Batch Culture Fermentation Research Articles

An In Vitro Approach to Study Effects of Prebiotics and Probiotics on the Faecal Microbiota and Selected Immune Parameters Relevant to the Elderly

The aging process leads to alterations of gut microbiota and modifications to the immune response, such changes may be associated with increased disease risk. Prebiotics and probiotics can modulate microbiome changes induced by aging; however, their effects have not been directly compared. The aim of this study was to use anaerobic batch culture fermenters to assess the impact of various fermentable carbohydrates and microorganisms on the gut microbiota and selected immune markers. Elderly volunteers were used as donors for these experiments to enable relevance to an aging population. The impact of fermentation supernatants on immune markers relevant to the elderly were assessed in vitro. Levels of IL-1β, IL-6, IL-8, IL-10 and TNF-α in peripheral blood mononuclear cell culture supernatants were measured using flow cytometry. Trans-galactooligosaccharides (B-GOS) and inulin both stimulated bifidobacteria compared to other treatments (p<0.05). Fermentation supernatants taken from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus and Ba. coagulans inhibited LPS induced TNF-α (p<0.05). IL-10 production, induced by LPS, was enhanced by fermentation supernatants from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus, Ba. coagulans and Bac. thetaiotaomicron (p<0.05). To conclude, prebiotics and probiotics could lead to potentially beneficial effects to host health by targeting specific bacterial groups, increasing saccharolytic fermentation and decreasing inflammation associated with aging. Compared to probiotics, prebiotics led to greater microbiota modulation at the genus level within the fermenters.

Optimizing strategy for Escherichia coli growth and hydrogen production during glycerol fermentation in batch culture: Effects of some heavy metal ions and their mixtures

Hydrogen (H2) is well-known effective, ecologically clean and renewable fuel. Bacterial H2 production is a promising one and its use in industrial level is expected to increase in the nearest future to establish sustainable and renewable energy source. Escherichia coli wild type BW25113 growth yield was shown to be stimulated 1.3–1.5-fold by nickel (Ni2+), iron (Fe2+, Fe3+) ions and by some metal ion mixtures: Ni2++Fe2+ + molybdenum (Mo6+), Ni2++Fe3+, Ni2++Fe2+ and Mo6++Fe3+ in low concentrations (<0.05mM) stimulated the growth during glycerol (10gL−1) fermentation up to stationary phase at pH 6.5; Ni2++Fe2+ mixture showed the maximal effect. However, the same concentrations of these metals and their mixtures had no effects or slightly inhibited bacterial specific growth rate: it was suppressed ∼1.2-fold upon Ni2+, Fe3+, Mo6+ and Ni2++Mo6+ mixture supplementation. H2 production by E. coli from glycerol was observed with the yield of 0.75±0.02mmolL−1. Moreover, H2 yield was markedly stimulated 1.7–3-fold in the presence of Ni2++Fe3+, Ni2++Fe3++Mo6+ and Fe3++Mo6+ mixtures, but not sole metals: maximal stimulation was established by Fe3++Mo6+ mixture with the concentrations of 0.05mM and 0.02mM, respectively. While copper (Cu+, Cu2+) ions in low concentration (0.1mM) had H2 production suppressing effect. The results point out that some heavy metal ions and their mixtures can stimulate E. coli growth, as well as enhance bio-hydrogen production. Discrimination between Fe2+ and Fe3+ was important for H2 production. Some interaction of Ni2+ with Fe2+ was suggested to be effective factor increasing bacterial biomass and determining activity of H2 producing hydrogenases together with Fe3+. Mo6+ was significant for H2 production. The results obtained were important to develop H2 production biotechnology using glycerol as cheap substrate and optimizing the technological conditions by some heavy metals and their mixtures at low concentrations.

Alternative fermentation pathway of cinnamic acid production via phenyllactic acid.

