Although the resident bacteria of the colon depend to a large extent on exogenous substrates for energy and growth, it has proved experimentally difficult to produce consistent changes by dietary manipulation (Keush and Gorbach 1985). However, it could be of interest to manipulate colonic flora because it is supposed that some intestinal bacteria could be beneficial to health whereas others coud be harmful (Metchnikoff 1908). Prebiotics are mainly oligosaccharides which, on oral ingestion, could lead to changes in the colonic bacterial ecosystem. In healthy humans, these oligosaccharides are virtually not digested by the intestinal enzymes and reach the colon, where they are fermented by the bacterial flora (Gibson et al. 1995). Unlike other di- or oligosaccharides not digested in the small intestine such as lactose or inulin (Bouhnik et al. 1996, Ito and Kimura 1993), they are usually fermented by only a limited number of colonic bacteria and for this reason might specifically stimulate the growth of certain microorganisms (Rowland 1992, Tanaka et al. 1983). They might therefore alter the intracolonic bacterial balance and foster microorganisms such as bifidobacteria, which might have beneficial effects on the host (Gibson et al. 1995, Mitsuoka 1984).

Transgalacto-oligosaccharides consist of a mixture of indigestible oligosides obtained by biosynthesis from lactose, by the action of a transfer enzyme -galactosidase from Aspergillus orizae (Kan et al. 1989). In vitro experiments in continuous cultures showed that transgalacto-oligosaccharides were extensively converted to short-chain fatty acids with a large acetate molar proportion (Durand et al. 1992). Other in vitro investigations showed that transgalacto-oligosaccharides were preferentially consumed by most of the bifidobacterial species and by a few strains of enterobacteria (Minami et al. 1985, Sumihara 1987, Tanaka et al. 1983). In fact, galactose could represent an optimal substrate because the enzymes of galactose metabolism are constitutive in cells of Bifidobacterium (Lee et al. 1980). In addition, transgalacto-oligosaccharides have useful technological properties and are already used for animal feeds. In Japan, they are also used to make food for human consumption, chiefly with the claim of bifidobacteria promoters (Ito et al. 1990, Sumihara 1987).

The aim of the present work was to assess, in healthy humans, clinical tolerance of transgalacto-oligosaccharides and the effects of prolonged administration of 10 g/d on fecal concentrations of bifidobacteria and respiratory excretion of fermentation gases. Because of the difficulty of studying intracolonic fermentation products in vivo, an additional study was conducted in vitro to examine the consequences of prolonged administration of transgalacto-oligosaccharides on the bacterial metabolism.

SUBJECTS AND METHODS

To assess tolerance of transgalacto-oligosaccharides, subjects filled in a diary sheet on which the following symptoms were graded from 0 to 3: excess rectal gases, bloating, borborygmi and abdominal pains. Stool frequency and consistency were also noted.

The experimental fermentor was sampled at given times after inoculation of transgalacto-oligosaccharides on d 1, 2, 7 and 14. The control fermentor was also sampled at the same times. The pH and the concentrations of adenosine triphosphate were measured at 0, 1, 2, 3, 5 and 8 h after inoculation. Short-chain fatty acids and D- and L-lactic acids were measured at 0, 2 and 5 h after inoculation. The volume of gases produced was measured hourly for 5 h and at h 8. To measure residual transgalacto-oligosaccharides, 24-h effluents were collected at the fermentor outlet on the first 6 d after the addition of transgalacto-oligosaccharides, and then on d 12 and 13. 

In vitro study. Short-chain fatty acid concentrations in the fermentors were determined by gas chromatography (Ottenstein and Bartley 1971), and adenosine triphosphate concentrations by bioluminescence (Komisarczuk et al. 1984). Concentrations of residual transgalacto-oligosaccharides in the effluents were measured by colorimetry after hexose hydrolysis with phenolsulfuric acid (Dubois et al. 1956). A sample of effluent was first deproteinized with HClO4; then 0.5 mL was mixed with 0.5 mL phenol (50 g/L) and 2.5 mL concentrated sulphuric acid. The mixture was left at 20°C for 30 min before shaking and the absorbance was determined at 490 nm.

