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.     

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.     

Cell-surface modification of non-GMO without chemical treatment by novel GMO-coupled and -separated cocultivation method

We developed a novel method to coat living non-genetically modified (GM) cells with functional recombinant proteins. First, we prepared GM yeast to secrete constructed proteins that have two domains: a functional domain and a binding domain that recognizes other cells. Second, we cocultivated GM and non-GM yeasts that share and coutilize the medium containing recombinant proteins produced by GM yeasts using a filter-membrane-separated cultivation reactor. We confirmed that GM yeast secreted enhanced green fluorescent protein (EGFP) fusion proteins to culture medium. After cocultivation, EGFP fusion proteins produced by GM yeast were targeted to non-GM yeast (Saccharomyces cerevisiae BY4741ΔCYC8 strain) cell surface. Yeast cell-surface engineering is a useful method that enables the coating of GM yeast cell surface with recombinant proteins to produce highly stable and accumulated protein particles. The results of this study suggest that development of cell-surface engineering from GM organisms (GMOs) to living non-GMOs by our novel cocultivation method is possible.     

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     

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.     

Monitoring the human β1, β2, β3 adrenergic receptors expression and purification in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using the fluorescence properties of the enhanced green fluorescent protein

The three beta adrenergic receptor subtypes, β1-, β2- and β3-, were expressed in the methylotrophic yeast Pichia pastoris. These receptors were N-terminally fused to the enhanced green fluorescent protein (EGFP) and the fluorescent properties of EGFP were used: (1) to select the recombinant strains, (2) to monitor the expression of the fluorescent receptors, and (3) to monitor the purification of the receptors by immobilized metal affinity chromatography. We demonstrate here that Pichia pastoris can be an alternative host to express and purify milligram amounts of human beta adrenergic receptors.     

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.     

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.     

粉质质量指数在冬小麦面团品质及烘烤品质中的应用价值研究

为了进一步了解粉质质量指数在小麦面团品质和烘烤品质评价中的作用,对90份冬小麦样品的粉质质量指数(FQN)与揉混仪参数、拉伸仪参数和面包品质参数之间的相关关系进行了研究。结果表明：FQN与揉混仪参数(峰值时间、峰值面积、8min尾高和斜率)有极显著的正相关关系,且以线性相关为主,可以用于评价小麦粉的耐揉性和筋力;FQN与拉伸曲线面积和最大拉伸阻力有较好的相关关系,但与延伸度相关性较小,且以非线性相关为主。因此,FQN不能很好地进行评价面团的拉伸特性;FQN与面包体积、面包评分和面包坚实度有一定的非线性相关关系,FQN可以用作评价面包烘烤品质的参数指标。     

Experimente mit Hefe

Why bread dough rises, although yeast cannot metabolize starch: Experiments with yeast Yeast is ideal for simple experiments to visualize metabolic processes, such as those known from baking bread, for example. Thus the metabolism of different sugars to carbon dioxide can be qualitatively demonstrated by inflating balloons or quantitatively by gas production in a fermentation tube (glass airlock for fermentation). The experiments presented can be used to answer the question of why yeast is added to bread dough even though it is not able to convert the starch present in the flour.     

High Molecular Weight Glutenin Subunit Composition of Indian Wheats and Their Relationships with Dough Strength

One hundred and seventy two wheat varieties including twenty-five durum wheat cultivars were evaluated for high molecular weight glutenin subunit (HMW-GS) composition using SDS-PAGE. The relationship between HMW-GS and sedimentation tests for dough strength was studied. Three alleles were present at the Glu-A1 locus, eight at Glu-B1 and two at Glu-D1 in bread wheat. The data indicated the prevalence of the Glu-A1b allele (63.5%) at the Glu-A1 and Glu-D1a (71.4%) at Glu-D1 loci. Three alleles, namely Glu-B1b (30.61%), Glu-B1c (25.85%) and Glu-B1i (34.00%) represented about 90% of the alleles at Glu-B1 locus. The combination of Glu-A1b, Glu-B1i and Glu-D1d alleles exhibited highest dough strength as measured by sedimentation value in comparison to other combinations (p<0.001). However, this combination was present only in 7% of the samples evaluated. In durum wheat, the null allele (Glu-A1c) was observed more frequently (76%) than the Glu-A1b allele (24%). Glu-B1f and Glu-B1e alleles represented equally (32% each). Protein subunits 13+16 and 6+8 were found correlated positively (p<0.05) with improved dough strength as compared to subunit 20 in durum wheat. This information can be a valuable reference for designing breeding programme for the improvement of bread and pasta making quality of bread and durum wheats, respectively in India.     

A note on the leavening activity of yeasts isolated from Nigerian palm wine

The role of the yeast flora of Nigerian palm wine in the leavening activity of the beverage was investigated by subjecting organisms from the wine to dough-raising tests. Those with appreciable leavening activity were identified as Saccharomyces cerevisiae and Candida spp. They produced maximum dough volumes in 3–4 h at 37°C. The study has provided experimental evidence that yeasts contribute to the leavening activity of palm wine and has identified strains which have potential utility in commercial bread baking.     

