Technical problems related to in vitro study of colon flora.

The complexity of colon flora and the technical problems encountered in the sampling techniques and their processing limit the study of its composition and activities. First, we list the main limitations related to the sampling procedure-transport and storage. We show that (i) use of a cryoprotective medium is necessary for sample storage and (ii) that storage has to be at -40 degrees C. Second, bacterial analysis and enzymatic activities are examined. The lack of specificity of the culture media generally used means that systematic studies are difficult to carry out and that bacterial identification at species level requires genetic techniques. Third, we show that activities of procarcinogenic enzymes are significantly affected by any kind of storage. Finally, for statistics, the problem of the size and the nutritional habits of the studied population is examined.

Effects of lactulose and lactitol on colonic microflora and enzymatic activity.

The effects of lactulose and lactitol (2 x 10 g/d) were studied in 36 healthy volunteers in comparison to placebo. All parameters studied were affected by both treatments, lactulose in general leading to more pronounced changes compared to lactitol. Probiotic bacteria were increased, and putrefactive bacteria and potential pathogens were significantly reduced. These variations in colonic flora had the following consequences: (i) a reduced activity of pro-carcinogenic enzymes: azoreductase, 7 alpha-dehydroxylase, beta-glucuronidase, nitroreductase and urease activity; (ii) a global increase of short-chain fatty acids in faeces; (iii) an effect on pH and moisture of faeces, and (iv) also on aromatic compounds such as phenol, cresol, indole and skatol. The findings suggest that lactulose and lactitol are not comparable in their effect on the colonic microflora, its metabolism, and the consequent probiotic effects on human health. The differences found may also be of clinical relevance suggesting that neither compound is equipotent.

Adhesion of different bifidobacteria strains to human enterocyte-like Caco-2 cells and comparison with in vivo study.

The validity of the in vitro adhesion tests performed with cultured cell lines, was determined in this study by comparison with results obtained in vivo, in a previous study. To make this experiment the in vitro adhesion tests were performed during a long period by utilization of an appropriate medium, to determine the capacity of the adhered strain to colonize the intestinal tract. It was demonstrated that the ability of the strain to adhere and colonize the intestinal cell in vivo or the cultured intestinal cells in intro was similar.

Effects of three strains of bifidobacteria on cholesterol.

To determine the validity of the hypothesis of assimilation or precipitation of cholesterol by Bifidobacterium species, resting cell assays and cultures were undertaken in TPY medium containing oxgall. With resting cell assays (pH 5), cholesterol was precipitated and redissolved in phosphate buffer (pH 7). At the end of the cultures, only part of the removed cholesterol from the culture medium was found in the phosphate buffer, while the missing cholesterol was in cell extracts. It appeared that removal of cholesterol during culturing was not solely due to its precipitation. It is concluded that growing bifidobacteria cells are able to remove cholesterol both by precipitation and assimilation.

Bifidobacteria and probiotic effects: action of Bifidobacterium species on conjugated bile salts.

The effect of six different conjugated bile salts (two trihydroxyconjugated bile salts: tauro and glycocholic acids; and four dihydroxyconjugated bile salts: tauro- and glycochenodeoxycholic, tauro- and glycodeoxycholic acids) on eight bifidobacteria strains were studied. A strong growth-inhibitory effect was observed (80% at 0.95 mM) for each bile salt and strain. This phenomenon was explained by the production of deconjugated bile salt during bifidobacteria growth. The deconjugation phenomenon was concurrent with biomass production, and deconjugated bile salts were the sole compound produced during bifidobacteria biotransformation. In resting cell experiments, differences appeared between the strains and the kind of bile salts, particularly concerning taurocholic acid. The Bifidobacterium longum strains were the most efficient among the bacteria tested.

Characterization of fructose 6 phosphate phosphoketolases purified from Bifidobacterium species.

Fructose 6 phosphate phosphoketolases (F6PPKs) were purified from Bifidobacterium longum BB536, B. dentium ATCC 27534, B. globosum ATCC 25864, and Bifidobacterium animalis ATCC 25527. Concerning ions (Cu++, Zn++, Ca++, Mg++, Fe++, Co++, Mn++) and common enzyme inhibitors (fructose, ammonium sulfate, iodoacetate, and parachloromercuribenzoic acid), no difference appeared between the enzymes. Cu++, parachloromercuribenzoic acid (pCMB), and mercuric acetate induced high enzymatic inhibition. The study of pCMB demonstrated a noncompetitive inhibition. Additional results showed that the sulfhydryl group was not involved in catalytic reaction. Photooxidation experiments and determination of ionizable group pKas (5.16-7.17) suggested the presence of one or more histidines necessary for the catalytic reaction and explained the inhibition observed with pCMB. In light of the noncompetitive inhibition, this group was not directly involved in substrate binding. Determination of Km demonstrated that the affinities for fructose 6 phosphate in the case of animal and human origin strains were close. In addition, the same enzymatic efficiency (Kcat/Km) was obtained for each strain. The F6PPK activity was regulated by sodium pyrophosphate, ATP, and especially by ADP.

