Biomedical subjects

G T Macfarlane

Publications and source records attributed to G T Macfarlane.

65 records · Page 4Linked to original sources

PubMed1988

Effect of nitrate on methane production and fermentation by slurries of human faecal bacteria.

Most probable number counts showed that denitrifying species were the numerically predominant NO3- reducing bacteria in the faeces of five methanogenic individuals [about 10(10) bacteria (g dry wt faeces)-1]. In faecal slurries, however, denitrification was a relatively minor route of NO3- dissimilation, since only about 3% of the NO3- was converted to gaseous products, with NO3- being mainly reduced to NO2- and NH4+. When KNO2 was added to the slurries, denitrification became quantitatively more significant with approximately 23% of the NO2- being lost as gaseous products. The addition of KNO3 (10 mM) to slurries containing either starch or casein significantly decreased H2 and CH4 production. The effect of NO3- on methanogenesis was twofold: firstly, H2 accumulation decreased due to diversion of electrons towards NO3-/NO2- reduction, and as a result of H2 being used as an electron donor for NO3- reduction, resulting in the removal of the methanogenic substrate; secondly, there was direct inhibition of methane-producing bacteria by NO3- and NO2-. In starch-containing slurries, acetate: butyrate molar ratios were increased when NO3- was added but this effect was not observed when casein replaced starch. These results show that the ability of NO3-/NO2- to act as an electron sink can significantly influence the major products of the human colonic fermentation.

Bacteria↗

PubMed1988

Characterization of proteases formed by Bacteroides fragilis.

Bacteroides fragilis NCDO 2217 produced three major proteases, P1, P2 and P3 of estimated molecular masses 73, 52 and 34 kDa respectively. Protease P1 weakly hydrolysed azocasein but strongly hydrolysed valyl-alanine p-nitroanilide (VAPNA), glycyl-proline p-nitroanilide (GPRPNA), and to a lesser extent leucine p-nitroanilide (LPNA), indicating it to be an exopeptidase. Proteases P2 and P3 hydrolysed only azocasein and LPNA. The high protease:arylamidase ratios of these enzymes indicated that they were probably endopeptidases. Experiments with protease inhibitors suggested that P1 and P2 had characteristics of serine and metalloproteases respectively and that P3 was a cysteine protease. The proteolytic activity of whole cells was stimulated by divalent metal ions such as Mn2+, Ca2+ and Mg2+, but was strongly inhibited (about 95%) by Cu2+ and Zn2+. The temperature optimum for protein hydrolysis was 43 degrees C. Proteolysis was temperature sensitive, however (90% reduction at 60 degrees C) and was maximal at alkaline pH, with two broad peaks at pH 7.9 and pH 8.8. Cell fractionation showed that P1 was located intracellularly and in the periplasm, whereas P2 and P3 were largely associated with the outer membrane. Release of the membrane-bound proteases by treatment with 1 M-NaCl suggested that ionic interactions were involved in the association of these enzymes with the membranes.

Aminopeptidases↗

PubMed1988

Occurrence of sulphate-reducing bacteria in human faeces and the relationship of dissimilatory sulphate reduction to methanogenesis in the large gut.

Sulphate-reducing bacteria (SRB) were enumerated in 40 faecal samples obtained from two different human populations in the United Kingdom and rural South Africa. Species able to metabolize acetate, lactate, propionate, butyrate, H2/CO2, succinate, pyruvate, valerate, ethanol and a glutamate/serine/alanine mixture were found in faeces from both populations. Although a variety of nutritionally and morphologically distinct species of SRB belonging to the genera Desulfotomaculum, Desulfobacter, Desulfomonas and Desulfobulbus were identified, Desulfovibrio types always predominated. Significant numbers of SRB were present only in faecal samples from subjects whose breath methane excretion was low or undetectable. Reduced or absent methanogenesis in the presence of SRB was confirmed in fermentation studies with faecal slurries. Fourteen of 20 (70%) British faecal samples contained SRB and the remainder produced methane. The reverse was the case with 20 rural black South Africans, where only three (15%) of the samples had significant levels of SRB; the remaining 85% produced methane. These results suggest that to a large extent, dissimilatory sulphate reduction and methanogenesis are mutually exclusive in the human large gut.

Breath Tests↗

PubMed1988

Competition for hydrogen between sulphate-reducing bacteria and methanogenic bacteria from the human large intestine.

