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PubMed2005

The effect of enzyme treatment on the in vitro fermentation of lucerne incubated with equine faecal inocula.

A series of experiments was conducted to determine the effects of a fibrolytic enzyme preparation (enzyme 1; E1) on the in vitro fermentation of lucerne incubated with equine faecal inocula. In experiment 1, high-temperature-dried (HT) lucerne was treated with five levels of E1 (0 to 2.4 ml/g DM) and incubated at 50 degrees C for 20 h. Samples then received a simulated foregut digestion (SFD) treatment before DM and NSP analysis. In experiment 2, HT lucerne was treated with the same enzyme levels used in experiment 1. Samples were then split into two groups; plus or minus an SFD treatment before in vitro fermentation using an equine faecal inoculum. In experiment 3, fresh and wilted lucerne were treated with the same levels of E1 as experiments 1 and 2, incubated at 50 degrees C for 20 h, then fermented in vitro. For experiment 4, fresh and wilted lucerne were treated with low levels (0 to 0.008 ml/g DM) of E1 before fermentation. E1 significantly (P<0.05) enhanced DM and NSP losses from HT lucerne following SFD treatment compared with the control. High levels of E1 significantly (P<0.05) enhanced the rate, but not extent, of fermentation of HT, wilted and fresh lucerne; however, low levels of E1 were ineffective. At higher application levels, E1 appears to have considerable potential to enhance the nutritive value of lucerne for horses. Information on the fermentation kinetics of the substrates was valuable; all end-point measurements showed no effect of enzyme treatment.

Animal Feed↗

PubMed1997

Effects of cellobiose and monensin on in vitro fermentation of organic acids by mixed ruminal bacteria.

The objective of this study was to determine the effects of cellobiose and monensin on the in vitro fermentation of organic acids (L-aspartate, fumarate, and DL-malate) by mixed ruminal bacteria. Ruminal fluid was collected from a steer fed 36.7 kg of forage and 4.5 kg of concentrate supplement once per day. Ruminal fluid was centrifuged to sediment feed particles and protozoa, and the resulting supernatant, which contained bacteria, was added (33%, vol/vol) to anaerobic media (500 ml). Incubations (n = 2) were performed in batch culture at 39 degrees C and sampled at 0, 2, 4, 6, 8, 12, and 24 h. Organic acids were added to achieve a final concentration of 7.5 mM. Cellobiose was added to obtain a final concentration of 5 mM, and monensin dissolved in ethanol was included at concentrations of 0 or 5 ppm. Addition of cellobiose to organic acid fermentations increased the rate of organic acid utilization by the mixed bacterial population. Total concentrations of volatile fatty acids were increased by the addition of cellobiose to all fermentations. A lag period (< or = 8 h) occurred in fermentations that were treated with monensin before organic acids were utilized. Total concentrations of volatile fatty acids were increased, and the acetate to propionate ratio was decreased, by monensin treatment. When cellobiose and monensin were added together, propionate production and organic acid utilization were increased. Both cellobiose and monensin affected the in vitro fermentation of organic acids by mixed ruminal bacteria by providing a carbon and energy source and by influencing electron disposal.

Acetates↗

PubMed1998

The comparison of in vitro fermentation kinetics estimated by three different methods.

Three different methods for the estimation of in vitro fermentation kinetics are compared. The glass syringe, flow gasometer and pressure transducer methods were used for measurement of gas production. The rumen fluid from fistulated Merino sheep mixed with McDougall's buffer (1:1) was used as an inoculum and added at an amount of 35 ml into the fermentation vessels containing 0.25 g of meadow hay. The total gas produced was recorded after 12, 24, 36, 48, 60, 72 and 96 h of incubation. Hay dry matter degradability was the same with all three methods and achieved 56.5 to 58.2%. Total volatile fatty acids were significantly lower with the pressure transducer method than with the syringe and flow gasometer method. Lower values of mol% of butyrate and valerate obtained with the flow gasometer and pressure transducer methods in comparison with the syringe were also observed. Total gas production estimated by the flow gasometer method was lower than that stated by the two other methods. With regard to precision of the used methods syringe method was the best followed by the pressure transducer and flow gasometer method. It can be concluded that in spite of some limitations the pressure transducer method used in this experiment can be regarded as suitable for total gas estimation in in vitro rumen fermentation experiments.

