Influence of growth conditions on bacteriocin production by vaginal Lactobacillus salivarius CRL 1328

MARÍA SILVINA JUÁREZ TOMÁS; ELENA BRU; BIRGITT WIESE; AIDA A. P. DE RUIZ HOLGADO; MARÍA ELENA NADER-MACÍAS

Roma. Italia.

Congreso; Probiotics, prebiotics and new foods; 2001

Idea Congress Srl

Objectives. The main objective of our group is to select probiotic strains for local application in the human vagina. In the present work the influence of pH, temperature and culture media on the kinetics of growth, decreasement of pH, and bacteriocin production by vaginal Lactobacillus salivarius CRL 1328 was examined in order to a) evaluate the growth parameters through the Gompertz model, b) identify the optimal pH, temperature and culture media for growth, acid and bacteriocin production, c) study the kinetics of bacteriocin production and its relationship with the growth of the producer organism. Methods. The bacterial strains used were Lactobacillus salivarius subsp. salivarius CRL 1328 (bacteriocin-like substance producer) and Enterococcus faecalis (indicator strain) both isolated from vaginal swabs. The micro-organisms were stored in milk-yeast extract at –70°C and propagated in MRS or LAPTg broths at 37ºC before experiments. The study was performed by using a complete factorial experimental design 2x32, repited two times. The factors taken into consideration were: culture media with two levels (LAPTg and MRS broth); temperature with three levels (30ºC, 37ºC and 44ºC) and initial pH also with three levels (5.0, 6.5 y 8.0). L. salivarius CRL 1328 was inoculated (2% v/v) into 100 ml of each medium (250-ml Erlenmeyer flasks) and  incubated in a water bath, without agitation, at different temperatures. Samples were taken at specific time intervals to determine the O.D. at 540 nm (Spectrophotometer Model 250; Gilford Instrument Lab, U.S.A.); pH (pH meter Digimeter IV; Luftman; Argentina); and the titer of the bacteriocin by the plate method assay. The bacterial growth curves were described with the modified 4-parameter-Gompertz model. Results. For all the conditions assayed, the highest growth was always obtained in MRS broth, being pH 6,5 and a temperature of 37°C  the optimal conditions (the highest final optical density and growth rate, together with the shortest lag phase and exponential phase time). Good growth conditions were also obtained at 37ºC and both initial pH: 5.0 and 8.0, whereas at lower temperature (30°C) and pH 6.5 and 8.0 high values of final optical densities, longer lag phases and lower growth rates were observed. The growth in LAPTg was always lower than in MRS with their concomitant decreasement of the maximal optical densities and growth rates. The pH values reached after the growth in MRS and LAPTg broths were very similar, for the same temperature and initial pH. The difference between final and at time 0 pH was directly related to the initial pH of the broth, in both growth media at different temperatures. The bacteriocin production during the growth of  L. salivarius CRL 1328 was higher in LAPTg than in MRS, although the cell mass and growth rates were greater in MRS broth. Optimal conditions for the production of this substance were: temperature: 37°C, at both initial pH: 6,5 and 8.0, and culture medium: LAPTg, producing the highest value (1280 AU ml-1) at 6h of culture. On the other hand, 320 AU ml-1  was the maximum bacteriocin activity obtained in MRS, at 6h of culture at 37ºC (initial pH 6.5 and 8.0), and at 9h and 12h at 30ºC, initial pH 6.5 and 8.0, respectively. At a higher temperature, as 44°C, there was no production of bacteriocin, at the three pH assayed in both media. The maximal activity was always detected during the second part of the exponential phase, which means that is a substance produced while the microorganisms are actively dividing and before entering to the stationary phase. Conclussions. Lact. salivarius CRL 1328 grew more in MRS than in LAPTg. Despite the higher growth in MRS, the final pH was not lower than in LAPTg because of the buffering capacity of first medium. The optimum bacteriocin production was observed in LAPTg (a relatively simple and chip medium)at 37ºC and initial pH 6.5 and 8.0. This strain showed no obvious correlation between the cell number of the culture and bacteriocin production. It appears that Lact. salivarius CRL 1328 makes better use of the medium ingredients at 37ºC and initial pH 6.5-8.0, which were the optimal temperature and pH for growth and for bacteriocin production. The properties of the bacteriocin-like-substance-producing Lact. salivarius CRL 1328 allow us to further study its probable application in a probiotic for the prevention of urogenital infections.