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Pectin methylesterase, metal ions and plant cell-wall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase.

The hydrolysis of p-nitrophenyl acetate catalysed by pectin methylesterase is competitively inhibited by pectin and does not require metal ions to occur. The results suggest that the activastion by metal ions may be explained by assuming that they interact with the substrate rather than with the enzyme. With pectin used as substrate, metal ions are required in order to allow the hydrolysis to occur in the presence of pectin methylesterase. This is explained by the existence of 'blocks' of carboxy groups on pectin that may trap enzyme molecules and thus prevent the enzyme reaction occurring. Metal ions may interact with these negatively charged groups, thus allowing the enzyme to interact with the ester bonds to be cleaved. At high concentrations, however, metal ions inhibit the enzyme reaction. This is again understandable on the basis of the view that some carboxy groups must be adjacent to the ester bond to be cleaved in order to allow the reaction to proceed. Indeed, if these groups are blocked by metal ions, the enzyme reaction cannot occur, and this is the reason for the apparent inhibition of the reaction by high concentrations of metal ions. Methylene Blue, which may be bound to pectin, may replace metal ions in the 'activation' and 'inhibition' of the enzyme reaction. A kinetic model based on these results has been proposed and fits the kinetic data very well. All the available results favour the view that metal ions do not affect the reaction through a direct interaction with enzyme, but rather with pectin.

Calcium

[Retention of apple starch in production of pectin by the aluminum pectinate procedure].

Apple pectin produced by means of the aluminum pectinate procedure contains less starch than apple pectin obtained by precipitation with ethanol. The degradatin of the starch which occurs during production depends to a great extent upon the conditions of pomace digestion. The absorption maxima of the amylose-iodine complexes range in general from 550 to 570 nm. Model experiments with highly esterified pectin and partially degraded (by mechanolysis) apple starches were performed to establish favourable conditions for fractionating the pectin and starch components which are obtained when the pectin is precipitated as aluminum pectinate and when the granular coagulate is washed with water.

Aluminum

Cloning and expression of a second Aspergillus niger pectin lyase gene (pelA): indications of a pectin lyase gene family in A. niger.

Using the previously cloned Aspergillus niger N756 pectin lyase D gene as a probe, the corresponding pelD gene has been isolated from a genomic library of the laboratory strain A. niger N400. This gene encodes PLD, previously described as PLI, which is one of the two major pectin lyases isolated from the commercial pectinase preparation Ultrazym. Heterologous hybridization of the A. niger N400 genomic library with the pelD gene led to the isolation of another five genes: pelA, B, C, E, and F. These genes differ in their hybridization patterns with probes containing either the entire pelD gene, or 5' or 3' parts thereof. By partial sequencing, and expression in an A. niger transformant containing multiple copies of the pelA gene, we show that this gene, which hybridizes strongest with the pelD gene, encodes the other major pectin lyase from Ultrazym, PLII.

Amino Acid Sequence

The effect of pectin on hepatic lipogenesis in the enterally-fed rat.

The fermentation of pectin by colonic bacteria produces short-chain fatty acids (SCFA) which are then absorbed by the host. The purpose of this study was to determine whether pectin, added to a chemically defined diet, would increase hepatic lipogenesis and whether this effect is mediated by intestinal bacteria. Eighteen Sprague-Dawley rats underwent placement of a feeding gastrostomy and a swivel apparatus. Postoperatively, rats were randomly assigned to one of three groups: 1) No Pectin received a fat-free chemically defined diet, 2) Pectin received the same diet with the addition of 1% (w/v) pectin, and 3) Neomycin received the same diet with 1% w/v pectin and neomycin (80 mg/kg of body weight daily). On the 5th postoperative d, all diets included 12.5% (v/v) deuterium as D2O. After the infusion of the labeled diets for 24 hr, the content and deuterium enrichment of liver palmitate, stearate and oleate were measured and the production rates calculated. The liver content and production rates of these fatty acids were higher in Pectin animals than in either the No Pectin or Neomycin animals. Since the effect of pectin on hepatic lipogenesis was reduced by the concomitant administration of the intestinal antibiotic neomycin, it appears that this effect depends on the bacterial fermentation of pectin. It is postulated that the SCFA produced during pectin fermentation promote lipogenesis via a direct stimulatory effect, in addition to being carbon donors.

