# Manufacture of a Potential Antifungal Ingredient Using Lactic Acid Bacteria from Dry-Cured Sausages
## 3 Results and Discussion
### In Vitro Antifungal Study of the MB Formulated  
Two methods were performed to determine the fungal sensitivity to bacterial isolates: agar diffusion test and microdilution of BFS in 96-well plates. The MB was formulated by changing the composition of the MRSb by subtracting the yeast extract and meat extract and adding freeze-dried pork loin as a protein source at 2.00, 4.00, 8.00, and 10.00 g/L (MB2, MB4, MB8, and MB10, respectively). These new formulations were fermented with the 14 bacteria strains identified in Section 3.1, and a first qualitative evaluation was performed using the agar-diffusion test to assess the antifungal properties. Moreover, the fermented MRSb was also prepared with the same bacteria strains to compare the effectiveness of the newly formulated ingredient. After fermentation, only the MB10 exhibited antifungal activity similar to the fermented MRSb used as a control reference. As presented in Table 3, the inhibition halos obtained varied according to the fungal strain tested and the bacteria used as inoculum. In particular, the most sensitive strains to the MB10 were _P. commune_ CECT 20767, _P. nortacum_ CECT 2320, and _P. verruccosum_ VTT-D01847. These strains were susceptible to all the formulated MB, with inhibition halos observed on the PDA plates ranging from 0.2 cm (+) to inhibition halos greater than 0.4 cm (++). The fungal strains with the most significant resistance were those of the _Aspergillus_ genera, in which only _Pediococcus_ C12, _Pediococcus_ Peptosacus_ C15, and _Lactiplantiobacillus plantarum_ C60 showed inhibitory activity. This finding agrees with the previous characterization of the LAB isolates by overlay method since _Aspergillus_ strains showed higher resistance to the LAB than _Penicillium_ strains. For this reason, the formulation MB10, _P. pentosacus_ C12, _P. pentosacus_ C15, and _L. plantarum_ C60 isolates were selected, and the preliminary results of the antifungal activity were confirmed through the MIC50, MIC100, and MFC determination in vitro. The MB10 and MRSb that were fermented for 48 h obtained the highest inhibition halo zone. The antifungal effect of MB10 fermented for 24, 48, and 72 h is plotted in Table S2. Similar findings in the literature indicated that a range of theories might explain LAB's antifungal activities; [PERSON] and [PERSON] (2008) found that the suppression of mold \begin{table}
\begin{tabular}{c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Selected**} & \multicolumn{8}{c}{**Antifungal Activity of the BFS of the Fermented MRS Broth and the Meat Broth (MB10)**} \\ \cline{2-13}  & _A. flavus_ & _A. parasticus_ & _P. commune_ & _P. griseofulvum_ & _P. nordicum_ & _P. verruccosum_ \\ \cline{2-13}  & **MRSb** & **MB10** & **MRSb** & **MB10** & **MRSb** & **MB10** & **MRSb** & **MB10** & **MRSb** & **MB10** \\ \hline C11 & + & - & - & - & + & + & + & + & + & + & + & + \\ C12 & + & + & ++ & + & ++ & ++ & ++ & ++ & + & ++ & ++ \\ C13 & - & - & + & - & + & + & + & + & ++++ & ++++ & ++++ \\ C15 & ++ & ++ & ++ & ++++ & ++++ & ++++ & ++++ & ++++ & ++++ & ++++ \\ C20 & - & - & + & - & + & + & + & + & ++++ & ++ & + & + \\ C28 & + & - & - & - & + & + & + & + & ++++ & ++++ & ++++ \\ C56 & - & - & + & - & + & + & + & + & + & + & + & + \\ C58 & - & - & - & - & + & + & ++ & ++ & ++++ & ++++ & ++++ \\ C60 & + & + & ++ & ++ & ++ & ++++ & ++ & ++++ & ++++ & ++ & ++ \\ C66 & + & - & - & - & ++ & ++ & ++ & ++++ & ++ & ++++ & ++ \\ C69 & - & - & + & - & + & + & - & - & + & + & + & + \\ C71 & - & - & + & - & + & + & - & - & + & + & + & + \\ C72 & + & - & - & - & ++ & + & ++ & ++ & ++ & ++++ & ++++ \\ C79 & + & - & - & - & ++ & + & ++ & ++ & ++ & ++ & ++++ & ++ \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Antifungal activity of bacterial free supernatant (BFS) extract in agar diffusion method. LAB fermented MRS broth (MRSb) and a meat broth (MB10) for 48 h at 37 \({}^{\circ}\)C. Then, the supernatant was freeze-dried and resuspended at a concentration of 250 g/L.