J. Anim. Sci. 2004. 82:2615-2622
© 2004 American Society of Animal Science
In vitro fermentation of various fiber and starch sources by pig fecal inocula1
J. F. Wang*,
,2,
Y. H. Zhu,
D. F. Li*,
Z. Wang* and
B. B. Jensen
* College of Veterinary Medicine, China Agricultural University, Beijing 100094, P.R. China; and
and
Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Research Center Foulum, Tjele, Denmark
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Abstract
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Freeze-dried ileal effluent (1% wt/vol) from cannulated pigs fed rice-based diets with the inclusion of either animal protein (CON), animal protein plus potato starch (PS), animal protein plus sugar beet pulp (SBP), or animal protein plus wheat bran (WB) was incubated anaerobically at pH 6.0 in fermenters containing 5% (wt/vol) fecal slurry comprising mineral salts medium and 50 g/L of fresh feces from pigs fed the same diets as the cannulated pigs. Samples were collected from the fermenters at 0, 2, 4, 12, 24, and 48 h during in vitro fermentation for measuring nonstarch polysaccharides (NSP), starch, and short-chain fatty acids (SCFA). Results showed that the major SCFA produced were acetate, propionate, and butyrate. The inclusion of soluble dietary fiber (diet SBP) caused the highest concentrations of acetate, propionate, butyrate, and total SCFA, whereas the increase in the production of propionate resulting from the addition of insoluble dietary fiber (diet WB) only occurred at the initial stages during 48 h in vitro fermentation. At all sampling occasions (except for 4 h), the levels of butyrate were increased (P < 0.01) by resistant starch compared with fiber sources, showing that a higher level of butyrate can be achieved through microbial fermentation by potato starch. Lowered (P < 0.05) butyrate concentrations were observed with diet WB during in vitro fermentation. With the inclusion of fiber sources, the energy originating from SCFA was similar to that from NSP disappearance, whereas the values were lower (P < 0.05) from NSP disappearance than for SCFA generated without fiber sources supplemented. We conclude that more substrate is available in ileal effluent with the addition of soluble dietary fiber, and an increased level of butyrate could be achieved through microbial fermentation by resistant starch.
Key Words: Dietary Fiber • In Vitro Fermentation • Pig • Short-Chain Fatty Acids • Starch
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Introduction
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The large intestine provides a chamber for the final phase of digestion in the pig, and this digestive function principally involves the breakdown of carbohydrates by microbes under anaerobic conditions to short-chain fatty acids (SCFA) and gases (H2, CO2, CH4; Cummings and Macfarlane, 1991
). The major SCFA are acetate, propionate, and butyrate, accounting for 90 to 95% of total fatty acids (Christensen et al., 1999). A number of factors (e.g., the type and chemical structure of polysaccharides fermented, activities of the colonic microbial population, and gastrointestinal tract transit time) can affect the composition and molar ratios of SCFA produced in the large intestine (Englyst et al., 1987).
Due to the physical inaccessibility of the large intestine, numerous investigations on SCFA production by colonic bacteria have been undertaken with feces; however, 95% of the SCFA generated in the colon are absorbed from the large intestine during transit of effluent through the gut (Cummings, 1981). As a consequence, determinations of fecal SCFA cannot be directly related to the events taking place in the large intestine itself (Cummings and Macfarlane, 1991). In the pig, the amount of SCFA absorbed per kilogram of BW is 95 mmol/d (Stevens et al., 1980). One approach to solving this problem is to use an in vitro fermentation method and animal models. The pig is the most reliable animal model with which to study SCFA production and gut physiology in man (Flemming and Arce, 1986), although the length and the capacity of the large intestine in pigs are approximately 1.5 to 3 times larger than in humans (Jensen, 1990). The aim of the current study was to investigate how dietary polysaccharide sources affect SCFA production in an in vitro fermentation system.
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Materials and Methods
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This experiment was carried out at the Danish Institute of Agricultural Sciences, Foulum, Denmark. Experimental protocols for the care and use of animals were in accordance with the guidelines established by the Danish Ethical Commission.
Experimental Diets, Animals, and Feeding
Rice was cooked daily (1 atm, 121°C, 20 min) with water in a 1:2 (wt/wt) proportion. The four experimental diets (Table 1) consisted mainly of cooked rice with the addition of protein sources (CON), partial replacement of cooked rice with potato starch (PS), partial replacement of cooked rice with sugar beet pulp (SBP), or partial replacement of cooked rice with wheat bran (WB).