Animals

Effect of pectin on gastric emptying and gastroduodenal motility in normal subjects.

The effects of pectin ingestion on gastric emptying, gastroduodenal motility, and plasma levels of glucose, insulin, and glucagon were studied. Initial studies demonstrated that 15 g of pectin was the optimal dose. Subsequently 6 healthy male volunteers were studied on 4 separate days at random. On day 1, gastric emptying of a liquid and a solid meal was assessed by radioisotope technique using 99mTc-dithiopropylthiomine. On day 2, the gastric emptying study was repeated with the addition of pectin to each meal. Plasma levels of glucose, insulin, and glucagon also were determined during these 2 days. On day 3, the effects of liquid and solid meals on gastroduodenal motility were assessed by means of a perfused catheter system. On day 4, the motility study was repeated with the addition of pectin to each meal. Pectin supplementation caused a significant prolongation of gastric emptying half-time of both liquid and solid meals (p less than 0.05). The addition of pectin, however, did not have any significant effect on gastroduodenal motility other than increasing the duodenal motility index 10 min after the liquid meal. The addition of pectin to the liquid meal lowered plasma levels of insulin at 15, 30, and 45 min, and glucagon levels 15 min after the meal. No effect was noted on blood sugar levels. On the other hand, the addition of pectin to the solid meal had no effect on plasma levels of glucose, insulin, and glucagon. We conclude that pectin supplementation delays gastric emptying of both liquid and solid meals in normal human subjects without causing notable changes in gastroduodenal motility or significant variations in pancreatic hormone plasma levels. The pectin effect on gastric emptying may be caused solely by increasing the viscosity of the meals.

Adult

Effect of pectin, a soluble dietary fiber, on functional and morphological parameters of the small intestine in rats.

We investigated the effects of pectin, a soluble dietary fiber, on functional and morphological parameters of the small intestine in rats. A control group and a pectin-fed group were given a fiber-free elemental liquid diet and an elemental liquid diet containing 2.5% (w/w) pectin, respectively, for 2 weeks. The ileal mucosal specific activities of maltase, sucrase and alkaline phosphatase increased significantly in the pectin-fed group. Maltose absorption of the ileum, studied in vitro by the method of everted sacs and disaccharide-dependent potential difference, increased significantly in the pectin-fed group. The length of the small intestine as well as the villus height and crypt depth of both the jejunum and the ileum were significantly greater in the pectin-fed group. The crypt cell production rate of the jejunum and the ileum was also significantly greater in the pectin-fed group. Plasma enteroglucagon, but not gastrin, increased significantly in the pectin-fed group. These data suggest that pectin feeding results in hyperplasia of the small-intestinal mucosa and a significant increase in the enzyme activities of the brush border membrane of the ileum.

Alkaline Phosphatase

Temperature-independent pectin gel method for aerobic plate count in dairy and nondairy food products: collaborative study.

