The Journal of Nutrition Vol. 128 No. 1 January 1998, pp. 85-90

Changes in Production of Ethanol, Acids and H₂ from Glucose by the Fecal Flora of a 16- to 158-d-Old Breast-Fed Infant¹

Meyer J. Wolin^{*, 2}, Susan Yerry^*, Terry L. Miller^*, Yongchao Zhang, and Shelton Bank

^* Wadsworth Center for Laboratories and Research, New York State Department of Health, Empire State Plaza, Albany, NY 12201-0509 and  Department of Chemistry, State University of New York-Albany, Albany, NY 12222

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Microbes in the adult human colon ferment dietary substrates chiefly to acetic, propionic and butyric acids and CO₂, H₂ and CH₄. How this fermentation evolves after microbial colonization of the neonate is unknown. We examined the fermentation of glucose by fecal suspensions of a breast-fed infant from d 16 to 158 and found that the fermentation changed with age. Acetate, ethanol, succinate, lactate, formate and H₂ were formed up to 117 d of age. Production of succinate, lactate, formate and H₂ ceased after 117 d and acetate production increased. Butyrate and propionate were minor products up to 117 d. Afterwards, there was a slight increase in propionate production with no change in butyrate formation. Acetate was always the major product of glucose fermentation by the fecal suspensions. Approximately the same amounts of ethanol were formed throughout the study period. The fermentations were similar to fermentations of Escherichia coli and streptococci through 117 d. Nuclear magnetic resonance (NMR) analysis of the acetate formed from 1-¹³C- and 3-¹³C-glucose showed that the dominant fermentation pathway used by the colonic microbes switched from the Embden-Meyerhof-Parnas pathway at 16 d of age to the Bifidobacterium pathway at 158 d of age. An increase in the contribution of the Bifidobacterium fermentation to the overall colonic fermentation after 117 d would account for the increase in the formation of acetate from glucose. Chemical and NMR analyses of products of fecal fermentations from two other breast-fed infants <1 mo old were similar to those of the infant examined between 16 and 158 d.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Microbes in the adult human large intestine ferment dietary substrates chiefly to acetic, propionic and butyric acids and CO₂, H₂ and CH₄ (Cummings 1985, McNeil 1984, Wolin and Miller 1983). How this fermentation evolves after colonization of the neonate is unknown. Presumably, a fecal inoculum in the neonatal colon gives rise to a progression of species changes. As the composition of the colonic microbial community changes, the colonic fermentation also changes until the species that produce the adult fermentation become established as the dominant microbial community. Minor species can become major species as the colonic environment changes with time. At any age, only the most abundant species govern the characteristics of the colonic fermentation.

Changes in the colonic fermentation between birth and the onset of the adult fermentation have not been extensively studied. However, several studies show differences from the adult fermentation for at least 2-3 mo after gestation. Major differences are the preponderance of acetate, low concentrations of propionate and the almost total absence of butyrate in infant feces (Bullen et al. 1976, Edwards et al. 1994, Lifschitz et al. 1990, Parrett and Edwards 1997, Siigur et al. 1993) and as products of in vitro sugar fermentation (Lifschitz et al. 1990, Parrett and Edwards 1997). Fecal concentrations of short-chain fatty acids of premature infants (gestation age 33 wk) also suggest a preponderance of acetate (Stansbridge et al. 1993). Lactate and ethanol, found in infant feces (Lifschitz et al. 1990, Parrett and Edwards 1997, Stansbridge et al. 1993), are not normal products of the colonic fermentation of adults. Methane production in the colon is usually not large enough to be detected by breath measurements until the age of 2-10 y (Bond et al. 1971).

We examined the fermentation of glucose by fecal suspensions of a breast-fed infant to determine whether fermentation changes occurred between 16 and 158 d of age. This report shows that the products of the fermentation changed after 117 d of age. Acetate, ethanol, succinate, lactate, formate and H₂ and traces of butyrate and propionate were formed up to 117 d of age. Afterwards, a change in the dominant species was indicated because the fermentation products were exclusively acetate, ethanol, trace amounts of butyrate and increased amounts of propionate. Acetate was the major product before and after 117 d. A change in the population that governed the fermentation was confirmed by nuclear magnetic resonance (NMR) analysis of the acetate formed from 1-¹³C-and 3-¹³C-glucose. The results showed that the Embden-Meyerhof-Parnas pathway was the primary colonic fermentation pathway when the infant was 16 d old, whereas the Bifidobacterium pathway was dominant when the infant was 158 d old. Results of chemical and NMR studies of colonic fermentation of two other breast-fed infants <1 mo old were similar to those of the early fermentations of the infant examined between 16 and 158 d.

