Fermentation Products in Feces of Rats Fed High-Fiber or Fiber-Free Diets

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1993, p /93/3657-6$2./ Copyright 1993, American Society for Microbiology Vol. 59, No. 3 Amounts of Viable Anaerobes, Methanogens, and Bacterial Fermentation Products in Feces of Rats Fed High-Fiber or Fiber-Free Diets ANNE E. MACZULAK,t MEYER J. WOLIN, AND TERRY L. MILLER* Wadsworth Center for Laboratories and Research, New York State Department of Health, Box 59, Albany, New York Received 2 October 1992/Accepted 9 December 1992 We investigated the impact of dietary fiber on the fecal output of microorganisms and microbial fermentation products of rats. Two groups of five male Wistar rats were fed high-fiber (HF) and fiber-free (FF) diets in the following order (group 1) lab chow -- HF -* FF -* HF and (group 2) lab chow -+ FF -- HF -- FF. Daily fecal output of total viable anaerobes was 71 times higher with the HF diet. Daily output of methanogens was 1.4 times higher for the HF diet than for the FF diet. Daily excretion of total fermentation acid products (acetate, propionate, butyrate, lactate, succinate, and formate) was 2.4 and.1 mmol for HF and FF diets, respectively. The ratios of acetate/propionate/butyrate were 69:21:1 for the HF diet and 92:7:1 for the FF diet. The results show that an HF diet significantly increases microbial growth in the colon and influences the proportions of organic acid products. The HF diet did not increase the ratio of methanogens to total anaerobes. We suggest that the contribution of host-derived substrates to colonic microbial growth and fermentation is insignificant. High-fiber (HF) diets are recommended to prevent the development of some diseases, e.g., arthersclerosis and colon cancer (2), and for controlling glucose assimilation by diabetics (7). Plant polysaccharides, e.g., cellulose, hemicellulose, pectin, and starch, are major constituents of HF diets (21). Of these, only starch is digested by host enzymes (in saliva and the small intestine). Some starch escapes host digestion and enters the colon, along with the other polysaccharides that are not digested by host enzymes. The polysaccharides are then used as substrates for fermentation by the microbial species that constitute the colonic microbial community. The polysaccharides are converted to acetic, propionic, and butyric acids, which are used by the host as sources of carbon and energy (8). Butyric acid is a major energy source for colonic epithelial cells (19). Recent studies show that butyric acid enemas decrease the rate of defecation and bleeding in patients with inflammatory bowel disease (22). The production of microorganisms and their fermentation products in the colon and their excretion in feces presumably increase as dietary fiber consumption increases because of the increase in the availability of substrates for microbial growth. Increased fecal output would be expected because bacterial dry matter constitutes about 25% of the dry matter of feces (4, 23). However, there are no direct measurements of the relationship between dietary fiber ingestion and the production of microbial biomass. In this study, we determined the output of bacteria and feces of rats that received no dietary fiber and the increase caused by the addition of large amounts of fiber. We compared the daily output of fecal dry matter, total viable anaerobes, and fermentation acids. We fed the semidefined HF and fiber-free (FF) diets designed by Demigne and Remesy (9). They used the diets to investigate the effects of HF diets on the production of * Corresponding author. t Present address: Department of Dermatology, School of Medicine, University of California, San Francisco, CA microbial fermentation products in the ceca of rats. They measured the concentrations of acetate, propionate, and butyrate in the cecum and their rates of transport to blood. Our results complement their results on production and transport of fermentation products. We used a Wistar rat colony that we had previously shown to harbor colonic methanogens (15) to investigate the possibility that an HF diet might specifically increase the concentration of fecal methanogens. The cause of the large differences in colonic methane production (1) and concentrations of methaneforming bacteria in human feces (25) is unknown. MATERIALS AND METHODS Animals. Rats were from a Wistar colony maintained by the central animal facility of the Wadsworth Center. The colony was established in 1959 from animals obtained from the Walter Reed Army Hospital and maintained by random breeding. Previous studies showed that these rats harbor methanogens (15, 