Role of bifidobacteria in the activation of the lignan secoisolariciresinol diglucoside

Lignans are ubiquitous plant polyphenols, which have relevant health properties being the major phytoestrogens occurring in Western diets. Secoisolariciresinol (SECO) is the major dietary lignan mostly found in plants as secoisolariciresinol diglucoside (SDG). To exert biological activity, SDG requires being deglycosylated to SECO and transformed to enterodiol (ED) and enterolactone (EL) by the intestinal microbes. The involvement of bifidobacteria in the transformation of lignans glucosides has been investigated for the first time in this study. Twenty-eight strains were assayed for SDG and SECO activation. They all failed to transform SECO into reduced metabolites, excluding any role in ED and EL production. Ten Bifidobacterium cultures partially hydrolyzed SDG, giving both SECO and the monoglucoside with yields < 25%. When the cell-free extracts were assayed in SDG transformation, seven additional strains were active in the hydrolysis. Cellobiose induced β-glucosidase activity and caused the enhancement of both the rate of SDG hydrolysis and the final yield of SECO only in the strains capable of SDG bioconversion. The highest SDG conversion to SECO was achieved by Bifidobacterium pseudocatenulatum WC 401, which exhibited 75% yield in cellobiose-based medium after 48 h. These results indicate that SDG hydrolysis is not a common feature in Bifidobacterium genus, but selected probiotic strains can be combined to β-glucoside-based prebiotics to enhance the release of SECO, thus improving its bioavailability for absorption by colonic mucosa and/or the biotransformation to ED and EL by other intestinal microorganisms.     

Differences in Composition and Mucosal Adhesion of Bifidobacteria Isolated from Healthy Adults and Healthy Seniors

Fifty-one Bifidobacterium strains were isolated from the feces of healthy adults (30–40 years old) and seniors (older than 70 years of age). B. adolescentis, B. breve, B. infantis, and B. longum were isolated from the healthy adults and B. adolescentis and B. longum from elderly subjects. The tested bacteria bound, in vitro, to intestinal mucus in a strain dependent manner. The strains isolated from healthy adults, and especially B. adolescentis, bound better to intestinal mucus than those isolated from seniors. These results indicate that the mucosal adhesive properties of the human Bifidobacterium flora were reduced with the aging of the host. This shift to a Bifidobacterium flora with reduced adhesive abilities may explain the decrease in bifidobacteria levels in the intestinal microflora of aging people. Received: 7 February 2001 / Accepted: 3 April 2001     

Stereospecific microbial production of isoflavanones from isoflavones and isoflavone glucosides

A Gram-negative anaerobic microorganism, MRG-1, isolated from human intestine showed high activities of deglycosylation and reduction of daidzin, based on rapid TLC analysis. A rod-shaped strain MRG-1was identified as a new species showing 91.0% homology to Coprobacillus species, based on 16S rRNA sequence analysis. The strain MRG-1 showed β-glucosidase activity toward daidzin and genistin, and daidzein and genistein were produced, respectively. However, the strain MRG-1 did not react with flavone glycosides, flavanone glycosides, and isoflavone C-glucoside. Besides, MRG-1 showed stereoselective reductase activity to isoflavone, daidzein, genistein, 7-hydroxyisoflavone, and formononetin, resulting in the formation of corresponding R-isoflavanone enantiomers. The new isoflavanones of 7-hydroxyisoflavanone and dihydroformononetin were characterized by NMR, and the absolute configurations of the enantiomers were determined with CD spectroscopy. The kinetic study of the anaerobic biotransformation showed both activities were exceptionally fast compared to the reported conversion by other anaerobic bacteria.     

Formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempe

Five tempe-derived bacterial strains identified as Micrococcus or Arthrobacter species were shown to transform the soybean isoflavones daidzein and glycitein to polyhydroxylated isoflavones by different hydroxylation reactions. All strains converted glycitein and daidzein to 6,7,4′-trihydroxyisoflavone (factor 2) and the latter substrate also to 7,8,4′-trihydroxyisoflavone. Three strains transformed daidzein to 7,8,3′,4′-tetrahydroxyisoflavone and 6,7,3′,4′-tetrahydroxyisoflavone. In addition, two strains formed 6,7,8,4′-tetrahydroxyisoflavone from daidzein. Conversion of glycitein by these two strains led to the formation of factor 2 and 6,7,3′,4′-tetrahydroxyisoflavone. The structures of these transformation products were elucidated by spectroscopic techniques and chemical degradation. Revision received: 9 September 1995 / Accepted: 21 September 1995     

