Buckwheat Fagopyritols
Flatulence Report, June 30, 1999
prepared by
Ralph L. Obendorf, P.I.
Bertha A. Lewis, collaborator
Kathryn J. Steadman, Postdoctoral Associate
Monica S. Burgoon and Hutton J. John, graduate students
Department of Crop and Soil Sciences and Division of Nutritional Sciences
Cornell University, Ithaca, NY 14853-1901
Part 9
Breakdown of fagopyritols by human fecal bacteria
Summary
Human fecal bacteria readily digest fagopyritols. Fagopyritols were prepared from whole groats as described in Part 10. The concentrated extract containing fagopyritols was digested by dry granular Baker=s yeast to remove large quantities of sucrose from the extract. Trehalose in the preparation of fagopyritols was a byproduct of fermentation by yeast. Fermentation with fecal bacteria was conducted under anaerobic conditions in vitro. Fermentation of the fagopyritol extract by human fecal bacteria in vitro resulted in the digestion of all fagopyritols in the samples within six hours (and probably much sooner). d-chiro-Inositol, myo-inositol, trehalose, galactinol, and digalactosyl myo-inositol were also metabolized by the bacteria. The controls without bacteria did not show degradation of fagopyritols indicating that fecal bacteria were responsible for fagopyritol degradation. Upon analysis by gas chromotography, no trace of any of these compounds was present in the samples with bacteria. Consumption of d-chiro-inositol by the bacteria occurred in the absence of other fermentable materials as would normally be present in a human diet and in the presence of a vast excess of microorganisms.
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Objective: To present the human digestive tract and potential for utilization of fagopryitols after oral dietary consumption and degradation of fagopyritols by human fecal bacteria.
Introduction:
Buckwheat is a rich natural source of d-chiro-inositol, mostly in the form of fagopyritols, galactosyl derivatives of d-chiro-inositol (Horbowicz et al., 1997). Buckwheat and buckwheat products were claimed to be a useful dietary treatment for lowering blood glucose in persons with diebetes (Wang et al., 1992; Kasatkina and Odud, 1993). Medical research has linked a faulty metabolism of d-chiro-inositol to type II diabetes mellitus (Kennington et al., 1990; Asplin et al., 1993). Use of d-chiro-inositol as a dietary treatment for type II diabetes mellitus has been patented (Larner and Kennington, 1992). Reports of research with obese Rhesus monkeys that were spontaneously diabetic (type II, non-insulin dependent) has provided some evidence that d-chiro-inositol, when added to the diet and fed orally, is effective in lowering blood glucose (Ortmeyer et al., 1995; Hansen and Ortmeyer, 1996; Ortmeyer, 1996). Concentrated extracts from buckwheat provide a rich source of fagopyritols, that are readily hydrolyzed by α-galactosidase (Horbowicz et al., 1998). Since cells in the human digestive tract do not produce this enzyme, the present experiment was designed to verify that fagopyritols can be degraded by microorganisms normally present in the human digestive tract.
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Review of the human digestive system: Foodstuffs ingested by mouth pass through the esophagus to the stomach. Partially digested foods pass from the stomach to the small intestine. The first part of the small intestine is the duodenum, a C-shaped 25-cm long tube that curves around the pancreas. The upper part (40%) of the small intestine is the jejunum (100 cm long) and the lower part of the small intestine (60%) is the ileum (160 cm long). The internal surface area of the small intestine is increased by circular folds of mucosa (the cells lining the lumen or internal cavity). The mucosa has many villi (invaginations), and cells of the villi have microvilli (microinvaginations) with a greatly increased surface area. There are 20 to 40 villi per square centimeter of mucosa. The mucosa cells produce secretions and are active in uptake of digested foodstuffs in the watery contents. Enzymes found in the mucosa membrane include many disaccharidases, peptidases, and enzymes involved in the breakdown of nucleic acids. The ileum terminates at the ileocecal junction where food residues enter the large intestine (colon). The colon is about 110 cm long and includes the ascending colon, the transverse colon, the descending colon, and the S-shaped colon that leads to the anus. Unlike the small intestine, the colon does not have mucosa and the fecal contents become progressively drier during passage through the large intestine.
