Method of preparation, visualization and ultrastructural analysis of a formulation of probiotic Bacillus subtilis KATMIRA1933 produced by solid-phase fermentation

Probiotic preparations are used in medical treatment and in agricultural practice. They modulate numerous activities in eukaryotic hosts, such as: inhibition of pathogenic microbiota; stimulation of immunological responses; and production of antioxidants, anti-mutagens, and DNA protectors. Also, probiotic bacteria are used as a preventive measure to prevent bacterial diseases of the gastrointestinal tract. Solid-phase fermentation is reported as being used in the production of probiotic formulations where a solid substratum, such as soy and oil meal, is utilized for the growth of beneficial microorganisms. However, there are insufficient reports in the literature related to methodological approaches enabling evaluation of the final products of solid-phase fermentation. We suggest a novel method enabling evaluation of probiotic solid-state fermentation dry powders and observation of their morphology, ultrastructure, and elucidation of the quantitative distribution of probiotic microorganisms in solid substrates using electron microscopy. • The method is intended for ultrastructure microphotography of dry substances - for example, ultrastructure of solid-phase fermentation products. • The method allows preserving the ultrastructure of substrates that are damaged when soaking. • The method does not require additional equipment and reagents and can be used in all laboratories using electron microscopy.