Oat β-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains

Novel oligomers that resist digestion in the upper gut were prepared from oat mixed-linked β-glucan and xylan by enzymatic hydrolysis with lichenase of Bacillus subtilis and xylanase of Trichoderma reesei respectively. The low-molecular-mass hydrolysis products of β-glucan and xylan were compared with fructooligomers and raffinose in their ability to provide growth substrates for probiotic (Lactobacillus and Bifidobacterium) and intestinal (Bacteroides, Clostridium and Escherichia coli) strains in vitro. A degradation profile of each carbohydrate and total sugar consumption were analysed with HPLC, and bacterial growth rate with an automatic turbidometer, the Bioscreen C system. β-Glucooligomers and xylooligomers both enhanced the growth of health-promoting probiotic strains as compared with intestinal bacterial growth, but not to a significant level. Raffinose stimulated the probiotic strains significantly, whereas fructooligomers induced high average growth for intestinal bacteria also. Received: 16 May 1997 / Received revision: 12 September 1997 / Accepted: 19 September 1997     

Exopolysaccharide production of <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> BCRC 14759 and <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> BCRC 14615

In the present study, the production of exopolysaccharides (EPS) by 13 strains of Lactobacillus and 6 strains of Bifidobacterium in a chemical defined medium (CDM) supplemented with 30 g lactose/l was first compared. The highest EPS production of the Lactobacillus strains was found in L. salivarius BCRC 14759 while among the Bifidobacterium strains examined, B. bifidum BCRC 14615 showed the highest EPS production. Analyzes of the effect of lactose concentration and cultivation temperature on EPS production revealed that L. salivarius produced the highest amount of EPS (45.3 mg/l) in CDM supplemented with 5 g lactose/l at 40°C while B. bifidum produced the highest EPS (17.0 mg/l) in CDM supplemented with 40 g lactose/l at 35°C. α-Phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose-4-epimerase exhibited a markedly notable activity compared with other enzymes examined in the cell extract of both test organisms. This indicates their possible involvement in the biosynthesis of EPS.     

Conditions affecting cell surface properties of human intestinal bifidobacteria

The cell surface properties of human intestinal bifidobacteria have been characterized for 30 strains isolated from a fecal sample. Strain identification to the species level was obtained by restriction analysis of the amplified 16S rRNA gene and confirmed by DNA/DNA reassociation experiments. The isolates were grouped in four genetically homogeneous clusters whose members belonged to Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum species. Cell surface properties of Bifidobacterium strains were evaluated by determining the level of hydrophobicity, adhesion to hydrocarbons and contact angle measurements, and their autoaggregation ability. The results showed high and homogeneous level of hydrophobicity in all tested strains when contact angle measurements values were considered. On the contrary, autoaggregation assays and bacterial adhesion to hydrocarbons detected interesting differences in cell surface properties among the tested Bifidobacterium strains. The highest levels of autoaggregation, detected in B. bifidum and B. adolescentis strains, were strictly dependent on the pH of the medium. Moreover, protease treatment experiments suggested that proteins had a key role in the autoaggregating ability of B. bifidum and B. adolescentis strains.     

Production of phytoestrogen <Emphasis Type="Italic">S</Emphasis>-equol from daidzein in mixed culture of two anaerobic bacteria

An anaerobic incubation mixture of two bacterial strains Eggerthella sp. Julong 732 and Lactobacillus sp. Niu-O16, which have been known to transform dihydrodaidzein to S-equol and daidzein to dihydrodaidzein respectively, produced S-equol from daidzein through dihydrodaidzein. The biotransformation kinetics of daidzein by the mixed cultures showed that the production of S-equol from daidzein was significantly enhanced, as compared to the production of S-equol from dihydrodaidzein by Eggerthella sp. Julong 732 alone. The substrate daidzein in the mixed culture was almost completely converted to S-equol in 24 h of anaerobic incubation. The increased production of S-equol from daidzein by the mixed culture is likely related to the increased bacterial numbers of Eggerthella sp. Julong 732. In the mixture cultures, the growth of Eggerthella sp. Julong 732 was significantly increased while the growth of Lactobacillus sp. Niu-O16 was suppressed as compared to either the single culture of Eggerthella sp. Julong 732 or Lactobacillus sp. Niu-O16. This is the first report in which two metabolic pathways to produce S-equol from daidzein by a mixed culture of bacteria isolated from human and bovine intestinal environments were successfully linked under anaerobic conditions.     

