Stereochemical Control in Microbial Reduction. Part 10. Asymmetric Reduction of Alkyl 3-Methyl-2-Oxobutanoate with Immobilized Bakers' Yeast in Hexane

Esters of 3-methyl-2-oxobutanoic acid are reduced with bakers' yeast by three methods: free bakers' yeast in water, immobilized bakers' yeast in water, and immobilized bakers' yeast in hexane. Although (R)-hydroxy esters are obtained in all cases, the enantiomeric excess varies from 3% (reduction of the methyl ester with free bakers' yeast in water) to 93% (reduction of the butyl ester with immobilized bakers' yeast in hexane) depending on the structure of substrate and on the reaction conditions. The mechanism of the present stereochemical control is discussed.     

Comparison of fermentative capacities of industrial baking and wild-type yeasts of the species Saccharomyces cerevisiae in different sugar media

AIMS: To compare the fermentative capacity of wild and domesticated isolates of the genus Saccharomyces. METHODS AND RESULTS: The fermentative capacity of yeasts from a variety of wild and domesticated sources was tested in synthetic dough media that mimic major bread dough types. Domesticated yeast strains were found to have better maltose-utilizing capacity than wild yeast strains. The capacity to ferment sugars under high osmotic stress was randomly distributed amongst wild and baking strains of Saccharomyces. CONCLUSION: The domestication of bakers' yeast has enhanced the ability of yeasts to ferment maltose, without a similar impact on the fermentative capacity under high osmotic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, combined with molecular studies of both wild and domesticated yeast, showed that domestication of bakers' yeast has resulted in improved maltose utilization, apparently via the duplication and mutation of the MAL genes.     

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     

Improving the freeze tolerance of bakers' yeast by loading with trehalose

We examined the freeze tolerance of bakers' yeast loaded with exogenous trehalose. Freeze-tolerant and freeze-sensitive compressed bakers' yeast samples were soaked at several temperatures in 0.5 M and 1 M trehalose and analyzed. The intracellular trehalose contents in both types of bakers' yeast increased with increasing soaking period. The initial trehalose-accumulation rate increased with increasing exogenous trehalose concentration and soaking temperature. The maximum trehalose content was almost identical (200-250 mg/g of dry cells) irrespective of the soaking temperature and the type of bakers' yeast, but depended on the exogenous trehalose concentration. The leavening ability of both types of bakers' yeast loaded with trehalose was almost identical to that of the respective original cells, irrespective of the soaking conditions. The freeze-tolerant ratio (FTR) of both types of bakers' yeast increased with increasing intracellular trehalose content. However, FTR decreased during over-soaking after the maximum amount of trehalose had accumulated. FTR of the freeze-sensitive bakers' yeast was more efficiently improved than that of the freeze-tolerant type.     

The use of marine yeast (Candida sp.) and bakers' yeast (Saccharomyces cerevisiae) in combination with Chlorella sp. for mass culture of the rotifer Brachionus plicatilis

Use of marine yeast and bakers' yeast in combination with Chlorella sp. for the large-scale production of the rotifer Brachionus plicatilis was investigated. The culture density of marine yeast fed rotifers was significantly higher than rotifers fed bakers' yeast. Rotifer production was significantly higher and the doubling time was lower for marine yeast fed rotifers than for bakers' yeast fed rotifers. It appears that the addition of marine yeast to the feed enhances the birth rate and overall production of rotifers in the culture system. The nutritional quality of rotifers is discussed.     

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.     

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.     

Cloning and expression in Escherichia coli of the gene for an Arthrobacter beta-(1----3)-glucanase.

When inserted in the correct orientation at the BamHI site of plasmid YRp7, an 8.6-kilobase BamHI fragment of Arthrobacter sp. strain YCWD3 DNA gave Escherichia coli HB101 cells harboring the recombinant plasmid pBX20 the ability to lyse bakers' yeast cell walls or bakers' yeast glucan in agar medium. An extract of the transformed E. coli cells contained an endo-beta-(1----3)-glucanase with the same activity pattern as that of glucanase I produced by Arthrobacter sp. strain YCWD3. Although part of the glucanase activity was contributed by apparently defective molecules, two protein species were found which had high lytic activity on yeast cell walls and adsorbed to microcrystalline cellulose, and both had a single constituent polypeptide with a molecular weight of about 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these properties the protein species were indistinguishable from those glucanase I protein species of Arthrobacter sp. strain YCWD3 which we believe are nearly the intact molecule. We conclude that the cloned fragment of Arthrobacter sp. strain YCWD3 DNA contains the structural gene for glucanase I. A recombinant plasmid obtained by subcloning a PstI fragment of pBX20 into pBR322 caused the transformed E. coli cells to produce apparently defective glucanase molecules only. This observation serves as additional supporting evidence for our conclusion.     

