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PubMed2005

Production of high-purity isomalto-oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells.

A method for the production of high-purity isomalto-oligosaccharides (IMO) involving the transglucosylation by transglucosidase and yeast fermentation was proposed. The starch of rice crumbs was enzymatically liquefied and saccharified, and then converted to low-purity IMO syrup by transglucosylation. The low-purity IMO produced either from rice crumbs or tapioca flour as the starch source could be effectively converted to high-purity IMO by yeast fermentation to remove the digestible sugars including glucose, maltose, and maltotriose. Both Saccharomyces carlsbergensis and Saccharomyces cerevisiae were able to ferment glucose in the IMO syrup. Cells of S. carlsbergensis harvested from the medium of malt juice were also able to ferment maltose and maltotriose. A combination of these two yeasts or S. carlsbergensis alone could be used to totally remove the digestible sugars in the IMO, coupled with the production of ethanol. The resultant high-purity IMO, including mainly isomaltose, panose, and isomaltotriose made up more than 98% w/w of the total sugars after a 3-day fermentation. When the low-purity IMO was produced from the starch of tapioca flour, 3-day fermentation under the same conditions resulted in IMO with purity lower than that from rice crumbs. For low-purity IMO from rice crumbs, fermentation with washed S. carlsbergensis cells harvested at log phase was the most effective. However, for the low-purity IMO from tapioca flour, incubation with S. cerevisiae for the first 24 h and then supplementing with an equal amount of S. carlsbergensis cells for further fermentation was the most effective approach for producing high-purity IMO.

Chromatography, High Pressure Liquid↗
PubMed2005

Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation.

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.

Algorithms↗
PubMed2006

Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering.

The worldwide surplus of glycerol generated as inevitable byproduct of biodiesel fuel and oleochemical production is resulting in the shutdown of traditional glycerol-producing/refining plants and new applications are needed for this now abundant carbon source. In this article we report our finding that Escherichia coli can ferment glycerol in a pH-dependent manner. We hypothesize that glycerol fermentation is linked to the availability of CO(2), which under acidic conditions is produced by the oxidation of formate by the enzyme formate hydrogen lyase (FHL). In agreement with this hypothesis, glycerol fermentation was severely impaired by blocking the activity of FHL. We demonstrated that, unlike CO(2), hydrogen (the other product of FHL-mediated formate oxidation) had a negative impact on cell growth and glycerol fermentation. In addition, supplementation of the medium with CO(2) partially restored the ability of an FHL-deficient strain to ferment glycerol. High pH resulted in low CO(2) generation (low activity of FHL) and availability (most CO(2) is converted to bicarbonate), and consequently very inefficient fermentation of glycerol. Most of the fermented glycerol was recovered in the reduced compounds ethanol and succinate (93% of the product mixture), which reflects the highly reduced state of glycerol and confirms the fermentative nature of this process. Since glycerol is a cheap, abundant, and highly reduced carbon source, our findings should enable the development of an E. coli-based platform for the anaerobic production of reduced chemicals from glycerol at yields higher than those obtained from common sugars, such as glucose.

Anaerobiosis↗
PubMed1996

Effect of beer, yeast-fermented glucose, and ethanol on pancreatic enzyme secretion in healthy human subjects.

The effect of beer, ethanol (4% v/v), and corresponding volumetric (water), caloric (glucose 5.76% w/v), and osmotic (glucose 11.5% w/v) control solutions on pancreatic enzyme output and release of gastrin and cholecystokinin (CCK) were studied in six healthy human subjects. As a simpler model of beer, yeast-fermented glucose solution (11.5% w/v) was also studied and compared with unfermented glucose (11.5% w/v). Among the control solutions, the two glucose solutions, but not water, significantly (P < 0.05) increased the 150-min integrated trypsin and amylase output over basal levels. Beer and fermented glucose caused a significantly higher increase in trypsin and amylase output compared to water or glucose. Ethanol (4% v/v) failed to stimulate pancreatic enzyme output. Fermented glucose and beer, but not the control solutions, significantly increased plasma gastrin levels above basal values. Isotonic and hypertonic glucose, beer, and fermented glucose significantly increased plasma levels of cholecystokinin (CCK), but the effect was significantly higher after hypertonic glucose than after isotonic glucose, beer, or fermented glucose. Ethanol and water had no effect on plasma levels of gastrin and CCK. We conclude that: (1) in the doses studied intragastric beer and fermented glucose but not ethanol (4% v/v) stimulate pancreatic enzyme output and release of gastrin and CCK; (2) the lack of effect of ethanol indicates that nonalcoholic ingredients of beer and fermented glucose are responsible for this stimulatory effect; and (3) CCK could be one of the major mediators of the stimulation of pancreatic enzyme output after ingestion of beer and fermented glucose.

