Jason S Peters

The effect of condensed tannins in Lotus pedunculatus on the solubilization and degradation of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39; Rubisco) protein in the rumen and the sites of Rubisco digestion

Three experiments were undertaken to determine the effect of condensed tannin (CT) in Lotus pedunculatus (45-55 g extractable CT/kg DM) on the digestion of the principal leaf protein, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39; Rubisco; fraction 1 leaf protein). In two of the experiments Lotus pedunculatus was fed to sheep, with one group receiving a continuous intraruminal infusion (per fistulum) of PEG (molecular weight 3500) to bind and inactivate the CT (PEG group). The other group, which did not receive PEG, was termed the control sheep (CT acting). Expt 3 involved in vitro incubations of Lotus pedunculatus in buffered rumen fluid, with and without PEG added. In all experiments the results have been interpreted in terms of the effects of CT on Rubisco solubilization and degradation. Disappearance of N and Rubisco from Lotus pedunculatus suspended in polyester bags in the rumen was used as a measure of solubilization. Degradation was defined as the disappearance of Rubisco from in vitro incubations of Lotus pedunculatus in rumen fluid. In Expt 1, CT reduced the digestion of Rubisco in the rumen from 0.96 to 0.72 of intake (P < 0.01). Rubisco digestion in the small intestine was 0.27 of intake in control sheep and 0.04 of intake in PEG sheep. In Expt 2, PEG had no effect on the loss of Rubisco from Lotus pedunculatus contained in polyester bags which were incubated in the rumen, hence CT did not affect the solubilization of Rubisco. Observations in Expt 1 were confirmed by in vitro incubations in Expt 3, where PEG addition substantially increased the rate of degradation of plant protein to NH3. Addition of PEG decreased the period of time taken to degrade 50% of the Rubisco from about 13.8 h to about 3.0 h. It was concluded that the action of CT reduced the digestion of Rubisco in the rumen of sheep fed on fresh Lotus pedunculatus, and that this was primarily due to the ability of CT to slow its degradation by rumen micro-organisms, without affecting its solubilization. Both fresh-minced, and freeze-dried and ground lotus were used for in sacco and in vitro incubations; however, fresh-minced lotus was more suitable for the evaluation of protein solubilization and degradation in fresh forages.

Solubilization and degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39; Rubisco) protein from white clover (Trifolium repens) and Lotus corniculatus by rumen microorganisms and the effect of condensed tannins on these processes

In situ and in vitro rumen incubations were used to determine the effect of condensed tannins (CT) on the solubilization and degradation of the plant protein from white clover ( Trifolium repens ) and Lotus corniculatus . These forages contained, respectively 0·3 and 22·1 g CT/kg dry matter (DM). The sheep used for the experiments were also fed either white clover or L. corniculatus . Effects of CT were determined by making measurements in the presence and absence of polyethylene glycol (PEG; molecular weight 3500), which binds and inactivates CT. The loss of DM, neutral detergent fibre (NDF), total nitrogen (N) and Rubisco (ribulose-1,5- bis phosphate carboxylase/oxygenase; EC 4.1.1.39; fraction I leaf protein) from polyester bags suspended in the rumen of sheep was measured. The loss of these constituents from polyester bags suspended in the rumen was used as a measurement of their solubilization. Degradation was defined as the disappearance of Rubisco from white clover and L. corniculatus added to in vitro incubations with rumen fluid obtained from the same fistulated sheep fed either white clover or L. corniculatus . In the absence of PEG, the in situ loss of Rubisco from L. corniculatus was less rapid than the loss of this protein from white clover when each forage was incubated in the rumen of sheep fed the same diet. Addition of PEG tended to increase the loss of Rubisco from L. corniculatus , suggesting that CT slowed the rates of solubilization of Rubisco from this forage. Effects of rumen fluid were small, but there was some evidence that the rumen fluid in sheep fed L. corniculatus reduced the solubilization of Rubisco from white clover. The action of CT did not inhibit the in situ loss of NDF from either white clover or L. corniculatus . In the absence of PEG, the in vitro degradation of Rubisco from L. corniculatus was slower when compared to the degradation of this protein from white clover; PEG addition increased the degradation of Rubisco from L. corniculatus , but not from white clover, showing that CT was the causal agent. The addition of CT extracted from L. corniculatus markedly depressed the degradation of Rubisco from white clover, with the effect being completely reversible by PEG. The large subunit (LSU) of Rubisco was consistently degraded at a faster rate than the small subunit (SSU) and added CT had a greater effect in slowing the degradation of the LSU compared to the SSU. There was little difference in the degradation of Rubisco when rumen fluid from sheep fed either white clover or L. corniculatus was used for in vitro incubations. It was concluded that the action of CT from L. corniculatus reduces the digestion of protein in the rumen of sheep. This effect is predominantly due to the action of CT reducing the degradation of plant protein, although CT also reduced the solubilization of plant protein. The main effects of CT on protein solubilization and degradation seemed to be produced locally by CT present in plant tissue; transfer of these effects through rumen fluid was small in magnitude.

