Introduction

Lactulose is a synthetic disaccharide composed of galactose and fructose ((1,4)-galactosido-fructose). Lactulose is not metabolised or absorbed in the human small intestine. Once in the caecum, lactulose is fermented by the colonic microflora resulting in changes in bacterial composition and metabolic activities of the colonic flora. After ingestion of a large dose of lactulose ranged 20–60 g/day (Berge Henegouwen et al, 1987), the following has been shown: (a) an increase of bifidobacterial counts (Ballongue et al, 1997); (b) an inhibition of bacterial degradation of primary to secondary bile acids (Nair, 1988) and (c) an inhibition of colon cancer in rats when administered alone or in conjunction with bifidobacteria (Hennigan et al, 1995; Challa et al, 1997).

For these reasons, lactulose could be perceived as a prebiotic, defined as a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of colonic bacteria, and thus improving host health (Gibson & Roberfroid, 1995). At such large doses, lactulose may however induce inconvenient digestive symptoms, and it is unclear whether faecal bifidobacteria and lactobacilli are increased using low and well-tolerated doses of lactulose in humans.

The aim of this double-blind randomised study was to assess in healthy volunteers the effects of prolonged low-dose lactulose administration on faecal bifidobacteria and selected metabolic indexes potentially involved in colonic carcinogenesis and on clinical tolerance.

Subjects and methods

Subjects

In total, 16 healthy volunteers (five males and 11 females), aged 19–42 y, participated in the study. None had any history of gastrointestinal disease, except for appendectomy. No antibiotics or laxatives had been taken during the 3 months before the study. No other medication was allowed during the investigation period. Subjects gave written informed consent to the protocol, which was approved by the Ethics Committee of Saint Louis Hospital.

Experimental design

The volunteers were randomised into two parallel groups (eight subjects per group). They ingested in a double-blind manner for 6 weeks at the end of lunch and dinner a powder mixture containing 5 g of lactulose or placebo (sucrose). We used lactulose from Duphalac^® (Laboratories Solvay Pharma, 92151 Suresnes Cedex, France). Throughout the study, subjects consumed their usual diet, except for fermented dairy products containing viable bifidobacteria because they have been shown to lead to an increase in faecal bifidobacteria within a few days of consumption (Bouhnik et al, 1992).

Tolerance was evaluated using a daily chart where the symptoms (excess flatus, borborygmi, bloating, abdominal pain) were graded from 0 (no symptom) to 3 (severe symptoms). Frequency and consistency of stools were also noted and diarrhoea was defined as one or more watery stool, or more than three stools per day.

Stool collection

Stools were collected thrice, on the day before the sugar consumption (d0), and after 3 (d21) and 6 (d42) weeks of the lactulose or placebo ingestion. They were collected in plastic containers under anaerobic conditions (Anaerocult A^®; Merck, Darmstadt, Germany), immediately stored at 4°C and analysed within 3 h.

Bacterial counts and pH

Faecal samples were homogenised with a high-speed blender (Ultraturax, Labortechnik, Staufer, Germany) and serially diluted 10-fold in solution for anaerobes (saline, glucose and cysteine). A volume of 100 l of each dilution was inoculated in appropriate agars. Total anaerobic bacteria were counted on Wilkins-Chalgren agar (DIFCO, Détroit, MI, USA); bifidobacteria were counted on the Beerens medium (Beerens, 1991); lactobacilli were counted on the Rogosa medium. Total anaerobes, bifidobacteria and lactobacilli were cultured for respectively 7, 5 and 3 days at 37°C in anaerobic conditions (Gas Pak system, BBL, Cockeysville, MD, USA) with Anaerocult A. An aliquot of stools was diluted five-fold in distilled water, homogenised, and the pH measured with a pH meter (Bioblock, Illkirch, France).

Neutral sterols and bile acids

Lipids from the dry lyophilised faeces were extracted with ethanol for 24 h in a Soxhlet apparatus. Lipid fractions were saponified in boiling ethanolic 2 M potassium hydroxide for 3 h. The sterols were extracted with hexane, and after acidification to pH 2, deconjugated bile acids were extracted with diethyl ether. Total bile acids and neutral sterols were measured on a gas chromatograph equipped with an OV 1701 capillary column (25 m length, 0.32 mm i.d. 0.2 m film thickness).

Data analysis

Faecal concentrations of bacteria were expressed as log colony forming unit (CFU)/g wet weight. The results were expressed as meanss.e.m. One-way ANOVA, with treatment as factor, was used to compare the bacterial, biliary acids and neutral sterol concentrations and pH at d0 in each treated group. Two-way ANOVA, with time and treatment as factors, was used to compare the bacterial concentrations and pH between d0, d21 and d42 in each group. Following a significant F test (P<0.05), the Newman–Keuls test was used to identify differences between individual means. Symptoms experienced with lactulose were compared to those with placebo using the Wilcoxon signed-rank test.

Results

Faecal bacterial counts and pH

Faecal bifidobacterial counts were higher after lactulose ingestion than after placebo ingestion (P=0.03) (Figure 1). Lactulose ingestion led to an increase in faecal bifidobacterial counts from d0 to d21 and d42 (8.250.53, 8.960.40 and 9.540.28 log CFU/g wet wt, respectively (P=0.048)). Placebo ingestion did not lead to any faecal bifidobacterial count change. Total anaerobes, Lactobacillus and pH were not significantly changed throughout the study in the two groups (Table 1).