Cinnamic acid (CA) is the chemical basis for bulk production of flavoring reagents and chemical intermediates, and it can be fermented from biomass. Phenylalanine ammonia lyase (PAL) has been used exclusively in the bacterial fermentation of sugar biomass in which the fermentation intermediate phenylalanine is deaminated to CA. Here, we designed an alternative metabolic pathway for fermenting glucose to CA. An Escherichia coli strain that generates phenylalanine in this pathway also produces Wickerhamia fluorescens phenylpyruvate reductase and ferments glucose to D-phenyllactate (D-PhLA) (Fujita et al. Appl Microbiol Biotechnol 97: 8887-8894, 2013). Thereafter, phenyllactate dehydratase encoded by fldABCI genes in Clostridium sporogenes converts the resulting D-PhLA into CA. The phenyllactate dehydratase expressed by fldABCI in the D-PhLA-producing bacterium fermented glucose to CA, but D-PhLA fermentation and phenyllactate dehydration were aerobic and anaerobic processes, respectively, which disrupted high-yield CA fermentation in single batch cultures. We overcame this disruption by sequentially culturing the two strains under aerobic and anaerobic conditions. We optimized the incubation periods of the respective aeration steps to produce 1.7g/L CA from glucose, which exceeded the yield from PAL-dependent glucose fermentation to CA 11-fold. This process is a novel, efficient alternative to conventional PAL-dependent CA production.

Quantification of organic acids in ruminal in vitro batch culture fermentation supplemented with fumarate using a herb mix as a substrate

Two 24 h in vitro batch culture experiments were conducted to investigate the effects of fumarate addition (10 mmol L−1) on the ruminal fermentation parameters of selected medicinal herbs, and the effects of different doses of fumarate (0, 10, or 30 mmol L−1) on ruminal metabolism of organic acids when a high-concentrate diet (meadow hay and barley grain, 400/600, w/w) was supplemented with a mix of medicinal herbs (Artemisia absinthium L., Melissa officinalis L., Malva sylvestris L., Matricaria chamomilla L., Plantago lanceolata L., Foeniculum vulgare Mill., and Althaea officinalis L.). Depending on the concentration, fumarate treatment decreased methane production (by 10–11%) and increased propionate proportions (by 5–13%) with high-concentrate diets. The organic acid (fumaric, succinic, malic, and lactic acid) concentrations in the batch culture were measured at intervals of 0, 4, 6, 12, and 24 h. The time and organic acid concentrations with 10 mmol L−1 fumarate were well correlated (R 2 = 0.846). The amount of succinate was accumulated and metabolized more slowly than that of fumarate (&gt;24 h). The addition of fumarate and a herb mix could positively influence in vitro ruminal fermentation parameters of high-concentrate diets by increasing the levels of propionate and succinate as well as the pH, and by decreasing of methane emissions.

Enhanced butanol production by optimization of medium parameters using Clostridium acetobutylicum YM1.

A new isolate of the solvent-producing Clostridium acetobutylicum YM1 was used to produce butanol in batch culture fermentation. The effects of glucose concentration, butyric acid addition and C/N ratio were studied conventionally (one-factor-at-a-time). Moreover, the interactions between glucose concentration, butyric acid addition and C/N ratio were further investigated to optimize butanol production using response surface methodology (RSM). A central composite design was applied, and a polynomial regression model with a quadratic term was used to analyze the experimental data using analysis of variance (ANOVA). ANOVA revealed that the model was highly significant (p < 0.0001) and the effects of the glucose and butyric acid concentrations on butanol production were significant. The model validation experiment showed 13.82 g/L butanol was produced under optimum conditions. Scale up fermentation in optimized medium resulted in 17 g/L of butanol and 21.71 g/L of ABE. The experimental data of scale up in 5 L bioreactor and flask scale were fitted to kinetic mathematical models published in the literature to estimate the kinetic parameters of the fermentation. The models used gave the best fit for butanol production, biomass and glucose consumption for both flask scale and bioreactor scale up.

