The biodiversity of predominant lactic acid bacteria in dolo and pito wort for the production of sorghum beer.

AIM: To quantify and identify the predominant lactic acid bacteria (LAB) in dolo and pito wort processing, and to examine their biodiversity at strain level. MATERIALS AND RESULTS: The processing of dolo and pito wort was studied at four production sites in Burkina Faso and Ghana. The succession of dominant micro-organisms, pH and titratable acidity were determined from sorghum malt through mashing and acidification to final wort. In the sorghum malt and during mashing, the LAB counts were 5.7-7.5 log CFU g(-1). Similar levels of yeasts and gram-negative, catalase-positive bacteria were observed. These levels decreased to 3.7-4.5 log CFU g(-1) and

Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin.

AIMS: To identify the dominant micro-organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development. METHODS AND RESULTS: Samples of sorghum gowé produced twice at three different production sites were taken at different fermentation times. DNA amplification by internal transcribed spacer-polymerase chain reaction of 288 lactic acid bacteria (LAB) isolates and 16S rRNA gene sequencing of selected strains revealed that the dominant LAB responsible for gowé fermentation were Lactobacillus fermentum, Weissella confusa, Lactobacillus mucosae, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella kimchii. DNA from 200 strains of yeasts was amplified and the D1/D2 domain of the 26S rRNA gene was sequenced for selected isolates, revealing that the yeasts species were Kluyveromyces marxianus, Pichia anomala, Candida krusei and Candida tropicalis. CONCLUSIONS: Gowé processing is characterized by a mixed fermentation dominated by Lact. fermentum, W. confusa and Ped. acidilactici for the LAB and by K. marxianus, P. anomala and C. krusei for the yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The diversity of the LAB and yeasts identified offers new opportunities for technology upgrading and products development in gowé production. The identified species can be used as possible starter for a controlled fermentation of gowé.

Biodiversity of Predominant Bacillus Isolated from Afitin; Iru and Sonru at Different Fermentation Time

Two hundred (200) presumptive isolates of Bacillus collected at different fermentation time from spontaneous fermented samples of afitin; iru and sonru produced in three different regions of Benin were identified at species and strains levels. ITS-PCR-RFLP revealed that 79of the isolates were really identified as Bacillus; 5as Staphylococcus and 16as unidentified bacteria. The 16S rDNA sequencing showed that 74.7of the Bacillus belonged to the B. subtilis group and 25.3to the B. cereus group. Additional biochemical tests and API 50 CHB system applied to 50 isolates randomly selected from the B. subtilis group divided the latter into 80typical B. subtilis and 20typical B. licheniformis; which showed different PFGE-RFLP band patterns. Strains belonging to the B. cereus group were differentiated by PCR with specific primers BCW1F and BCW1R and specific enzymes EcoRI; Sau3AI and RsaI. All strains of the B. cereus group; except one; were found to be typical B. cereus. This work showed the predominance of B. subtilis in afitin; iru and sonru along the fermentation process

Analgesic efficacy and safety of lornoxicam quick-release formulation compared with diclofenac potassium: randomised, double-blind trial in acute low back pain.

BACKGROUND: NSAIDs are widely used for patients presenting with low back pain. A quick-release formulation of lornoxicam, a potent NSAID from the chemical class of oxicams, offers a faster onset of pain relief compared with the standard tablet formulation. METHODS: Time to onset of pain relief with lornoxicam was compared with the quick-release formulation of diclofenac potassium in acute low back pain in a randomised, double-blind, multicentre study. 220 patients received either lornoxicam 24 mg or diclofenac potassium 150 mg on day 1 followed by lornoxicam 8 mg twice daily or diclofenac potassium 50 mg twice daily for 5 days. Efficacy outcomes included time to onset of pain relief, as measured by the stopwatch method (primary outcome), pain intensity, pain relief, rescue medication, ability to perform daily activities and global evaluation of the study medication. RESULTS: The time to onset of pain relief ratios between diclofenac potassium/lornoxicam was 1.03 (95% CI 0.91, 1.26) and 1.05 (95% CI 0.93, 1.29) in the intention-to-treat (ITT) and per-protocol (PP) analyses, respectively, demonstrating the non-inferiority of lornoxicam (defined by lower limits of the 95% CIs >0.80). Time to onset of pain relief was shorter with lornoxicam (30 minutes) compared with diclofenac potassium (36 minutes). The difference was not statistically significant (ITT analysis). A higher magnitude of analgesic effect associated with better global evaluation of the study medication for lornoxicam was also demonstrated. The drugs were equally well tolerated. CONCLUSION: Lornoxicam administered as a quick-release formulation was shown to be non-inferior to the equivalent formulation of diclofenac potassium in terms of onset of pain relief and more effective on most of the major standard efficacy outcomes.

