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The contribution of moulds and yeasts to the fermentation of 'agbelima' cassava dough.

Agbelima, a fermented cassava meal widely consumed in Ghana, Togo and Benin, is produced by fermenting grated cassava with one of several types of traditional cassava dough inoculum. During fermentation a smooth textured sour dough is produced, the toxicity of cassava is reduced and there is a build up of volatile aroma compounds. Four types of inocula were included in the present investigation. In one type moulds were found to form a dominant part of the microbiota, the species present being Penicillium sclerotiorum, P. citrinum, P. nodulum, Geotrichum candidum and a basidiomycete. All these moulds were found to possess cellulase activity which was responsible for the hydrolysis of cassava tuber cellulose during fermentation leading to a breakdown of the coarse texture of cassava dough. The yeasts Candida krusei, C. tropicalis and Zygosaccharomyces spp. were present in high numbers in the four types of inocula including the moudly inoculum. The yeasts C. tropicalis and some strains of Zygosaccharomyces, all of which possessed cellulase activity, were also found to contribute to the modification of cassava texture during fermentation. All yeasts and moulds exhibited linamarase activity and were therefore capable of breaking down the cyanogenic glucosides present in cassava.

Basidiomycota↗

Characterization of Candida krusei strains from spontaneously fermented maize dough by profiles of assimilation, chromosome profile, polymerase chain reaction and restriction endonuclease analysis.

Several isolates of Candida krusei from indigenous spontaneously fermented maize dough have been characterized for the purpose of selecting appropriate starter cultures and methods for their subspecifies typing. The present work describes the occurrence of C. krusei in Ghanaian fermented maize dough. For detailed pheno- and genotyping, 48 representative isolates were selected and comparison was made with clinical isolates of C. krusei and reference cultures. The techniques applied included the assimilation of carbon compounds by the API ID 32 C kit, determination of chromosome profile by pulse field gel electrophoresis, polymerase chain reaction (PCR) profiles, restriction endonuclease analysis (REA) and Southern blot hybridization. For the 48 isolates tested, 82% had the same assimilation profiles, being able to assimilate N-acetyl-glucosamine, DL-lactate, glycerol and to ferment glucose. Chromosome and PCR profiles, REA and Southern blot hybridization techniques all had a high discriminatory power and revealed DNA polymorphism, which allowed for discrimination among the strains and hence subspecific typing. On the basis of PCR and REA profiles, isolates were grouped into clusters. Southern blot hybridization appeared to be the most sensitive with respect to strain specificity. Our results demonstrated that the three methods, PCR, REA and Southern blot hybridization, were suitable tools, easy to analyse, fast (with regard to PCR) and reliable methods for the typing of C. krusei isolates to species and below species level. Based on the use of these techniques, we demonstrated that several strains of C. krusei were involved in the fermentation of maize dough from the onset and remain dominant throughout the fermentation.

Blotting, Southern↗

The stress response is repressed during fermentation in brewery strains of yeast.

Yeast cells encounter a variety of environmental stresses during brewing and must respond to ensure cell survival. Cells can respond to stress by inducing a Heat Shock Response in which heat shock proteins (Hsps) are synthesized. In laboratory strains of Saccharomyces cerevisiae, the heat shock protein, Hsp104, plays a major role in the acquisition of tolerance to a variety of stresses such as heat, ethanol and sodium arsenite, and as such acts as an excellent stress indicator. The induction of Hsp104 in bottom-and top-fermenting brewery strains was examined when grown under laboratory and industrial fermentation conditions, and it was found that each brewing strain exhibits its own unique pattern of Hsp104 expression. During industrial fermentations, brewery strains are capable of mounting a stress response at the early stages of fermentation. However, as the fermentation proceeds, the response is repressed. The results suggest that conditions experienced in industrial brewing prevent the activation of the stress response. This study increases our understanding of alterations in gene expression patterns during the brewing process, and yields information that will aid in the definition of best practice in yeast management.

Aerobiosis↗

Nutritional requirements and simplified cultivation medium to study growth and energetics of a sourdough lactic acid bacterium Lactobacillus fermentum Ogi E1 during heterolactic fermentation of starch.