Ten laboratories participated in a collaborative study to compare the pectin-based plate count (PC) Redigel method with the aerobic plate count and standard plate count agar-based standard methods for the estimation of total bacterial counts in 9 different nondairy food and dairy food products. The foods were cream, homogenized milk, raw milk, cheese, raw chicken, raw oysters, frozen broccoli, flour, and spices. Each laboratory analyzed 6 samples (3 sample pairs) of each food group. Counts obtained by the pectin-based plate count and agar-based plate count methods differed significantly (P less than 0.05) only for homogenized milk, where the pectin gel method resulted in higher counts. The actual counts were higher in the pectin gel method in 8 of the 9 food groups. The log means for pectin gel and agar-based media, respectively, for the 9 food groups were: cream 8.106 and 7.844; homogenized milk 8.642 and 8.231; raw milk 8.711 and 8.423; chicken 7.654 and 7.645; oysters 7.201 and 7.180; broccoli 7.102 and 6.798; cheese 8.045 and 8.055; flour 4.112 and 3.988; spice 5.379 and 5.314. The repeatability standard deviations favored the pectin gel method in 6 of the 9 foods tested. The reproducibility standard deviations favored the pectin gel method in 7 of the 9 foods tested. These results strongly support the suitability of the pectin gel method as an alternative to agar-based plate count and other methods for total bacterial counts in nondairy and dairy food products. The pectin gel method has been adopted official first action.

Aerobiosis

Comparison of kaolin-pectin and activated charcoal for inhibition of aspirin absorption.

The effects of kaolin-pectin suspension and of activated charcoal on aspirin absorption were compared. Ten fasting volunteers each received on five separate occasions three 325-mg aspirin tablets with: (1) 240 ml of water, (2) 10 g of activated charcoal in a slurry with 240 ml of water, (3) 30 ml of kaolin-pectin suspension with 210 ml of water, (4) 60 ml of kaolin-pectin with 180 ml of water, and (5) 90 ml of kaolin-pectin with 150 ml of water. Aspirin bioavailability was estimated from spectrophotometric assay of total 48-hour urinary salicylate recovery. The mean urine salicylate recovery following administration of activated charcoal (69.5%) was significantly less (p less than 0.01) than that following administration of 30, 60 or 90 ml of kaolin-pectin (90.6, 94.6 and 95.3%, respectively) or of water only (98.6%). The mean percent aspirin recoveries for the 30-ml and 60-ml kaolin-pectin treatments were significantly less than that for water only (p less than 0.05). Neither activated charcoal nor kaolin-pectin delayed the rate of aspirin absorption. Although kaolin-pectin reduces the absorption of aspirin, the effect would be of marginal clinical importance. Kaolin-pectin suspension is not recommended as a treatment for aspirin poisoning.

Adult

Neomycin has no persistent sparing effect on vitamin B-12 status in pectin-fed rats.

In the present study, rats were depleted of vitamin B-12 with fiber-free or 5% pectin diets, with or without neomycin. Through use of this intestinal antibiotic reported to "spare" vitamin B-12, we sought to determine if bacterial fermentation of pectin might explain our previous observations of negative effects of pectin on vitamin B-12 status. However, neomycin did not lessen interference by pectin with vitamin B-12 metabolism. Pectin increased urinary methylmalonate and decreased propionate oxidation to a greater extent in the presence than in the absence of neomycin. Also, regardless of the presence of neomycin, the biologic half-life of injected [57Co]vitamin B-12 was 58 d for rats fed the fiber-free diets and only 38 d for rats fed 5% pectin diets. Neomycin delayed early fecal excretion of 57Co but had no persistent effect. Thus, neomycin-sensitive bacteria do not mediate the negative effects of pectin on vitamin B-12 status. Pectin may interfere directly with vitamin B-12 absorption or may stimulate vitamin B-12 uptake or propionate production by microbial species that have adapted to neomycin.

Animals

Dietary pectin shortens the biologic half-life of vitamin B-12 in rats by increasing fecal and urinary losses.

As little as 5% of pectin added to a fiber-free diet elevates urinary methylmalonic acid (MMA) severalfold in vitamin B-12--deprived rats. The present study examines whether increased urinary MMA reflects lower vitamin B-12 status or occurs only because of fermentation of pectin by intestinal bacteria and increased production of propionate, a precursor of MMA. By monitoring urinary and fecal excretion of 57Co after a tracer dose of [57Co]vitamin B-12, we found the biologic half-life of vitamin B-12 to be 59 d for rats fed a fiber-free diet and only 19 d for rats fed a 5% pectin diet. Also, pectin-fed rats oxidized only 12% of a 1-mmol dose of [14C]propionate to 14CO2 in 2 h, whereas rats fed the fiber-free diet expired 33% of the dose. Finally, high urinary MMA persisted even after the removal of pectin from the diet. We conclude that dietary pectin accelerates vitamin B-12 depletion in rats, possibly by interfering with enterohepatic recycling of vitamin B-12. By stimulating microbial propionate production, pectin and other fermentable fibers may also contribute to increased urinary MMA in vitamin B-12 deficiency, but a larger propionate pool does not account for the other effects of pectin on vitamin B-12 status.

