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Nutrient Metabolism

Dietary Fiber-Rich Barley Products Beneficially Affect the Intestinal Tract of Rats¹

Gerhard Dongowski², Mario Huth, Erich Gebhardt^* and Wilhelm Flamme

Department of Food Chemistry and Preventive Nutrition, German Institute of Human Nutrition Potsdam-Rehbrücke, Bergholz-Rehbrücke, Germany; ^* Institute of Nutritional and Environmental Research, Bergholz-Rehbrücke, Germany; Institute of Stress Physiology and Quality of Raw Materials, Federal Center for Breeding Research on Cultivated Plants, Groß Lüsewitz, Germany

²To whom correspondence should be addressed. E-mail: dongo{at}www.dife.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The objective of this study was to investigate the effects of barley-rich diets in the intestinal tract of rats. Four test groups (A–D) of 10 young male Wistar rats were fed diets containing 50 g/100 g barley extrudates (A, B and D) or mixtures (C) for 6 wk; the control diet contained no barley. The barley-containing supplements in the test diets were: A = cultivar "HiAmi"; B = "HiAmi" and "Prowashonupana" (50:50); C = "Prowashonupana" and Novelose (50:50); D = "Prowashonupana" and amylose from maize (60:40). These supplements contained 7–12 g/100 g ß-glucan and 7–24 g/100 g resistant starch. Additionally, 5 g microcrystalline cellulose/100 g was present in all diets. Carbohydrate utilization (indirect calorimetry) was lower (P < 0.05) in rats fed the barley-containing diets C or D than in the controls. In the test groups, the following differences from the controls were found: greater food intake in the last 2 wk (P < 0.05); increased weight gain in wk 6 (P < 0.05); greater mass of the ceca (groups B–D; P < 0.05) and colons (P < 0.001) as well as masses of cecal (groups C and D; P < 0.01) and colon contents (P < 0.001); greater concentrations of resistant starch in cecal and most of the colon contents (P < 0.05); and more ß-glucan in the small intestine, cecum and colon (P < 0.05). The numbers of coliforms and Bacteroides were lower than in the controls in groups B–D and those of Lactobacillus were greater in all test groups (P < 0.05). Short-chain fatty acids (SCFA) were higher in the cecal contents of the test groups ( 800 µmol/g DM; P < 0.001) compared with the controls ( 200 µmol/g DM). Similarly, SCFA were higher in colon and feces of the test groups. The concentrations of excreted bile acids increased up to 30% during the feeding period. The proportions of secondary bile acids were lower and the amounts of neutral sterols (P < 0.001) were greater in feces of rats fed the barley-containing diets for 6 wk than in the controls. Diets containing more soluble macromolecular dietary fibers such as ß-glucans affected the excretion of bile acids and neutral sterols the most, whereas the fermentation of dietary fiber, including resistant starch, influenced the steroids in feces. These results suggest that dietary fiber-rich barley-containing diets have beneficial physiologic effects.

KEY WORDS: • barley • physiologic effects • rats • resistant starch • ß-glucan

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Dietary fibers (DF)³ are essential components in human and animal nutrition. A high intake of DF is positively related to different physiologic and metabolic effects. The daily intake of DF is below the recommended levels in most developed countries (1 ). Cereal products, particularly from whole grains, are the most important source of DF in the Western diet.

Barley (Hordeum vulgare L.) grains are relatively rich in DF such as ß-glucan, arabinoxylans and cellulose. The consumption of ß-glucan-rich diets results in several beneficial physiologic effects due to a relatively high concentration, soluble state and high molecular weight of this polysaccharide. The extractability and viscosity of ß-glucan are influenced by both the technological conditions during food preparation and the physiologic conditions in the gastrointestinal tract (2 ,3 ). In several studies it has been shown that blood cholesterol and lipoprotein concentrations can be reduced in humans (4 ) and animals (5 ) by ß-glucan from barley or oats. Furthermore, ß-glucan or ß-glucan-enriched diets lowered the postprandial blood glucose and insulin responses in normal individuals (6 ). Zhang et al. (7 ) found that administration of brewer’s spent grain (with only 1.05 g/100 g ß-glucan) increased the daily cholesterol output and decreased serum LDL cholesterol and apolipoprotein B levels when the bile acid (BA) output was low in ileostomy subjects. Other DF components present in barley products also are involved in lowering serum LDL cholesterol. Generally, the greater excretion of bile acids may be responsible for the cholesterol-lowering effects of ß-glucan-rich diets (8 ). ß-Glucans are fermented by the intestinal microflora in vivo and in vitro, resulting in the formation of short-chain fatty acids (SCFA) (9 ,10 ).

