Study on Media Formulation and Production Process of Bacillus subtilis Spores for Animal Probiotics

Abstract

The utilization of probiotic microorganism in animal feed has gained considerable attention becauseit helps improving animal health, increasing animal production and reducing antibiotic utilization. Bacillussubtillis (BS) is microorganism used in this study because it is a spore-forming bacteria and it can growrapidly. The aims of this study were to achieve high spore concentration by using media formulation andmode of cultivation. In addition, the antagonistic activity of BS against some pathogens was investigated.

Two sets of media formulations were studied in shake flask culture, i.e., molasses based formulaand defatted soy bean meal based formula. The molasses based media contained 19.85 g/L molasses (asreducing sugar), 0.35 g/L CaCI₂ .2H₂O and 0.15 g/L MnSO₄ .H₂O gave the highest spore concentrationof 5.5 x 10⁹ CFU/ml whereas the soy bean meal based media contained 20 g/L soy bean meal, 3 g/Lmolasses and 0.5 g/L K₂HPO₄ gave the highest spore concentration of 1.3 x 10⁹ CFU/ml. However, molassesbased media led to foaming problem in a fermentation process. Soy bean meal based media was thereforeused for fermentor cultivation. In 2 L-fermentor, spore concentrations at 48 h were 1.78 x 10⁹ and4.03 x 10⁸ CFU/ml for batch and fed-batch process respectively. Molasses added during cultivation couldenhance cell growth but it could impede spore formation. Therefore, in 75-L fed-batch cultivation, nomolasses was fed after the stationary phase of growth, resulting in the spore concentration of 5.73 x 10⁹CFU/ml at 48 h. The results of well diffusion assay and paper disc method show that this probiotic doesnot have an antagonistic activity against several pathogens tested.

Keywords : Bacillus subtillis / Probiotics / Batch Cultivation / Fed-batch Cultivation /Media Formulation