Condensed tannins from Lotus corniculatus and Lotus pedunculatus exert different effects on the in vitro rumen degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein

Condensed tannins (CT) or proanthocyanidins (PA), which occur in a restricted range of forages, have the ability to interact with proteins and enzymes and can influence the digestion of plant protein in the rumen. We compared the effects of CT extracts from Lotus corniculatus and pedunculatus on degradation of the principal leaf protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), by rumen microorganisms. Total soluble leaf protein extracted from white clover (Trifolium repens ) was incubated with fresh rumen fluid from sheep and a range of concentrations of each CT extract. The rate of degradation of the large (LSU) and small subunit (SSU) of Rubisco was quantified by fractionating the proteins in samples taken from in vitro rumen incubations using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and imaging densitometry. To deduce the effects of the CT extracts, experiments were performed in the presence (CT inactive) and absence (CT active) of polyethylene glycol (PEG; MW 3350). The two CT extracts differed markedly in their effects on the degradation of the LSU and SSU of Rubisco. At concentrations of 0.89 and 1.79 mg CT mg −1 total soluble leaf protein, the CT extract from L pedunculatus was more effective at preventing the degradation of the LSU and SSU by rumen microorganisms than the CT extract from L corniculatus. At a concentration of 1.79 mg CT mg −1 total soluble leaf protein, the CT extracts from L corniculatus and pedunculatus prevented about 0.75 and 0.83 of the LSU and about 0.69 and 0.86 of the SSU, respectively, from being degraded. Addition of PEG removed the inhibition and almost complete degradation of these proteins occurred, as was the case in incubations without CT extracts. The results of this study suggest that the concentration of CT in the diet and the chemical structure which affects the activity of the CT needs to be considered when assessing the effects of CT on protein metabolism in ruminants. © 1999 Society of Chemical Industry

The Condensed Tannin Content of a Range of Subtropical and Temperate Forages and the Reactivity of Condensed Tannin with Ribulose‐ 1,5‐bis‐phosphate Carboxylase (Rubisco) Protein

A series of subtropical grasses and temperate grasses, herbs and legumes were analysed for the presence of extractable and bound condensed tannin (CT) using colorimetric analysis by the butanol–HCl method. Condensed tannins are routinely purified using affinity chromatography with Sephadex LH-20 as a matrix. Therefore, Sephadex LH-20 extracts were further analysed for the presence of CT by 13C nuclear magnetic resonance, for anthocyanidin formation after butanol–HCl treatment and for their ability to precipitate ribulose-1,5-bisphosphate carboxylase (Rubisco) protein from lucerne, at pH 7·0. Criteria for the presence or absence of CT were defined. Trace amounts of CT (0·2–2·5 g kg−1 dry matter; DM) were identified and confirmed in summer grass (Digiteria sanguinalis), perennial ryegrass (Lolium perenne) and red clover (Trifolium pretense), with chicory (Chicorium intybus), lucerne (Medicago sativa) and plantain (Plantago lanceolata) identified as probably containing CT. It was concluded that the subtropical grasses kikuyu (Pennisetum clandestinum), paspalum (Paspalum diatatum), smooth witchgrass (Panicum dichotomiflorum) and crowfoot (Eleusine indica) and the temperate grass, Yorkshire fog (Holcus lanatus) probably did not contain CT. Analysis of the extractable fractions by vanillin–HCl gave higher values for CT than analysis by butanol–HCl and wrongly identified some forages as containing trace levels of CT. It was concluded that vanillin–HCl was not specific enough for the detection of trace levels of CT in forages. These results raise the possibility of plant selection programmes to increase the level of CT in grazed forages to approximately 5 g kg−1 DM, the suggested minimum level required to prevent bloat in cattle and to increase wool growth in sheep. It is suggested that this be considered for perennial ryegrass, chicory, red clover and lucerne.

