SEQUENTIAL FACTORIAL DESIGN FOR THE FORMULATION OF A CULTURE MEDIA TO OPTIMIZE THE BIOMASS AND BACTERIOCIN PRODUCTION OF LACTOBACILLUS SALIVARIUS SUBSP. SALIVARIUS CRL 1328

ELENA BRU; MARIA SILVINA JUAREZ TOMAS; BIRGITT WIESSE; MARIA ELENA FÁTIMA NADER

Florianopolis

Conferencia; XXVth International Biometric Conference 2010; 2010

The International Biometric Society

Lactic acid bacteria (LAB) are a group of microorganisms very exigent nutritionally. They need a wide range of nutrients to grow and to synthesize their byproducts, being some of their nutritional requirements usually strain specific. The standard culture media commonly used to support the growth of lactobacilli includes MRS (De Man-Rogosa-Sharpe) and LAPTg (yeast autolysate-peptone-tryptone-tween 80-glucose). These media contain carbon and energy sources (carbohydrates, e.g. glucose or lactose), complex nitrogen sources (yeast extract, meat extract, tryptone and peptone) and supplements derived from oleic acid (Tween 80). MRS also includes inorganic and organic salts with stimulating effect or essential for the growth of most of the species of the genus. The modifications of the various components of these culture media strongly affect the growth and bacteriocin production of several microorganisms that are being considered for food or pharmaceutical applications. The complex composition of the commercial culture media, together with their high prizes, require the formulation of novel culture media less expensive to support the highest biomass and bacteriocin production in a probiotic L. salivarius CRL 1328 strain for pharmaceutical use. An optimization strategy based on the desirability function approach together with factorial (fractional, composite, central) design methodology have been used to optimize the growth and bacteriocin production. Three different types of experiments were carried out sequentially. The first one was performed to determine the effects of different carbon and nitrogen sources on the growth and bacteriocin production of this microorganism. A fractional factorial design 35–1 was applied to evaluate the effects of five nutrients (glucose, lactose, yeast extract, tryptone and meat peptone), at three different concentrations (0, 1 and 2%). Results were statistically analyzed for linear and quadratic effects of nutrients, along with their interactions. In the second set of experiment, the combined effects of salts were assayed in a 26 complete factorial design. The six factors considered were: basal culture medium (BCM) at two levels [LAPTg or carbon and nitrogen sources of MRS (CNS-MRS), which were employed to test the resulting salt combinations] and the five salts present in standard MRS medium, each one at two different concentrations. To evaluate the effect of the BCM assayed (qualitative variable) statistically, CNS-MRS was arbitrarily considered as ‘low level’ and LAPTg as ‘high level’. In each BCM, a central point was also assayed, which represents half of the concentrations of each one of the salts tested. The combination of the central point was assayed twice and a total of 68 growth curves were analyzed. Finally, in the third experiment, the objective was to formulate a culture medium of lower cost than conventional laboratory media, in order to simultaneously obtain high amounts of both biomass and bacteriocin of vaginal Lactobacillus salivarius CRL 1328. The growth assays under different culture conditions were performed by using a 28-2 central composite experimental design, with a central point and sixteen additional points. The factors taken into consideration and their levels were taken from the results obtained in the previous experiments, being assayed glucose, lactose, yeast extract, tryptone, ammonium citrate, sodium acetate, MgSO4 and MnSO4. The optimized growth media obtained from these set of experiments allow a cost reduction of around 25 to 40% compared with conventional broths. The results obtained represent an advance in the search of the most suitable strategies for the production of bioactive compounds, including biomass and bacteriocin, to be used in the design of novel pharmaceutical products to prevent or treat female urogenital infections.