Estimation of short-chain fatty acid production from protein by human intestinal bacteria based on branched-chain fatty acid measurements

Abstract The importance of protein breakdown and amino acid fermentation in the overall economy of the large intestine has not been quantitated. We have therefore measured the production of branched chain-fatty acids (BCFA) both in vitro and in vivo in order to estimate the contribution of protein to fermentation.
In vitro batch-culture studies using human faecal inocula showed that short-chain fatty acids (SCFA) were the principal end products formed during the degradation of protein by human colonic bacteria. Approximately 30% of the protein broken down was converted to SCFA. Branched-chain fatty acids (BCFA) constituted 16% of the SCFA produced from bovine serum albumin and 21% of the SCFA generated when casein was the substrate. BCFA concentrations in gut contents taken from the human proximal and distal colons were on average, 4.6 and 6.3 mmol kg⁻¹ respectively, corresponding to 3.4% and 7.5% of the total SCFA. These results suggest that protein fermentation could potentially account for about 17% of the SCFA found in the caecum, and 38% of the SCFA produced in the sigmoid/rectum. Measurements of BCFA in portal and arterial blood taken from individuals undergoing emergency surgery indicated that net production of BCFA by the gut microflora was in the region of 11.1 mmol day⁻¹, which would require the breakdown of about 12 g of protein. These data highlight the role of protein in the colon and may explain why many colonic diseases affect mainly the distal bowel.     

Engineering Escherichia coli to produce branched-chain fatty acids in high percentages

Branched-chain fatty acids (BCFAs) are key precursors of branched-chain fuels, which have cold-flow properties superior to straight chain fuels. BCFA production in Gram-negative bacterial hosts is inherently challenging because it competes directly with essential and efficient straight-chain fatty acid (SCFA) biosynthesis. Previously, Escherichia coli strains engineered for BCFA production also co-produced a large percentage of SCFA, complicating efficient isolation of BCFA. Here, we identified a key bottleneck in BCFA production: incomplete lipoylation of 2-oxoacid dehydrogenases. We engineered two protein lipoylation pathways that not only restored 2-oxoacid dehydrogenase lipoylation, but also increased BCFA production dramatically. E. coli expressing an optimized lipoylation pathway produced 276 mg/L BCFA, comprising 85% of the total free fatty acids (FFAs). Furthermore, we fine-tuned BCFA branch positions, yielding strains specifically producing ante-iso or odd-chain iso BCFA as 77% of total FFA, separately. When coupled with an engineered branched-chain amino acid pathway to enrich the branched-chain α-ketoacid pool, BCFA can be produced from glucose at 181 mg/L and 72% of total FFA. While E. coli can metabolize BCFAs, we demonstrated that they are not incorporated into the cell membrane, allowing our system to produce a high percentage of BCFA without affecting membrane fluidity. Overall, this work establishes a platform for high percentage BCFA production, providing the basis for efficient and specific production of a variety of branched-chain hydrocarbons in engineered bacterial hosts.     

Evidence for the synthesis of the multi-positional isomers of monounsaturated fatty acid in Methylococcus capsusatus by the anaerobic pathway

Abstract The biosynthesis of the positional isomers of the monounsaturated fatty acids of Methylococcus capsulatus (Bath) has been investigated by studying the incorporation of [2-¹⁴C]malonyl CoA into long-chain fatty acids in vitro. The major unsaturated products were Δ ⁹ 16:1 and Δ ¹¹ 18:1; however, Δ ⁸, Δ ¹⁰ and Δ ¹¹ 16:1, as well as, Δ ¹⁰, Δ ¹² and Δ ¹³ 18:1 were also synthesized. The exclusion of O₂ from the reaction vessel did not affect the synthesis of unsaturated fatty acids or the double bonds positions. Cerulenin inhibited the synthesis of unsaturated fatty acid more than saturated fatty acid. The use of both [1-¹⁴C] octanoate and [1-¹⁴C] decanoate as substrate resulted in the synthesis of long-chain fatty acids, however, unsaturates were only synthesized from octanoate. These results imply that the unique positional isomers of M. capsulatus are not synthesized by an aerobic mechanism.     

