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PubMed1983

Effects of dietary fiber on fecal mucinase and beta-glucuronidase activity in rats.

Mucinase and beta-glucuronidase enable colon bacteria to degrade protective mucins and recycle glucuronide conjugates of toxins and carcinogens. The response of these bacterial enzymes to dietary fiber was studied in the laboratory rat. Fiber-free basal diet was mixed with guar gum, pectin, carrageenan, or cellulose at levels of 5 and 15%. These diets were fed for 21 days to groups of six male Fischer-344 rats having an average weight of 150 g. Mucinase and beta-glucuronidase activities were assayed in fresh rat feces. Rats fed 15% guar gum or pectin gained significantly (P less than 0.05) less weight than the other rats. Mucinase specific activity was highest in the fiber-free diet group and lowest in the 15% guar gum group. Total daily output of mucinase was highest in rats fed fiber-free diet or cellulose and lower in rats fed more readily fermentable fiber. Specific activity and total output of beta-glucuronidase were highest in rats fed fiber-free diet and significantly lower in those fed 15% fiber diets. These data are consistent with the hypothesis that some kinds of dietary fiber may play a role in the etiology of intestinal disease.

Animals↗
PubMed1983

Effects of dietary fibers on mineral status of chicks.

We investigated the effects of replacing dietary starch with dietary fibers of various types and particle sizes on utilization of bivalent cations by chicks. Inclusion of polyethylene powder, alfalfa cell walls, corn bran, oat bran and wheat bran into diets resulted in lower serum and tibia Zn levels. However, addition of pectin, cellulose and a xylose:gum arabic (50:50) mixture did not affect tissue Zn levels, whereas addition of 4% lignin increased serum Zn level from 2.50 to 3.44 micrograms/ml. Ingestion of fibers with particle sizes less than 300 micrometers resulted in higher serum Zn levels compared with those of particle sizes greater than 300 micrometers. Incorporation of 4 and 8% pectin decreased (P less than 0.05) serum and tibia Mg levels. When brans were fed, kidney Mg levels were lower due to fiber additions. Inclusion of lignin, the xylose:gum arabic mixture and alfalfa cell walls resulted in lower liver Cu levels; polyethylene and solka floc had no effect on liver Cu status. Fiber did not affect tissue Ca or Fe status. Each mineral influenced by fiber type, level or particle size appeared to be affected through different mechanisms.

Animals↗
PubMed1983

Influence of dietary fiber on fecal excretion of volatile fatty acids by human adults.

The ability of dietary fibers to influence the excretion of volatile fatty acids (VFA) in feces was evaluated in five healthy men. Throughout the 63-day study subjects were confined to a metabolic unit and fed semipurified diets. Each diet was consumed for 9 days. Feces were homogenized, and aliquots were analyzed for VFA by a steam distillation procedure followed by gas-liquid chromatography. Individuals showed wide variability in the extent to which they excreted VFA, and there is evidence of some subject-diet interaction. Acetate, propionate and butyrate were generally present in the ratios of 35:25:20, but the proportion of acetate was increased when xylan and corn bran were consumed. The excretion of VFA during the last 3 days of each metabolic period increased with diet as follows: xylan, fiber free, cellulose, pectin and corn bran. Corn bran caused a significant increase in total VFA excretion over the fiber-free diet. Concentration of VFA in excreted feces (or expressed as percentage of fecal solids) increased by diet in the following order: cellulose, xylan, corn bran, pectin and fiber free. Thus, the concentration of VFA in feces showed no relationship to the total quantity of VFA excreted.

Adult↗
PubMed1989

Effects of feeding fermentable carbohydrates on the cecal concentrations of minerals and their fluxes between the cecum and blood plasma in the rat.

