ABSTRACT
In natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. These natural conditions can be simulated using retentostat cultivations. The present study describes the physiological and proteome adaptations of the probiotic Bifidobacterium breve NRBB57 from high (0.4 h-1) to near-zero growth rates. Lactose-limited retentostat cultivations were carried out for 21 days in which the bacterial growth rate progressively reduced to 0.00092 h-1, leading to a 3.4-fold reduction of the maintenance energy requirement. Lactose was mainly converted into acetate, formate, and ethanol at high growth rates, while in the retentostat, lactate production increased. Interestingly, the consumption of several amino acids (serine, aspartic acid, and glutamine/arginine) and glycerol increased over time in the retentostat. Morphological changes and viable but nonculturable cells were also observed in the retentostat. Proteomes were compared for all growth rates, revealing a downregulation of ribosomal proteins at near-zero growth rates and an upregulation of proteins involved in the catabolism of alternative energy sources. Finally, we observed induction of the stringent response and stress defense systems. Retentostat cultivations were proven useful to study the physiology of B. breve, mimicking the nutrient scarcity of its complex habitat, the human gut. IMPORTANCE In natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. In this study we used retentostat cultivation to investigate how the probiotic Bifidobacterium breve adapts its physiology and proteome under severe nutrient limitation resulting in near-zero growth rates (<0.001 h-1). We showed that the nutrient limitation induced a multifaceted response including stress defense and stringent response, metabolic shifts, and the activation of novel alternative energy-producing pathways.
Subject(s)
Bifidobacterium breve , Proteome , Humans , Lactose , Ecosystem , Adaptation, PhysiologicalABSTRACT
En el presente estudio se evaluó la capacidad probiótica in vitro de una cepa nativa de Saccharomyces cerevisiae (A) y se comparó con una cepa comercial (B) utilizada como probiótico. Para esto se determinó la concentración de melaza de caña (10, 20 y 30 por ciento (p/v)) que permitiera obtener la mayor cantidad de biomasa de las cepas, así mismo se determinaron parámetros cinéticos. La concentración de melaza que arrojó mejores resultados fue 20por ciento (p/v) y se encontró diferencia en la producción de biomasa para la cepa en estudio A (28g/L) y la cepa control B (3g/L) en medio melaza. Se realizaron pruebas in vitro como resistencia a sales biliares, tolerancia a rangos de pH y jugos gástricos, donde no se observaron diferencias entre la cepa A y B al medir el crecimiento. La reducción del colesterol en presencia de sales biliares después de 12 horas de incubación fue de 54 por ciento para la cepa A y 58 por ciento para la B. Por último se realizó una prueba de adherencia en células Caco-2, encontrando adherencia a estas por parte de ambas cepas. De acuerdo con los resultados anteriores se demostró que la cepa A tiene propiedades probióticas in vitro que pueden ser corroboradas con posteriores estudios in vivo que confirmen su utilización como probiótico en nutrición animal.
The in vitro probiotic capacity of a native strain of Saccharomyces cerevisiae (A) was evaluated and compared with a commercial strain (B) used as a probiotic. The effect of the concentration of sugarcane molasses (10, 20 and 30% (w/v)) on the biomass production was investigated and kinetic parameters were determined. The best molasses concentration was 20% (w/v) and differences in biomass production on molasses medium between strain A (28 g/L) and control strain B (3 g/L) were observed. In vitro tests such as tolerance to bile salts, pH and gastric juices were carried out, and no differences in growth between strain A and B were found. Cholesterolreduction on presence of bile salts after 12 hours of incubation was of 54% for strain A and 58% for strain B. Both strains showed adherence to Caco-2 cells. Results reveal that strain A possesses in vitro probiotic propertiesthat can be verified with further in vivo studies to confirm its suitability as probiotic in animal nutrition.
