A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria

Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.     

Identification and characterization of the serine/threonine protein kinases in Bifidobacterium

Six genes encoding the bifidobacterial Hanks-type (eukaryote-like) serine/threonine protein kinases (STPK) were identified and classified. The genome of each bifidobacterial strain contains four conserved genes and one species-specific gene. Bifidobacterium longum and Bifidobacterium bifidum possess the unique gene found only in these species. The STPK genes of Russian industrial probiotic strain B. longum B379M were cloned and sequenced. The expression of these genes in Escherichia coli and bifidobacteria was observed. Autophosphorylation of the conserved STPK Pkb5 and species-specific STPK Pkb2 was demonstrated. This is the first report on Hanks-type STPK in bifidobacteria.     

Live and heat-inactivated lactobacilli from feces inhibit <Emphasis Type="Italic">Salmonella typhi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> adherence to caco-2 cells

A quantitative approach has been proposed to evaluate the competitive inhibition of Escherichia coli and Salmonella typhi by live and heat-inactivated laboratory isolated Lactobacillus sp. on adhesion to monolayer of Caco-2 cells. Three species of Lactobacillus (L. casei, L. acidophilus, L. agilis) isolated from human neonate feces and two commercial probiotic strains (L. casei, L. acidophilus) have been compared for probiotic activity. All lactobacilli were able to attach to the Caco-2 cells, however, the degree of adhesion was bacterial strain-dependent. The adhesion indices of the two commercial probiotic strains were not significantly different from the values obtained for the other two similar fecal strains (p > 0.01). The inhibition of attachment of the pathogenic bacteria by inactivated cells of fecal L. acidophilus was examined and compared to the results of live bacteria. The inhibition pattern was similar for live and heat-inactivated L. acidophilus (p > 0.01). The number of attached pathogenic bacteria to the Caco-2 cells decreased when the number of L. acidophilus increased from 10⁶ to 10⁹ CFU/mL. The heat-inactivated L. acidophilus displayed similar probiotic activity compared to the live bacteria.     

Effect of Encapsulation on Viability of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> CFR815j and Physiochemical Properties of Ice Cream

The health beneficial attributes of bifidobacteria and its safe association with the host gut has increased its significance as a probiotic. However delivering probiotic bifidobacteria with Minimum Biological Value (MBV) through product has always been a challenge. In the present study, an attempt was made to maintain the viability of native isolate of Bifidobacterium longum CFR 815j and deliver through ice-cream. B. longum CFR815j was microencapsulated in alginate starch capsules by emulsification followed by evaluation of bead stability in simulated gastrointestinal conditions. After incorporation in ice-cream, the effect on chemical properties, sensory parameters and meltdown characteristics of the product were also evaluated. Survival studies of B. longum revealed higher counts than 10⁷ in the product which is essential for probiotic bacteria to exhibit beneficial effect. Further, all the properties of this ice-cream were comparable to the regular ice-cream. Our studies conclude that encapsulation was able to maintain the requisite MBV of bifidobacteria in ice-cream without affecting the sensory characteristics.     

Proteomic comparison of the cytosolic proteins of three <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> human isolates and <Emphasis Type="Italic">B. longum</Emphasis> NCC2705

Background  

Bifidobacteria are natural inhabitants of the human gastrointestinal tract. In full-term newborns, these bacteria are acquired from the mother during delivery and rapidly become the predominant organisms in the intestinal microbiota. Bifidobacteria contribute to the establishment of healthy intestinal ecology and can confer health benefits to their host. Consequently, there is growing interest in bifidobacteria, and various strains are currently used as probiotic components in functional food products. However, the probiotic effects have been reported to be strain-specific. There is thus a need to better understand the determinants of the observed benefits provided by these probiotics. Our objective was to compare three human B. longum isolates with the sequenced model strain B. longum NCC2705 at the chromosome and proteome levels.     

