Experimente mit Hefe (2)

The so‐called iodine test is ideal for demonstrating the role of the enzyme “amylase” in simple experiments based on a colour reaction when a yeast dough rises, when carbohydrates are digested or when dishes are washed in the dishwasher. The experiments presented can be used to clarify the question of which everyday substances are capable of degrading starch or whether a food contains starch or not. The central question of the experiments is, how yeast makes the bread dough rise despite of its “starch intolerance”.     

固态发酵降低上部低次烟叶中淀粉及蛋白质的含量

利用固态发酵的方法降低上部低次烟叶中淀粉和蛋白质的含量,并对发酵过程中的厌氧细菌和酵母的数量进行检测。采用单因素和正交试验对固态发酵的条件进行优化,结果表明:各因素对发酵上部低次烟叶影响显著性主次次序依次为发酵时间(C)、发酵温度(A)和发酵水分质量分数(B),固态发酵的最佳条件为A2B2C3,即温度45℃、水分质量分数50%、发酵时间9 d。在该发酵条件下,上部低次烟叶的淀粉降解率为20.41%,蛋白质降解率为12.35%。通过固态厌氧发酵的方法可取得较好的、短期内快速降解上部低次烟叶中淀粉和蛋白质含量的效果。     

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.     

Engineering Saccharomyces cerevisiae for direct conversion of raw,uncooked or granular starch to ethanol

The production of raw starch-degrading amylases by recombinant Saccharomyces cerevisiae provides opportunities for the direct hydrolysis and fermentation of raw starch to ethanol without cooking or exogenous enzyme addition. Such a consolidated bioprocess (CBP) for raw starch fermentation will substantially reduce costs associated with energy usage and commercial granular starch hydrolyzing (GSH) enzymes. The core purpose of this review is to provide comprehensive insight into the physiological impact of recombinant amylase production on the ethanol-producing yeast. Key production parameters, based on outcomes from modifications to the yeast genome and levels of amylase production, were compared to key benchmark data. In turn, these outcomes are of significance from a process point of view to highlight shortcomings in the current state of the art of raw starch fermentation yeast compared to a set of industrial standards. Therefore, this study provides an integrated critical assessment of physiology, genetics and process aspects of recombinant raw starch fermenting yeast in relation to presently used technology. Various approaches to strain development were compared on a common basis of quantitative performance measures, including the extent of hydrolysis, fermentation-hydrolysis yield and productivity. Key findings showed that levels of α-amylase required for raw starch hydrolysis far exceeded enzyme levels for soluble starch hydrolysis, pointing to a pre-requisite for excess α-amylase compared to glucoamylase for efficient raw starch hydrolysis. However, the physiological limitations of amylase production by yeast, requiring high biomass concentrations and long cultivation periods for sufficient enzyme accumulation under anaerobic conditions, remained a substantial challenge. Accordingly, the fermentation performance of the recombinant S. cerevisiae strains reviewed in this study could not match the performance of conventional starch fermentation processes, based either on starch cooking and/or exogenous amylase enzyme addition. As an alternative strategy, the addition of exogenous GSH enzymes during early stages of raw starch fermentation may prove to be a viable approach for industrial application of recombinant S. cerevisiae, with the process still benefitting from amylase production by CBP yeast during later stages of cultivation.     

Effect of flocculation on performance of arming yeast in direct ethanol fermentation

In the direct ethanol fermentation of raw starch by arming yeast with α-amylase and glucoamylase, it is preferable to use a flocculent yeast because it can be recovered without centrifugation. Three types of arming yeast system, I (nonflocculent), II (mildly flocculent), and III (heavily flocculent), were constructed and their fermentation performances were compared. With an increase in the degree of flocculation, specific ethanol production rate for soluble starch decreased (0.19, 0.17, and 0.12 g g-dry-cell⁻¹ h⁻¹ for systems I, II, and III, respectively), but that for raw starch did not decrease as much as expected (0.06, 0.06, and 0.04 g g-dry-cell⁻¹ h⁻¹ for systems I, II and III, respectively). Microscopic observation revealed that many starch granules were captured in the yeast flocs in system III during the direct ethanol fermentation of raw starch. It was suggested that the capture of starch granules increases apparent substrate concentration for amylolytic enzymes in arming yeast cell flocs; thus, the specific ethanol production rate of system III was kept at a level comparable to those of the other systems.     

Optimal quality control of bakers' yeast fed-batch culture using population dynamics

An optimal quality control policy for the overall specific growth rate of bakers' yeast, which maximizes the fermentative activity in the making of bread, was obtained by direct searching based on the mathematical model proposed previously. The mathematical model had described the age distribution of bakers' yeast which had an essential relationship to the ability of fermentation in the making of bread. The mathematical model is a simple aging model with two periods: Nonbudding and budding. Based on the result obtained by direct searching, the quality control of bakers' yeast fed-batch culture was performed and confirmed to be experimentally valid.     