Animal Feed↗

PubMed1992

Impact of in vitro fermentation techniques upon kinetics of fiber digestion.

Three in vitro fermentation experiments were conducted to examine the impact on kinetics of fiber digestion of microminerals and tryptone addition, media reduction, fermentation vessel, CO2 gassing regimen, and buffer type. Alfalfa and bromegrass hays were incubated for 0, 4, 8, 12, 18, 24, 30, 36, 48, 72, and 96 h and analyzed for NDF. Kinetic measures of fiber digestion were estimated using nonlinear regression with iteratively reweighted least squares. In Experiment 1, continuous CO2 gassing increased rate and decreased lag time prior to NDF digestion compared with purging a non-CO2-saturated buffer at inoculation. Vessel type (50-ml polypropylene tube, 125-ml pyrex Erlenmeyer flask), use of additives (microminerals, tryptone), and media reduction had no effect on kinetics of NDF digestion. In Experiment 2, elimination of both media reduction and nutritive additives increased the lag time prior to NDF digestion. In Experiment 3, continuous CO2 gassing of buffer in 125-ml Erlenmeyer flasks resulted in faster rates of NDF digestion than CO2-saturated buffer in 50-ml screw-cap polypropylene tubes. The method that yielded the fastest rates and shortest lag times of NDF digestion consisted of continuous CO2 gassing, reduction, and use of additives to ensure that no nutrient limited fiber digestion.

Animals↗

PubMed2000

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↗

PubMed2002

Structure, physicochemical properties and in vitro fermentation of enzymatically degraded cell wall materials from apples.

Cell wall materials (CWM) prepared from apple parenchyma tissue by treatment with commercial enzymes for maceration, mash fermentation and liquefaction were characterised with regard to their composition and structure as well as their physicochemical and physiological properties. Increasing enzymatic degradation of the CWM resulted in growing loss of the pectin matrix, decreasing porosity as well as increasing particle aggregation. Due to these structural alterations the water binding, the viscoelastic properties of the CWM-water-suspensions and the in vitro fermentation, forming short chain fatty acids, were reduced. The investigations showed that interrelations exist between enzymatic treatment and changes of (i) structure and state of matrices (evaluated by means of thermal analysis), (ii) physicochemical properties and (iii) physiological properties. So the application of liquefying enzymes can lead to a complete removal of the pectin matrix, causing an essentially improved thermal stability of the CWM preparation, but strongly reduced water binding and reduced structure-forming properties into the CWM-water-suspensions. The formation of short-chain fatty acids during in vitro fermentation of the CWM preparations by fresh human faeces flora depended on the portion and the state of the pectin matrix and the cellulose network, respectively.

Cell Wall↗

PubMed2001

Electrospray ionisation mass spectrometric study of degradation products of quercetin, quercetin-3-glucoside and quercetin-3-rhamnoglucoside, produced by in vitro fermentation with human faecal flora.

Eight phenolic compounds, obtained by in vitro fermentation of quercetin, quercetin-3-glucoside and quercetin-3-rhamnoglucoside were analysed by electrospray ionisation mass spectrometry (ESI-MS). Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) was performed on the [M - H]- precursor ions to obtain specific fragmentation. Typical fragmentation of the phenolic acids was loss of 44 (CO2) and 18 (H2O) u. Production of m/z 108 by loss of neutral radicals, e.g. HCO2, CH3 or HCO, was also favoured. Structures of the compounds, numbered 1-8, were suggested based on the fragmentation patterns.

