RESUMEN
The phase behavior of the ternary unsaturated monoglycerides (UMG)-DL-α-tocopheryl acetate-water system has been studied. The effects of lipid composition in both bulk and dispersed lyotropic liquid crystalline phases and microemulsions were investigated. In excess water, progressive addition of DL-α-tocopheryl acetate to a binary UMG mixture results in the following phase sequence: reversed bicontinuous cubic phase, reversed hexagonal (H(II)) phase, and a reversed microemulsion. The action of DL-α-tocopheryl acetate is then compared to that of other lipids such as triolein, limonene, tetradecane, and DL-α-tocopherol. The impact of solubilizing these hydrophobic molecules on the UMG-water phase behavior shows some common features. However, the solubilization of certain molecules, like DL-α-tocopherol, leads to the presence of the reversed micellar cubic phase (space group number 227 and symmetry Fd3m) while the solubilization of others does not. These differences in phase behavior are discussed in terms of physical-chemical characteristics of the added lipid molecule and its interaction with UMG and water. From an applications point of view, phase behavior as a function of the solubilized content of guest molecules (lipid additive in our case) is crucial since macroscopic properties such as molecular release depend strongly on the phase present. The effect of two hydrophilic emulsifiers, used to stabilize the aqueous dispersions of UMG, was studied and compared. Those were Pluronic F127, which is the most commonly used stabilizer for these kinds of inverted type structures, and the partially hydrolyzed emulsifier lecithin (Emultop EP), which is a well accepted food-grade emulsifier. The phase behavior of particles stabilized by the partially hydrolyzed lecithin is similar to that of bulk sample at full hydration, but this emulsifier interacts significantly with the internal structure and affects it much more than F127.
Asunto(s)
Agua/química , alfa-Tocoferol/química , Alcanos/química , Ciclohexenos/química , Emulsionantes/química , Emulsiones , Interacciones Hidrofóbicas e Hidrofílicas , Lecitinas/química , Limoneno , Micelas , Transición de Fase , Poloxámero/química , Solubilidad , Terpenos/química , Trioleína/químicaRESUMEN
Low molecular weight surfactants, for example monoglycerides and phospholipids, form a multitude of self-assembled structures, such as inverted cubic or hexagonal mesophases, if brought into contact with water/oil. These mesophases can be dispersed in water using adequate surface-active materials such as low molecular weight surfactants or surface active polymers. In order to use such mesophase particles for incorporating drugs and aromas, it is essential to determine their internal crystallographic structure and to understand their mechanism of stabilization. Cryo-transmission electron microscopy was used to investigate the internal structure of different dispersed particles at various temperatures and oil contents. It is shown here that cryo-transmission electron microscopy, in combination with fast Fourier transform and tilting experiments, is effective in obtaining information on crystallographic structure, space group and morphology of particles with reversed bicontinuous cubic and hexagonal structures. In particular, using the presence or the absence of the {111} reflections and viewing the same particle under different axes of observation allows one to discriminate between the Im3m and Pn3m space groups. A major advantage of cryo-transmission electron microscopy is the ability to analyse single particles. This allows the identification of particles present at very low concentrations and the coexistence of particles with different internal self-assembly structures. With this technique we have obtained strong evidence for the presence of two cubic internal self-assembly structures with different space groups within the same dispersion. In addition, we found that cryo-transmission electron microscopy combined with tilting experiments enables the analysis of internal particle morphology, allowing the discussion of mechanisms for hexosome stabilization.
RESUMEN
The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent. Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G(1) phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation of Giardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.
