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.

Effect of two levels of transgalactosylated oligosaccharide intake in rats associated with human faecal microflora on bacterial glycolytic activity, end-products of fermentation and bacterial steroid transformation.

The effects of two levels of transgalactosylated oligosaccharide (TOS) intake on bacterial glycolytic activity, end products of fermentation and bacterial steroid transformation were studied in rats associated with a human faecal flora. Rats were fed a human-type diet containing 0, 5 or 10% TOS. Caecal pH decrease correlated with the amount of TOS in the diet. Intake of the TOS diet induced a decrease in blood cholesterol and a strong increase in beta-galactosidase activity in the hindgut. TOS fermentation led to production of hydrogen and short chain fatty acids, whereas ammonia and branched-chain fatty acids were decreased. A diet containing 10% TOS increased caecal lactic acid concentrations and reduced beta-glucuronidase activities and steroid transformation.

Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora.

Washed bacterial suspensions obtained from the pig hindgut were incubated under 13CO2 in a buffer containing NaH13CO3 and carbohydrates. Incorporation of 13C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H2 added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H2 markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H2 significantly increased the incorporation of 13CO2 into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H2 partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO2 and H2 (represented mostly by double labelling). A highly significant interaction between BES and H2 concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.

Effects of galacto-oligosaccharide and bacterial status on mucin distribution in mucosa and on large intestine fermentation in rats.

The purpose of the present paper was to study the effects of a dietary undigestible carbohydrate and intestinal microflora on mucin distribution (neutral, acid, sulphonated), glycolytic activities: beta-D-galactosidase (EC 3.2.1.23), N-acetyl-beta-D-galactosaminidase (EC 3.2.1.43), N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), alpha-L-fucosidase (EC 3.2.1.51) and bacterial metabolism (gas production, short-chain fatty acids (SCFA) and lactic acid caecal concentration) in germ-free (GF), conventional (CV) and heteroxenic (HE) rats (GF rats associated with a human flora). Rats were fed on either a control diet or a diet containing 40 g trans-galactosylated oligosaccharide (TOS)/kg. In GF rats fed on the control diet caecal pH was almost neutral and glycolytic activities negligible. The number of mucus-containing cells increased from the caecum to the colon for the three types of mucin. TOS had no effect in the caecum but it modified mucin cell repartition in the colon. In CV and HE rats fed on the control diet caecal pH was similar (6.8), but caecal SCFA and lactic acid concentrations (mumol/g) and gas production (ml/24 h) were higher in CV (70, 5.9 and 2.3 respectively) than in HE rats (32, 4.6 and 0.4 respectively). In CV, as in HE rats, acid-mucin-containing cells increased from the caecum to the colon and glycolytic activities were similar. TOS reduced acid-mucin-containing cells in the caecum of CV rats by twofold but had no effect in either the caecum or the colon of HE rats. TOS strongly increased beta-galactosidase activity and slightly modified the other glycolytic activities. Its effect on bacterial metabolites depended on bacterial status. However, comparison between CV and HE rats showed no evident relationship between the number of mucus-containing cells and measured bacterial metabolites. Differences between CV and HE rats might be due to bacterial microflora specificity. TOS had an intrinsic effect on mucus cell distribution in the colon of GF rats. In CV and HE rats the presence of the flora abolished this effect.

NMR study of 13CO2 incorporation into short-chain fatty acids by pig large-intestinal flora.

The nuclear magnetic resonance technique was used to study carbon dioxide reduction by the pig large-intestinal flora. Washed bacterial cell suspensions were incubated for 6 and 15 h under 13CO2 and H2 as the gas phase and with a buffer containing NaH13CO3 and cellobiose and amino acids (casein hydrolysate) as substrates. Methane was produced in all incubation media. Significant amounts of single- as well as multiple-labelled acetate and butyrate were formed, demonstrating synthesis of acetate from H2 + CO2. Propionate was labelled mainly on the carboxyl group, which was attributed to an enzymatic exchange of the carboxyl group of propionate with 13CO2. These results indicate that the reduction of CO2 to acetate may be an important pathway for microbial production of acetate in the pig large intestine even in the presence of methanogenesis.