Adult↗
PubMed1997

Assessment of a bacteriocin-producing Lactobacillus strain in the control of spoilage of a cereal-based African fermented food.

As a part of a program to develop starter cultures aiding in the spoilage control and sanitation of African fermented foods, a cereal-based food ('ogi' and its solid form 'agidi' or 'eko') was prepared using a bacteriocin-producing Lactobacillus strain as the starter culture. The survival of an enterotoxigenic Escherichia coli strain was investigated in the naturally fermented food and in food fermented with the starter bacteriocin-producing Lactobacillus strain. An inhibition of E. coli was observed within 2 h of incubation in 'ogi' fermented with the bacteriocin producing strain. After 6 h, the viable count of E. coli in locally fermented 'ogi' was log 6.41 (2.54 X 10(6) CFU/mL), whereas in 'ogi' fermented with the bacteriocin producer it was reduced to log 1.70 (0.5 x 10(2) CFU/mL). Comparison of the shelf life of 'agidi' prepared from the naturally fermented food with that bacteriocin-producing starter culture showed that the latter had a better shelf life (kept for 11 d before spoilage occurred as compared with 7 d for the natural one). The results are discussed in terms of the potential of bacteriocin-producing cultures in the control and retardation of spoilage and food-forne infections in some African fermented foods.

Africa↗
PubMed1991

Ethanolic fermentation of pentoses in lignocellulose hydrolysates.

In the fermentation of lignocellulose hydrolysates to ethanol, two major problems are encountered: the fermentation of the pentose sugar xylose, and the presence of microbial inhibitors. Xylose can be directly fermented with yeasts, such as Pachysolen tannophilus, Candida shehatae, and Pichia stipis, or by isomerization of xylose to xylulose with the enzyme glucose (xylose) isomerase (XI; EC 5.3.1.5), and subsequent fermentation with bakers' yeast, Saccharomyces cerevisiae. The direct fermentation requires low, carefully controlled oxygenation, as well as the removal of inhibitors. Also, the xylose-fermenting yeasts have a limited ethanol tolerance. The combined isomerization and fermentation with XI and S. cerevisiae gives yields and productivities comparable to those obtained in hexose fermentations without oxygenation and removal of inhibitors. However, the enzyme is not very stable in a lignocellulose hydrolysate, and S. cerevisiae has a poorly developed pentose phosphate shunt. Different strategies involving strain adaptation, and protein and genetic engineering adopted to overcome these different obstacles, are discussed.

Aerobiosis↗
PubMed2005

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source.

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h(-1)) and 2.54 g l(-1) (D=0.15 h(-1)), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol(-1) were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.

Acetic Acid↗
PubMed2005

Use of oxidoreduction potential as an indicator to regulate 1,3-propanediol fermentation by Klebsiella pneumoniae.