18). All rats were housed in cages lined with wood shavings (two to three rats per cage). Fecal samples were collected from individual rats 1 to 2 h after they were placed in metabolism cages. Fecal samples from groups of rats were collected 1 to 2 h after they were placed in cages fitted with raised wire floors. Rat fecal and colonic experimental protocols were approved by the Wadsworth Animal Welfare Committee in June 1983, and protocols have been reviewed and approved yearly thereafter. Diet study. The laboratory chow (special mix 521-3) was from the Ralston Purina Co., St. Louis, Mo. The HF (diet 191, lot no. 1346) and FF (diet 192, lot no. 1366) diets were prepared by Bioserve, Inc., Frenchtown, N.J. They were identical to those described by Demigne and Remesy (9). Both diets contained 5% each of corn oil and a salts mixture and 1% of a vitamin mixture. The FF diet contained 24% soya protein and 65% wheat starch. The HF diet contained 4% soya cake, 2% crude potato starch, 19% wheat bran, and 5% each of apple pectin and carob gum. Demigne and Remesy (9) found that higher production and absorption of Downloaded from on December 31, 218 by guest 657

2 658 MACZULAK ET AL. APPL. ENvIRON. MICROBIOL. n E Cu ) l L-. D 25-5 l Age (days) FIG. 1. Rats' weights during the experiment. Each point is the mean of the individual weights of five rats in each group. Group 1 (e) and group 2 (A) rats were fed lab chow from weaning. Group 1 diet changes were made when their ages were 62 (HF), 84 (FF), and 119 (HF) days. Group 2 diet changes were made when their ages were 66 (FF), 92 (HF), and 114 (FF) days. The arrows indicate the days that the diets were switched. fermentation acids occurred when animals were fed mixtures of fibers rather than high concentrations of a single fiber. Two groups of five male Wistar rats each were used. The rats were maintained on lab chow from weaning until the beginning of the experiment. A switchback design of diets was used as follows: (group 1) lab chow -- HF -- FF -- HF and (group 2) lab chow -- FF -+ HF -- FF. Each diet was fed for a 3- to 4-week period. The rats were provided water ad libitum and adapted to a 12-h light-12-h dark cycle and a temperature of 2 to 22 C. Body weight, 24-h feed intake, and 24-h fecal output and fecal dry matter were determined for each rat twice weekly during each diet period. Fecal samples from the five rats of each group were pooled every day for at least 5 days during and after the third week of a diet. The pooled samples were analyzed for total viable anaerobic bacteria, viable methanogenic bacteria, soluble fermentation products, and dry matter. Enumeration procedures. The anaerobic techniques and media for enumeration of total viable anaerobe and methanogen concentrations in feces were as described by Maczulak et al. (15). The 1-1 (wt/vol) dilution was prepared by the Stomacher method (16). Serial dilutions (1-fold) were prepared in anaerobic dilution solution (3). Media and dilution solution were reduced with 1.25% each of dithiothreitol and Na2S. 1OH2. For total anaerobe counts,.5 ml of appropriate dilutions was inoculated into duplicate roll tubes containing a complex agar medium with 1% rumen fluid and a 1% CO2 atmosphere. Methanogens were enumerated in a similar fashion in the complex medium with the addition of cephalothin (6.7,ug/ml), clindamycin (1.7,ug/ml), and an 8% H2-2% C2 atmosphere. All incubations were done at 37 C. At 14 days, roll tube colonies were counted and portions of the headspace of antibiotic-containing roll tubes were analyzed for CH4. The remainder of the 11 dilution (ca. 1 ml) was used for analysis of soluble organic fermentation products. Fecal dry matter was determined as previously described (15). Analytical methods. Soluble fermentation products were determined by high-performance liquid chromatography procedures (1). We used an Aminex ion-exclusion column (HPX-87H; Bio-Rad Laboratories, Richmond, Calif.) at 35 C, which was eluted with.13 N H2SO4 (.55 ml/min, 77 kg. cm2). Compounds were detected by refractive index and identified and quantified with a C-R3A integrator (Shimadzu Scientific, Baltimore, Md.) and the absolute calibration curve method. CH4 was quantified by using previously described gas chromatographic procedures (5). Statistics. Student's t test for unpaired samples was used to determine the significance of differences between measured values. The calculations were performed by using the RS/1 release 4 software program (BBN Software Products Corp., Cambridge, Mass.). RESULTS Weight gains were essentially the same for rats fed the lab chow -> HF -- FF -- HF sequence (group 1) and rats fed the lab