AIMS: Nutritional requirements of Lactobacillus fermentum Ogi E1 were studied in order to define a simplified fermentation medium. METHODS AND RESULTS: When grown with MRS-medium in 2l bioreactors, a biphasic pattern of growth and metabolite production was observed. Study of nutritional requirements resulted in a simplified medium (SYAM) that allowed, under anaerobiosis, similar results to be obtained as in MRS medium, but without biphasic fermentation kinetics. The best substrates for both growth and amylase production were starch and maltose. Although melibiose, raffinose, fructose, sucrose and glucose also supported growth, lower amylase activity was observed. CONCLUSION: The physiology of the strain can be investigated with SYAM medium, using either starch or maltose as substrate. The strain also presented potential for alpha-galactoside fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus fermentum was one of the dominant bacteria of African maize dough fermentations. Amylolytic strains with activity against other compounds (i.e. raffinose) suggested a potential to be used as starter for cereal fermentation.

Acetates↗

The effect of oxygen on the survival of non-Saccharomyces yeasts during mixed culture fermentations of grape juice with Saccharomyces cerevisiae.

AIMS: The effect of oxygen on the survival of Torulaspora delbrueckii and Kluyveromyces thermotolerans during mixed culture fermentations in grape juice with Saccharomyces cerevisiae was investigated. METHODS AND RESULTS: Fermentations were carried out in two simple fermentation systems differing in the availability of oxygen. At low available oxygen conditions, T. delbrueckii and K. thermotolerans began to die off after two days of mixed culture fermentation. In filtrates from 2-day-old mixed cultures, single cultures of T. delbrueckii and K. thermotolerans survived and actively produced ethanol to concentrations of approx. 65 and 70 g l-1, respectively, at low available oxygen conditions. Oxygen clearly increased the survival time and decreased the death rate of T. delbrueckii and K. thermotolerans in mixed cultures, whereas it did not affect the growth and survival of S. cerevisiae. CONCLUSION: Our results show that the deaths of T. delbrueckii and K. thermotolerans in mixed cultures at low available oxygen conditions are not due to toxic metabolites produced by the yeasts but rather to the lack of oxygen. Furthermore, they indicate that T. delbrueckii and K. thermotolerans are less tolerant to low available oxygen conditions than S. cerevisiae. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals new knowledge on the mechanisms underlying the succession of yeasts during wine fermentations. This knowledge may be of importance when creating defined, mixed starter cultures for the controlled production of wines with a wide range of flavour compositions.

Carbohydrate Metabolism↗

Transcription profile of brewery yeast under fermentation conditions.

AIMS: Yeast strains, used in the brewing industry, experience distinctive physiological conditions. During a brewing fermentation, yeast are exposed to anaerobic conditions, high pressure, high specific gravity and low temperatures. The purpose of this study was to examine the global gene expression profile of yeast subjected to brewing stress. METHODS AND RESULTS: We have carried out a microarray analysis of a typical brewer's yeast during the course of an 8-day fermentation in 15 degrees P wort. We used the probes derived from Saccharomyces cerevisiae genomic DNA on the chip and RNA isolated from three stages of brewing. This analysis shows a high level of expression of genes involved in fatty acid and ergosterol biosynthesis early in fermentation. Furthermore, genes involved in respiration and mitochondrial protein synthesis also show higher levels of expression. CONCLUSIONS: Surprisingly, we observed a complete repression of many stress response genes and genes involved in protein synthesis throughout the 8-day period compared with that at the start of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This microarray data set provides an analysis of gene expression under brewing fermentation conditions. The data provide an insight into the various metabolic processes altered or activated by brewing conditions of growth. This study leads to future experiments whereby selective alterations in brewing conditions could be introduced to take advantage of the changing transcript profile to improve the quality of the brew.

Anaerobiosis↗

Fermentation properties of gentio-oligosaccharides.

AIMS: To investigate the fermentation properties of gentio-oligosaccharides (GOS), as compared to fructo-oligosaccharides (FOS) and maltodextrin in mixed faecal culture. METHODS AND RESULTS: The substrates were incubated in 24 h batch culture fermentations of human faecal bacteria. Fluorescent in situ hybridization was used to determine changes in populations of bifidobacteria, lactobacilli, clostridia, bacteroides, streptococci and Escherichia coli. Gas and short-chain fatty acid (SCFA) production was also measured. GOS gave the largest significant increases in bifidobacteria, lactobacilli and total bacterial numbers during the incubations. However, FOS appeared to be a more selective prebiotic as it did not significantly stimulate growth of bacterial groups which were not probiotic in nature. GOS and maltodextrin produced the highest levels of SCFA. Lowest gas production was seen with GOS and highest with FOS. CONCLUSIONS: GOS possessed bifidogenic activity in vitro. Although fermentation of GOS was not as selective as FOS, gas production was lower. Gas production is often seen as an undesirable side effect of prebiotic consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided the first data on fermentation of GOS in mixed faecal culture. The study has also used molecular microbiology methods (FISH) to quantify bacterial groups. The data extend our knowledge of the selectivity of fermentation of oligosaccharides by the gut microflora.