Anaerobic fermentation was relatively difficult to optimize due to lack of monitoring parameters. In this paper, a new method was reported using extracellular oxidoreduction potential (ORP) to monitor 1,3-propanediol (1,3-PD) biosynthesis process by Klebsiella pneumoniae. In batch fermentation, cell growth, 1,3-propanediol production and by-products distribution were studied at four different ORP levels: 10, -140, -190 and -240 mV. From the results, the ORP level of -190 mV was preferable, which resulted in fast cell growth and high 1,3-propanediol concentration. The NAD+/NADH ratio was determined at different ORP levels, and a critical NAD+/NADH ratio of 4 was defined to divide fermentation environments into two categories: relatively oxidative environment (NAD+/NADH>4) and relatively reductive environment (NAD+/NADH<4). The former was correlative with high 1,3-propanediol productivity and high specific growth rate. The mechanism of ORP regulation was discussed. It is suggested that ORP regulation of fermentation might be due to its influence on the ratio of NAD+/NADH, which determined metabolic flux. Furthermore, a batch fermentation of modulating ORP following a profile in different levels corresponding to different fermentation stage was tested. The 1,3-PD concentration was 22.3% higher than that of constant ORP fermentation at -190 mV. Therefore, ORP is a valuable parameter to monitor and control anaerobic fermentation production.

Anaerobiosis↗
PubMed2005

The importance of aeration strategy in fuel alcohol fermentations contaminated with Dekkera/Brettanomyces yeasts.

Whole corn mash fermentations infected with industrially-isolated Brettanomyces yeasts were not affected even when viable Brettanomyces yeasts out-numbered Saccharomyces yeasts tenfold at the onset of fermentation. Therefore, aeration, a parameter that is pivotal to the physiology of Dekkera/Brettanomyces yeasts, was investigated in mixed culture fermentations. Results suggest that aeration strategy plays a significant role in Dekkera/Brettanomyces-mediated inhibition of fuel alcohol fermentations. Although growth of Saccharomyces cerevisiae was not impeded, mixed culture fermentations aerated at rates of > or =20 ml air l(-1) mash min(-1) showed decreased ethanol yields and an accumulation of acetic acid. The importance of aeration was examined further in combination with organic acid(s). Growth of Saccharomyces occurred more rapidly than growth of Brettanomyces yeasts in all conditions. The combination of 0.075% (w/v) acetic acid and contamination with Brettanomyces TK 1404W did not negatively impact the final ethanol yield under fermentative conditions. Aeration, however, did prove to be detrimental to final ethanol yields. With the inclusion of aeration in the control condition (no organic acid stress) and in each fermentation containing organic acid(s), the final ethanol yields were decreased. It was therefore concluded that aeration strategy is the key parameter in regards to the negative effects observed in fuel alcohol fermentations infected with Dekkera/Brettanomyces yeasts.

Acetic Acid↗
PubMed2006

Proteomic analysis of a distilling strain of Saccharomyces cerevisiae during industrial grain fermentation.

The fermentation performance of industrial yeast strains is influenced, among other things, by their genetic composition and the nature of the fermentable sugar, availability of nitrogen, and temperature. Therefore, to manipulate the fermentation process, it is important to understand, at a molecular level, the changes occurring in the yeast cell throughout industrial fermentation processes. With this aim in mind, using two-dimensional gel electrophoresis and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), we have examined the proteome of distillers yeast in an industrial context. Using yeast sampled from a local grain whisky distillery, we have prepared a detailed reference map of the proteome of distillers yeast and have examined in some detail the alterations in protein levels that occur throughout fermentation. In particular, as fermentation progresses, there is a significant increase in the levels of a variety of proteins involved in protecting against stress and nitrogen limitation. These results therefore give an insight into the stresses that yeast are exposed to in industrial fermentations and reveal some of the proteins and enzymes that are either necessary or important for efficient fermentation.

Carbohydrates↗
PubMed1995

Fermentable carbohydrates exert a more potent cholesterol-lowering effect than cholestyramine.

