-PHARMACEUTICAL BIOTECHNOLOGY: METHODS TO OPTIMIZE THE PRODUCTION OF METABOLITES FROM LACTOBACILLUS PHARMACOLOGICALLY ACTIVES

ALBERTO N RAMOS; MARIA DE LOS ANGELES LINDON; MARIA EMILIA CRUZ; MARIA E SESTO CABRAL; CABRERA CARLA AGOSTINA; JUAN C VALDEZ

Tucuman

Simposio; IV Simposio Internacional de Bacterias Lácticas SIBAL; 2013

CERELA

Our medical team applied Lactobacillus plantarum (whole culture and supernatants) in chronic wounds (diabetic foot ulcers, venous ulcers, burns, etc) with very encouraging results. In previous works we demonstrated that L. plantarum culture supernatants (LpS) have antipathogenic activities (on bacteria typically isolated from chronic wounds), pro-healing and anesthetic properties. Also was determined the bioactive metabolites responsible for the aforementioned properties and its action mechanism was hypothesized. The aim of this work was to design methods and culture media that maximize the production of the above metabolites to increase the therapeutic effectiveness of the supernatants. Modifications were made in MRS broth on its composition and concentration of carbohydrates, proteins, boron, cations and surfactants sources. L. plantarum was cultivated in the different media for 12 h at 37 °C with distinct physicochemical conditions (CO2 concentration, agitation, etc.). Modified supernatants (LpSm) were obtained by centrifugation and filtration. Antipathogenic activity of LpSm was quantified using LpS as control: 1) bacteriostatic and bactericidal (growth curves in microplates), 2) inhibition of biofilm formation and disruption of pre-formed biofilm (crystal violet technique in microplates and polypropylene beads). The tested pathogenic strains represent 90% of the aerobic isolates in chronic wounds. The concentration of bioactive metabolites in LpSm was quantified by gas chromatography in tandem with mass spectrometry (GCMS). In healthy volunteers, we evaluated the LpSm relative anesthetic potency and effect duration compared to lidocaine 2%. The LpSm obtained from media with high concentrations of yeast extract possessed the greatest anesthetic power due to a greater production of barbiturates (GCMS). Those containing greater amount of meat extract, cations and surfactants had the highest capacity of biofilm disruption. When the glucose and galactose concentration were increased in media, the LpSm had the greatest bacteriostatic and bactericidal power as well as those grown with higher concentrations of CO2. This was due to increased production of organic acids, mainly lactic acid. Finally, media with higher concentrations of boric acid had higher inhibitory capacity of biofilm formation by increased production of furanosyl borate diester (autoinducers type 2). The identification of the precursor substrates of the bioactive metabolites allows in the future an LpSm manufacture with greater therapeutic power than LpS and even custom properties for each type of wound.