Prevalência e caracterização de espécies de lactobacilos vaginais em mulheres em idade reprodutiva sem vulvovaginites

OBJETIVO: identificar espécies de lactobacilos isolados do conteúdo vaginal de mulheres saudáveis e assintomáticas; determinar as espécies mais prevalentes e caracterizá-las fenotipicamente. MÉTODOS: lactobacilos foram isolados em meio seletivo a partir de amostras de conteúdo vaginal de 135 mulheres, sem queixa de corrimento e com diagnóstico laboratorial negativo para infecções vaginais, acompanhadas em um ambulatório de Planejamento Familiar. Os isolados foram identificados por PCR multiplex e, quando necessário, submetidos ao sequenciamento do gene RNAr 16S. Foram também avaliados quanto à acidificação do meio de cultura, à produção de ácido láctico, de H2O2, bacteriocinas e a capacidade de adesão às células epiteliais. RESULTADOS: oitenta e três cepas de lactobacilos foram isoladas e identificadas, sendo as espécies predominantes L. crispatus (30,1%), L. jensenii (26,5%), L. gasseri (22,9%) e L. vaginalis (8,4%). Apenas 20 destes isolados não produziram H2O2 em quantidades detectáveis. Das 37 linhagens selecionadas para teste de adesão a células epiteliais, 12 apresentaram adesão entre 50 a 69%, 10 apresentaram 70% ou mais, e as restantes pouca ou nenhuma adesão. Nenhum dos isolados produziu bacteriocinas. CONCLUSÕES: as espécies de lactobacilos mais prevalentes em mulheres sem vulvovaginites, isoladas em meio de cultura seletivo e identificadas por métodos moleculares, foram L. crispatus, L. jensenii e L. gasseri. Além de mais frequentes, tais linhagens também apresentaram melhor produção de H2O2 e atingiram menores valores de pH em meio de cultura.

The effect of L-arginine on guinea-pig and rabbit airway smooth muscle function in vitro

We have investigated the effects of L-arginine, D-arginine and L-lysine on airway smooth muscle responsiveness to spasmogens in vitro. Both L-arginine and D-arginine (100 mM) significantly reduced the contractile potency and maximal contractile response to histamine but not to methacholine or potassium chloride in guinea-pig epithelium-denuded isolated trachea. Similarly, the contractile response to histamine was significantly reduced by L-arginine (100 mM) in rabbit epithelium-denuded isolated bronchus. The amino acid L-lysine (100 mM) failed to significantly alter the contractile potency of histamine in guinea-pig isolated trachea (P>0.05). In guinea-pig isolated trachea precontracted with histamine, both L-arginine and D-arginine produced a concentration-dependent relaxation which was not significantly altered by epithelium removal or by the presence of the nitric oxide synthase inhibitor, NG-nitro L-arginine methyl ester (L-NAME; 50 µM). Thus, at very high concentrations, arginine exhibit a non-competitive antagonism of histamine-induced contraction of isolated airway preparations that was independent of the generation of nitric oxide and was not dependent on charge. These observations confirm previous studies of cutaneous permeability responses and of contractile responses of guinea-pig isolated ileal smooth muscle. Taken together, the data suggest that high concentrations of arginine can exert an anti-histamine effect.

Avaliação do capim-elefante (Pennisetum purpureum Schum.) cultivar taiwan A-148, ensilado com diferentes técnicas de redução de umidade

O objetivo deste trabalho foi avaliar o efeito de diferentes técnicas de redução de umidade sobre a composição química, digestibilidade in vitro da matéria seca (DIVMS) e degradação ruminal da matéria seca da silagem de capim-elefante (Pennisetum purpureum Schum.) cv. Taiwan A-148. Os tratamentos (A, Controle; B, 20,0; C, 30,0; D, 40,0% de sabugo de milho; E, emurchecimento por 12 horas; F, emurchecimento por 24 horas; e G, esmagamento + emurchecimento por 24 horas) foram distribuídos em delineamento inteiramente casualizado com três repetições. Os tratamentos da silagem B e E não foram eficientes em reduzir a umidade excessiva da forragem. O emurchecimento por 24 horas aumentou o teor de MS, sem influir no teor de PB e na DIVMS. Os tratamentos C e D favoreceram o desenvolvimento da população de clostrídeos, aumentaram a concentração de N-NH3 e reduziram a concentração de ácido lático e a DIVMS das silagens. O tratamento G aumentou o teor de MS e reduziu a concentração de N-NH3 e DIVMS. A adiçao de sabugo de milho (tratamentos B, C e D) reduziu a degradação ruminal da MS das silagens. O sabugo de milho reduziu a umidade, mas apresentou efeitos negativos na qualidade da silagem, enquanto o esmagamento e/ou emurchecimento foram procedimentos que mostraram bons resultados em relação à conservação do material ensilado.