The effect of the addition of proteases and glucanases during yeast autolysis on the production and properties of yeast extracts

Yeast extracts (YE) were produced with the addition of proteases or glucanases during bakers' yeast (Saccharomyces cerevisiae) autolysis. Chemical composition, physical properties, and biological value of the YE were examined. Proteases had the highest impact on the turbidity and filterability of YE. All 11 proteases and two glucanases increased YE yields (% yeast solids solubilized) obtained from heated (80 degrees C/15 min) bakers' yeast creams (BYC). However, when proteases were added to native (unheated) BYC during autolysis, few increased YE yields, with papain being the most effective. The increased yields were generally related to increased levels of total nitrogen (TN) and alpha-amino nitrogen (alpha-AN) in the YE. Media were supplemented with the various yeast extracts, and the highest growth rates (mumax) and biomass values (ODmax) of Lactobacillus acidophilus were noted. The best growth was obtained with YE produced with native BYC treated with a fungal protease, and results of this study show that some enzymes could be used to produce improved YE for microbiological media.     

Use of immobilized cell biocatalysts in baking

Baking using baker's yeast immobilized in a starch–gluten–milk matrix (traditional fermented cereal food trahanas), containing viable lactic acid bacteria (LAB), and kefir (natural co-culture of yeasts and LAB) immobilized on orange peel, were investigated. The use of immobilized cells increased shelf life, delayed staling, and improved overall the quality of bread, compared with the traditional baker's yeast bread. These improvements were attributed to the reduction of pH, the lower moisture loss rates, and the presence of LAB, which are known to exhibit antimould properties. Better results were obtained using the sourdough method compared to the straight dough bread-making method. Headspace SPME GC–MS analysis showed that the use of immobilized cells increased the number of bread aroma volatiles, especially esters. The best results, including shelf life and overall bread quality, were obtained in the case of baker's yeast immobilized on trahanas, although kefir immobilized on orange peel seems to be a more cost effective biocatalyst.     

Effect of ultrafiltration of yeast extracts on their ability to promote lactic acid bacteria growth

Five yeast extracts (YE) were fractionated by ultrafiltration (UF) with 1, 3, and 10 kDa molecular weight cutoff membranes, concentrated by freeze-drying, and the resulting powders of yeast extract filtrates (YEF) were evaluated for their growth-promoting properties on nine cultures of lactic acid bacteria (LAB). There was an increase in alpha-amino nitrogen content of the YEF powders as the pore size of the UF membranes used to filter the YE solutions decreased. The source of YE had a much greater effect than UF on the growth of LAB. This was also the case for the YEF contents in total and alpha-amino nitrogen. Growth curves of the LAB showed that maximum growth rate (mumax) data were on average 30% higher with bakers' YE than with brewers' YE, while maximum optical density (ODmax) values were on average 16% higher with bakers' YE. This could be related to the higher nitrogen content of the bakers' YE used in this study. Modification by UF of the YE had no significant influence on the growth of 4 of the 9 LAB strains. The three strains of Lactobacillus casei were negatively influenced by UF, as they did not grow as well in the media containing the YEF obtained after filtering with 1 and 3 kDa membranes. On a total solids basis, the 2.5 x retentates from the 10 kDa membrane gave, on average, 4% lower mumax and 5% lower ODmax values as compared to cultures where the corresponding YEF was used as medium supplement. This could also be partially related to the different nitrogen contents of the filtrates and retentates.     

Monitoring cell concentration and activity by multiple excitation fluorometry.

Four key cellular metabolic fluorophores--tryptophan, pyridoxine, NAD(P)H, and riboflavin--were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems--bakers' yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cerevisiae RTY110/pRB58 growing on glucose. The measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. The results indicate that the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for the monitoring of cell concentration in bakers' yeast and C. utilis fermentations. Pyridoxine fluoresce is the best signal for the monitoring of cell concentration in the S. cerevisiae RTY110/pRB58 fermentation. In bakers' yeast fermentations the pyridoxine fluorescence signal can be used to monitor cellular activity. The NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used to control ethanol feeding in a fed-batch process.     

Industrial Application of Artificially Induced Diploid Strains of Torulaspora delbrueckii

Diploid strains of Torulaspora delbrueckii were tested for industrial application. Because the cell volume of the diploid strain was three times as large as that of the parental haploid strain, collection and subsequent dehydration to make compressed yeast cakes were greatly improved with the diploid YL3. The time required for dehydration of the diploid strain was shortened to 1/2.5 that of the parent strain under conventional conditions. Moreover, for the diploid cells frequent filter changes for dehydration were not required, which was the case with parental cells. Fermentation activity and tolerance to freeze-thawing in dough were succesfully inherited by the diploid strains. The diploid YL3 showed nearly the same activity as the diploid F31 in bread making. However, the endurance period of yeast cakes when stored at 30°C without softening to lead to liquefaction was much longer in YL3 (199 h) than in F31 (132 h). This superiority was ascribed to the fact that YL3 was induced through direct diploidization and had no genetic defect on chromosomes because the wild-type strain was employed as the parent, whereas F31 was obtained through protoplast fusion from two auxotrophic mutants and carried at least two mutagenized genes that were masked by heterolallelism.     