Inhibitory Impact of Bifidobacteria on the Transfer of β-Lactam Resistance among Enterobacteriaceae in the Gnotobiotic Mouse Digestive Tract

While looking for new means to limit the dissemination of antibiotic resistance, we evaluated the role of potentially probiotic bifidobacteria on the transfer of resistance genes between enterobacteria. Transfers of bla genes encoding extended-spectrum β-lactamases (SHV-5 and CTX-M-15) were studied in the absence or presence of bifidobacteria. In vitro, transfer frequencies of these bla genes decreased significantly in the presence of three of five tested strains, i.e., Bifidobacterium longum CUETM-89-215, Bifidobacterium bifidum CIP-56.7T, and Bifidobacterium pseudocatenulatum CIP-104168T. Four transfer experiments were conducted in the digestive tract of gnotobiotic mice, the first three observing the effect of B. longum CUETM-89-215, B. bifidum CIP-56.7T, and B. pseudocatenulatum CIP-104168T on bla_SHV-5 transfer and the fourth experiment studying the effect of B. bifidum CIP-56.7T on bla_CTX-M-15 transfer. These experiments revealed significant decreases in the transconjugant levels (up to 3 logs) in mice having received B. bifidum CIP-56.7T or B. pseudocatenulatum CIP-104168T compared to control mice. Bifidobacteria appear to have an inhibitory impact on the transfer of antibiotic resistance genes. The inhibitory effect is associated to specific bifidobacterial strains and may be related to the production of thermostable metabolites by these strains.     

Effect ofKluyveromyces marxianus on the growth and survival of bifidobacteria in milk

Four strains of bifidobacteria (B. bifidum 93,B. infantis ATCC 17 930,B. longum ATCC 15 707, andB. longum JR) cultivated in MRS broth modified by the addition of cysteine-hydrochloride (0.05 %) were serially subcultured in unsupplemented cow milk alone or in combination withKluyveromyces marxianus var.marxianus 269 under aerobic conditions. In monoculture, bifidobacteria did not multiply after the second subculture. In contrast, in coculture with yeast bifidobacteria reached counts about 8 log CFU/g even during 15 subcultures. In addition,K. marxianus var.marxianus 269 significantly prolonged the survival of bifidobacteria in milk at 4 °C. The most susceptible strains (B. longum Jr andB. infantis ATCC 17 930) completely lost their viability within 5 and 12 d of storage, respectively, while in coculture with yeasts all bifidobacteria cultures tested survived for at least 40 d. The results could be useful in producing kefir-like fermented milks containing bifidobacteria. The study was supported by grant no. 238/10/12596/0 of theInternal Grant Agency of Czech University of Agriculture ion Prague.     

Cholesterol-lowering probiotics: in vitro selection and in vivo testing of bifidobacteria

Thirty-four strains of bifidobacteria belonging to Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium pseu-docatenulatum were assayed in vitro for the ability to assimilate cholesterol and for bile salt hydrolase (BSH) against glycocholic and taurodeoxycholic acids (GCA and TDCA). Cholesterol assimilation was peculiar characteristic of two strains belonging to the species B. bifidum (B. bifidum MB 107 and B. bifidum MB 109), which removed 81 and 50 mg of cholesterol per gram of biomass, being the median of specific cholesterol absorption by bifidobacteria 19 mg/g. Significant differences in BSH activities were not established among bifidobacterial species. However, the screening resulted in the selection of promising strains able to efficiently deconjugate GCA and TDCA. No relationship was recognized between BSH phenotype and the extent of cholesterol assimilation. On the basis of cholesterol assimilation or BSH_GCA and BSH_TDCA activities, B. bifidum MB 109 (DSMZ 23731), B. breve MB 113 (DSMZ 23732), and B. animalis subsp. lactis MB 2409 (DSMZ 23733) were combined in a probiotic mixture to be fed to hypercholesterolemic rats. The administration of this probiotic formulation resulted in a significant reduction of total cholesterol and low-density cholesterol (LDL-C), whereas it did not affect high-density cholesterol (HDL-C) and HDL-C/LDL-C ratio.     

Effect of Milk Whey and Its Fermentation Products by Lactic Acid Bacteria on Mitochondrial Lipid Peroxide and Hepatic Injury in Bile Duct-ligated Rats