Acetates↗

PubMed1995

In vitro fermentation of selected fibrous substrates by dog and cat fecal inoculum: influence of diet composition on substrate organic matter disappearance and short-chain fatty acid production.

Two in vitro fermentation experiments were conducted to evaluate the influence of source of dietary fiber fed to dogs and cats on fermentative activity of their fecal microflora. In Exp. 1, six English Pointer dogs were fed a diet containing either a non-fermentable fiber (Solka Floc) or a fermentable fiber (citrus pulp). A fecal sample from each dog was used as the inoculum source to determine in vitro OM disappearance (OMD) and short-chain fatty acid (SCFA) production from selected fibrous substrates. When data were pooled across substrates and fermentation times, a lower (P = .02) OMD (24.8 vs 29.4%) and a higher (P = .01; 3.8 vs 2.2) acetate to propionate ratio (A:P) occurred for the Solka Floc than for the citrus pulp diet. In Exp. 2, six short-hair cats were fed a diet containing no supplemental fiber (NF) or a diet containing beet pulp (BP). When data were pooled across substrates and fermentation times, NF resulted in a greater (P < .01) A:P than the BP diet (3.4 vs 1.5). The BP treatment resulted in a slightly higher (P = .07) OMD (42.0 vs 39.3%) and a higher (P = .07) propionate production (.74 vs .47 mmol/g of OM) than the NF diet. In summary, in vitro substrate OMD increased and A:P decreased when fecal inoculum from dogs and cats fed diets containing a supplemental source of fermentable fiber was used. In vitro fermentation of fibrous substrates by fecal microflora from dogs and cats increased with inclusion of fermentable fiber in the diet.

Animal Feed↗

PubMed2000

In vitro fermentation of swine ileal digesta containing oat bran dietary fiber by rat cecal inocula adapted to the test fiber increases propionate production but fermentation of wheat bran ileal digesta does not produce more butyrate.

This experiment evaluated three hypotheses: i) production of propionate is increased during fermentation of substrate containing oat bran (OB)(6); ii) production of butyrate is increased during fermentation of substrate containing wheat bran (WB) and iii) results of in vitro fermentations using physiological substrates and inocula agree with in vivo data. Ileal digesta collected from swine fed OB and WB were the substrates. Digesta was fermented for 0-96 h in an anaerobic in vitro system using inocula prepared from ceca of rats fed the same fiber sources. Carbohydrate and short-chain fatty acid (SCFA) contents in the fermentations were measured by gas chromatography. Fermentation of WB digesta did not produce more n-butyrate (P > 0.05) and was significantly slower (P < 0.05) than fermentation of OB digesta. OB digesta fermentation produced a significantly greater (P < 0.05) molar proportion of SCFA as propionate. Bacterial mass increased more and was maintained longer during fermentation of OB digesta than the WB digesta. Our results indicate that dilution of undigested WB fiber and not n-butyrate production is one mechanism by which WB may protect colonic mucosa; propionate production is increased during fermentation of beta-glucan in OB; and an in vitro system using physiological sources of inoculum and substrate containing WB and OB yields results that agree with in vivo findings in humans and rats.

Analysis of Variance↗

PubMed2003

In vitro fermentation characteristics of two mushroom species, an herb, and their polysaccharide fractions, using chicken cecal contents as inoculum.

In vitro fermentabilities of two mushrooms (Lentinus edodes--LenS; Tremella fuciformis--TreS), an herb (Astragalus membranaceus--AstS), and their polysaccharide fractions (LenE, TreE, and AstE) were investigated using microflora from chicken ceca. Polysaccharides were extracted using the hot water method. The mushrooms had lower polysaccharide yields (8 to 10%) than the herb (31%). Fermentation kinetics were determined using the in vitro cumulative gas production technique. End-products, such as gas, volatile fatty acids (VFA), and ammonia, were also determined. The gas profiles of intact materials were similar for AstS and LenS. The TreS had a diphasic digestion pattern. The extracts had similar profiles to the intact materials though gas production rates were faster. Intact materials tended to produce less VFA than the extracts though LenS and AstE had the highest total VFA production overall. Intact materials contained more protein than the extracts, and therefore resulted in more branched-chain fatty acids and ammonia. Fermentation kinetics and end-point products demonstrated differences in availability of substrates between the mushrooms and herb. These medicinal mushroom and herb materials, particularly their polysaccharide extracts, show promise in altering microbial activities and composition in chicken ceca. In vivo experiments are necessary for confirmation of this hypothesis.