This study was conducted to determine in rats to what extent fermentable carbohydrates alter the mineral composition of cecal contents and the absorption of the major cations. The carbohydrates studied were as follows: an oligosaccharide (lactulose, 10%); a soluble fiber (pectin, 10%); and an amylose-rich starch, incompletely broken down in the small intestine (amylomaize starch, 25 or 50%). All of these carbohydrates elicited a marked enlargement of the cecum, a drop of cecal pH and an increase in the volatile fatty acids (VFA) pool. With the lactulose diet, the VFA concentration was the lowest, whereas VFA absorption was similar to that observed with the 10% pectin or 25% amylomaize diets. From comparisons between germfree and conventional rats adapted to a fiber-free diet, it appears that VFA are required as counter anions to maintain high concentrations of cations, especially sodium. In conventional rats fed fermentable carbohydrates, sodium concentration in the cecal fluid was approximately 80 mM, except with the lactulose diet (49.5 mM), due to osmotic effects of lactulose. There was, compared to the fiber-free diet, an increase in the cecal concentrations of potassium, calcium and phosphate, but not of magnesium; nevertheless, the cecal pool of all of these minerals was considerably increased. Potassium absorption was increased by fermentable carbohydrates in the cecum, which also appears to be a major site of magnesium and calcium absorption. Thus, fermentable carbohydrates shift aborally the absorption of the major cations, and this point is especially interesting in regard to calcium, since an enhanced supply of calcium in the large bowel has been invoked for fiber effects on colonic carcinogenesis.

Animals↗
PubMed1993

Relationship of colonic luminal short-chain fatty acids and pH to in vivo cell proliferation in rats.

The mechanism by which fermentable fibers may stimulate colonic cell proliferation was tested using two types of fiber (highly fermentable pectin, and less fermentable wheat bran), measuring in vivo concentrations of short-chain fatty acids (SCFA) in the cecum and the proximal and distal colon of rats and correlating these concentrations with cell proliferation indices at the same locations within the intestine. Pectin supplementation resulted in higher concentrations of propionate in proximal and distal colon as compared with fiber-free controls, whereas wheat bran resulted in a higher concentration of butyrate at every site. In the cecum, pH had the strongest correlation to indices of cell proliferation. The lower the pH the greater the number of cells per crypt column (P < 0.05), cells per crypt circumference (P < 0.01), and total number of cells per crypt (P < 0.001). Butyrate had the strongest correlation between a specific SCFA and indices of cell proliferation. In the distal colon, butyrate concentration was positively correlated with number of cells per crypt column (P < 0.05) and total number of cells per crypt (P < 0.05). This study shows that different fibers are fermented to different SCFA in different amounts and that the in vivo concentration of certain of these SCFA is significantly correlated with cell proliferation indices.

Analysis of Variance↗
PubMed1998

A low-viscosity soluble-fiber fruit juice supplement fails to lower cholesterol in hypercholesterolemic men and women.

This study was designed to determine whether a soluble dietary fiber supplement containing gum arabic and pectin in apple juice would lower serum lipids in 110 hypercholesterolemic men and women. Subjects were stabilized on an American Heart Association Phase I Diet for 8 wk. Those with elevated low density lipoprotein cholesterol levels, despite dietary modification, continued to follow the diet and were randomly assigned to receive 720 mL/d of apple juice containing 0 (control), 5, 9 or 15 g of gum arabic and pectin (4:1 ratio) for 12 wk, followed by a 6-wk apple juice-only washout phase. Serum lipid profiles, body weight and 3-day diet records were collected at 3-wk intervals. No significant differences among groups were observed in serum lipid responses during treatment or washout. During the treatment phase, mean serum total cholesterol and triglyceride concentrations increased by 3.5 and 28.5%, respectively (all groups combined, P < 0.0001). The high density lipoprotein cholesterol level did not change significantly from baseline in any group. During washout, mean total cholesterol concentration rose by an additional 2.4% (P < 0.05) compared with the value at the end of the treatment period, suggesting that the apple juice used to deliver the fiber supplement may have contributed to the adverse changes observed in the serum lipid profile. These findings do not support the hypothesized hypocholesterolemic effect of the gum arabic/pectin (4:1) mixture studied, but do underline the importance of selecting appropriate vehicles for delivery of dietary fiber mixtures.