In Vitro Prebiotic Properties of Partially Purified Asparagus Falcatus and Taraxacum Javanicum Inulins

In Vitro Prebiotic Properties of Partially Purified Asparagus Falcatus and Taraxacum Javanicum Inulins The in vitro prebiotic effects of Asparagus falcatus (AF) and Taraxacum javanicum (TJ) inulins were investigated and compared with commercially available chicory inulin using anaerobic batch culture fermenters, inoculated with human faecal slurries. The effect of three concentrations (1%, 2% and 3%) of each inulin type and fructose were examined. Changes in the growth of four genera of intestinal microbiota namely; Bifidobacteria, Lactobacilli, Clostridia, Coliform, total aerobes and, total anaerobes were determined using a range of selective media after 0, 6, 24 and 48 hours of in vitro fermentation. Short chain fatty acids (acetate, propionate, and butyrate) and lactic acid development during in vitro fermentation were measured using HPLC. Inulin contents before (0 hr) and after fermentation (48 hrs) were measured using enzymatic HPLC method. Data revealed that counts of Bifidobacteria and Lactobacilli were increased, while coliform decreased significantly (P<0.05) after 48 hr fermentation and in the presence of 3% inulin (AF, TJ and Chicory inulin) compared with fructose. All inulins at 3% and after 48 hr of fermentation reduced the Clostridium counts significantly (P<0.05). The highest in vitro production of propionate and butyrate were achieved using 2-3% TJ inulin. Results revealed, up to 68% of added inulin and up to 87% of added fructose was utilized by faecal microflora after 48 hr of in vitro fermentation. This study suggests that newly isolated inulins from Asparagus falcatus and Taraxacum javanicum plants have the potential to exert similar prebiotic effects to the commercial Chicory inulin.

In vitro faecal fermentation of novel oligosaccharides enzymatically synthesized using microbial transglycosidases acting on sucrose

Fermentation profiles of five enzymatically synthesized purified oligosaccharides (kojibiose, 4-galactosyl-kojibiose, lactulosucrose, and sets of lactosyl- (DP 4-6) and raffinosyl-oligofructosides (DP 4-7)) were studied using an in vitro batch-culture fermentation system inoculated with human faecal slurries, simulating the events in the distal colon. Selectivity index for bifidobacteria and lactobacilli was calculated for each oligosaccharide to obtain a comparative relationship between the growth of these beneficial bacteria with respect to the other bacterial groups studied. Results showed a strong bifidogenic effect for all oligosaccharides, with similar Bifidobacterium levels compared to those produced by well-established prebiotics such as lactulose and fructo-oligosaccharides. Accordingly, acetic acid was the most prevalent SCFA produced. Oligosaccharides sharing structural features such as monomer composition and type of glycosidic linkage were grouped by dendrogram analysis, thus, underlining the potential correlation between the structural features of prebiotic candidates and their impact on the composition and metabolism of the human faecal microbiota.

Evaluation on prebiotic properties of β-glucan and oligo-β-glucan from mushrooms by human fecal microbiota in fecal batch culture

