ABSTRACT:Lactobacillus plantarum culture supernatants (LAPS) have antimicrobial, pro-healing and anesthetic properties, so our medical team applied whole cultures and LAPS in chronic wounds with encouraging results. Metabolites responsible for LAPS properties and its action mechanism were previously determined. Objective: to design methods and culture media to increase the production of LAPS metabolites and optimize it´s therapeutic effectiveness. Modifications in MRS broth composition were made and L. plantarum was cultivated in the resulting different media with different physicochemical conditions to obtain modified supernatants (LAPS m ). Antimicrobial and anti-biofilm activity (crystal violet) of LAPS m on strains typically isolated from chronic wounds (Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Serratiamarcescens), was tested. Concentration of barbiturates in LAPS m was quantified by GCMS and its relative anesthetic potency in healthy volunteers was evaluated. DNAase and auto inducers type 2biological activity in LAPS m were quantified. In addition, a safety assessment of LAPS m application on healthy skin of 200 volunteers was performed. LAPS m obtained from media with high concentrations of yeast extract possessed the greatest anesthetic power. Those containing greater amount of proteins, cations and surfactants had the highest capacity of biofilm disruption (p<0.01). When glucose and galactose concentration were increased in media, LAPS m had the greatest antimicrobial power (p<0.05). Finally, there is great safety in the application of LAPS m as no edema or erythema was observed. The results obtained will allow the manufacture of LAPS m with greater therapeutic effectiveness and even custom properties for each type of wound.