Inhibitory potential of probiotic spore formers grown as mono- and mix-cultures under conditions of different nutrient availability
Authors: Knysh O. V. , Martynov A. V. , Osolodchenko T. P. 
Published: January 2025

In many clinical cases, the use of probiotic microorganisms is a safer alternative to antibiotic therapy. Spore-forming probiotics, due to their high resistance to damaging environmental factors, stand out among other beneficial microorganisms and, therefore, deserve special attention. Determining the conditions that enhance the inhibitory potential of probiotic bacilli is an important step towards increasing their effectiveness against pathogenic bacteria and pathobionts.
The aim of this study was to determine whether the inhibitory potential of spore-forming probiotic species: Alkalihalobacillus clausii (previously known as B. clausii), Heyndrickxia coagulans (formerly B. coagulans) and B. subtilis increases under conditions of co-cultivation or nutrient depletion (starvation).
Materials and methods. The commercial strains of bacilli from three probiotic preparations: Enterogermina (A. clausii), Lactovit forte (H. coagulans) and Subalin (B. subtilis) were used in this study. The ability of mono- and mix-cultures of the studied probiotic species cultured on conventional and semi-starvation nutrient media to affect the growth of indicator bacteria-pathobionts (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa) was investigated by the agar block method (using 1.5 % nutrient agar) and the spot-on-lawn assay with wells (using 0.7 % nutrient agar).
Results. Inhibitory activity against indicator bacteria-pathobionts was characteristic of both mono- and mixed probiotic test cultures. Using the agar block method, probiotic monocultures grown on nutritive agar demonstrated moderate inhibitory activity against S. aureus and P. aeruginosa, but weak inhibitory activity against E. coli. Probiotic monocultures grown on “semi-starvation” agar and mix-cultures regardless of the culture medium showed pronounced inhibitory activity against S. aureus and P. aeruginosa, but moderate inhibitory activity against E. coli. Co-cultivation and cultivation on semi-starved media were accompanied by an increase in the number of isolated colonies of probiotic bacilli (disseminates) in the transparent zone of no growth and the pathobiont growth zone. Using spot-on-lawn assay revealed moderate inhibition of staphylococcus growth by both mono- and mixed probiotic cultures, regardless of their cultivation conditions. However, the inhibition indicators of probiotic mix-cultures were statistically significantly higher than those of monocultures. The inhibitory activity of probiotic monocultures against E. coli and P. aeruginosa was moderate, while probiotic mix-cultures had a pronounced inhibitory effect on these gram-negative bacteria.
Conclusions. The inhibitory activity and dissemination ability of spore-forming probiotic species: A. clausii, H. coagulans and B. subtilis increases under conditions of co-cultivation or nutrient depletion (starvation). The demonstrated effect of increasing the inhibitory potential of probiotic spore-forming bacteria by co-cultivation and application of nutrient depletion conditions requires further study and clarification of the underlying molecular mechanisms.