Background: β-glucan is dietary fiber, a structural polysaccharide, β-linked linear chains of D-glucose polymers with variable frequency of branches. β-glucan is isolated from different sources such as cell walls of baker’s yeast (Saccharomyces cerevisiae), cereals (oat and barley) and various species of mushrooms. Among 8 mushrooms in the study, Schizophylum commune Fr and Auricularia auricula Judae had the highest in β-glucan contents and the cheapest cost of mushroom per content of β-glucan, respectively. Even the function of β-glucan on immune modulation has been known however no report on interaction between β-glucan and human gut microbiota. Gut microbiota is thought to have health effects by interaction with non-digestible component particular fermentable dietary fiber. It is important to correlate the specific groups of the microbial communities associated with β-glucan fermentation and the consequential SCFA profiles. β-glucan from mushroom may has potential prebiotic function similar to those from commercial yeast (Saccharomyces cerevisiae) β-glucan. Objective: To evaluate on prebiotic properties of soluble β-glucans and oligo-β-glucans from Schizophylum commune Fr and Auricularia auricula Judae by fecal fermentation in batch culture. Methods: In vitro fecal fermentation in anaerobic batch cultures under simulated conditions similar to human colon with human faecal samples from three donors were performed. Comparison on 3 β-glucans and 2 oligo-β-glucans have been studied. Sample was taken at 0 h, 24 h and 48 h to analyze the numbers of bacterial changes by fluorescent in situ hybridization (FISH) technique. Short chain fatty acids (SCFA) were analyzed by HPLC. The prebiotic index (PI) was calculated according to the change of 5 specific bacterial genus within 48 h fermentation. Results: Soluble β-glucan from Auricularia auricula Judae increased numbers of bifidobacteria and lactobacillus significantly (P&lt;0.05). The PI of soluble β-glucan and oligo-β-glucan from Schizophylum commune Fr were 0.01 and -0.01, respectively. β-glucan and oligo-β-glucan from Auricularia auricula Judae were 0.11 and -0.07, respectively. Whereas PI of β-glucan from commercial yeast (Saccharomyces cerevisiae) was 0.03. Acetate was the most prevalent SCFA found in all treatments followed by propionate, butyrate and lactate, respectively. Conclusion: The study confirmed that β-glucan from Schizophylum commune Fr and Auricularia auricula Judae are candidate prebiotics. Keywords: β-glucan, oligo-β-glucan, prebiotic, mushroom, fecal batch culture

Effect of chito-oligosaccharides over human faecal microbiota during fermentation in batch cultures

Chitosan with high number of deacetylated units, its reacetylated derivative and COS obtained through an enzymatic treatment with chitosanase were tested in pH controlled batch cultures to investigate the ability of the human faecal microbiota to utilise them.Chitosan derivatives with high number of deacetylated units decreased the bacterial populations: Bifidobacterium spp., Eubacterium rectale/Clostridium coccoides, C. Histolyticum and Bacteroides/Prevotella. On the other hand, chitosan derivatives with high content of acetylated residues maintained the tested bacterial groups and could increase Lactobacillus/Enterococcus. Regarding short chain fatty acids (SCFA), only low Mw COS increased the production in similar levels than fructo-oligossacharides (FOS). The acetylated chitosans and their COS do not appear as potential prebiotics but did not affect negatively the faecal microbiota, while derivatives with high number of deacetylated units could induce a colonic microbiota imbalance.

The effects of essential oil and condensed tannin on fermentation and methane production under in vitro conditions

The potential of five different essential oils (EO) and quebracho condensed tannin extract (QCT) as antimethanogenic additives in ruminant feeds were investigated. The first experiment was conducted to screen the effects rosemary oil, sage oil, cinnamon oil (CNO), eucalyptus oil and myrrh oil at 500 mg/L of culture fluid on methane (CH4) production under in vitro conditions. Rumen contents were collected from a cannulated Holstein dairy cow and used for a 24-h batch-culture experiment. Treatments were a control (CON) or CON plus EO at 500 mg/L. Results showed that CNO decreased CH4 production and, therefore, was selected for Experiment 2. The second experiment was designed to test the effects of CNO at three different dose levels on CH4 production and fermentation in 24-h batch-culture experiments. Treatments were CON or CON plus CNO supplemented at 125, 250 and 500 mg/L. Relative to CON, CNO decreased total gas production at the 250 and 500 mg/L doses. All doses of CNO decreased CH4 production. Total volatile fatty acid production was lower in cultures incubated with CNO at the 500 mg/L. Ammonia-N concentration decreased in cultures incubated with CNO at the 500 mg/L. The third experiment was designed to test the effects of QCT on CH4 production and fermentation in 24-h batch cultures. Treatments were CON or CON plus QCT at 25, 50 and 75 g/kg of diet DM. Relative to CON, total volatile fatty acid concentration increased with the 50 g/kg QCT, but was similar to the 25 and 75 g/kg QCT. The proportions of acetate decreased, while the proportions of propionate increased with the 25 g/kg QCT compared with CON. Methane production was not affected in cultures incubated with QCT. Relative to CON, all doses of QCT decreased ammonia-N concentration. In conclusion, results from the present study showed that except for CNO, EO tested in the study had no effects on rumen CH4 production. Addition of CNO to rumen cultures at 125 and 250 mg/L reduced CH4 production without negative effects on rumen fermentation. Quebracho condensed tannin-extract supplementation had no effects on CH4 production and fermentation parameters except for ammonia-N concentration.

