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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, Ltd., Tsukuba, Ibaraki 300-2698, Japan
1To whom correspondence should be addressed. E-mail: k_ushida{at}kpu.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • gluconic acid • butyrate enhancer • Megasphaera elsdenii • lactic acid bacteria • pig cecal digesta
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). SCFA are rapidly absorbed from the mucosa of the large intestine (2
) to provide energy to epithelial cells (3
). Among SCFA, butyrate is considered to be the most effective for promoting epithelial growth (4
,5
) and sodium and water absorption (6
). Moreover, the inhibitory effect of butyrate on tumor development has been frequently discussed (7
–9
). Stimulation of butyrate production in the hindgut by means of dietary components is thus thought to be desirable.
Many saccharides are indigestible materials that reach the large intestine (10
–17
). However, in only a few cases have saccharides been shown to stimulate butyrate production in the large intestine (11
–13
,17
). We are, therefore, seeking novel stimulators of butyrate production. It has been shown that gluconic acid, which may be fermented in the large intestine, can reach the large intestine of rats (18
). However, the fermentation pattern induced by gluconic acid has not been elucidated.
Accordingly, the aim of the present study was to investigate the fermentation properties induced by gluconic acid in a batch culture of pig cecal digesta. Glucose and sorbitol were also tested because these sugars are structurally related to gluconic acid and their fermentations have been studied previously in in vitro human fecal and pig cecal models (14
,17
). According to these reports, sorbitol can reach the large intestine and may stimulate butyrate or propionate production. The fermentation rate, which may be a factor that affects the fermentation pattern of these sugars, was also reported (14
). We thus compared the effect of gluconic acid with that of sorbitol (slowly fermented sugar) and glucose (rapidly fermented sugar) on fermentation in the large intestine.
Gluconic acid–utilizing bacteria were isolated from pig cecal digesta and identified to explain how gluconic acid was used and metabolized. Their roles in gluconic acid fermentation and, particularly, in butyrate production were investigated. Pigs are often considered to be one of the better human nutritional and physiological models in terms of the large intestine (19
–23
). Therefore, the possible effects of gluconic acid on fermentation in the large intestine of humans were also investigated in the present study.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Three adult cross-bred (Landrace x Large white x Duroc) sows weighing 185, 180 and 163 kg, fitted with permanent cecal cannulas, were used as donors of the cecal digesta. The pigs were fed 500 g meat-bone meal, 500 g alfalfa (Medicago sativa) meal and 1640 g cracked maize supplemented with 8 g NaCl, 14 g CaCO3 and 8 g vitamin–mineral premix (Kokin Kagaku, Osaka, Japan) daily, as described (24
). Pigs consumed the food3
completely and drank water ad libitum.
Chemicals
All chemicals were obtained from Wako Pure Chemicals (Osaka, Japan) and Nacalai tesque (Kyoto, Japan) unless otherwise stated.
Experiment 1
Sodium gluconate (Fujisawa Pharmaceutical, Tokyo, Japan), glucose and sorbitol were used as substrates in this experiment.
The sampling procedure was the same as that used by Ushida and Sakata (26
). Briefly, cecal digesta were sampled from three sows by suction through the cannula, just after morning feeding, and diluted with four volumes of an anaerobic sodium phosphate buffer (50 mmol/L, pH 6.5). Diluted digesta were squeezed through four layers of surgical gauze. A portion (25 mL) of strained digesta was inoculated into a 120-mL serum bottle that contained substrate. The initial concentration of the substrate was adjusted to 40 mmol/L. The bottles were filled with a gas mixture composed of 80% N2 and 20% CO2 and closed with butyl rubber stoppers and aluminum clips. Blank cultures (without substrate) were also performed to evaluate the organic acid production from the endogenous substrate in the sampled cecal digesta. The bottles were incubated at 39°C for 0, 4, 8, 12 and 24 h, and were laid on their sides to increase the surface of the sediment. Three bottles were allotted to each incubation period; therefore, 15 bottles were used in total for each pig, and 45 bottles in total were used in this experiment. All bottles were agitated manually at the time of sampling. At the end of the incubation, fermentation was stopped by the addition of 1 mL of 6 mol/L HCl. After centrifugation at 12,000 x g for 15 min, the organic acid concentrations of the supernatant were analyzed by ion-exclusion HPLC (26
). The apparent production of organic acids from the substrates was calculated by subtraction of the 0-time concentration from that at the end of the incubation. The blank incubations were used to evaluate the effect of endogenous substrates. The values of apparent production for the blank incubations were subtracted from those with experimental substrates.
