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Rat cecal inocula produce different patterns of short-chain fatty acids than fecal inocula in in vitro fermentations.

The effects of bacterial collection site, bacterial adaption to substrate and duration of fermentation on short-chain fatty acid (SCFA) production were studied using an in vitro fermentation system. Cecal and fecal inocula from rats fed purified diets containing 100 g/kg dietary fiber from canned peas or psyllium seed husk, or a nonpurified diet containing 170 g/kg dietary fiber fermented ileal excreta from colectomized rats fed a purified diet containing 100 g/kg dietary fiber from canned peas. The SCFA concentration, measured by gas chromatography, in anaerobic fermentations of 0, 3, 6, 12, 24, 48, 72 and 96 h, increased (P < 0.05) through 72 h. Compared with fecal inocula, cecal inocula produced SCFA at greater (P < 0.05) initial rates, more (P < 0.05) total SCFA and a greater (P < 0.05) proportion as n-butyrate. Inocula from rats fed the pea or nonpurified diets produced SCFA at greater (P < 0.05) molar proportions as acetate and less (P < 0.05) as propionate than inocula from rats fed psyllium seed husk at most time points. We conclude that collection site, adaptation of bacteria to the substrate to be fermented and duration of fermentation significantly influence the results of in vitro fermentation studies.

Animals↗

Different substrates and methane producing status affect short-chain fatty acid profiles produced by In vitro fermentation of human feces.

Five different substrates, i.e., lactulose, rhamnose, cornstarch, guar and ileostomy effluent, were used to determine whether methane producing status alters the production of short-chain fatty acids (SCFA) in methane producers (MP; n = 6) and nonproducers (MNP; n = 5). Fecal samples from MP and MNP were fermented with the five substrates using an in vitro fermentation method. Subjects with a mean breath methane concentration > 0.045 micromol/L above ambient air were classified as MP. Fermentation was stopped and samples were obtained at 3, 5 and 24 h. An HPLC method was used to measure the SCFA, acetate, propionate, isobutyrate, butyrate, valerate and isocaproate. A significant interaction between methane producing status and time for acetate production from lactulose was observed. There were no differences in fermentation of the four remaining substrates between MP and MNP, but there were significant differences among substrates in the two groups combined. Acetate production from lactulose was significantly greater than from the four other substrates, whereas that from ileostomy effluent was significantly less than the four other substrates. The amount of propionate produced from rhamnose was significantly higher than from the other substrates. The amount of butyrate produced from lactulose and cornstarch was significantly higher than from the other substrates. We conclude that differences exist in the fermentation patterns of lactulose, rhamnose, cornstarch, guar and ileostomy effluent. Methane producing status may influence fermentation patterns only of substrates that are largely fermented to acetate and not others.

Adult↗

Fermentation of soybean meal with Aspergillus usamii reduces phosphorus excretion in chicks.

Fermentation with Aspergillus usamii almost completely degrades phytate phosphorus in soybean meal. Phosphorus excretion was investigated in chicks fed a fermented soybean meal-based diet. Thirty chicks were fed one of three experimental diets; a control soybean meal-based diet (total phosphorus, 5.2 g/kg; nonphytate phosphorus, 2.3 g/kg), a control soybean meal-based diet with added inorganic phosphorus (total phosphorus, 7.1 g/kg; nonphytate phosphorus, 4.0 g/kg), or a fermented soybean meal-based diet without supplemental inorganic phosphorus (total phosphorus, 5.8 g/kg; nonphytate phosphorus, 3.9 g/kg) for 4 wk. Body weight gain, the amount of retained phosphorus, and femoral phosphorus content were lower in the control group than in the phosphorus-supplied group and the fermented soybean meal group. The latter two groups showed similar body weight gain and femoral phosphorus content. On the other hand, phosphorus excretion was markedly more in the phosphorus-supplied group than in the other groups. As a result, phosphorus retention (percentage of intake) was lower in the phosphorus-supplied group than in the fermented soybean meal group. In conclusion, fermentation improved phosphorus bioavailability in soybean meal and supplemental inorganic phosphorus was not necessary for the fermented soybean meal-based diet, which remarkably reduced phosphorus excretion.

Animals↗

Recycling dead hens by enzyme or sodium hydroxide pretreatment and fermentation.