Branched-chain fatty acid biosynthesis in Escherichia coli

β-Ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) catalyzes the first elongation step in straight-chain fatty acid (SCFA) biosynthesis in Escherichia coli. Overproduction of the corresponding KASIII gene, or the Brassica napus KASIII gene has previously been observed to lead to an increase in the amount of shorter-chain fatty acids produced by E. coli. In this study it is shown that overexpression of the KASIII gene, which initiates branched-chain fatty acid (BCFA) in Streptomyces glaucescens, does not lead to a change in the fatty acid profiles of E. coli. E. coli produces trace levels of BCFAs when grown in the presence of isobutyric acid, but the amounts of these are not significantly altered by expression of the S. glaucescens KASIII gene. In contrast, the amounts of BCFAs produced from isobutyryl CoA in vitro by E. coli cell-free extracts can be increased at least four-fold by the presence of the S. glaucescens KASIII. These observations suggest that in vivo production of isopalmitate by E. coli expressing the S. glaucescens KASIII is limited by availability of the appropriate BCFA biosynthetic primers. Journal of Industrial Microbiology & Biotechnology (2001) 27, 246–251. Received 10 January 2001/ Accepted in revised form 13 July 2001     

Comparison of fermentation reactions in different regions of the human colon

Colonic contents were obtained from two human sudden-death victims within 3 h of death. One of the subjects (1) was methanogenic, the other (2) was a non-CH, producer. Measurements of bacterial fermentation products showed that in both individuals short-chain fatty acids, lactate and ethanol concentrations were highest in the caecum and ascending colon. In contrast, products of protein fermentation, such as ammonia, branched chain fatty acids and phenolic compounds, progressively increased from the right to the left colon, as did the pH of gut contents. In Subject 1, cell population densities of methanogenic bacteria (MB) increased distally through the gut and methanogenic activity was lower in the right (0.78–1–18 μmol CH₄ produced/h/g dry wt contents) than in the left colon (1.34 μmol CH₄ produced/h/g dry wt contents). Methane production rates did not correlate with MB numbers.
Sulphate-reducing bacteria (SRB) were not found and dissimilatory sulphate reduction was not detected in any region of the colon. Methanogenic bacteria did not occur in subject 2, but high numbers of SRB were present throughout the gut ( ca 10⁹/g dry wt contents). Sulphate reduction rates were maximal in the ascending and transverse colons (0.24 and 0.22 μmol ³⁵SO^2–₄ reduced/h/g dry wt contents, respectively). Short-chain fatty acid production by caecal contents was up to eight-fold higher than contents from the sigmoid/rectum. These findings demonstrate significant differences in fermentation reactions in different regions of the large gut.     

Release of Neurotransmitter Amino Acids from Synaptosomes: Enhancement of Calcium-Independent Efflux by Oleic and Arachidonic Acids

Abstract: The release of preloaded [¹⁴C]neuroactive amino acids (glutamic acid, proline, γ-aminobutyric acid) from rat brain synaptosomes can occur via a time-dependent, Ca²⁺ -independent process. This Ca²⁺-independent efflux is increased by compounds that activate Na⁺ channels (veratridine, scorpion venoms), by the ionophore gramicidin D, and by low concentrations of unsaturated fatty acids (oleic acid and arachidonic acid). Saturated fatty acids have no effect on the efflux process. Neither saturated nor unsaturated fatty acids have an effect on the release of [¹⁴C]leucine, an amino acid not known to possess neurotransmitter properties. The increase in the efflux of neuroactive amino acids by oleic and arachidonic acids can also be demonstrated using synaptosomal membrane vesicles. Under conditions in which unsaturated free fatty acids enhance amino acid efflux, no effect on ²²Na⁺ permeability is observed. Since Na⁺ permeability is not altered by fatty acids, the synaptosomes are not depolarized in their presence and, thus, the Na⁺ gradient can be assumed to be undisturbed. We conclude that unsaturated fatty acids represent a potentially important class of endogenous modulators of neuroactive amino acid transport in nerve endings and further postulate that their action is the result of an uncoupling of amino acid transport from the synaptosomal Na⁺ gradient.     