Resistant starch (RS), i.e., the starch or starch degradation products that are not absorbed in the small intestine of healthy individuals, is a major substrate for colonic fermentation and is a good source of butyrate (11 ). RS should be classified as a DF (12 ). A previous study showed that the amylose component of barley meal could be partly converted into RS by extrusion technology under optimized conditions followed by a freeze-storage treatment, whereas the macromolecular state of ß-glucan was preserved (13 ). The processes used during the extrusion influenced the functional and physicochemical properties of the preparations obtained and of their extracts (e.g., water binding, viscosity, flow behavior, molecular weight of polymers). Further, the barley extrudates interacted with glycoconjugated BA in vitro. During their in vitro fermentation with human fecal flora, the production of SCFA and the molar proportion of butyrate were increased compared with nonextruded barley meal (13 ).

The objective of this study was to evaluate the physiologic effects of DF-rich barley-containing diets (particularly extrudates) from the whole-grain type in the intestinal tract. Male Wistar rats were chosen as the animal model. The test diets differed in their concentrations of total DF and of individual DF (ß-glucan, RS). Our hypothesis was that barley-rich diets with an optimized DF composition would cause beneficial direct or indirect physiologic effects in the intestinal tract. Therefore, the effects of the barley-containing diets particularly on food intake and weight gain, on organ weights and mass of intestinal contents, on composition of the microflora, on formation of SCFA and on the concentration and composition of excreted steroids were investigated.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Preparation and composition of the barley samples.

The following source materials were used for the preparation of barley samples A–D: 1) barley mutant "(HiAmi x Cheri) x Cheri" (Institute for Stress Physiology and Quality of Raw Materials, Groß Lüsewitz, and Saatzucht Dr. h.c. Carsten, Bad Schwartau, Germany) with 6.5 g/100 g ß-glucan, 56.9 g/100 g starch, 33.8 g/100 g amylose (in the starch component) and 9.7 g/100 g protein (referred to here as HiAmi); 2) waxy barley cultivar "Prowashonupana" (ConAgra, Omaha, NE) with 17.6 g/100 g ß-glucan, 30.1 g/100 g starch, 5 g/100 g amylose (in the starch component), 17 g/100 g protein, 8.3 g/100 g insoluble DF and 21.5 g/100 g soluble DF; 3) Novelose 330 (National Starch and Chemical GmbH, Hamburg, Germany) with 30 g/100 g RS type III; 4) amylose from maize (Sigma, Deisenhofen, Germany).

Barley samples A (HiAmi), B (HiAmi/Prowashonupana; 50:50, wt/wt), and D (Prowashonupana/Amylose; 60:40, wt/wt) were prepared by extrusion using a feed moisture content of 20%, a mass temperature of 150°C, a speed of 350 rpm, and a diameter of the dies of 2 x 3.5 mm in the twin-screw extruder APV 50 (Baker-Perkins, Peterborough, UK). All extrudates were stored for 3 d at -18°C before use. Barley sample C was prepared by mixing Prowashonupana and Novelose (50:50, wt/wt). Table 1 summarizes the composition of the barley samples.

Table 2 shows the composition of the diets (prepared as pellets in the German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany). Experimental diets A–D containing 500 g/kg of the barley samples were prepared by a partial replacement of wheat starch (used in the control diet).

Analytical methods.

For the determination of ß-glucan, sample material was suspended in phosphate buffer (pH 6.5) and mixed for 5 min at 100°C. The diluted suspension was hydrolyzed with lichenase (Megazyme International, Bray, Ireland) for 60 min at 40°C (16 ). After dilution and centrifugation (10 min at 1500 x g), a part of the supernatant was incubated with ß-glucosidase (Megazyme) in acetate buffer at pH 4.5 and 40°C for 15 min. The glucose liberated was determined with the hexokinase/glucose-6-phosphate dehydrogenase kit from Boehringer (Mannheim, Germany).

