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* Laboratory of Animal Science, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan;
Kyodoken Institute, Kyoto 612-8073, Japan; and
** Chemical Products Research Laboratories, Fujisawa Pharmaceutical Company, Ibaraki 300-2698, Japan
3To whom correspondence and reprint requests should be addressed. E-mail: k_ushida{at}kpu.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • Megasphaera elsdenii JCM1772T • hyperlactate accumulation • butyrate producers • rat cecum
In patients with short-bowel syndrome and those who have undergone jejuno-ileal by-pass surgery, a rapid and large influx of digestible carbohydrates causes accumulation of lactate in the large intestine, leading to acidosis (1). Large amounts of fecal lactate in diarrheic feces in porcine dyspepsia are due to the same mechanism (2). The accumulation of lactate in the large intestine lowers the luminal pH (3), loosens intestinal tissue and causes diarrhea (4). Lactate is often the predominant acid in the feces of patients with inflammatory bowel disease (IBD)2 (5,6). Thus, the accumulation of lactate in the large intestine is apparently related to many disorders in that organ. In normal colonic fermentations, lactate is seldom detected because it is an intermediate compound that is further metabolized to SCFA, such as acetate, propionate and butyrate, by a range of acid-utilizing bacteria. This process is particularly important in the large intestine; SCFA are absorbed and utilized as nutrients by colonocytes and promote moisture and electrolyte absorption, whereas lactate is not well absorbed and, therefore, is not utilized by colonocytes (7). Among SCFA, the role of butyrate has been emphasized repeatedly (8–10). Indeed, low butyrate concentration in the large intestine has been reported in patients with IBD (5,6). Therefore, a higher concentration of lactate and a lower concentration of butyrate are conditions that may exacerbate the above-mentioned diseases. Conversion of lactate to butyrate may cure or alleviate these diseases.
Megasphaera elsdenii, one of the major acid-utilizing intestinal bacteria, produces butyrate from lactate in the rumen (11,12). The potential of this bacterium to cure rumen lactic acidosis has been suggested (12). In addition, the importance of this bacterium in butyrate production from lactate in the large intestine was also suggested in a previous experiment (13).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This experiment was conducted in accordance with the guidelines for studies with laboratory animals of the Kyoto Prefectural University Experimental Animal Committee. Twelve specific pathogen–free (SPF) Sprague-Dawley male rats (6 wk old) were obtained from the Japan SLC (Shizuoka, Japan). The rats were free of M. elsdenii as determined by M. elsdenii-specific PCR detection. They were housed individually in steel wire cages under a controlled temperature (25°C) and a 12-h light:dark cycle. After their arrival, the rats had free access to water and a standard nonpurified diet (Labo-MR Stock; Nihon Nosan, Tokyo, Japan) for 3 d. Food was offered to the rats at 0900 h each day.
Induction of lactate production in the large intestine.
After completing this 3-d adaptation period, rats consumed a semipurified diet containing fructooligosaccharide (100 g/kg of diet; FOS, Meiji Seika Kaisha, Tokyo, Japan) (14) ad libitum to induce lactate production in the large intestine. The experimental diet (15) contained (g/kg): 
-cornstarch, 540; casein, 200; soybean oil, 50; cellulose, 50; mineral mixture, 50; vitamin mixture, 10; and FOS, 100. The mineral and vitamin mixtures were purchased from oriental Yeast (Yokohama, Japan). The compositions of the mineral and vitamin mixtures were described by Harper (16). Fresh feces were collected daily from d 0 (the last day of the adaptation period) into sterile 1.5-mL microfuge tubes from the rectum under gentle stimulation with a sterile wet cotton swab at 1700 h throughout the experiment, and immediately analyzed for organic acid (SCFA, lactate, succinate) concentrations by ion-exclusion HPLC as described elsewhere (17) and moisture content by drying at 80°C for 48 h.
Lactate in feces of all rats was >30 mmol/kg wet feces as early as d 2 of FOS feeding. We considered this much fecal lactate to be a sign of hyperlactate production in the hindgut. In a previous experiment (2), young pigs with dyspeptic diarrhea excreted feces containing amounts of lactate no >30 mmol/kg. Therefore, we considered a fecal lactate concentration > 30 mmol/kg to reflect "hyperlactate production."
Preparation of M. elsdenii.
