Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures
Introduction
The human colonic microbiota is a complex ecosystem dominated by obligate anaerobes that can reach levels of over 1011 cells per gram of contents (Bäckhed et al., 2005). Dietary compounds not digested by the host are the main carbon source from which gut microorganisms obtain energy. These substrates, which mainly include plant oligo and polysaccharides, can be subjected to gut bacterial metabolic transformations and hence they can have a major influence on human nutrition and health (Flint et al., 2008). Short chain fatty acids (SCFA) and lactate are the main products arising from the microbial fermentation of carbohydrates. SCFA can provide energy to the colonic epithelium, modulate cholesterol and lipid metabolism, suppress pathogenic intestinal bacteria and modulate the immune system (Cummings and Macfarlane, 1997, Topping and Clifton, 2001). Chemical composition and availability of fermentable carbohydrates in the colon have a strong influence on the gut microbiota composition. Specific carbohydrates are now being used as prebiotics based on the concept that they stimulate selected beneficial bacteria in the colon (Gibson and Roberfroid, 1995). The most studied prebiotics are inulin and fructooligosaccharides (FOS) (Roberfroid, 2007), and they have a recognised bifidogenic effect (Bouhnik et al., 2004, Gibson et al., 1995, Kruse et al., 1999, Rossi et al., 2005).
Many strains of lactic acid bacteria and bifidobacteria are able to produce exopolysaccharides (EPS). The physiological functions of these carbohydrate polymers have not yet been clearly determined (Ruas-Madiedo et al., 2008). Among the beneficial effects attributed to these biopolymers such as cholesterol lowering capability and immunomodulating ability, the possibility of acting as fermentable substrates in vitro has been demonstrated to date by Korakli et al. (2002) for a fructan-type EPS produced by a strain of Lactobacillus sanfranciscensis. There was also evidence of an in vitro bifidogenic effect for the levan-type EPS produced by another strain of the same species (Dal Bello et al., 2001). We have recently demonstrated in a non pH-controlled faecal slurry culture that EPS isolated from different human intestinal bifidobacteria can act as fermentable substrates for microbial intestinal populations (Salazar et al., 2008). This promoted shifts in SCFA profiles, moderate increases in bifidobacteria populations, and changes in banding patterns when cultures were analysed by PCR-DGGE using universal primers, which indicated microbial population shifts.
Bifidobacteria are intestinal microorganisms that are being extensively used as probiotics, based on health-promoting benefits attributed to them (Masco et al., 2005). In order to gain further insight into the changes that EPS from bifidobacteria may mediate in the intestinal microbiota, in the present work we quantified modifications in the levels of selected microbial groups by fluorescent in situ hybridisation (FISH) and established possible relationships between metabolic changes and variations in microbial populations by using pH-controlled faecal batch cultures. Compared to our previous non pH-controlled experiments (Salazar et al., 2008), this design is a more realistic simulation of the conditions in the human distal colon with regard to local pH, substrate availability and transit time, as the semisolid luminal contents of the distal colon are almost static therein. Two microbial polymers were used in this study as carbon sources; EPS E44 was produced by a human faecal isolate of Bifidobacterium longum subsp. longum, one of the most abundant species of Bifidobacterium found in the faecal samples of donors from our region (Asturias, North of Spain) (Delgado et al., 2006). This EPS was chosen on the basis of the high reduction of acetic to propionic acid ratio promoted by polymers of the species B. longum and particularly by E44 (Salazar et al., 2008). The EPS R1 was produced by a strain of Bifidobacterium animalis subsp. lactis, species widely used as adjunct culture in functional dairy products. Cultures of B. animalis subsp. lactis IPLA R1 present a ropy phenotype which could be an interesting technological property (Salazar et al., 2009). Both strains were considered in order to ascertain if the origin of the EPS-producing strain and their different monosaccharide composition could influence microbiota dynamics. The use of bifidobacteria producing fermentable EPS may have the advantage over other known prebiotic substrates of the use/administration of both, a potential probiotic and a fermentable substrate, in a single step.
Section snippets
EPS-producing strains and culture conditions
B. longum subsp. longum IPLA E44 was previously isolated from faeces of a healthy adult volunteer with the approval of the Regional Ethics Committee (Asturias, Spain; Delgado et al., 2006). B. animalis subsp. lactis IPLA R1 was of dairy origin (Ruas-Madiedo et al., 2006). Both strains are held in the IPLA's culture collection. Strain identity was confirmed by partial amplification of the 16S rRNA gene using primers Y1–Y2 (Young et al., 1991), sequencing and alignment with sequences from
Susceptibility of EPS to degradation under simulated gastrointestinal conditions
Susceptibility of the EPS E44 and R1 to simulated gastric and intestinal juices was evaluated by possible modifications on the amount of EPS recovered before and after submission to treatments. The amount of the highest peak of both EPS fractions (7.4 ± 2.50 × 105 g/mol and 1.55 ± 0.20 × 106 g/mol, for E44 and R1, respectively) recovered from SEC did not show significant variation after any treatment (Fig. 1). Thereby, E44 and R1 were not degraded during submission to simulated gastric and intestinal
Discussion
We recently demonstrated that EPS isolated from bifidobacteria of human origin could act as fermentable substrates for intestinal bacteria and qualitative rearrangements of several microbial populations were evidenced by PCR-DGGE (Salazar et al., 2008). However, EPS could only act as fermentable substrates for colonic bacteria if they arrive not degraded to the colon. Under the conditions used in our study, EPS E44 and R1 did not suffer an in vitro breakdown by simulated gastric and intestinal
Acknowledgements
This work was financially supported by the European Union FEDER funds and by the Plan Nacional de I + D under project AGL2007-62736. N. Salazar was the recipient of a predoctoral fellowship from the Spanish Ministry of Science and Innovation (FPI Program).
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2022, Food and Bioproducts ProcessingCitation Excerpt :After 24 h of cultures, environmental challenges were then performed independently for 3 h as shown in Table 1. EPS were extracted from cell suspensions collected according to the method described by Salazar et al. (2009) and Nguyen et al. (2014). EPS total produced by L. plantarum VAL6 was harvested after 24 h by mixing 100 mL of supernatant with an equal amount of 2 M NaOH and stirring gently overnight at room temperature.