The study was carried out to assess whether bovine milk whey and its products fermented by lactic acid bacteria could ameliorate the lipid peroxidation of hepatic mitochondria associated with cholestatic liver injury due to bile duct ligation. Rats were maintained on one of five diets for 3 weeks before being operated upon and killed 3 weeks after bile duct ligation. The diets included one deficient in vitamin E (control diet) and others supplemented with either 5% milk whey or 5% milk whey fermented with Bifidobacterium longum (B. longum), Lactobacillus acidophilus (L. acidophilus), and Streptococcus salivarius subsp. thermophillus (S. thermophillus). Bile duct-ligated rats, compared with sham-operated rats, had higher organ weights (liver and spleen), higher serum alkaline phosphatase activity, higher serum bilirubin concentration, and higher content of hepatic mitochondrial lipid hydroperoxide. The rats fed on diets containing milk whey fermented with B. longum ameliorated the elevation of organ weights, enzyme activity, bilirubin concentration, and content of mitochondrial lipid hydroperoxide. Milk whey and milk whey fermented with L. acidophilus and S. thermophillus also suppressed the elevation of mitochondrial lipid hydroperoxide, but had no ameliorating effects on organ weights, enzyme activity, and bilirubin concentration. The elevation of serum lipid hydroperoxide was ameliorated in rats fed on diets containing milk whey and milk whey fermented with B. longum and S. thermophillus. The reduction in plasma α-tocopherol due to bile duct ligation was ameliorated in those rats fed on diets containing milk whey fermented with B. longum as well as by S. themophillus. These results suggest that a milk whey fermented with lactic acid bacteria exerts a beneficial effect on free radical-mediated hepatic injury.     

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine metabolism in <Emphasis Type="Italic">Bifidobacterium</Emphasis>

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism. S. Foley and E. Stolarczyk contributed equally to this work     

Bacteria competing with the adhesion and biofilm formation by <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The activity of antagonistic substances produced by Pseudomonas aeruginosa and Lactobacillus acidophilus against the planktonic and sessile populations of Staphylococcus aureus strains was demonstrated. The strongest effects were caused by probiotic L. acidophilus strain — bacteriocin-like inhibitory substances (BLIS) positive. However, the S. aureus A3 growth, adhesion and biofilm formation was also limited by cell-free supernatant of L. acidophilus H-1 (BLIS negative). Moreover, competitive direct interactions were observed between staphylococci and the above bacteria, which influenced the formation of dualspecies aggregates on the surface.     

Growth-promoting Effects of N-Acetylneuraminic Acid-containing Substances on Bifidobacteria

The use of N-acetylneuraminic acid, sialyl-lactose, and glyco-macropeptide by bifidobacteria and lactobacilli, and their growth-promoting effects on B. longum, B. breve, B. bifidum, and B. infantis were investigated. The data presented here suggest that fortification with N-acetylneuraminic acid-containing substances of infant formula may provide formula-fed infants with a function that human milk possesses.     

Immune Response of Mice to Orally Administered Lactic Acid Bacteria

In order to elucidate the interaction of lactic acid bacteria with the immune system, immune responses to the lactic acid bacteria, Bifidobacterium longum and Lactobacillus acidophilus, were examined in mice fed with each organism. In mice fed with B. longum for more than 8 weeks, an antibody response was detected to the cytoplasm of B. longum, but not to the cell wall. On the other hand, in mice fed with L. acidophilus for more than 6 weeks, an antibody response was detected to both the cytoplasm and cell wall of L. acidophilus. Moreover, feeding each organism for 2 weeks enhanced the proliferative response of Peyer’s patch (PP) cells to the cell fraction against which the serum antibody was detected. However, this was not found with spleen cells. These results suggest that mucosal stimulation by lactic acid bacteria may induce a systemic immune response to them.     

More Protection of Lactobacillus acidophilus Than Bifidobacterium bifidum Probiotics on Azoxymethane-Induced Mouse Colon Cancer

Based on the ability of the probiotics in the gut microflora modification, they can have the beneficial effects on diseases in the short and/or the long term. In previous study, we revealed that unlike Bifidobacterium bifidum, the amount of Lactobacillus acidophilus remained almost unchanged in mice gut microflora in the long term, indicating more stability of L. acidophilus than B. bifidum which can be used to prevent some incurable diseases such as cancer. Thirty-eight male BALB/c mice were divided into four groups, control, azoxymethane (AOM), L. acidophilus, and B. bifidum probiotics, to evaluate the protective effects of the probiotics on AOM-induced mouse colon cancer. Except for the control group, the rest of the animals were weekly given AOM (15 mg/kg, s.c) in three consecutive weeks. Colon lesion incidence was 74% in the AOM group in comparison with the control (0%) (P < 0.05). The lesions were varied from mild to severe dysplasia and colonic adenocarcinoma. Administration of the probiotics inhibited the incidence of colonic lesions by about 57% in L. acidophilus (P < 0.05) and 27% in B. bifidum (P > 0.05) compared to the AOM group. The serum levels of CEA and CA19-9 tumor markers were significantly decreased in L. acidophilus in comparison with the AOM group (P < 0.05). Moreover, the serum levels of IFN-γ and IL-10 and the number of CD4⁺ and CD8⁺ cells were significantly increased in L. acidophilus compared to AOM (P < 0.05). Our study highlighted the more potential effects of L. acidophilus probiotic than B. bifidum on mouse colon cancer.