View larger version (12K): [in this window] [in a new window]   Fig 1. Production of acetate, ethanol, propionate and butyrate from glucose by fecal suspensions prepared at different ages of Baby 1. Fecal suspensions (10%) were incubated with 27.7 mmol/L glucose for 24 h and analyzed for products using HPLC. Ethanol was also measured enzymatically with alcohol dehydrogenase and the enzymatically determined values were used for the graph.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Fecal suspensions.  Suspensions of feces were prepared in anaerobic dilution solution under CO₂ as described previously (Miller and Wolin 1982, Weaver et al. 1986). The suspensions were held at 4°C and used within 24 h of collection. Anaerobic conditions were maintained by use of the serum bottle-modification of the Hungate technique (Miller and Wolin 1974). Duplicate portions (5 mL) were dried to constant weight to determine dry matter content. Fecal samples were collected from one male (Baby 1) and two female (Baby 2 and Baby 3) exclusively breast-fed infants at the ages indicated in the text. Human fecal fermentation protocols were reviewed and approved by the New York State Department of Health Institutional Review Board.

Fermentations.  Fermentations of glucose were performed in 10-mL serum bottles. Glucose (25 mg) was added as a dry powder before insertion of butyl rubber stoppers and sealing with aluminum seals. The bottles were gassed with 80% N₂:20% CO₂. A portion (2.5 mL) of 76 mmol/L NaHCO₃ from a serum bottle with 100% N₂ was injected and the bottles were cooled to 0°C in ice water. After the addition of 2.5 mL of suspension and incubation for 24 h at 37°C, the bottles were boiled for 10 min. The contents were either analyzed immediately or frozen at 20°C and thawed before removing gas samples for gas chromatography and mass-spectrometric (GC-MS) analyses. Suspensions were then acidified by the addition of 0.5 mL of 2.5 mol/L H₂SO₄ and centrifuged at 16,000 × g for 15 min. Supernatants were analyzed for fermentation products and residual glucose.

Chromatographic methods.  Soluble fermentation products and glucose were determined by HPLC procedures (Ehrlich et al. 1981) as described in the companion paper (Wolin et al. 1998). H₂ and CH₄ were quantified by using described gas chromatographic procedures (Miller and Wolin 1996). The presence of ethanol was confirmed with the use of a commercial ethanol dehydrogenase test kit (no. 176290, Boehringer Mannheim, Mannheim, Germany).

Mass spectrometry.  Mass spectral analyses for ¹³CO₂ were conducted on a Hewlett-Packard (Palo Alto, CA) 5890A Gas Chromatography-Hewlett-Packard 5970 Series Mass Selective Detector. Helium was used as the carrier gas. Temperatures were as follows: GC oven 50°C, transfer line 200°C, source 200°C. Electron ionization was 70 eV, and the mass range was 40-50. Five microliter samples of gas were used for each GC-MS analysis.

NMR methods.  Acidified fermentation supernatants were added to the NMR tube without adding any solvents. The same reference capillary was also put in the tube each time. NMR spectra were acquired on a Varian (Walnut Grove, CA) XL-300 spectrometer operating at 75.43 MHz. Details of the NMR analyses are presented in the companion paper (Wolin et al. 1998).

	RESULTS

Abstract Introduction Methods Results Discussion References

Fermentations by fecal suspensions.  Acetate, propionate, butyrate, formate, lactate, succinate, H₂ and CO₂ were formed when glucose was added to the infant's fecal suspensions. Succinate, lactate, formate and H₂ were produced before but not after 117 d of age (Fig. 1). Figure 2 shows products that were produced between 16 and 158 d of age. Acetate concentrations ranged from 25 to 35 mmol/L between 16 and 117 d of age and increased to ~60 mmol/L from 149 to 158 d of age. Ethanol was also produced by all samples and ranged from 5 to 18 mmol/L. Butyrate was barely detectable until 80 d of age; the highest concentration formed was 5 mmol/L at 108 d. Propionate concentrations were variable but tended to increase with time and were 28 and 15 mmol/L at 149 and 158 d, respectively.