Kiwifruit fermentation drives positive gut microbial and metabolic changes irrespective of initial microbiota composition

It is well established that individuals vary greatly in the composition of their core microbiota. Despite differing ecology, we show here that metabolic capacity converges under the pressure of kiwifruit substrates in a model gut system. The impact of pre-digested green and gold kiwifruit on the human colonic microbiota and their metabolic products was assessed using in vitro, pH-controlled, anaerobic batch culture fermenters. Phylogenetic analyses revealed that bacterial composition changed over time, irrespective of whether a substrate was added or not, indicating a natural adjustment period to the gut model environment. Adding kiwifruit substrate caused additional changes in terms of growth of specific bacterial groups, bacterial diversity and metabolite profiles. Relative abundance of Bacteroides spp. increased with both green and gold kiwifruit substrate while Bifidobacterium spp. increased only with green kiwifruit. NMR spectroscopy and GC demonstrated an increase in organic acids (primarily acetate, butyrate, and propionate) and a concomitant decrease in several amino acids and oligosaccharides following addition of green and gold kiwifruit substrate. The experiments demonstrated that despite markedly different baseline profiles in individual donor inoculum, kiwifruit components can induce substantive change in microbial ecology and metabolism which could have consequences for human health.

In vitro fermentation of anthocyanins encapsulated with cyclodextrins: Release, metabolism and influence on gut microbiota growth

Anthocyanins are thought to exert protective influences on chronic gut disorders such as colon cancer and inflammatory bowel disease. They may also positively modulate intestinal bacterial populations. However, their bioavailability in the gastrointestinal tract is an important determinant factor for their in vivo activity. In this study, we attempted to increase their stability by encapsulation with cyclodextrins. From anaerobic batch-culture fermentation experiments with gut bacteria, release of anthocyanins and the formation of phenolic microbial metabolites were quantified. Despite a rapid release of anthocyanins observed within the first 30 min, encapsulation allowed anthocyanin degradation to be slowed down. As a consequence of anthocyanin degradation phenolic acids were formed. Quantitative analysis of bacterial populations revealed that there was a significant growth of members of the domain Bacteria in vessels with malvidin-3-glucoside, compared to the negative control, and there was inhibition of the Clostridium histolyticum group in those vessels where delphinidin-3-glucoside or cyanidin-3-glucoside was added. These results illustrate the ability of encapsulation to increase bioavailability of anthocyanins, allowing them to be released in the colon and to exert their potential health benefits.

In vitro colonic metabolism of coffee and chlorogenic acid results in selective changes in human faecal microbiota growth.

Coffee is a relatively rich source of chlorogenic acids (CGA), which, as other polyphenols, have been postulated to exert preventive effects against CVD and type 2 diabetes. As a considerable proportion of ingested CGA reaches the large intestine, CGA may be capable of exerting beneficial effects in the large gut. Here, we utilise a stirred, anaerobic, pH-controlled, batch culture fermentation model of the distal region of the colon in order to investigate the impact of coffee and CGA on the growth of the human faecal microbiota. Incubation of coffee samples with the human faecal microbiota led to the rapid metabolism of CGA (4 h) and the production of dihydrocaffeic acid and dihydroferulic acid, while caffeine remained unmetabolised. The coffee with the highest levels of CGA (P<0·05, relative to the other coffees) induced a significant increase in the growth of Bifidobacterium spp. relative to the control vessel at 10 h after exposure (P<0·05). Similarly, an equivalent quantity of CGA (80·8 mg, matched with that in high-CGA coffee) induced a significant increase in the growth of Bifidobacterium spp. (P<0·05). CGA alone also induced a significant increase in the growth of the Clostridium coccoides-Eubacterium rectale group (P<0·05). This selective metabolism and subsequent amplification of specific bacterial populations could be beneficial to host health.