The total starch content was analyzed enzymatically using amyloglucosidase and the Boehringer glucose kit after its extraction with dimethyl sulfoxide/HCl or 1 mol/L NaOH. RS was measured in vitro according to Berry (17 ). First, the digestible starch was hydrolyzed by incubation with pancreatin (Merck, Darmstadt, Germany) in acetate buffer (pH 7.0) for 16 h at 37°C, simulating starch hydrolysis in small intestine. After addition of the fourfold amount of 96% ethanol and centrifugation (10 min at 3000 x g), the twice extracted (with 80% ethanol) and then freeze-dried RS-containing residue was solubilized in 1 mol/L NaOH. The diluted solution was hydrolyzed with amyloglucosidase at pH 4.6, and the liberated glucose was determined enzymatically using the hexokinase and glucose-6-phosphate dehydrogenase kit (Boehringer). Dietary fiber (total, insoluble and soluble) was analyzed by the enzymatic-gravimetric AOAC method (18 ).

Rat experiment.

Young male Wistar rats (Tierzucht Schönwalde) weighing 67.2 ± 4.2 g (n = 50) were housed in pairs in temperature- and humidity-controlled cages (22 ± 2°C and 55 ± 5%) on a normal light cycle (0600 to1800 h, light; 1800 to 0600 h, dark). All rats were fed the control diet for 7 d after arrival (wk 0). Then they were randomly divided into 5 groups of 10 rats each and fed the barley-containing diets A–D or the control diet for 6 wk. The rats had free access to food and water.

Growth of the rats and food intake were determined weekly. Feces were collected completely for 24 h at wk 0, 2, 4 and 6. At the end of the experiments, selected organs (stomach, small intestine, cecum and colon) and the contents of ileum, cecum and colon were prepared for analysis. The small intestine was divided into equal length upper (UP) and lower (LP) parts. The microbial counts were determined in fresh feces before the test phase, on d 3 and at the end of the experiment.

Determination of SCFA, microbial counts and steroids.

The SCFA were analyzed in the intestinal contents and feces using gas chromatography (19 ). The microbial counts were determined as described elsewhere (19 ).

Before extraction of the steroids, 7,12ß-dihydroxy-5ß-cholanic acid and 5-cholesten-3ß,25-diol or 5-cholestan were added as internal standards for BA and neutral sterols (NS), respectively, to 50 mg of the freeze-dried feces. The mixtures were treated for 1 h at 80°C by shaking with a mixture of 2.55 mL of 0.5 mol/L NaOH and 23 mL of 96% ethanol. After centrifugation (15 min at 4°C and 5000 x g), the supernatant was concentrated to 3 mL in a Speed-Vac, mixed first with 7 mL of 1 mol/L NaOH and 2 mL of methanol and then with 10 mL hexane. The nonpolar NS fractions were separated from the BA using extraction with hexane (three times) by shaking and centrifugation (15 min at 4°C and 5000 x g). The purified NS-containing hexane phases were dried in a vacuum and redissolved in ethanol. After removal of the organic solvents in a Speed-Vac, the BA containing phases were diluted with water and the BA were purified by solid phase extraction on Bakerbond SPE C18 columns using the Baker SPE-12G system (J. T. Baker, Gross Gerau, Germany).

Bile acids were estimated by HPLC using precolumn derivatization and fluorescence detection (20 ). The free and glycine-conjugated BA were directly derivatized with 4-bromomethyl-7-methoxycoumarin (BMC) in the presence of 18-Crown-6 as a catalyst. The taurine conjugates, which cannot react with BMC, were hydrolyzed enzymatically with cholylglycine hydrolase (Sigma) before their derivatization and analysis as free BA. The BMC-labeled derivatives were analyzed on a nonpolar stationary phase (Nucleosil 100 µm; C18; 5 µm; 250 x 4.6 mm) at 40°C in HPLC equipment from Gynkotek (Germering, Germany) with online-degasser DG 1310, gradient pump M 480, injection automate GINA 160, column oven (Peltier), fluorescence detector RF 1002 (excitation 320 nm; emission 385 nm) (Shimadzu Europe, Duisburg, Germany) and GynkoSoft software. Linear gradients consisting of acetonitrile (30–100%), methanol (40–0%) and water (30–0%) were applied.