Eight 10-wk-old castrated pigs (Danish Landrace x Large White) weighing 30 to 33 kg, obtained from the Danish Institute of Agricultural Sciences swine herd (Foulum, Denmark), were used in the current study. The pigs were fitted with a permanent T-cannula placed approximately 150 mm anterior to the ileocecal junction (Bach Knudsen et al., 1993).
For the preparation of freeze-dried ileal effluent, the study was carried out with a repeated 4 x 4 Latin square design with eight cannulated pigs fed one of the four experimental diets. Experimental periods lasted 15 d and comprised 7 d of adaptation to each diet, followed by 2 d of feces and urine collection, 3 d of ileal effluent collection, and 2 d of recording gas exchange in four consecutive periods. A detailed description of the test diets, feeding, and collection of ileal effluent has previously been presented by Wang et al. (2002). Briefly, ileal effluent samples were collected for 12 h, comprising 0900 to 1100 and 1300 to 1500 on d 13, 0800 to 1000 and 1200 to 1400 on d 14, and 0700 to 0900 and 1100 to 1300 on d 15. A total of 32 ileal effluent samples (four ileal effluent samples per pig) was used to predict the production of SCFA. The composition of ileal contents is indicated in Table 2.
In Vitro Fermentation System
In the present in vitro fermentation study, fresh feces were taken directly from the rectum of four pigs (35 to 45 kg BW) fed the test diets for providing the respective fermenting microbial flora (Christensen et al., 1999). The feces were rapidly suspended under a constant flow of CO2 gas in sterile anaerobic salt medium to give a final fecal slurry of 100 g of feces/L as described by Jensen et al. (1995). The sterile anaerobic salt medium comprised the following constituents in distilled water (g/L): NaHCO3, 5.0; NaCl, 0.9; (NH4)2SO4, 0.9; KH2PO4, 0.45; K2HPO4•3H2O, 0.45; CaCl2•2H2O, 0.03; CaCl2•2H2O, 0.03; MgCl2, 0.02; MnSO4•4H2O, 0.01; CoCl2•6H2O, 0.01; FeSO4•7H2O, 0.01; and cysteine, 0.25. Sterile anaerobic salt medium was autoclaved (1 atm, 121°C, 15 min) and then cooled under high-purity N gas, and the filter-sterilized cysteine was then added to the cooled anaerobic salt medium. The fecal slurry was rapidly transferred to a CO2-flushed plastic bag and homogenized in a stomacher laboratory blender (Seward Medical, London, U.K.) for 2 min.
The fermenters (1.5-L volume) were autoclaved before use. Three hundred fifty milliliters of the respective fecal slurry was added to a fermenter containing 350 mL of sterile anaerobic salt medium and 7 g of the respective freeze-dried ileal effluent, thereby giving a total volume of 700 mL containing 50 g of feces/L and 10 g of freeze-dried ileal content/L. As a control, a fermenter with a working volume of 400 mL containing 200 mL of fecal slurry of the respective treatment and 200 mL of sterile anaerobic salt medium was used to measure the fecal contribution to the production of SCFA. The incubation temperature was kept at 38°C by a circulating water bath, and the culture pH was automatically adjusted to 6.0 (±0.3) by a pH controller (Braun Diessel Biotech, Melsungen, Germany) connected to a pH-electrode (Radiometer, Copenhagen, Denmark) using 5 M NaOH and 5 M HCl as titrants. The slurry was stirred magnetically and maintained under anoxic conditions by sparging with high-purity N gas.
Samples of 10 mL were aseptically removed from the fermenters at 0, 2, 4, 12, 24, and 48 h for measurements of SCFA. Total nonstarch polysaccharides (NSP) and starch contents of samples at 0 h were also measured. After removing the 48-h samples, the contents of total NSP and starch were determined in the remaining suspension. All samples were stored at –20°C until analysis.