Inoculante enzimático-bacteriano, composição química e parâmetros fermentativos das silagens dos capins Tanzânia e Mombaça

Objetivou-se, com o presente trabalho, avaliar os efeitos de idades de colheita e do uso de inoculante enzimático-bacteriano sobre os parâmetros de fermentação, de composição química e digestibilidade in vitro da matéria seca (DIVMS) das silagens dos cultivares Tanzânia e Mombaça (Panicum maximum Jacq.). As forragens foram colhidas aos 45 e 60 dias após o corte de uniformização, submetidas a dois tratamentos (A - Ensilagem sem aditivo [Controle]; B - Ensilagem com adição de inoculante enzimático-bacteriano) e distribuídas em um delineamento inteiramente casualizado em esquema fatorial (dois capins x duas inoculações x duas idades de corte), com três repetições. O conteúdo de MS das silagens confeccionadas com as plantas colhidas aos 60 dias de rebrota foi superior àquele das silagens colhidas com 45 dias. As silagens confeccionadas com as plantas colhidas aos 45 dias de rebrota apresentaram os maiores teores de PB (11,9%). Não se observaram efeitos das silagens, dos tratamentos e das idades de corte sobre os teores de hemicelulose, celulose e DIVMS e valores de pH e N-NH3. As silagens do capim-tanzânia apresentaram os maiores teores de MS, FDN, FDA, ácido lático e ácido butírico, enquanto as de capim-mombaça, os de PB e de ácido acético. A adição de inoculante enzimático-bateriano promoveu silagens com menores teores de MS, PB e lignina que as não-tratadas. Os capins tanzânia e mombaça não apresentaram limitações ao processo de ensilagem. O inoculante enzimático-bacteriano não melhorou as características qualitativas, fermentativas e nutricionais das silagens avaliadas.

Influência da utilização de aditivos químicos no perfil da fermentação, no valor nutritivo e nas perdas de silagens de cana-de-açúcar

Avaliou-se o efeito do uso de aditivos químicos nas perdas de matéria seca, no perfil fermentativo e no valor nutritivo de silagens de cana-de-açúcar. O experimento foi realizado em delineamento inteiramente casualizado com quatro repetições por tratamento (sete tratamentos). Os seguintes aditivos foram utilizados na confecção das silagens: controle (sem aditivo), L. buchneri, óxido e carbonato de cálcio em doses de 1,0 e 1,5% da massa verde e sulfato de cálcio a 1,0% da massa verde, diluídos em 40 litros de água por tonelada de forragem. As variáveis analisadas foram: perdas totais e gasosas, produção de efluente, recuperação de matéria seca, composição química e valor nutritivo. As menores perdas fermentativas e gasosas foram observadas nas silagens com óxido ou carbonato de cálcio, que resultaram em maior taxa de recuperação de matéria seca. da mesma forma, as silagens tratadas com estes aditivos apresentaram maior teor de carboidratos solúveis residuais e de ácido lático e reduzida fermentação alcoólica. As silagens tratadas com óxido e carbonato apresentaram, no momento da abertura, maior teor de cinzas, menor concentração de componentes fibrosos e maiores coeficientes digestibilidade da matéria seca e da matéria orgânica. O teor de proteína encontrado nessas silagens foi semelhante ao observado na forragem fresca. A ensilagem de cana-de-açúcar com L. buchneri e sulfato de cálcio possibilitou a obtenção de desempenho similar ao determinado com a silagem sem aditivos. Silagens tratadas com carbonato e óxido de cálcio apresentam maior desempenho durante o processo fermentativo.

Antibiosis associated to Lactobacillus acidophilus growth .1. Strain selection and study of an alternative medium using whey

The production of antimicrobial compounds of some strains of Lactobacillus acidophilus has been studied. They have been grown in whey supplemented with soy milk. It has been found that the production of compounds is able to inhibit the growing of both target bacteria analysed: Staphylococcus aureus and Escherichia coli. The results showed a significant variation (p>0.05) depending on the strain of L. acidophilus and on the level of supplementation utilized. Most of the inhibition observed resulted from the presence of the lactic acid produced. It has also been found the production of other antimicrobial compounds showing inhibitory capacity. The action of these compounds was influenced by the substract pH.