M. elsdenii JCM1772T was of rumen origin and obtained from the Japan Collection of Microorganisms (RIKEN, Wako, Japan). This strain was cultured on an anaerobic medium at 37°C as described in a previous report (18). A portion of M. elsdenii culture was transferred to the same medium (1 L) and incubated for 24 h at 37°C.
The cells were counted under a phase-contrast microscope, and cell densities of 6.5 x 1013/L were obtained. Whole cells (6.5 x 1013) were harvested by centrifugation (25,000 x g, 10 min) and washed with saline to eliminate the residual medium. The volume of the final bacterial pellet was 
2.5 mL. The M. elsdenii pellet was prepared just before use.
Because anaerobiosis cannot be guaranteed during this preparation, some portions of M. elsdenii were inevitably lost. In a preliminary experiment, we examined whether the inclusion of FOS in the diet could produce hyperlactate fermentation in the large intestine of rats. FOS feeding did not always induce hyperlactate production. Moreover, this FOS-induced hyperlactate production did not persist for as long as 7 d. Therefore, this system should be regarded as an acute hyperlactate production model. The effect of M. elsdenii on lactate and butyrate in the large intestine was examined during this period, in which the difference between the groups in fecal lactate was clearly maintained.
Administration of M. elsdenii.
The rats were randomly divided into two groups on the morning of d 4 of FOS feeding. At first, we intended to prepare a sufficient amount of cell pellet for 6 rats. However, only 5 rats were treated due to a shortage of pellets. The remaining 7 rats were used as the control. Cells (1.3 x 1013) of M. elsdenii in a 0.5-mL pellet were administrated to the 5 rats through a stomach tube with a sterile feeding needle (1.2 mm o.d. x 80 mm long; Fuchigami, Tokyo, Japan). The other rats were dosed orally with a sterile saline solution as the vehicle control. Rats were treated without anesthesia and a new feeding needle was used on each rat. The oral dose was given at 1000 h on d 4, 5 and 6. Feces were sampled and analyzed for organic acids and moisture as indicated above.
Sampling of cecal contents and histological study.
On d 6, the large intestine was removed from all rats under general anesthesia with pentobarbital sodium (Nembutal; Dainippon Seiyaku, Osaka, Japan) at least 2 h after the final administration of M. elsdenii. Cecal contents were immediately and gently collected into sterile microfuge tubes after a midline incision with sterile scissors. A portion of the contents was analyzed for organic acids as indicated above and for pH.
After removal of the contents, cecal tissues were fixed in neutralized formalin. The fixed ceca were further cut into cross sections of 3 mm length. These cross-sectioned tissue samples were embedded in paraffin wax; 3-µm thick cross sections were then prepared and stained with hematoxylin and eosin (HE) and alcian green-counterstained with hematoxylin (AG). The pH of the AG staining solution was 3.5. The numbers of columnar epithelial cells and mitotic cells per longitudinal section of the left side of the crypt column were counted on HE-stained preparations. The mucin-containing cells in the left side of the crypt column were enumerated on the AG-stained preparations. The mitotic zone was estimated as described by Ichikawa and Sakata (19).
Quantitative competitive (QC)-PCR of cecal M. elsdenii.
Bacterial DNA was extracted from cecal contents according to Godon et al. (20). The extracted DNA was coamplified with the serially diluted competitor DNA for the 16S rDNA of M. elsdenii using primers 5'-ggaggctcttcggagcttt-3' (E. coli 16S rDNA position 202–221) and 5'-cccgtcaattcatttgagttt-3' (906–926). The competitor DNA for QC-PCR was constructed using a Takara Competitive DNA Construction Kit (Takara Shuzo, Kyoto, Japan) according to the manufacturer’s instructions. The concentration of the DNA competitor was 6.85 g/L. Twenty-five thermal cycles for QC-PCR, 94°C for 30 s, 60°C for 30 s and 72°C for 30 s with 5 min of an initial denaturation step at 94°C and 10 min of a final elongation step at 72°C, were performed using Ex taq polymerase. The reaction mixture was constructed in accordance with the manufacturer’s instructions (Takara Shuzo). After agarose gel electrophoresis and ethidium bromide staining, the intensities of the amplified products were analyzed by Kodak 1D Image Analysis Software (Kodak Digital Science, Rochester, NY). The concentration of the target template in the sample was estimated according to the method described by Li and Drake (21) using the serially 10-fold diluted competitor (6.85 x 10-4, 6.85 x 10-5, 6.85 x 10-6 and 6.85 x 10-7 g/L).