Ammonia↗

PubMed1986

In vitro fermentation of sugars, grains, and by-product feeds in relation to initiation of ruminal lactate production.

In vitro fermentations of various sugars, grains, and by-products were conducted to investigate the relationships between soluble carbohydrates and initiation of ruminal lactate production. Fermentation of hexose sugars, both monosaccharides and disaccharides, resulted in greater accumulation of lactate than did fermentation of pentoses. Results of fermentation of grains and by-products, in order of greatest to least potential to produce lactic acid, were steam-flaked barley = barley = wheat greater than moisture corn = sorghum grain. Water-soluble fractions of the grains and by-products were more rapidly fermented to lactate than the insoluble fractions. Combining 10% soluble fractions from wheat, barley, and steam-flaked barley with 90% insoluble fractions from corn resulted in significant increases in lactate concentration. Analysis of sugar composition of these water soluble materials indicated that monosaccharides and disaccharides constitute 23 to 46% by weight. Data suggest that water-soluble materials play a role in initiation of lactate production from grains, and further production is dependent on starch fermentability.

Animal Feed↗

PubMed2006

Influence of the amount of dietary fiber on the available energy from hindgut fermentation in growing pigs: use of cannulated pigs and in vitro fermentation.

Available energy from hindgut fermentation to pigs fed various amounts of dietary fiber was investigated using an in vivo-in vitro methodology. Six growing pigs fitted with a simple T-shaped cannula at the terminal ileum, and following a Latin-square design, were fed 3 diets differing in the content of non-starch polysaccharides (NSP): a low fiber diet (LFD, 77 g/kg of DM), a standard fiber diet (SFD, 160 g/kg of DM), and a high fiber diet (HFD, 240 g/kg of DM). After adaptation to the diet for 10 d, samples from feces and ileum were collected and analyzed for DM, energy, NSP, and chromic oxide; feces were also analyzed for short chain fatty acids (SCFA). Freeze-dried ileal samples (10 g/L) were fermented in vitro in a fecal slurry consisting of an anaerobic mineral salt medium and feces (50 g/L) from cannulated pigs fed the same diets. Available energy was calculated from the amount of SCFA produced in vitro after 48 h of incubation. Nonstarch polysaccharide content in the fermented material was measured to assess the in vitro degradation of this fraction. Increasing dietary NSP from 77 to 240 g/kg of feed DM increased (P < 0.001) ileal flow from 199 to 468 g/kg of feed, leading to a reduction in the energy digested at the terminal ileum, from 15 to 11 MJ/kg of feed DM and an increment in energy digested in the hindgut, from 1.6 to 3.5 MJ/kg of feed DM. Total in vitro production of SCFA/kg of feed DM was dependent on the amount of ileal substrate available for fermentation; that is, increased concentrations of NSP in the diet led to an increase in the SCFA that may be available to the animal (P < 0.001). The molar ratio of SCFA produced in vitro was affected by diet; the high fiber diet showed the greatest (P = 0.004) proportion of acetic acid, and the low fiber diet showed a tendency (P = 0.081) to an increased butyric acid proportion compared with the other 2 diets. Net disappearance of NSP during fermentation in vivo and in vitro were compared and showed a close relationship (P < 0.001, slope = 0.906, r = 0.960). In our experimental conditions, available energy as SCFA to the animal from hindgut fermentation increased with the concentration of dietary NSP (P < 0.001) and provided between 7.1 and 17.6% of the total available energy.