Adult↗
PubMed1998

Energy metabolism of rat colonocytes changes during the tumorigenic process and is dependent on diet and carcinogen.

Alterations in ATP production, intracellular energy levels and mitochondrial function have been shown to trigger cytokinetic events in vitro, including inhibition of cell division, abnormal or blocked differentiation and inhibition of apoptosis. Changes in colonic cytokinetics are directly related to colon tumorigenesis but alterations in energy metabolism during the tumorigenic process have never been reported. We conducted a 2 x 2 x 3 factorial design study in 120 male Sprague-Dawley rats with two diets (pectin or cellulose-supplemented), two injected subgroups (with or without the carcinogen azoxymethane, AOM) and three termination time points (6, 16 and 36 wk post-second injection). Colonocytes were isolated and incubated with their primary energy substrates (radiolabeled butyrate, glucose, glutamine and beta-hydroxybutyrate) for 60 min. Production of lactate, ketone bodies and CO2 were determined. At 6 wk, there were no significant differences in metabolism among treatments. In contrast, at 16 wk, AOM-injected rats had dramatically lower rates of CO2 production (P < 0.001) from both glucose and butyrate and lower rates of lactate and ketone body production than their saline counterparts. At 36 wk, when tumors developed, the depressed production of lactate and ketone bodies seen in AOM-injected rats at 16 wk returned to control values. However, in AOM-injected rats, CO2 production from glucose and butyrate remained depressed. Cellulose feeding resulted in decreased oxidation of glucose, butyrate and glutamine and an increased production of ketone bodies from butyrate by colonocytes compared with pectin feeding at 36 wk. We conclude that colonocyte energy metabolism differs in AOM-injected rats vs. saline controls and changes during tumorigenesis, and suggest a relationship between intracellular energy status and changes in cell kinetics. This is the first report that such a relationship may exist in vivo.

3-Hydroxybutyric Acid↗
PubMed2004

European, Chinese and Japanese pear fruits exhibit differential softening characteristics during ripening.

Softening characteristics were investigated in three types of pear fruit, namely, European pear 'La France', Chinese pear 'Yali', and Japanese pear 'Nijisseiki'. 'La France' fruit softened dramatically and developed a melting texture during ripening, while 'Yali' fruit with and without propylene treatment showed no change in flesh firmness and texture during ripening. Non-treated 'Nijisseiki' did not show a detectable decrease in flesh firmness, whereas continuous propylene treatment caused a gradual decrease in firmness resulting in a mealy texture. In 'La France', the analysis of cell wall polysaccharides revealed distinct solubilization and depolymerization of pectin and hemicellulose during fruit softening. In 'Nijisseiki', propylene treatment led to the solubilization and depolymerization of pectic polysaccharides to a limited extent, but not of hemicellulose. In 'Yali', hemicellulose polysaccharides were depolymerized during ripening, but there was hardly any change in pectic polysaccharides except in the water-soluble fraction. PC-PG1 and PC-PG2, two polygalacturonase (PG) genes, were expressed in 'La France' fruit during ripening, while only PC-PG2 was expressed in 'Nijisseiki' and neither PC-PG1 or PC-PG2 was expressed in 'Yali'. The expression pattern of PC-XET1 was constitutive during ripening in all three pear types. PG activity measured by the reducing sugar assay increased in all three pears during ripening. However, viscometric measurements showed that the levels of endo-PG activity were high in 'La France', low in 'Nijisseiki', and undetectable in 'Yali' fruits. These results suggest that, in pears, cell wall degradation is correlated with a decrease in firmness during ripening and the modification of both pectin and hemicellulose are essential for the development of a melting texture. Furthermore, the data suggest that different softening behaviours during ripening among the three pear fruits may be caused by different endo-PG activity and different expression of PG genes.

