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Physicochemical properties of extracellular (1-->4)-beta-D-glucuronan produced by the Rhizobium meliloti M5N1CS strain during fermentation: evidence of degradation by an exoenzyme activated by Mg2+.

The mutant Rhizobium meliloti M5N1CS (NCIMB 40472) produced a partially acetylated (1-->4)-beta-D-glucuronan during fermentation. The polysaccharide (EPS) extracted from broth during fermentation by microfiltration and ultrafiltration (using a 100 kDa cut-off membrane) was characterized by size exclusion chromatography. The weight-average molecular weight (Mw) of the EPS decreased from 7.5 x 10(5) to 5 x 10(5) between 27 and 75 h of fermentation. When MgSO4.7H2O (0.8 gl-1 per day) was present in the medium during the same period, the Mw of the EPS decreased to 1.5 x 10(5). Since EPS degradation was detected after microfiltration of the fermentation medium supplemented with magnesium, the Mw decrease of the EPS extracted during fermentation may be explained by enzymic degradation of the polymer activated by Mg2+ ions.

Bacterial Proteins↗

Changes in chemical composition and sensory qualities of peanut milk fermented with lactic acid bacteria.

The effects of fermentation of aqueous extracts of peanuts (peanut milk) with Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus, separately and in combination, on selected chemical and sensory qualities were investigated. Changes in pH, titratable acidity and viable cell populations indicated that there was a synergistic interaction between L. delbrueckii ssp. bulgaricus and S. salivarius ssp. thermophilus during fermentation. Analysis of headspace volatiles revealed that hexanal, which is one of the compounds responsible for undesirable green/beany flavor in peanut milk, completely disappeared as a result of fermentation. S. salivarius ssp. thermophilus was more effective than L. delbrueckii ssp. bulgaricus in reducing the hexanal content. The acetaldehyde content of peanut milk increased during fermentation. Changes in concentrations of these volatile compounds were correlated with sensory evaluation scores which showed that a significant (P less than or equal to 0.05) decrease in green/beany flavor and a significant increase in creamy flavor occurred as a result of fermentation.

Acetaldehyde↗

Sapal: a traditional fermented taro [Colocasia esculenta (L.) Schott] corm and coconut cream mixture from Papua New Guinea.

Sapal is a traditional fermented food made by mixing cooked, grated taro [Colocasia esculenta (L.) Schott] corm with coconut cream and allowing it to ferment at ambient temperature. The fermentation was primarily due to heterofermentative lactic acid bacteria, which reached 10(10) cfu/ml. Seven out of 10 isolated bacterial strains were identified as Leuconostoc mesenteroides or Leuc. paramesenteroides. The initial microbial flora was derived from the coconut cream. Yeasts grew on the surface of the sapal in the later stages of the fermentation. Overnight storage of the grated taro corm resulted in the glucose concentration increasing from 1.1 to about 5 g/l. During the fermentation the glucose concentration decreased to undetectable levels. The pH value fell from an initial value of 6.1 to 4.1 after 24 h.

Cocos↗

The fermentation pathways of Escherichia coli.

Under anaerobic conditions and in the absence of alternative electron acceptors Escherichia coli converts sugars to a mixture of products by fermentation. The major soluble products are acetate, ethanol, acetate and formate with smaller amounts of succinate. In addition the gaseous products hydrogen and carbon dioxide are produced in substantial amounts. The pathway generating fermentation products is branched and the flow down each branch is varied in response both to the pH of the culture medium and the nature of the fermentation substrate. In particular, the ratio of the various fermentation products is manipulated in order to balance the number of reducing equivalents generated during glycolytic breakdown of the substrate. The enzymes and corresponding genes involved in these fermentation pathways are described. The regulatory responses of these genes and enzymes are known but the details of the underlying regulatory mechanisms are still obscure.

Acetates↗

Use of Saccharomyces cerevisiae cells immobilized on orange peel as biocatalyst for alcoholic fermentation.

A biocatalyst was prepared by immobilizing a commercial Saccharomyces cerevisiae strain (baker's yeast) on orange peel pieces for use in alcoholic fermentation and for fermented food applications. Cell immobilization was shown by electron microscopy and by the efficiency of the immobilized biocatalyst for alcoholic fermentation of various carbohydrate substrates (glucose, molasses, raisin extracts) and at various temperatures (30-15 degrees C). Fermentation times in all cases were low (5-15 h) and ethanol productivities were high (av. 150.6 g/ld) showing good operational stability of the biocatalyst and suitability for commercial applications. Reasonable amounts of volatile by-products were produced at all the temperatures studied, revealing potential application of the proposed biocatalyst in fermented food applications, to improve productivities and quality.

Alcohols↗

Fermentability of eastern gamagrass, big bluestem and sand bluestem grown across a wide variety of environments.