A polyphasic study on the taxonomic position of industrial sour dough yeasts

The sour dough bread making process is extensively used to produce wholesome palatable rye bread. The process is traditionally done using a back-slopping procedure. Traditional sour doughs in Finland comprise of lactic acid bacteria and yeasts. The yeasts present in these doughs have been enriched in the doughs due to their metabolic activities, e.g. acid tolerance. We characterized the yeasts in five major sour bread bakeries in Finland. We found that most of the commercial sour doughs contained yeasts which were similar to Candida milleri on the basis of 18S rDNA and EF-3 PCR-RFLP patterns and metabolic activities. Some of the bakery yeasts exhibited extensive karyotype polymorphism. The minimum growth temperature was 8 degrees C for C. milleri and also for most of sour dough yeasts.     

Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long‐pass emission filter with single‐wavelength excitation, which is technically easier to use than dual‐emission fluorescent proteins that require dual‐wavelength excitation. Copyright © 2013 John Wiley & Sons, Ltd.     

A new method using enhanced green fluorescent protein (EGFP) to determine grazing rate on live bacterial cells by protists

A new method was developed for estimating the grazing rate of live bacteria by protists. Bacterial cells (Escherichia coli bearing plasmid pEGFP) expressing enhanced green fluorescent protein (EGFP) were used as a live bacterial tracer. Ciliates (Tetrahymena thermophila) were fed with EGFP-tagged bacterial cells, and the individual cells taken up by the ciliates were detected by epifluorescence microscopy. The EGFP fluorescence was stable during the storage of samples fixed with glutaraldehyde. Comparison of clearance rates based on the uptake of EGFP-tagged live cells and fluorescently-labeled heat-killed cells suggested that the use of heat-killed cells underestimates the clearance rates. We suggest that EGFP-tagged bacteria are a useful tracer for determining protist bacterivory in culture and aquatic environments. Received: January 22, 2001 / Accepted: October 27, 2001     

Isolation of Marine Yeasts Collected from the Pacific Ocean Showing a High Production of γ-Aminobutyric Acid

Marine yeasts were collected from coastal and deep sea areas in the Pacific Ocean and the Sea of Japan around central and northern Japan to prepare a novel type of natural seasoning. It was found that one of the marine yeasts collected from the Pacific Ocean off Hachinohe showed a high concentration of γ-aminobutyric acid (GABA) in its extract, about 7–10 times higher than those of commercially available bread yeast and other marine yeasts. The marine yeast isolated and named Hachinohe No. 6 catalyzed the reaction from monosodium glutamate to GABA only in the presence of glucose. Subsequently, several marine yeasts belonging to the genera Pichia and Candida were found to have such catalytic activities, but not those belonging to the genus Saccharomyces. Isolate Hachinohe No. 6 was found to have the highest catalytic activity among the yeasts examined in this study.     

Microbiology of pozol,a Mexican fermented maize dough

Mexican fermented maize dough, pozol, including traditional banana leaf-wrapped samples and material in plastic bags, was purchased. All samples were pH 4.7 to 5.7 approx. 12 h after preparation, pH declining to 3.6 to 3.9 after 6 to 9 days storage at ambient temperature. These latter samples had dry matter contents of 31% to 48% (w/w), 0.35% to 0.75% titratable acidity as lactic acid and lactic acid bacteria as predominant microbial flora at about 10⁸ c.f.u./ml. The lactic acid bacteria included strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus confusus, Lactococcus lactis and Lactococcus raffinolactis. Fungi were not found in the samples stored in plastic bags. The samples wrapped in banana leaf, however, developed a large surface mycoflora within 2 days. This included Geotrichum candidum, yeasts and moulds. The majority of the lactic acid bacteria and approx. 50% of yeasts hydrolysed starch to some extent. No Geotrichum isolate hydrolysed starch. Lactate was assimilated by all the Geotrichum isolates and by 17 of 39 yeast strains.     

Aerobic and Facultative Microflora of Fresh and Spoiled Refrigerated Dough Products

The microbial flora of fresh, unsterile, dough products held at refrigeration temperatures was compared with the microbial flora of the same products that had spoiled spontaneously. Various methods based on selective media were used to determine molds, yeasts, and bacteria present. Except for two special cases in which a yeast and Penicillium roqueforti induced spoilage, all of the samples deteriorated because of bacterial growth. A total of 1,132 bacterial isolates was subjected to further classification. In the spoiled products, 92% of the isolates belonged to the Lactobacillaceae. More than one-half of these (53%) belonged to the genus Lactobacillus, and an additional 36% were in the genus Leuconostoc. In the genus Leuconostoc almost all of the strains (94%) were L. mesenteroides. The third most common genus present was Streptococcus, represented by 3% of the total isolates. A preliminary taxonomic study of the microflora of refrigerated dough products revealed none of the isolates to be indicators of fecal contamination and none to be forms known to produce toxins. The highest counts encountered in the moist, fresh products were up to 200 million lactic acid bacteria per g in buttermilk biscuits, with a psychrophilic count as high as 4.8 million. In the spoiled samples, the highest total counts were 820 million in buttermilk biscuits. Mold counts were no higher than 1,800, except in the sample ruined by P. roqueforti where the count was 130,000 mold colonies.     

Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.