The NS were determined using high performance thin-layer chromatography (HPTLC). The solutions (500–3000 nL) were applied 8 mm from the bottom of 20 x 10 cm HPTLC silica gel 60 F₂₅₄ plates (Merck) by spraying (6 mm streaks) with the automatic TLC sampler III (Camag, Muttenz, Switzerland). The chromatograms were developed with ether/heptane (55:45, v/v) in an automatic developing chamber (Camag), with a run distance of 80 mm. The plates were then dipped for 3 s in a copper sulfate/phosphoric acid reagent using the chromatogram immersion device III (Camag) and heated for 5 min at 180°C. After cooling, the spots were measured at 405 nm using a TLC scanner II with CATS software (Camag).

Steroids.

The reference steroids were obtained from the following sources. Steraloids (Wilton, NH): -, ß- and -muricholic acids (MCA), 7-ketodeoxycholic acid (KDCA) and 12-ketolithocholic acid (KLCA), 5-cholesten-3ß,25-diol; Sigma (St. Louis, MO): cholic acid (CA), lithocholic acid (LCA), hyodeoxycholic acid (HDCA), 5-cholestan and 5-cholestan-3-one (cholestanone); Fluka (Neu-Ulm, Germany): chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), cholesterol, 5ß-cholestan-3-one (coprostanone); Serva (Heidelberg, Germany): 5ß-cholestan-3ß-ol (coprostanol); Calbiochem (La Jolla, CA): 7,12ß-dihydroxy-5ß-cholanic acid.

Statistical methods.

Statistical analysis was performed using Statistical Package for Social Sciences software SPSS 11.0 (SPSS, Chicago, IL). All values are given as arithmetical means ± SEM. Data were analyzed by one-way ANOVA, and differences between the diet groups and the control group were evaluated by Dunnett’s t test and Dunnett’s T3-test for multiple post-hoc comparisons. When variances were heterogeneous, data were log-transformed before analyses. Effects of diet on SCFA and steroid concentrations in feces were estimated by generalized linear models with repeated measures corrected by Greenhouse-Geisser Epsilon. Effects were given as time effects and as interactions between time and diet. Differences with P < 0.05 were considered significant.

Ethical considerations.

The experimental protocol was performed according to international and national guidelines. All treatments and diets were formally approved by the Animal Welfare Committee of the State Brandenburg (Ministry of Nutrition, Agriculture, and Forestry), Germany (permissions 48–3560-0/3).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Barley samples and diets.

Extrusion of barley meals (whole-grain type) from HiAmi or from the mixture of HiAmi and Prowashonupana under optimal technological conditions (13 ) followed by short-term storage of the extrudates at -18°C generated 7 g/100 g RS. Higher RS concentrations were obtained when an amylose-Prowashonupana mixture was extruded (barley sample D). Additionally, a mixture of Prowashonupana and the commercial RS preparation Novelose 330 was used (without extrusion). These prepared barley samples were relatively rich in ß-glucan (7–12 g/100 g) (Table 1) . It was not the aim of the extrusion experiments to generate a maximum of RS, but we tried to find an optimum between the RS content obtained in the extrudate and the preservation of advantageous properties of whole-grain barley products such as viscosity.

The extruded barley samples had a higher viscosity in aqueous systems and had greater water-binding properties than the samples that were not extruded. Such functional properties of barley samples may play a role during their passage through the gastrointestinal tract of humans or animals (e.g., binding and holding of water, viscosity and rheological properties, influences on the unstirred layer or on the absorption of nutrients).

In contrast to the control diet, the test diets A–D consisted of 500 g/kg of the barley products. Therefore, they contained more total DF, ß-glucan and RS as well as less wheat starch than the control diet. Furthermore, 50 g/kg of microcrystalline cellulose, a low fermentable DF, was present in all diets. Most of the DF in the diets was classified as insoluble based on the AOAC method (18 ) (Table 2) .