Phialophora graminicola, a dark septate fungus, is a beneficial associate of the grass Vulpia ciliata ssp. ambigua

The influence of three organic compounds and bakers' dry yeast on growth of external mycelium and phosphorus uptake of the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith (BEG 87) was examined. Two experiments were carried out in compartmentalized growth systems with root-free sand or soil compartments. The sand and soil in the root-free compartments were left untreated or uniformly mixed with one of the following substrates (0.5 mg g⁻¹ soil): bakers' dry yeast, bovine serum albumin, starch or cellulose. Effects of the organic substrates on biomass and hyphal length density of the arbuscular mycorrhizal fungus were examined by using specific fatty acid signatures in combination with direct microscopy. Micro-organisms other than the arbuscular mycorrhizal fungus were measured by fatty acid signatures, and radioactive ³³P labelling of the root-free soil was used to determine arbuscular mycorrhizal hyphal phosphorus uptake. In general, hyphal growth of G. intraradices was enhanced by yeast and bovine serum albumin, whereas the carbon sources, starch and cellulose, depressed fungal growth. By analysing the fatty acid 16:1ω5 from phospholipids (indicating mycelium) and neutral lipids (indicating storage structures) it was shown that increased fungal growth due to yeast was mainly in vegetative hyphae and less in storage structures. Arbuscular mycorrhizal hyphal phosphorus uptake was decreased by cellulose, but unaffected by the other substrates compared with the control. This means that both growth and phosphorus transport by the arbuscular mycorrhizal fungus were decreased under cellulose treatment. However, the composition of the microbial community varied under different substrate conditions indicating a possible interactive component with arbuscular mycorrhizal hyphal growth and phosphorus uptake.     

Production of bakers' yeast in cheese whey ultrafiltrate

A process for the production of bakers' yeast in whey ultrafiltrate (WU) is described. Lactose in WU was converted to lactic acid and galactose by fermentation. Streptococcus thermophilus was selected for this purpose. Preculturing of S. thermophilus in skim milk considerably reduced its lag. Lactic fermentation in 2.3x-concentrated WU was delayed compared with that in unconcentrated whey, and fermentation could not be completed within 60 h. The growth rate of bakers' yeast in fermented WU differed among strains. The rate of galactose utilization was similar for all strains, but differences in lactic acid utilization occurred. Optimal pH ranges for galactose and lactic acid utilization were 5.5 to 6.0 and 5.0 to 5.5, respectively. The addition of 4 g of corn steep liquor per liter to fermented WU increased cell yields. Two sources of nitrogen were available for growth of Saccharomyces cerevisiae: amino acids (corn steep liquor) and ammonium (added during the lactic acid fermentation). Ammonium was mostly assimilated during growth on lactic acid. This process could permit the substitution of molasses by WU for the industrial production of bakers' yeast.     

Nucleoprotein complexes of yeast and their biological effect on T-lymphocytes

Complexes of nucleic acids and acid nuclear proteins that are active toward human T-lymphocytes were isolated from cells of bakers' yeast Saccharomyces cerevisiae. The conditions of isolation of nucleoprotein complexes by acid extraction followed by microfiltration for concentration of macromolecular components were optimized. Gel filtration and electrophoresis were used to study the composition and molecular weights of components of the preparations obtained. It was shown that nucleoprotein complex had a molecular weight of 1430 kDa. However, only one zone was determined by electrophoresis of the protein component with a molecular weight of 30 kDa.     

Generation of 5-fluorouracil from 5-fluorocytosine by monoclonal antibody-cytosine deaminase conjugates.

Cytosine deaminase (CDase) catalyzes the conversion of cytosine to uracil and is also able to convert the clinically used antifungal agent 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU). The enzyme was purified from bakers' yeast in a six-step procedure. Studies indicated that bakers' yeast CDase had a molecular weight of approximately 32 kDa and was composed of two subunits of equal molecular weights. Monoclonal antibodies were covalently attached to CDase, forming conjugates that could bind to antigens on tumor cell surfaces. The combination of L6-CDase and 5FC was equivalent in cytotoxic activity to 5FU when tested against the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). 5FC alone was noncytotoxic. The activation of 5FC was immunologically specific since 1F5-CDase did not enhance 5FC activity.     

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.     

Rapid Method for Measuring Extracellular Water in Yeast Preparations

A rapid procedure for the quantitative determination of extracellular water in bulk bakers' yeast was developed on the basis of the solute dilution principle. A reagent is prepared by synthesizing the diazonium ion of p-aminobenzoic acid and coupling it to peptone. This "azopeptone reagent" permits direct colorimetric measurement, which accounts for the rapidity and simplicity of the test. Potential errors due to osmotic effects are avoided by supplementing the reagent with saline and, more importantly, minimizing the duration of contact between reagent and cells. The new method has acceptable accuracy and precision, and may also be suitable for use with other microorganisms.