Probiotic Enterococcus faecium CRL 183 inhibit chemically induced colon cancer in male Wistar rats

Several animal studies have shown that supplementation with specific strains of lactic acid bacteria could prevent the establishment, growth, and metastasis of transplantable and chemically induced tumors. The goal of this study was to determine the effect of Enterococcus faecium CRL 183 on the incidence of colorectal tumors induced experimentally by dimethylhydrazine (DMH). We used thirty 4-week old male Wistar rat. The animals belonging to the DMH groups were injected s.c 20 mg/kg body weight of 1,2 dimethylhydrazine and 1 mM EDTA (pH 6.5), in a weekly dose, for 14 weeks. Three groups were used: (1) Control (not initiated); (2) Initiated with DMH; (3) Initiated with DMH + intake of E. faecium CRL 183. At the end of the 42nd week, all the animals were euthanized; the colons were removed and analyzed histologically. All the groups were compared histologically and IL-4, IFN-gamma and TNF-alpha cytokines. The control group did not develop pre-neoplastic lesions. The E. faecium CRL 183-DMH group showed a 50% inhibition in incidence in average number of tumors (P < 0.001), reduced the formation of ACF (P < 0.001), the lowest number of adenocarcinoma being found in this group (P < 0.001) and enhanced the immune response by increasing IL-4, IFN-gamma and TNF-alpha (P < 0.001) when compared with the DMH group.

Effects of adding polyclonal antibody preparations on ruminal fermentation patterns and digestibility of cows fed different energy sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Rosmarinic acid, a new snake venom phospholipase A(2) inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. The methanolic extract from Cordia verbenacea (Cv) significantly inhibited paw edema induced by Bothrops jararacussu snake venom and by its main basic phospholipase A(2) homologs, namely bothropstoxins I and II (BthTXs). The active component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identified as rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2-O-cafeoil-3-(3,4-di-hydroxy-phenyl)-R-lactic acid]. This is the first report of RA in the species C. verbenacea ('baleeira', 'whaler') and of its anti-inflammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the edema and myotoxic activity induced by the basic PLA(2)s BthTX-I and BthTX-II. It was, however, less efficient to inhibit the PLA(2) activity of BthTX-II and, still less, the PLA(2) and edema-inducing activities of the acidic isoform BthA-1-PLA(2), from the same venom, showing therefore a higher inhibitory activity upon basic PLA(2)s. RA also inhibited most of the myotoxic and partially the edema-inducing effects of both basic PLA(2)s, thus reinforcing the idea of dissociation between the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA(2)s in experimental models. CD data presented here suggest that, after binding, no significant conformation changes occur either in the Cv-RA or in the target PLA(2). A possible model for the interaction of rosmarinic acid with Lys49-PLA(2) BthTX-I is proposed. (c) 2005 Elsevier Ltd. All rights reserved.

Inhibitory effect of essential oils against Lactobacillus rhamnosus and starter culture in fermented milk during its shelf-life period

The use of essential oils in foods has attracted great interest, due to their antagonistic action against pathogenic microorganisms. However, this action is undesirable for probiotic foods, as products containing Lactobacillus rhamnosus. The aim of the present study was to measure the sensitivity profile of L. rhamnosus and a yogurt starter culture in fermented milk, upon addition of increasing concentrations of cinnamon, clove and mint essential oils. Essential oils were prepared by steam distillation, and chemically characterised by gas chromatography-mass spectrometry (GC-MS) and determination of density. Survival curves were obtained from counts of L. rhamnosus and the starter culture (alone and in combination), upon addition of 0.04% essential oils. In parallel, titratable acidity was monitored over 28 experimental days. Minimum inhibitory concentration values, obtained using the microdilution method in Brain Heart Infusion medium, were 0.025, 0.2 and 0.4% for cinnamon, clove and mint essential oils, respectively. Cinnamon essential oil had the highest antimicrobial activity, especially against the starter culture, interfering with lactic acid production. Although viable cell counts of L. rhamnosus were lower following treatment with all 3 essential oils, relative to controls, these results were not statistically significant; in addition, cell counts remained greater than the minimum count of 10(8)CFU/mL required for a product to be considered a probiotic. Thus, although use of cinnamon essential oil in yogurt makes starter culture fermentation unfeasible, it does not prevent the application of L. rhamnosus to probiotic fermented milk. Furthermore, clove and mint essential oil caused sublethal stress to L. rhamnosus.

Amperometric biosensor for ascorbic acid

Desenvolveu-se um biossensor para ácido L-ascórbico empregando ascorbato oxidase. A enzima foi extraída do mesocarpo de pepino com solução tampão fosfato 0,05 mol L-1, pH 5,8 contendo NaCl 0,5 mol L-1. Após diálise versus solução tampão fosfato 0,05 mol L-1, pH 5,8 a enzima foi imobilizada em rede de nylon através de ligação covalente com glutaraldeído. A membrana foi acoplada em eletrodo de O2 e a reação monitorada pelo consumo de oxigênio a -600 mV em análise em fluxo (solução tampão fosfato 0,05 mol L-1, pH 5,8 como carregador e vazão 0,5 mL min-1). A curva analítica apresentou-se linear entre 1,2x10-4 a 1,0x10-3 mol L-1. O tempo de vida do biossensor foi de 500 análises. Amostras de medicamentos foram analisadas com a metodologia proposta e os resultados comparados com os obtidos com HPLC.

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.