The purpose of this work was to assess the respective role of bile acid excretion and of the end-products of cecal fermentations in the cholesterol-lowering effect of complex carbohydrates. The effects of two different fermentable carbohydrates (guar gum, beta-cyclodextrin), and sequestrant resin (cholestyramine) have been investigated in male Wistar rats. Guar gum and beta-cyclodextrin are broken down in the large bowel, with fermentation rich in propionic acid (37% against 26% for control), whereas cholestyramine did not enhance cecal fermentation. beta-Cyclodextrin and guar gum were less potent than cholestyramine to enhance bile acids and sterol excretion. Nevertheless, fermentable carbohydrates exerted a more potent cholesterol-lowering effect than cholestyramine. beta-Cyclodextrin also depressed triacylglycerol-rich lipoprotein (TGRLP). Fermentable carbohydrates lowered cholesterol of LDL and HDL1 fractions. The induction of hepatic HMG-CoA reductase was practically proportional to rate of fecal steroid excretion. Moreover, with beta-cyclodextrin, hepatic HMG-CoA reductase induction was concomitant to a decrease in fatty acid synthase (FAS) activity. Thus, the cholesterol-lowering effect of fermentable carbohydrates could be related to a depressed lipogenesis, as well as to an accelerated removal of HDL1, in relation to an elevated hepatic demand of cholesterol. In conclusion, fermentable carbohydrates could favour cholesterol elimination and have a general lipid-lowering effect by exerting more complex physiological effects than cholestyramine.

Animals↗
PubMed2005

Improvement of nutritive value of grass pea (Lathyrus sativus) seed meal in the formulated diets for rohu, Labeo rohita (Hamilton) fingerlings after fermentation with a fish gut bacterium.

Eight isonitrogenous (35% crude protein approximately) and isocaloric (4.0 kcalg(-1) approximately) diets were formulated incorporating raw and fermented grass pea (Lathyrus sativus) seed meal at 10%, 20%, 30% and 40% levels by weight into a fish meal based diet and fed to rohu, Labeo rohita, fingerlings for 80 days and fish performance was studied. A particular bacterial strain (Bacillus sp.) isolated from the intestine of adult common carp (Cyprinus carpio) reared in the wild having significant amylolytic, cellulolytic, lipolytic and proteolytic activities were used for fermentation of seed meal for 15 days at 37 degrees C. Fermentation of grass pea seed meal was effective in significantly reducing the crude fibre content and anti-nutritional factors, such as tannins, phytic acid and the neurotoxin, beta-ODAP and enhancing the available free amino acids and fatty acids. In terms of growth response, feed conversion ratio and protein efficiency ratio, 30% fermented grass pea seed meal incorporated diet resulted in significantly (P < 0.05) better performance of rohu fingerlings. In general, growth and feed utilization efficiencies of fish fed diets containing fermented seed meal were superior to those fed diets containing raw seed meal. The apparent protein digestibility (APD) values decreased with increasing levels of raw seed meal in the diets. The APD for raw seed meal was lower at all levels of inclusion in comparison to those for the fermented seed meals. The highest deposition of carcass protein was recorded in fish fed the diet containing 40% fermented seed meal. The results indicated that fermented grass pea seed meal can be incorporated in carp diets up to 30% level compared to 10% level of raw seed meal.

Amino Acids↗
PubMed2006

Diversity of Saccharomyces strains on grapes and winery surfaces: analysis of their contribution to fermentative flora of Malbec wine from Mendoza (Argentina) during two consecutive years.

Spontaneous fermentations are still conducted by several wineries in different regions of Argentina as a common practice. Native Saccharomyces strains associated with winery equipment, grape and spontaneous fermentations of Malbec musts from "Zona Alta del Río Mendoza" region (Argentina) were investigated during 2001 and 2002 in the same cellar. Low occurrence of Saccharomyces on grapes and their limited participation during fermentation were confirmed. Strain sequential substitution during fermentation was observed. Between 30% and 60% of yeast population at the end of fermentation was coming from yeasts already present in the winery. A stable and resident Saccharomyces micro-flora in the winery was confirmed. It exhibited a dynamic behaviour during season and between years. Commercial strains were found during fermentation in different percentages, but their presence on winery equipment was low. The present work represents a first approach to winery yeast and spontaneous fermentation Saccharomyces population dynamics in an important viticultural region from Argentina that has never been characterized before. The results obtained have an important significance for the local industry, showing for the first time the real situation of the microbial ecology of alcoholic fermentation in an industrial winery from Mendoza, Argentina.

Argentina↗
PubMed2006

Microbiological and chemical changes during the fermentation of African locust bean (Parkia biglobosa) to produce afitin, iru and sonru, three traditional condiments produced in Benin.