Screening of antioxidative activity of<Emphasis Type="Italic">Bifidobacterium</Emphasis> species isolated from Korean infant feces and their identification

Among 59 Korean isolated, 20 were confirmed as members of the genusBifidobacterium species based on gram staining, microscopic examination of cell morphology and the TLC method. The oxygen tolerance and antioxidative activities of these 20Bifidobacterium strains and 5 standardBifidobacterium strains were tested. All the strains demonstrated antioxidative activities as regards inhibiting linoleic acid peroxidation. The antioxidative activities of isolated and standard strains were found to range from 10.7–46.4% and from 10.7–22.2%, respectively. In addition, all tested strains exhibited a scavenging ability on DPPH free radicals, range from 15–41% for the isolated strains and 8.3–22% for the standard strain. Accordingly, the isolatedBifidobacterium strains demonstrated higher antioxidative activities than the 5 standardBifidobacterium strains. On the base of grades for each test, HJL 7511 was identified as the best strain, followed by HJL 7501. 2 strains were identified with Polymerase Chain Reaction (PCR) assay using group-specific primers designed from the nucleotide sequences of the 16S rRNA and internal transcribed spacer (ITS) regions of the Bifidobacteria. Based on the sequencing results, HJL 7511 and HJL 7501 were identified asBifidobacterium infantis.     

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g⁻¹ dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.     

Production of conjugated linoleic acid and conjugated linolenic acid isomers by Bifidobacterium species

Conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers have attracted great interest because of their potential health benefits. Formation of CLA and CLNA takes place in the rumen during biohydrogenation. Several studies have indicated that certain types of intestinal bacteria, including bifidobacteria, are able to convert linoleic acid (LA) to CLA. The role of intestinal bacteria in the formation of CLNA isomers is largely unknown. In the present study, a screening of 36 different Bifidobacterium strains for their ability to produce CLA and CLNA from free LA and α-linolenic acid (LNA), respectively, was performed. The strains were grown in MRS broth, to which LA or LNA (0.5 mg ml⁻¹) were added after 7 h of bacterial growth. Cultures were further incubated at 37°C for 72 h. Six strains (four Bifidobacterium breve strains, a Bifidobacterium bifidum strain and a Bifidobacterium pseudolongum strain) were able to produce different CLA and CLNA isomers. Conversion percentages varied from 19.5% to 53.5% for CLA production and from 55.6% to 78.4% for CLNA production among these strains. The CLA isomers produced were further identified with Ag⁺-HPLC. LA was mainly converted to t9t11-CLA and c9t11-CLA. The main CLNA isomers were identified with GC-MS as c9t11c15-CLNA and t9t11c15-CLNA.     

Influence of cell surface properties on adhesion ability of bifidobacteria

Adhesion ability of bifidobacteria to the intestinal mucosa is considered to be the prerequisite for colonization of bifidobacteria and can protect against gastrointestinal pathogens infection. The aim of this study was to investigate bifidobacterial surface traits related to adhesion ability in vitro and characterize the cell surface substances that may be involved in the adhesion process of bifidobacteria. Twelve strains of Bifidobacterium spp. were studied for the correlation among their adhesion ability, autoaggregation ability and surface hydrophobicity. The strain that exhibited good adhesion ability also showed high degree of hydrophobicity and strong autoaggregation ability. Pepsin treatment had negative effect on the surface traits and adhesion ability of B. bifidum KLDS2.0603 (P < 0.01), it revealed that hydrophobicity, autoaggregation and adhesion process maybe mediated by proteinaceous components on the surface of cell. Moreover, the adhesion and autoaggregation ability decreased after extraction of B. bifidum KLDS2.0603 with 5 mol l⁻¹ LiCl, and an unreported 50-kDa surface protein which can bind to Caco-2 cell was observed by western blotting. Our results indicated that surface hydrophobicity and autoaggregation ability can be used together for preliminary screening the strains with high adhesion ability, and the present of the surface proteinaceous components would contribute to understand the interactions between bifidobacteria and human intestinal mucosa.     

Effect of the Volume-to-Surface Ratio of Cultures on Escherichia coli Growth: An Experimental and Theoretical Analysis