View larger version (12K): [in this window] [in a new window]   Fig 2. Production of hydrogen, formate, succinate and lactate from glucose by fecal suspensions prepared at different ages of Baby 1. Fecal suspensions (10%) were incubated with 27.7 mmol/L glucose for 24 h and analyzed for soluble products using HPLC. H2 was measured by gas chromatography.

Fermentation of lactose.  Lactose is the major carbohydrate in the diet of breast-fed infants. Some lactose reaches the colon where it is fermented by the microbial flora. We compared lactose and glucose fermentation with the fecal suspension of Baby 1 at 50 d of age to see if there was a difference in product formation. Lactose was added at half the millimolar concentration of glucose to allow for fermentation of equimolar amounts of hexose units. The same products were formed from both sugars with negligible differences in the concentration of the products (Table 1).

Fermentation products of other babies.  The fermentations of glucose by fecal suspensions of two additional breast-fed babies <30 d of age and Baby 1 (16 d of age) are shown in Table 2. All suspensions formed succinate, acetate, ethanol, formate and propionate in varying amounts. Butyrate was a negligible fermentation product with all three suspensions. Lactate was found only with the suspension from Baby 1, which was also the only suspension that did not produce hydrogen during the fermentation.

Ethanol formation.  The production of ethanol as a substantial product of the fecal fermentations was especially interesting because Krebs and Perkins (1970) suggested that the physiologic role of liver alcohol dehydrogenase was to detoxify ethanol produced by microbes in the intestinal tract. Ethanol is not thought to be a normal product of the adult colonic fermentation. We confirmed the HPLC results with the suspensions of Baby 1 (16 d) and Baby 2 (14 d) by NMR analysis of the products of fermentation of 1-¹³C-glucose (Fig. 3). Peaks at chemical shifts of 58.4 and 17.7 ppm were the only peaks of the spectrum that were augmented when authentic ethanol was added to the sample and a new spectrum was determined. Confirmation of ethanol production was also obtained by enzymatic determination of ethanol with ethanol dehydrogenase.

View larger version (18K): [in this window] [in a new window]   Fig 3. Nuclear magnetic resonance (NMR) spectrum of products formed by fermentation of 1-13C-glucose by fecal suspensions of Baby 2 (A) and Baby 1 (B). Fecal suspensions (10%) were incubated with 27.7 mmol/L of 1-13C-glucose for 24 h and analyzed for products using HPLC and for labeling of the carbon atoms by NMR spectroscopy. Ethanol was also measured enzymatically with alcohol dehydrogenase and the enzymatically determined values were used. The enriched carbon giving rise to the signal numbered in the spectra is highlighted in bold. (A): l) 13CH3CH20H, 2) 13CH3COOH, 3) HOOC13CH213CH2COOH, 4) Dioxane (standard). (B): 1) 13CH3CH20H, 2) 13CH3CHOHCOOH, 3) 13CH3COOH, 4) HOOC13CH213CH2COOH, 5) Dioxane (standard).

Fermentation products in feces.  The concentrations of colonic fermentation products excreted in feces collected from Baby 1 were determined between 50 and 158 d of age. Figure 4 shows the fecal concentrations of acetate, ethanol, propionate and butyrate, and Figure 5 shows the fecal concentrations of acetate, lactate, succinate and formate. The acetate concentrations are shown in both figures for reference purposes. Fecal acetate concentrations increased dramatically after 110 d of age. Small amounts of ethanol were excreted at all ages. We also measured the concentrations of products in feces collected from Baby 3 (<30 d old). The products found were (µmol/g dry feces): succinate (155), acetate (409), ethanol (40) and propionate (194). Lactate, formate and butyrate were not detected.

View larger version (15K): [in this window] [in a new window]   Fig 4. Concentrations of acetate, ethanol, propionate and butyrate in freshly excreted feces of Baby 1. Analyses for products was by HPLC. Ethanol was also measured enzymatically with alcohol dehydrogenase and the enzymatically determined values were used.

