Functional properties of exopolysaccharide (EPS) extract from L. fermentum Lf2 and their analysis when combined with B. animalis INL1 in yogurt

ALE, C. ELISA; BURNS, PATRICIA; PERALTA, GUILLERMO H.; ÁVILA, O.B.; CONTINI, L.; REINHEIMER, JORGE A.; BINETTI, ANA G.

Congreso; VII Congreso Internacional de Ciencia y Tecnología de Alimentos (CICYTAC 2018); 2018

Lactobacillus fermentum Lf2 is an autochthonous strain which was isolated as non-starter culture from a local semi-hard cheese. This strain produces high amounts of EPS when it grows under controlled conditions of temperature and pH, reaching 0.8 g/L, approximately, in SDM medium. In the present study, the functional roles of an EPS extract from L. fermentum Lf2 (obtained by ethanol precipitation) were studied by means of in vitro and in vivo assays, isolated or combined with a probiotic strain, Bifidobacterium animalis subsp. lactis INL1, and both applied as food ingredients in yogurt. First, a preliminary in vitro analysis was done with THP-1 cell line. Macrophages derived from THP-1 were stimulated with crude or purified EPS: 60 μg/mL of the crude extract and 12.6 μg/mL of the purified form. This last concentration was proposed taking into account that, after purification of the crude EPS extract, 21% (approximately) is recovered. A positive control treated with lipopolysaccharide (LPS, 0.5 μg/mL) was also included. The cells were incubated at 37 °C, with 5% CO2 for 4 h for the detection of TNF-α, and 8 h for IL-6 and IL-10 determinations. The cytokine analysis was performed from the culture supernatants using the DuoSet ELISA kits (R&D Systems, Minneapolis, United States). A negative control with untreated cells was also included. On the other hand, an in vivo trial was done, in which BALB/c mice (7/group) received yogurt with EPS (600 mg/L, YE), yogurt with bifidobacteria at a concentration of 5x108 CFU/mL (YB), a combination of both ingredients (YEB), or yogurt with no additives (Y, control group), for 25 days. During treatment, different bacterial groups (Lactobacillus, Bifidobacterium, B. animalis, B. bifidum, B. breve, B. catenulatum, C. leptum, C. coccoides, Staphylococcus, Enterobacteriaceae, Streptococcus, general microbial load with universal primers) and short chain fatty acids (SCFA) were determined in faeces by qPCR and HPLC, respectively (at 0, 8, 18 and 25 days). Besides, IgA levels were quantified in intestinal fluid and cytokines IL-10, IFN-γ, IL-6 and TNF-α were measured in small intestine by ELISA. Histological analyses were carried out to in order to evaluate the morphology of small and large intestines. The group YE presented increased concentrations of acetic, butyric and total SCFA (the sum of acetic, butyric and propionic acids) at the end of the treatment, and increasing levels of the C. coccoides cluster (SCFA producing group) over time (p< 0.05). The group YEB presented a possible symbiotic role reflected mainly in the levels of the species B. animalis at all times evaluated. These results highlight the potential application of the EPS of L. fermentum Lf2 as a functional ingredient, alone or together with B. animalis subsp. lactis INL1, both autochthonous strains