African locust bean (Parkia biglobosa) was processed to produce afitin, iru and sonru, three different types of condiment from Benin. Whereas the fermentation of African locust bean to produce afitin is carried out for 24 h without using any additive, the fermentation for iru and sonru production takes place for 48 h with adding respectively "iku-iru" and "yanyanku" two traditional malvacene bean-based (Hibiscus sabdariffa) additives. The main microorganisms involved in the fermentation of the three condiments were Bacillus spp., although Staphylococcus spp. was found in lower number. The use of additive seems to enhance the initial Bacillus counts in iru and sonru, but also a slightly higher Staphylococcus count was observed. The number of staphylococci did not exceed 10(2) cfu/g and it was below the detection level at the end of the fermentation (24 h) in afitin; it reached a level of about 10(6) cfu/g at 12-18 h in iru and sonru followed by a decrease to about 5 x 10(4) cfu/g after 48 h of fermentation. The final pH and total basic volatile nitrogen content of iru and sonru are higher than those of afitin. Proximate composition changes are similar in both fermenting iru and sonru, but different in afitin, the latter showing a slight decrease of proteins of about 5% whereas for iru and sonru they increased to about 6%. Ash, crude fat and fibre contents also increased during the fermentation in the three products whereas total carbohydrates decreased significantly. Due to their long fermentation time, iru and sonru are very soft products as they are preferred by some consumers whereas afitin is preferred with cotyledons having less soft texture.

Bacillus↗
PubMed2006

Antimicrobial properties of lactic acid bacteria and yeast-LAB cultures isolated from traditional fermented milk against pathogenic Escherichia coli and Salmonella enteritidis strains.

The survival and growth of Escherichia coli 3339 and Salmonella enteritidis 949575 isolated from human clinical samples, in milk fermented with lactic acid bacteria (LAB) and yeast strains previously isolated from Zimbabwean naturally fermented milk (NFM) was studied. The LAB starter cultures used were Lactococcus lactis subsp. lactis biovar. diacetylactis C1 alone (C1) or in combination with Candida kefyr 23 (C1/23), L. lactis subsp. lactis Lc261 alone (LC261) or in combination with C. kefyr 23 (Lc261/23). The growth of the same pathogens in milk fermented with a commercial DL culture (CH-N 22) and spontaneously fermented raw milk was also monitored. The C1 and C1/23 cultures significantly (P<0.05) inhibited the growth of both pathogens. When inoculated at the beginning of the fermentation, both E. coli 3339 and S. enteritidis 949575 counts were significantly (P<0.05) reduced by about two log cycles in C1 and C1/23 cultured milk. However, in naturally fermented milk and the DL cultured milk, both E. coli 3339 and S. enteritidis 949575 grew and reached high populations of about 9 and 8.8 log cfu ml(-1), respectively, after 18 h. When E. coli 3339 was inoculated into previously fermented milk, the viable counts were significantly (P<0.05) reduced in the presence of C1 and C1/23 from 7 log cfu ml(-1) to 3 log cfu ml(-1) after 48 h. S. enteritidis 949575 could not be recovered from these cultures after 48 h. The addition of the yeast did not enhance or diminish the inhibitory capacity of the LAB cultures. The pathogens survived in high numbers when inoculated into pre-fermented NFM and the commercial DL- (CH-N 22) cultured milk. The C1 strain, therefore, offered the best protection against the pathogens. Its inhibitory effect was mainly related to fast acid production.

Animals↗
PubMed2000

In vitro digestibility of bacillus fermented soya bean.

Bacillus fermented legume products include among others dawadawa and soumbala made from African locust bean, and natto and kinema made from soya bean. Bacillus subtilis is the dominant species involved in the fermentation. During Bacillus fermentation for 48 h of autoclaved soya bean the quantity of soluble and dialyzable matter increased from 22% and 6% up to 65% and 40%, respectively. Protein and carbohydrate degradation during fermentation of soya bean with several Bacillus spp. was investigated and appeared to be substantial during the first 18 h of fermentation resulting in the release of high levels of peptides and oligosaccharides. In vitro digestibility was increased from 29% up to 33-43% after Bacillus fermentation for 48 h. It was shown that Bacillus spp. were able to degrade soya bean macromolecules to a large extent resulting in water-soluble low molecular weight compounds. In vitro digestion of Bacillus fermented soya bean using gastrointestinal enzymes only slightly increased the amount of dialyzable matter, which clearly demonstrated the beneficial effect of Bacillus fermentation on food nutrient availability.

