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Aim: Probiotic microorganisms, primarily lactic acid bacteria (LAB) and bifidobacteria, are able to solve most of the problems of animal by-products that hinder their use in the food industry. The most important property of LAB is their antagonistic activity against pathogenic and opportunistic microorganisms. The aim of the study is to compare the antimicrobial activity of microorganisms from commercial starters, medicinal preparations, and newly isolated strains. It is important to evaluate two alternative methods for determining antimicrobial activity in terms of their interchangeability. Methods: A total of 11 microorganisms and consortia from various sources were studied, including five newly isolated strains. Their antagonistic activity against 8 strains of pathogenic and opportunistic microorganisms was evaluated by two in vitro methods: agar diffusion and co-cultivation. Their interchangeability was assessed using the linear Pearson correlation coefficient. Results: The 12th hour of cultivation, corresponding to the maximum specific growth rate of the studied newly isolated strains and consortia were determined and used to take a supernatant sample for co-cultivation with test pathogens. Of the studied cultures, lactobacilli and pediococci showed the greatest antagonistic activity against the tested pathogens, while Staphylococcus spp. showed minimal activity. Conclusions: The highest inhibition index was observed in consortia containing Lactobacillus and Pediococcus. The antagonistic activity of the newly isolated strains is lower than that of meat starter cultures and medicinal products. The evaluation of the comparability of analytical methods for determining antimicrobial activity demonstrates a high positive correlation of the results, but requires further research to resolve the issue of their interchangeability.