Choline Uptake in Cultured Human Y79 Retinoblastoma Cells: Effect of Polyunsaturated Fatty Acid Compositional Modifications

Abstract: Choline uptake in Y79 human retinoblastoma cells occurs through two kinetically distinguishable processes. The high-affinity system shows little sodium or energy dependence, and it does not appear to be linked to acetyl CoA: choline O -acetyltransferase. When the cells are grown in a culture medium containing 10% fetal bovine serum, the high-affinity system has a K' _m= 2.16 ± 0.13 μ m and V' _max= 27.0 ± 2.9 pmol min⁻¹ mg⁻¹, whereas the low-affinity system has K' _m= 20.4 ± 1.3 μ m and V' _max= 402 ± 49 pmol min⁻¹ mg⁻¹. Under these conditions, the polyunsaturated fatty acid content of the cell membranes is relatively low. When the polyunsaturated fatty acid content of the microsomal membrane fraction was increased by supplementing the culture medium with linolenic or docosahexaenoic acids (n-3 polyunsaturated fatty acids) or arachidonic acid (n-6 polyunsaturated fatty acid), the K' ^m of the high-affinity choline transport system was reduced by 40–60%. The V' _max also was reduced by 20–40%. Supplementation with oleic acid, the most prevalent monounsaturated fatty acid, did not affect either kinetic parameter. The results suggest that one functional effect of the high unsaturated fatty acid content of neural cell membranes is to facilitate the capacity of the high-affinity choline uptake system to transport low concentrations of choline. This effect appears to be specific for polyunsaturated fatty acids but not for a single type, for it is produced by members of both the n-3 and n-6 classes of polyunsaturated fatty acids.     

Microbial digestion in the herbivorous lizard Uromastyx aegyptius (Agamidae)

The utilization of a diet rich in plant cell walls was studied in a large, desert-dwelling, herbivorous lizard, Uromastyx aegyptius (Agamidae). The diet eaten by U. aegyptius in spring in the 'Arava Valley, Israel, consisted almost entirely of leaves and fruits of short-lived annual plant species. The leaves contained only moderate levels of fibre compared with grasses and tree leaves, but those fruits eaten were markedly higher in fibre and lignin. All items had notably high contents of ash.
Following oral doses of [¹⁴C] cellulose, ¹⁴CO₂ was detected in respired air from U. aegyptius , demonstrating that the cellulose was digested and that the lizards gained oxidative energy from cellulose degradation. The hind gut was the principal site of microbial activity and the apparent digestibility of the cell-wall fraction was 69%. Similarly, the caecum/proximal colon had the highest concentrations of SCFA (76–120 mM).
The mean rate of SCFA production at 40 C in vitro was 31 mmol/1 h^-1. Assuming that this is representative of daily production rate, 69 kJ/kg d^-1 would be made available to the animal. This is 47% of the mean digestible energy intake estimated in free-living animals. Microbial fermentation contributes an important part of the energy budget of U. aegyptius but the effects of variation in body temperature on digestion and fermentation need further consideration.     

Altered fatty acid metabolism and composition in cultured astrocytes under hyperammonemic conditions