Bacteria↗

Comparison of fermentative capacities of industrial baking and wild-type yeasts of the species Saccharomyces cerevisiae in different sugar media.

AIMS: To compare the fermentative capacity of wild and domesticated isolates of the genus Saccharomyces. METHODS AND RESULTS: The fermentative capacity of yeasts from a variety of wild and domesticated sources was tested in synthetic dough media that mimic major bread dough types. Domesticated yeast strains were found to have better maltose-utilizing capacity than wild yeast strains. The capacity to ferment sugars under high osmotic stress was randomly distributed amongst wild and baking strains of Saccharomyces. CONCLUSION: The domestication of bakers' yeast has enhanced the ability of yeasts to ferment maltose, without a similar impact on the fermentative capacity under high osmotic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, combined with molecular studies of both wild and domesticated yeast, showed that domestication of bakers' yeast has resulted in improved maltose utilization, apparently via the duplication and mutation of the MAL genes.

Bread↗

Fermentation of cellulose to acetic acid by Clostridium lentocellum SG6: induction of sporulation and effect of buffering agent on acetic acid production.

AIMS: To determine the growth, correlation between sporulation and acetic acid production and effect of buffering agent at high substrate cellulose concentrations of the strain Clostiridium lentocellum SG6. METHODS AND RESULTS: The strain SG6 was grown in cellulose mineral salt medium containing cellulose (Whatman No. 1 filter paper, Whatmore International Ltd., Maidstone, UK) or cellobiose. The strain fermented cellulose even after several transfers on cellobiose medium. The formation of endospores on third day onwards indicated the lowering of pH in the medium because of the formation of acetic acid. Maintaining the pH 7.2 at higher substrate concentrations resulted in increase of biomass, cellulose fermentation, acetic acid production, etc. CONCLUSIONS: The strain SG6, with its high fermentation yields and sporulating character can become a potential strain for acetic acid production and also as a probiotic strain in animal nutrition. SIGNIFICANCE AND IMPACT OF THE STUDY: The direct conversion of cellulosic biomass to acetic acid can eliminate expensive three-step saccharification, fermentation processes. The strain SG6 can ferment cellulose at high substrate concentrations.

Acetic Acid↗

Fermentation of green alga sea-lettuce (Ulva sp) and metabolism of its sulphate by human colonic microbiota in a semi-continuous culture system.

The green alga, sea-lettuce (Ulva sp), could be considered as a new source of dietary fibre. Ulva, however, contains high levels of sulphate, part of which is chemically bound in soluble polymers (ulvan). The purpose of this study was to assess the fermentation characteristics and sulphate metabolism of Ulva and ulvan by human faecal bacteria fermentation system using a semi-continuous fermenter. Ulva and ulvan were poorly fermented, even after adaptation of the microbiota. Only 16.6% and 8.9% of Ulva and ulvan organic matter, respectively, were recovered as short chain fatty acids. Nevertheless, 40% of the sulphate in Ulva was dissimilated to sulphide by sulphate-reducing bacteria. Supplementation of Ulva with more fermentable polysaccharides, such as algal xylan and resistant starch, though decreasing the ammonia production originating from Ulva protein degradation, did not significantly reduce the sulphide levels. It is postulated that unless crude Ulva is desulphated, its daily consumption at a level of 20 g of dry product could stimulate colonic microbial sulphate reduction, which may have detrimental effects for the host.

Acetates↗

Fermentable carbohydrate and digestive nitrogen excretion.

