Screening in a Lactobacillus delbrueckii subsp. bulgaricus Collection to select a strain able to survive to the human intestinal tract / Estudio de una colección de Lactobacillus delbrueckii subsp. bulgaricus y selección de una cepa capaz de sobrevivir en el tracto digestivo humano

Objectives: Genetic diversity and resistance of Lactobacillus bulgaricus sbsp. delbrueckii collection with 100 isolates from different home-made yogurt in rural Bulgarian areas were determined. Methods: The strain K98 was the most resistant to bile salts and low pH. Survival and effects on short chain fatty acids production were tested in 20 healthy volunteers. High genetic diversity was observed in the L. bulgaricus collection by RAPD, whereas the ability of tolerate high deoxycholic acid concentrations, and different acid pHs was variable. The strain K98 was selected and used to prepare a homemade yogurt which was administered to 20 healthy volunteers (500 ml/day during 15d). A basal faecal sample and another after yogurt intake were recovered. Results: DGGE experiments, using both universal and Lactic Acid Bacteria (LAB) primers, demonstrated no significant changes in the qualitative composition of gut microbiota. A band corresponding to L. bulgaricus was observed in all 20 samples. Viable L. bulgaricus K98 strain was only recovered in one volunteer. After yogurt intake we found an increase of LAB and Clostridium perfringens, and a decrease of Bacteroides-Prevotella-Porphyromonas. In addition, increases of acetic, butyric and 2-hydroxybutyric acids in faeces were detected. Conclusions: Genetic diversity of L. delbrueckii subsp. bulgaricus especie is high We have isolated a probiotic resistant strain to bile and high acidity, L. delbrueckii subsp. bulgaricus-K98. Qualitative and quantitative changes in the intestinal microbiota are found after ingestion of a homemade yogurt containing this strain, with a concomitant increase in faecal SCFA. Our findings support the interest in developing further studies providing different amounts of L. delbrueckii subsp. bulgaricus-K98, and should evaluate its clinical effects in human disease (AU)

Objetivos: Se determinaron la diversidad genética y la resistencia de una colección de más de 100 cepas de Lactobacillus bulgaricus subespecie delbrueckii, aisladas de diferentes yogures caseros de las áreas rurales de Bulgaria. Métodos: La cepa K98 fue la más resistente a las sales biliares y al pH bajo. La supervivencia y los efectos sobre la producción de ácidos grasos de cadena corta se evaluó en 20 voluntarios sanos. Se observó una alta diversidad genética en la colección de L. bulgaricus mediante RAPD, mientras que la capacidad de tolerar concentraciones altas del ácido desoxicólico y de diferentes niveles de pH fue variable. Se seleccionó la cepa K98 y se usó para preparar un yogur casero que se administró a los 20 voluntarios (500 ml/día durante 15 días). Se recogieron muestras fecales basales y tras la ingesta del yogur. Resultados: Los experimentos DGGE, empleando cebadores universales y para bacterias ácidolácticas (BAL) demostraron que no hubo cambios significativos en la composición cualitativa de la composición de la microflora intestinal. Se observó una banda correspondiente a L. bulgaricus en las 20 muestras. Sólo se recuperó una cepa viable de L. bulgaricus K98 en un único voluntario. Tras la ingesta de yogur, hallamos un aumento de BAL y de Clostridium perfringens y una disminución de Bacteroides-Prevotella-Porphyromonas. Además, se detectó un aumento en las heces de los ácidos acético, butírico y 2-hidroxibutírico. Conclusiones: La diversidad genética de L. delbrueckii subespecie bulgaricus es alta. Hemos aislado una cepa probiótica resistente a la bilis y a la acidez elevada, L. delbrueckii subesp. bulgaricus-K98. Se hallaron cambios cualitativos y cuantitativos en la microflora intestinal tras la ingesta de yogur casero que contenía esta cepa, con un aumento concomitante en las heces de AGCC. Nuestros hallazgos apoyan el interés por desarrollar estudios futuros con cantidades variables de L. delbrueckii subesp. bulgaricus-K98, y que evalúen sus efectos clínicos en la enfermedad humana (AU)

Screening in a Lactobacillus delbrueckii subsp. bulgaricus collection to select a strain able to survive to the human intestinal tract.