Effects of High-voltage Electric Field Treatment on the Water Activity of Bread

Treating bread dough by a high-voltage electric field (HVEF) during the first fermentation enabled the bread dough to retain water in the gluten fibers. The microstructure of the gluten fibers was still visible even with an HVEF treatment at 50 kV for 20 min, but could no longer be observed when the gluten was treated for more than 30 min. The crumb temperature of the HVEF treated bread during baking began to rise a few minutes sooner than that of the untreated bread, but the maximum temperature reached was the same in both cases: 99°C. The fact that the water activity of the HVEF-treated bread was 0.987 ± 0.0056, being higher than that of the untreated bread by 0.011, is in good agreement with the growing tests of Rhizopus nigricans. Furthermore, a distinct decrease in the water loss of the baked gluten was observed after the HVEF treatment. These results suggest that the HVEF treatment increased the ability of the gluten fibers, rather than the starch granules, to absorb and retain water; the state of the remaining water is considered to have become immobile, so that migration of moisture to the starch granules in the bread could be minimized.     

Repeated fermentation from raw starch using Saccharomyces cerevisiae displaying both glucoamylase and α-amylase

A diploid yeast strain displaying both α-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both glucoamylase and α-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100g/l of raw starch for 23 cycles without the loss of α-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.     

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.     

Yeasts in sustainable bioethanol production: A review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.     

High-level ethanol production from starch by a flocculent <Emphasis Type="Italic">Saccharomyces cerevisiae </Emphasis>strain displaying cell-surface glucoamylase

A Strain of host yeast YF207, which is a tryptophan auxotroph and shows strong flocculation ability, was obtained from SaccharomYces diastaticus ATCC60712 and S. cerevisiae W303-1B by tetrad analysis. The plasmid pGA11, which is a multicopy plasmid for cell-surface expression of the Rhyzopus oryzae glucoamylase/alpha-agglutinin fusion protein, was then introduced into this flocculent yeast strain (YF207/pGA11). Yeast YF207/pGA11 grew rapidly under aerobic condition (dissolved oxygen 2.0 ppm), using soluble starch. The harvested cells were used for batch fermentation of soluble starch to ethanol under anaerobic condition and showed high ethanol production rates (0.71 g h(-1) l(-1)) without a time lag, because glucoamylase was immobilized on the yeast cell surface. During repeated utilization of cells for fermentation, YF207/pGA11 maintained high ethanol production rates over 300 h. Moreover, in fed-batch fermentation with YF207/pGA11 for approximately 120 h, the ethanol concentration reached up to 50 g l(-1). In conclusion, flocculent yeast cells displaying cell-surface glucoamylase are considered to be very effective for the direct fermentation of soluble starch to ethanol.     

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.     

Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein

Recombinant Saccharomyces cerevisiae YPB-G strain secreting a fusion protein displaying both BsAAase/GAase activities was grown in 1.5 l YPS media containing single (starch) and mixed carbon sources (glucose+starch) using a 2.5 l New Brunswick BiofloIII fermenter. Ethanol and biomass formation, starch utilisation, secretion of the amylolytic enzymes (-amylase and glucoamylase), accumulation of reducing sugars and glucose were followed during the fermentation of YPB-G under different conditions. Moreover, a model has been developed for the growth of recombinant yeast on substitutable substrates using cybernetic framework principles and incorporating product formation. In the present work, both the biphasic and the diauxic growth patterns observed experimentally in batch culture of recombinant yeast cells were simulated successfully by modifying the cybernetic framework to include ethanol formation and the degradation kinetics of starch which is not directly utilised by yeast. The model can further be expanded to fed-batch systems.     

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.     

The effect of Saccharomyces cerevisiae on the stability of the herbicide glyphosate during bread leavening

AIMS: To investigate the ability of baker's yeast (Saccharomyces cerevisiae) to degrade the herbicide glyphosate during the fermentation cycle of the breadmaking process. METHODS AND RESULTS: Aqueous glyphosate was added to bread ingredients and kneaded by commercially available breadmaking equipment into dough cultures. Cultures were incubated in the breadmaker throughout the fermentation cycle. The recovery of glyphosate levels following fermentation was determined, thus allowing an estimation of glyphosate degradation by yeast. CONCLUSIONS: It was shown, for the first time, that S. cerevisiae plays a role in metabolizing glyphosate during the fermentation stages of breadmaking. Approximately 21% was degraded within 1 h. SIGNIFICANCE AND IMPACT OF THE STUDY: As a result of projected increases in the glyphosate use on wheat and the role of bread as a dietary staple, this may contribute to more informed decisions being made relating to the use of glyphosate on glyphosate-resistant wheat, from a public health/regulatory perspective.     