In vitro ¹H- and ¹³C-NMR spectroscopy was used to investigate the effect of ammonia on fatty acid synthesis and composition in cultured astrocytes. Cells were incubated 3 and 24 h with 5 mM ammonia in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. An increase of de novo synthesized fatty acids and the glycerol subunit of lipids was observed after 3 h treatment with ammonia (35% and 40% over control, respectively), the initial time point examined. Both parameters further increased significantly to 85% and 60% over control after 24 h ammonia treatment. Three hours incubation with ammonia increased the synthesis of diacylglycerides, while formation of triacylglycerides was decreased (40% over and 15% under control, respectively). The degradation of fatty acids was not affected by ammonia treatment. Furthermore, ammonia caused alterations in the composition of fatty acids, e.g. increased mono- and decreased polyunsaturated fatty acids (85% over and 15% under control concentrations, respectively). The decrease of polyunsaturated fatty acids was even more pronounced in isolated astrocytic mitochondria (39% lower than controls). Our results suggest ammonia-induced abnormalities in astrocytic membranes, which may be related to astrocytic mitochondrial dysfunction in hyperammonemic states. Most of the observed effects of ammonia on fatty acid synthesis and composition were ameliorated when glutamine synthetase was inhibited by methionine sulfoximine, supporting a pathological role of glutamine in ammonia toxicity. This study further emphasizes the importance of investigating the relative contribution of exogenous ammonia, effects of glutamine and of glutamine-derived ammonia on astrocytes and astrocytic mitochondria.     

Segmental differences in short-chain fatty acid transport in rabbit colon: Effect of pH and Na

Short-chain fatty acids (SCFAs) are the predominant luminal anion in the mammalian colon. Although they are rapidly absorbed in vivo, little is known about the mechanisms of transepithelial transport in vitro. Previous studies have suggested that SCFA transport may be linked to Na absorption or an anion exchange mechanism. We compared the transport of propionate under short-circuit conditions in rabbit proximal and distal colon to determine whether there were segmental differences, how SCFAs may be linked to either Na absorption or anion transport, and whether SCFAs, as weak electrolytes, may be affected by transepithelial pH gradients. In distal colon, propionate transport was not significantly altered by stimulation of electrogenic Na absorption, epinephrine or Cl removal. However, a modest transepithelial pH gradient (luminal 6.8/serosal 7.4) stimulated propionate absorption. In proximal colon, propionate transport was significantly altered by manuevers that either stimulated (lowered [Na] in the bathing media) or inhibited (theophylline) apical Na−H exchange. Neither Cl removal, nor the anion exchange inhibitor DIDS, nor a transepithelial bicarbonate gradient, altered propionate transport. A transepithelial pH gradient inhibited propionate secretion, but not in a manner entirely consistent with the effect of pH on the distribution of a weak electrolyte. These results suggest that there is significant segmental heterogeneity in colonic SCFA transport; that transepithelial propionate fluxes are altered by changes in pH or electroneutral Na absorption (Na−H exchange), but not by chloride removal, bicarbonate gradients or electrogenic Na absorption. Regulation of SCFA transport may be an important factor in the physiology of colonic fluid balance.     

Improved method for simultaneous determination of alcohols, volatile fatty acids, lactic acid or 2,3-butanediol in biological samples

A rapid and sample procedure was developed to determine, by gas-liquid chromatography, the concentrations of C₂---C₄ alcohols, C₂---C₆ volatile fatty acids (VFA) and lactic acid or 2,3-butanediol in fermentation liquids. both lactic acid and 2,3-butanediol are oxidized to acetaldehyde by periodic acid and acetaldehyde was eluted before ethanol. A complete separation of the alcohols and acids was performed in <15 min on a column packed with 80/100 Chromosorb WAW, having GP 10% SP-1200/1% H₃PO₄ as the liquid phase. The method was suitable for the analysis of rumen fluid and fermentation products from microbial cultures. The detection limits for all compounds were <0.13 nmol · injection⁻¹.     

In vitro gas production kinetics and short-chain fatty acid production from rumen incubation of diets supplemented with hop cones (Humulus lupulus L.)

The aim of this study was to assess the effects of hop cones (Humulus lupulus L.) from two varieties Aurora and Dana, differing in their α- and β-acid contents, on rumen microbial activity measured with in vitro gas production kinetics and short-chain fatty acids (SCFA) production. Hop cones were added to the total mixed dairy cow ration (CONT) in concentrations simulating a cow’s daily intake of 50, 100 and 200 g of hop cones – the concentrations of hop cones expressed on a substrate basis were 43, 82 and 153 mg/g of substrate. Substrates were anaerobically incubated in glass syringes, and gas production kinetic parameters were determined by fitting data with the Gompertz model. Gas produced after 24 h (Gas24), maximum fermentation rate (MFR) and time of maximum fermentation rate (TMFR) were calculated from the estimated gas production kinetic parameters. After 24 h of incubation, the fermentation liquids of each substrate were taken for the determination of SCFA. Increasing the hop cone concentration decreased the total potential gas production, Gas24, MFR and shortened TMFR. The highest hop cone concentration significantly decreased acetic and butyric acid productions and total SCFA production after 24 h of incubation, but not propionic acid production, resulting in a decreased ratio between acetic acid and propionic acid.     