View larger version (15K): [in this window] [in a new window]   Fig 5. Concentrations of acetate, succinate, lactate and formate in freshly excreted feces of Baby 1. Analyses for products was by HPLC.

Fermentation pathways.  Pathways of glucose fermentation by fecal suspensions of Baby 1 were determined at 16 and 158 d. We examined the labeling of the carbon atoms of acetate when ¹³CO₂ and ¹²C-glucose, 1-¹³C-glucose, or 3-¹³C glucose and ¹²CO₂ were fermented. The use of NMR analysis of products from these ¹³C substrates to distinguish between bacterial fermentation pathways is described in the companion paper (Wolin et al. 1998). Enrichment by ¹³CO₂ was minor at 16 and 158 d and no double-labeled acetate was formed. This showed that there was no reduction of CO₂ to acetate. Acetate formation from CO₂ reduction is a major feature of the colonic fermentation of adults (Miller and Wolin 1996). The NMR spectra of the 16-d supernatants of the 1-¹³C-glucose fermentation (Fig. 3) show that ¹³CH₃¹²COOH was formed and that ¹²CH₃¹³COOH and ¹³CH₃¹³COOH were absent. This is characteristic of the Embden-Meyerhof-Parnas pathway.

A singlet signal at 21.3 ppm is produced when ¹³C is only in the methyl group, and a singlet is produced at 177.6 ppm when only the carboxyl group contains ¹³C. When both the methyl and carboxyl groups are labeled with ¹³C, doublet signals are produced at both 21.3 and 177.6 ppm. Major amounts of double-labeled acetate were formed at 158 d but not at 16 d when 3-¹³C-glucose was supplied.The enrichment and concentrations of acetate carbons formed from ¹³C-glucose by fecal suspensions of Baby 1 at 16 and 158 d are compared in Table 3. The Bifidobacterium pathway is the only bacterial fermentation pathway that forms double-labeled acetate from 3-¹³C-glucose. Only minor amounts of single labeling of the methyl and carboxyl groups occurred at both ages when 3-¹³C-glucose was fermented. Labeling in the methyl but not the carboxyl group of acetate was observed at both ages when 1-¹³C-glucose was used. No double-labeled acetate was produced from 1-¹³C-glucose.

Production of labeled CO₂ was also measured to distinguish between possible microbial pathways of fermentation. The only microbial pathway that yields CO₂ from C-3 of glucose is the Embden-Meyerhof-Parnas pathway (Fig. 6). Major amounts of ¹³CO₂ were formed from 3-¹³C-glucose only with the fecal suspension prepared at 16 d of age. After fermentation, the percentage of ¹³CO₂ detected by mass spectrometry was 11.7 for the 16-d suspension and 1.8 for the 158-d suspension. Therefore, both the CO₂ and the acetate labeling patterns show that the Embden-Meyerhof-Parnas pathway was the major pathway at 16 d and the Bifidobacterium pathway was the major pathway at 158 d.

View larger version (12K): [in this window] [in a new window]   Fig 6. Embden-Meyerhof-Parnas pathway.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Microbiological studies of colonization, summarized by Cooperstock and Zedd (1983), indicate that Escherichia coli and Streptococcus species rapidly grow in the neonate's colon after inoculation via the fecal-oral route. The products of glucose fermentation by fecal suspensions of three breast-fed infants at <1 mo of age are similar to those expected of these early colonizers of the infant colon. Ethanol, succinate, lactate, acetate and formate are produced by E. coli (Wood 1961) and, except for succinate, by streptococci. Although streptococci produce only lactic acid when environmental pH drops because of acid formation, they also form acetate, formate and ethanol when the pH is kept around neutrality (Gunsalus and Niven 1942). Yeast fermentation might be another source of ethanol. Escherich (1885 translated in 1988) found yeasts in the feces of a 2-mo-old healthy, breast-fed child. H₂, another product of E. coli fermentation, was not produced at 16 d by fecal suspensions from Baby 1, but was formed from d 50 through 120. Fecal suspensions of the other two babies prepared at <1 mo of age produced H₂.