HTST-extrusion cooking in ethanol production from starchy materials

Starch syrup for ethanol fermentation is conventionally produced by acid or enzymatic hydrolysis. Recently, however, promising results have been obtained using HTST-extrusion cooking in starch liquefaction. The starchy material was pregelatinized and preliquefied in a Creusot-Loire BC45 twin-screw HTST-extrusion cooker before simultaneous saccharification by amyloglucosidase and fermentation by Saccharomyces cerevisiae or Zymomonas mobilis. With pretreatment of milled whole grain or starch by HTST-extrusion cooking a significantly shorter fermentation time could be achieved. Maximum ethanol yield was obtained in 45 h using conventional yeast and amyloglucosidase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) dosage, even without addition of Termamyl α-amylase (1,4-α-d-glucan glucanohydrolase, EC 3.2.1.1) during thermomechanical liquefaction. Immobilized yeast could also be used to produce ethanol both by a batch or continuous process. In this case, for a continuous process the DE-value of the syrup should be sufficiently high. A model for ethanol production as a function of dry matter, fermentation time, and yeast and Termamyl quantities has been developed.     

利用枣汁生产面包酵母

面包酵母是一类用来提高面包质量的重要添加剂。目前,不同国家主要采用分批培养、补料分批培养或连续培养的方式来生产面包酵母。酿酒酵母是用来发酵面团中淀粉的理想微生物,除了提升食品香味,增加口感之外,这一发酵过程可以产生多种维生素和蛋白质。用于生产酵母生物量的主要成分包括各种碳源,如甜菜糖蜜和甘蔗糖蜜等。由于甜菜糖蜜可用于高产率地生产乙醇,加之其带来的生物环境污染和废水处理问题,因此需要考虑用其他糖类来生产面包酵母。其中一个代替性糖源是枣。由于各种原因,伊朗每年都浪费大量的枣。研究了用枣作为培养基碳源的可行性。将废枣榨成汁,然后研究了酵母的产量和生长速率。结果发现,在pH3.4,温度30℃,通风量1.4vvm,发酵罐搅拌速度500r/min时,酵母对底物的产率接近50%。     

Engineering yeasts for raw starch conversion

Next to cellulose, starch is the most abundant hexose polymer in plants, an import food and feed source and a preferred substrate for the production of many industrial products. Efficient starch hydrolysis requires the activities of both α-1,4 and α-1,6-debranching hydrolases, such as endo-amylases, exo-amylases, debranching enzymes, and transferases. Although amylases are widely distributed in nature, only about 10?% of amylolytic enzymes are able to hydrolyse raw or unmodified starch, with a combination of α-amylases and glucoamylases as minimum requirement for the complete hydrolysis of raw starch. The cost-effective conversion of raw starch for the production of biofuels and other important by-products requires the expression of starch-hydrolysing enzymes in a fermenting yeast strain to achieve liquefaction, hydrolysis, and fermentation (Consolidated Bioprocessing, CBP) by a single organism. The status of engineering amylolytic activities into Saccharomyces cerevisiae as fermentative host is highlighted and progress as well as challenges towards a true CBP organism for raw starch is discussed. Conversion of raw starch by yeast secreting or displaying α-amylases and glucoamylases on their surface has been demonstrated, although not at high starch loading or conversion rates that will be economically viable on industrial scale. Once efficient conversion of raw starch can be demonstrated at commercial level, engineering of yeast to utilize alternative substrates and produce alternative chemicals as part of a sustainable biorefinery can be pursued to ensure the rightful place of starch converting yeasts in the envisaged bio-economy of the future.     

A model for continuous fermentations with amylolytic yeasts

A two-stage, associative fermentation process is more effective for continuous yeast biomass production from starch than a single-stage mixed culture fermentation process. By operating two stages, competition for the same growth limiting substrate is reduced leading to efficient starch utilization. In this article, a mathematical model has been proposed for continuous, two-stage fermentation with a pure culture, amylolytic yeast in the first stage and a mixed culture second stage with a faster growing, nonamylolytic yeast. The model parameters were determined for Saccharomycopsis fibuligera and Candida utilis in continuous, single-stage, pure cultures. In the two-stage model, the effects of changes in dilution rate on biomass, amylase, reducing sugar, and starch concentration, and ratio of stage volumes on microbial composition are discussed and compared with experimental data.