Cells (mucosa) lining the lumen of the duodenum, jejunum, and ileum of the small intestine have several disaccharidases that degrade maltose, sucrose, cellobiose, lactose, trehalose, and others (Auricchio et al., 1963; Gitzelmann and Auricchio, 1965) but not α-galactosidase (Gitzelmann and Auricchio, 1965). Thus, most monosaccharides and disaccharides are digested by the human system. However, some α-galactosides such as raffinose, stachyose and verbascose, that are commonly found in grain legume seeds and in oilseeds, are not degraded by the human digestive system since it lacks α-galactosidase, but these α-galactosides pass through the system to the intestinal bacteria that have α-galactosidase (Gherardini et al., 1985). Raffinose, stachyose and verbascose are readily degraded by bacteria and promptly fermented to hydrogen and carbon dioxide gas (and methane in 20-40% of the people (Hill, 1986a)) resulting in increasing flatulence production in the jejunum, ileum, and colon (Richards and Steggerda, 1966). Masses of bacteria are present in the human digestive tract, mostly in the colon (large intestine) where they may constitute 40-50% of the total fecal mass. Bacteria are present in the small intestine in the jejunum and increasing in number in the ileum, the distal small intestine (Hawksworth et al., 1971). Flatulence production from fermentation of the raffinose series oligosaccharides and other soluble α-galactosides can start in the jejunum, increasing in the illeum and increasing more when passing into the colon leading to extensive production of flatulence as observed in dogs (Richards and Steggerda, 1966). Flatulence occurs as a result of fermentation of soluble oligosaccharides by intestinal bacteria containing α-galactosidase (Price et al., 1988). Flatulence may also occur as a result of fermentation of degradation products of dietary fiber from cell walls, including β-galactosides and β-glucosides (Champ et al., 1990). The population of different species of intestinal bacteria change in response to the nature of the foodstuffs ingested. Bacteria can produce one form of α-galactosidase in response to dietary fiber and a second form of α-galactosidase in response to raffinose and stachyose (Gherardini et al., 1985). Many hydrolytic enzymes are secreted by the bacteria, but bacterial α-galactosidase is not released into the intestinal lumen. About 70% of the bacterial α-galactosidase is soluble (cytosolic; in the bacterial cell) and about 30% is membrane bound. It is not known if the enzyme is external to the membrane for hydrolysis of the α-galactosyl bonds before uptake into the bacteria or if the α-galactosides must be taken up by the bacterial cell before hydrolysis.
d-chiro-Inositol and fagopyritols in the human digestive tract: Ingestion of buckwheat products or fagopyritols extracted from buckwheat introduces d-chiro-inositol, mostly in the form of α-galactosides of d-chiro-inositol, into the human digestive tract. The fagopyritols are mono-, di- and tri- α-galactosides of d-chiro-inositol and are readily hydrolyzed by α-galactosidase (Horbowicz et al., 1998). The possible fate of fagopyritols and d-chiro-inositol in the gut must be considered as part of the dietary effect of buckwheat.
Hawksworth (1971) notes three factors that determine the degree of metabolism of ingested or bile-excreted compounds:
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1. The region of the gut from which the compound is absorbed (if it is absorbed).
2. The distribution of bacteria along the gut.
3. The occurrence of the necessary enzyme or enzyme systems in bacteria.
General consideration of each of these factors, along with specific reference to the d-chiro-inositol, myo-inositol and fagopyritols, follow.
Absorption
The small intestine is the main site of absorption of products of hydrolysis that result from digestion. However, α-galactosides of d-chiro-inositol in buckwheat, being similar to the α-galactosides of sucrose (raffinose and stachyose) found in legumes, probably escape human digestion due to the absence of α-galactosidase production in the gut mucosa (Gitzelmann and Auricchio, 1965; Price et al., 1988). Therefore, like raffinose and stachyose, fagopyritols remain in the digestive tract and are likely degraded when they contact the microflora. Experiments with animal systems indicate that 80% of seed αgalactosides are degraded by the end of the small intestine (Gdala et al., 1997). It is likely that fagopyritols are mostly utilized also before the end of the small intestine.
Free d-chiro-inositol is probably absorbed like myo-inositol in the small intestine. Feeding a mixtutre of free d-chiro-inositol and free D-pinitol to humans with NIDDM resulted in an increase in plasma d-chiro-inositol and D-pinitol and a decrease in plasma insulin and glucose (Ostlund and Sherman, 1998), and feeding d-chiro-inositol to hyperinsulinemic Rhesus monkeys resulted in decreased plasma glucose (Ortmeyer et al., 1995), providing evidence of uptake and utilization of d-chiro-inositol by the digestive system. When myo-inositol was fed to humans, plasma myo-inositol was significantly increased, providing evidence for uptake. Feces contained only small amounts of myo-inositol (Clements and Reynertson, 1977). Free myo-inositol has been shown to be actively transported by the small intestine of hamsters though glucose and galactose acted as non-competitive inhibitors of the transport of myo-inositol (Caspary and Crane, 1970). A stereospecific myo-inositol-d-chiro-inositol transporter in human liver cells selectively uptakes d-chiro-inositol, but not L-chiro-inositol, in the presence of excess myo-inositol (Ostlund et al., 1996). Presence of such a stereospecific transporter in other tissues has not been reported, but free d-chiro-inositol is clearly taken up by the digestive system.