Daidzein (4′,7-dihydroxyisoflavone), a phytoestrogen found in soybeans mainly in the form of its glycoside daidzin, is metabolized by colonic bacteria to compounds with altered estrogenic activities, which may affect human health. Antibacterial agents used for the treatment of infections can alter the composition of bacterial populations in the colon and therefore can affect daidzein metabolism. To rapidly detect the effects of different concentrations of antibiotics on daidzein metabolism by colonic bacteria of monkeys and identify the subpopulation involved in daidzein metabolism, Etest strips containing antibacterial agents from three classes (tetracyclines, fluoroquinolones, and β-lactams) were used to eliminate the colonic bacteria that were susceptible to 0–32 μg/ml of each antibacterial agent and test the surviving bacteria for their ability to metabolize daidzein. The metabolism of daidzein by the colonic microflora was measured before and after the colonic bacterial population was exposed to antibacterial agents. The metabolites were detected by high performance liquid chromatography and mass spectrometry after incubation of the cultures for various times. Exposure of colonic microflora to antibiotics had various effects on daidzein metabolism. Tetracycline completely removed the bacteria metabolizing daidzein, metabolism of daidzein was not changed in cultures of bacteria after ceftriaxone treatment, and ciprofloxacin enriched for the bacteria metabolizing daidzein. In liquid cultures treated with various concentrations of ciprofloxacin, 4 μg/ml of ciprofloxacin favored the growth of bacteria that metabolized daidzein. This is the first time in which the Etest has been used to show that, whereas some antibiotics eliminate phytoestrogen-metabolizing bacteria in colonic microflora, others enrich them by eliminating the non-metabolizing strains in the population.     

Prevention of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 infection in gnotobiotic mice associated with <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains

Previous reports have shown that Escherichia coli O157:H7 infection is strongly modified by intestinal microbes. In this paper, we examined whether bifidobacteria protect against E. coli O157:H7 infections using gnotobiotic mice di-associated with Bifidobacterium strains (6 species, 9 strains) and E. coli O157:H7. Seven days after oral administration of each Bifidobacterium strain, the mice were orally infected with E. coli O157:H7 and their mortality was examined. Bifidobacterium longum subsp. infantis 157F-4-1 (B. infantis 157F) and B. longum subsp. longum NCC2705 (B. longum NS) protected against the lethal infection, while mice associated with all other Bifidobacterium strains, including type strains of B. longum subsp. infantis and B. longum subsp. longum, died. There were no significant differences in the numbers of E. coli O157:H7 in the faeces among the Bifidobacterium-associated mouse groups. However, the Shiga toxin concentrations in the cecal contents and sera of the GB mice associated with B. infantis 157F and B. longum NS were significantly lower than those of the other groups. However, there were no significant differences in the volatile fatty acid concentrations and histopathological lesions between these two groups. These data suggest that some strains of B. longum subsp. longum/infantis can protect against the lethal infections of E. coli O157:H7 by preventing Shiga toxin production in the cecum and/or Shiga toxin transfer from the intestinal lumen to the bloodstream.     

Changes of Fecal <Emphasis Type="Italic">Bifidobacterium</Emphasis> Species in Adult Patients with Hepatitis B Virus-Induced Chronic Liver Disease

The beneficial effects of Bifidobacteria on health have been widely accepted. Patients with chronic liver disease have varying degrees of intestinal microflora imbalance with a decrease of total Bifidobacterial counts. Since different properties have been attributed to different Bifidobacterium species and there is no information available for the detailed changes in the genus Bifidobacterium in patients with chronic liver disease heretofore, it is meaningful to investigate the structure of this bacterium at the species level in these patients. The aim of this study was to characterize the composition of intestinal Bifidobacterium in patients with hepatitis B virus-induced chronic liver disease. Nested-PCR-based denaturing gradient gel electrophoresis (PCR-DGGE), clone library, and real-time quantitative PCR were performed on the fecal samples of 16 patients with chronic hepatitis B (CHB patients), 16 patients with hepatitis B virus-related cirrhosis (HBV cirrhotics), and 15 healthy subjects (Controls). Though there was no significant difference in the diversity among the three groups (P = 0.196), Bifidobacterium dentium seems to be specifically enhanced in patients as the PCR-DGGE profiles showed, which was further validated by clone library and real-time quantitative PCR. In contrast to the B. dentium, Bifidobacterium catenulatum/Bifidobacterium pseudocatenulatum were detected less frequently in the predominant profile and by quantitative PCR in HBV cirrhotics than in the controls, and the level of this species was also significantly different between these two groups (P = 0.023). Although having no quantitative difference among the three groups, Bifidobacterium longum was less commonly detected in HBV cirrhotics than in CHB patients and Controls by quantitative PCR (P = 0.011). Thus, the composition of intestinal Bifidobacterium was deeply altered in CHB and HBV cirrhotic patients with a shift from beneficial species to opportunistic pathogens. The results provide further insights into the dysbiosis of the intestinal microbiota in patients with hepatitis B virus-induced chronic liver disease and might potentially serve as guidance for the probiotics interventions of these diseases.     