chow -- FF -- HF -- FF sequence (group 2). Figure 1 shows the mean weights of the two groups for the 16-week period of the experiment. Daily feed intake was the same for rats fed either diet (Fig. 2). The mean intake was 22. g for group 1 and 18.4 g for group 2 up to 9 days and 2.5 g for group 1 and 2.3 g for group 2 for the remaining days, with no significant differences between the means. Since the order of feeding an HF or FF diet did not affect weight gain or daily feed intake, further comparisons were between all HF- and all FF-diet-fed rats of groups 1 and 2. Feces from FF-diet-fed rats had a much higher percentage of dry matter than those from HF-diet-fed rats. The percentages (standard deviations) of pooled samples were 36.5 (2.5) for the HF diet and 6. (3.) for the FF diet; those for the HF diet differed significantly from those for the FF diet (P =.1). Total daily fecal dry matter excretion was much higher with the HF diet than with the FF diet. Total dry matter (grams of dry matter [gdm]) excreted (standard deviation) was 5.6 g (.5) with the HF diet and 1.4 g (.2) with the FF diet (45 separate measurements for each). The amounts of dry matter excretion of HF- versus FF-diet-fed rats were significantly different (P =.1). The concentrations of total viable anaerobes were much Downloaded from on December 31, 218 by guest

3 VOL. 59, 1993 FECAL BACTERIA AND FERMENTATION ACIDS DL 3-t ) -._ D Age (days) FIG. 2. Feed intake during the experiment. Each point is the mean of the individual intakes of five rats in each group. Down and up arrows show when diet changes were made for group 1 (-) and group 2 (A) rats, respectively. See the legend to Fig. 1 for details about the diet changes. greater in the feces of rats fed the HF diet than in feces of rats fed the FF diet. There was no difference in the concentrations of methanogens in feces of rats fed the two diets. Table 1 shows the number of methanogens and total anaerobes per gram of dry matter of feces on the days that fecal samples were pooled for bacteriological and fermentation product analyses. The data for samples from group 1 and group 2 rats fed an HF or FF diet were averaged. Figure 3 shows a comparison of the average total anaerobe and methanogen concentrations (per gram of dry matter) and standard deviations. The total anaerobe concentrations with the HF diet were significantly higher (18 times; P =.1) than those with the FF diet. There were no significant differences in the concentration of methanogens (HF diet versus FF diet). The different diets produced differences in the amounts of fermentation acids per gram of dry matter of feces (Table 2). Concentrations of all fermentation acid products except formate were significantly different between the HF and FF diets. Acetate, propionate, butyrate, and lactate were 3.9, 17.2, 39.6, and 8.5 times higher with the HF diet than with the FF diet. Succinate was 4.2 times higher with the FF diet. E ' cm _ co CD co -C LL 1 1 E. Q 1. o 9 :Y I cm HF FF FIG. 3. Means and standard deviations of methanogens ( m ) and total anaerobes ( E3 ) per gram of dry matter of feces of rats fed an HF or FF diet. TABLE 1. Methanogen (M) and total anaerobe (TA) concentrations in the feces of rats fed an HF or FF diet Diet Age Concna (days) M TA Group 1 HF FF HF Group 2 FF HF FF a Values (log1o per gram of dry matter) are given for pooled fecal pellets of five rats. Downloaded from on December 31, 218 by guest

4 66 MACZULAK ET AL. APPL. ENvIRON. MICROBIOL. Diet TABLE 2. Concentrations of acid products in the feces of rats fed an HF or FF diet Acetate Propionate Butyrate Lactate Succinate Formate Concna %b Concn % Concn % Concn % Concn % Concn % HF 267.5c (67.5) 63.9c (5.) 82.4c (28.5) 19.4c (3.2) 39.6c (22.8) 9.1c (3.4) 29.6c (32.5) 7. (7.6) 1.3c (1.8).3c (.4) 1.4 (3.4).3 (.8) FF (35.1) 8.5- (13.7) 4.8- (5.1) 6.1- (6.1) 1.- (1.7) 1.2- (2.1) 3.5C (5.4) 5. (8.1) 5.5C (4.2) 6.1- (3.3).7 (1.2) 1.1 (2.1) a Concentrations (micromoles per gram of dry matter) are the means with standard deviations in parentheses. b %, percentage of total acid products, with the standard deviation in parentheses. c P of <.1 by t test for unpaired samples (HF [n = 13] versus FF [n = 15]). The proportions of acid products also differed between diets (Table 2). Propionate and butyrate were significantly higher with the HF diet than with the FF diet. Acetate and succinate were significantly higher with the FF diet than with the HF diet. All pooled fecal samples from the HF-diet-fed rats contained acetate, propionate, and butyrate. Lactate concentrations were highly variable with the HF diet. Four samples contained no detectable lactate, two