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Unité d
Ecologie et de Physiologie du Système Digestif, Équipe Métabolites Bactériens et Santé, INRA, 78352 Jouy en Josas Cedex, France and * CIRDC, Danone, 92350 Le Plessis-Robinson, France
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
The aim of this study was to compare the effects of milk and of various fermented milks on the composition and metabolic activities of the intestinal microflora. Groups of eight rats were fed for 6 wk a diet containing 30% nonfermented milk (M), yogurt (Y), milk fermented with Lactobacillus casei (LcFM) or milk fermented with the association of L. casei DN 114.001 and yogurt starters (LcYFM). In the first study, the survival of the lactic acid bacteria from the fermented milks was assessed by bacterial enumeration in feces of germ-free rats (GF rats) fed milk or fermented milks. The metabolic activities of the lactic acid bacteria were studied in these rats by the measurement of glycolytic activities and products of bacterial fermentation, i.e., acetate and lactate (isoforms L and D). In a second study, the effects of fermented milks on the composition and metabolism [gas, glycolytic activities, short-chain fatty acids (SCFA), alcohol and ammonia] of human flora were studied using human flora-associated rats (HF rats). In GF rats, the survival of L. casei in the feces did not differ between those fed the LcFM and LcYFM diets. L. bulgaricus was detected in the feces of the rats fed Y, whereas Streptoccus thermophilus was found in the feces of the LcYFM group. In HF rats, fecal concentration of Bifidobacteria was greater in the LcFM group than in the others. 
-Glucuronidase (EC 3.2.1.31) activity was lower in rats fed LcFM and Y than in those fed M and LcYFM, whereas -galactosidase (3.2.1.23), 
-glucosidase (EC 3.2.1 20) and -glucosidase (EC 3.2.1.21) activities were higher in the LcYFM group compared with the others. Methane excretion was higher in rats fed Y than in other groups. Cecal SCFA concentrations did not differ in LcFM, Y and M groups, but total SCFA, acetate, propionate and butyrate were significantly greater in the LcYFM group. These results suggest that milk fermented with the combination of L. casei and yogurt starters leads to specific effects that are different from the simple addition of the effects found with yogurt and milk fermented with L. casei. These specific effects are potentially beneficial to human health.
INTRODUCTION
The intestinal microflora is a complex ecosystem composed of a large variety of bacteria. The metabolic capacity of the flora is extremely diverse and can produce both positive and negative effects on the gut physiology (Gorbach 1986
, Macfarlane and Cummings 1991). There is therefore great interest in the possibility of altering the intestinal microbiota in a beneficial way with the goal of improving the health of the host. Lactic acid bacteria have been considered potentially useful in this respect (Sanders 1993).
Lactic acid bacteria have been used traditionally in food fermentation for thousands of years. In fermented milks, these bacteria produce lactic acid as the primary end-product, along with acetic acid and ethanol, in the case of heterofermentative species. They also produce acetaldehyde, peptoglycan, peptides, vitamins and antimicrobial substances that contribute to the taste, texture, and potential health benefits of the products (Hartley and Denariaz 1993). Yogurt remains a staple food and is fermented using a combination of Lactobacillus bulgaricus and Streptococcus thermophilus. Other lactic acid bacteria are often combined with yogurt starters to produce fermented milks with specific desirable characteristics related to flavor or health properties (Robinson 1991).
Upon consumption, fermented milks deliver a large number of lactic acid bacteria into the gastrointestinal tract. These transiting microorganisms are capable of partially resisting gastric and bile acids and can therefore deliver enzymes and other substances into the intestines (Marteau and Rambaud 1993). Yogurt's ability to provide -galactosidase and to decrease lactose intolerance symptoms in lactose maldigesters has been frequently reported (Marteau and Rambaud 1993, Sanders 1993).
