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.     

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.     

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.     

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.     

Some enzymological factors influencing the leavening capacity and keeping quality of baker's yeast

Summary In tests with different brands of baker's yeast, no correlation was found between the -glucosidase activity and either the leavening power or the ability to ferment maltose.During storage of pressed baker's yeast, the carbohydrate reserves were consumed, the fermentation ability of the yeast disappeared, and the decomposition of acid soluble nucleotides was followed by a discharge of proteins and nucleic acids, RNA and DNA. The contents of the RNA fractions increased initially during storage. Under extreme conditions (at 35°C), the amount of rRNA attained its maximum value after 5 days, followed by DNA (7 days), then tRNA (10–12 days) and lastly 5S RNA; thereafter they all decreased. The mRNA fraction diminished rapidly: after only two day's storage the mRNA declined by 90%.Paper presented at the 8th International I.C.C. (International Association for Cereal Chemistry) Working and Discussion Meetings, 16–18th May, 1974.Dedicated to Professor Otto Hoffmann-Ostenhof on the celebration of his sixtieth birthday.     

Sourdough Bread Made from Wheat and Nontoxic Flours and Started with Selected Lactobacilli Is Tolerated in Celiac Sprue Patients

This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients. Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients. This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE). A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli. After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally. Proteins were extracted from sourdough and used to produce a peptic-tryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin. The minimal agglutinating activity was ca. 250 times higher than that of doughs chemically acidified or started with baker's yeast. Two types of bread, containing ca. 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients. Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread. When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values. The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread. These results showed that a bread biotechnology that uses selected lactobacilli, nontoxic flours, and a long fermentation time is a novel tool for decreasing the level of gluten intolerance in humans.     

Glycerol Production by Fermenting Yeast Cells Is Essential for Optimal Bread Dough Fermentation

Glycerol is the main compatible solute in yeast Saccharomyces cerevisiae. When faced with osmotic stress, for example during semi-solid state bread dough fermentation, yeast cells produce and accumulate glycerol in order to prevent dehydration by balancing the intracellular osmolarity with that of the environment. However, increased glycerol production also results in decreased CO₂ production, which may reduce dough leavening. We investigated the effect of yeast glycerol production level on bread dough fermentation capacity of a commercial bakery strain and a laboratory strain. We find that Δgpd1 mutants that show decreased glycerol production show impaired dough fermentation. In contrast, overexpression of GPD1 in the laboratory strain results in increased fermentation rates in high-sugar dough and improved gas retention in the fermenting bread dough. Together, our results reveal the crucial role of glycerol production level by fermenting yeast cells in dough fermentation efficiency as well as gas retention in dough, thereby opening up new routes for the selection of improved commercial bakery yeasts.     

Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients

This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients. Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients. This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE). A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli. After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally. Proteins were extracted from sourdough and used to produce a peptic-tryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin. The minimal agglutinating activity was ca. 250 times higher than that of doughs chemically acidified or started with baker's yeast. Two types of bread, containing ca. 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients. Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread. When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values. The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread. These results showed that a bread biotechnology that uses selected lactobacilli, nontoxic flours, and a long fermentation time is a novel tool for decreasing the level of gluten intolerance in humans.     

Genetic Analysis of Haploids from Industrial Strains of Baker's Yeast

Strains of baker's yeast conventionally used by the baking industry in Japan were tested for the ability to sporulate and produce viable haploid spores. Three isolates which possessed the properties of baker's yeasts were obtained from single spores. Each strain was a haploid, and one of these strains, YOY34, was characterized. YOY34 fermented maltose and sucrose, but did not utilize galactose, unlike its parental strain. Genetic analysis showed that YOY34 carried two MAL genes, one functional and one cryptic; two SUC genes; and one defective gal gene. The genotype of YOY34 was identified as MATalpha MAL1 MAL3g SUC2 SUC4 gall. The MAL1 gene from this haploid was constitutively expressed, was dominant over other wild-type MAL tester genes, and gave a weak sucrose fermentation. YOY34 was suitable for both bakery products, like conventional baker's yeasts, and for genetic analysis, like laboratory strains.     

Disruption of the CAR1 gene encoding arginase enhances freeze tolerance of the commercial baker's yeast Saccharomyces cerevisiae

The effect of intracellular charged amino acids on freeze tolerance in dough was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.     