Animals

Adhesion of bacteria on pectin casted films.

Pectin and non-pectin degrading bacteria were tested for their ability to adhere to a film casted of low methoxylated pectin (polygalacturonic acid). Klebsiella oxytoca and a newly isolated strain of Escherichia coli adhered to the film, whereas only K. oxytoca was able to utilize pectin as a sole carbon source. Other E. coli strains, containing plasmids with pectinolytic enzymes, did not adhere to the solid pectin film. Therefore, the ability of the bacteria to adhere to the films was not correlated with their ability to degrade pectin. When the solubilization (dissolution) of pectin matrices (tablets) was analysed with and without K. oxytoca, a significant retardation in the dissolution rate was observed in the presence of K. oxytoca, suggesting the formation of a biofilm on the matrix or sedimentation of insoluble pectin salts.

Bacterial Adhesion

Effects of pectin on fatty acid and glucose absorption and on thickness of unstirred water layer in rat and human intestine.

Effects of pectin, a soluble dietary fiber, on fatty acid and glucose absorption were studied in vivo in rats and humans by perfusing the intestine with linoleic acid and glucose solutions with and without pectin. Linoleic acid and glucose absorption decreased with increasing concentrations of pectin. The reduction in linoleic acid absorption was not caused by binding of linoleic acids by pectin or impaired micelle formation due to binding of bile acid by pectin. The unstirred water layer expanded with increasing concentrations of pectin. These results suggest that enlargement of the unstirred water layer is closely associated with the reduction of absorption of fatty acid and glucose ingested with pectin.

Adult

Pectin delays gastric emptying and increases satiety in obese subjects.

As pectin delays gastric emptying in normal subjects and satiety may be linked to the rate of gastric emptying, we designed this study to evaluate, in a group of obese subjects, the effect of adding pectin to a meal on gastric emptying, sensation of satiety, and postprandial plasma cholecystokinin and pancreatic polypeptide levels. We studied gastric emptying of solids in 9 adult obese subjects on 2 separate days in a randomized fashion. On day 1, 15 g of pectin was added to the meal, and on day 2 15 g of methylcellulose was added and served as control. Satiety was evaluated by an analogue rating scale. Pectin significantly delayed gastric emptying time [t1/2 = 116 +/- 23 min vs. 71 +/- 17 min observed with methylcellulose (p less than 0.001)]. Pectin also significantly increased subjects' sensation of satiety [98 +/- 7 vs. 74 +/- 17 (p less than 0.001)]. Postprandial release of cholecystokinin and pancreatic polypeptide was not modified by pectin. As pectin induces satiety and delays gastric emptying in obese patients, it may be a useful adjuvant in the treatment of disorders of overeating.

Adult

Effect of wheat bran and pectin on the absorption and retention of phosphorus, calcium, magnesium and zinc by the growing pig.