Plant biomass has attracted interest as a feedstock for biofuels production, but much of this work has been focused on relatively few plant species. In this study, three relatively-unstudied species of warm-season perennial grasses, grown at multiple locations in the eastern and central US and harvested over a three year period, were examined for fermentability via in vitro ruminal gas production and dry matter digestibility assays, and near-infrared reflectance calibrations were developed for these fermentation parameters. Big bluestem (Andropogon gerardii Vitman) displayed greater fermentability than did sand bluestem (Andropogon hallii Hack) or eastern gamagrass [Tripsacum dactyloides (L.) L.], but displayed lower biomass yields. The bluestems also displayed lower N contents and less variation in fermentability over different growth environments (geographic locations and harvest years), suggesting a more consistent biomass quality than for eastern gamagrass. Thus, in addition to their use as forage for ruminant animals, bluestems may be of particular interest as feedstocks for bioconversion to ethanol and other products via direct microbial fermentation (consolidated bioprocessing) schemes, and thus merit additional efforts to enhance biomass yield potential.

Agriculture↗

Regulation of rumen fermentation during seasonal fluctuations in food intake of muskoxen.

We studied rumen fermentation of castrated adult muskoxen (Ovibos moschatus; n=4) during periods of low (May) and high (August) food intake. Turnover time (17+/-1.8 h) and volume (26+/-3.9 L) of rumen fluid were consistent between May and August and among days within each season. Rumen temperature did not vary significantly during the day (38.8+/-0.29 degrees C) in either season. Rumen osmolality (271.9+/-16.4 vs. 245.9+/-11.4 mOsm kg(-1)) and pH (6.81+/-0.31 vs. 6.39+/-0.15) were higher in May than in August indicating a shift in the allostatic set point. Rumen fluid pH was more variable in May than in August both before and after a single meal of fermentable substrate even though fermentation acids were lower in May than in August (101.0+/-11.0 vs. 126.0+/-8.74 mM). Changing proportions of minor fermentation acids indicated a shift in metabolic pathways even though bacterial numbers were similar between seasons (6.4+/-5.8x10(9) mL(-1)). Allostatic set points probably alter the homeostatic range of conditions and the microbial diversity of fermentations in herbivores from highly seasonal environments.

Animals↗

A distinct type of alcohol dehydrogenase, adh4+, complements ethanol fermentation in an adh1-deficient strain of Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, only one alcohol dehydrogenase gene, adh1(+), has been identified. To elucidate the influence of adh1(+) on ethanol fermentation, we constructed the adh1 null strain (delta adh1). The delta adh1 cells still produced ethanol and grew fermentatively as the wild-type cells. Both DNA microarray and RT-PCR analysis demonstrated that this ethanol production is caused by the enhanced expression of a Saccharomyces cerevisiae ADH4-like gene product (SPAC5H10.06C named adh4(+)). Since the strain lacking both adh1 and adh4 genes (delta adh1 delta adh4) showed non-fermentative retarded growth, only these two ADHs produce ethanol for fermentative growth. This is the first observation that a S. cerevisiae ADH4-like alcohol dehydrogenase functions in yeast ethanol fermentation.

Alcohol Dehydrogenase↗

Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation: both low- and high-affinity Hxt transporters are expressed.

The transport of glucose and fructose into yeast cells is a critical step in the utilization of sugars during wine fermentation. Hexose uptake can be carried out by various Hxt carriers, each possessing distinct regulatory and transport-kinetic properties capable of influencing yeast fermentation capacity. We investigated the expression pattern of the hexose transporters Hxt1 to 7 at the promoter and protein levels in Saccharomyces cerevisiae during wine fermentation. The Hxt1p carrier was expressed only at the beginning of fermentation, and had no role during stationary phase. The Hxt3p carrier was the only one to be expressed throughout fermentation, displaying maximal expression at growth arrest and slowly decreasing in abundance over the course of the stationary phase. The high-affinity carriers Hxt6p and Hxt7p displayed similar expression profiles, with expression induced at entry into stationary phase and persisting throughout the phase. The expression of these two carriers occurred despite the presence of high amounts of hexoses, and the proteins were stably expressed when the cells were starved for nitrogen. The Hxt2p transporter was only transiently expressed during lag phase, which suggests a role for the protein in growth initiation. Characterization of glucose transport kinetics indicated the presence of a shift in the low-affinity component that is consistent with a predominant expression of Hxt1p during growth phase and of Hxt3p during stationary phase. In addition, a high-affinity uptake component consistent with functional expression of Hxt6p/Hxt7p was identified during stationary phase.

Culture Media↗

Evolution of the population of Saccharomyces cerevisiae from grape to wine in a spontaneous fermentation.