Compared with the control diet (carbohydrate utilization = 100%), carbohydrate utilization was reduced in the test diets as assessed by indirect calorimetric examination in rats. Carbohydrate utilization was significantly diminished in diets C and D to 51.2 ± 5.3 and 66.1 ± 6.1% of control (P < 0.05), respectively. In diets B and A, values were 75.2 ± 12.7% (P = 0.1) and 83.1 ± 11.3%, (P > 0.1) of control, respectively.

Behavior, food intake, and weight gain of the rats.

All diets were well accepted by the rats. There were no treatment-related changes in appearance or behavior of the rats during the experiment. All rats remained healthy during the experimental period. No mortality was caused by the diets.

With some exceptions, food intake increased in all groups from wk 1 [between 85.3 ± 4.1 and 98.7 ± 2.9 g/(rat · wk)] to wk 5 [between 130.4 ± 1.3 and 145.9 ± 2.8 g/(rat · wk)] of the experiment. Generally, food intake was significantly greater in groups fed barley-containing diets in the last 2 wk of the experiment (P < 0.05). Food intake was 118.9 ± 5.5 g/rat in the control group and between 135. 9 ± 3.2 and 142.9 ± 0.7 g/rat at wk 6.

Relatively small differences were measured in the weight gain of all rat groups. The weight of the rats increased from 67 g to 290 g between wk 0 and 6. During wk 6 of the experiment, weights of the barley-fed rats were greater (between 291.4 ± 3.6 g and 298.3 ± 8.1 g) than those of the control group (266.8 ± 5.0) (P < 0.05). The food efficiency (g gain/g feed) did not differ between the control and test groups during the 6-wk experiment (data not shown).

Organ mass, intestinal contents and feces mass.

The masses of the stomach and both parts of small intestine were generally heavier in the control group than in the experimental groups (Table 3 ). In contrast, the masses of the ceca (groups B–D) (P < 0.05) and colons of all groups fed the barley-containing diets (P < 0.001) were greater than those of the control group. This effect was greatest in rats fed the RS-rich diets C and D.

The total dry mass of the intestinal contents, calculated from the wet mass and their dry matter (DM), were greater in the test groups. For instance, the dry colon contents were between 0.463 ± 0.019 and 0.699 ± 0.023 g in the test groups but only 0.301 ± 0.006 g in the control group.

The amounts of feces were determined after freeze-drying all feces collected over 24 h at wk 0, 2, 4 and 6. Excretions were between 2.62 and 2.94 g DM/d in the control group and up to 4.24 g DM/d in the test groups.

Starch and ß-glucans in gastrointestinal contents and feces.

Both starch and ß-glucan were determined at wk 6 in gastrointestinal contents and in feces. The high concentrations of starch were present in the stomach. Although intestinal enzymes degraded the digestible starch, RS remained in the small intestine contents. Therefore, the greater amounts of starch found in the LP of the small intestine in groups C and D (P < 0.001) were due to the RS preparation, Novelose, or resulted from the RS formed during the extrusion of the barley amylose. The starch found in the cecum (P < 0.001) and colon contents (groups A, C and D; P < 0.05) of the test groups consisted exclusively of RS. The disappearance of RS in the feces (including group C) indicated their complete fermentability by the intestinal microflora (Table 4 ).

Microbial counts.

The microflora (in colon, determined in fresh feces) of the rats remained relatively stable throughout the experiment. Only small differences were found in the starting phase (d 3) or at the end of the experiment (d 42) in total aerobic and anaerobic microorganisms (Table 5 ). On the other hand, numbers of coliforms and Bacteroides decreased in test groups B–D (P < 0.05) at wk 6, and higher numbers of Lactobacillus were found in groups B–D at d 3 as well as at the end of the experiment in all test groups (P < 0.05).