Purification and Characterization of Conjugated Bile Salt Hydrolase from Bifidobacterium longum BB536.

Bifidobacterium species deconjugate taurocholic, taurodeoxycholic, taurochenodeoxycholic, glycocholic, glycodeoxycholic, and glycochenodeoxycholic acids. The enzyme level increases in the growth phase. No increase in activity is observed for the cytoplasmic enzyme after addition of conjugated bile acids to a stationary-phase culture. Conjugated bile salt hydrolase (BSH) was purified from Bifidobacterium longum BB536. Its apparent molecular mass in denaturing polyacrylamide gel electrophoresis was ca. 40,000 Da. The intact enzyme had a relative molecular weight of ca. 250,000 as determined by gel filtration chromatography, suggesting that the native BSH of B. longum is probably a hexamer. The purified enzyme is active towards both glycine and taurine conjugates of cholate, deoxycholate, and chenodeoxycholate. The pH optimum is in the range of 5.5 to 6.5. A loss of BSH activity is observed after incubation at temperatures higher than 42(deg)C; at 60(deg)C, 50% of the BSH activity is lost. The importance of free sulfhydryl groups at the enzyme active center is suggested. For B. longum BB536, no significant difference in the initial rate of deconjugation and enzymatic efficiency appears between bile salts. The enzymatic efficiency is higher for B. longum BB536 than for other genera. In this paper, a new method which permits a display of BSH activity directly on polyacrylamide gels is described; this method confirms the molecular weight obtained for B. longum BB536 BSH.

Effect of bifidobacteria on nitrites and nitrosamines.

The effects of six different bifidobacteria strains were studied on two procarcinogens: nitrite and nitrosamines. Growth of bifidobacteria was not affected by nitrite concentrations below 50 mumol l-1. At nitrite concentrations greater than 2000 mumol l-1, total growth inhibition was observed. Nitrite elimination by a non-enzymic mechanism was noted for six strains of bifidobacteria. Acids produced by the bacteria seem to be involved in nitrite elimination. Nitrosamines tested had no effect on growth of bifidobacteria. Only one strain (Bifidobacterium longum BB 536) was able to metabolize nitrosamines by an intracellular mechanism.

[Role of acetate and butyrate in the induction of NADH: rubredoxin oxidoreductase in Clostridium acetobutylicum]. / Rôle de l'acétate et du butyrate dans l'induction de la NADH: rubrédoxine oxydoréductase chez Clostridium acetobutylicum.

Study of the biosynthesis of NADH: rubredoxin oxidoreductase in resting cells of Clostridium acetobutylicum shows that this enzyme is synthesized at a maximal rate in the presence of acetic acid at a concentration of 3 g . l-1 and at pH 4.8. Protons do not play any role in this biosynthesis since no induction is observed in a medium without acetate for the same values of pH. Butyric acid at a concentration of 0.5 g . l-1 gives 50% induction and formic acid, isobutyric acid and propionic acid have no inductive action on NADH: rubredoxin oxidoreductase. These results are confirmed by studies using a dialysis bag. Only a culture against acetic acid at an initial concentration of 2 g . l-1 gives maximal biosynthesis of the enzyme, whereas a culture in which all products of metabolism are eliminated gives an activity which is 80% lower.

Differential levels of ferredoxin and rubredoxin in Clostridium acetobutylicum.

Ferredoxin and rubredoxin levels have been determined in DEAE-cellulose treated extracts of Clostridium acetobutylicum using specific enzymatic assays. In contrast to ferredoxin, the content of rubredoxin is affected by various culture conditions; it fluctuates in the proportions of 1 to 3 according to the growth phase, 1 to 8 according to the medium composition, and 1 to 40 according to the pH. Highest rubredoxin level is obtained at the end of the acid phase when the cells grow in chemically defined medium, the pH of which is not controlled. Such variations suggest that rubredoxin as well as rubredoxin-reductase take part in an electron transport chain system inducible by the culture conditions.

[Effect of growth phase and composition of the medium on the rate of biosynthesis of NADH: rubredoxin oxidoreductase in Clostridium acetobutylicum]. / Influence de la phase de croissance et de la composition du milieu sur le taux de biosynthèse de la NADH: rubrédoxine oxydoréductase chez Clostridium acetobutylicum.

Among the three strains of Clostridium acetobutylicum we investigated: NRRL 592, NCIB 619 and ATCC 824, only the last was shown to contain a NADH: rubredoxin oxidoreductase activity. We report that the biosynthesis rate of this enzyme fluctuated in the proportions of 1 to 50 according to the growth phase and medium composition. These variations reflect a mode of regulation adjusted to the metabolism of bacteria.