China↗
PubMed2002

Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa.

Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-(3)H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.

Carbon-Carbon Double Bond Isomerases↗
PubMed2001

In vitro evaluation of nonstarch polysaccharide digestibility of feed ingredients by enzymes.

Some of the commonly used feed ingredients for poultry (corn, sorghum, finger millet, deoiled ricebran, soybean meal, peanut meal, sunflower meal, and rapeseed meal) were screened for pentosans, cellulose, pectin, and total nonstarch polysaccharides. The ingredient in vitro digestibilities by enzymes were evaluated. Cereal samples screened contained mainly pentosans. Pectin content was rich in oilseed meals. Sunflower meal, soybean meal, deoiled rice bran, and a broiler starter diet were subjected to a two-stage in vitro digestion assay with three different enzyme mixtures viz., Enzyme-I (xylanase + cellulase from Trichoderma viridae), Enzyme-II (xylanase + cellulase + beta-glucanase from Huminicola insolens), and Enzyme-III (xylanase + cellulase + pectinase + beta-glucanase from Aspergillus aculeatus) by incubating 0.1 g of the sample with 3 mL of a pepsin-HCl mixture (2,000 U pepsin/mL of 0.1N HCl) for 45 min to simulate the peptic phase of bird digestion. A pancreatin-NaHCO3 mixture (2 mg pancreatin/mL of 1 M NaHCO3) was used for 2 h at 40 C to simulate the pancreatic phase. Digestibility was assessed by measuring the relative viscosity of the digesta supernatent and the total sugars released. Enzyme-I produced the least relative viscosity and highest total sugars in sunflower meal, deoiled rice bran, and broiler starter diet, whereas Enzyme-III was very effective in soybean meal subjected to in vitro digestion. The assay was a convenient and rapid method of screening for effective and stable enzymes.

Animal Feed↗
PubMed1996

Fecal short-chain fatty acids in patients with diarrhea-predominant irritable bowel syndrome: in vitro studies of carbohydrate fermentation.

Colonic bacterial production of short-chain fatty acids (SCFA) plays an important role in the salvage of unabsorbed carbohydrate and in colonic absorption of electrolytes and water. The objective of this study was to determine whether patients with diarrhea-predominant irritable bowel syndrome (DP-IBS) have a different pattern and rate of fermentation of carbohydrate and fiber to SCFA compared with controls. Fecal homogenates from 10 patients with DP-IBS and 10 age-matched controls were studied. SCFA were measured by gas chromatography in baseline fecal samples and in fecal homogenates in an in vitro anaerobic fermentation system after incubation with no additional substrate, lactulose, potato starch, citrus pectin, and hemicellulose over a 24-hour period. Net SCFA production rates were calculated for the first 6 h of the incubation period. Patients with DP-IBS had a consistently different pattern of less total SCFA, a lower percentage of acetate (p < 0.05), and a higher proportion of n-butyrate (p < 0.05) than controls. In stool homogenates from both controls and DP-IBS patients, lactulose fermentation resulted in the highest rate of SCFA production followed by pectin, starch, and hemicellulose. However, at all time points, the fecal homogenates from controls generated a higher concentration of total SCFA, acetate, and propionate with all substrates tested. SCFA production rates were higher in controls incubated with lactulose, starch, and hemicellulose. The fecal SCFA profile of patients with DP-IBS is characterized by lower concentrations of total SCFA, acetate, and propionate and a higher concentration and percentage of n-butyrate. Fecal flora from these patients produced less SCFA in an in vitro fermentation system in response to incubations with various carbohydrates and fibers. Differences in SCFA production by colonic bacterial flora in patients with DP-IBS may be related to the development of gastrointestinal symptoms.

Acetates↗
PubMed1980

A comparison of the properties of Klebsiella strains isolated from different sources.