Animal Feed↗

PubMed2005

[In vitro fermentation characteristics of crop straws and their combined utilization].

By the technique of in vitro gas production, this paper studied the in vitro fermentation characteristics of different kinds of crop straws and their combined utilization. The results showed that the theoretical maximum gas production and the gas production rate of silage corn were increased by 19.1% and 8.2%, respectively, compared to corn stalk. These two indices of mixed crop straws were higher than those of single crop straw, and significant positive interactions in gas production were observed after 48 h of incubation. It was concluded that a combined utilization of silage corn with different kinds of crop straws could be an efficient way to improve the nutritional characteristics. The optimum ratio of the mixture was 25/75 or 50/50 when silage corn mixed with wheat or rice straw.

Animal Feed↗

PubMed1995

Effect of hydrothermal treatment of rice straw on its composition and in sacco digestibility and in vitro fermentation by rumen microorganisms.

Chemical composition, in sacco rumen disappearance of various cell wall constituents (CWC) and in vitro fermentation pattern of hydrothermally treated (1 to 14 kp/cm2 pressure for 5 min) rice straw was examined. At 10 kp/cm2 pressure treatment (maximum effect) the contents of dry matter (DM), organic matter (OM), neutral detergent fibre (NDF), acid detergent fibre (ADF), hemicellulose (HC) and cellulose (CE) were decreased by 32.5, 35.3, 27.8, 10.2, 61.2 and 25.1%, respectively (P < 0.05), over the untreated control. The in sacco rumen disappearance of DM, OM, HC and CE from rice straw treated at 8 kp/cm2 pressure (maximum effect) and incubated for 48 h was increased from 53.2 to 77.7, 52.4 to 80.3, 49.5 to 82.0 and 49.2 to 79.3%, respectively (P < 0.01). In vitro production of total volatile fatty acids and the content of TCA-insoluble protein was also significantly higher (P < 0.05) on treated compared with untreated straw.

Animal Feed↗

PubMed1993

Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine.

The in vitro fermentability of oligofructose and inulin was compared with a range of reference carbohydrates by measuring bacterial end-product formation in batch culture. Short chain fatty acid and gas formation indicated that these substrates, which occur naturally in the diet and reach the colon in a largely intact form, were utilized by mixed populations of gut bacteria. Bacterial growth data showed that oligofructose and inulin exerted a preferential stimulatory effect on numbers of the health-promoting genus Bifidobacterium, whilst maintaining populations of potential pathogens (Escherichia coli, Clostridium) at relatively low levels. Pure culture studies confirmed the enhanced ability of bifidobacteria to utilize these substrates in comparison with glucose. Batch culture experiments demonstrated that the growth of Bifidobacterium infantis had an inhibitory effect towards E. coli and Clostridium perfringens. Potentially, an increase in the concentration of these substrates in the diet may therefore improve the composition of the large intestinal microflora and have positive effects on the quality of the Western diet.

Adult↗

PubMed1995

In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using fecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle.

We evaluated the influence of gastrointestinal tract microflora from several species on fiber fermentation characteristics in vitro. Selected fibrous substrates (cellulose, beet pulp, citrus pulp, and citrus pectin) were incubated for 6, 12, 24, and 48 h with ruminal fluid from cattle or feces from dogs, cats, pigs, horses, or humans. When data were pooled across all substrates and fermentation times, OM disappearance (29.4%) and acetate, propionate, butyrate, and total short-chain fatty acid (SCFA) production (1.09, .41, .12, and 1.61 mmol/g of OM, respectively) were lowest (P < .05), and lactate production (.23 mmol/g of OM) was greatest (P < .05) for horse fecal microflora compared with samples from the other species. The greatest (P < .05) acetate production resulted when substrates were fermented by cat fecal microflora (2.38 mmol/g of OM). The greatest (P < .05) propionate productions resulted from pig fecal and cattle ruminal microflora (.88 and .83 mmol/g of OM, respectively), and the greatest (P < .05) butyrate productions resulted from human and pig fecal microflora (.39 and .40 mmol/g of OM, respectively). Total SCFA production was greatest (P < .05) for cat fecal microflora (3.38 mmol/g of OM). When data were pooled across the species, substrate OM disappearance and SCFA production ranked from least to greatest in the following order: cellulose < beet pulp < citrus pulp < citrus pectin. The fermentability of different fibrous substrates by fecal or ruminal microflora from various species seems to be dependent not only on the fermentative activity of the microbial population but on other factors as well, perhaps lag time and rate of digesta passage.

