycerol and especially to retain it.     

Transcriptional regulation of fermentative and respiratory metabolism in Saccharomyces cerevisiae industrial bakers' strains

Bakers' yeast-producing companies grow cells under respiratory conditions, at a very high growth rate. Some desirable properties of bakers' yeast may be altered if fermentation rather than respiration occurs during biomass production. That is why differences in gene expression patterns that take place when industrial bakers' yeasts are grown under fermentative, rather than respiratory conditions, were examined. Macroarray analysis of V1 strain indicated changes in gene expression similar to those already described in laboratory Saccharomyces cerevisiae strains: repression of most genes related to respiration and oxidative metabolism and derepression of genes related to ribosome biogenesis and stress resistance in fermentation. Under respiratory conditions, genes related to the glyoxylate and Krebs cycles, respiration, gluconeogenesis, and energy production are activated. DOG21 strain, a partly catabolite-derepressed mutant derived from V1, displayed gene expression patterns quite similar to those of V1, although lower levels of gene expression and changes in fewer number of genes as compared to V1 were both detected in all cases. However, under fermentative conditions, DOG21 mutant significantly increased the expression of SNF1 -controlled genes and other genes involved in stress resistance, whereas the expression of the HXK2 gene, involved in catabolite repression, was considerably reduced, according to the pleiotropic stress-resistant phenotype of this mutant. These results also seemed to suggest that stress-resistant genes control desirable bakers' yeast qualities.     

Trehalose levels and survival ratio of freeze-tolerant versus freeze-sensitive yeasts.

Five freeze-tolerant yeast strains suitable for frozen dough were compared with ordinary commercial bakers' yeast. Kluyveromyces thermotolerans FRI 501 cells showed high survival ability after freezing when their resting cells were fermented for 0 to 180 min in modified liquid medium, and they grew to log and stationary phases. Among the freeze-tolerant strains of Saccharomyces cerevisiae, FRI 413 and FRI 869 showed higher surviving and trehalose-accumulating abilities than other S. cerevisiae strains, but were affected by a prolonged prefermentation period and by growth phases. The freeze tolerance of the yeasts was, to some extent, associated with the basal amount of intracellular trehalose after rapid degradation at the onset of the prefermentation period. In the freeze-sensitive yeasts, the degree of hydrolysis of trehalose may thus be affected by the kind of saccharide, unlike in freeze-tolerant yeasts.     

Trehalose levels and survival ratio of freeze-tolerant versus freeze-sensitive yeasts

Five freeze-tolerant yeast strains suitable for frozen dough were compared with ordinary commercial bakers' yeast. Kluyveromyces thermotolerans FRI 501 cells showed high survival ability after freezing when their resting cells were fermented for 0 to 180 min in modified liquid medium, and they grew to log and stationary phases. Among the freeze-tolerant strains of Saccharomyces cerevisiae, FRI 413 and FRI 869 showed higher surviving and trehalose-accumulating abilities than other S. cerevisiae strains, but were affected by a prolonged prefermentation period and by growth phases. The freeze tolerance of the yeasts was, to some extent, associated with the basal amount of intracellular trehalose after rapid degradation at the onset of the prefermentation period. In the freeze-sensitive yeasts, the degree of hydrolysis of trehalose may thus be affected by the kind of saccharide, unlike in freeze-tolerant yeasts.     

Improvement of maltotriose fermentation by Saccharomyces cerevisiae

AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts.     

Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters

In many yeast species, including Kluyveromyces lactis, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions. If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place. This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step. We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth. By introducing additional sugar permease genes, we have obtained K. lactis strains that were capable of growing on galactose and raffinose in the absence of respiration. High dosages of both the permease and maltase genes were indeed necessary for K. lactis cells to grow on maltose in the absence of respiration. These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K. lactis and probably in many other yeasts.     

Leavening ability and freeze tolerance of yeasts isolated from traditional corn and rye bread doughs.

Strains of Saccharomyces cerevisiae and Torulaspora delbrueckii isolated from traditional bread doughs displayed dough-raising capacities similar to the ones found in baker's yeasts. During storage of frozen doughs, strains of T. delbrueckii (IGC 5321, IGC 5323, and IGC 4478) presented approximately the same leavening ability for 30 days. Cell viability was not significantly affected by freezing, but when the dough was submitted to a bulk fermentation before being stored at -20 degrees C, there was a decrease in the survival ratio which depended on the yeast strain. Furthermore, the leavening ability after 4 days of storage decreased as the prefermentation period of the dough before freezing increased, except for strains IGC 5321 and IGC 5323. These two strains retained their fermentative activity after 15 days of storage and 2.5 h of prefermentation, despite showing a reduction of viable cells under the same conditions. The intracellular trehalose content was higher than 20% (wt/wt) in four of the yeasts tested: the two commercial strains of baker's yeast (S. cerevisiae IGC 5325 and IGC 5326) and the two mentioned strains of T. delbrueckii (IGC 5321 and IGC 5323). However, the strains of S. cerevisiae were clearly more susceptible to freezing damages, indicating that other factors may contribute to the freeze tolerance of these yeasts.     

Selection of Wine Yeasts for Growth and Fermentation in the Presence of Ethanol and Sucrose

To optimize the conversion of carbohydrates to ethanol, strains of several Saccharomyces species were examined for the ability to grow and ferment in a range of sucrose and ethanol concentrations. A total of 632 wine yeasts, most of them isolated from wineries in Andalusia and Extremadura, southwestern Spain, were subjected to screening and selection. Growth and fermentative capacity in different ethanol and sucrose concentrations varied from one strain to another. There was no correlation between growth and fermentative capacity. The best 35 strains grew in 15% ethanol and fermented in 18% ethanol. Ethanol accumulated, although at a reduced rate, after the cells stopped growing. Most yeast strains were highly fermentative in 50% sucrose. Some of them effectively utilized the carbohydrates of the culture, yielding final ethanol concentrations of > 14%. Of the 35 selected strains, 16 were promising for genetic analysis and breeding because of their capacity to sporulate. These strains were homothallic, and their spores were viable. The meiotic products analyzed so far were also homothallic.     