Measurements, Calculations, and Statistical Analyses
The GE content of diets was determined with an adiabatic bomb calorimeter (IKA Calorimeter C 400; Janke and Kunkel, Staufen, Germany). Crude protein was measured in a Kjell-Foss 1620 autoanalyzer (Foss Electric A/S, Hillerød, Denmark) by the Kjeldahl method. Hydrochloric acid-hydrolyzed fat (HCl-fat) was extracted with diethyl ether after acid hydrolysis (Stoldt, 1952). Ash was analyzed by the method of the AOAC (1990) Method 942.05. Dry matter contents of ileal effluent and feces were analyzed after freeze-drying followed by drying at 105°C for 20 h. Chromic oxide was determined according to Fenton and Fenton (1979). Total NSP and constituent sugars were determined as alditol acetates by GLC and uronic acids by colorimetry as described by Bach Knudsen (1997). Cellulose was estimated as the difference in NSP-glucose content obtained for total NSP and that obtained after hydrolyzing starch-free residues directly with 2 M H2SO4. Starch was measured enzymatically as described by Bach Knudsen (1997). Short-chain fatty acids and lactate were determined as described by Jensen et al. (1995). An internal standard (100 µL of 100 mmol of 2-ethylbutyric acid/L) was added to 1 mL of sample (or a standard solution) and the acids were extracted by the addition of 0.5 mL of concentrated HCl and 2 mL of diethyl ether, followed by vortex mixing. After centrifugation (1,000 x g, 4°C, 10 min), 50 µL of the ether layer was removed and transferred to a gas chromatography microvial, and 10 µL of derivatization reagent (N-(tert-butyldimethylsilyl)-N-methyl-trifluoracetamid, Fluka 19918, Fluka Chemie AG, Buch, Switzerland) was added and the mixture heated to 80°C for 20 min. The reaction mixture was left at room temperature for a further 48 h to ensure complete derivatization. Total ileal effluent was calculated relative to the chromic oxide content as described by Bach Knudsen et al. (1993). The amounts of SCFA produced from in vitro fermentation were calculated as:
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where SCFA(substrate, diet) is the individual or total SCFA produced per gram of ileal effluent from the in vitro fermentation of 1% (wt/vol) ileal effluent and 5% (wt/vol) feces, and SCFA(feces, diet) is the individual or total SCFA produced from the in vitro fermentation of 5% (wt/vol) feces alone. The energy equivalents of each class of SCFA (kJ/mol) and of NSP (kJ/g) were determined as follows: 877 acetate, 1,533 propionate, 2,185 butyrate (CRC, 1983), and 17.6 NSP (Christensen et al., 1999).
The statistical analyses were performed using the mixed model procedure of SAS (SAS Inst., Inc., Cary, NC). The model included diet, time, and diet x time. When there was an overall effect of diet (P < 0.05), differences between means were compared by the Fisher’s least significant difference. Results are presented as mean values and standard error of means.
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Results and Discussion
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The pigs behaved normally and remained in good health throughout the experimental period. The ileal effluent collection was generally completed within 15 min, and no complications or potential problems such as leakage from the cannula occurred during the experiment.
Effect of the Test Diets on SCFA Molar Ratios, and Degradation Coefficients of NSP and Starch After Incubation for 48 h
The results of SCFA determinations from the in vitro fermentation of freeze-dried ileal effluent with fresh fecal inocula show that colonic bacteria formed different fermentation products from different substrates. After 48 h of incubation, the molar ratios of acetate, propionate, and butyrate were 52:23:14 with diet CON, 54:22:16 with diet PS, 60:23:12 with diet SBP, and 55:28:10 with diet WB (Table 3), suggesting that different fermentation products from different substrates could be achieved by the colonic bacteria. This is in agreement with the findings of Macfarlane and Macfarlane (1993) using different substrates (starch, pectin, arabinogalactan and xylan). The rates of breakdown of the polysaccharides were different: starch was degraded most rapidly, followed by pectin, arabinogalactan and xylan, and the molar ratios of acetate, propionate, and butyrate were 50:22:29 with starch, 84:14:2 with pectin, 50:42:8 with arabinogalactan, and 82:15:3 with xylan (Macfarlane and Macfarlane, 1993). Because these substrates must be depolymerized before they can be fermented, the rates of substrate hydrolysis will strongly affect the rate at which carbohydrates become available to the bacteria (Macfarlane and Macfarlane, 1993). In the current study, the production of lactate only occurred at 0 to 2 h (data not shown); up to 4 h, the concentration of lactate decreased dramatically and the decreases in the later stages should be due to its conversion into SCFA. Similarly, a large amount (11 to 15 mmol/L) of lactate was only detected in the initial stages of the incubation (3 to 12 h) during 96 h fermentation of starch by human colonic bacteria (Macfarlane and Englyst, 1986). After incubation for 96 h, acetate, propionate, and butyrate as the only SCFA produced corresponded to 44, 18, and 24 mmol/L, respectively, and the molar ratios of acetate, propionate, and butyrate were 51:21:28 (Macfarlane and Englyst, 1986).
