Anthony J. Richardson

Hydroxycinnamic acids in the digestive tract of livestock and humans

Hydroxycinnamic acids are consumed as water-soluble conjugates and in larger amounts bound to plant cell walls. Bound acids are primarily released by microbial action in the modified forestomach of ruminants and the hindgut of non-ruminant species, including humans. In the rumen, rapid hydrogenation of p-coumaric, ferulic and caffeic acids, followed by dehydroxylation at C4 and more slowly at C3 yields 3-phenylpropionic acid. Phenylpropionate is absorbed and undergoes β-oxidation in the liver to benzoic acid which is then excreted predominately (75-95%) as its glycine conjugate (hippuric acid), but also as the free acid or glucuronide. In non-ruminants, hydroxycinnamates may be absorbed unchanged in the upper digestive tract via a Na + -dependent saturable transport system or escape to the hindgut where they are subject to microbial transformations with further absorption of metabolites. Metabolites of p-coumaric acid found in rat urine are the unchanged compound and its glycine conjugate, the reduced derivative and the β-oxidation product, 4-hydroxybenzoic acid. Caffeic acid and its methyl ethers (ferulic and iso-ferulic acids) are interconvertable and share metabolites. As in the rumen, reduction of the C 3 side-chain, demethylation of ferulate and dehydroxylation at C4 are products of microbial action. Dehydroxylation at C3 is more rarely encountered. The resulting 3-hydroxyphenylpropionic acid is commonly found in the urine of all species and is the major metabolite in rats where relatively little chain-shortening occurs. A larger range of metabolites including C 6 -C 1 compounds have been detected in human urine. Metabolism of hydroxycinnamate dimers found as cross-links between polysaccharide chains has been little studied although evident differences in the ability to metabolise such compounds exist between the human and rumen microflora.

Anti-Inflammatory Implications of the Microbial Transformation of Dietary Phenolic Compounds

Due to the success of therapeutic anti-inflammatory compounds to inhibit, retard, and reverse the development of colon cancer, the identification of dietary compounds as chemopreventives is being vigorously pursued. However, an important factor often overlooked is the metabolic transformation of the food-derived compounds in the gut that may affect their bioactivity. Commonly consumed dietary phenolics (esterified ferulic acid and its 5-5′-linked dimer), which have the potential to undergo predominant microbial transformations (de-esterification, hydrogenation, demethylation, dehydroxylation, and dimer cleavage), were incubated with human microbiota. The metabolites were identified (high-performance liquid chromatography and nuclear magnetic resonance) and confirmed to be present in fresh fecal samples from 4 human volunteers. The potential anti-inflammatory properties were compared by measuring the ability of the parent compounds and their metabolites to modulate prostanoid production in a cell line in which the inflammatory pathways were stimulated following a cytokine-induced insult. The compounds were readily de-esterified and hydrogenated, but no dimer cleavage occurred. Only the monomer underwent demethylation and selective de-hydroxylation. The resultant metabolites had differing effects on prostanoid production ranging from a slight increase to a significant reduction in magnitude. This suggests that the microbial transformation of dietary compounds will have important inflammatory implications in the chemoprevention of colon cancer.

Effects of esculin and esculetin on the survival of Escherichia coli O157 in human faecal slurries, continuous-flow simulations of the rumen and colon and in calves

The human pathogen Escherichia coli O157:H7 is thought to be spread by direct or indirect contact with infected animal or human faeces. The present study investigated the effects of the plant coumarin esculin and its aglycone esculetin on the survival of a strain of E. coli O157 under gut conditions. The addition of these compounds to human faecal slurries and in vitro continuous-flow fermenter models simulating conditions in the human colon and rumen caused marked decreases in the survival of an introduced strain of E. coli O157. When four calves were experimentally infected with E. coli O157 and fed esculin, the pathogen was detected in five of twenty-eight (18 %) of faecal samples examined post-inoculation, compared with thirteen of thirty-five (37 %) of faecal samples examined from five control calves not fed esculin. Coumarin compounds that occur naturally in dietary plants or when supplemented in the diet probably inhibit the survival of E. coli O157 in the gut.

Attachment to cellulose and maturation of attached thalli in the anaerobic rumen fungus Neocallimastix frontalis strain RE1

Observations are presented showing that mature thalli of the rumen anaerobic fungal isolate Neocallimastix frontalis strain RE1 attach to cellulose. This process and subsequent development of the thalli have been studied. Thalli matured without attachment and then attached directly by means of rhizoids to the surface of the substrate. This process was quantified by the measurement of attachment of 14 C-radiolabelled fungal cells pregrown on [UL- 14 C]- d -glucose to filter paper. Washed cell suspensions were separated into zoospores and thalli by filtration and the time-course of attachment of each fraction to filter paper strips was determined. Both thalli and zoospores made a significant contribution to the colonization process. It is suggested that the previously unrecognized ability of mature thalli to attach to cellulose could be of importance in the degradation of plant material in the rumen. After attachment to filter paper the formation of dark brown pigmented sporangia was observed after incubation for periods in excess of 4 d. These had some similarities to resistant sporangia which have been described in aerobic and anaerobic chytrids, but they differed in some important respects. The pigmented cytoplasm retracted, leaving a clear margin within an unthickened cell wall. Histochemical tests showed that the pigment was not melanin and strain RE1 was able to grow and form the pigment in the presence of each of three inhibitors, tricyclazole, PP389 and glyphosate, which block melanin biosynthesis pathways in fungi.

Hydrogen Transfer in Mixed Cultures of Anaerobic Bacteria and Fungi with Methanobrevibacter Smithii

The processes involved in the reduction of the oxides of sulphur, nitrogen and carbon compete for electrons. The free-energy changes involved, and the nature of the environment influence the outcome of this competition. Although methanogenesis is normally seen as the ultimate electron acceptor in anaerobic fermentations, methanogenesis may be competitively inhibited under some conditions, for example in anaerobic sediments containing high concentrations of sulphates (Widdel, 1986), or when methanogenic faecal slurries are supplemented with nitrates (Allison & Macfarlane, 1988).

Hydrogen Transfer Between Neocallimastix Frontalis and Selenomonas Ruminantium Grown in Mixed Culture

The anaerobic fungus Neocallimastix frontalis ferments cellulose to H2, C02, formate, acetate, lactate and ethanol (Bauchop & Mountfort, 1981). Succinate is also a fermentation product of some anaerobic fungi (Prins & Marvin-Sikkema, personal communication, Richardson et al., 1989). Theoretically, all of these products could be utilised by other microorganisms. However not all of these potential interactions are likely to occur in the gut. For example, acetate supports the growth of some methanogens in anaerobic ecosystems with slow turnover times, but in the gut acetate is not an important substrate for methanogenesis (Reviewed by Stewart et al., this symposium). Some other potential interactions based on the utilisation of fungal fermentation products are summarised schematically in Fig 1, which is compiled from the known properties of the named microorganisms (Hungate, 1966; Stewart & Bryant, 1988).

Effect of coumarin on glucose uptake by anaerobic rumen fungi in the presence and absence of

The effect of coumarin (1,2 benzopyrone) on glucose utilisation by the anaerobic rumen fungi Neocallimastix frontalis and N. patriciarum has been compared with the effect of p-coumaric acid. Both compounds largely inhibited glucose utilisation by N. patriciarum strain Cx when present in the medium at a concentration of 2.5 mM, and had a similar effect on N. frontalis strain RE1 at 5 mM. Although in earlier studies co-culturing rumen fungi with Methanobrevibacter smithii enhanced resistance to ionophores, no comparable protective effect of M. smithii was found in the present study.