     

Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota

A large number of commensal bacteria inhabit the intestinal tract, and interbacterial communication among gut microbiota is thought to occur. In order to analyze symbiotic relationships between probiotic strains and the gut microbiota, a ring with a membrane filter fitted to the bottom was used for in vitro investigations. Test strains comprising probiotic nitto strains (Lactobacillus acidophilus NT and Bifidobacterium longum NT) and type strains (L. acidophilus JCM1132^T and B. longum JCM1217^T) were obtained from diluted fecal samples using the membrane filter to simulate interbacterial communication. Bifidobacterium spp., Streptococcus pasteurianus, Collinsella aerofaciens, and Clostridium spp. were the most abundant gut bacteria detected before coculture with the test strains. Results of the coculture experiments indicated that the test strains significantly promote the growth of Ruminococcus gnavus, Ruminococcus torques, and Veillonella spp. and inhibit the growth of Sutterella wadsworthensis. Differences in the relative abundances of gut bacterial strains were furthermore observed after coculture of the fecal samples with each test strain. Bifidobacterium spp., which was detected as the dominant strain in the fecal samples, was found to be unaffected by coculture with the test strains. In the present study, interbacterial communication using bacterial metabolites between the test strains and the gut microbiota was demonstrated by the coculture technique. The detailed mechanisms and effects of the complex interbacterial communications that occur among the gut microbiota are, however, still unclear. Further investigation of these relationships by coculture of several fecal samples with probiotic strains is urgently required.     

Probiotic Strawberry Yogurts: Microbiological,Chemical and Sensory Properties

This study was performed to determine the viability of Lactobacillus acidophilus and Bifidobacterium bifidum in yogurt made with strawberry marmalade (SM) and to examine the quality properties of probiotic yogurt. Acidity, pH, bacterial counts and sensory analysis of the yogurt samples were investigated on days 1, 3, 5, 7, 10 and 14 during storage at 4 °C. The survival rate of L. acidophilus was greater than that of B. bifidum. The viability of L. acidophilus decreased during the storage period, but B. bifidum numbers remained stable during the storage period. The highest L. acidophilus count (7.20 log cfu/g) was found in L. acidophilus + B. bifidum SM yogurt on day 1. The highest B. bifidum count (6.13 log cfu/g) was detected in yogurt containing L. acidophilus + B. bifidum SM yogurt on day 7. Yeast and mould counts of all yogurts increased during the storage period. Coliform bacteria and Staphylococcus aureus were not detected in the yogurt samples. The highest overall acceptance sensory score was observed in yogurts containing L. acidophilus. Considering the sensory and probiotic characteristics of all yogurt samples, this study suggested that strawberry yogurt with a suitable 5–7 day storage period can be produced with single L. acidophilus addition or single B. bifidum addition.     

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.     

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.     

Kinetics of microbial hydrogenation of free linoleic acid to conjugated linoleic acids

Aims: To investigate the ability of selected probiotic bacterial strains to produce conjugated linoleic acid (CLA) and also to estimate the biohydrogenation kinetics of Lactobacillus acidophilus on the production of CLA from free linoleic acid (LA). Methods and Results: Six probiotic bacteria, Lact. paracasei, Lact. rhamnosus GG, Lact. acidophilus ADH, and Bifidobacterium longum B6, Lact. brevis, and Lact. casei, were used to examine their ability to convert LA to CLA. LA tolerance was evaluated by addition of different LA concentrations in MRS broth. Lact. acidophilus showed the major tolerant to LA and the greatest CLA‐producing ability (36–48 μg ml^?1 of CLA). The rate‐controlling steps were k₂ and k₁ for the addition of 1 and 3 mg ml^?1 of LA, respectively. The percentage of CLA conversion was higher in MRS broth supplemented with 1 mg ml^?1 (65%) than 3 mg ml^?1 (26%). Conclusion: The results provide useful information and new approach for understanding the biohydrogenation mechanisms of CLA production. Significance and Impact of the Study: This study would help elucidate the pathway from LA to stearic acid (SA), known as biohydrogenation. In addition, the use of selected probiotic bacteria might lead to a significant improvement in food safety.