Absorption↗
PubMed2001

Selection and characterization of mixed starter cultures for lactic acid fermentation of carrot, cabbage, beet and onion vegetable mixtures.

An evaluation of various lactic acid bacteria (LAB) for the fermentation of cabbage, carrot and beet-based vegetable products was carried out. As part of a screening process, the growth of 15 cultures in a vegetable juice medium (VJM) was characterized by automated spectrophotometry. Acidification patterns as well as viability during storage of the LAB were also established. There were greater differences between the pure cultures than the mixed ones with respect to growth in VJM and viability during storage. Reductions in viable cell counts during storage of the fermented VJM occurred more rapidly with a Leuconostoc strain than for pediococci or lactobacilli. Inoculation of vegetables was carried out with cultures of Lactobacillus plantarum NK-312, Pediococcus acidilactici AFERM 772 and Leuconostoc mesenteroides BLAC which were rehydrated in a brine. This rehydration procedure was not detrimental to viability. During fermentation of a carrot/cabbage vegetable mix, sugar metabolism was characterized by the assimilation of both glucose and fructose, but sugars remained in the fermented vegetables when acidification stopped. The pH in the LAB-inoculated vegetables after 72 h at 20 degrees C was significantly lower (by 0.2 units) than the uninoculated control. Inoculation with LAB designed for silage fermentation resulted in the inhibition of acetic acid production, and reduced the production of ethanol during fermentation. The selection process on VJM enabled the preparation of a mixed culture that was more rapid than the silage inoculants in acidifying the medium and was more effective in reducing the production of gas during the fermentation and storage of the fermented vegetables.

Colony Count, Microbial↗
PubMed2001

Fermentative capacity after cold storage of baker's yeast is dependent on the initial physiological state but not correlated to the levels of glycolytic enzymes.

Growth and starvation of baker's yeast was monitored by on-line microcalorimetry and cells originating from four different physiological states were stored at low temperature (4 degrees C) for up to 26 days. The different physiological states were designated F (respiro-Fermentative phase of growth), R (initial Respiratory phase of growth), -N (non-growing state because of Nitrogen depletion), and -NC (non-growing state because of both Nitrogen and Carbon depletion). The cells were tested before and after cold storage for their fermentative capacity, and characterised by 2D gel analysis (and subsequent quantitative silver staining and image analysis with software PDQUEST) for their levels of six enzymes of the glycolytic pathway (hexokinase 2 (Hxk2p), fructose bisphosphate aldolase (Fba1p), glyceraldehyde-3-phosphate dehydrogenase (Tdh3p), enolase A (Enolp), enolase B (Eno2p), and triose phosphate isomerase (Tpi1p)) and two enzymes of the fermentative branch (pyruvate decarboxylase (Pdc1p) and alcohol dehydrogenase (Adh1p)). The enzymes Hxk2p, Tdh3p, Eno2p, Pdc1p and Adh1p were down-regulated by 25-80% during the transition between the F and R states. During the transition to non-growing states (-N and -NC states), the levels of Hxk2p, Tdh3p and Eno2p were further reduced. However, after cold storage, the glycolytic and fermentative enzymes of the different physiological states were expressed to the same extent. In contrast, the fermentative capacity differed between the states; the R-state cells were superior compared to cells from the other states tested and preserved more than 50% of their initial fermentative capacity (6 mmol ethanol per gram dry weight and hour). Our data therefore clearly demonstrate that persistence of fermentative capacity during total starvation at low temperature after as long as 1 month is strongly dependent on the physiological state from which the cells originate. However, the level of expression of the glycolytic enzymes could not explain the difference in fermentative capacity of the different physiological states after cold storage.

Calorimetry↗