The SCFA were determined in fresh feces at wk 2, 4 and 6 as well as in the LP of the small intestine, cecum and colon contents at the end of the experiment. No SCFA were found in the LP contents. The greatest fermentation of the DF components occurred in the cecum where the greatest concentrations of SCFA appeared. In contrast to the control group ( 200 µmol total SCFA/g DM), the total SCFA concentration was 800 µmol/g DM or greater in the cecal contents of the test groups (P < 0.001) (Table 6 ). The greatest formation of SCFA was in groups B and D, consistent with the high concentrations of fermentable DF in diets B and D. Acetic acid was the major SCFA, followed by propionic and butyric acids. Further, small amounts of valeric acid and iso-valeric acid were present in the cecum. The butyrate concentration was > 100 µmol/g DM in the test groups. With the exception of the propionate level in group C (P < 0.2), all individual cecal SCFA were greater in the test groups than in the controls (acetate and butyrate: P < 0.001; propionate: P < 0.05).

In fresh feces, SCFA were determined every 2 wk. The concentrations of the three main SCFA were greater in all test groups than in the control group (P < 0.001) (Table 7 ). The greatest SCFA concentrations were in groups C and D fed diets with the highest contents of RS and insoluble DF. In the test groups, the molar proportion of butyrate was greater than in the control group. The following fecal molar proportions of butyrate were calculated after 6 wk: control, 8.6%; group A, 14.4%; group B, 11.2%; group C, 13.7%; and group D, 13.0%.

From the concentrations of SCFA present in the cecum and colon contents and in feces, absorbed SCFA were estimated. The difference between the SCFA (acetate, propionate and butyrate) in the cecal contents and in feces was 160 µmol/g DM in the control group and between 680 and 770 µmol/g DM in groups A, B and D.

pH values.

As a result of the increased concentrations of SCFA, the pH in the fresh fecal materials were lower in all groups fed the barley-containing diets at wk 3 and 6 as well as in the cecum at the end of the experiment (P < 0.001) (Fig. 1 ). The decrease in pH was greatest in groups B and D (up to one pH unit). Differences in pH between wk 3 and 6 were minimal.

View larger version (44K): [in this window] [in a new window]   FIGURE 1 Cecal content and fecal pH of rats fed control or barley-containing diets for 6 wk. Values are means ± SEM, n = 5 (cecal contents) or 10 (feces). *Different from the control group, P < 0.001.

At wk 0, 2, 4 and 6, BA and NS were analyzed in feces collected for 24 h. As a result of enzymatic actions by intestinal bacteria, conjugated BA were hydrolyzed yielding free BA. Therefore, tauroconjugated BA, which appear mainly in bile, were not detected in the feces. Tables 8 and 9 summarize the concentrations of the individual and total BA. In the control group, no changes in the total excreted BA were observed between wk 0 and 6.

View larger version (53K): [in this window] [in a new window]   FIGURE 2 Primary bile acids in feces of rats fed control or barley-containing diets for 6 wk. Values are means ± SEM, n = 10. *Different from the control group, P < 0.01.

View larger version (48K): [in this window] [in a new window]   FIGURE 3 Secondary bile acids proportions in feces of rats fed control or the barley-containing diets for 6 wk. Values are means ± SEM, n = 10. *Different from the control group, P < 0.001.

Concentrations of all individual BA as well as of primary and secondary BA or of total BA in feces changed during the 6-wk experiment (P < 0.001). The changes differed among the test groups for each BA as well as for primary, secondary and total BA (P < 0.001), with the exception of LCA (P = 0.1). In summary, the proportion of primary BA was increased in the presence of greater amounts of highly fermentable resistant starch and ß-glucan, whereas the total amounts of the excreted BA were positively related to the concentration of ß-glucan in the diets.

Generally, greater concentrations of neutral sterols were found in the feces of the test groups compared with the controls. Feeding the ß-glucan-rich diets B and D resulted in the greatest levels of total NS (Table 10 ). The relationship between the individual NS was relatively constant in all groups. Coprostanol, at 60%, was the dominating NS. Greater excretions of all individual and, therefore, of the total NS were found in the test groups B–D in wk 2, 4 and 6 (P < 0.005) compared with the control. In group A, differences from the control group were found for coprostanone throughout the experiment (P < 0.05) and for total NS (P < 0.001) in wk 2 and 4.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Physiologically, barley is a cereal with an interesting composition (starch, cell wall polysaccharides, lipids, secondary metabolites, vitamins) (21 ,22 ). At present, this cereal is used only to a small extent for human food in most of the developed countries (< 5% of the total production) (21 ). Barley is used particularly in the brewing industry and in animal nutrition.