BACKGROUND: Interventions that restrict protein intake lower plasma urea concentration and may slow the progression of renal failure. The question arises whether the effect of a dietary protein restriction could be reinforced by enrichment of the diet with fermentable carbohydrate because these carbohydrates may stimulate the extra-renal route of nitrogen (N) excretion through the digestive route. METHODS: The influence of fermentable carbohydrate and moderate protein restriction on N metabolism was investigated in a rat model of renal failure with ablation of 70% of renal mass compared with control rats with intact kidneys. Animals were adapted to diets varying with respect to nondigestible fermentable carbohydrate (0% or 10% fructooligosaccharide [FOS]) and with respect to protein content (10% or 18% casein). RESULTS: Feeding FOS led to a considerable enlargement of the cecum (increase in contents, wall thickness, and blood flow). These changes resulted in a concomitant enhancement of urea N uptake into the cecum and a decrease in plasma urea concentration (-30%). The extent of urea uptake by the cecum was influenced by plasma urea level that was determined by the dietary protein level and by the renal function. Thus, compared with control rats, the rate of urea uptake by the cecum and the total N excreted by the uremic rats was greater under all nutritional conditions. It is noteworthy that, when expressed as a percentage of total N excretion, fecal N excretion nearly doubled in rats adapted to the low-protein diets containing FOS. These effects occurred in both control rats and in uremic rats, in which a 22% decrease in urinary N was recorded as a result of FOS in addition to the low-protein diet. Globally, decreasing the amount of protein in the diet and adding a fermentable carbohydrate led to a decrease in urinary N excretion of more than 65% in uremic rats. CONCLUSION: These results suggest a possible usefulness for combining fermentable carbohydrate, such as FOS, with a low-protein diet to increase N excretion through the digestive route in detriment of the renal route. This may represent an efficient preventive measure to relieve the renal function in case of renal failure.

Ammonia↗

Large enrichments in fatty acid 2H/1H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae.

Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2&#x3b5;fattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2&#x3b5;fattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550&#x2030; 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200&#x2030;. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2&#x3b5;fattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2&#x3b5;fattyacid/water variations (<30&#x2030;) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.

Saccharomyces cerevisiae↗

Persistence and functional impact of a microbial inoculant on native microbial community structure, nutrient digestion and fermentation characteristics in a rumen model.

Small sub-unit (SSU) rRNA-targeted oligonucleotide probes were used to monitor the persistence of a genetically engineered bacterium inoculated in model rumens. Eight dual flow continuous culture fermenters were operated with either standard artificial saliva buffer or buffer with chondroitin sulfate (0.5 g/l) added. After 168 h of operation, fermenters were inoculated with Bacteroides thetaiotaomicron BTX (BTX), at approximately 1% of total bacteria. B. thetaiotaomicron was quantified using a species-specific probe and shown to persist in fermenters 144 h after inoculation (relative abundance 0.48% and 1.42% of total SSU rRNA with standard and chondroitin sulfate buffers, respectively). No B. thetaiotaomicron SSU rRNA was detected in fermenter samples prior to inoculation with strain BTX. Relative abundances of Bacteria, Eucarya and Archaea were not affected by either inoculation or buffer type. Fiber digestion, in particular the hemicellulose fraction, increased after strain BTX addition. Chondroitin sulfate addition to the buffer increased bacterial nitrogen flow in fermenters, but did not alter fiber digestion. Neither inoculum nor buffer type altered total short chain fatty acid (VFA) concentrations but proportions of individual VFA differed. In model rumens, B. thetaiotaomicron BTX increased fiber digestion when added to mixed ruminal microbes, independent of chondroitin sulfate addition; but further study is needed to determine effects on other fiber-digesting bacteria.

Animals↗

Effect of in vitro fermentation using human faecal inoculum on the water-holding capacity of dietary fibre.

The water-holding capacities (WHC) of four sources of fibre were measured using dialysis membranes and osmotic-suction pressures of 45, 89 and 178 mosmol/1 (1, 2 and 4 atm). At all pressures, pectin had the highest WHC, followed by cabbage (Brassica oleracea) and lucerne (Medicago sativa) and then cellulose. A suction pressure of 89 mosmol/1 (2 atm) was used in the subsequent fermentation study since it had the lowest standard error of the mean and most closely approximated physiological conditions. The four fibres were anaerobically fermented in vitro with human faecal inoculum for 24 h. The WHC of the fermentation residues were measured. The potential water-holding capacity (PWHC), a function of the extent of fermentability and the WHC of the fermentation residues, was highest for lucerne, followed by cellulose, then cabbage and, finally, pectin. Only the PWHC values ranked the four fibres in the same order as in vivo values. It was concluded that the ethanol-insoluble residues containing unfermented fibre organic matter and microbial organic matter, both of which hold water, should be used to calculate PWHC and to predict the effect of fibre on rate of passage and faecal mass in humans.

Body Water↗

Dietary fibre fermentation in the rat intestinal tract: effect of adaptation period, protein and fibre levels, and particle size.