Onset of acetaminophen analgesia: comparison of oral and intravenous routes after third molar surgery.

BACKGROUND: The purpose of this randomized double-blind study was to compare the efficacy and safety of propacetamol 2 g (an i.v. acetaminophen 1 g formulation) administered as a 2-min bolus injection (n=50) or a 15-min infusion (n=50) with oral acetaminophen 1 g (n=50) or placebo (n=25) for analgesia after third molar surgery in patients with moderate to severe pain after impacted third molar removal. METHODS: All patients were evaluated for efficacy during the initial 6 h period after treatment administration (T(0)) and for safety during the entire week after T(0). RESULTS: The onset of analgesia after propacetamol was shorter (3 min for bolus administration, 5 min for 15-min infusion) than after oral acetaminophen (11 min). Active treatments were significantly better for all parameters (pain relief, pain intensity, patient's global evaluation, duration of analgesia) than placebo (P<0.05). Adverse events were more frequent after propacetamol, especially pain at the injection site. Propacetamol bolus resulted in a much higher incidence of local adverse events than the infusion (propacetamol bolus 90% vs propacetamol infusion 52%) with no clinically significant benefits in terms of analgesic efficacy. CONCLUSION: I.V. propacetamol, administered as a 15-min infusion, is a fast-acting analgesic agent. It is more effective in terms of onset of analgesia than a similar dose of oral acetaminophen.

Case study of the distribution of mucosa-associated Bifidobacterium species, Lactobacillus species, and other lactic acid bacteria in the human colon.

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.

DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses.

The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses. In total 60 isolates were included. Of these, 40 strains of the following species, Debaryomyces hansenii var. hansenii, D. hansenii var. fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D. hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses. ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections. Ten strains of each variety of D. hansenii and five strains of each of the above mentioned species were analysed. For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed. Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses. ITS-PCR RFLP was investigated for the purpose of strain typing of D. hansenii. Ten CBS strains of each variety of D. hansenii were analysed. Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains. This enzyme divided the 20 strains in three groups. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D. hansenii showed an identity of 99.84%, corresponding to a difference in one basepair. Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D. hansenii. MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D. hansenii on the 20 CBS strains of D. hansenii. The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D. hansenii at subspecies level. The 20 dairy isolates showed a lower genetic variability than the CBS strains as they were divided into eight groups. Cluster analysis of the 20 CBS strains and the 20 dairy isolates based on their mtDNA restriction profiles showed (max. similarity level = 52%) that the dairy isolates only clustered with the CBS strains of D. hansenii var. hansenii. For some of the dairies more than one strain of D. hansenii were found to be involved in the ripening process, indicating that the method could be useful for subspecies typing and investigation of the microbial succession between strains of D. hansenii during the ripening process of surface ripened cheeses.

Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization.

Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta-galactosidases were highly specific for hydrolysis of beta-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.

16S to 23S rRNA spacer fragment length polymorphism of Salmonella enterica at subspecies and serotype levels.

The variation in the lengths of the internal transcribed spacer (ITS) between 16S and 23S rRNA genes of 101 strains representing 58 serotypes of Salmonella enterica (used for Salm. choleraesuis) subsp. enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV) and indica (VI) was used for typing by PCR amplification. Ten fragment lengths were observed by denaturing polyacrylamide gel electrophoresis on an automatic DNA sequencer resulting in 21 unique fragment patterns. Ten out of the 58 serotypes showed specific patterns but 48 serotypes were not fully differentiated. More than one ITS pattern was observed in seven serotypes. Five of the 21 fragment patterns contained representatives of more than one subspecies. Under non-denaturing electrophoresis conditions, serotype specificity was obtained but precise ITS fragment length determination was not possible. DNA sequence comparison between ITSs of individual rrn operons is needed to further interpret ITS diversity within Salm. enterica at serotype and subspecies levels.

Time to onset of analgesia and analgesic efficacy of effervescent acetaminophen 1000 mg compared to tablet acetaminophen 1000 mg in postoperative dental pain: a single-dose, double-blind, randomized, placebo-controlled study.