Fecal suspensions of Baby 1 did not produce succinate, lactate, H₂ or formate from glucose after 120 d. Acetate, small amounts of propionate and ethanol, and trace amounts of butyrate were the only products detected after 120 d. Acetate concentrations increased above the amounts found before 120 d. This indicates a change in the microbial community that produces the dominant fermentation. This was confirmed by studies of fermentation pathways using NMR analysis of products from ¹³C-labeled substrates. At 16 d, the labeling of acetate and CO₂ showed that the major colonic fermentation pathway used for glucose catabolism was the Embden-Meyerhof-Parnas pathway (Fig 6). The same labeling pattern was obtained after fermentation of glucose by Baby 2 at 14 d of age (Fig. 3). E. coli and streptococci and, as we recently showed, the adult microbial community (Miller and Wolin 1996) use the Embden-Meyerhof-Parnas pathway to degrade glucose. When the infant reached 158 d of age, the NMR data showed that the Embden-Meyerhof-Parnas pathway was a minor pathway for glucose degradation, whereas the major pathway of glucose fermentation was a unique pathway used by Bifidobacterium species (Wolin et al. 1998). Bifidobacteria are also early colonizers of the colon (Cooperstock and Zedd 1983 ), but they apparently were not sufficiently numerous to have a major effect on the fermentation when the infant was 16 d old. We suspect that the transition to dominance by the bifidobacteria pathway occurred in the colon of Baby 1 after 120 d and corresponded to the period of disappearance of succinate, lactate, formate and H₂ as end products of fermentation.

Concentrations of acetate in excreted feces of Baby 1 increased with age in parallel with the increased production of acetate by suspensions prepared from the same fecal specimen. The ratios of acetate concentrations to the sum of the concentrations of all organic fermentation products were strikingly similar at all ages for fecal samples per se and the products of glucose fermentation. This suggests that fermentation of dietary substrates in vivo produces the same products as glucose fermentation by suspensions in vitro.

The changes of fermentation products and pathways reflect a slow change in the dominant microbial community despite the maintenance of a constant diet of breast milk. One explanation for the slow transition is rapid colonization of the aerobic postnatal ecosystem by facultative anaerobes such as E. coli and streptococci followed by slow colonization by anaerobes. Bullen et al. (1976) suggested that the facultative anaerobes produce a falling pH and Eh, which favor the growth of bifidobacteria. Anaerobes such as bifidobacteria present in the inoculum grow slowly until the establishment of anaerobiosis. Once established, their growth rates increase and become greater than the growth rate of the primary colonizers. Development of the microbial community that produces the characteristic adult fermentation probably occurs after weaning.

Ethanol is not considered a normal product of the adult colonic fermentation (Cummings 1985). However, studies of fermentation products primarily use gas-chromatography procedures that detect short-chain fatty acids. Ethanol has usually not been evaluated in feces or as an in vitro fermentation product. This study shows that the fecal fermentations of three breast-fed infants produce ethanol. Fecal suspensions of Baby 1 produced ethanol from 16 to 158 d and excreted feces of Baby 1, 2 and 3 contained ethanol. Other studies also suggest that ethanol can be a product of the infant colonic fermentation. Stansbridge et al. (1993) found ethanol in 34 of 52 fecal samples of 1- to 28-d-old premature infants fed with a formula containing a Lactobacillus species. The median was 6.3 µmol/g dry feces. In a control group reared without the Lactobacillus, 31 of the 83 samples had ethanol with a median of 3.3 µmol/g dry feces. All infants had a gestation age <33 wk Krebs and Perkins (1970) suggested that the physiologic role of liver alcohol dehydrogenase was to detoxify ethanol produced by microbes in the intestinal tract. The cited published studies and our results support the hypothesis that the primary function of alcohol dehydrogenase in humans is the detoxification of ethanol produced by the colonic microbial fermentation of milk constituents in infants.