Statistical analyses.
Statistical analyses were performed using Statcel (22), which is an add-in application of Microsoft-Excel (version 5.0, Microsoft, Seattle, WA). We conducted a repeated-measures ANOVA (two experimental groups x sampling day) to detect the effect of the oral administration of M. elsdenii on the fecal variables. The effect of the two-way interaction (Day x M. elsdenii; P < 0.05) on the concentrations of butyrate was significant. Therefore, statistical analysis was applied separately to the results of each day. Depending on the results of the F-test, Student’s t test or Welch’s t test was used to analyze the differences among means for each day. Differences were considered significant at P < 0.05. Values presented are mean ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The lactate concentration of cecal contents was lower in rats administered M. elsdenii than in controls (P < 0.05) and the butyrate concentration was greater (P < 0.05; Table 1). The other two major SCFA, acetate and propionate, were not affected, but the sum of acetate, propionate, and butyrate was greater in rats administered M. elsdenii than in controls (P < 0.05). Succinate, another major intermediate in hindgut fermentation (23) did not differ between the groups (data not shown). The pH of the cecal contents also did not differ (Table 1).
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The QC-PCR specific for M. elsdenii detected that band in rats administered M. elsdenii but not in the control group, indicating that there was no M. elsdenii in those rats. The concentration of the target DNA prepared from the 4 x 108 of M. elsdenii cells was 0.3 ng. Therefore, the concentration of the target DNA in cecal contents was 27 µg/g. The weight of the cecal contents was 3.6 ± 0.9 g in rats administered M. elsdenii and 3.2 ± 1.2 g in the control rats. Therefore, the amount of M. elsdenii in the cecal contents was estimated to be 3.6 x 107 cells/g, which corresponded to 1.3 x 108 cells/pouch.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Improvement of hindgut fermentation affected the morphometric variables of the cecal mucosa as well as feces. An increase in the number of epithelial cells in crypts was likely a result of increased butyrate production. Other variables such as absorption of water and electrolytes might also be affected by the conversion of lactate to butyrate. Recovery from diarrhea was also likely due to such a conversion.
Probiotics are defined as live health-promoting bacteria. However, a probiotic preparation inevitably contains dead bacteria. Therefore, the effect of a probiotic is due not only to the activity of the live bacteria but also, at least in part, to the dead bacteria. In this experiment, the effect of M. elsdenii on the host physiology may have been due to both live and dead bacteria. Further work is warranted to clarify the effect of dead M. elsdenii.
The importance of acid-utilizing bacteria, such as M. elsdenii, has not been as well recognized in monogastric animals, including humans, as it has in ruminants (11,12). Therefore, the major objective of this study of rats was to assess whether acid-utilizing bacteria such as M. elsdenii could potentially be used to improve the hindgut environment in humans. Lactate can be oxidized to acetate and can be converted to propionate by the hindgut microbiota (30). We found in a previous experiment (13) that, among the three major SCFA, butyrate was the least thermodynamically favored product from lactate in the hindgut. As discussed in this paper, butyrate is the ideal SCFA in terms of hindgut function and health. However, it is difficult to enhance the conversion of lactate to butyrate in a natural system. M. elsdenii, a lactate-utilizing, butyrate-producing bacterium is especially effective for this purpose, particularly in cases of hyperlactate production.
In a previous study, this bacterium was isolated with several Lactobacilli when cecal butyrate production was enhanced by gluconic acid (13). It is also plausible that a mixture of this bacterium with the lactic acid bacteria usually applied as a probiotic will improve their combined beneficial effect.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Fujisawa Technical Service, Co., Ltd., 2–1-6 Kashima, Yodogawa, Osaka 532-0031, Japan.
4 Abbreviations used: AG stain, alcian green counterstained with hematoxylin stain; FOS, fructooligosaccharide; HE stain, hematoxylin and eosin stain; IBD, inflammatory bowel disease; (QC)-PCR, quantitative competitive-PCR; SPF, specific pathogen–free.
Manuscript received 24 March 2003. Revision accepted 30 June 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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