1. The fermentative breakdown of one resistant type of dietary fibre (wheat bran) and one easily-fermented fibre (low-methoxyl pectin) was studied with respect to the length of the adaptation period and fibre level in the diet. The breakdown of the resistant fibre was also studied regarding the protein level in the diet and particle size of the fibre. 2. Prolongation of the adaptation period from 4 to 18 d decreased the faecal dry weight considerably. The excretion of dietary fibre however, was similar, whereas a decrease in faecal nitrogen excretion could be seen. 3. A level of dietary protein of less than 50 g/kg impaired the fermentation of wheat-bran fibre, whereas a level higher than 100 g protein/kg did not further increase the degree of fermentation of the fibre. 4. The particle size did not change the fermentability of the fibre, equal amounts of the main components of coarse and milled bran being excreted in faeces. 5. Two different levels of wheat-bran fibre (48 and 96 g/kg) in the diet did not influence the fibre breakdown. Similar results were obtained with two levels of fibre from low-methoxyl pectin (42 and 84 g/kg), but a tendency towards a decreased percentage of faecal excretion of uronic acids was seen at the lower level of low-methoxyl pectin.

Adaptation, Physiological↗

Influence of the pattern of peptide supply on microbial activity in the rumen simulating fermenter (RUSITEC).

The source and pattern of N supply was varied in the rumen simulation technique (RUSITEC) in order to determine if continuous, rather than transient, availability of peptides was required for optimum ruminal fermentation. The energy source was fibre prepared from sugar-beet pulp. N was added as NH3 continuously infused (AC) or peptides (Bacto(R) Casitone, a pancreatic hydrolysate of casein; Difco Laboratories, Detroit, MI, USA) continuously infused (PC) or added as a single dose at the time of feeding (PS). Free peptides were detected in the fermenter liquid for 4 h after feeding in the AC treatment, for 10 h in the PS treatment, and at all times with the PC treatment. Treatments had no effect on DM degradation. Approximately 40 % of the degradation occurred during the time no peptides were detected in the PS treatment. Microbial N flow tended to be higher with the peptide additions (P<0.061), with no significant difference between the two peptides treatments. The production of liquid-associated micro-organisms (LAM) was higher in the PC treatment (P<0.05) and the proportion of LAM derived from NH3 lower (P<0.05). However, LAM only accounted for 20-30 % total microbial population. Our main conclusion was that peptides had a small stimulatory effect on the fermentation, but there was no indication that synchrony of supply of energy and amino acid-N in the fermenter promoted a more efficient fermentation than non-synchronous supply. This conclusion must be qualified, however, because some N remained in the fibre and may have become available progressively as the fibre was digested by the micro-organisms.

Animal Nutritional Physiological Phenomena↗

Fermentative characteristics of cereal brans and vegetable fibers.

Dietary fiber has been suggested to alter the colonic environment and protect against colorectal diseases, but several recent epidemiological studies indicate that cereal and vegetable fibers may not confer the same benefit. To address this issue, five common cereal brans (barley, corn, oat, rice, and wheat) and five vegetable fiber isolates (broccoli, cabbage, carrot, cauliflower, and lettuce) were fermented in vitro with human fecal microbiota for 4, 8, 12, and 24 hours. Short-chain fatty acid (SCFA), hydrogen (H2), and methane (CH4) productions and neutral-detergent fiber (NDF) digestibility differed significantly with fiber source. The vegetable fibers were significantly more fermentable than were the cereal brans, as evidenced by greater NDF digestibility and increased productions of SCFA, H2, and CH4, despite the presence of fermentable starch and protein in the latter. We concluded that vegetable fibers may more effectively stimulate colonic fermentation than cereal brans do. These findings suggest an attribute (i.e., fermentability) that may partially explain previously observed differences between vegetable and cereal fiber consumption patterns and colorectal carcinogenesis.

Colorectal Neoplasms↗

Effect of fermentation on naturally occurring deoxynivalenol (DON) in Argentinean bread processing technology.

The stability of naturally occurring DON was evaluated during the fermentation stage of the bread-making process on a pilot scale. Two different products, French bread and Vienna bread, were prepared with naturally contaminated wheat flour (150 mg kg(-1)) under controlled experimental conditions. Dough was fermented at 30, 40 and 50 degrees C according to standard procedures employed in Argentinean low-technology bakeries. When the dough was fermented at 50 degrees C, the maximum reduction was 56% for the Vienna bread, with French bread being reduced by 41%. DON reduction during bread-making occurs not only in the baker due to thermal decomposition, but also during the fermentation step. The Argentinean traditional bread-making process might reduce DON levels during the fermentation stages if the dough is leavened at temperatures > 30 degrees C.

Analysis of Variance↗