OBJECTIVES: Genetic diversity and resistance of Lactobacillus bulgaricus sbsp. delbrueckii collection with 100 isolates from different home-made yogurt in rural Bulgarian areas were determined. METHODS: The strain K98 was the most resistant to bile salts and low pH. Survival and effects on short chain fatty acids production were tested in 20 healthy volunteers. High genetic diversity was observed in the L. bulgaricus collection by RAPD, whereas the ability of tolerate high deoxycholic acid concentrations, and different acid pHs was variable. The strain K98 was selected and used to prepare a homemade yogurt which was administered to 20 healthy volunteers (500 ml/day during 15d). A basal faecal sample and another after yogurt intake were recovered. RESULTS: DGGE experiments, using both universal and Lactic Acid Bacteria (LAB) primers, demonstrated no significant changes in the qualitative composition of gut microbiota. A band corresponding to L. bulgaricus was observed in all 20 samples. Viable L. bulgaricus K98 strain was only recovered in one volunteer. After yogurt intake we found an increase of LAB and Clostridium perfringens, and a decrease of Bacteroides- Prevotella-Porphyromonas. In addition, increases of acetic, butyric and 2-hydroxy-butyric acids in faeces were detected. CONCLUSIONS: Genetic diversity of L. delbrueckii subsp. bulgaricus especie is high We have isolated a probiotic resistant strain to bile and high acidity, L. delbrueckii subsp. bulgaricus-K98. Qualitative and quantitative changes in the intestinal microbiota are found after ingestion of a homemade yogurt containing this strain, with a concomitant increase in faecal SCFA. Our findings support the interest in developing further studies providing different amounts of L. delbrueckii subsp. bulgaricus-K98, and should evaluate its clinical effects in human disease.

Objetivos: Se determinaron la diversidad genética y la resistencia de una colección de más de 100 cepas de Lactobacillus bulgaricus subespecie delbrueckii, aisladas de diferentes yogures caseros de las áreas rurales de Bulgaria. Métodos: La cepa K98 fue la más resistente a las sales biliares y al pH bajo. La supervivencia y los efectos sobre la producción de ácidos grasos de cadena corta se evaluó en 20 voluntarios sanos. Se observó una alta diversidad genética en la colección de L. bulgaricus mediante RAPD, mientras que la capacidad de tolerar concentraciones altas del ácido desoxicólico y de diferentes niveles de pH fue variable. Se seleccionó la cepa K98 y se usó para preparar un yogur casero que se administró a los 20 voluntarios (500 ml/día durante 15 días). Se recogieron muestras fecales basales y tras la ingesta del yogur. Resultados: Los experimentos DGGE, empleando cebadores universales y para bacterias ácido-lácticas (BAL) demostraron que no hubo cambios significativos en la composición cualitativa de la composición de la microflora intestinal. Se observó una banda correspondiente a L. bulgaricus en las 20 muestras. Sólo se recuperó una cepa viable de L. bulgaricus K98 en un único voluntario. Tras la ingesta de yogur, hallamos un aumento de BAL y de Clostridium perfringens y una disminución de Bacteroides- Prevotella-Porphyromonas. Además, se detectó un aumento en las heces de los ácidos acético, butírico y 2-hidroxibutírico. Conclusiones: La diversidad genética de L. delbrueckii subespecie bulgaricus es alta. Hemos aislado una cepa probiótica resistente a la bilis y a la acidez elevada, L. delbrueckii subesp. bulgaricus-K98. Se hallaron cambios cualitativos y cuantitativos en la microflora intestinal tras la ingesta de yogur casero que contenía esta cepa, con un aumento concomitante en las heces de AGCC. Nuestros hallazgos apoyan el interés por desarrollar estudios futuros con cantidades variables de L. delbrueckii subesp. bulgaricus-K98, y que evalúen sus efectos clínicos en la enfermedad humana.