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Table 3. Molar ratios (%) of short-chain fatty acids after incubation for 48 h using in vitro fermentation systema
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It is generally thought that NSP and starch, which are not hydrolyzed by the small intestinal enzymes, subsequently become available for fermentation by the colonic microflora and provide the major sources of SCFA in the large intestine (Cummings, 1981; Macfarlane and Englyst, 1986). These SCFA are physiologically active in the large intestine and absorbed through the gut wall, thus providing an additional source of energy for the host (Cummings, 1981). Short-chain fatty acids are rapidly absorbed from the large intestine (McNeil et al., 1978), from which they pass into the portal vein (Cummings and Macfarlane, 1991). In humans, cell population densities of anaerobic starch-hydrolyzing bacteria in the stools corresponded to 1.1 x 1010 to 3.3 x 1012/g of feces (Macfarlane and Englyst, 1986). Approximately 60% of starch utilized and entering the colon was converted to SCFA, which would be potentially available for absorption in the large intestine of man (Macfarlane and Englyst, 1986). The major SCFA occurring in the human large intestine are acetate, propionate, and butyrate, which amount to 46, 17, and 15 mmol/L, respectively (Rubinstein et al., 1969). For comparison, in our previous study with growing pigs fed rice-based diets with the inclusion of dietary fiber and starch sources, we found that the concentrations of acetate, propionate, and butyrate were 64 to 86, 19 to 26, and 11 to 24 mmol/kg of feces, respectively (Wang et al., 2004b). A higher level of acetate in pigs was observed compared with humans. This might be explained by the fact that the length and the capacity of the large intestine in pigs are approximately 1.5 to 3 times larger than in humans (Jensen, 1990), allowing for enhanced microbial fermentation in the pig large intestine.
In the current investigation, the energy calculated from NSP disappearance after incubation for 48 h is negative (–0.6 MJ) with diet PS, whereas the values corresponded to 19.9, 45.1, and 29.9 MJ with diets CON, SBP, and WB, respectively (Table 4). The total energy values derived from SCFA produced during the 48-h incubation amounted to 30.8, 34.7, 44.8, and 28.4 MJ with diets CON, PS, SBP, and WB, respectively. This indicated that the energy value from NSP disappearance was similar to that from SCFA for diet SBP based on soluble dietary fiber (45.1 vs. 44.8 MJ) or for diet WB based on insoluble dietary fiber (29.9 vs. 28.4 MJ). However, the energy value derived from SCFA was markedly higher than that based on NSP disappearance for diet CON (30.8 vs. 19.9 MJ) and for diet PS (34.7 vs. –0.6 MJ), respectively. It is well known that resistant starch will pass into the large intestine, where it may act as substrate for colonic microflora (Wang et al., 2004a). It should be noted that the negative value might be due to a lowered level of NSP in the ileal effluent with diet PS.
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Table 4. Production of short-chain fatty acids, total SCFA energy (TESCFA), nonstarch polysaccharides (NSP) energy (ENSP), and degradation coefficients (dc) of NSP and starch after incubation for 48 h using in vitro fermentation systema,b
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The current study shows that the starch degradation coefficients were higher with diets CON and PS than with diets SBP and WB (on average 0.73 vs. on average 0.15; Table 4
). This further confirms that starch, escaping digestion in the small intestine, could act as the substrate for colonic microbial fermentation. An extremely lowered value with the addition of wheat bran might be the result of limited starch content in the terminal ileum.
In the present investigation, on diet CON, the SCFA were produced by microbial fermentation of NSP escaping small intestine digestion and starch resisting breakdown in the small intestine (i.e., resistant starch). Nevertheless, on diet PS with potato starch, SCFA were mainly originated from microbial fermentation of resistant starch due to a low level of NSP in the ileal effluent. This implies that a substantial part of potato starch passed into the hindgut, where it was acting as a main source of carbon for the colonic microflora (Jensen, 1990; Macfarlane and Macfarlane, 1993). However, it should be noted that the overall amount of energy available from microbial fermentation in the hindgut might be underestimated by the in vitro fermentation method due to the fact that endogenous secretion of glycoproteins and exfoliated epithelial cells in the large bowel were not included (Christensen et al., 1999).
