Classification of a moderately oxygen-tolerant isolate from baby faeces as Bifidobacterium thermophilum

<p>Abstract</p> <p>Background</p> <p>Bifidobacteria are found at varying prevalence in human microbiota and seem to play an important role in the human gastrointestinal tract (GIT). Bifidobacteria are highly adapted to the human GIT which is reflected in the genome sequence of a <it>Bifidobacterim longum </it>isolate. The competitiveness against other bacteria is not fully understood yet but may be related to the production of antimicrobial compounds such as bacteriocins. In a previous study, 34 <it>Bifidobacterium </it>isolates have been isolated from baby faeces among which six showed proteinaceous antilisterial activity against <it>Listeria monocytogenes</it>. In this study, one of these isolates, RBL67, was further identified and characterized.</p> <p>Results</p> <p><it>Bifidobacterium </it>isolate RBL67 was classified and characterized using a polyphasic approach. RBL67 was classified as <it>Bifidobacterium thermophilum </it>based on phenotypic and DNA-DNA hybridization characteristics, although 16S rDNA analyses and partial <it>gro</it>EL sequences showed higher homology with <it>B. thermacidophilum </it>subsp. <it>porcinum </it>and <it>B. thermacidophilum </it>subsp. <it>thermacidophilum</it>, respectively. RBL67 was moderately oxygen-tolerant and was able to grow at pH 4 and at a temperature of 47°C.</p> <p>Conclusion</p> <p>In order to assign RBL67 to a species, a polyphasic approach was used. This resulted in the classification of RBL67 as a <it>Bifidobacterium thermophilum </it>strain. To our knowledge, this is the first report about <it>B. thermophilum </it>isolated from baby faeces since the <it>B. thermophilum </it>strains were related to ruminants and swine faeces before. <it>B. thermophilum </it>was previously only isolated from animal sources and was therefore suggested to be used as differential species between animal and human contamination. Our findings may disapprove this suggestion and further studies are now conducted to determine whether <it>B. thermophilum </it>is distributed broader in human faeces. Furthermore, the postulated differentiation between human and animal strains by growth above 45°C is no longer valid since <it>B. thermophilum </it>is able to grow at 47°C. In our study, 16S rDNA and partial <it>gro</it>EL sequence analysis were not able to clearly assign RBL67 to a species and were contradictory. Our study suggests that partial <it>gro</it>EL sequences may not be reliable as a single tool for species differentiation.</p

EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of health claims related to a combination of Lactobacillus helveticus CNCM I-1722 and Bifidobacterium longum subsp. Longum CNCM I-3470 and alleviation of psychological stress (ID 938) and “maintains the balance of healthy microbiota that helps to strengthen the natural defence” (ID 2942) (further assessment) pursuant to Article 13(1) of Regulation (EC) No 1924/2006

&lt;p&gt;Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to provide a scientific opinion on health claims pursuant to Article 13 of Regulation (EC) No 1924/2006 in the framework of further assessment related to a combination of &lt;em&gt;Lactobacillus &lt;/em&gt;&lt;em&gt;helveticus&lt;/em&gt; CNCM I-1722 and &lt;em&gt;Bifidobacterium longum&lt;/em&gt; subsp. &lt;em&gt;longum &lt;/em&gt;CNCM I-3470 and alleviation of psychological stress and “maintains the balance of healthy microbiota that helps to strengthen the natural defence”. The food constituent that is the subject of the health claims, a combination of &lt;em&gt;L. helveticus&lt;/em&gt; CNCM I-1722 and &lt;em&gt;B. longum&lt;/em&gt; subsp. &lt;em&gt;longum &lt;/em&gt;CNCM I-3470, is sufficiently characterised. The claimed effect, alleviation of psychological stress, is a beneficial physiological effect. No human intervention studies were provided from which conclusions could be drawn for the scientific substantiation of the claim. On the basis of the data provided, the Panel concludes that a cause and effect relationship has not been established between the consumption of a combination of &lt;em&gt;L. helveticus&lt;/em&gt; CNCM I-1722 and &lt;em&gt;B. longum&lt;/em&gt; subsp. &lt;em&gt;longum &lt;/em&gt;CNCM I-3470 and alleviation of psychological stress. From the information provided for the claimed effect “maintains the balance of healthy microbiota that helps to strengthen the natural defence” it was not possible to establish the specific effect which is the subject of the claim. The Panel considers that the claimed effect is general and non-specific, and does not refer to any specific health claim as required by Regulation (EC) No 1924/2006.&lt;/p&gt