Further studies of breast-fed and formula-fed babies should be conducted to ascertain the frequency and extent of colonic microbial production of ethanol in the infant colon. We calculate that substantial amounts of ethanol could be formed in the colon. Estimates of the amount of ingested lactose fermented in the colon of premature infants range from 15 to 66% (Kien et al. 1989). Lactose is also available for fermentation in the colon of breast-fed full-term infants (Lifschitz et al. 1983). Because lactose intake is about 12.6 g/(d·kg body weight) for premature or full-term infants, large amounts of sugar can become available for microbial production of ethanol in the colon. The microflora of a 4.54 kg baby (10 lb) could ferment 5.72 g of lactose per day if 10% of the ingested lactose reached the colon. Because we found a mean ratio of 12.5 mol of ethanol to 27.7 mol of hexose units for in vitro glucose fermentation, ~0.7 g of ethanol could be produced per day in the infant colon On a per kilogram basis, this would be 2.5 times the amount that a 68 kg (150 lb) adult would imbibe in 750 mL (25 oz) of beer containing 4% (v/v) ethanol. Ethanol clears from the blood of infants at the same rate as from adult blood (Simon et al. 1994), suggesting that the activities of liver alcohol dehydrogenase are the same at all ages. Rates of alcohol production in different parts of the colon and in feces may vary. Different features of microbial activity in different colonic segments of adult cadavers have been reported (Macfarlane et al. 1992).

Only small amounts of butyrate are formed from glucose by in vitro infant fecal fermentations. Other investigators also showed that butyrate concentrations in feces of breast-fed infants are low in relation to concentrations of acetate (Bullen et al. 1976, Edwards et al. 1994, Lifschitz et al. 1990, Siigur et al. 1993) and as products of in vitro sugar fermentation (Lifschitz et al. 1990). Apparently, butyrate is not a major energy source for the colonic epithelial cells of infants. Butyrate is a major energy source for the colonic epithelial cells of adults (Roediger 1982) and causes cell differentiation (Kruh 1982). Low concentrations of butyrate produce differentiation of human colon carcinoma cells (Tanaka et al. 1990). Transition to use of butyrate as an energy source by infants may occur after weaning. An intriguing possibility is that a butyrate-dependent differentiation of mucosal cells produces the capability of using butyrate as an energy source.

The exact timing of fermentation changes will undoubtedly differ among breast-fed babies. Host (mother and child) differences and environmental variables, including the characteristics of the flora that initially colonize the colon, will influence the temporal sequence. In vitro studies of fermentation product formation and pathway changes using NMR techniques in infants can elucidate the temporal sequences in breast- and formula-fed infants including premature infants. Fermentation measurements as indicators of microbial population changes are less time consuming than conventional microbiological analyses. Fermentation analysis may aid in designing formulas and other methods for beneficial control of the colonic flora of infants.