The ability to form gas in lactose bile-salt broth at 44.5 degrees C (the "faecal coliform" or FC test), growth in nutrient broth at 10 degrees C, indole production and pectin liquefaction were studied in 480 strains of Klebsiella isolated from human and animal infections, from various sites in the hospital environment and hospital food, and from river water and flowers. A positive FC response was correlated inversely with the ability to grow at 10 degrees C. Most strains of human and animal clinical origin were FC positive, whereas strains from water and flowers were mainly FC negative. The frequency of a positive FC response in strains from the hospital environment fell between these two extremes. The production of indole and liquefaction of pectin by klebsiellas was correlated directly with the ability to grow at 10 degrees C and a negative FC response. Nearly all of the strains could be allocated to one of four groups on the basis of these tests. The capsular serotype, bacteriocine-inhibition patterns and antibiotic sensitivities of the strains were examined. No correlation was evident between the first two properties and klebsiellas from any particular source. Strains of clinical origin were more often resistant to five or more antibiotics than were strains from the hospital environment, which in turn showed a greater frequency of antibiotic resistance than did strains from river water and flowers.

Anti-Bacterial Agents↗
PubMed2005

Effect of carbon source on the cellulosomal subpopulations of Clostridium cellulovorans.

Clostridium cellulovorans produces a cellulase enzyme complex called the cellulosome. When cells were grown on different carbon substrates such as Avicel, pectin, xylan, or a mixture of all three, the subunit composition of the cellulosomal subpopulations and their enzymic activities varied significantly. Fractionation of the cellulosomes (7-11 fractions) indicated that the cellulosome population was heterogeneous, although the composition of the scaffolding protein CbpA, endoglucanase EngE and cellobiohydrolase ExgS was relatively constant. One of the cellulosomal fractions with the greatest endoglucanase activity also showed the highest or second highest cellulase activity under all growth conditions tested. The cellulosomal fractions produced from cells grown on a mixture of carbon substrates showed the greatest cellulase activity and contained CbpA, EngE/EngK, ExgS/EngH and EngL. High xylanase activity in cellulose, pectin and mixed carbon-grown cells was detected with a specific cellulosomal fraction which had relatively larger amounts of XynB, XynA and unknown proteins (35-45 kDa). These results in toto indicate that the assembly of cellulosomes occurs in a non-random fashion.

Blotting, Western↗
PubMed2006

Quantitative trait loci analysis of primary cell wall composition in Arabidopsis.

Quantitative trait loci (QTL) analysis was used to identify genes underlying natural variation in primary cell wall composition in Arabidopsis (Arabidopsis thaliana). The cell walls of dark-grown seedlings of a Bay-0 x Shahdara recombinant inbred line population were analyzed using three miniaturized global cell wall fingerprinting techniques: monosaccharide composition analysis by gas chromatography, xyloglucan oligosaccharide mass profiling, and whole-wall Fourier-transform infrared microspectroscopy. Heritable variation and transgression were observed for the arabinose-rhamnose ratio, xyloglucan side-chain composition (including O-acetylation levels), and absorbance for a subset of Fourier-transform infrared wavenumbers. In total, 33 QTL, corresponding to at least 11 different loci controlling dark-grown hypocotyl length, pectin composition, and levels of xyloglucan fucosylation and O-acetylation, were identified. One major QTL, accounting for 51% of the variation in the arabinose-rhamnose ratio, affected the number of arabinan side chains presumably attached to the pectic polysaccharide rhamnogalacturonan I, paving the way to positional cloning of the first gene underlying natural variation in pectin structure. Several QTL were found to be colocalized, which may have implications for the regulation of xyloglucan metabolism. These results demonstrate the feasibility of combining fingerprinting techniques, natural variation, and quantitative genetics to gain original insight into the molecular mechanisms underlying the structure and metabolism of cell wall polysaccharides.

Arabidopsis↗
PubMed1995

Expansin mode of action on cell walls. Analysis of wall hydrolysis, stress relaxation, and binding.