Effect of growth temperature and salt concentration on the fatty acid composition of Flavobacterium halmephilum CCM2831

Abstract In this study the fatty acid composition of Flavobacterium halmephilum CCM2831 grown at different temperatures and salt concentrations is reported. At elevated growth temperatures the amount of cellular saturated long-chain acids (C_18:0 and C_20:0) and the branched-chain acid br-C_17:0 increased, and the concentrations of both cyclic acids and the branched chain acid br-C_15:0 decreased. Increasing the salt concentration in the medium resulted in a gradual increase in cellular cyclopropanoic acids and a concomitant decrease in the monounsaturated fatty acid content.     

SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human intestinal C2/bbe cells

Short-chain fatty acids (SCFA), produced by colonic bacterial flora fermentation of dietary carbohydrates, promote colonic Na absorption through mechanisms not well understood. We hypothesized that SCFA promote increased expression of apical membrane Na/H exchange (NHE), serving as luminal physiological cues for regulating colonic Na absorptive capacity. Studies were performed in human colonic C2/bbe (C2) monolayers and in vivo. In C2 cells exposed to butyrate, acetate, proprionate, or the poorly metabolized SCFA isobutyrate, apical membrane NHE3 activity and protein expression increased in a time- and concentration-dependent manner, whereas no changes were observed for NHE2. In contrast, no significant changes in brush-border hydrolase or villin expression were noted. Analogous to the in vitro findings, rats fed the soluble fiber pectin exhibited a time-dependent increase in colonic NHE3, but not NHE2, protein, mRNA, and brush-border activity. These changes were region-specific, as no changes were observed in the ileum. We conclude that luminal SCFA are important physiological cues for regulating colonic Na absorptive function, allowing the colon to adapt to chronic changes in dietary carbohydrate and Na loads.     

The Effect of Temperature on the Fatty Acid Composition of Tetrahymena pyriformis WH-14

SYNOPSIS. A reduction in the growth temperature of Tetrahymena pyriformis strain WH-14 from 35 C to 15 C resulted in distinct alterations in the fatty acid composition of the glycerophospholipids. The proportion of normal saturated acids declined from 26 to 19%; palmitoleic acid increased by 6%, and the composition of the polyunsaturated fatty acids increased in 18:2 Δ^6,11(n) and decreased in 18:2 Δ^9,12(n) and 18:3 Δ^6,9,12(n). The unsaturation index (the average number of double bonds/100 molecules) did not change with a shift in temperature.
Two biosynthetic pathways exist in Tetrahymena for the formation of unsaturated fatty acids. The observed changes in fatty acid composition that accompany a lowering of the environmental temperature can be accounted for by a reduction in the accumulation of products of the fatty acid pathway leading to the formation of γ-linolenic acid [16:0(n) → 18:0(n) → 18:1 Δ⁹(n) → 18:2 Δ^9,12(n) → 18:3 Δ^6,9,12(n)] and an increase in the components of the pathway leading to the formation of 18:2 Δ^6,11(n) [16:0(n) → 16:1 Δ⁹(n) → 18:1 Δ¹¹(n) → 18:2 Δ^6,11(n)]. The data suggest that the regulatory mechanism in Tetrahymena differs from that found in some bacteria where a simple substitution of unsaturated fatty acids for saturated fatty acids occurs at low culture temperatures.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Transmission of pea bacterial blight (Pseudomonas syringae pv. pisi) from seed to seedling: effects of inoculum dose, inoculation method, temperature and soil moisture