A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86–34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH4)2SO4 precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15–35 °C and pH 5–9, with the optimal conditions being 15–25 °C and pH 5–6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold–active lipase. Its activity was found to increase in the presence of Zn2+, but it was strongly inhibited by Fe2+, Fe3+, Hg2+ and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short-and medium-chain length p-nitrophenyl (C4 and C8 acyl group) esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group) with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil

Antibacterial Activities of Nisin Z Encapsulated in Liposomes or Produced In Situ by Mixed Culture during Cheddar Cheese Ripening

This study investigated both the activity of nisin Z, either encapsulated in liposomes or produced in situ by a mixed starter, against Listeria innocua, Lactococcus spp., and Lactobacillus casei subsp. casei and the distribution of nisin Z in a Cheddar cheese matrix. Nisin Z molecules were visualized using gold-labeled anti-nisin Z monoclonal antibodies and transmission electron microscopy (immune-TEM). Experimental Cheddar cheeses were made using a nisinogenic mixed starter culture, containing Lactococcus lactis subsp. lactis biovar diacetylactis UL 719 as the nisin producer and two nisin-tolerant lactococcal strains and L. casei subsp. casei as secondary flora, and ripened at 7°C for 6 months. In some trials, L. innocua was added to cheese milk at 10(5) to 10(6) CFU/ml. In 6-month-old cheeses, 90% of the initial activity of encapsulated nisin (280 ± 14 IU/g) was recovered, in contrast to only 12% for initial nisin activity produced in situ by the nisinogenic starter (300 ± 15 IU/g). During ripening, immune-TEM observations showed that encapsulated nisin was located mainly at the fat/casein interface and/or embedded in whey pockets while nisin produced by biovar diacetylactis UL 719 was uniformly distributed in the fresh cheese matrix but concentrated in the fat area as the cheeses aged. Cell membrane in lactococci appeared to be the main nisin target, while in L. casei subsp. casei and L. innocua, nisin was more commonly observed in the cytoplasm. Cell wall disruption and digestion and lysis vesicle formation were common observations among strains exposed to nisin. Immune-TEM observations suggest several modes of action for nisin Z, which may be genus and/or species specific and may include intracellular target-specific activity. It was concluded that nisin-containing liposomes can provide a powerful tool to improve nisin stability and availability in the cheese matrix

Inactivation of Adhesion and Invasion of Food-Borne Listeria monocytogenes by Bacteriocin-Producing Bifidobacterium Strains of Human Origin

Three bacteriocin-producing bifidobacterial isolates from newborns were identified as Bifidobacterium thermacidophilum (two strains) and B. thermophilum (one strain). This study was undertaken to evaluate the ability of these strains to compete with food-borne Listeria monocytogenes for adhesion and invasion sites on Caco-2 and HT-29 cells. The bifidobacteria adhered at levels ranging from 4% to 10% of the CFU added, but none of the bifidobacteria were able to invade cells. The abilities of Listeria to adhere to and to invade cells varied widely depending on the strain tested. Three groups of Listeria were identified based on invasiveness: weakly invasive, moderately invasive, and highly invasive strains. One strain from each group was tested in competition with bifidobacteria. B. thermacidophilum RBL70 was the most effective in blocking invasion of Listeria, and the decreases in invasion ranged from 38% to 90%. For all three bifidobacterial strains, contact between the cell monolayer and the bifidobacteria for 1 h before exposure to Listeria increased the degree of inhibition. Finally, visualization of competition for adhesion sites on cells by fluorescent in situ hybridization suggested that the two bacteria tended to adhere in close proximity

Encapsulation of a Lactic Acid Bacteria Cell-Free Extract in Liposomes and Use in Cheddar Cheese Ripening

A concentrated form of cell free extract (CFE) derived from attenuated Lactococcus lactis supsb. lactis 303 CFE was encapsulated in liposomes prepared from two different proliposome preparations (Prolipo Duo and Prolipo S) using microfluidization. Entrapment efficiencies of 19.7 % (Prolipo S) and 14.0 % (Prolipo Duo) were achieved and the preparations mixed in the ratio 4 (Prolipo Duo):1 (Prolipo S). Cheddar cheese trials were undertaken evaluating the performance of CFE entrapped in liposomes, empty liposomes and free CFE in comparison to a control cheese without any CFE or liposomes. Identical volumes of liposome and amounts of CFE were used in triplicate trials. The inclusion of liposomes did not adversely impact on cheese composition water activity, or microbiology. Entrapment of CFE in liposomes reduced loss of CFE to the whey. No significant differences were evident in proteolysis or expressed PepX activity during ripening in comparison to the cheeses containing free CFE, empty liposomes or the control, as the liposomes did not degrade during ripening. This result highlights the potential of liposomes to minimize losses of encapsulated enzymes into the whey during cheese production but also highlights the need to optimize the hydrophobicity, zeta potential, size and composition of the liposomes to maximize their use as vectors for enzyme addition in cheese to augment ripening