Construction from a single parent of baker's yeast strains with high freeze tolerance and fermentative activity in both lean and sweet doughs.

From a freeze-tolerant baker's yeast (Saccharomyces cerevisiae), 2,333 spore clones were obtained. To improve the leavening ability in lean dough of the parent strain, we selected 555 of the high-maltose-fermentative spore clones by using a method in which a soft agar solution containing maltose and bromocresol purple was overlaid on yeast colonies. By measuring the gassing power in the dough, we selected 66 spore clones with a good leavening ability in lean dough and a total of 694 hybrids were constructed by crossing them. Among these hybrids, we obtained 50 novel freeze-tolerant strains with good leavening ability in all lean, regular, and sweet doughs comparable to that of commercial baker's yeast. Hybrids with improved leavening ability or freeze tolerance compared with the parent yeast and commercial baker's yeasts were also obtained. These results suggest that hybridization between spore clones derived from a single parent strain is effective for improving the properties of baker's yeasts.     

<Emphasis Type="Italic">Cachaça</Emphasis> yeast strains: alternative starters to produce beer and bioethanol

This work was performed to verify the potential of yeast strains isolated from cachaça distilleries for two specific biotechnological applications: beer and bioethanol production. In the beer production, the strains were tested for characteristics required in brewery practices, such as: capacity to ferment maltose and maltotriose, ability to grow at lowest temperatures, low H₂S production, and flocculation profile. Among the strains tested, two of them showed appropriate characteristics to produce two different beer styles: lager and ale. Moreover, both strains were tested for cachaça production and the results confirmed the capacity of these strains to improve the quality of cachaça. In the bioethanol production, the fermentation process was performed similarly to that used by bioethanol industries: recycling of yeast biomass in the fermentative process with sulfuric acid washings (pH 2.0). The production of ethanol, glycerol, organic acids, dry cell weight, carbohydrate consumption, and cellular viability were analyzed. One strain presented fermentative parameters similar to PE2, industrial/commercial strain, with equivalent ethanol yields and cellular viability during all fermentative cycles. This work demonstrates that cachaça distilleries seem to be an interesting environment to select new yeast strains to be used in biotechnology applications as beer and bioethanol production.     

Air drying optimization of Saccharomyces cerevisiae through its water-glycerol dehydration properties

AIMS: This study describes the different stages of optimization in an original drying process for yeasts, which allows the retrieval of dried samples of Saccharomyces cerevisiae CBS 1171 with maximum viability. METHODS AND RESULTS: The process involves the addition of wheat flour to yeast pellets, followed by mixing and then air-drying in a fluidized bed dryer. The sensitivity to the osmotic stress was first studied in a water-glycerol solution and the observed results were then applied to the drying process. This study have shown that the yeast was quite resistant to osmotic stress and pointed out the existence of zones of sensitivity where viability dramatically decrease as function of final osmotic pressure and temperature of the treatment. Thus, for dehydration until low osmotic pressure (133 MPa, i.e. a(w) = 0.38) results have shown that viability was better when temperature of the treatment was less than 8 degrees C or higher than 25 degrees C. Moreover, kinetic of dehydration was found to greatly influence cells recovery. CONCLUSIONS: These observations allowed the choice of parameters of dehydration of yeasts with an original drying process which involve the mix of the yeasts with wheat flour and then drying in a fluidized bed. SIGNIFICANCE AND IMPACT OF THE STUDY: This process dried rapidly the yeasts to less than 220 MPa (aw < or = 0.2) with whole cell recovery and good fermentative capabilities.     

Production of Ethanol from d-Xylose by Using d-Xylose Isomerase and Yeasts

d-Xylulose, an intermediate of d-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for d-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from d-xylulose. Fermenting yeasts are also able to produce ethanol from d-xylose when d-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from d-xylose in a yield of greater than 80% by a two-step process. First, d-xylose is converted to d-xylulose by xylose isomerase. d-Xylulose is then fermented to ethanol by yeasts.     

A population genomics insight into the Mediterranean origins of wine yeast domestication

The domestication of the wine yeast Saccharomyces cerevisiae is thought to be contemporary with the development and expansion of viticulture along the Mediterranean basin. Until now, the unavailability of wild lineages prevented the identification of the closest wild relatives of wine yeasts. Here, we enlarge the collection of natural lineages and employ whole‐genome data of oak‐associated wild isolates to study a balanced number of anthropic and natural S. cerevisiae strains. We identified industrial variants and new geographically delimited populations, including a novel Mediterranean oak population. This population is the closest relative of the wine lineage as shown by a weak population structure and further supported by genomewide population analyses. A coalescent model considering partial isolation with asymmetrical migration, mostly from the wild group into the Wine group, and population growth, was found to be best supported by the data. Importantly, divergence time estimates between the two populations agree with historical evidence for winemaking. We show that three horizontally transmitted regions, previously described to contain genes relevant to wine fermentation, are present in the Wine group but not in the Mediterranean oak group. This represents a major discontinuity between the two populations and is likely to denote a domestication fingerprint in wine yeasts. Taken together, these results indicate that Mediterranean oaks harbour the wild genetic stock of domesticated wine yeasts.