Gene Expression Changes in the Colon Epithelium Are Similar to Those of Intact Colon during Late Inflammation in Interleukin-10 Gene Deficient Mice

In addition to their role in absorption and secretion, epithelial cells play an important role in the protection of the colon mucosa from the resident microbiota and are important for the maintenance of homeostasis. Microarray analysis of intact colon samples is widely used to gain an overview of the cellular pathways and processes that are active in the colon during inflammation. Laser microdissection of colon epithelial cells allows a more targeted analysis of molecular pathways in the mucosa, preceding and during inflammation, with potentially increased sensitivity to changes in specific cell populations. The aim of this study was to investigate the molecular changes that occur in early and late inflammation stages in colon epithelium of a mouse model of inflammatory bowel diseases. Microarray analysis of intact colon samples and microdissected colon epithelial cell samples from interleukin-10 gene deficient and control mice at 6 and 12 weeks of age was undertaken. Results of gene set enrichment analysis showed that more immune-related pathways were identified between interleukin-10 gene deficient and control mice at 6 weeks of age in epithelial cells than intact colon. This suggests that targeting epithelial cells could increase sensitivity for detecting immune changes that occur early in the inflammatory process. However, in the later stages of inflammation, microarray analyses of intact colon and epithelium both provide a similar overview of gene expression changes in the colon mucosa at the pathway level.

Promotility Action of the Probiotic Bifidobacterium lactis HN019 Extract Compared with Prucalopride in Isolated Rat Large Intestine

Attention is increasingly being focussed on probiotics as potential agents to restore or improve gastrointestinal (GI) transit. Determining mechanism of action would support robust health claims. The probiotic bacterium Bifidobacterium lactis HN019 reduces transit time, but its mechanisms of action and effects on motility patterns are poorly understood. The aim of this study was to investigate changes in GI motility induced by an extract of HN019 on distinct patterns of colonic motility in isolated rat large intestine, compared with a known promotility modulator, prucalopride. The large intestines from male Sprague Dawley rats (3–6 months) were perfused with Krebs buffer at 37°C in an oxygenated tissue bath. Isometric force transducers recorded changes in circular muscle activity at four independent locations assessing contractile propagation between the proximal colon and the rectum. HN019 extract was perfused through the tissue bath and differences in tension and frequency quantified relative to pre-treatment controls. Prucalopride (1 μM) increased the frequency of propagating contractions (by 75 ± 26%) in the majority of preparations studied (10/12), concurrently decreasing the frequency of non-propagating contractions (by 50 ± 11%). HN019 extract had no effect on contractile activity during exposure (n = 8). However, following wash out, contraction amplitude of propagating contractions increased (by 55 ± 18%) in the distal colon, while the frequency of non-propagating proximal contractions decreased by 57 ± 7%. The prokinetic action of prucalopride increased the frequency of synchronous contractions along the length of colon, likely explaining increased colonic rate of transit in vivo. HN019 extract modified motility patterns in a different manner by promoting propagating contractile amplitude and inhibiting non-propagations, also demonstrating prokinetic activity consistent with the reduction of constipation by B. lactis HN019 in humans.

Intestinal amino acid absorption in lambs fed fresh Lucerne (Medicago sativa) during an established Trichostrongylus colubriformis infection

The effects of an established Trichostrongylus colubriformis infection on amino acid (AA) absorption from the small intestine and their availability to other tissues were determined in lambs 48 days post infection. The lambs were fed fresh Lucerne (Medicago sativa; 800 g dry matter (DM)/day) and dosed with 6000 L3 T. colubriformis larvae for 6 days (n = 5) or kept as parasite free controls (n = 6). Faecal egg production was monitored every second day from day 22 to day 48. A nitrogen (N) balance was conducted on days 35 to 43 after infection, and digesta flow and AA concentration measurements were made on day 44. On day 48 after infection, blood was continuously collected from the mesenteric artery and vein, plasma harvested and AA concentrations measured. Faecal egg production peaked on the 26th day after infection (P < 0.001) and intestinal worm burdens on day 48 were greater (P < 0.001) in the infected lambs. Feed intake and liveweight gain were similar (P > 0.10) between control and infected lambs. Digestibility and flow of DM and N through the digestive tract were also unaffected (P > 0.10) by parasite infection. Despite a trend towards higher abomasal AA flux in the parasitised lambs (P < 0.10), apparent AA absorption from the small intestine and AA availability to other tissues were unaffected (P > 0.10) by infection. These results suggest that an established parasite infection had little effect on the intestinal absorption and availability of AA to other tissues in lambs fed fresh Lucerne.