Sucrose fermentation by Saccharomyces cerevisiae lacking hexose transport

Sucrose is the major carbon source used by Saccharomyces cerevisiae during production of baker's yeast, fuel ethanol and several distilled beverages. It is generally accepted that sucrose fermentation proceeds through extracellular hydrolysis of the sugar, mediated by the periplasmic invertase, producing glucose and fructose that are transported into the cells and metabolized. In the present work we analyzed the contribution to sucrose fermentation of a poorly characterized pathway of sucrose utilization by S. cerevisiae cells, the active transport of the sugar through the plasma membrane and its intracellular hydrolysis. A yeast strain that lacks the major hexose transporters (hxt1-hxt7 and gal2) is incapable of growing on or fermenting glucose or fructose. Our results show that this hxt-null strain is still able to ferment sucrose due to direct uptake of the sugar into the cells. Deletion of the AGT1 gene, which encodes a high-affinity sucrose-H(+) symporter, rendered cells incapable of sucrose fermentation. Since sucrose is not an inducer of the permease, expression of the AGT1 must be constitutive in order to allow growth of the hxt-null strain on sucrose. The molecular characterization of active sucrose transport and fermentation by S. cerevisiae cells opens new opportunities to optimize yeasts for sugarcane-based industrial processes.     

Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding

Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (alpha-galactosidase) activity. To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker's yeast by both classical breeding and recombinant DNA technology. Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture. Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.     

Disruption of the CAR1 Gene Encoding Arginase Enhances Freeze Tolerance of the Commercial Baker's Yeast Saccharomyces cerevisiae

The effect of intracellular charged amino acids on freeze tolerance in doughs was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.     

Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.     

Osmotolerance and leavening ability in sweet and frozen sweet dough. Comparative analysis between Torulaspora delbrueckii and Saccharomyces cerevisiae baker's yeast strains

The response of Saccharomyces cerevisiae and freeze-tolerant Torulaspora delbrueckii strains to osmotic stress and their CO₂ production capacity in sweet and frozen-sweet dough has been examined. T. delbrueckii strains, IGC5321 and IGC5323 showed higher leavening ability than Saccharomyces, specially after exposure to hyperosmotic stress of bread dough containing 20% sucrose and 2% salt added. In addition, Torulaspora and especially T. delbrueckii IGC5321 exhibited no loss of CO₂ production capacity during freeze-thaw stress. Overall, these results appeared to indicate that Torulaspora cells are more tolerant than Saccharomyces to osmotic stress of bread dough. This trait correlated with a low invertase activity, a slow rate of trehalose mobilisation and the ability to respond rapidly to osmotic stress. Growth behaviour on high osmotic synthetic media was also examined. Cells of the IGC5321 strain showed intrinsic osmotolerance and ion toxicity resistance. However,T. delbrueckii IGC5323 exhibited a clear phenotype of osmosensitivity. Hence, this characteristic may not be essential or the only determinant for leavening ability in salted high-sugar dough. This revised version was published online in August 2006 with corrections to the Cover Date.     

Superior molasses assimilation, stress tolerance, and trehalose accumulation of baker's yeast isolated from dried sweet potatoes (hoshi-imo)

Yeast strains were isolated from dried sweet potatoes (hoshi-imo), a traditional preserved food in Japan. Dough fermentation ability, freeze tolerance, and growth rates in molasses, which are important characteristics of commercial baker's yeast, were compared between these yeast strains and a commercial yeast derivative that had typical characteristics of commercial strains. Classification tests including pulse-field gel electrophoresis and fermentation/assimilation ability of sugars showed that almost the stains isolated belonged to Saccharomyces cerevisiae. One strain, ONY1, accumulated intracellular trehalose at a higher level than commercial strain T128. Correlated with intracellular trehalose contents, the fermentation ability of high-sugar dough containing ONY1 was higher. ONY1 also showed higher freeze tolerance in both low-sugar and high-sugar doughs. The growth rate of ONY1 was significantly higher under batch and fed-batch cultivation conditions using either molasses or synthetic medium than that of strain T128. These results suggest that ONY1 has potential commercial use as baker's yeast for frozen dough and high-sugar dough.     

Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeasts.

To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken. When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6%. Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30 degrees C were checked, and the cells were collected from presporulation media in either exponential or stationary phase. Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22 degrees C. Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected. Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts. When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters. This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)