Shifts in metabolic hydrogen sinks in the methanogenesis-inhibited ruminal fermentation: a meta-analysis

Maximizing the flow of metabolic hydrogen ([H]) in the rumen away from CH4 and toward volatile fatty acids (VFA) would increase the efficiency of ruminant production and decrease its environmental impact. The objectives of this meta-analysis were: (i) To quantify shifts in metabolic hydrogen sinks when inhibiting ruminal methanogenesis in vitro; and (ii) To understand the variation in shifts of metabolic hydrogen sinks among experiments and between batch and continuous cultures systems when methanogenesis is inhibited. Batch (28 experiments, N = 193) and continuous (16 experiments, N = 79) culture databases of experiments with at least 50% inhibition in CH4 production were compiled. Inhibiting methanogenesis generally resulted in less fermentation and digestion in most batch culture, but not in most continuous culture, experiments. Inhibiting CH4 production in batch cultures resulted in redirection of metabolic hydrogen toward propionate and H2 but not butyrate. In continuous cultures, there was no overall metabolic hydrogen redirection toward propionate or butyrate, and H2 as a proportion of metabolic hydrogen spared from CH4 production was numerically smaller compared to batch cultures. Dihydrogen accumulation was affected by type of substrate and methanogenesis inhibitor, with highly fermentable substrates resulting in greater redirection of metabolic hydrogen toward H2 when inhibiting methanogenesis, and some oils causing small or no H2 accumulation. In both batch and continuous culture, there was a decrease in metabolic hydrogen recovered as the sum of propionate, butyrate, CH4 and H2 when inhibiting methanogenesis, and it is speculated that as CH4 production decreases metabolic hydrogen could be increasingly incorporated into formate, microbial biomass, and perhaps, reductive acetogenesis in continuous cultures. Energetic benefits of inhibiting methanogenesis depended on the inhibitor and its concentration and on the in vitro system.

Effect of fibrolytic enzymes added to a Andropogon gayanus grass silage-concentrate diet on rumen fermentation in batch cultures and the artificial rumen (Rusitec)

In vitro batch cultures were used to screen four fibrolytic enzyme mixtures at two dosages added to a 60 : 40 silage : concentrate diet containing the C4 tropical grass Andropogon gayanus grass ensiled at two maturities – vegetative stage (VS) and flowering stage (FS). Based on these studies, one enzyme mixture was selected to treat the same diets and evaluate its impact on fermentation using an artificial rumen (Rusitec). In vitro batch cultures were conducted as a completely randomized design with two runs, four replicates per run and 12 treatments in a factorial arrangement (four enzyme mixtures×three doses). Enzyme additives (E1, E2, E3 and E4) were commercial products and contained a range of endoglucanase, exoglucanase and xylanase activities. Enzymes were added to the complete diet 2 h before incubation at 0, 2 and 4 μl/g of dry matter (DM). Gas production (GP) was measured after 3, 6, 12, 24 and 48 h of incubation. Disappearance of DM (DMD), NDF (NDFD) and ADF (ADFD) were determined after 24 and 48 h. For all four enzyme mixtures, a dosage effect (P<0.05) was observed for NDFD and ADFD after 24 h and for DMD, NDFD and ADFD after 48 h of incubation of the VS diet. For the FS diet, a dosage effect was observed for GP and NDFD after 24 h and for GP, DMD, NDFD and ADFD after 48 h of incubation. There was no difference among enzyme mixtures nor was there an enzyme×dose interaction for the studied parameters. Because of the greatest numerical effect on NDF disappearance and the least cost price, enzyme mixture E2 at 4 µl/g of diet DM was selected for the Rusitec experiment. The enzyme did not impact (P>0.05) DM, N, NDF or ADF disappearance after 48 h of incubation nor daily ammonia-N, volatile fatty acids or CH4 production. However, enzyme application increased (P<0.05) microbial N production in feed particle-associated (loosely-associated) and silage feed particle-bound (firmly associated) fractions. With A. gayanus silage diets, degradation may not be limited by microbial colonization, but rather by the ability of fibrolytic enzymes to degrade plant cell walls within this recalcitrant forage.

