## 2 Materials and Methods
### Antimicrobial Activity Assay  
Four standardized ATCC strains from laboratory stock cultures were used in the evaluation of the antimicrobial activity of the CFSs, i.e., _Salmonella enterica_ ATCC14028, _Staphylococcus aureus_ ATCC 29213, _Escherichia coli_ ATCC 25922 and _Klebsiella pneumoniae_ ATCC 700603. Testing media (Selective Chromoagar for each pathogen) and Mueller-Hinton Agar (MHA) were used in order to evaluate the antimicrobial activity. The antimicrobial activity of CFSs filtrate was determined against the pathogen's organisms. The CFSs incubated for 30 days at 37 \({}^{\circ}\)C. Fifty microliters of the incubated CFSs was removed on different days in order to find the best antimicrobial activity. One hundred microliters of microbial culture of an approximate inoculums size of 1.0 \(\times\) 10\({}^{8}\) CFU/mL was added to all wells. Fifty microliters of the CFSs, on different days of incubation, were loaded into the microtiter plate containing each of the tested microbial strains. The plate was then incubated at 37 \({}^{\circ}\)C for 24 h. The lowest effective dose of each selected supernatant, which still significantly inhibited the growth of target pathogens, was determined as described in Table 4. The antimicrobial activity was confirmed using selective agar plates for each pathogen, as shown in Figures 1-4. All measurements were performed in triplicate [27]. Figure 1: Chromatogram at 255 nm of 14 standard mixes (50 ppm; 10 mL injection) of previously reported antifungal compounds from LAB prepared in water–methanol (90/10) and MRS broth MRS broth (filtered 0.22 μm; LLE extraction; 20 μm; direct injection). (A) 4-hydroxybenzoic acid (19.3 min); (B) DL-p-Hydrooxyphenyllactic acid (16.3 min); (D) 3-(4-Hydroxyphenyl) propionate acid (26.4 min); (F) Methylcinnamic acid (41.3 min); (G) Salicylic acid (36 min); (H) 1,2-Dihydroxybenzene (15.7 min); (I) 3,4-Dihydrocinnamic acid (21.7 min); (J) Vanillic acid (21.3 min); (L) Ferulic acid (28.7 min); (M) salicylic acid (34.2 min); (O) 4-Hydrocinnamic acid (27.4 min). Figure 2: Chromatogram at 280 nm of 11 standard mixes (50 ppm; 10 mL injection) of previously reported antifungal compounds from LAB prepared in water–methanol (90/10) and MRS broth (filtered 0.22 μm; LLE extraction; 20 μm; direct injection). (A) 4-hydroxybenzoic acid (19.3 min); (B) DL-p-Hydroxyphenyllactic acid (16.3 min); (D) 3-(4-Hydroxyphenyl) propionate acid (26.4 min); (F) Methylcinnamic acid (41.3 min); (G) Salicylic acid (36 min); (H) 1,2-Dihydroxybenzene (15.7 min); (I) 3,4-Dihydrocinnamic acid (21.7 min); (J) Vanillic acid (21.3 min); (L) Ferulic acid (28.7 min); (M) salicylic acid (34.2 min); (O) 4-Hydrocinnamic acid (27.4 min). such as organic acids [28; 29; 30; 31; 32]. Certain strains of _L. plantarum_ from sourdough have been reported to produce 3-(4-hydroxyphenyl)lactic acid (HPLA) and PLA and have been reported to be antifungal compounds. Many reports have suggested that antifungal activity is a combination of organic acids such as lactic, acetic, phenyllactic acids or bacteriocins and low-molecular-weight antimicrobial agents and peptides [26; 33]. The objective of this study was to optimize the HPLC-UV/DAD method for the qualitative and quantitative determination of 14 different LAB metabolites. Sample preparation (extraction and clean-up) is a prerequisite for analyzing the majority of food matrices, and in the area of LAB, this process is appropriate. The high accuracy, the time and the cost of our method in this article were the same compared to an earlier study undertaken by [PERSON] [23; 24]. According to [PERSON] et al., 2014, HPLC-UV/DAD, GC-MS and LC-MS were used to identify LAB-producing compounds. These methods allow the compounds to be determined in a single chromatographic test. The retention time of these methods is about 40 min. Elution was performed using a gradient flow of water containing formic acid (solvent A) and acetonitrile containing formic acid (solvent B). The cost of analysis for all methods mentioned above is the same price for every single run.