Quantitative analyses of the utilization of amino acids by Lactococcus lactis subsp. cremoris FD1 in yeast extract medium (YE) and in casein peptone medium (CP) have been performed. Both free and peptide-bound amino acids were measured. In the CP most amino acids are peptide-bound and some amino acids are virtually only present in peptides. Thirty-six per cent of all peptide bonds in CP are hydrolysed during fermentation (6·3 mmol peptide bonds per gram biomass formed) and there is a transition of the growth rate related ATP consumption Y _xATP (mmol ATP g biomass^-1) from 25 mmol g^-1 to 71 mmol g^-1 coincident with a decrease of the peptide consumption. In YE most of the amino acids are on the free form and only 26% of the peptide bonds are hydrolysed during fermentation (1·5 mmol peptide bonds per gram biomass formed). A constant Y _xATP= 38 mmol g^-1 prevails throughout the fermentation in YE.     

Fermentation of prebiotics by human colonic microbiota in vitro and short-chain fatty acids production: a critical review

Prebiotics are known for their health benefits to man, including reducing cardiovascular disease and improving gut health. This review takes a critical assessment of the impact of dietary fibres and prebiotics on the gastrointestinal microbiota in vitro. The roles of colonic organisms, slow fermentation of prebiotics, production of high butyric and propionic acids and positive modulation of the host health were taken into cognizance. Also, the short-chain fatty acids (SCFAs) molecular signalling mechanisms associated with their prebiotic substrate structural conformations and the phenotypic responses related to the gut microbes composition were discussed. Furthermore, common dietary fibres such as resistant starch, pectin, hemicelluloses, β-glucan and fructan in context of their prebiotic potentials for human health were also explained. Finally, the in vitro human colonic fermentation depends on prebiotic type and its physicochemical characteristics, which will then affect the rate of fermentation, selectivity of micro-organisms to multiply, and SCFAs concentrations and compositions.     

Curdlan intake changes gut microbial composition,short-chain fatty acid production,and bile acid transformation in mice

Indigestible polysaccharides, such as dietary fibers, benefit the host by improving the intestinal environment. Short-chain fatty acids (SCFAs) produced by gut microbial fermentation from dietary fibers exert various physiological effects. The bacterial polysaccharide curdlan benefits the host intestinal environment, although its effect on energy metabolism and SCFA production remains unclear. Hence, this study aimed to elucidate the effect of curdlan intake on gut microbial profiles, SCFA production, and energy metabolism in a high-fat diet (HFD)-induced obese mouse model. Gut microbial composition of fecal samples from curdlan-supplemented HFD-fed mice indicated an elevated abundance of Bacteroidetes, whereas a reduced abundance of Firmicutes was noted at the phylum level compared with that in cellulose-supplemented HFD-fed mice. Moreover, curdlan supplementation resulted in an abundance of the family Bacteroidales S24-7 and Erysipelotrichaceae, and a reduction in Deferribacteres in the feces. Furthermore, curdlan supplementation elevated fecal SCFA levels, particularly butyrate. Although body weight and fat mass were not affected by curdlan supplementation in HFD-induced obese mice, HFD-induced hyperglycemia was significantly suppressed with an increase in plasma insulin and incretin GLP-1 levels. Curdlan supplementation elevated fecal bile acid and SCFA production, improved host metabolic functions by altering the gut microbial composition in mice.     

Utilisation of protein by human gut bacteria

Abstract Mixed populations of human gut bacteria degraded cas casein by producing a variety of cell-bound and extracellular proteolytic enzymes. Casein was initially hydrolysed to TCA soluble peptides which were subsequently broken down to volatile fatty acids, ammonia, dicarboxylic acids and a range of phenolic compounds. Amino acids did not accumulate to any extent during casein breakdown, suggesting that the rate of peptide hydrolysis was the limiting step in protein utilisation by these bacteria. Similar fermentation products were produced from bovine serum albumin, however, the insoluble protein collagen was considerably more resistant to degradation by the colonic microflora, as evidenced by the reduced quantities of fermentation end-products formed.