Diversity of Intestinal Bifidobacteria in Patients with Japanese Cedar Pollinosis and Possible Influence of Probiotic Intervention

This study was conducted to evaluate the potential association between intestinal bifidobacteria and Japanese cedar pollinosis (JCPsis) and possible influences of probiotic intervention. In this study, fecal samples were the collected from 29 JCPsis patients. The qualitative and quantitative analyses of fecal bifidobacteria were conducted by quantitative real-time PCR with 16S rRNA-gene-targeted species-specific primers before cedar pollen spread and after a 10-week intervention with fermented milk prepared with Lactobacillus GG and L. gasseri TMC0356 during pollen spread. Each JCPsis patient had a unique diversity of bifidobacteria, which varied qualitatively and quantitatively in an individual-dependent manner during pollen spread. The serum IgE concentration of JCPsis patients with more than 3 detectable Bifidobacterium species was significantly lower than that of patients with less than 2 detected species. The prevalence of B. adolescentis, B. longum, and B. catenulatum increased after probiotic intervention, although the changes were not statistically significant. These results suggest that lower diversity of intestinal Bifidobacterium species might be a pathological aspect of JCPsis. The diversity of intestinal bifidobacteria could be a prospective target for using probiotics in the management of IgE-mediated allergic disorders including JCPsis.     

Biotransformation of soybean isoflavones by a marine <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 060524 and cytotoxicity of the products

A marine Streptomyces sp. 060524 capable of hydrolyzing the glycosidic bond of isoflavone glycosides, was isolated by detecting its β-glucosidase activity. 5 isoflavone aglycones were isolated from culture filtrates in soybean meal glucose medium. They were identified as genistein (1), glycitein (2), daidzein (3), 3′,4′,5,7-tetrahydroxyisoflavone (4), and 3′,4′,7-trihydroxyisoflavone (5), based on UV, NMR and mass spectral analysis. The Streptomyces can selectively hydroxylate at the 3′-position in the daidzein and genistein to generate 3′-hydroxydaidzein and 3′-hydroxygenistein, respectively. The Strain biotransformed more than 90% of soybean isoflavone glycosides into their aglycones within 108 h. 3′-hydroxydaidzein and 3′-hydroxygenistein exhibited stronger cytotoxicity against K562 human chronic leukemia than daidzein and genistein.     

Oral Administration of Live <Emphasis Type="Italic">Bifidobacterium</Emphasis> Substrains Isolated from Centenarians Enhances Intestinal Function in Mice

We studied the effects of two bifidobacteria strains isolated from healthy centenarians on intestinal function in mice. Bifidobacterium adolescentis BBMN23 and Bifidobacterium longum BBMN68 were orally administrated to specific pathogen-free BALB/c mice at different doses (2 × 10¹¹, 2 × 10⁹, or 2 × 10⁷ CFU/kg body weight) each day for 4 weeks. Villus height, crypt depth, villus width, and villus/crypt ratio (V/C) were determined. The content of duodenal secreted immunoglobulin A (sIgA) was also evaluated. There were clear increases in height and width of duodenal villi in both treated groups. Crypt depths were deeper in animals treated with BBMN23 than in controls, while depths were reduced in animals receiving BBMN68. The V/C ratio was increased after feeding with BBMN68, while BBMN23 had no significant effect. Both strains improved the sIgA content of the duodenum. These results suggest that BBMN23 and BBMN68 may improve intestinal digestion and ability and enhance immune barrier function in the intestine.     

Screening ofBifidobacterium strains for bacteriocin production

Summary Thirteen strains of bifidobacteria (Bifidobacterium) were examined for bacteriocin production. One strain among these produced bacteriocin which is active against lactic streptococci strains and against other species ofBifidobacterium, Clostridium andLactobacillus, but not against Gram-negative bacteria such asPseudomonas, Klebsiella, Serratia andEscherichia coli.Bacteriocin activity was inhibited by proteolytic enzymes, but not by heating 15 min or30 min at 100°C, and it was resistant for 15 min at 121°C and active at pH between 2 to 10.     

Characterization of a β-glucosidase from Sulfolobus solfataricus for isoflavone glycosides

The specific activity of a recombinant β-glucosidase from Sulfolobus solfataricus for isoflavones was: daidzin > glycitin > genistin > malonyl genistin > malonyl daidzin > malonyl glycitin. The hydrolytic activity of this enzyme for daidzin was highest at pH 5.5 and 90°C with a half-life of 18 h, a K _m of 0.5 mM, and a k _cat of 2532 s⁻¹. The enzyme converted 1 mM daidzin to 1 mM daidzein after 1 h with a molar yield of 100% and a productivity of 1 mM h⁻¹. Among β-glucosidases, that from S. solfataricus β had the highest thermostability, k _cat, k _cat/K _m, conversion yield, and productivity in the hydrolysis of daidzin.