	FOOTNOTES

Manuscript received 11 March 1997. Initial reviews completed 25 April 1997. Revision accepted 26 September 1997.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Bond J. H., Engel R. F., Levitt M. D. Factors influencing pulmonary methane excretion in man. An indirect method of studying the in situ metabolism of the methane producing colonic bacteria. J. Exp. Med. 1971; 133:572-588 [Abstract]
• Bullen C. L., Tearle P. V., Willis A. T. Bifidobacteria in the intestinal tract of infants. J. Med. Microbiol. 1976; 9:325-333 [Abstract/Free Full Text]
• Cooperstock, M. S. & Zedd, A. Z. (1983) Intestinal flora of infants. In: Human Intestinal Flora in Health and Disease (Hentges, D. A., ed.), pp. 79-99. Academic Press, New York, NY.
• Cummings J. H. Fermentation in the human large intestine: evidence and implications for health. Lancet 1985; 1:1206-1208 ^[Medline]
• Edwards C. A., Parrett A. M., Balmer S. E., Wharton B. A. Faecal short chain acids in breast-fed and formula-fed babies. Acta. Paediatr. 1994; 83:459-462 [Medline]
• Ehrlich G. G., Goerlitz D. F., Bourell J. H., Eisen G. V., Godsy E. M. Liquid chromatographic procedure for fermentation product analysis in the identification of anaerobic bacteria. Appl. Environ. Microbiol. 1981; 42:878-885 ^{[Abstract/Free Full Text]}
• Escherich T. Classics in infectious disease. The intestinal bacteria of the neonate and breast-fed infant. Rev. Infect. Dis. 1988; 10:1220-1225 [Medline]
• Gunsalus I. C., Niven C. F. Jr. The effect of pH on the lactic acid fermentation. J. Biol. Chem. 1942; 145:131-136 ^{[Free Full Text]}
• Kien C. L., Heitlinger L. A., Li B. U., Murray R. D. Digestion, absorption and fermentation of carbohydrates. Semin. Perinatol. 1989; 13:78-87 [Medline]
• Krebs H. A., Perkins J. R. The physiological role of alcohol dehydrogenase. Biochem. J. 1970; 118:635-644 [Medline]
• Kruh J. Effects of sodium butyrate, a new pharmacological agent, on cells in culture. Mol. Cell. Biochem. 1982; 42:65-82 [Medline]
• Lifschitz C. H., Smith E. O., Garza C. Delayed complete functional lactase sufficiency in breast-fed infants. J. Pediatr. Gastroenterol. Nutr. 1983; 2:478-482 [Medline]
• Lifschitz C. H., Wolin M. J., Reeds P. J. Characterization of carbohydrate fermentation in feces of formula-fed and breast-fed infants. Pediatr. Res. 1990; 27:165-169 [Medline]
• Macfarlane G. T., Gibson G. R., Cummings J. H. Comparison of fermentation reactions in different regions of the human colon. J. Appl. Bacteriol. 1992; 72:57-64 [Medline]
• McNeil N. I. The contribution of the large intestine to energy supplies in man. Am. J. Clin. Nutr. 1984; 39:338-342 [Abstract/Free Full Text]
• Miller, T. L. &Wolin, M. J. A serum bottle modification of the Hungate technique for cultivating obligate anaerobes. Appl. Microbiol. 1974; 27:985-987 [Medline]
• Miller T. L., Wolin M. J. Enumeration of Methanobrevibacter smithii in human feces. Arch. Microbiol. 1982; 131:14-18 [Medline]
• Miller T. L., Wolin M. J. Pathways of acetate, propionate, and butyrate formation by the human fecal microbial flora. Appl. Environ. Microbiol. 1996; 62:1589-1592 [Abstract]
• Parrett A. M., Edwards C. A. In vitro fermentation of carbohydrate by breast fed and formula fed infants. Arch. Dis. Child. 1997; 76:249-253 [Abstract/Free Full Text]
• Roediger W.E.W. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 1982; 83:424-429 ^[Medline]
• Siigur U., Ormisson A., Tamm A. Faecal short-chain fatty acids in breast-fed and bottle-fed infants. Acta Paediatr. 1993; 82:536-538 [Medline]
• Simon H. K., Cox J. M., Sucov A., Linakis J. G. Serum ethanol clearance in intoxicated children and adolescents presenting to the ED. Academic Emergency Medicine. 1994; 1:520-524 [Medline]
• Stansbridge E. M., Walker V., Hall M. A., Smith S. I., Millar M. R., Bacon C., Chen S. Effects of feeding premature infants with Lactobacillus GG on gut fermentation. Arch. Dis. Child. 1993; 69:488-492 [Abstract/Free Full Text]
• Tanaka Y., Bush K., Eguchi T., Ikekawa N., Takaguchi T., Kobayashi Y., Higgins P. J. Effects of 1,25-dihydroxyvitamin D₃ and its analogs on butyrate-induced differentiation of HT-29 human colonic carcinoma cells and on the reversal of the differentiated phenotype. Arch. Biochem. Biophys. 1990; 276:415-423 [Medline]
• Weaver G. A., Krause J. A., Miller T. L., Wolin M. J. Incidence of methanogenic bacteria in a sigmoidoscopy population: an association of methanogenic bacteria and diverticulosis. Gut 1986; 27:698-704 [Abstract/Free Full Text]
• Wolin, M. J. & Miller, T. L. (1983) Carbohydrate fermentation. In: Human Intestinal Flora in Health and Disease (Hentges, D. A., ed.), pp. 147-165. Academic Press, New York, NY.
• Wolin M. J., Zhang Y., Bank S., Yerry S., Miller T. L. NMR detection of ¹³CH₃¹³COOH from 3-¹³C-glucose: a signature for Bifidobacterium fermentation in the intestinal tract. J. Nutr. 1998; 128:81-86
• Wood, W. A. (1961) Fermentation of carbohydrates and related compounds. In: The Bacteria (Gunsalus, I. C. & Stanier, R. Y., eds.), vol. 2, pp. 59-149. Academic Press, New York, NY.

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