To determine the grape or winery origin of the Saccharomyces cerevisiae involved in spontaneous fermentation, musts were collected at different stages of wine-making process and fermented. First, grapes were collected in two different vineyards and crushed at the laboratory. Second, musts were collected after crushing and clarification in the cellar. Third, musts collected in the cellar were sterilized and inoculated with tartar deposit collected in the vats. The fourth fermentation was in the cellar. For the two vineyards, two hundred of S. cerevisiae clones were isolated for each of the four fermentations, driving to a library of 1600 clones. All the library was analysed by inter-delta PCR with a basic set of primers and about 20% of the library was further analysed by inter-delta PCR with an improved set of primers. Six, and more than 30 different PCR patterns were obtained from basic- and improved-PCR analysis, respectively. The amounts of each family were analysed at the different stages of wine making. Our study demonstrates that the two vineyards present different S. cerevisiae populations. Moreover the S. cerevisiae strains involved in spontaneous fermentation in the cellar originate partly from the vineyard and partly from the winery, in amounts varying with the must.

Biodiversity↗

Survival of Escherichia coli O157:H7 in dry fermented sausages containing micro-encapsulated probiotic lactic acid bacteria.

Escherichia coli O157:H7 is capable of surviving the rigorous processing steps during the manufacture of dry fermented sausages. The effect of adding two probiotic organisms, Lactobacillus reuteri and Bifidobacterium longum as co-cultures with the meat starter cultures Pediococcus pentosaceus and Staphylococcus carnosus on the viability of E. coli O157:H7 in dry fermented sausages was studied. A 5 strain cocktail of E. coli O157:H7 was added at 7.4 log cfu/g to the sausage batter and challenged with either or both Lb. reuteri or B. longum before or after they were micro-encapsulated. Sausages were fermented at < or = 26 degrees C and 88% relative humidity (RH) followed by drying at 75% RH and 13 degrees C for 25 d. The pH, water activity (aw), protein, moisture, and numbers of all inoculated organisms were monitored during processing. The pH and aw decreased from 5.7 and 0.98 to 4.9 and 0.88 at the end of fermentation and drying, respectively. These processes reduced E. coli O157:H7 by 1.0 and 0.7 log cfu/g at the end of fermentation and drying, respectively. Unencapsulated Lb. reuteri with or without B. longum reduced E. coli O157:H7 by 3.0 log cfu/g and B. longum caused a 1.9 log cfu/g reduction. While micro-encapsulation increased survival of Lb. reuteri and B. longum, it reduced their inhibitory action against E. coli O157:H7.

Animals↗

Yeast species associated with the spontaneous fermentation of cider.

This paper reports the influence of cider-making technology (pneumatic and traditional pressing) on the dynamics of wild yeast populations. Yeast colonies isolated from apple juice before and throughout fermentation at a cider cellar of Asturias (Spain), during two consecutive years were studied. The yeast strains were identified by restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the two flanking internal transcribed sequences (ITS). The musts obtained by pneumatic pressing were dominated by non-Saccharomyces yeasts (Hanseniaspora genus and Metschnikowia pulcherrima) whereas in the apple juices obtained by traditional pressing Saccharomyces together with non-Saccharomyces, were always present. The species Saccharomyces present were S. cerevisiae and S. bayanus. Apparently S. bayanus, was the predominant species at the beginning and the middle fermentation steps of the fermentation process, reaching a percentage of isolation between 33% and 41%, whereas S. cerevisiae took over the process in the final stages of fermentation. During the 2001 harvest, with independence of cider-making technology, the species Hanseniaspora valbyensis was always isolated at the end of fermentations.

Base Sequence↗

Rhizopus oligosporus and yeast co-cultivation during barley tempeh fermentation--nutritional impact and real-time PCR quantification of fungal growth dynamics.

Barley tempeh was produced by fermenting barley kernels with Rhizopus oligosporus. The potential of the yeasts Saccharomyces cerevisiae (three strains), S. boulardii (one strain), Pichia anomala (one strain) and Kluyveromyces lactis (one strain) to grow together with R. oligosporus during barley tempeh fermentation was evaluated. All yeast strains grew during the fermentation and even during cold storage of tempeh (P<0.01). The growth of yeasts slightly increased the ergosterol contents, but did not influence amino acid contents and compositions, and did not reduce phytate contents. Slight increases of vitamins B(6) and niacinamide, and slight decreases of B(1) and biotin were observed. Quantification of fungal growth is difficult during mixed species fermentations because ergosterol is found in all fungal species, and colony-forming-unit (cfu) estimations are not reliable for R. oligosporus and other sporulating fungi. Therefore, we developed a quantitative real-time PCR method for individually quantifying S. cerevisiae and R. oligosporus growth in barley tempeh. The PCR results were highly correlated with the ergosterol content of R. oligosporus and with the number of cfu of S. cerevisiae. Thus, real-time PCR is a rapid and selective method to quantify yeasts and R. oligosporus during mixed species fermentation of inhomogenous substrate such as barley tempeh.