Mason (1980) assumed an efficiency of 75% for converting carbohydrates into organic acids. The values of the net absorption of energy derived from fermentative breakdown of carbohydrates in the large intestine amounted to 10 to 24% of the ME required for maintenance (MEm; Bach Knudsen and Hansen, 1991). Mason (1980) reported even higher values for energy contribution from large intestine (33 to 44% of MEm). For comparison, by an in vitro fermentation method the SCFA produced in the large intestine corresponded to 11.6 and 9.6% of MEm with low- and high-carbohydrate diets, respectively (Imoto and Namioka, 1978).
Results of the current study clearly indicate that higher concentrations of total SCFA were found with the inclusion of dietary fiber (sugar beet pulp or wheat bran) compared with diets CON and PS after incubation for 4 h (see Figure 4), suggesting that the levels of all the SCFA produced in the hindgut could be increased through enhanced microbial fermentation by addition of the fiber sources in the initial stages of fermentation. After 4 h, however, a marked increase in the concentration of total SCFA only occurred with sugar beet pulp supplementation. This indicates that more substrate was available in ileal contents from diet SBP, and that there is a difference in the utilization by colonic microflora between various fiber sources. Moreover, this may have implications for the gut environment (e.g., pH and bacteria) in pigs. For comparison, an earlier study shows that dietary fiber (sugar beet pulp and wheat bran) led to increased amounts of all the SCFA and increased populations of various bacteria (e.g., lactic acid bacteria, lactobacilli, yeasts, coliforms) in the hindgut, suggesting that the enhanced microbial fermentation takes place by the addition of dietary fiber (Wang et al., 2004b).
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Figure 4. The concentration of total short-chain fatty acid during 48 h of in vitro fermentation of ileal effluent by fecal bacteria from pigs fed the test diets CON, PS, SBP, and WB. Values are means of eight observations with the standard error of means represented by vertical bars. Within the same time point: *P < 0.05; **P < 0.01. CON = control diet, PS = potato starch-supplemented diet, SBP = sugar beet pulp-supplemented diet, and WB = wheat bran-supplemented diet.
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Effect of the Test Diets on Acetate, Propionate, Butyrate, and Total SCFA Production During In Vitro Fermentation for 48 h
Figure 1 depicts the concentrations of acetate during in vitro fermentation for 48 h. There was no diet x time interaction (P = 0.33). No differences were found in the level of acetate between diets CON and PS during in vitro fermentation for 48 h. At 2 h, the level of acetate was higher (P < 0.001) with the inclusion of either sugar beet pulp or wheat bran, showing that an enhanced microbial fermentation could be achieved by the addition of dietary fiber in the initial stages of the in vitro incubation. High levels of acetate were observed throughout the 48-h fermentation for diet SBP. The high level of acetate occurring in the initial phase with the addition of insoluble dietary fiber suggests that there was a difference in the production of SCFA between various fiber sources, which may be of importance for the kinetics in microbial fermentation.
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Figure 1. The concentration of acetate during 48 h of in vitro fermentation of ileal effluent by fecal bacteria from pigs fed the test diets CON, PS, SBP, and WB. Values are means of eight observations with the standard error of means represented by vertical bars. Within the same time point: *P < 0.05; **P < 0.01; ***P < 0.001. CON = control diet, PS = potato starch-supplemented diet, SBP = sugar beet pulp-supplemented diet, and WB = wheat bran-supplemented diet.
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There was no diet x time interaction (P = 0.29) affecting the concentrations of propionate during in vitro fermentation for 48 h (Figure 2). In the initial stages (within 4 h) during fermentation, propionate concentrations tended (P < 0.10) to increase with diet WB compared with diet CON. Thereafter, higher propionate concentrations (P < 0.05) were observed with the inclusion of sugar beet pulp compared with the other test diets. It has been suggested that propionate, generated during colonic fermentation, may be gluconeogenic and inhibit cholesterol synthesis in the liver in animals (Thacker et al., 1981). In a study performed with humans, rectally infused acetate increased serum cholesterol, glucagon, and acetate concentrations, and decreased serum FFA concentrations within 0.5 h, whereas propionate infused alone resulted in increased serum propionate, glucose, and glucagon levels with no influence on cholesterol and a delayed fall in FFA. However, a mixture of acetate (180 mmol) and propionate (60 mmol) caused no increase in serum cholesterol (Wolever et al., 1991). This suggests that colonic propionate is a gluconeogenic substrate and inhibits the utilization of acetate for cholesterol synthesis (Wolever et al., 1991).