Effects of phosphorus deficiency on rumen microbial activity associated with the solid and liquid phases of a fermentor (Rusitec).

The rumen simulation technique (Rusitec) has been used to study the effects of phosphorus (P) deficiency on bacterial protein synthesis and chemical composition and on adenosine triphosphate (ATP) concentrations in the solid and liquid phases of fermentors. 16 g DM of a P-deficient mixed diet was put into each vessel daily and the vessels were infused with about 1 l of a P-deficient (0 mg of P) or P-supplemented (120 mg of P/l) buffer. Two vessels per treatment were used, and during a second experimental period the deficient and supplemented buffers were interchanged to determine the effects of P repletion and depletion. The proportion of bacterial N directly incorporated from dietary amino acids or peptides was greater in solid-associated bacteria (SAB) than in liquid-associated bacteria (LAB) (46 vs 19%). P deficiency increased this proportion in both bacterial populations. Protein synthesis associated with the solid phase represented about 30% of the total protein synthesis in the system. P deficiency induced a marked decrease in microbial protein synthesis in both phases. Microbial yield declined by about 5 points (g of N/kg OMF) in P-deficient conditions. ATP concentrations were greatly reduced in both phases but P deficiency had no effect on protozoal numbers. The effects of P depletion during period II were similar to those of P deficiency in period I, and P repletion showed that the effects of P deficiency were almost entirely reversible.

[In vitro estimation using radioactive phosphorus of the phosphorus requirements of rumen microorganisms]. / Estimation in vitro à l'aide du phosphore radioactif des besoins en phosphore des microorganismes du rumen.

Microbial requirements for P were assumed to be a function of the amount of microbial protein synthesis (microbial growth) and of the quantity of organic matter (OM) fermented in the rumen. The relationships among P incorporation into microbial matter and protein synthesis, ammonia utilization, volatile fatty acid (VFA) production and organic matter fermented (OMF) were studied in short-term incubations (3 h) using 32P-labelled phosphate. The amount of P incorporated was calculated from extracellular phosphate pool specific activity and the radioactivity incorporated into the microbial sediment during incubation (table 1). The inocula came from sheep fed a protein-free purified diet. In order to vary the intensity of fermentation, carbohydrates with a wide range of degrees of enzymatic susceptibility were used as substrates and the medium was either provided or was deficient in S and trace elements (table 4). Nitrogen was supplied as ammonium salts. Linear regression analyses showed that P incorporation was positively correlated with the criteria of protein synthesis and OM fermentation (figs. 1, 2, 3, 4). However, there was significant phosphorus incorporation when the value for nitrogen incorporation was zero (equation A: (Pi (mg) = 0.162 NH3-N + 0.376; r = 0.9). This was assumed to result either from energetic uncoupling (fermentation without concomitant bacterial growth) or from the lysis of cold microbial cells only. Equation A would reflect total P incorporation and equation A' Pi (mg) = 0.162 NH3-N (mg), net P incorporation. It was assumed that in vitro microbial requirements for P were in the range of 30-70 mg of P/liter of medium for 3-hour incubation, depending on the intensity of fermentation. From a mean value of microbial N yield of 30 g/kg of DOMR (organic matter apparently digested in the rumen), it was calculated that the total and net P requirements in vivo were 6 and 4.9 g/kg of DOMR, respectively, corresponding to 3.9 and 3.2 g/kg of DOM (digestible organic matter). From equation D, relating Pi to OMF, the P requirements were about 4.4 g/kg of DOM. It is suggested that microbial requirements for P varied from 3 to 5 g of P/kg of DOM, depending on the efficiency of microbial synthesis and the extent of carbohydrate fermentation. These results, considered as indicative, should be checked in in vivo experiments.