A survey of modulation of gut microbiota by dietary polyphenols.

Dietary polyphenols present in a broad range of plant foods have been related to beneficial health effects. This review aims to update the current information about the modulation of the gut microbiota by dietary phenolic compounds, from a perspective based on the experimental approaches used. After referring to general aspects of gut microbiota and dietary polyphenols, studies related to this topic are presented according to their experimental design: batch culture fermentations, gastrointestinal simulators, animal model studies, and human intervention studies. In general, studies evidence that dietary polyphenols may contribute to the maintenance of intestinal health by preserving the gut microbial balance through the stimulation of the growth of beneficial bacteria (i.e., lactobacilli and bifidobacteria) and the inhibition of pathogenic bacteria, exerting prebiotic-like effects. Combination of in vitro and in vivo models could help to understand the underlying mechanisms in the polyphenols-microbiota-host triangle and elucidate the implications of polyphenols on human health. From a technological point of view, supplementation with rich-polyphenolic stuffs (phenolic extracts, phenolic-enriched fractions, etc.) could be an effective option to improve health benefits of functional foods such as the case of dairy fermented foods.

Use of tomato and cucumber waste fruits in goat diets: effects on rumen fermentation and microbial communities in batch and continuous cultures

SUMMARYTwoin vitroexperiments were conducted to analyse the effects of replacing dietary barley grain with wastes of tomato and cucumber fruits and a 1 : 1 tomato : cucumber mixture on rumen fermentation characteristics and microbial abundance. The control (CON) substrate contained 250 g/kg of barley grain on a dry matter (DM) basis, and another 15 substrates were formulated by replacing 50, 100, 150, 200 or 250 g of barley grain/kg with the same amount (DM basis) of tomato or cucumber fruits or 1 : 1 tomato : cucumber mixture. In Expt 1, all substrates were incubated in batch cultures with rumen micro-organisms from goats for 24 h. Increasing amounts of tomato, cucumber and the mixture of both fruits in the substrate increased final pH and gas production, without changes in final ammonia-nitrogen (NH3-N) concentrations, substrate degradability and total volatile fatty acid (VFA) production, indicating that there were no detrimental effects of any waste fruits on rumen fermentation. Therefore, in Expt 2 the substrates including 250 g of waste fruits (T250, C250 and M250 for tomato, cucumber and the mixture of both fruits, respectively) and the CON substrate were incubated in single-flow continuous-culture fermenters for 8 days. Total VFA production did not differ among substrates, but there were differences in VFA profile. Molar proportions of propionate, isobutyrate and isovalerate were lower and acetate : propionate ratio was greater for T250 compared with CON substrate. Fermentation of substrates containing cucumber (C250 and M250) resulted in lower proportions of acetate, isobutyrate and isovalerate and acetate : propionate ratio, but greater butyrate proportions than the CON substrate. Carbohydrate degradability and microbial N synthesis tended to be lower for substrates containing cucumber than for the CON substrate, but there were no differences between CON and T250 substrates. Abundance of total bacteria,Fibrobacter succinogenesandRuminococcus flavefaciens, fungi, methanogenic archaea and protozoa were similar in fermenters fed T250 and CON substrates, but fermenters fed C250 and M250 substrates had lower abundances ofR. flavefaciens, fungi and protozoa than those fed the CON substrate. Results indicated that tomato fruits could replace dietary barley grain up to 250 g/kg of substrate DM without noticeable effects on rumen fermentation and microbial populations, but the inclusion of cucumber fruits at 250 g/kg of substrate DM negatively affected some microbial populations as it tended to reduce microbial N synthesis and changed the VFA profile. More studies are needed to identify the dietary inclusion level of cucumber which produces no detrimental effects on rumen fermentation and microbial growth.