Lactic acid bacteria have also been reputed to modify the intestinal milieu. Some studies have shown that L. acidophilus, L. casei and Bifidobacterium bifidum can modify potentially harmful bacterial activities such as those of -glucuronidase and nitroreductase (Goldin and Gorbach 1984, Marteau et al. 1990). Few reports exist, however, that investigate the ability of lactic acid bacteria to alter the main fermentative processes of endogenous microflora. One recent study in humans showed that yogurt with or without B. longum did not modify fecal short-chain fatty acid (SCFA)3 concentrations (Bartram et al. 1994).
An in vitro model simulating the various compartments of the gastrointestinal tract recently demonstrated that the survival rate of lactic acid bacteria in the gut varied, depending on whether the bacteria were associated with other bacteria or were tested alone (Havenaar et al. 1994). Thus, the ability of the lactic acid bacteria in fermented milks to have an effect on the intestinal tract in vivo may depend on their combination in the product.
The objective of this study was to compare the influence of the consumption of milk fermented with L. casei and yogurt starters (LcYFM) to those of yogurt (Y), milk fermented with only L. casei (LcFM) and nonfermented milk (M) on the composition and metabolic activities of the intestinal microflora. Two experiments were performed successively. In the first study, germ-free (GF) rats were used to evaluate both the survival of ingested bacteria through the gastrointestinal tract and their metabolic profile in vivo. In the second study, rats born germ-free and inoculated with human intestinal microflora (HF rats) were used to determine the effects of the ingested fermented milks on the composition and metabolism of the intestinal microbiota.
MATERIALS AND METHODS
2 dilution of fresh human feces from a methane producer (HF rats). Fecal dilution was performed in an anaerobic cabinet (N2/H2/CO2, 85:10:5). All procedures were conducted in accordance with the institute's guide for the care and use of laboratory animals.
Experimental design.
For 1 wk before the experiments, rats were given a human-like diet with the following composition (g/kg): mashed potatoes, 460; fish meal, 230; cellulose, 50; corn oil, 40; lard, 100; sucrose, 100; cholesterol, 0.15; and mineral and vitamin mixture, 20 (Andrieux and Sacquet 1986). The diet was sterilized by gamma irradiation at 45 kGy in plastic vacuum bags. When experiments started, GF or HF rats were assigned to four groups of eight and were transferred into sterile isolators, one isolator for each group. Rats were then given free access to diet offered as a paste containing 70% of powdered human-like diet that contain 30% M or one of three fermented milks, Y, LcFM or LcYFM. Milk and fermented milks were obtained from Danone (CIRDC, Le Plessis-Robinson, France). The products were conditioned in sterile pots and sealed with a double cover. The pots were provided on a daily basis to the isolators through a lock sterilized by peracetic acid (100 g/L). Milk and fermented milks contained the following (g/kg): protein, 3.7 and fat, 3.3. The fermented milks contained 5.2 g/kg and milk 12.7 g/kg of carbohydrates. The lactic acid bacteria composition of fermented milks is listed in Table 1.
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Table 1. Concentration of lactic acid bacteria in fermented milks, diets and in feces of germ-free rats fed the fermented milks diets1,2 |
). After a 6-wk period, both GF and HF rats were killed by ether anesthesia, and their ceca were removed and weighed. Cecal pH was measured, and the contents were immediately frozen in liquid N2 and stored at 
80oC until analyzed for enzymatic activities and bacterial metabolites.
Analyses.
Lactic acid bacteria were analyzed in triplicate; three fecal samples from each GF rat were diluted from 106 to 109 in LCY medium4 and then plated (0.1 mL) on selective agars. For L. bulgaricus, MRS (Man Rogosa and Sharpe) medium was incubated anaerobically at 44oC for 72 h. MRS with bactoxgall was used for L. casei, and M17 medium was used for S. thermophilus; plates for both were incubated aerobically at 37oC for 72 h. After incubation, colonies were counted and observed microscopically. Fecal samples of HF rats were diluted to 109 in an anaerobic Freter's chamber in the prereduced liquid medium LCY. Dilutions of 107, 108 and 109 were plated (0.1 mL) on the following selective agars: BHI (brain heart infusion) neomycin medium for Bacteroides, Beerens medium for Bifidobacteria, GAPTS1 with azide for Enterococci, and DCA (desoxycholate acid) medium for enterobacteria. Plates containing BHI or Beerens medium were incubated at 37oC for 48 h in an anaerobic chamber, and those containing GAPTS1 and DCA were incubated aerobically at 37oC for 48 h. Cultures were prepared in triplicate. Colonies were counted and bacteria were observed microscopically.