samples had less than 3,umol/gdm, two had 17 to 24,umol/gdm, and five ranged in concentration from 61 to 78,umol/gdm. Succinate and formate were also variable. The concentrations in the 5 samples that contained detectable succinate ranged from 3 to 5,umol/gdm. Formate was detected in only two samples, which had concentrations of 7 and 11,umol/gdm. All samples from FF-diet-fed rats had acetate. Eight of 15 samples had no detectable propionate, and the range of propionate in the remaining samples was 5 to 13,umol/gdm. Only four samples contained butyrate, with a range of 3 to 5,umol/gdm. Lactate, 7 to 12,umol/gdm, was found in 5 of 15 samples, and formate, 2 to 3,umol/gdm, was found in 4 of 15 samples. Only one sample did not contain detectable succinate. The daily excretions of total anaerobes and methanogens were calculated by multiplying concentrations per gram of dry matter shown in Fig. 3 by the daily fecal output. The total number of anaerobes (2.44 x 111) excreted by HFdiet-fed rats was 71 times higher than that of FF-diet-fed rats (3.44 x 19). HF-diet-fed rats had higher concentrations per gram of dry matter and higher daily fecal output. There was no significant difference in methanogen concentrations per gram of dry matter between HF- and FF-diet groups. The log1 of the mean concentration of methanogens for the two diets was The higher methanogen concentration excreted by HF-diet-fed rats was due solely to a fourfold greater daily output of feces with the HF diet. Daily excretion of fermentation acids was also calculated by multiplying the concentrations per gram of dry matter shown in Table 2 by daily fecal (gram of dry matter) output. The results are shown in Table 3. Total daily fecal output of TABLE 3. Diet Daily fecal output of acid products of rats fed HF or FF diets Output product (,umol/24 h) of: Acetate Propionate Butyrate Lactate Succinate Formate HF 1, FF Ratio of HF/FF the carbon in fermentation acids of rats fed the FF diet was 4.6% of the amount for the rats fed the HF diet. DISCUSSION We used the same HF and FF diets described by Demigne and Remesy (9). They found that the cecal pool of acetic, propionic, and butyric acids was 1.6 times higher with the HF diet. Also, the rate of absorption of the acids in the cecal vein was about 17 times higher with the HF diet. The HF diet supplies substrates that significantly increase microbial fermentation and growth in the cecum. About 7 times more total viable anaerobic bacteria are excreted daily in the feces of HF-diet-fed rats than in the feces of FF-diet-fed rats. Fecal concentrations of total anaerobes and fecal output were significantly higher with the HF diet. Although the data are not reported, we also analyzed the feces of lab chow-fed rats for all of the parameters examined for HF and FF diets. The results were essentially the same as those obtained with the HF diet, i.e., the commercial lab chow used is an HF diet. As indicated above, total daily viable anaerobe output with the FF diet is 1/7th (1.4%) of the output with the HF diet. This suggests that the FF diet supplied the colonic community with only 1.4% of the amount of substrate provided by the HF diet. Also as indicated above, Demigne and Remesy showed that acid absorption into the cecal veins of rats fed the FF diet was 1/17th (5.9%) of the venous absorption of rats fed the HF diet (9). Their study and ours indicate that the amount of substrate available for fermentation and growth of the microbial community in the FF diet is much lower than the amount in the HF diet. The small amount of substrate entering the colons of FF-diet-fed rats may be host derived (e.g., mucins) or food that escapes monogastric digestion. These results do not rule out the possibility that HF diets stimulate the excretion of hostderived substrates into the lumen and increase their contribution to microbial growth and fermentation. Methanogen concentrations are about the same in the feces of rats fed either diet. However, methanogen output is higher in the HF group because of the increase in daily fecal output. The ratio of total anaerobic bacteria to methanogens was the same for HF and FF diets. The predominant colonic methanogen of these rats uses H2 to reduce CO2 to CH4 (17, 18). The growth and final concentration of methanogens are limited by the amount of H2 produced by the nonmethanogenic flora and the ability of methanogens to compete for H2 with other biological and physical processes for removing the gas from the ecosystem. The results suggest that no selective alteration of the balance between H2 production and utilization by methanogens occurred when HF substrates dramatically increased the production of the nonmethanogenic and methanogenic flora in the colon. Downloaded from on December 31, 218 by guest