The biochemical mechanisms underlying cell wall expansion in plants have long been a matter of conjecture. Previous work in our laboratory identified two proteins (named "expansins") that catalyze the acid-induced extension of isolated cucumber cell walls. Here we examine the mechanism of expansin action with three approaches. First, we report that expansins did not alter the molecular mass distribution or the viscosity of solutions of matrix polysaccharides. We conclude that expansins do not hydrolyze the major pectins or hemicelluloses of the cucumber wall. Second, we investigated the effects of expansins on stress relaxation of isolated walls. These studies show that expansins account for the pH-sensitive and heat-labile components of wall stress relaxation. In addition, these experiments show that expansins do not cause a progressive weakening of the walls, as might be expected from the action of a hydrolase. Third, we studied the binding of expansins to the cell wall and its components. The binding characteristics are consistent with this being the site of expansin action. We found that expansins bind weakly to crystalline cellulose but that this binding is greatly increased upon coating the cellulose with various hemicelluloses. Xyloglucan, either solubilized or as a coating on cellulose microfibrils, was not very effective as a binding substrate. Expansins were present in growing cell walls in low quantities (approximately 1 part in 5000 on a dry weight basis), suggesting that they function catalytically. We conclude that expansins bind at the interface between cellulose microfibrils and matrix polysaccharides in the wall and induce extension by reversibly disrupting noncovalent bonds within this polymeric network. Our results suggest that a minor structural component of the matrix, other than pectin and xyloglucan, plays an important role in expansin binding to the wall and, presumably, in expansin action.

Cell Wall↗
PubMed1995

Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pelA, pelB, pelC, pelD and pelE genes. Recently, a new set of pectate lyases was identified in E. chrysanthemi mutants deleted of those pel genes. We cloned the pelL gene, encoding one of these secondary pectate lyases of E. chrysanthemi 3937, from a genomic bank of a strain deleted of the five major pel genes. The nucleotide sequence of the region containing the pelL gene was determined. The pelL reading frame is 1275 bases long, corresponding to a protein of 425 amino acids including a typical amino-terminal signal sequence of 25 amino acids. Comparison of the amino acid sequences of PelL and the exo-pectate lyase PelX of E. chrysanthemi EC16 revealed a low homology, limited to 220 residues of the central part of the proteins. No homology was detected with other bacterial pectinolytic enzymes. Regulation of pelL transcription was analysed using gene fusion. As shown for the other pel genes, the transcription of pelL is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, temperature, iron starvation, osmolarity, anaerobiosis, nitrogen starvation and catabolite repression. Regulation of pelL expression appeared to be independent of the KdgR repressor, which controls all the steps of pectin catabolism. In contrast, the pecS gene, which is involved in regulation of the synthesis of the major pectate lyases and of cellulase, also appeared to be involved in pelL expression. The PelL protein is able to macerate plant tissue. This enzyme has a basic isoelectric point, presents an endo-cleaving activity on polygalacturonate or partially methylated pectin, with a basic pH optimum and an absolute requirement for Ca2+. The pelL mutant displayed a reduced virulence on potato tubers and Saintpaulia ionantha plants, demonstrating the important role of this enzyme in soft-rot disease.

Amino Acid Sequence↗
PubMed2006

An Arabidopsis cell wall-associated kinase required for invertase activity and cell growth.

The wall-associated kinases (WAK), a family of five proteins that contain extracellular domains that can be linked to pectin molecules of the cell wall, span the plasma membrane and have a cytoplasmic serine/threonine kinase domain. Previous work has shown that a reduction in WAK protein levels leads to a loss of cell expansion, indicating that these receptor-like proteins have a role in cell shape formation. Here it is shown that a single wak2 mutation exhibits a dependence on sugars and salts for seedling growth. This mutation also reduces the expression and activity of vacuolar invertase, often a key factor in turgor and expansion. WAKs may thus provide a molecular mechanism linking cell wall sensing (via pectin attachment) to regulation of solute metabolism, which in turn is known to be involved in turgor maintenance in growing cells.

Arabidopsis↗