This study was conducted to evaluate the recycling of whole dead hens into feed ingredients by enzyme or sodium hydroxide pretreatment and fermentation. Evaluation criteria included nutrient preservation, pathogenic microorganism elimination, and assays of nutritional quality. The pH levels of enzyme- and NaOH-treated hen carcasses decreased from 6.01 and 7.66 to 4.18 and 4.24, respectively, during the 21-d fermentation. Hydrogen sulfide levels were not detected on Days 1 and 3 from the enzyme treatment; however, high levels (800 ppm) were measured from the NaOH treatments. By Day 21, H2S levels of both treatments had decreased to 78 ppm. The control, enzyme, and NaOH treatments before fermentation contained high levels of Escherichia coli and Staphylococcus aureus; however, after fermentation, these potential pathogens were eliminated in the enzyme and NaOH treatments. Levels of CP, EE, and ash of the control product were higher than either the enzyme or NaOH treatment. NaOH reduced pepsin digestibility by 11% compared to the enzyme treatment. In a bioassay, the chicks fed control autoclaved hen meal (CHM) had higher (P < 0.05) feed intake, weight gain, protein efficiency ratio (PER), and net protein ratio (NPR) than enzyme-treated, fermented, and autoclaved hen meal (EHM) or NaOH-treated, fermented, and autoclaved hen meal (NHM). However, the AMEn of the CHM and EHM were higher than the NHM (P < 0.05) when evaluated using mature cockerels. These results indicated that fermentation processing of dead hens reduced the concentration of some nutrients and depressed growth performance when hen meals were fed to young chicks.

Amino Acids↗

In situ proton-NMR analyses of Escherichia coli HB101 fermentations in 1H2O and in D2O.

Experiments using one-dimensional Fourier-transform proton-NMR spectrometry for non-invasive analyses of microbial fermentations in situ, in vivo and in normal aqueous buffer are described. Analyses of the 'mixed acid' fermentation during growth of Escherichia coli on glucose and citrate were performed to identify and quantitatively estimate the concentrations of the two substrates provided and of the six products formed without sampling from the NMR tube. Identification of fermentation substrates and products was achieved by coincidence of selected diagnostic proton signals of individual compounds in the same solvent. The complete time course of growth of E. coli in the NMR tube correlated well with that of the same culture grown outside the magnet, with samples taken for proton-NMR analyses. The entire course of these in situ proton measurements during growth over 16-24 h was obtained automatically, usually unattended overnight. Thus, the utilization and formation of eight substances in the fermentation were monitored simultaneously, in normal 1H2O, without sampling and individual analysis. Several metabolic changes could be readily detected during the fermentations. Additionally, the pH changes were estimated from the chemical shifts of the acetate signal as growth progressed. The effect of varying D2O concentrations in the solvent on growth rates and product yields was examined, and the increase in the complexity of signals given by these fermentations is described. This versatile and rapid method for the simultaneous, direct and automatic analysis of mixtures of many compounds has the potential to be extended to routine on-line analyses of industrial fermentations.

Citric Acid↗

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products.

The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20 degrees C for 24 h and then stored at either 20 degrees C or 5 degrees C for 96 h. Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20 degrees C and 5 degrees C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10(7)-10(9)/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10(3)-10(6) and 10(2)-10(5) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Higher maximum numbers, 10(9)-10(10), of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10(5)-10(8) and 10(4)-10(7) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.

Animals↗

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry.

AIMS: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. METHODS AND RESULTS: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l(-1)) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. CONCLUSIONS: The yeast strain required a minimum of 267 mg N l(-1) to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality.

Ammonium Sulfate↗

Influence of grape treatment on the wine yeast populations isolated from spontaneous fermentations.

AIM: To study the influence of different methods of grape treatment in wineries on the diversity of the yeast species in spontaneous fermentations. METHODS AND RESULTS: Grapes were crushed and pressed in three different ways followed by spontaneous fermentation. The same grape material picked and crushed aseptically directly in the vineyard served as control. Yeasts isolated at different stages of the fermentation were characterized by 5.8S-ITS-RFLP. Yeasts of the Saccharomyces sensu stricto complex were additionally analysed by microsatellite polymerase chain reaction fingerprinting. The diversity of yeast species isolated from winery fermentations was much greater than from the vineyard fermentation in respect to yeasts of the genus Saccharomyces as well as non-Saccharomyces. CONCLUSIONS: Oenonogical methods alter significantly the yeast diversity in spontaneous fermentations of grape juice. SIGNIFICANCE AND IMPACT OF THE STUDY: Managing spontaneous fermentations successfully depends not only on choosing the suitable grapes but also on the crushing and pressing techniques leading to different yeast populations.

Fermentation↗

Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation.

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.

Culture Media↗

Use of flow cytometry to follow the physiological states of microorganisms in cider fermentation processes.