Experiment 2
Cecal digesta were sampled and squeezed as in expt. 1. Squeezed digesta were diluted with nine volumes of an anaerobic dilution solution (27
). A portion (1 mL) of strained digesta was inoculated into 120-mL serum bottles with 24 mL of peptone yeast extract broth containing Fildes solution (PYF broth, pH 7.0) (28
). Two bottles were allotted to each pig. The bottles were filled with a gas phase at 100% CO2 and closed with butyl rubber stoppers and aluminum clips. The bottles were stored at 39°C for 144 h, with a shaking at the time of the sampling. Sterile gluconic acid solution (1 mL of 0.46 mol/L) was added to a bottle at the beginning (0 h), at 48 h and at 96 h of the incubation. The same amount of distilled water (1 mL) was added to another bottle and regarded as the negative control. Samples (1 mL) taken at 96 and 144 h were analyzed for major bacteria in gluconic acid cultures. For this purpose, the sampled incubated medium was subjected to a 10-fold serial dilution with an anaerobic dilution solution (27
) up to 10-8 under 100% CO2 and used for bacterial colony counts on blood liver agar (BL agar) (Nissui, Tokyo, Japan). BL agar was supplemented with defibrinated horse blood (5% v/v). BL agar plates were incubated in an anaerobic chamber (Coy Laboratory, Ann Arbor, MI) with a mixture of gas phase composed of N2 (80%), CO2 (10%) and H2 (10%). The colonies were counted after a 48-h incubation period at 37°C. The colonies, which were morphologically distinct (color, shape, size and texture) when compared with the colonies on the BL agar plates from the negative control, were isolated and further checked for their gluconic acid utilization in the PYF broth. Gluconic acid utilizers were obtained if grown on the gluconic acid–supplemented PYF broth.
The ability of gluconic acid utilization was further checked if the isolate produced a substantially larger amount of organic acids in 46 mmol/L gluconic acid–supplemented PYF broth (n = 4) than in PYF broth without gluconic acid (n = 4). Organic acids in the culture supernatant were analyzed as described above after a 48-h incubation. For the identification of the gluconic acid utilizer, 16S rDNA was partially sequenced after PCR amplification. Genomic DNA was isolated from the cells grown in the PYF broth, and the 16S rDNA gene was amplified using a forward primer (27f: 5'-AGAGTTTGATCCTGGCTCAG-3'), a reverse primer (1492r: 5'-GGCTACCTTGTTACGACTT-3') and rTaq polymerase (Toyobo, Tokyo, Japan) according to Hiraishi (29
). The amplified products were cloned using a pGEM-T Easy Vector (Promega, Madison, WI) and Escherichia coli JM 109 (Toyobo) as indicated by the manufacturers. Inserts within plasmids were sequenced with an LI-COR autosequencer (LIC-4200L; Aloka, Tokyo, Japan) using a Thermo Sequenase Cycle Sequencing Kit (Amersham Pharmacia Biotech, Tokyo, Japan). The obtained sequences were subjected to a BLAST search with known sequences in DDBJ.
Experiment 3
A strain of the gluconic acid–utilizing bacteria obtained from expt. 2 was used to examine the utilization of lactate and acetate in the production of butyrate. The standard strain of Megasphaera elsdenii JCM 1772T was also used for comparison. These two bacteria were preincubated at 37°C for 24 h in a peptone yeast extract broth (PY broth) (30
) supplemented with lactate (10 mmol/L) and acetate (10 mmol/L) or with lactate (10 mmol/L) alone. All broths in this experiment were adjusted to pH 6.8. The bacteria were transferred to the same fresh media and incubated for another 24 h. After incubation was completed, the organic acids in the culture supernatant were analyzed as described above.