Sulphate-reducing activity in human faecal slurries was followed by measuring sulphide production. Sulphate-reducing bacteria (SRB) were found to outcompete methanogenic bacteria (MB) for the mutual substrate hydrogen in faecal slurries from methane- and non-methane-producing individuals mixed together. When molybdate (20 mmol/l) was added to these slurries, sulphate reduction was inhibited and methanogenesis became the major route of electron disposal. Sulphide production was stimulated by the addition of 20 mmol/l sulphate in non-methanogenic but not in methanogenic slurries. In methanogenic slurries that contained the methanogen inhibitor 2-bromoethanesulphonic acid (BES), hydrogen accumulated whilst sulphide levels were unaffected, confirming the absence of SRB in methanogenic faeces. The addition of nitrate (10 mmol/l) to faecal slurries completely inhibited methanogenesis but only slightly reduced sulphate reduction. The sulphated mucopolysaccharides, chondroitin sulphate and mucin, strongly stimulated sulphide production in non-methanogenic faecal slurries only, suggesting that these substances may be a potential source of sulphate in the large gut.

Alkanesulfonates↗

PubMed1988

Contribution of the microflora to proteolysis in the human large intestine.

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p-nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis-type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.

Aminopeptidases↗

PubMed1988

Use of a three-stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria.

A mixed culture of human fecal bacteria was grown for 120 days in a three-stage continuous culture system. To reproduce some of the nutritional and pH characteristics of the large gut, each vessel had a different operating volume (0.3, 0.5, and 0.8 liter) and pH (6.0, 6.5, and 7.0). A mixture of polysaccharides and proteins was used as carbon and nitrogen sources. Measurements of H2, CH4, S2-, sulfate reduction rates, sulfate-reducing bacteria (SRB), and volatile fatty acids were made throughout the experiment. After 48 days of running, porcine gastric mucin (5.8 g/day) was independently fed to vessel 1 of the multichamber system. The mucin was extensively degraded as evidenced by the stimulation of volatile fatty acid production. In the absence of mucin, sulfate-reducing activity was comparatively insignificant and methanogenesis was the major route for the disposal of electrons. The reverse occurred upon the addition of mucin; sulfate reduction predominated and methanogenesis was completely inhibited. This was attributed to release of sulfate from the mucin which enabled SRB to outcompete methanogenic bacteria for H2. SRB stimulated by mucin were acetate-utilizing Desulfobacter spp., lactate- and H2-utilizing Desulfovibrio spp., and propionate-utilizing Desulfobulbus spp. When the mucin pump was switched off, the multichamber system reverted to a state close to its original equilibrium. These data provide further evidence that sulfated polysaccharides such as mucin may be a source of sulfate for SRB in the human large gut.

Bacteria, Anaerobic↗

PubMed1987

Short chain fatty acids in human large intestine, portal, hepatic and venous blood.

Evidence for the occurrence of microbial breakdown of carbohydrate in the human colon has been sought by measuring short chain fatty acid (SCFA) concentrations in the contents of all regions of the large intestine and in portal, hepatic and peripheral venous blood obtained at autopsy of sudden death victims within four hours of death. Total SCFA concentration (mmol/kg) was low in the terminal ileum at 13 +/- 6 but high in all regions of the colon ranging from 131 +/- 9 in the caecum to 80 +/- 11 in the descending colon. The presence of branched chain fatty acids was also noted. A significant trend from high to low concentrations was found on passing distally from caecum to descending colon. pH also changed with region from 5.6 +/- 0.2 in the caecum to 6.6 +/- 0.1 in the descending colon. pH and SCFA concentrations were inversely related. Total SCFA (mumol/l) in blood was, portal 375 +/- 70, hepatic 148 +/- 42 and peripheral 79 +/- 22. In all samples acetate was the principal anion but molar ratios of the three principal SCFA changed on going from colonic contents to portal blood to hepatic vein indicating greater uptake of butyrate by the colonic epithelium and propionate by the liver. These data indicate that substantial carbohydrate, and possibly protein, fermentation is occurring in the human large intestine, principally in the caecum and ascending colon and that the large bowel may have a greater role to play in digestion than has previously been ascribed to it.

Acetates↗

PubMed1986

Protein degradation by human intestinal bacteria.