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Figure 2. The concentration of propionate during 48 h of in vitro fermentation of ileal effluent by fecal bacteria from pigs fed the test diets CON, PS, SBP, and WB. Values are means of eight observations with the standard error of means represented by vertical bars. Within the same time point: *P < 0.05; **P < 0.01. CON = control diet, PS = potato starch-supplemented diet, SBP = sugar beet pulp-supplemented diet, and WB = wheat bran-supplemented diet.
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Using human fecal inocula, the fermentative fates of various fiber sources (cellulose, sugar beet fiber, soybean fiber, corn bran, and pectin) varied by measuring the residual NSP and SCFA production during 24 h of in vitro fermentation of fiber sources (Barry et al., 1995). The results show that after incubation for 24 h, the degradability coefficients of cellulose and corn bran were very low (0.072 and 0.062, respectively), whereas pectin and soybean fiber were highly degraded (0.97 and 0.91, respectively). The degradability coefficient of sugar beet fiber was in an intermediate level (0.60). Furthermore, SCFA production was closely related to NSP degradability (r = 0.99). In the current work, the degradability coefficient of sugar beet fiber after incubation for 48 h was 0.51, which is lower than that found by Barry et al. (1995). This may be explained by the fact that the substrate used in the current study was freeze-dried ileal effluent, in which NSP was more difficultly degraded by the colonic microflora compared with using the fiber source itself as a substrate.
As shown in Figure 3, there was no diet x time interaction (P = 0.22) for the levels of butyrate formation during in vitro fermentation for 48 h. The levels of butyrate formation were higher at 12 (P < 0.01) and 24 h (P < 0.001) with the inclusion of potato starch compared with diets CON and WB. After incubation for 48 h, the level of butyrate was higher with diets PS and SBP than with diet WB, suggesting that potato starch resulted in increased levels of butyrate, and decreased butyrate concentrations by inclusion of wheat bran. High levels of butyrate formation from starch have also been reported in several in vivo studies in man (Scheppach et al., 1988) and in pig (Wang et al., 2004b). Because butyrate is the preferred fuel for colonic epithelial cells (Roediger, 1980) and has been suggested as beneficial in the prevention of colon cancer (van Munster and Nagengast, 1993) and to inhibit the growth of certain neoplastic cell lines in some circumstances (Kim et al., 1982), the relationship between starch fermentation and butyrate production is of special interest (Roediger, 1980).
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Figure 3. The concentration of butyrate during 48 h of in vitro fermentation of ileal effluent by fecal bacteria from pigs fed the test diets CON, PS, SBP, and WB. Values are means of eight observations with the standard error of means represented by vertical bars. Within the same time point: **P < 0.01; ***P < 0.001. CON = control diet, PS = potato starch-supplemented diet, SBP = sugar beet pulp-supplemented diet, and WB = wheat bran-supplemented diet.
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Implications
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The current study suggests that the in vitro fermentation system, in which freeze-dried ileal effluent was used as the fermentative substrate and fresh feces as inocula, was a reliable model to simulate colonic fermentation. The study indicates that the main end products of colonic microbial in vitro fermentation were acetate, propionate, and butyrate. The inclusion of soluble dietary fiber enhanced concentrations of short-chain fatty acids. Potato starch increased butyrate formation. The study also revealed that types and amount of residues escaping small intestine digestion could affect microbial fermentation in the hindgut.
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Footnotes
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1 The authors thank S. Thomke for valuable discussion and comments. We are also grateful to the Danish Ministry of Food, Agriculture, and Fisheries, and the China Agricultural University for financial support. 
2 Correspondence: No.2 Yuanmingyuan West Road (phone: 86-10-62893053; fax: 86-10-62891274; e-mail: jiufeng_wang{at}hotmail.com).
Received for publication December 26, 2003.
Accepted for publication May 17, 2004.
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M. Anguita, N. Canibe, J. F. Perez, and B. B. Jensen
Influence of the amount of dietary fiber on the available energy from hindgut fermentation in growing pigs: Use of cannulated pigs and in vitro fermentation
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October 1, 2006;
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[Full Text]
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Abstract
Introduction
