Chemical and structural characterization of the exopolysaccharides (EPS) produced by Lactobacillus fermentum Lf2

ELISA C. ALE; JORGE A. REINHEIMER; ANA G. BINETTI

Córdoba

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

Lactobacillus fermentum Lf2 is an autochthonous strain which was isolated from regional semihard cheese. Its interest resides on the ability to produce high amounts of EPS (~ 0.8 g/L) when it grows under controlled conditions. The crude EPS extract was obtained by ethanol precipitation. Briefly, bacteria were grown in SDM broth in a 2 L fermentor (Sartorius Biostat A plus, Goettingen, Germany) at 30°C for 72 h, with continuous agitation (6 g) and sparging with CO2 (0,2 L/min). pH was kept automatically constant at a value of 6 with sterile NaOH 8 M. Cells were removed by centrifugation (19000 g, 30 min, 4 ºC) and the EPS was precipitated from the supernatant by the addition of 2 volumes of chilled absolute ethanol. After 48 h, EPS was obtained by centrifugation (4000 g, 30 min, 4 ºC), then it was resuspended in bidistilled water, dialyzed against 5 L of distilled water (MWCO 12000 Da) for 3 days and the extract was finally freeze-dried. In order to obtain the purified EPS, the crude extract was treated with DNAse I and Pronase E (both from Sigma Aldrich, St. Louis, MO, USA), and a precipitation with 12% (m/v) of TCA was done to eliminate peptides and proteins. The supernatant was dialyzed and freeze dried as described previously. This purified extract was analyzed by different techniques: NMR (Nuclear Magnetic Resonance), SEC-MALLS (Size Exclusion Chomatography- Multi-Angle Laser Light Scattering), GC-MS (Gas Chromatography-Mass Spectrometry) and HPLC (High Performance Liquid Chromatography) in order to study the chemical structure and composition. It was possible to elucidate that the extract was composed of two fractions of homo and hetero-polysaccharides: a β-glucan of relatively high molecular weight (1.8x103 kDa), whose repetitive unit was a trisaccharide composed of glucose (two glucoses linked by β(1,3) bonds and a branch composed of a glucose molecule linked by β(1,2) bonds to the second monosaccharide) and another, of medium molecular weight (90 kDa), whose repetitive unit was a disaccharide formed by glucose and galactose (the backbone consists of galactose molecules linked by α(1,6) bonds, all molecules with glucose branches linked at position 2). Both fractions were present in similar proportions, approximately 40 and 45%, respectively, in the purified EPS extract. Also, it was possible to detect another fraction of medium molecular weight that represented only 10%, and whose composition and structure are currently under study. All this knowledge is of great value because relationships of this EPS extract with its functional and technological roles could be disclosed.