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Table 2. Cecal weight, pH, and bacterial metabolites in the cecal contents of germ-free rats fed milk or fermented milks1 |
-galactosidase EC 3.2.1.23 and -glucosidase EC 3.2.1.20) or were related to the release and enterohepatic recirculation of toxic substances (-glucosidase EC 3.2.1.21 and -glucuronidase EC 3.2.1.31). Enzyme assays were performed in an anaerobic chamber in 2-mL Eppendorf tubes. Glycolytic activity was measured by the rate of release of p-nitrophenols from p-nitrophenylglucoside. The reaction mixture contained 0.1 mL of a 5 mmol/L substrate solution and 0.2 mL of a 1:20 (v/v) dilution of the cecal sample in 0.1 mol/L phosphate buffer at pH 6.4. Incubation was at 37oC, and paranitrophenol concentration was measured according to the optical absorbency at 400 nm after the addition of 1.6 mL of 0.25 mol/L sodium carbonate. Enzyme activity was expressed as micromoles of product hydrolyzed per minute per gram of cecal sample.
Isle dAbeau Chesnes, France) impregnated with SP 1000. Carrier gas (He) flow-rate was 10 mL/min, inlet temperature 175oC, column termperature 100oC, detector temperature 280oC, hydrogen and compressed air flow-rate 40 mL/min; 2ethylbutytrate was used as an internal standard.
RESULTS
-glucosidase and -galactosidase were detected. In the Y group, -galactosidase activity was significantly higher than in the M group, and -glucuronidase was detected (Table 3). In rats fed the LcFM diet, -galactosidase activity was as low as in the group fed the M diet, -glucuronidase activity was not different from rats fed the Y diet and -glucosidase was detected. In the LcYFM group, -galactosidase, -glucosidase and -glucuronidase activities did not differ from those in rats fed the Y diet, whereas -glucosidase activity was not different from that in rats µ fed the LcFM diet.
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Table 3. Bacterial glycolytic activities in the cecal content of germ-free rats fed milk or fermented milks1 |
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Table 4. Composition of the fecal microflora in human flora-associated rats fed milk or fermented milks diets1 |
-galactosidase, -glucosidase and -glucosidase were significantly higher in the LcYFM group than in all other groups. -Glucuronidase activity was significantly lower in rats fed the Y and LcFM diets than in those fed M or LcYFM.
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Table 5. pH and cecal concentrations of short-chain fatty acids (SCFA), L- and D-lactate, ethanol and ammonia in human flora-associated rats fed milk or fermented milks diets1 |
DISCUSSION
). With the use of an in vitro model, Havenaar et al. (1994) demonstrated that different strains of L. casei were able to survive transit through the gastrointestinal tract and reach the ileum in quantities sufficient to exert a physiologic effect.
).
).
, Ling et al. 1992, Saxelin et al. 1991). The ability of other lactic acid bacteria to survive is less clear. Bianchi Salvadori et al. (1978) found live L. bulgaricus and S. thermophilus in the feces of humans after yogurt consumption, whereas Pedrosa et al. (1995) did not. Hargrove and Alford (1978) found that in conventional rats, L. bulgaricus was not always present and S. thermophilus never survived beyond the upper small intestine.