Asunto(s)
Adhesión Celular , Giardia lamblia/crecimiento & desarrollo , Mucosa Intestinal/parasitología , Lactobacillus/metabolismo , Animales , Células CACO-2 , Gatos , Medios de Cultivo Condicionados , Perros , Citometría de Flujo , Giardia lamblia/efectos de los fármacos , Giardia lamblia/fisiología , Giardiasis/parasitología , Interacciones Huésped-Parásitos , Humanos , Lactobacillus/crecimiento & desarrollo , Microscopía Electrónica de RastreoRESUMEN
The carbohydrate-binding specificities of the probiotic lactic acid bacterium Lactobacillus johnsonii La1 (a health-beneficial bacterial strain able to be incorporated into the human intestinal microflora) were investigated in vitro. First various soluble complex carbohydrates were tested as potential inhibitors of the strain adhesion onto Caco-2 intestinal epithelial cells, and then bacterial binding to glycolipids immobilized on TLC plates was probed. Two major carbohydrate-binding specificities of Lactobacillus johnsonii La1 were identified. A first one for an Endo-H treated yeast cell wall mannoprotein carrying mainly O:-linked oligomannosides, and a second one for the gangliotri- and gangliotetra-osylceramides (asialo-GM1). Similar carbohydrate-binding specificities are known to be expressed on cell surface adhesins of several enteropathogens, enabling them to adhere to the host gut mucosa. These findings corroborate the hypothesis that selected probiotic bacterial strains could be able to compete with enteropathogens for the same carbohydrate receptors in the gut.
Asunto(s)
Adhesión Bacteriana/fisiología , Metabolismo de los Hidratos de Carbono , Enterobacteriaceae/fisiología , Enterobacteriaceae/patogenicidad , Lactobacillus/fisiología , Adhesinas Bacterianas/fisiología , Adhesión Bacteriana/efectos de los fármacos , Sitios de Unión , Unión Competitiva , Células CACO-2 , Secuencia de Carbohidratos , Carbohidratos/farmacología , Cromatografía en Capa Delgada , Glucolípidos/metabolismo , Glucolípidos/farmacología , Humanos , Técnicas In Vitro , Intestinos/microbiología , Datos de Secuencia Molecular , ProbióticosRESUMEN
BACKGROUND: Specific strains of Lactobacillus acidophilus are known to inhibit intestinal cell adhesion and invasion by enterovirulent bacteria. As L. acidophilus can survive transiently in the human stomach, it may downregulate Helicobacter pylori infection. METHODS: The ability of L. acidophilus (johnsonii) La1 supernatant to interfere with H. pylori bacterial growth, urease activity, and adhesion to epithelial cells was tested in vitro. Its effect on H. pylori infection in volunteers was monitored in a randomized, double-blind, controlled clinical trial, using a drinkable, whey-based, La1 culture supernatant. H. pylori infected volunteers were treated 14 days with 50 ml of La1 supernatant four times a day combined with either omeprazole 20 mg four times a day or with placebo. Infection was assessed by breath test, endoscopy, and biopsy sampling, performed at inclusion, immediately at the end of the treatment (breath test only), and 4 weeks after the end of the treatment. RESULTS: La1 supernatant inhibited H. pylori growth in vitro, regardless of previous binding of H. pylori to epithelial cells. In 20 subjects (8 females, 12 males, mean age 33.1 years) a marked decrease in breath test values was observed immediately after treatment with La1 supernatant, both in the omeprazole and in the placebo group (median 12.3 vs. 28.8 and 9.4 vs. 20.4, respectively; p < 0.03). In both treatment groups, breath test values remained low 6 weeks after treatment (omeprazole treated 19.2, placebo treated 8. 3; p < 0.03 vs. pretreatment), but the persistence of H. pylori infection was confirmed in gastric biopsies. CONCLUSION: La1 culture supernatant shown to be effective in vitro has a partial, acid-independent long-term suppressive effect on H. pylori in humans.
Asunto(s)
Gastritis/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/crecimiento & desarrollo , Lactobacillus acidophilus/fisiología , Adolescente , Adulto , Anciano , Anticuerpos Antibacterianos/análisis , Adhesión Bacteriana , Pruebas Respiratorias , Recuento de Colonia Microbiana , Método Doble Ciego , Femenino , Estudios de Seguimiento , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Gastritis/tratamiento farmacológico , Gastritis/metabolismo , Células HT29/microbiología , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/enzimología , Helicobacter pylori/inmunología , Humanos , Ácido Láctico/metabolismo , Masculino , Persona de Mediana Edad , Omeprazol/uso terapéutico , Resultado del Tratamiento , Ureasa/metabolismoRESUMEN
The influence of pH on the adhesion of two Lactobacillus strains to Caco-2 human intestinal cells was investigated. One strain, Lactobacillus johnsonii La1, was adherent at any pH between 4 and 7. The other one, L. acidophilus La10, did not attach to this cell line under the same experimental conditions. On the basis of these results, we used the monoclonal antibody technique as a tool to determine differences on the surface of these bacteria and to identify a factor for adhesion. Mice were immunized with live La1, and the hybridomas produced by fusion of spleen cells with ONS1 cells were screened for the production of antibodies specific for L. johnsonii La1. A set of these monoclonal antibodies was directed against a nonproteinaceous component of the L. johnsonii La1 surface. It was identified as lipoteichoic acid (LTA). This molecule was isolated, chemically characterized, and tested in adhesion experiments in the same system. The adhesion of L. johnsonii La1 to Caco-2 cells was inhibited in a concentration-dependent way by purified LTA as well as by L. johnsonii La1 culture supernatant that contained LTA. These results showed that the mechanism of adhesion of L. johnsonii La1 to human Caco-2 cells involves LTA.
