The gastrointestinal tracts of breast-fed infants are colonized more easily with bifidobacteria than are those of formula-fed infants. Colonization is thought to reduce infant diarrhea. Amendments to formulas that improve colonization by bifidobacteria are being actively investigated. Colonization studies almost invariably require measurements of the concentration of the bifidobacteria in feces to assess their importance in the colon. We investigated the use of nuclear magnetic resonance (NMR) analysis of products of fermentations of 1- and 3-13C-glucose to evaluate the importance of bifidobacteria in the colonic ecosystem. Bifidobacteria use a unique pathway of hexose catabolism to produce primarily acetate and lactate. The fermentation yields 3 mol of acetate from 2 mol of glucose. Two of the acetates are formed from C1 and C2 of glucose and the third is formed entirely from C3 of glucose. We first employed high resolution NMR to verify the pathway used by a pure culture of Bifidobacterium bifidum. The major products of fermentation of 1- and 3-13C-glucose were acetate and lactate. Most of the 13C from 3-13C-glucose was in 13CH313COOH with equal enrichment in the methyl and carboxyl groups. The 13C-acetate from 1-13C-glucose was almost entirely enriched in the methyl of acetate and no 13CH313COOH was produced. NMR analysis of glucose fermentation by the colonic flora of a 158-d-old strictly breast-fed infant showed production of 13CH313COOH from 3-13C-glucose. The amount of 13CH313COOH formed established that the Bifidobacterium pathway was the major pathway used for glucose fermentation by this infant's colonic microbes.

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Fecal suspensions from humans were incubated with 13Co2 and H2. The suspensions were from subjects who harbored 108 and 1010 methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 1010 per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and "3CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.

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