This randomized, double-blind, placebo-controlled study compared the time to onset of analgesia and the analgesic efficacy of two formulations of acetaminophen 1000 mg--an effervescent solution and tablet--in 242 patients with moderate or severe pain following dental surgery. Onset of analgesia was determined using a two-stopwatch procedure. Analgesia was assessed over a 4-hour period. Treatments were compared using standard indexes of pain intensity and pain relief and summary measures. Both acetaminophen formulations were significantly more effective than their corresponding placebo for all efficacy assessments. The median time to onset of analgesia was significantly shorter with effervescent acetaminophen (20 minutes) compared to tablet acetaminophen (45 minutes). During the first 45 minutes after administration, effervescent acetaminophen was significantly more effective at each scheduled assessment time than tablet acetaminophen. The median time to meaningful pain relief was significantly shorter with effervescent acetaminophen (45 minutes) compared to tablet acetaminophen (60 minutes). At 4 hours after administration, the pain relief was significantly better with tablet acetaminophen than with effervescent acetaminophen. No other significant differences were observed between the active treatments. In conclusion, effervescent acetaminophen produces a significantly faster onset of analgesia than tablet acetaminophen.

Sequence variation of the 16S to 23S rRNA spacer region in Salmonella enterica.

The possibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was investigated by direct sequencing of polymerase chain reaction-amplified DNA from all operons simultaneously in a collection of 25 strains of 18 different serotypes of S. enterica, and by sequencing individual cloned operons from a single strain. It was only possible to determine the first 117 bases upstream from the 23S rRNA gene by direct sequencing because of variation between the rrn operons. Comparison of sequences from this region allowed separation of only 15 out of the 18 serotypes investigated and was not specific even at the subspecies level of S. enterica. To determine the differences between internal transcribed spacers in more detail, the individual rrn operons of strain JEO 197, serotype IV 43:z4,z23:-, were cloned and sequenced. The strain contained four short internal transcribed spacer fragments of 382-384 bases in length, which were 98.4-99.7% similar to each other and three long fragments of 505 bases with 98.0-99.8% similarity. The study demonstrated a higher degree of interbacterial variation than intrabacterial variation between operons for serotypes of S. enterica.

Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans.

The probiotic potential of 47 selected strains of Lactobacillus spp. was investigated. The strains were examined for resistance to pH 2.5 and 0.3% oxgall, adhesion to Caco-2 cells, and antimicrobial activities against enteric pathogenic bacteria in model systems. From the results obtained in vitro, five strains, Lactobacillus rhamnosus 19070-2, L. reuteri DSM 12246, L. rhamnosus LGG, L. delbrueckii subsp. lactis CHCC 2329, and L. casei subsp. alactus CHCC 3137, were selected for in vivo studies. The daily consumption by 12 healthy volunteers of two doses of 10(10) freeze-dried bacteria of the selected strains for 18 days was followed by a washout period of 17 days. Fecal samples were taken at days 0 and 18 and during the washout period at days 5 and 11. Lactobacillus isolates were initially identified by API 50CHL and internal transcribed spacer PCR, and their identities were confirmed by restriction enzyme analysis in combination with pulsed-field gel electrophoresis. Among the tested strains, L. rhamnosus 19070-2, L. reuteri DSM 12246, and L. rhamnosus LGG were identified most frequently in fecal samples; they were found in 10, 8, and 7 of the 12 samples tested during the intervention period, respectively, whereas reisolations were less frequent in the washout period. The bacteria were reisolated in concentrations from 10(5) to 10(8) cells/g of feces. Survival and reisolation of the bacteria in vivo appeared to be linked to pH tolerance, adhesion, and antimicrobial properties in vitro.

Carbon metabolism regulates expression of the pfl (pyruvate formate-lyase) gene in Escherichia coli.

The anaerobic expression of pfl is reduced both in a strain mutated in the pgi gene and in a pfkA pfkB double mutant strain when cells are grown in medium supplemented with glucose. When cells are grown in medium supplemented with either fructose or pyruvate, no reduction is observed in these strains. The amount of pyruvate in the cells may be responsible for the reduced expression of pfl in the strains mutated in the genes encoding the glycolytic enzymes. Because of the lowered oxygen concentration in the medium, the expression of pfl is induced when an exponentially growing culture enters the stationary phase. This induction is increased when the Casamino Acid concentration is raised 10-fold or when the medium is supplemented with NaCl. Superhelicity of DNA is decreased in a pgi mutant strain grown in medium supplemented with glucose. The superhelicity is also changed, but the opposite way, in a wild-type strain grown in medium supplemented with Casamino Acids at a high concentration or 0.3 M sodium chloride. Our data show that changes in superhelicity do not affect the aerobic expression of pfl but might be important for the anaerobic induction of pfl.