Asunto(s)
Adhesión Bacteriana , Lactobacillus/fisiología , Lipopolisacáridos/metabolismo , Ácidos Teicoicos/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Células CACO-2 , Humanos , Concentración de Iones de Hidrógeno , Inmunización , Inmunohistoquímica , Lactobacillus/química , Lactobacillus acidophilus/química , Lactobacillus acidophilus/fisiología , Lipopolisacáridos/análisis , Proteínas de la Membrana/inmunología , Ratones , Ácidos Teicoicos/análisisRESUMEN
The protective effects of milk and milk products against dental caries have been demonstrated in many animal studies. We have shown that this effect was mediated by micellar casein or caseinopeptide derivatives. A reduction in the Streptococcus sobrinus population in the oral microbiota of animals fed diets supplemented with these milk components was consistently observed. A possible explanation for these findings is that milk components are incorporated into the salivary pellicle, thereby reducing the adherence of S. sobrinus. This hypothesis was tested in vitro by the incubation of bovine enamel discs with unstimulated saliva. The resulting pellicle was washed and incubated with caseinoglycomacropeptide (CGMP) and/or caseinophosphopeptide (CPP) labeled with 17- and 12-nm gold particles. All samples were prepared for electron microscopy by high-pressure freezing followed by freeze-substitution. It was demonstrated by high-resolution scanning electron microscopy with back-scattered electron imaging, as well as by transmission electron microscopy, that both peptides were incorporated into the pellicle in exchange for albumin, confirming previous findings. This protein was identified with a mouse anti-human serum albumin followed by goat anti-mouse IgG labeled with 25-nm gold particles. Incorporation of CGMP and/or CPP into salivary pellicles reduced the adherence of both S. sobrinus and S. mutans significantly. It is suggested that the calcium and phosphate-rich micellar casein or caseinopeptides are incorporated into the pellicle. The resulting ecological shifts, together with the increased remineralization potential of this biofilm, may explain its modified cariogenic potential.
Asunto(s)
Adhesión Bacteriana , Caseínas/metabolismo , Caries Dental/etiología , Esmalte Dental/microbiología , Fragmentos de Péptidos/metabolismo , Fosfopéptidos/metabolismo , Saliva/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Streptococcus mutans/fisiología , Streptococcus sobrinus/fisiología , Adulto , Animales , Bovinos , Interpretación Estadística de Datos , Caries Dental/prevención & control , Depósitos Dentarios/microbiología , Película Dental , Durapatita , Congelación , Humanos , Técnicas In Vitro , Ratones , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Microtomía , Persona de Mediana Edad , LecheRESUMEN
Bovine caseinate, derivatives of its glycosylated moiety [caseinoglycomacropeptide (CGP)], and caseinophosphopeptides were evaluated as inhibitors of adhesion of oral bacteria to saliva-coated hydroxyapatite beads (S-HA). All milk casein-derived components behaved as potent inhibitors of Streptococcus sanguis OMZ 9 and Streptococcus sobrinus OMZ 176 adhesion to S-HA, whereas neither bovine serum albumin nor polyethyleneglycol were able to interfere with the adhesion of these strains. By contrast, none of the molecular species tested was able to inhibit the attachment of Actinomyces viscosus Ny 1 to S-HA. On the other hand, casein derivatives were shown to displace human serum albumin from S-HA beads. They were also able to bind to the bacterial cell surface of all strains examined. Collectively, these findings suggest that interactions between acidic casein-derived milk components and the biological surfaces involved in bacterial adhesion to S-HA result in an inhibitory effect that is selective for the oral streptococci examined.