The content of ß-glucan, the most important DF, is relatively low in most barley varieties. The high viscosity of this cell wall polysaccharide is undesirable in malting and brewing processes. It was shown in several studies that the presence of ß-glucan in the diet is associated with advantageous physiologic effects. Therefore, we prepared extrudates from barley cultivars with relatively high (HiAmi) or very high concentrations of ß-glucan (Prowashonupana) (13 ). The naked barley cultivar Prowashonupana is known for its high DF content ( 15 g/100 g ß-glucan; 12 g/100 g arabinoxylans) (23 ). In addition to the DF components present in whole-grain barley products, we generated RS (preferentially from a part of the amylose components) using extrusion technology. Optimization of this extrusion process resulted in products with up to 7 g/100 g RS, whereas the ß-glucans remained in a macromolecular form (13 ).

The presence of 500 g barley product/kg test diet resulted in a lower carbohydrate utilization in two of the test diets as shown by indirect calorimetry. Recently, a similar effect was found in calorimetric experiments in rats using isolated DF and RS (15 ).

Our experiment was done with young rather than adult rats. The DF-enriched barley-containing diets did not negatively affect the growth and development of these rats. It is interesting that the food intake of the test groups was higher in the last 2 wk of the experiment. This effect may be due to the lower energy content of the test diets compared with the control diet. Food efficiency was not affected by consumption of the barley-containing diets. In contrast, Hecker et al. (24 ) reported lower feed consumption and body weights but no change in feed/gain ratios in rats fed ß-glucan-enriched tortillas.

In an earlier study with pectin, alterations in the intestinal mass and thickness of the intestinal walls were found as a result of the physicochemical actions of the viscous DF in the intestinal tract (25 ). In this study, the masses of the cecum and colon (without contents) were significantly greater in the test groups. The main reason for this effect was the presence of viscous DF such as ß-glucan in the diets. Additionally, the masses of some cecal contents and of all colon contents were increased in the test groups. Much more material must be transported through the lower parts of the intestinal tract if DF-enriched diets are fed. Therefore, the motility of the cecum and large intestine is increased and the weights and thickness of the walls (not shown) were greater.

Although digestible starch disappears in the ileum, resistant starch reaches the lower parts of the intestinal tract. RS is preferentially fermented in the cecum of rats by the microflora. Our results showed that some of the RS in the test groups reached the colon where complete fermentation occurred, resulting in the formation of SCFA. Obviously, ß-glucan was highly fermentable in the cecum. In feces, RS and ß-glucan were not found using sensitive enzymatic methods.

Effects of DF on the intestinal microflora depend on both the type and structure of the fibers and their concentration. Thus, stimulation of beneficial bacteria and inhibition of the growth of harmful bacteria were reported for inulin and oligofructose (26 ). In our experiments, only small effects of the diets on the microbial counts and the composition of the microflora were observed. An interesting effect was the increase in numbers of the beneficial microorganisms (Lactobacillus) in all test groups.

Like other DF (including RS), isolated ß-glucan is rapidly fermented by the intestinal microflora in vivo (27 ) and in vitro, with formation of SCFA as the most important end products. It was shown that the structure of the DF components is essential for both the degree of fermentation and the relationship between the individual SCFA (28 ). Thus, a rapid fermentation of ß-glucan and starch in vitro was reported in the literature (29 ). RS is a good source for the production of high levels of butyrate (30 ). The lower concentrations of SCFA in the ceca of rats in group C was associated with the greater content of unfermented RS in the cecal contents of this group and an increased amount of SCFA in feces. Therefore, it seems to be possible to shift the fermentation site toward the lower parts of the colon if more slowly fermentable DF (e.g., Novelose 330 compared with the generated RS) are present in the diet. Related effects were described for the fermentation of RS (high amylose cornstarch) in the presence of psyllium (31 ) or for pectins with higher degrees of methylation (19 ).