RESULTS

Table 1. Fecal data in healthy volunteers during ingestion of 10 g transgalacto-oligosaccharides per day for 21 d1 [View Table]

Bifidobacteria concentrations were significantly greater on d 7, 14 and 21 after transgalacto-oligosaccharides ingestion compared with d 1 (Fig. 1). Before ingestion, these concentrations were 8.6 ± 0.6 log CFU/g vs. 9.7 ± 0.5, 9.7 ± 0.6 and 9.5 ± 0.6 on d 7, 14 and 21, respectively (P < 0.05). However, the concentrations of enterobacteria were not affected during transgalacto-oligosaccharides ingestion.

Breath H₂ excretion was significantly lower on d 7, 14 and 21 than on d 1 (P < 0.01) and methane excretion was unchanged (Table 2).

Table 2. Volume of H2 and CH4 in gases expired by healthy volunteers after ingestion of 10 g transgalacto-oligosaccharides per day for 21 d1 [View Table]

Kinetic study in the fermentors. In the control fermentor, the concentrations of ATP, which reflect fermenting activity, remained stable, at a mean of 2.8 mmol/L. After the addition of transgalacto-oligosaccharides, ATP concentrations rose to a peak of nearly 12 mmol/L (Fig. 2). However, the peak was maximal at h 5, 3 and 1 on d 1, 2 and 7, respectively. On d 14, it was again observed at the first hour.

The pH of the control fermentor remained stable and was close to neutral (6.9). After the addition of transgalacto-oligosaccharides, the acidification kinetics showed that as early as d 2, the pH dropped to 6.0 at h 3, and by d 7 fell to 5.2 (Fig. 3). At d 14, its level was close to that of d 7. 

The concentrations of total short-chain fatty acids in the control fermentor were 50 mmol/L, with proportions of acetate, propionate and butyrate of 50, 24 and 12%, respectively. After the addition of transgalacto-oligosaccharides, the initial total short-chain fatty acid concentrations (time 0) gradually rose, between d 1 and 7, from 50 to 100 mmol/L (Fig. 4). On d 1 and 2, the rise in these concentrations occurred between h 2 and 5. On d 7 and 14, the concentrations measured at h 2 were close to those measured at h 5. The mean proportion of acetate rose from 51 to 65% between d 1 and 7 at all of the times measured. This rise occurred at the expense of the proportion of propionate, which dropped from 25 to 17% between d 1 and 7. 

In the control fermentor, and also during d 1 and 2 after the addition of transgalacto-oligosaccharides, no lactate was detected. On the other hand, it was detected on d 7 when its concentration at h 0, 2 and 5 ranged from 1.1 to 1.7 mmol/L for L-lactate, and from 1 to 1.2 mmol/L for D-lactate. At d 14, L- and D-lactate (1 mmol/L) were observed only in the sample taken 2 h after transgalacto-oligosaccharides inoculation.

The volumes of total gases produced during the 8 h after transgalacto-oligosaccharides inoculation (Fig. 5) were close on d 1, 2, 7 and 14 (about 2500 mL compared with 340 mL in the control fermentor). However, the initial rate of gas production, calculated from the values for the first 2 h, rose from 187 mL/h on d 1 post-inoculation to 580 mL/h on d 7 and 14. 

