Molecular microbial analysis of Bifidobacterium isolates from different environments by the species-specific amplified ribosomal DNA restriction analysis (ARDRA).

One hundred and six isolates of the genus Bifidobacterium, isolated from different environments (mainly gastrointestinal), were identified and classified taxonomically to species level by amplified ribosomal DNA restriction analysis. Two restriction endonucleases (Sau3AI and BamHI) were chosen for aligning the 16S rRNA sequences of 16 bifidobacterial species retrieved from various databases, to obtain species-specific restriction patterns. A rapid and accurate identification scheme was obtained by comparing the resulting 16S rDNA digestion profiles of 16 Bifidobacterium type-strains and 90 strains of various origins. All of the investigated strains were previously confirmed at the species level as belonging to the genus Bifidobacterium by fluorescence in-situ hybridisation and by polymerase chain reaction amplification with genus- and species-specific primers. The present work demonstrates that species-specific detection of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium coryneforme, Bifidobacterium cuniculi, Bifidobacterium dentium, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium suis, Bifidobacterium magnum, Bifidobacterium pseudolongum, Bifidobacterium pseudocatenulatum and Bifidobacterium pullorum present in different micro-ecological environments (e.g. gastrointestinal tract) can be accomplished in a reliable, rapid and accurate manner, circumventing the recognised deficiencies of traditional identification techniques.

Specific identification and targeted characterization of Bifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach.

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.

Iron requirement of Lactobacillus spp. in completely chemically defined growth media.

A completely chemically-defined growth medium, containing guanine, thymine, cytidine, 2'-deoxyadenosine and 2'-deoxyuridine as DNA precursors, was developed for Lactobacillus johnsonii, on the basis of statistically designed techniques suitable for other lactobacilli. Particular focus was given to the nucleotide composition of different defined media, and to the specific nucleotide requirements of Lact. johnsonii. Most of the lactobacilli tested grew in a medium containing five free bases, four ribonucleosides or five deoxyribonucleosides. Adenine and guanine were replaceable by inosine. The requirement for thymine and cytosine was satisfied with uracil. The presence of inosine and uracil was identified as being essential for the growth of different Lactobacillus species, displaying their inability to synthesize purines and pyrimidines de novo. Defined recipes with different nucleotide composition were used to investigate iron requirements of lactobacilli. Only marginal differences in growth were observed in iron-depleted media supplemented with five free bases, four ribonucleosides or five deoxyribonucleosides; iron depletion had a greater effect on growth when inosine and uracil were supplied as the only nucleotide sources. The results suggest that iron plays a role in the pyrimidine and purine metabolism of lactobacilli. Lactobacillus spp., particularly Lact. johnsonii, require iron under particular environmental conditions with limited or specific nucleotide sources.

The development of functional foods: lessons from the gut.

Functional foods have resulted from the gradual recognition that healthy diets result from eating nutritious foods and from the identification of the mechanisms by which foods modulate metabolism and health. After initial successes with foods that reduce blood cholesterol level, probiotic bacteria and prebiotic carbohydrates have now also demonstrated added health benefits. As ingredients become more complex, the need to stabilize such ingredients in foods become increasingly important to the success of functional foods. Modern biotechnologies such as genomics, genetic expression and biomarkers of health and performance will be applied to this increasingly visible portion of human diets.

Effect of restricted and ad libitum feeding on semen production and fertility in broiler breeder males.

1. The effect of food restriction on the quality of semen production and fertility in broiler breeder males was studied. 2. Seventy-two Ross broiler breeder males, from 20 to 54 weeks of age, were divided into 4 groups and fed as follows: group 1 = 110 g/bird/d; group 2 = 120 g/bird/d; group 3 = 130 g/bird/d; group 4 = ad libitum. 3. Body weight, sperm quality (volume, concentration, % motility and % live cells) and fertility were measured. The birds were slaughtered at 55 weeks of age; the abdominal fat pad and testicles were weighted. 4. Groups 2 and 3 produced the highest volume of semen. The quality of semen was very similar in all the restricted groups. 5. Males fed ad libitum produced semen with the best motility and percentage of live cells. Groups 3 and 4 showed the best fertility percentage (79%) against group 1 and 2 (59 and 72% respectively).