Absorption of some substances occurs in the large intestine also, though it does not have the extensive surface area by which the small intestine increases its absorptive capacity. Besides water, electrolytes and other minerals, some B vitamins and Vitamin K are absorbed in the colon; other nutrients are known to be used by bacteria, namely, ascorbic acid, choline and cyanocobalamin (Ganong, 1995). It is important to note that inositol fermentation can occur as is evident by several studies which used inositol for biotyping bacteria, some of which occur in the microflora (Ganong, 1995). However, inositol is also produced by intestinal bacteria in myo-inositol deficient rats (Hayashi et al., 1974) so that it is difficult to predict the fate of free inositols in the lower digestive tract (colon or large intestine).
Bacteria in the Gut
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Each gram of gut contents is estimated to contain up to 1012 microorganisms resulting in the bacterial biomass making up about 40% of fecal material. Stomach acid is bacteriostatic and bactericidal; also, bile acids, pancreatic enzymes, rapid transit time and peristalysis contribute to control bacterial growth in the proximal small intestine (duodenum, jejunum and proximal ileum). Gram-positive cocci are the primary group detected there. In the distal small intestine the microflora resembles that of the large bowel in that there are more Gram-negative and anaerobic bacteria. The total number, however, is still considerably less than that of the large intestine which harbors what has been described as a complex ecosystem of microorganisms influencing host physiology in ways that have only recently been investigated (Hill, 1986b). The smaller numbers of bacteria in the small intestine may be sufficient for hydrolysis of fagopyritols since bacterial enzymes are highly active on α-galactosides (Gherardini et al., 1985). The massive numbers of bacteria in the colon are Aoverkill@, designed to scavenge nutrients remaining in the fecal materials.
In general, in the healthy human, the main site of bacterial colonization is the large intestine with the lower small intestine showing similar but fewer bacteria and less colonization. In regards to fermentation, most occurs in the cecum and ascending colon where substrate concentration (undigested material) is highest, bacterial growth rate fastest, pH most acidic and transit time most rapid. Substrate concentration and bacterial growth rate are known to decrease and pH increase along the length of the colon from the cecum to the rectum (Gibson and Wang, 1994). Fagopyritols are soluble and therefore are probably fermented quickly in the cecum, or possibly earlier in the distal ileum. In the pig, β-glucans were degraded mainly in the distal ileum (Knudsen and Johansen, 1995).
It should be noted that the rodent model is questionable in studies involving the digestive tract because rodents are coprophagic and secrete less stomach acid (Hawksworth et al., 1971). Therefore, the total number and distribution of bacteria is greater in rodents and the metabolic effects they produce can be expected to differ from what occurs in the human.
Enzymes of the microfloral bacteria
Gut bacteria have an array of enzymes at their disposal; most activity is saccharolytic since energy is derived mainly from carbohydrates. The production of enzymes has been shown to be influenced by colonic pH, oxygen tension and substrate concentration. Also, enzymes useful in the colonic environment are either cell-associated or extracellular. Enzyme location has been studied; it appears that bacterial extracellular enzymes mainly produce partially-degraded polysaccharides from high molecular weight substrates (dietary fiber) while mono- and disaccharides are the major products of cell-associated enzymes. In this way, it is less likely that competing microorganisms would benefit from the enzyme activity since the mono- and disaccharides are directly transported into the cells for metabolism (Salyers, 1979; Egli, 1975; Murashige and Skoog, 1962).
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Specifically, -galactosidase activity has been demonstrated in many of the dominant bacterial genera of the gut. In fact, people eating a corn-bean diet have increased fecal -galactosidase activity compared to those eating the normal low-fiber American diet. The galactose that is freed is readily transported into bacterial cells and used for energy or biomass (Salyers, 1979) which likely explains why Gitzelmann and Aurrichio (1965) found no indication that galactose from soybean -galactosides is absorbed by the human gut.