The flow cytometry (FC) technique used with certain fluorescent dyes (ChemChrome V6 [CV6], DRAQ5, and PI) has proven useful to label and to detect different physiological states of yeast and malolactic bacterium starters conducting cider fermentation over time (by performing sequential inoculation of microorganisms). First, the technique was tested with pure cultures of both types of microorganisms grown in synthetic media under different induced stress conditions. Metabolically active cells detected by FC and by the standard plate-counting method for both types of microorganisms in fresh overnight pure cultures gave good correlations between the two techniques in samples taken at this stage. Otherwise, combining the results obtained by FC and plating during alcoholic and malolactic fermentation over time in the cider-making process, different subpopulations were detected, showing significant differences between the methods. A small number of studies have applied the FC technique to analyze fermentation processes and mixed cultures over time. The results were used to postulate equations explaining the different physiological states in cell populations taken from fresh, pure overnight cultures under nonstress conditions or cells subjected to stress conditions over time, either under a pure-culture fermentation process (in this work, corresponding to alcoholic fermentation) or under mixed-fermentation conditions (for the malolactic-fermentation phase), that could be useful to improve the control of the processes.

Alcoholic Beverages↗

Influence of the rate of ethanol production and accumulation on the viability of Saccharomyces cerevisiae in "rapid fermentation".

Whereas "rapid fermentation" of diluted clover honey (25 degrees Brix) fortified with yeast nutrients using 8 X 10(8) brewers' yeast cells per ml resulted in an ethanol content of 9.5% (wt/vol; 12% vol/vol) in 3 h at 30 C, death rate of the yeast cells during this period was essentially logarithmic. Whereas 6 h was required to reach the same ethanol content at 15 C, the yeast cells retained their viability. Using a lower cell population (6 X 10(7) cells/ml), a level at which the fermentation was no longer "rapid," the yeast cells also retained their viability at 30 C. Ethanol added to the medium was much less lethal than the same or less quantities of ethanol produced by the cell in "rapid fermentation." It was considered possible that ethanol was produced so rapidly at 30 C that it could not diffuse out of the cell as rapidly as it was formed. The hypothesis was postulated that ethanol accumulating in the cell was contributing to the high death rate at 30 C. It was found that the intracellular ethanol concentration reached a level of approximately 2 X 10(11) ethanol molecules/cell in the first 30 min of fermentation at 30 C. At 15 C, with the same cell count, intracellular ethanol concentration reached a level of approximately 4 X 10(10) ethanol molecules/cell and viability remained high. Also, at 30 C with a lower cell population (6 X 10(7) cells/ml), under which conditions fermentation was no longer "rapid," intracellular ethanol concentration reached a similar level (4 X 10(10) molecules ethanol/cell) and the cells retained their viability. Alcohol dehydrogenase (ADH) lost its activity in brewers' yeast under conditions of "rapid fermentation" at 30 C but retained its activity in cells under similar conditions at 15 C. ADH activity was also retained in fermentations at 30 C with cell populations of 6 X 10(7)/ml. It would appear that an intracellular level of about 5 X 10(10) ethanol molecules/cell is normal and that this level does not damage either cell viability or ADH activity. Higher intracellular ethanol concentrations, such as 2 X 10(11) molecules ethanol/cell (a fourfold increase in intracellular ethanol concentration), are accompanied by inactivation of ADH and loss of cell viability.

Alcohol Oxidoreductases↗

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.

Ethanol↗

Inhibition of ruminal cellulose fermentation by extracts of the perennial legume cicer milkvetch (Astragalus cicer).

Cicer milkvetch (Astragalus cicer L.) is a perennial legume used as a pasture or rangeland plant for ruminants. A study was undertaken to determine whether reported variations in its ruminal digestibility may be related to the presence of an antinutritive material. In vitro fermentation of neutral detergent fiber (NDF) of cicer milkvetch by mixed rumen microflora was poorer than was the fermentation of NDF in alfalfa (Medicago sativa L.). Fermentation of cicer milkvetch NDF was improved by preextraction of the ground herbage with water for 3 h at 39 degrees C. Such water extracts selectively inhibited in vitro fermentation of pure cellulose by mixed ruminal microflora and by pure cultures of the ruminal bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85. Inhibition of the cellulose fermentation by mixed ruminal microflora was dependent upon the concentration of cicer milkvetch extract and was overcome upon prolonged incubation. Pure cultures exposed to the extract did not recover from inhibition, even after long incubation times, unless the inhibitory agent was removed (viz., by dilution of inhibited cultures into fresh medium). The extract did not affect the fermentation of cellobiose by R. flavefaciens but did cause some inhibition of cellobiose fermentation by F. succinogenes. Moreover, the extracts did not inhibit hydrolysis of crystalline cellulose, carboxymethyl cellulose, or p-nitrophenylcellobioside by supernatants of these pure cultures of cellulolytic bacteria or by a commercial cellulase preparation from the fungus Trichoderma reesei. The agent caused cellulose-adherent cells to detach from cellulose fibers, suggesting that the agent may act, at least in part, by disrupting the glycocalyx necessary for adherence to, and rapid digestion of, cellulose.

Animals↗

Differential importance of trehalose in stress resistance in fermenting and nonfermenting Saccharomyces cerevisiae cells.