Statistical analyses
Values in expt. 1 are given as means ± SD for individual pigs (n = 3). We first conducted repeated-measure ANOVA (3 experimental groups x sampling time), to decide whether we could pool the results of different sampling times, or whether we should analyze results from different sampling times separately. Effects of both the sampling time (P < 0.05) and the two-way interaction (experimental groups x sampling time; P < 0.05) on the concentrations of all detected organic acids were significant. Therefore, we applied statistical analysis to results of each sampling time separately. Either a one-way ANOVA or the Kruskal–Wallis test, depending on the results of the Bartlett test, was used to analyze the differences among means at each time. Dunn’s post hoc comparison (parametric or nonparametric) was used for multiple comparisons when needed.
Values in expt. 2 are given as means ± SD for the cultures of each bacteria (n = 4). Either a Student’s t test or a Welch’s t test, depending on the results of the F test, was used to analyze the differences among means. Values in expt. 3 are given as means ± SD for the cultures of each bacteria (n = 3). Either a one-way ANOVA or the Kruskal–Wallis test, depending on the results of the Bartlett test, was used to analyze the differences among means. Post hoc comparison was as in expt. 1. The difference between means was considered significant at P < 0.05 in all statistical analyses. All data were analyzed by Statcel (31
), which is an add-in application of Microsoft Excel (version 5.0; Microsoft, Seattle, WA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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)In all cases, irrespective of whether they increased or decreased relative to 0-h concentration, only trace amounts of succinate, formate, iso-butyrate and iso-valerate were detected. Therefore, these acids are not discussed in this report.
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Isolation and identification of gluconic acid–utilizing bacteria from the enrichment culture in expt. 2
Eighty-four morphologically distinct colonies were isolated from the gluconic acid–supplemented cultures. They were analyzed for gluconic acid utilization in the PYF broth. Ten bacteria grew in the gluconic acid–supplemented PYF broth. When these isolates were further tested for their ability to produce larger amounts (P < 0.05) of organic acid in gluconic acid–supplemented PYF broth than in that without gluconic acid, seven isolates were obtained (Table 2
). Isolates 2, 9 and 10 did not produce significantly larger amounts of organic acid from gluconic acid. Therefore, these isolates were not subjected to further analysis. Isolate 1, whose number in the culture (log CFU/mL) was 6.8, produced lactate and acetate from gluconic acid. Isolate 3 produced formate, acetate and n-butyrate, and its number was 6.3. Isolate 4 produced lactate, formate and acetate, and its number was 9.6. Isolates 5, 6 and 7 all produced lactate and acetate, and their numbers were all 9.3. Isolate 8 produced succinate, lactate, acetate, propionate and n-butyrate from gluconic acid, and its number was 7.3.
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Fermentation end products in Megasphaera elsdenii pure culture (Table 3
)
M. elsdenii JCM 1772T and isolate 3 were used to examine their ability to use lactate and acetate for butyrate production. Lactate supplementation stimulated apparent succinate, formate, acetate, propionate and valerate production (P < 0.05) in M. elsdenii JCM 1772T. Additional acetate further increased apparent n-butyrate production (P < 0.05) in M. elsdenii JCM 1772T. On the other hand, isolate 3 was less able to utilize lactate, and a relatively small increase (P = 0.17 PYL vs. PY, P = 0.19 PYLA vs. PY) in butyrate was detected.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,17
). In this investigation, sorbitol elevated production of propionate, rather than of butyrate, and this is consistent with Kiriyama et al. (14
). The effect of sorbitol on butyrate production remains inconclusive and needs further in vivo evaluation. The effect of gluconic acid had been confirmed by an in vivo feeding trial. We observed an increase in butyrate concentration in the cecal contents (from 9.6 to 30.0 mmol/kg digesta) when pigs (n = 3) were fed a diet supplemented with 0.5 g/100 g gluconic acid for 35 d (Tsukahara and Ushida, unpublished observations, 2002). We thought that the characteristic patterns of SCFA concentration in expt. 1 were principally the same, at least for the stimulation of butyrate production, as those in the preliminary in vivo trial where gluconic acid was fed to the pigs.