Adult↗

PubMed2000

In vitro fermentation pattern of D-tagatose is affected by adaptation of the microbiota from the gastrointestinal tract of pigs.

Knowledge of the fermentation pattern of D-tagatose is important for the assessment of energy value and compliance of D-tagatose. In vitro fermentation experiments with pig intestinal contents and bacteria harvested from the gastrointestinal tract of pigs were used to investigate the degradation of D-tagatose and the formation of fermentation products. Two groups of eight pigs were fed either a control diet containing 150 g/kg sucrose or a diet which had 100 g/kg of the sucrose replaced by D-tagatose. After 18 d the pigs were killed and the gastrointestinal contents collected for in vitro studies. No microbial fermentation of D-tagatose occurred in the stomach or in the small intestine, whereas the sugar was fermented in the cecum and colon. Formate, acetate, propionate, butyrate, valerate, caproate and some heptanoate were produced by the microbial fermentation of D-tagatose by gut microbiota. Hydrogen and methane were also produced. The population of D-tagatose-degrading bacteria in fecal samples and the capacity of bacteria from the hindgut to degrade D-tagatose were higher in the pigs adapted to D-tagatose compared with unadapted pigs. In unadapted pigs, the major fermentation product from D-tagatose was acetic acid. Much more butyric and valeric acids were produced from D-tagatose by bacterial slurries of tagatose-adapted pigs compared with unadapted pigs; this was especially the case for samples from the colon. We conclude that D-tagatose is not fermented in the upper gastrointestinal tract, and the ability of the large intestinal microbiota to ferment D-tagatose is dependent on adaptation.

Acetic Acid↗

PubMed1993

The dependence of the in vitro fermentation of dietary fibre to short-chain fatty acids on the contents of soluble non-starch polysaccharides.

The fermentability of cellulose and dietary fibre in common clinical use (Inolaxol, Fiberform, Vi-Siblin, Lunelax, pectin) was measured as the in vitro production of short-chain fatty acids, lactate, and ammonia in 16.6% faecal homogenates from 18 healthy volunteers. The results were compared with the contents of soluble and insoluble non-starch polysaccharides as determined by the method of Englyst. The amounts of soluble non-starch polysaccharides in the fibre were closely associated with the mean productions of short-chain fatty acids after 6 h (R = 0.94, p < 0.002) and 24 h (R = 0.98, p < 0.0002) of incubation. The mean production of ammonia was inversely related to the soluble fraction of the fibre (after 6 h, R = -0.93, p < 0.003; after 24 h, R = -0.90, p < 0.006). These variables were not dependent on the insoluble fractions of the fibre. The in vitro fermentability differed considerably among the fibres: cellulose and Inolaxol (sterculia gum) were almost non-fermentable, Fiberform (wheat bran-based) was low-grade fermentable, Vi-Siblin and Lunelax (both ispaghula husk) were intermediately fermentable, and pectin was highly fermentable. These findings support that the water solubility determines the degree of fermentability of dietary fibre and thereby the corresponding bacterial assimilation of ammonia. In vitro measurements of short-chain fatty acid production in faecal homogenates may hence supplement commonly used methods to classify dietary fibre.

Adult↗