Relationships and significance of lactate minimum, critical velocity, heart rate deflection and 3 000 m track-tests for running

The running velocities associated to lactate minimum (V-lm), heart rate deflection (V-HRd), critical velocity (CV), 3000 M (V-3000) and 10000 m performance (V-10km) were compared. Additionally the ability of V-lm and VHRd on identifying sustainable velocities was investigated.Methods. Twenty runners (28.5 +/- 5.9 y) performed 1) 3000 m running test for V3000; 2) an all-out 500 in sprint followed by 6x800 m incremental bouts with blood lactate ([lac]) measurements for V-lm; 3) a continuous velocity-incremented test with heart rate measurements at each 200 m for V-HRd; 4) participants attempted to 30 min of endurance test both at V-lm(ETVlm) and V-HRd(ETVHRd). Additionally, the distance-time and velocity-1/time relationships produced CV by 2 (500 m and 3000 m) or 3 predictive trials (500 m, 3000 m and distance reached before exhaustion during ETVHRd), and a 10 km race was recorded for V-10km.Results. The CV identified by different methods did not differ to each other. The results (m(.)min(-1)) revealed that V-.(lm) (281 +/- 14.8)< CV (292.1 +/- 17.5)=V-10km (291.7 +/- 19.3)< V-HRd (300.8 +/- 18.7)=V-3000 (304 +/- 17.5) with high correlation among parameters (P < 0.001). During ETVlm participants completed 30 min of running while on the ETVHRd they lasted only 12.5 +/- 8.2 min with increasing [lac].Conclusion. We evidenced that CV and Vim track-protocols are valid for running evaluation and performance prediction and the parameters studied have different significance. The V-lm reflects the moderate-high intensity domain (below CV), can be sustained without [lac] accumulation and may be used for long-term exercise while the V-HRd overestimates a running intensity that can be sustained for long-time. Additionally, V-3000 and V-HRd reflect the severe intensity domain (above CV).

Evaluation in vitro of the antagonistic substances produced by Lactobacillus spp. isolated from chickens

To determine the inhibitory capacity of lactic acid bacteria due to the action of antagonistic substances, we tested 474 isolates of Lactobacillus from the crop and cecum of chickens against gram-positive and gram-negative indicator microorganisms by the spot-on-the-lawn and well-diffusion antagonism methods. of the 474 isolates, 265 demonstrated antimicrobial activity against the indicator microorganisms. Isolates identified as L. reuteri, L. salivarius, or Lactobacillus spp. inhibited Enterococcus faecalis, E. faecium, Listeria monocytogenes, and Salmonella spp. but not L. casei, L. delbrueckii, L. fermentum, or L. helveticus by the well-diffusion simultaneous antagonism method under anaerobic incubation conditions. The antagonistic substances produced by some of the Lactobacillus isolates were inactivated after treatment by proteolytic enzymes, which suggested that the substances could be antimicrobial peptides or bacteriocins.

Atividade antimicrobiana de Lactobacillus acidophilus, contra microrganismos patogênicos veiculados por alimentos

The antimicrobial activity of a commercial probiotic culture, Lactobacillus acidophilus (La5), was tested against two foodborne pathogens, Escherichia coli and Staphylococcus aureus. The antagonistic effect of the probiotic culture in vitro was performed by applying both Multilayer Agar Plate and Agar Well Diffusion methods. The results indicated that the inhibitory substance present on 72 hours culture broth supernatant was extracellular and diffusible. The incubation period of the lactic acid bacteria on MRS Broth, at 3 7 degrees C in aerobic conditions, for the highest lactic acid production (1,08 g/%) was 72 hours, which gave a minimum pH value of the supernatant (3,90) and the best inhibition results by the Well Diffusion Agar Assay, showing inhibition zone diameters of 14,75mm and 15,0mm for E. coli and S. aureus, respectively. The inhibitor compound was not sensitive to proteolytic enzyme and freezing, but was totally inactivated when the supernatant was neutralized with NaOH 1 N solution. The results suggest that the inhibitory activity was due to the lactic acid concentration and the low pH of the probiotic culture broth.

Effects of recovery type after a judo combat on blood lactate removal and on performance in an intermittent anaerobic task

Aim. The objective of this study was to verify the effects of active (AR) and passive recovery (PR) after a judo match on blood lactate removal and on performance in an anaerobic intermittent task (4 bouts of upper body Wingate tests with 3-min interval between bouts; 4WT).Methods. The sample was constituted by 17 male judo players of different competitive levels: A) National (Brazil) and International medallists (n. 5). B) State (São Paulo) medallists (n. 7). Q City (São Paulo) medallists (n. 5). The subjects were submitted to: 1) a treadmill test for determination of VO2peak and velocity at anaerobic threshold (VAT); 2) body composition; 3) a 5-min judo combat, 15-min of AR or PR followed by 4WT.Results. The groups did not differ with respect to: body weight, VO2peak, VAT, body fat percentage, blood lactate after combats. No difference was observed in performance between AR and PR, despite a lower blood lactate after combat (10 and 15 min) during AR compared to PR. Groups A and B performed better in the high-intensity intermittent exercise compared to athletes with lower competitive level (C).Conclusion. The ability to maintain power output during intermittent anaerobic exercises can discriminate properly judo players of different levels. Lactate removal was improved with AR when compared to PR but AR did not improve performance in a subsequent intermittent anaerobic exercise.