5 VOL. 59, 1993 This study and that of Demigne and Remesy (9) indicate relative increases in butyrate and decreases in acetate production with HF diets. They found ratios of acetate/propionate/butyrate of 6:25:15 and 71:26:3 in the cecal pools of HF- and FF-diet-fed rats, respectively. We found ratios of 69:21:1 and 92:7:1 in the feces of HF- and FF-diet-fed rats, respectively. Specific increases in growth of bacteria that produce butyrate probably occur when the carbohydrates in the HF diet are provided to the colonic microbial community. Walter et al. (24) found increased proportions of butyrate and increased total volatile acids in rat ceca when a low-fiber diet was supplemented with wheat bran. Horn et al. also found higher concentrations of butyrate in the ceca of rats fed a commercial pellet diet than in those of rats fed an FF diet (13). Brighenti et al. found high proportions of butyrate in rat ceca with hazelnut supplementation of an FF diet, increased proportions of propionate when guar gum was the supplement, and little change with pectin supplementation (2). Only guar gum and pectin caused a significant increase in total volatile acids. Goodlad and Mathers demonstrated increasing molar percentages of butyrate in the ceca of rats fed increasing proportions of peas (11). Decreased proportions of butyrate in the feces compared with those in the cecum may be due to butyrate utilization by colonic epithelial cells. Butyrate oxidation accounts for 86% of the total oxygen consumption of colonic epithelial cells of the rat (19). Decreases between the cecum and feces would be more pronounced when small amounts of butyrate are produced in the cecum with FF diets because of the dearth of fermentable substrates. If epithelial cell utilization of butyrate is constant between diets, a larger percentage of the amount of butyrate formed in the cecum would be removed by cecal epithelial cells when an FF or low-fiber diet is fed. We also found lower proportions of propionate in the feces of FF-diet-fed rats than in the feces of rats fed the HF diet. Since Demigne and Remesy found essentially no differences in cecal propionate proportions between the diets (9), the results suggest the possibility of utilization of propionate by colonic epithelial cells. Demigne and Remesy did not report the presence of lactic, succinic, or formic acid (9). The gas chromatography procedure that they used does not detect these acids. In a subsequent report, large amounts of lactic acid were detected in the ceca of rats fed a diet rich in amylase-resistant starch (crude potato starch) (14). As the rats were adapted to the diet over a 3-day period, cecal lactate concentrations declined and the concentrations of acetate, propionate, and butyrate increased. Cecal lactate absorption was much lower than absorption of the other acid products. The lactate that we found in the feces of HF-diet-fed rats probably resulted from a similar fermentation. We also found low concentrations in the feces of FF-diet-fed rats. Small amounts of succinate were present in the feces of FF- and HF-diet-fed rats. Succinate or succinyl-coenzyme A is a precursor of propionate. Vitamin B12 is required for production of propionate by species of human intestinal Bacteroides (6). They accumulate succinate when the vitamin is not supplied. There is no information on the absorption of succinate from the rat cecum. Small amounts of formate were detected for HF and FF diets. Fermentative bacteria produce formate that is used by other bacteria to form CH4 or other products (12). Its presence in feces probably indicates that its rate of production is slightly higher than its rate of utilization. We suggest that it would be useful to supplement the usual measurements of acetate, propionate, and butyrate in studies of colonic fermentations FECAL BACTERIA AND FERMENTATION ACIDS 661 with measurements of lactate, succinate, and formate. Determination of the concentration of the latter acid products would help to clarify their significance as products of colonic fermentations and to indicate whether their production is associated with particular diets or diseases. Our results show that an HF diet significantly increases microbial growth in the colon and influences the proportions of organic acid products. The HF diet did not increase the ratio of methanogens to total anaerobes. We suggest that the contribution of host-derived substrates to colonic microbial growth and fermentation is insignificant. 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