CONICET | Buscador de Institutos y Recursos Humanos

Bovine mastitis (BM) is the responsible of the major economic losses in the dairy farm and industry. Focused on the design of a probiotic to prevent BM, we previously isolated lactic acid bacteria (LAB) from teat canal, foremilk and stripping milk samples. From 102 isolated LAB, 5 strains were selected based on their surface and inhibitory properties. One of the selected LAB was Enterococcus mundtii CRL 1656, isolated from a stripping bovine milk sample which is able to produce a bacteriocin designed mundticin CRL1656, active against the Gram-positive food-borne pathogen Listeria monocytogenes. The optimization of the physico-chemical conditions to produce antagonistic metabolites and biomass constitutes a valuable step to produce raw material for probiotic manufacture. For these reasons, the aim of the present work was to optimize the bacteriocin and biomass production by E. mundtii CRL 1656. A 3x24 complete factorial design with replication was performed. The analyzed factors were culture medium (MRS, LAPTg, M17), inoculum (2.5%, 5.0%), pH (5.0, 6.5), temperature (30 ºC, 37 ºC) and agitation (0 opm, 50 opm). E. mundtii CRL 1656 was grown in LAPTg broth at 37 ºC and the third subculture was washed with sterile saline solution. Optical density at 540 nm (OD540) was adjusted and the suspension was employed as inoculum for the growth experiment. The analyzed responses were OD540 (as growth measure) and bacteriocin titre. Samples were taken every 3 h during 24 h and OD540 were determined by using a microplate reader and the bacteriocin titre by using the agar plate bioassay with L. innocua as indicator strain. To estimate the growth parameters, the modified Gompertz model was applied. Standard errors were determined by the bootstrap model. To estimate the effects of medium, inoculum, pH, temperature and agitation, a non-linear mixed model was applied. Bacteriocin production was statistically evaluated by applying a linear mixed model. The statistic analysis indicates that medium, temperature, pH and inoculum had a significant effect on lag phase. Medium, temperature, pH and medium-pH interaction affected exponential phase (p<0.0001). Medium, temperature, pH, agitation and medium-pH interaction had significant influence in the stationary phase (p<0.0001). With regard to bacteriocin production, temperature, pH and medium had a significant influence. The optimal production of biomass, bacteriocin and bacteriocin/biomass ratio were obtained in different conditions. The use of statistical methodology allowed the simultaneous evaluation of the influence of several phisico-chemical factors on the growth and mundticin production by E. mundtii CRL 1656. Further studies are necessary to design low cost alternative media to achieve simultaneous high biomass and bacteriocin production.