Haemagglutination induced by Bordetella pertussis filamentous haemagglutinin adhesin (FHA) is inhibited by antibodies produced against FHA(430-873) fragment expressed in Lactobacillus casei

Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough.

The Lactate Minimum Test protocol provides valid measures of cycle ergometer VO2 peak

Aim. The aim of the present study was to investigate the validity of the Lactate Minimum Test (LMT) for the determination of peak VO2 on a cycle ergometer and to determine the submaximal oxygen uptake (VO2) and pulmonary ventilation (VE) responses in an incremental exercise test when it is preceded by high intensity exercise (i.e., during a LMT).Methods. Ten trained male athletes (triathletes and cyclists) performed 2 exercise tests in random order on an electromagnetic cycle ergometer: 1) Control Test (CT): an incremental test with an initial work rate of 100 W, and with 25 W increments at 3-min intervals, until voluntary exhaustion; 2) LMT: an incremental test identical to the CT, except that it was preceded by 2 supramaximal bouts of 30-sec (similar to120% VO(2)peak) with a 30-sec rest to induce lactic acidosis. This test started 8 min after the induction of acidosis.Results. There was no significant difference in peak VO2 (65.6+/-7.4 ml.kg(-1).min(-1); 63.8+/-7.5 ml.kg(-1).min(-1) to CT and LMT, respectively). However, the maximal power output (POmax) reached was significantly higher in CT (300.6+/-15.7 W) than in the LMT (283.2+/-16.0 W).VO2 and VE were significantly increased at initial power outputs in LMT.Conclusion. Although the LMT alters the submaximal physiological responses during the incremental phase (greater initial metabolic cost), this protocol is valid to evaluate peak VO2, although the POmax reached is also reduced.

Physicochemical properties of maize starch obtained from intermittent milling and dynamic steeping (IMDS) under various steeping conditions

Physicochemical properties of maize starch obtained under different steeping conditions by intermittent milling and dynamic steeping process (IMDS) were studied. Brazilian dent maize (hybrid XL 606) was milled using a 2x2x3 factorial experimental design with two lactic acid levels (0.0 and 0.55%, v/v), two SO2 levels (0.05 and 0.1%, w/v), and three temperatures (52, 60, and 68degreesC). Properties of starch obtained by conventional wet-milling process (36 hr at 52degreesC, 0.55% lactic acid, and 0.2% SO2) were used for comparison. Starch protein content and solubility increased with presence of lactic acid, while swelling power decreased. Higher SO2 concentration (0.1%) had the same effect as lactic acid on some properties. Steeping temperatures of 60 and 68degreesC increased solubility and most of the thermal properties but reduced swelling power, suggesting stronger starch annealing during IMDS at these temperatures. Some thermal changes on starch granules were visualized by scanning electron microscopy (SEM) at 60 and 68degreesC. Amylose content as well as pasting properties were affected by steeping factors and interactions. Starches from IMDS and conventional wet-milling processes were similar in most properties, indicating that IMDS provides starch with quality similar to that from conventional milling.

Validação de métodos para determinação do teor de 3-hidroxibutirato e 3-hidroxivalerato em polímero biodegradável

The efficiency of two methods for determining 3-hydroxybutyrate and 3-hydroxyvalerate content in the biopolymer (poly(3-hydroxybutyrate-co-3-hydroxyvalerate) has been evaluated. Both methods are based on (a) the hydrolysis of polymers, (b) the esterification with methyl (method A) and isoamilic alcohol (method B) and (c) analysis of the esters by gas chromatography using flame ionization detection (GC-FID). After optimization of the analytical conditions, the main validation parameters (linearity, quantification limit, detection limit, recovery, sensitivity and precision) were determined showing that both analytical procedures can be applied to control the process of poly(3-hydroxybutyrate) production. However, method B gives better analytical results than method A and has the advantage of not using chlorinated solvents.

Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of Mycobacterium tuberculosis

The high incidence of tuberculosis around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against tuberculosis is needed. A single antigen, the mycobacterial heat shock protein hsp65, is sufficient to protect BALB/c mice against challenge infection when administered as DNA vaccine in a three-dose-based schedule. In order to simplify the vaccination schedule, we coencapsulated hsp65-DNA and trehalose dimicolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. BALB/c mice immunized with a single dose of DNA-hsp65/TDM-1oaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-gamma in the supernatant of spleen cell cultures. DNA-hsp65/TDM-loaded microspheres were also able to induce high IFN-gamma production in bulk lung cells from challenged mice and confer protection as effective as that attained after three doses of naked DNA administration. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against tuberculosis.