). The specific changes observed in this study in the endogenous bacterial population and in bacterial metabolism of HF rats suggest that the lactic acid bacteria modified the intestinal medium in specific ways. We found that LcFM significantly increased the amount of endogenous Bifidobacterium in the feces. Similar effects have been described in humans (Hayatsu and Hayatsu 1993, Saxelin et al. 1991, Sepp et al. 1993). This may be beneficial because Bifidobacteria have been associated with many health and nutritional benefits (Ballongue 1993). The low cecal pH observed in the LcFM group may have been due to the production of formic acid by Bifidobacteria, and not due to the survival of L. casei because lactate and acetate, the main products of L. casei, were not enhanced in cecal contents.
-glucuronidase activity, which has been implicated in colon carcinogenesis (Goldin and Gorbach 1984). Other reports have noted a decrease in -glucuronidase activity in rats (Goldin and Gorbach 1984) and in humans (Ling et al. 1994) during ingestion of L. casei.
-glucuronidase activity may be related to a change in intestinal pH and/or to a modification in the composition of the intestinal flora. In our experiment, -glucuronidase activity was lower in both LcFM and Y groups than in the M group; however, although the LcFM diet led to a decrease in pH and a change in Bifidobacterium population, the Y diet showed no influence on cecal pH or the composition of the endogenous intestinal microflora. L. bulgaricus was found in the feces of GF rats fed Y, and L. casei in those fed LcFM diet, and both bacteria had similar low levels of -glucuronidase but different levels of -galactosidase and fermentative metabolites, as shown in Table 3. These results suggest that lactic acid bacteria can potentially modify deleterious bacterial activity such as that of -glucuronidase by more than one mechanism, depending on the species and strains present in the fermented milk. These mechanisms remain to be found.
-glucuronidase activity was modified, and the amount of Bifidobacterium tended to be higher in only 50% of rats in this group. The particular effect of this new product was an increase in the activities of -galactosidase and - and -glucosidase. In addition, there was a concomitant increase in total and specific SCFA concentrations.
-galactosidase producers and are able to hydrolyze carbohydrates other than lactose; they might provide glycolytic enzymes in the intestinal tract. Glycolytic activities, however, were low in GF rats compared with HF rats. Morevoer, the increase in acetate, propionate and butyrate in the ceca of HF rats fed the LcYFM diet without a concomitant change in lactate concentration is not characteristic of lactic acid bacterial metabolism. Thus, endogenous bacteria clearly are implicated in the glycolytic enzyme induction and fermentative process.
). In addition, the enhanced -galactosidase and -glucosidase activities found with the LcYFM diet offer a number of advantages for the host. The importance of -galactosidase in improving lactose digestion is well recognized, and -glucosidase may improve fermentation of resistant starch, which leads to butyrate production, improved bowel habits and increased stool output (Macfarlane and Cummings 1991). On the other hand, the increase in -glucosidase activity is less clear because this activity has been implicated in both the generation of toxins (Mallett and Rowland, 1988) and of bacterial derivatives, which might be protective against chemically induced cancer (Roland et al. 1993).
ACKNOWLEDGMENTS
FOOTNOTES
). GAPTS1 medium: yeast extract (Difco), 10; peptone, 15.0; tryptone, 10.0; sucrose, 1.0; sodium azide, 3.0 (pH 7.5). DCA agar medium: peptone, 10.0; lactose (Sigma, LIsle dAbeau Chesnes, France), 10.0; sodium deoxycholate (Sigma), 5.0; NaCl, 5.0; Na2HPO4, 2.0; iron citrate, 1.0; sodium citrate, 1.0; neutral red, 0.03.
Manuscript received 3 March 1997. Initial reviews completed 15 April 1997. Revision accepted 26 June 1997.
LITERATURE CITED
ingestion de yogurt sur lactivité lactasique intestinale chez des souris axéniques ou holoxéniques. Ann. Microbiol. 134A: 219-230.administration des cellules lactiques du yaourt. Lait 571/572: 17-42.une chambre respiratoire connectée sur lisolateur pour la mesure in vivo des gaz de fermentation chez lanimal gnotoxénique.
Sci. Technol. Anim. Lab.
1989;
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