Butyrate is the major energy source of the epithelial cells of the large intestine (5
) and it is considered to have an antitumorigenic effect in the large intestine (7
–9
). In this context, studies have focused on the dietary components that stimulate butyrate production in the large intestine (8
,9
,11
–13
).
Butyrate-producing bacteria are limited to several genera: sugar fermenters, such as Clostridium spp., Eubacterium spp., Fusobacterium spp. and Butyrivibrio spp., and acid utilizers, such as M. elsdenii. The former group of bacteria produces butyrate as a major end product of fermentation (30
). However, these bacteria were not stimulated by dietary gluconic acid. In humans, it was demonstrated that dietary gluconic acid increased the population of Bifidobacteria (32
). In the present study, Lactobacilli were isolated from the gluconic acid–enriched cultures as a major gluconic acid utilizer (Table 2)
. Obviously, Bifidobacteria and Lactobacilli do not produce butyrate (33
). A similar result was published concerning the effect of resistant starch on fecal flora composition and the SCFA pattern in rats (34
). In that report, butyrate production was significantly increased (7 times) by diets containing resistant starch. At the same time, the numbers of lactic acid bacteria increased significantly; Lactobacilli and Bifidobacteria increased 100 and 10 times, respectively. The relationship between increases in lactic acid bacteria and butyrate concentration seems to be indirect. It is plausible that an increased number of lactic acid bacteria induced a higher lactate production and that lactate was then efficiently converted to butyrate by acid-utilizing bacteria, such as M. elsdenii.
In the present study, M. elsdenii was indeed isolated as a unique colony from gluconic acid–enriched cultures, and their butyrate production was stimulated by lactate and acetate (Tables 2
and 3)
. This hypothesis was not fully substantiated in the present experiment, although it explains the following apparent contradiction about the effect of many bifidogenic prebiotics on colonic and cecal SCFA production patterns. The increase in lactic acid–producing bacteria did not induce lactic acid fermentation in the large intestine in most cases (11
,35
–37
) and often stimulated butyrate production (11
).
The fermentation rate of the substrate may be a key characteristic to determine the pattern of fermentation end products. In the present study, glucose was rapidly fermented in the batch culture of pig cecal digesta, whereas sorbitol and gluconic acid were fermented relatively slowly (Table 1)
. Lactate was produced at a higher rate than the lactate conversion rate during the first 8 h in the glucose culture. Rapid fermentation of glucose might cause rapid growth of Gram-positive cocci, such as Streptococci, which cause the accumulation of lactate (38
). Rapid accumulation of lactate often eliminates the lactate conversion system because of the decline in pH (26
,38
). Accumulation of lactate started at pH 5.8 under conditions similar to those in this study (26
). Because we added HCl to stop fermentation promptly, pH could not be measured. Accumulation of lactate at 4 h suggested a low pH at this point. Based on the lag times, sorbitol was fermented more slowly than glucose. Sorbitol was previously shown to be a slowly fermented saccharide, but gluconic acid was fermented far more slowly than was sorbitol. The production rate of each SCFA indicated the clear presence of a lag time for the metabolism of gluconic acid. In preliminary experiments, the pH of cultures was as low as 6.3 when gluconic acid or sorbitol was supplemented to the cultures (Tsukahara et al., unpublished observations, 2000). This level of decline in pH may not be harmful for the fermentation system as indicated by our previous report (26
). When 1 g/100 g gluconic acid was added to the daily diet, pH values of the cecal contents of pigs (n = 4) were 6.3 ± 0.3 and 6.5 ± 0.1, respectively, at 4 and 8 h after feeding (Tsukahara et al., unpublished observations, 2001). This suggests that gluconic acid at this level may not acidify the cecal contents. Slow fermentation might be favorable for lactate-utilizing butyrate producers. The roles of those bacteria in the effects of prebiotics likely are important. Moreover, M. elsdenii itself can use gluconic acid (32
; this study), and it is selectively increased in the large intestine when gluconic acid is ingested.