Coculture Techniques↗

Exploring the mechanism of aroma production in fermented cherry juice by L. brevis LD1.0600 using flavomics and whole genome analysis.

This study focused on L.brevis LD1.0600 with excellent fermentation traits: it analyzed genome-wide key regulatory genes for micro-metabolites, combined with fermented cherry juice flavor metabolomics data, and used machine learning to explore correlations between gene regulation, metabolite production, and flavor formation. The SVM model screened and verified fermented cherry juice VOCs; through OAV and flavor wheel analysis, LD1.0600 emerged as the top-performing strain, with a sweet, fruity dominant aroma. Key aroma-active components (OAV&#xa0;>&#xa0;100) included 2-methoxy-4-vinylphenol, benzaldehyde, 2-methyl-butanoic acid and hexanoic acid, and 2-methoxy-4-vinylphenol and hexanoic acid elevated by LD1.0600-regulated genes (Chrom1-001884, Chrom1-000925, fabF and Chrom1-000199). At the same time, through research, a "strain screening-SVM screening of DVCs-OAV screening of key aroma components-whole genome sequencing of flavor regulatory genes" system was established. This system can not only be applied to the screen fermentation strains, but also can be extended to the application of other fermentation products.

Fermentation↗

Use of RAPD-PCR as a method to follow the progress of starter cultures in sauerkraut fermentation.

DNA fingerprinting methods were used to follow the progress of unmarked starter cultures in laboratory sauerkraut fermentations (1.2 and 13 l). Random prime PCR (RAPD-PCR) was used for strain-specific identification of Leuconostoc mesenteroides cultures. A comparative analysis of RAPD banding patterns for fermentation isolates and starter cultures was carried out using both genetically marked and unmarked cultures. While some variation in the RAPD patterns was observed, the results showed that the starter cultures dominated the fermentation during early heterofermentative stage for up to 5 days after the start of fermentation. Results from marked and unmarked starter cultures were confirmed by intergenic transcribed spacer (ITS)-PCR, and strain identify was confirmed by pulse field gel electrophoresis (PFGE) patterns. The results demonstrate the utility of RAPD to follow the progression of unmarked starter cultures of L. mesenteroides in sauerkraut fermentations.

Brassica↗

Yeast ecology of Kombucha fermentation.

Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species.

Acetobacter↗

Partial 26S rDNA restriction analysis as a tool to characterise non-Saccharomyces yeasts present during red wine fermentations.

Restriction patterns of amplified regions of ribosomal large subunit RNA encoding genes (26S rDNA) were evaluated as a routine methodology to examine yeast species diversity during red wine fermentation. The results were confirmed by sequencing of D1/D2 region of 26S rDNA. Red wine production was carried out using a yeast starter culture together with different commercial products, namely enzymes, fermentation activators and tannins and their influence on the non-Saccharomyces yeast population was studied. Yeast strains were isolated using lysine agar as a selective medium for non-Saccharomyces yeasts, after morphological characterisation of colonies. Amplification of 26S rDNA followed by digestion with three restriction enzymes applied to the 121 isolates, generated 19 profiles and a very high correlation with sequencing results was achieved. Although a starter yeast culture was added, results showed that several yeast species were present during all stages of fermentation, independent of the conditions tested, emphasizing the diversity of microorganisms associated with winemaking. On the other hand commercial additives did not significantly influence the diversity of yeast population during the fermentation process. For non-Saccharomyces strains, restriction patterns of a PCR amplified 26S rDNA region proved to be an adequate tool for clustering strains at species level and enabled the monitoring of yeast population dynamics during red wine fermentation.

Base Sequence↗

Screening of 42 Bacillus isolates for ability to ferment soybeans into dawadawa.

Forty-two cultures of Bacillus species isolated from soybean dawadawa were screened for their proteolytic activity on Skim Milk Agar, amylolytic activity on Starch Agar, and ability to grow on Soybean Agar. Distinct differences were observed between the cultures for all the criteria. Eleven isolates were selected for laboratory fermentation trials and each produced soybean dawadawa which was found acceptable by a taste panel. The pH of the samples, which increased from 6.37-6.58 to 8.22-8.85 during fermentation, were significantly different at P< or =0.05 for the different cultures. In the fermented samples, Bacillus counts exceeded 10(9) cfu/g, with the population of only one sample being significantly different at P< or =0.05. A market focus group familiar with soybean dawadawa selected Bacillus subtilis 24BP(2) and B. subtilis FpdP(2) as the best potential starter cultures. A taste panel found no significant differences in overall acceptance between soybean dawadawa either fermented spontaneously or with B. subtilis 24BP(2) and also between soybean dawadawa fermented with either B. subtilis 24BP(2) or B. subtilis FpdP(2).

Bacillus↗