The trehalose content in laboratory and industrial baker's yeast is widely believed to be a major determinant of stress resistance. Fresh and dried baker's yeast is cultured to obtain a trehalose content of more than 10% of the dry weight. Initiation of fermentation, e.g., during dough preparation, is associated with a rapid loss of stress resistance and a rapid mobilization of trehalose. Using specific Saccharomyces cerevisiae mutants affected in trehalose metabolism, we confirm the correlation between trehalose content and stress resistance but only in the absence of fermentation. We demonstrate that both phenomena can be dissociated clearly once the cells initiate fermentation. This was accomplished both for cells with moderate trehalose levels grown under laboratory conditions and for cells with trehalose contents higher than 10% obtained under pilot-scale conditions. Retention of a high trehalose level during fermentation also does not prevent the loss of fermentation capacity during preparation of frozen doughs. Although higher trehalose levels are always correlated with higher stress resistance before the addition of fermentable sugar, our results show that the initiation of fermentation causes the disappearance of any other factor(s) required for the maintenance of stress resistance, even in the presence of a high trehalose content.

Fermentation↗

Effect of specific growth rate on fermentative capacity of baker's yeast.

The specific growth rate is a key control parameter in the industrial production of baker's yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial Saccharomyces cerevisiae strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates, D) below 0.28 h-1, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol . g of biomass-1 . h-1 at D = 0.40 h-1. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (D < 0.28 h-1). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol . g of dry yeast biomass-1 . h-1 at D = 0.025 h-1 to 20.5 mmol of ethanol . g of dry yeast biomass-1 . h-1 at D = 0.28 h-1. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol . g of dry yeast biomass-1 . h-1 at D = 0.40 h-1. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.

Acetates↗

Effect of fermented feed on the microbial population of the gastrointestinal tracts of pigs.

An in vivo experiment was performed with pigs to study the inhibitory effect of fermented feed on the bacterial population of the gastrointestinal tract. Results demonstrated a significant positive correlation between pH and lactobacilli in the stomach contents of pigs in dry feed as well as in the stomach contents of pigs fed fermented feed. Furthermore, a significant positive correlation between the pH and the numbers of bacteria in the family Enterobacteriaceae in the contents of the stomach of pigs fed dry feed was found. In the stomach contents of pigs fed fermented feed, a significant negative correlation was found between the concentration of the undissociated form of lactic acid and the numbers of Enterobacteriaceae. The numbers of Enterobacteriaceae in the contents of the stomach, ileum, cecum, colon, and rectum of pigs fed fermented feed were significantly lower compared with the contents of the stomach, ileum, caecum, colon, and rectum of pigs fed dry feed. The numbers of total lactobacilli were significantly higher in the stomach contents of pigs fed fermented feed and in the ileum contents of one pig group fed fermented feed compared with the contents of pigs fed dry feed. However, the influence of lactobacilli on numbers of Enterobacteriaceae could not be demonstrated. It was concluded that fermented feed influences the bacterial ecology of the gastrointestinal tract and reduces the levels of Enterobacteriaceae in the different parts of the gastrointestinal tract.

Animal Feed↗

Identification and characterization of Leuconostoc fallax strains isolated from an industrial sauerkraut fermentation.

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.

Brassica↗

Microbial and physiological characterization of weakly amylolytic but fast-growing lactic acid bacteria: a functional role in supporting microbial diversity in pozol, a Mexican fermented maize beverage.

Pozol is an acid beverage obtained from the natural fermentation of nixtamal (heat- and alkali-treated maize) dough. The concentration of mono- and disaccharides from maize is reduced during nixtamalization, so that starch is the main carbohydrate available for lactic acid fermentation. In order to provide some basis to understand the role of amylolytic lactic acid bacteria (ALAB) in this fermented food, their diversity and physiological characteristics were determined. Forty amylolytic strains were characterized by phenotypic and molecular taxonomic methods. Four different biotypes were distinguished via ribotyping; Streptococcus bovis strains were found to be predominant. Streptococcus macedonicus, Lactococcus lactis, and Enterococcus sulfureus strains were also identified. S. bovis strain 25124 showed extremely low amylase yield relative to biomass (139 U g [cell dry weight](-1)) and specific rate of amylase production (130.7 U g [cell dry weight](-1) h(-1)). In contrast, it showed a high specific growth rate (0.94 h(-1)) and an efficient energy conversion yield to bacterial cell biomass (0.31 g of biomass g of substrate(-1)). These would confer on the strain a competitive advantage and are the possible reasons for its dominance. Transient accumulation of maltooligosaccharides during fermentation could presumably serve as energy sources for nonamylolytic species in pozol fermentation. This would explain the observed diversity and the dominance of nonamylolytic lactic acid bacteria at the end of fermentation. These results are the first step to understanding the importance of ALAB during pozol fermentation.

Beverages↗