High levels of SCFA, particularly of butyrate, in the lower parts of the intestinal tract are important for a healthy large intestine mucosa. SCFA, which are the major anions in the colonic lumen, are rapidly absorbed by the colonic mucosa (32 ). Butyrate is the preferred energy source of the colonic epithelial cells and acts specifically as a signal metabolite, stimulating cell migration and proliferation (33 ). However, the influence of butyrate on the functions, metabolic activities and products of normal and malignant epithelial cells may be different (34 ). It was shown that the fermentable substrates, as well as the adaptive period, positively influence both butyrate production and the protection against carcinogenesis in rats (35 ).

The greater concentrations of BA in feces of rats fed the barley-containing diets confirm observations that ß-glucan increased BA excretion in ileostomy subjects (36 ). It has been shown in vitro that there are interactions between BA and ß-glucan (13 ,37 ), but the mechanisms are notknown. Direct linkages between BA and ß-glucan were not found (38 ). Likely, the viscosity in the gut, a disturbance of the micelle formation and lipid digestion as well as the hindrance of BA absorption caused by DF components of the diet are the main reasons for the presence of greater concentrations of BA in lower parts of the intestinal tract and also in feces. A relationship between the amount of BA excreted and the decrease of plasma cholesterol in (hypercholesterolemic) subjects and animals has been discussed (39 ,40 ). Further, the cholesterol-lowering effect may be mediated through a viscosity-associated specific reduction in cholesterol absorption (41 ). It should be mentioned that in our experiment, using young rats with normal plasma lipid levels, no alterations in cholesterol and its fractions were observed. Only the concentrations of phospholipids were significantly lower in groups fed the barley-containing diets (data not shown).

The reason for the reduced amounts and proportions of secondary BA is the partial inhibition of the bacterial enzyme 7-dehydroxylase, which is responsible for the formation of secondary BA from primary BA, by lower intestinal pH values (42 ). Secondary BA, particularly DCA and LCA, are considered to be promoting factors in the pathogenesis of colon cancer (43 ). Therefore, levels of secondary BA should be suppressed and the transit time of the gut contents should be decreased. DF can positively influence these effects. Further, unfermented, swollen DF "dilute" the BA in the colon. Butyrate and DCA appear to interact in a complex and antagonistic manner to selectively modulate crypt base and surface proliferation in the rat colon (44 ).

In conclusion, barley products enriched in DF show a variety of beneficial physiologic and protective actions in the intestinal tract of rats. They increase viscosity in the small intestine and are highly fermentable. The viscosity of the ß-glucans is associated with greater excretion of BA and NS. The more acidic conditions in the cecum and the colon lead to the smaller proportion of secondary BA. Both the significantly higher formation and the increased absorption of SCFA are important in the protection of the colon mucosa. Regardless of differences in anatomy, steroid pattern or microflora, for example, between humans and rats (45 –47 ), the results demonstrate the considerable physiologic benefits possible from RS-enriched barley products of the whole-grain type.

	ACKNOWLEDGMENTS

 
The authors thank Angelika Lorenz (Gesellschaft für Innovative Mikrobiologie mbH, Wildenbruch, Germany) for measuring microbial counts, Uwe Frenz for indirect calorimetry experiments and Corinna Koebnick for their assistance in statistical analysis. The skillful technical assistance of Monika Niehaus and Horst Maischack is gratefully acknowledged. We thank W. Bonner, ConAgra, Omaha, NE for providing the "Prowashonupana" sample.

	FOOTNOTES

 
¹ Supported by the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn), the AiF and the Ministry of Economics and Technology (Project no. 10751 B).

³ Abbreviations used: BA, bile acid; BMC, 4-bromomethyl-7-methoxycoumarin; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; DF, dietary fiber; DM, dry matter; HDCA, hyodeoxycholic acid; HPTLC, high performance thin-layer chromatography; KDCA, 7-ketodeoxycholic acid; KLCA, 12-ketolithocholic acid; LCA, lithocholic acid; LP, lower part of small intestine; MCA, muricholic acid; NS, neutral sterol; RS, resistant starch; SCFA, short-chain fatty acid; UDCA, ursodeoxycholic acid; UP, upper part of small intestine.

Manuscript received 4 June 2002. Initial review completed 5 July 2002. Revision accepted 3 September 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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