ASSESSMENT OF NEW OVULES CONTAINING FREEZE-DRIED PROBIOTIC Lactobacillus fermentum L23 (GENBANK GQ455406.1): CONTROL OF VIABILITY AND ANTIBIOFILM ACTIVITY DURING STORAGE TIME

RUIZ, FRANCESCA; ASURMENDI, PAULA; GARCÍA, MARÍA J; CAMILETTI ANA; PASCUAL, LILIANA; BARBERIS, LUCILA

Otro; IV Reunión Conjunta de Sociedades de Biología de la República Argentina; 2020

The growing interest in the development of bio-products containing safe microorganisms as a strategy to restore the vaginal ecosystem and prevent or combat pathogenic microorganisms in this niche has re-emerged in recent years. However, in most Latin American countries, including Argentina, there is low availability of these vaginal products in the market. In the development of new ovules containing probiotic strains, the control of viability and probiotic properties in storage is relevant. The aims of this work were (i) to select a vaginal formulation that ensures the best level of L. fermentum L23 viability over time and, (ii) to evaluate the maintenance of antibiofilm activity after recovering from the ovules through time. To obtain a high biomass production, successive cultures of L23 strain in MRS broth (pH 5.5) were made. Bacterial cells were concentrated 20-fold in 10% skimmed milk solution and subsequently, lyophilized. Three vaginal formulations (F1–F3), combining lyophilized powder of L23 with different proportions of glycerol, gelatin, skim milk, lactose or Tween80 were tested. Then, the ovules by fusion method were obtained. To determine bacterial viability recovered from the ovules over storage time, samples every 30 days for 270 days were taken. Bacterial counts on MRS agar plates were done, and their average (log CFU/mL) was used to calculate the survival rates (%). As an important antagonistic property from L23 strain, the antibiofilm activity over time was tested. Thus, the effect of bioactive metabolites produced by L23 after recovering from ovules was evaluated on pathogenic biofilm formation every 90 days until 270 days. Pure cell-free supernatants (CFS) and neutralized CFS containing (organic acid + bacteriocin) and (bacteriocin), respectively, were tested on two biofilm-producing pathogens, Streptococcus agalactiae (SGB) and Staphylococcusaureus. Then, the antibiofilm activity (%) was calculated by spectrophotometer measures. Formulations F1 (base components) and F2 (skimmed milk + lactose) allowed to maintain the viability of L23 strain for 180 days, whereas in F3 (skimmed milk, lactose + Tween80) only remained for 150 days. The survival achieved with F1 and F2 was meaningful in comparison to F3 until the end of the experience (P < 0.05). For F1, F2, and F3, the average (%) reduction on the SGB biofilm over time produced by the CFSs and NCFS of L23 ranged between 70–73% and between 57.3–66.33%, respectively. Similarly, on the S. aureus biofilm, the percent reduction ranged between 62–70% and 55–63% for CFS and NCFS, respectively. In conclusion, F1 and F2 formulations were the better options for reaching optimal viability levels of this strain over time. Likewise, the strong antibiofilm activity maintained over time confirms its technological potential.