Molecular analysis of yogurt containing Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in human intestinal microbiota.

BACKGROUND: Yogurt has traditionally been considered a probiotic-carrier food with health-promoting effects. Despite the universal assumption of this assertion, several researchers have evaluated the real capability of the yogurt bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to survive and proliferate in the human intestine and have found contradictory results. OBJECTIVE: This double-blind crossover study assessed the qualitative and quantitative effects of fresh and heat-treated yogurt on bacterial intestinal microbiota from healthy subjects. DESIGN: The subjects were divided into experimental (n=63) and control (n=16) groups. The experimental group consumed fresh and heat-treated yogurt for 15 d according to a crossover design with a washout period of 2 wk. Three different fecal samples per individual were recovered: at baseline, after fresh yogurt intake, and after heat-treated yogurt intake. Qualitative changes in microbiota were studied by denaturing gel gradient electrophoresis (DGGE) with universal and lactic acid bacteria (LAB) 16S-rRNA primers. Quantitative changes in LAB, Clostridium coccoides, Clostridium perfringens, and Bacteroides groups were analyzed by real-time polymerase chain reaction. RESULTS: A particular DGGE stable band pattern was observed in each sample. No significant qualitative differences were detected in any fecal sample. However, a significantly higher density of LAB and C. perfringens and a significant decrease in the density of Bacteroides was observed after consumption of both types of yogurt. Microbiota density was not significantly different between the fresh and heat-treated yogurt groups, except for LAB, which was significantly greater in the fresh yogurt group. CONCLUSION: The main change in human microbiota observed after yogurt consumption was an increase in the density of LAB and C. perfringens to the detriment of Bacteroides. Bacterial changes were not different after the consumption of fresh and heat-treated yogurt.

Molecular and microbiological analysis of caecal microbiota in rats fed with diets supplemented either with prebiotics or probiotics.

The potential health-improving effects of both a prebiotic and a probiotic infant formula have been evaluated in a rat model. Two groups of 10 rats were fed with either prebiotics containing fructo-oligosaccharides or probiotics containing viable Bifidobacterium lactis and Streptococcus thermophilus. The composition of their caecal microbiota was analyzed both by classical plate count of the main bacterial groups and by PCR amplification of a V3 fragment of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE). Both diets induced a significant reduction of clostridia and Bacteroides spp. compared to a control diet, whereas prebiotics were also able to reduce the number of coliforms and to increase the presence of bifidobacteria. DGGE analysis showed a significant increase of 16S rRNA gene fragments in rats fed with either probotics or prebiotics. Nineteen bands were sequenced and most of them showed similarity to cultured bacteria. Detection of Bifidobacterium spp. by this technique using genus-specific primers only permitted these bacteria to be detected in prebiotics-fed rats, whereas the use of Lactobacillus group-specific primers gave similar results in rats fed with any diet, in agreement with the plate count results.

Scarce evidence of yogurt lactic acid bacteria in human feces after daily yogurt consumption by healthy volunteers.

In a double-blind prospective study including 114 healthy young volunteers, the presence in human feces of the yogurt organisms Lactobacillus delbrueckii and Streptococcus thermophilus after repeated yogurt consumption (15 days) was analyzed by culture, specific PCR, and DNA hybridization of total fecal DNA. Detection of yogurt lactic acid bacteria in total fecal DNA by bacterial culture and PCR assay was consistently negative. DNA compatible with yogurt bacteria was found by hybridization experiments in only 10 (10.52%) of 96 individuals after consumption of fresh yogurt and in 2 (2.10%) of 96 individuals after consumption of pasteurized yogurt (P = 0.01).