Analysis of human gut contents showed that substantial quantities of soluble protein, ammonia and branched chain volatile fatty acids occurred throughout the large intestine [0.1-24.4 g (kg contents)-1, 7.7-66.0 mmol (kg contents)-1 and 1.5-11.1 mmol (kg contents)-1 respectively]. The presence of these metabolites suggested that substantial proteolysis was occurring. In vitro studies showed that casein and bovine serum albumin were partly degraded in slurries of human faeces over a 96 h incubation period, to produce TCA-soluble peptides, ammonia and volatile fatty acids. Proteolytic activity detected in the stools of five individuals ranged from 3.5 to 19.8 mg azocasein hydrolysed h-1 (g faecal material)-1. Washed cell and washed particulate faecal fractions accounted for 24-67% of total activity. The predominant proteolytic bacteria in the faecal samples examined were identified as Bacteroides spp. [1.0 X 10(11)-1.3 X 10(12) (g dry wt faeces)-1] and Propionibacterium spp. [1.2 X 10(8)-1.0 X 10(10) (g dry wt faeces)-1]. Other proteolytic bacteria which occurred in lesser numbers were identified as belonging to the genera Streptococcus, Clostridium, Bacillus and Staphylococcus. These results demonstrate that the gut microflora could potentially play a major role in proteolysis in the human colon.

Bacillus↗

PubMed1986

Starch utilization by the human large intestinal microflora.

High levels (2-565 units/g) of amylase activity were observed in human faeces. Over 92% of amylase activity in faeces obtained from healthy persons was extracellular, whereas only about 9% of activity was associated with particulate material and washed cells. Bacterial cell-bound amylases were considerably more efficient in breaking down starch, however, than were the soluble enzymes which occurred in cell-free faecal supernatant fluids. Cell population densities of anaerobic starch-hydrolysing bacteria in the stools of ten persons ranged from 1.1 X 10(10) to 3.3 X 10(12)/g of faeces. Identification of 120 starch-hydrolysing colonies isolated from the stools of six subjects showed that the predominant amylolytic bacteria belonged to the genera Bifidobacterium, Bacteroides, Fusobacterium and Butyrivibrio. Mixed populations of gut bacteria rapidly fermented starch with the production of volatile fatty acids and organic acids. Lactate was observed to be a major, though transient intermediate during starch fermentation by these cultures. Approximately 60% of starch utilized was converted to volatile fatty acids, which in the human colon would be potentially available for absorption.

Amylases↗

PubMed1980

Asparagine and glutamine metabolism in Rhodopseudomonas acidophila.

Rhodopseudomonas acidophila strain 7050 achieved balance growth when provided with either asparagine or glutamine as nitrogen source. Under these growth conditions R. acidophila synthesized a mixed amidase which exhibited similar activity (223--422 nmol/min . mg protein) against either nitrogen source. Determination of the free intracellular amino acid pools show that deamidation of asparagine and glutamine resulted in elevated levels of both aspartate and glutamate. Cell-free extracts of R. acidophila showed significant aminotransferase activity, particularly glutamine-oxaloacetate aminotransferase (89.7--209.3 nmol/min . mg protein), glycine oxaloacetate aminotransferase (135--227 nmol/min . mg protein), alanine glyoxylate aminotransferase (66.3--163.2 nmol/min . mg protein) and serine-glyoxylate aminotransferase (57.1--68.4 nmol/min . mg protein). Short term labelling experiments using 14C-glyoxylate show that glycine plays an important role in amino nitrogen transfer in R. acidophila and that the enzymes for the metabolism of glyoxylate via glycine, serine and hydroxypyruvate were present in cell-free extracts. These data confirm that R. acidophila can satisfy all its' nitrogen requirements by transamination.

Asparaginase↗

PubMedDate not supplied

Role of intestinal bacteria in nutrient metabolism.

The human large intestine contains a microbiota, the components of which are generically complex and metabolically diverse. Its primary function is to salvage energy from carbohydrate not digested in the upper gut. This is achieved through fermentation and absorption of the major products, short chain fatty acids (SCFA), which represent 40-50% of the available energy of the carbohydrate. The principal SCFA, acetate, propionate and butyrate, are metabolized by the colonic epithelium (butyrate), liver (propionate) and muscle (acetate). Intestinal bacteria also have a role in the synthesis of vitamins B and K and the metabolism of bile acids, other sterols and xenobiotics. The colonic microflora are also responsive to diet. In the presence of fermentable carbohydrate substrates such as non-starch polysaccharides, resistant starch and oligosaccharides, bacteria grow and actively synthesize protein. The amount of protein synthesis and turnover within the large intestine is difficult to determine, but around 15 g biomass is excreted in faeces each day containing 1 g bacterial-N. Whether bacterially synthesized amino acids are ever absorbed from the colon remains unclear. Finally, individual colonic micro-organisms such as sulphate-reducing bacteria, bifidobacteria and clostridia, respond selectively to specific dietary components in a way that may be important to health.

Bacteria↗