Organic acid profile of commercial sour cassava starch

A presença de ácidos orgânicos no polvilho azedo, além de contribuir com aspectos como sabor e aroma, tem, conforme a literatura indica, correlação com a propriedade de expansão, que é um fator determinante no uso alimentício. Amostras de polvilho azedo foram coletadas nas Regiões Sul e Sudeste diretamente nas empresas ou no comércio. Foram preparadas para análise em cromatografia líquida de alta eficiência (CLAE), sendo que o cromatógrafo estava equipado com coluna Biorad Aminex HPX-87H para análise de ácidos orgânicos e detector refratométrico. As condições de análise envolveram o emprego da fase móvel ácido sulfúrico 0,005M, fluxo de 0,6 ml/min e temperatura da coluna de 60oC. Os ácidos quantificados foram lático (0,036 a 0,813 g/100g), acético (0 a 0,068 g/100g), propiônico (0 a 0,013 g/100g) e butírico (0 a 0,057 g/100g), presentes na fermentação natural. Os resultados revelaram grande variação entre as amostras, com diferenças mesmo dentro das Regiões. Algumas amostras apresentaram quantidades elevadas de ácidos, especialmente do ácido lático, mas nestas amostras os ácidos propiônico e butírico não foram detectados. A ausência do ácido butírico não era esperada, uma vez que esse ácido está diretamente relacionado com o aroma característico do polvilho azedo. O fato pode sugerir que a obtenção de algumas das amostras estudadas pode ter ocorrido sem o processo fermentativo natural.

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.

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.

Effect of iron on inhibition of acid demineralisation of bovine dental enamel in vitro

Objectives: Iron ions (Fe2+) have been shown to be cariostatic in many studies particularly by their ability to reduce bacterial metabolism. Nevertheless, the role of iron ions on dissolution of enamel is unexplored. The aim of the present study was therefore to investigate the protective effect of increasing concentrations (0-120 mmol/L) of Fe2+ on the dissolution of enamel.Design: Enamel powder was subjected to acetic acid made with increasing concentrations with respect to FeSO4 center dot 7H(2)O. In order to determine the amount of enamel dissolved, the phosphate released in the medium was analysed spectrophotometrically using the Fiske-Subarrow method. Data were tested using Kruskall-Wall and Dunn's tests (p < 0.05). The degree of protection was found to approach maximum at about 15 mmol/L Fe2+. Higher concentrations of Fe2+ did not have an extra effect on inhibition of dissolution of enamel powder. In the next step, the protective effect of 15 mmol/L Fe2+ against mineral dissolution of the bovine enamel was evaluated using a simple abiotic model system. Enamel blocks were exposed to a sequence of seven plastic vials, each containing 1 mL of 10 mmol/L acetic acid. The acid in vial 4 was made 15 mmol/L with respect to FeSO4 center dot 7H(2)O. The mineral dissolved during each challenge was thus determined by phosphate released as described above. Data were tested using two-way ANOVA (p < 0.05). Results: Lower demineralisation (around 45%) was found in vial 4 (with Fe) that continued stable until vial 7.Conclusions: Thus, our data suggest that Fe2+, can be effective on inhibition of dissolution of enamel and that this effect may be durable. (c) 2006 Elsevier Ltd. All rights reserved.

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.

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.

Influence of free amino acids on clavulanic acid production by Streptomyces clavuligerus in synthetic medium

The effect of nitrogen source on clavulanic acid production was investigated in shake flasks. Media containing asparagine plus one of several different amino acids or a combination of two amino acids was tested. The best result, ca. 180 mg/L clavulanic acid, CA, in 60 h, was obtained with the lysine-tyrosine pair In an aerated and agitated fermentor this medium led to CA concentrations of ca. 210 mg/L, a remarkable production for synthetic medium utilization. Amino acids analysis during cultivation indicated that, while asparagine was consumed rapidly, lysine and tyrosine were metabolized slowly, promoting CA production.

Characterization of PLGA microparticles as a drug carrier for 3-ethoxycarbonyl-2H-benzofuro[3,2-f]-1-benzopyran-2-one. Ultrastructural study of cellular uptake and intracellular distribution

Here we describe the application of microparticles (MPs) for the delivery and release of the drug a benzopsoralen. We also evaluated the intracellular distribution and cellular uptake of the drug by using an encapsulation technique for therapeutic optimization. MPs containing the compound 3-ethoxycarbonyl-2H-benzofuro[3,2-f]-1-benzopyran-2-one (psoralen A) were prepared by the solvent evaporation technique, and parameters such as particle size, drug encapsulation efficiency, effect of the encapsulation process on the drug's photochemistry, zeta potential, external morphology, and < i > in vitro release behavior were evaluated. The intracellular distribution of MPs as well as their uptake by tissues were monitored. Size distribution studies using dynamic ligh scattering and scanning electron microscopy revealed that the MPs are spherical in shape with a diameter of 1.4 mu m. They present low tendency toward aggregation, as confirmed by their zeta potential (+10.6 mV). The loading efficiency obtained was 75%. As a consequence of the extremely low diffusivity of the drug in aqueous medium, the drug release profile of the MPs in saline phosphate buffer (pH 7.4) was much slower than that obtained in the biological environment. Among the population of peritoneal phagocytic cells, only macrophages were able to phagocytose poly-d,l-lactic-co-glycolic acid (PLGA) MP. The use of psoralen A in association with ultraviolet light (360 nm) revealed morphological characteristics of cell damage such as cytoplasmic vesiculation, mitochondria condensation, and swelling of both the granular endoplasmatic reticulum and the nuclear membrane. These results indicate that PLGA MP could be a promising delivery system for psoralen in connection with ultraviolet irradiation therapy (PUVA).