Figure 1.

Effect of 42-day ingestion of lactulose or placebo (10 g/d) on faecal bifidobacteria in healthy volunteers. Values are meanss.e.m., n=8 subjects per group.

Full figure and legend (56K)

Table 1 - Effect of 42-day ingestion of lactulose or placebo (10 g/d) on the faecal bacterial counts (ms.e.m.; log cfu/g) and pH (ms.e.m.) in healthy volunteers. Values are meanss.e.m., n=8 subjects per group.

Full table

Neither faecal bile acids nor neutral sterols (cholesterol and by-products) were quantitatively and qualitatively modified by lactulose or placebo ingestion (Table 2). Phytosterols and particularly beta-sistosterol, the main phytosterol in plant oil, were transformed by microbial reduction in coprophytosterols. The proportion of beta-sistosterol transformed into coprobeta-sistosterol was totally similar to the proportion of microbial sterol coming from cholesterol in each group.

Table 2 - Effect of 42-day ingestion of lactulose or placebo (10 g/d) on the faecal neutral sterols and bile acids in healthy volunteers. Values are meanss.e.m., n=8 subjects per group.

Full table

Digestive tolerance

The digestive symptoms reported by the volunteers during the 42-day consumption periods are shown in Table 3. Comparisons between the two groups showed that excess flatus was significantly more frequent in the lactulose group (P=0.03), but was very mild. For other symptoms (borborygmi, bloating, abdominal pain), there was a trend, but the difference was not significant between the groups. Diarrhoea was not reported in any of the volunteers.

Table 3 - Intensity of digestive symptoms reported during 42-day ingestion of lactulose or placebo (10 g/d). Values are means symptom score/day, n=8 subjects per group.

Full table

Discussion

This study shows that a low dose of lactulose (5 g b.i.d.) introduced in the human diet is well tolerated and can alter the balance of colonic bacteria, leading to a significant bifidobacterial count increase. Therefore, we confirmed the results recently reported by Tuohy et al (2002), which showed in a 30-day double-blind, placebo-controlled study in 20 healthy volunteers the bifidogenic effect of lactulose using similar doses. This effect was determined using culture-based methodologies like ours and also the fluorescent in situ hybridisation method.

We also show what other authors have reported using higher doses, which could lead to intolerance symptoms such as abdominal pain and diarrhoea (Hoffmann & Bircher, 1969; Ballongue et al, 1997). As there is no satisfactory definition of a bifidogenic effect and no data available about the absolute rise in bifidobacteria CFU counts to obtain a biological relevance, we considered, as most authors, that a substrate is bifidogenic if a statistically significant rise is observed. We did not find a significant increase in Lactobacillus spp after lactulose ingestion, neither faecal pH acidification, as has been previously reported (Riggio et al, 1990; Terada et al, 1992; Salminen & Salminen, 1997). However, results obtained in stool are not necessarily a good indicator for fermentation and acidity in the more proximal colon, which is very difficult to study in vivo due to its relative inaccessibility. Increasing bifidobacterial counts can be perceived as beneficial, and this saccharolytic genus could be of interest in the prevention of colon carcinogenesis (Buddington et al, 1996). Experimental studies in rat have shown that feeding of lactulose and Bifidobacterium longum singly and in combination had an additive antitumorigenic effect in the rat colon (Challa et al, 1997; Singh et al, 1997). Under our experimental conditions, the partial replacement of other anaerobes with bifidobacteria did not alter the faecal pH, concentrations of bile acids and neutral sterols, which are factors potentially implicated in colon carcinogenesis (Nair, 1988; Owen, 1997). The absence of modification of the microbial transformation of cholesterol and bile by oral lactulose results probably from the low level of the dose, which is the half or the sixth of that used by other authors (Berge Henegouwen et al, 1987; Nagengast et al, 1988). In fact, the effects of the poorly digestible carbohydrates or prebiotics such as lactose, lactulose and amylomaize starch on bile acid transformations depend linearly on the prebiotic dose (Andrieux et al, 1989). However, this low dose of lactulose is nevertheless sufficient stimulate bifidobacteria growth. Using similar experimental conditions as ours, Tuohy et al (2002) showed that faecal water genotoxicity, a putative marker of colon carcinogenesis, was not reduced with lactulose ingestion as compared to the placebo and to the pretreatment samples. We have not measured the effects of lactulose on other parameters potentially implicated in colonic cancer, such as procarcinogenic enzymes (azoreductase, 7 alpha-dehydroxylase, beta-glucuronidase and nitroreductase) and aromatic compounds (phenol, cresol, indole and skatol), but other authors found a modification of these activities in a beneficial way for the host (Ballongue et al, 1997) at a higher dose.

In conclusion, our results taken together with earlier findings strengthened the hypothesis that lactulose, even administered at a low dose of 10 g/day, increases faecal bifidobacterial counts. As these findings have been suggested to be beneficial in healthy humans, lactulose fulfils the criteria request to be considered as a prebiotic.

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