DISCUSSION

Although the colonic flora depend on substrates from food for energy, it is very difficult to demonstrate alterations in the flora equilibrium that occur after ingestion of food supplements in the form of indigestible carbohydrates such as dietary fibers (Drasar et al. 1976, Woods and Gorbach 1993). In our study, a slight alteration in diet led to a 10-fold rise in the concentration of bifidobacteria in the colonic flora. In vitro, transgalacto-oligosaccharides proved to be good substrates for enterobacteria (Minami et al. 1985, Sumihara 1987). In contrast, after transgalacto-oligosaccharides consumption, numbers of enterobacteria were significantly decreased in rats, results obtained by inoculating germ-free rats with a suspension of fresh human feces (Rowland and Tanaka 1993). In our study, no modifications were observed in the fecal concentrations of enterobacteria. This is a good illustration of the discrepancies obtained from in vitro and in vivo studies, which can be explained by the extreme complexity of colonic flora in humans (Ducluzeau 1989, Keush and Gorbach 1985). In our study, in vivo transgalacto-oligosaccharides may have had no effect on the enterobacteria because these microorganisms belong to the subdominant flora and are therefore 1000 to 10000 times less numerous than the bacteria of the predominant flora such as the bifidobacteria, which preferentially consume most or all of the substrate (Keush and Gorbach 1985). Ito et al. (1990) also showed a significant although moderate, increase in fecal bifidobacteria in volunteers who had ingested 10 g of transgalacto-oligosaccharides per day for a week. In their study, ingestion of transgalacto-oligosaccharides did not alter either the fecal concentrations of enterobacteria or those of total anaerobes, which also suggests that transgalacto-oligosaccharides might act specifically on the growth of bifidobacteria. In a subsequent study of rats harboring a human colonic flora, administration of transgalacto-oligosaccharides (5 g/100 g diet) for 4 wk led to a significant rise in cecal concentrations of bifidobacteria and also of total anaerobes (Rowland and Tanaka 1993). However, the proportion of bifidobacteria in the total bacterial mass diminished, suggesting that transgalacto-oligosaccharides also stimulated the growth of other bacterial anaerobes. This effect of transgalacto-oligosaccharides, which was not confined to bifidobacteria, might be attributable to the doses used in different studies because, for an equal body weight, the doses given to rats (Rowland and Tanaka 1993) were 20 to 30 times larger than in our study and that of Ito et al. (1990), both conducted in humans.

Prolonged administration of transgalacto-oligosaccharides led to a change in their fermentation metabolism. In vivo in humans, the composition of the fermentation metabolites in stools is a poor reflection of what really occurs in the colonic lumen in clinical situations in which there is no diarrhea. For instance, when volunteers ingest 20 g of lactulose in the morning and evening for a week, the cecal metabolism of this sugar and the resulting metabolites undergo great changes between the first and last doses, whereas stool composition remains largely unaffected (Florent et al. 1985). Fermentation metabolites are efficiently absorbed in the human colon, and if the time allowed for this process is not shortened, the fraction of metabolites eliminated in the stools does not correspond to their intracolonic production. On the other hand, in the lung emunctory, the fraction of breath H₂ expired is roughly proportional to the amount produced in the colon (Christl et al. 1992). In our study, prolonged transgalacto-oligosaccharides ingestion led to a significant drop in expired breath H₂ without altering expired breath methane. In view of the inaccessibility of the human colon, and for better understanding of what was happening in the colonic contents, we used an in vitro model. In view of its principle and the levels of acidification reached, the model does simulate fairly well what may occur in the human proximal colon, but it does have certain limits: it consists of only one compartment and can simulate neither motor activity nor the absorption and secretion processes which occur in the human colon. The results we obtained in vitro showed that transgalacto-oligosaccharides were fermented more efficiently and faster from d 7 after the beginning of daily transgalacto-oligosaccharides introduction. However, the changes that already appeared on d 2 suggested that the adaptation of bacterial metabolism to transgalacto-oligosaccharides occurred earlier, between d 2 and 7 of administration. After that, the degree of acidification, the evolution of the ATP and short-chain fatty acid concentrations, and gas production were stable between d 7 and 14 of transgalacto-oligosaccharides administration. In addition to more efficient and faster fermentation of transgalacto-oligosaccharides, the fermentation pathways were altered, exhibiting lactic acid production and a greater molar proportion of acetic acid among the short-chain fatty acids. These two metabolic changes are evidence that lactic acid-resistant flora developed in the fermentor to which transgalacto-oligosaccharides were added (Rasic and Kurmann 1983). These flora consist of bacteria known as lactic, which ferment hexoses via a heterolactic pathway, which does not produce H₂ and leads to the formation of acetate and lactate. Indeed, in our in vivo human experiments, there was a drop of H₂ production with maintained ingestion of transgalacto-oligosaccharides. Methane is produced from H₂ in the distal part of the human colon (Pochart et al. 1993). Consequently, although the concentrations of bifidobacteria increased throughout the entire colon, the absence of reduction in breath methane and in fecal pH suggests that the heterolactic metabolism of the bifidobacteria occurs only in the proximal part of the human colon.

In conclusion, our results show that the addition to the diet of small amounts of indigestible oligosaccharides, which do not induce digestive symptoms, alters the concentrations of bifidobacteria and the intracolonic fermentation metabolism. Such metabolic changes are those expected, but their real beneficial effects on the host have yet to be proved.