An in vitro study of the effect of probiotics, prebiotics and synbiotics on the elderly faecal microbiota

The use of dietary intervention in the elderly in order to beneficially modulate their gut microbiota has not been extensively studied. The influence of two probiotics (Bifidobacterium longum and Lactobacillus fermentum) and two prebiotics [isomaltooligosaccharides (IMO) and short-chain fructooligosaccharides (FOS)], individually and in synbiotic combinations (B. longum with IMO, L. fermentum with FOS) on the gut microbiota of elderly individuals was investigated using faecal batch cultures and three-stage continuous culture systems. Population changes of major bacterial groups were enumerated using fluorescent in situ hybridisation (FISH). B. longum and IMO alone significantly increased the Bifidobacterium count after 5 and 10 h of fermentation and their synbiotic combination significantly decreased the Bacteroides count after 5 h of fermentation. L. fermentum and FOS alone significantly increased the Bifidobacterium count after 10 h and 5, 10 and 24 h of fermentation respectively. B. longum with IMO as well as B. longum and IMO alone significantly increased acetic acid concentration during the fermentation in batch cultures. In the three-stage continuous culture systems, both synbiotic combinations increased the Bifidobacterium and Lactobacillus count in the third vessel representing the distal colon. In addition, the synbiotic combination of L. fermentum with scFOS resulted in a significant increase in the concentration of acetic acid. The results show that the elderly gut microbiota can be modulated in vitro with the appropriate pro-, pre- and synbiotics.

Fecal Metabolomics of Healthy Breast-Fed versus Formula-Fed Infants before and during In Vitro Batch Culture Fermentation

Nontargeted metabolomics analyses were used (1) to compare fecal metabolite profiles of healthy breast-fed (BF) and formula-fed (FF) infants before and during in vitro fermentation in batch culture and (2) to evaluate fecal metabolomics in assessing infant diet. Samples from healthy BF (n = 4) or FF (n = 4) infants were individually incubated at 37(°)C in anaerobic media containing 1% (wt/vol) galactooligosaccharides, 6'-sialyllactose, 2'-fucosyllactose, lacto-N-neotetraose, inulin, and gum arabic for up to 6 h, and supernatants were analyzed using GC/MS and LC/MS/MS to assess changes in various compounds. Comparison of over 250 metabolites prior to incubation showed that BF samples contained higher relative concentrations (P ≤ 0.05) of 14 compounds including human milk oligosaccharides and other metabolites presumably transferred through breast feeding (linoelaidate, myo-inositol) (P ≤ 0.05). Conversely, feces from FF infants contained 41 identified metabolites at higher levels (P ≤ 0.05) with many indicative of carbon limitation and protein fermentation. Our data are consistent with the notion that carbon-limited cultures catabolize protein and amino acids to obtain energy, whereas the provision of fermentable carbohydrate creates anabolic conditions relying on amino acids for bacterial growth. Results also suggest that fecal metabolomics can be a useful tool for studying interactions among diet, microbes, and host.

Robust Analysis and Parameters Identification of the Nonlinear Multistage Dynamical System in Batch Fermentation of Glycerol

This paper discusses a complex biological problem which is the fermentation of glycerol byKlebsiella pneumoniaein batch culture. We set up an improved multistage model involving the concentration of intracellular substances. Furthermore, the existence, uniqueness, and continuity of solutions with respect to the parameters are discussed. On the condition that glycerol and 1,3-propanediol are assumed to pass the cell membrane by passive diffusion coupled with facilitated transport, we take the relative errors between experimental data and computational values of the extracellular substances concentrations and the biological robustness of the intracellular substances concentrations as the performance index. Then we establish a parameter identification model and construct the particle swarm optimization algorithm to solve it. Finally, the numerical result shows that the improved model could describe the glycerol fermentation in batch culture well.