## 2 Materials and Methods
### Evaluation of Antimicrobial Effects of Selected Lactobacillus spp. Strains Isolated from Yoghurts  
The effects of _Lactobacillus_ spp. isolated from tested yoghurts on changes in the abundance of _L. monocytogenes, E. coli_ and S. Enteritidis were evaluated (one strain each). The pathogenic bacterial strains were the collection of the Department of Microbiology of Collegium Medicium in Bydgoszcz Nicolaus Copernicus University in Torun. The isolates were stored in a brain-heart infusion broth (BHI, Merck) with 15.0% glycerol (Avantor) at \(-80\) \({}^{\circ}\)C until the beginning of the research.  
Two species of _Lactobacillus_: _L. rhamnus_ and _L. paracasei_, which were previously isolated from the tested yoghurts, were selected for further study. _Lactobacillus_ spp. strains were inoculated onto the Rogosa medium and incubated for 72 h at 35 \({}^{\circ}\)C. Then, the suspensions were prepared (for each strain separately) in the LAPTg medium (L--lactose, A--tryptone, P--yeast extract, T--tomato juice, g--glucose) with a volume of 5 mL each and an optical density of 0.5 on the McFarland (McF) scale.  
The examined strains (from freezing) were plated on the Columbia agar with 5.0% sheep blood (BioMerieux, Marcy-I'Etoile, France). Incubation was carried out for 24 h at 37 \({}^{\circ}\)C. From the grown colonies, suspensions were prepared, for each strain separately, on the LAPTg medium (5 mL) and an optical density of 0.5 McF.  
The effect of _Lactobacillus_ spp. isolated from yoghurt (each strain individually and a mix of two strains) on _L. monocytogenes, E. coli_ and _S._ Enteritidis was then evaluated. For this purpose, mixed cultures were prepared. Cultures were obtained by combining 5 mL of the previously prepared suspension of a given _Lactobacillus_ spp. strain with 5 mL of the previously prepared suspension of the above-listed pathogenic strain for each tested strain separately. For the mix variant of the two _Lactobacillus_ spp. strains, 2.5 mL of _L. rhamnus_ and 2.5 mL of _L. paracasei_ suspension were combined with 5 mL of the suspension of the tested pathogenic strain, yielding a total volume of 10 mL. The negative control was 5 mL of a sterile LAPTg medium.  
All prepared mixtures were incubated for 72 h at 37 \({}^{\circ}\)C. Changes in the numbers of _Lactobacillus_ spp. and _L. monocytogenes_, _E. coli_ and \(S\). Enteritidis in the mixed cultures were evaluated immediately after establishment and after 24, 48 and 72 h, respectively. For this purpose, a series of 10-fold dilutions were prepared. Subsequently, 0.1 mL from each dilution was surface-inoculated (in duplicate) onto the Rogosa agar for _Lactobacillus_ spp., Oxford agar (OXID) for _L. monocytogenes_, MacConkey agar for _E. coli_ and XLD agar for \(S\). Enteritidis. The cultures were incubated under microaerophilic conditions for 72 h at 35 \({}^{\circ}\)C (_Lactobacillus_ spp.) and 48 h at 37 \({}^{\circ}\)C (other species), respectively. The grown colonies were counted, and their number was reported as log CFU per 1 cm\({}^{3}\) (log CFU \(\times\) cm\({}^{-3}\)).  
The changes in the number of all bacteria tested, both in monocultures and mixed cultures, after 72 h, were then counted according to the following formula:  
\[\text{CN}=\text{logB}-\text{logA},\]  
where  
CN--changes in the number of bacteria [log CFU \(\times\) cm\({}^{-3}\)];  
logA--logarithm of the initial number of bacteria in suspension;  
logB--logarithm of the number of bacteria after 72 h of incubation.