The present experiment suggests the potential of gluconic acid as a prebiotic that stimulates butyrate production in the large intestine, although the limitation of the presently used technique, the batch culture system, was obvious. Pigs are often considered to be one of the better human nutritional and physiological models in terms of the large intestine (19
–23
). Therefore, similar effects of gluconic acid on fermentation in the large intestine of humans may be obtained. Further large-scale in vivo experiments are warranted to determine the effect of gluconic acid on fermentation in the human large intestine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 The chemical composition of the diet (g/kg) expressed on a dry-matter basis (877 g/kg) was as follows: crude protein (190), crude fiber (58), crude fat (ether extracts) (51), nitrogen-free extract (599), and crude ash (102). The soluble and insoluble fiber concentrations of this feed (g/kg dry matter) were determined by the method of Prosky et al. (25
) to be 15 and 143, respectively. The vitamin–mineral premix contained vitamin A (4,500,000 IU/kg), cholecalciferol (450,000 IU/kg), vitamin E acetate (10 g/kg), sodium 2-methyl-1,4-naphthoquinone hydrogen sulfite (0.5 g/kg), riboflavin (2.1 g/kg), cyanocobalamin (10 mg/kg), D-calcium pantothenate (10 g/kg), nicotinamide (15 g/kg), chorine chloride (50 g/kg), d-biotin (50 mg/kg), MnCO3 (30 g/kg), FeSO4 (155.28 g/kg), CuSO4 (12.65 g/kg), (CH3COO)2Zn (26.6 g/kg), CaI2 (0.46 g/kg).
Manuscript received 12 September 2001. Initial review completed 31 October 2001. Revision accepted 15 April 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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P. Bikker, A. Dirkzwager, J. Fledderus, P. Trevisi, I. le Huerou-Luron, J. P. Lalles, and A. Awati The effect of dietary protein and fermentable carbohydrates levels on growth performance and intestinal characteristics in newly weaned piglets J Anim Sci, December 1, 2006; 84(12): 3337 - 3345. [Abstract] [Full Text] [PDF]
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G. Biagi, A. Piva, M. Moschini, E. Vezzali, and F. X. Roth Effect of gluconic acid on piglet growth performance, intestinal microflora, and intestinal wall morphology J Anim Sci, February 1, 2006; 84(2): 370 - 378. [Abstract] [Full Text] [PDF]
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 S. H. Duncan, P. Louis, and H. J. Flint Lactate-Utilizing Bacteria, Isolated from Human Feces, That Produce Butyrate as a Major Fermentation Product Appl. Envir. Microbiol., October 1, 2004; 70(10): 5810 - 5817. [Abstract] [Full Text] [PDF]
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T. B. Stanton, J. S. McDowall, and M. A. Rasmussen Diverse Tetracycline Resistance Genotypes of Megasphaera elsdenii Strains Selectively Cultured from Swine Feces Appl. Envir. Microbiol., June 1, 2004; 70(6): 3754 - 3757. [Abstract] [Full Text] [PDF]
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 C. Kameue, T. Tsukahara, K. Yamada, H. Koyama, Y. Iwasaki, K. Nakayama, and K. Ushida Dietary Sodium Gluconate Protects Rats from Large Bowel Cancer by Stimulating Butyrate Production J. Nutr., April 1, 2004; 134(4): 940 - 944. [Abstract] [Full Text] [PDF]
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K. Hashizume, T. Tsukahara, K. Yamada, H. Koyama, and K. Ushida Megasphaera elsdenii JCM1772T Normalizes Hyperlactate Production in the Large Intestine of Fructooligosaccharide-Fed Rats by Stimulating Butyrate Production J. Nutr., October 1, 2003; 133(10): 3187 - 3190. [Abstract] [Full Text] [PDF]
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