Grape seed proanthocyanidin extract inhibits DNA and protein damage and labile iron, enzyme, and cancer cell activities

Abstract Grape seed extract from (Vitis vinifera) (VGSE) is an excellent source of various polyphenols that exhibit highly potent antioxidant and disease prevention properties. Although numerous biological activities, with potential for improving human health, have been reported for VGSE, there is a lack of data relating to the health benefits of VGSE on DNA damage, protein damage, labile iron activity, and enzyme inhibitory effects. This investigation demonstrated, for the first time, that VGSE inhibits DNA and BSA damage and labile iron activity in-vitro. Moreover, VGSE also inhibited in-vitro activities of AChE, tyrosinase, and α-amylase. VGSE treatment significantly reduced viability of MCF-7, Hep-G2, Caco-2, and Huh-7 cells after 48-h treatments. The results obtained provide additional support for the purported health benefits of VGSE and reinforce its potential in disease prevention and therapy, especially in relation to cancer

Molecular characterization and lipase profiling of the yeasts isolated from environments contaminated with petroleum

WOS: 000340254400011PubMed ID: 23712936In the present study, 120 yeast isolates from different sources (active sludge, soil, and wastewater samples obtained from petroleum refinery and soil contaminated by petroleum) were obtained. The yeast isolates were screened for lipase production and twelve of the isolates (D3, D17, D24, D27, D30, D38, D40, D42, D44, D46, D56, and D57) exhibited lipase activity. Molecular characterization of the yeasts showing the lipase production was performed with RFLP of ITS15.8S- ITS2 and 18S rRNA and sequence analysis of D1/D2 domain of 26S rRNA. The 26S rRNA sequencing revealed that four new strains, D38, D40, D44 and D57 identified as Rhodotorula slooffiae, Candida davisiana, Cryptococcus diffluens, and Cryptococcus uzbekistanensis, respectively, are lipase producing yeast species. This study is the first report showed lipase production by these species. The other lipase producing strains identified as Candida parapsilosis (D3), Rhodotorula muciloginosa (D17 and D42), Cryptococcus albidus (D24, D27, D30, and D56), and Wickerhamomyces anomalus (D46).Scientific Research Unit of Ege UniversityEge University [11-FEN-015]This work was supported by the Scientific Research Unit of Ege University (11-FEN-015). We thank Msc. Digdem Tunali (Ege University) for providing some yeast strains. We also would like to extend our thanks to the Izmir Institute of Technology, Biotechnology and Bioengineering Central Research Laboratories (BIYOMER) for providing laboratory facilities for DNA sequencing

Antibiotic Susceptibility Profile of Bifidobacteria as Affected by Oxgall, Acid, and Hydrogen Peroxide Stress

The effects of acid, oxgall, and H(2)O(2) on susceptibilities to antibiotics and nisin were examined for 13 strains of bifidobacteria. Susceptibilities to ampicillin, cloxacillin, penicillin, vancomycin, kanamycin, neomycin, paramomycin, streptomycin, chloramphenicol, erythromycin, tetracycline, and nisin A were assayed by a microdilution broth method. Acid-, oxgall- and H(2)O(2)-stressed variants were produced and assayed. Exposure to a pH of 2.0 for 60 min reduced susceptibilities to cloxacillin and nisin A but increased susceptibilities to ampicillin, vancomycin, aminoglycosides, chloramphenicol, and erythromycin in a strain-dependent manner. Exposure to oxgall (0.3%) for 90 min increased susceptibilities to cell wall-directed antibiotics and aminoglycosides but increased resistances to tetracycline and nisin A. Oxidative stress increased the susceptibilities of 70% of the strains to ampicillin and chloramphenicol, of 50% of the strains to cloxacillin and tetracycline, and of 40% of the strains to erythromycin but did not affect susceptibilities to vancomycin, kanamycin, and nisin A. This study shows that exposure of bifidobacteria to stressful conditions resembling those in the gastrointestinal tract may substantially modify their susceptibilities to antibiotics and may thus affect their probiotic capacities, especially when they are used for the management of intestinal infections and antibiotic-associated diarrhea