# Manufacture of a Potential Antifungal Ingredient Using Lactic Acid Bacteria from Dry-Cured Sausages
## 3 Results and Discussion  
### Isolation, Screening, and Identification of Antifungal Bacteria Strains  
A total of 102 bacteria were isolated from different handmade dry Spanish sausages. Among these, 42 isolates were classified as LAB since they were gram-positive, could grow in micro-aerobic conditions, and lacked catalase activity (Table 1). Further characterization of bacteria strains highlighted that these isolates grew in the following conditions: temperature ranging from 15 to 45 \({}^{\circ}\)C; pH ranging from 3.5 to 6; and growth in MRS-NaCl at 3, 6.5, and 10%. Therefore, these bacteria were selected, and after the characterization, a first screening was performed using the overlay approach to assess the antifungal properties of the 42 bacteria isolated; they were assayed against six spoilage fungal strains in vitro that commonly contaminate cured sausage products. As presented in Table 2, only 14 bacteria isolates (C11, C12, C13, C15, C20, C28, C56, C58, C60, C66, C69, C71, C72, and C79) demonstrated inhibitory activity against all fungi examined, with varying degrees of inhibition depending on the fungal strain tested. In particular, _P. griseofulvum_ CECT 2605-T and _P. commune_ CECT 20767 were the most susceptible strains, with inhibition zones ranging from 1.5-3.0 cm and 1.0-3.0 cm, respectively. In contrast, _Aspergillus_ strains (_A. parasitic_s CECT 2681 and _A. flavus_ ITEM 8111) were the most resistant to the LAB, with inhibition zones ranging from 0.3-1.7 cm. An example of overlay methodology is given in Figure 1. These findings agree with those obtained by previous authors (2018), which noticed that _Aspergillus_ strains are more resistant to LAB than other fungal genera. \begin{table}
\begin{tabular}{c c c c c c c c} \hline  & \multicolumn{2}{c}{**Preliminary Characterization**} & \multicolumn{2}{c}{**Growth at**} & \multicolumn{2}{c}{**Growth in**} & \multicolumn{2}{c}{**Growth in MRS-NaCl**} \\  & & & **Temperature** & **pH** & **(\%)** & \\
**Bacteria** & **Morphology** & **Gram \({}^{*}\)** & **Catalase** & **15 to 45 \({}^{\circ}\)C** & **3.5 to 6** & **3 to 6.5** & **10** \\ \hline C1 & Coccoid & P & - & + & + & + & - \\ C4 & Coccoid & P & - & + & + & + & - \\ C5 & Coccoid & P & - & + & + & + & - \\ C6 & Coccoid rod & P & - & + & + & + & - \\ C11 & Coccoid & P & - & + & + & + & - \\ C12 & Coccoid & P & - & + & + & + & - \\ C13 & Coccoid & P & - & + & + & + & - \\ C14 & Coccoid rod & P & - & + & + & + & + \\ C15 & Coccoid & P & - & + & + & + & + \\ C16 & Rod-shaped & P & - & + & + & + & + \\ C17 & Rod-shaped & P & - & + & + & + & + \\ C19 & Coccoid & P & - & + & + & + & + \\ C20 & Rod-shaped & P & - & + & + & + & + \\ C27 & Rod-shaped & P & - & + & + & + & + \\ C28 & Coccoid & P & - & + & + & + & + \\ C29 & Rod-shaped & P & - & + & + & + & + \\ C30 & Rod-shaped & P & - & + & + & + & + \\ C31 & Coccoid & P & - & + & + & + & + \\ C35 & Coccoid & P & - & + & + & + & - \\ C36 & Coccoid & P & - & + & + & + & + \\ C37 & Coccoid & P & - & + & + & + & + \\ C38 & Coccoid & P & - & + & + & + & - \\ C39 & Coccoid & P & - & + & + & + & + \\ C44 & Coccoid & P & - & + & + & + & + \\ C45 & Coccoid & P & - & + & + & + & + \\ C46 & Coccoid rod & P & - & + & + & + & - \\ C47 & Coccoid & P & - & + & + & + & + \\ C48 & Coccoid & P & - & + & + & + & + \\ C50 & Coccoid & P & - & + & + & + & + \\ C56 & Coccoid & P & - & + & + & + & + \\ C57 & Coccoid & P & - & + & + & + & + \\ C58 & Coccoid & P & - & + & + & + & + \\ C59 & Coccoid & P & - & + & + & + & - \\ C60 & Rod-shaped & P & - & + & + & + & + \\ C66 & Coccoid & P & - & + & + & + & + \\ C68 & Coccoid & P & - & + & + & + & + \\ C69 & Coccoid & P & - & + & + & + & + \\ C70 & Coccoid & P & - & + & + & + & - \\ C71 & Coccoid & P & - & + & + & + & + \\ C72 & Coccoid & P & - & + & + & + & + \\ C79 & Coccoid & P & - & + & + & + & + \\ C80 & Coccoid & P & - & + & + & + & + \\ \hline \end{tabular}
* means no visual growth.  
\end{table} 
Table 1: Characterization of LAB isolated from dry-cured sausages.