Two separate balance experiments of P, Ca, Mg and Zn were carried out on 5 lots of 4 growing pigs each (35-40 kg) adapted for 3 weeks to one of the diets studied. In the first experiment, the control diet was compared with a diet containing 20% of coarse wheat bran, thus richer in minerals, the quantities ingested not being equalized. In the second experiment, three diets were compared: a control diet, a diet with 2.5% of high-methoxylated (HM) apple pectin, and a diet with 2.5% low-methoxylated (LM) apple pectin. The supplement of P and Mg provided by the wheat bran was well absorbed (apparent absorption) and retained by the pigs. On the contrary, in spite of higher intake of Ca and Zn with bran diet, the absorption of these minerals was not improved. The action of wheat bran phytase and the possible absorption of P and Mg (but not of Ca and Zn) in the large intestine could explain these results. Compared to HM pectin that had relatively little effect on mineral utilization, LM pectin drastically diminished the absorption and retention of the minerals studied and resulted in negative Ca, Mg and Zn balances. The degree of pectin esterification would thus be the main factor determining the effect of pectin on mineral availability. In conclusion, wheat bran is a source of available P and Mg for the pig but it might have an unfavorable effect on the utilization of Ca and Zn. LM pectin produces a deleterious influence on mineral balances.

Absorption

The effect of wheat bran and pectin on paracetamol absorption in the rat.

1. The absorption of an oral dose of paracetamol was measured in rats given either a basal fibre-free diet, or the basal diet with either pectin or bran added. 2. Urinary excretion of the oral dose was significantly greater during the first 8 h in the pectin-fed rats compared with those on basal diet, though cumulative excretion after 72 h was the same. 3. Free paracetamol levels in the plasma were significantly higher in the pectin-fed rats compared with those on basal diet at 30, 60, 90 and 120 min after the oral dose. 4. The plasma half-life of intravenously-injected paracetamol was shorter in the pectin-fed rats than in those on basal diet alone, but the antipyrine half-lives were not significantly different. 5. Pectin feeding had no effect on either the apparent volume of distribution of paracetamol and antipyrine, or on the rate of gastric empyting. 6. Perfusion of the whole length of the small bowel showed a significantly greater capacity for paracetamol absorption in the pectin-fed rats. 7. Bran had no effect on paracetamol absorption. 8. It was concluded that dietary fibre intake affects drug absorption and that the effect varies with the type of fibre. Unexpectedly pectin accelerates rather than retards absorption of paracetamol, though the mechansim of this effect is unknown.

Acetaminophen

Predominant conjugation with glycine of biliary and lumen bile acids in rats fed on pectin.

1. Bile acids were analysed in the bile and lumen samples of rats which received a cholesterol-free or cholesterol-enriched (5 g/kg) diet free from fibre, or containing cellulose or citrus pectin at the level of 100 g/kg. 2. Dietary pectin but not cellulose increased biliary bile acid concentration and excretion. Dietary cholesterol did not affect biliary bile acids quantitatively. 3. Biliary bile acids were almost exclusively conjugated with glycine or taurine in the various experimental situations. The predominant portion of bile acids in rats fed on the cholesterol-free diet was conjugated with taurine when the diet was either free from fibre or contained cellulose; the ratio of bile acids conjugated with glycine: those conjugated with taurine (G:T) was less than 0.2. In contrast, with pectin as a fibre source, the conjugation with glycine increased enormously (G:T increased to approximately 4). Cholesterol enrichment of the diet also increased the glycine conjugation in all groups of rats. Even in this situation, the G:T was highest in rats fed on pectin. 4. Pectin, but not cellulose, increased the bile acid content of the small intestine and caecum, both in rats fed on the cholesterol-free and cholesterol-enriched diets. Cholesterol feeding doubled the bile acid content of the caecum in rats fed on a fibre-free diet or a cellulose diet, but not in those fed on pectin. No such effect of cholesterol was observed in the small intestine, except for the ileal bile acid content in rats fed on cellulose. 5. A considerable portion of the bile acids in the small intestine was deconjugated. The extent of the deconjugation was higher in the ileum than in the jejunum. As in the bile, G:T in rats fed on pectin (3-5.5) were higher than those in the other groups (0.05-1.05) in various situations. Also, cholesterol feeding considerably increased the ratio in all groups of rats. 6. The observed dietary alteration of the partition of bile acids between glycine and taurine may be of physiological significance in regulating bile acid and lipid metabolism in rats.

Animals