# Effects of Preservative Agents on Quality Attributes of Dry-Cured Fermented Sausages
## 3 Results and Discussion
### In Vitro Effect of the Biocontrol Agents on the Growth of Beneficial Microorganisms  
The biocontrol agents Ef, Dh, and Pc did not affect the growth of Sv nor Dh46P in dual cultures in the four media (DFS-0.97, DFS-0.94, DFS-0.97P, and DFS-0.94P) at every combination of \(a_{\mathrm{w}}\) and temperature tested. In Figure 1, co-inoculated plates of Sv and Dh46P with the biocontrol agents incubated at 25 \({}^{\circ}\)C are shown as an example. The diameter of the colonies of Pn (Figure 2) and Pg222 (Figure 3) was slightly lower in the presence of the biocontrol agents than in their absence, but not completely impaired. Regarding the co-inoculation with Pc, mutual inhibition at contact was observed for both beneficial models in DFS-0.97 and DFS-0.94 (Figures 2 and 3). Figure 1: Interactions of three biocontrol agents (_Enterococcus faecium_ SE920, _Debaryomyces hansenii_ FSHCC 253H, and _Penicillium chrysogenum_ CECT 20922) with native dry-cured meat product mold _Penicillium nalgiovense_ FSHCC Pj261 at 0.97 and 0.94 water activity (\(a_{\mathrm{w}}\)) after 7 days at 25 \({}^{\circ}\)C in the presence and absence of the antifungal protein PgAFP in a dry-cured fermented sausage (salchichón)-based medium. Treatments: Control: _P. nalgiovense_ FSHCC Pj261 inoculated without biocontrol agents; Pn: _P. nalgiovense_ FSHCC Pj261; Pc20922. _P. chrysogenum_ CECT 20922; Dr: _D. hansenii_ FSHCC 253H; Ef: _E. faecium_ SE920. No PgAFP: PgAFP was not added; PgAFP: PgAFP was added. The biocontrol agent was inoculated on the left side of the plate and _P. nalgiovense_ FSHCC Pj261 on the right side of the plate. Figure 2: Interactions of the three biocontrol agents (_Enterococcus faecium_ SE920, _Debaryomyces hansenii_ FSHCC 253H, and _Penicillium chrysogenum_ CECT 20922) with the native dry-cured meat product mold _Penicillium nalgiovense_ FSHCC Pj261 at 0.97 and 0.94 water activity (\(a_{\mathrm{w}}\)) after 7 days at 25 \({}^{\circ}\)C in the presence and absence of the antifungal protein PgAFP in a dry-cured fermented sausage (salchichón)-based medium. Treatments: Control: _P. nalgiovense_ FSHCC Pj261; Pc20922. _P. chrysogenum_ CECT 20922; Dr: _D. hansenii_ FSHCC 253H; Ef: _E. faecium_ SE920. No PgAFP: PgAFP was not added; PgAFP: PgAFP was added. The biocontrol agent was inoculated on the left side of the plate and _P. nalgiovense_ FSHCC Pj261 on the right side of the plate. The marginal impact found could be attributed to the different mechanism of action reported for the studied biocontrol agents. Thus, the antagonistic effect of _D. hansenii_ Dh253H by nutritional competition, and the production of extracellular soluble and volatile antifungal compounds have previously been described [30]. The protective _P. chrysogenum_ CECT 20922 antagonism against Pg222 and Pn could be related to competition for nutrients and space, as previously described against _P. nordicum_[25]. However, this limited action of the protective cultures on the growth of both non-toxic molids did not completely inhibit their growth. Thus, they could coexist without affecting their contribution to the development of the sensory characteristics of sausages. The three biocontrol agents were then used for manufacturing dry-cured fermented sausages in the pilot plant assay. On the other hand, the presence of the antifungal protein PgAFP did not affect the growth of Sv nor Dh46P. However, the protein caused a decrease in the growth of Pn and Pg222, mainly at 0.97 \(a_{\mathrm{w}}\) (Figures 2 and 3, respectively). In addition, at this \(a_{\mathrm{w}}\) value, the colonies of Pn had an abnormal appearance in all conditions tested. The colonies lost their circular shape and seemed to be composed by multiple dots. When visualized under microscopy, the mycelia were brittle, not presenting the characteristic structure of the hyphae (Figure 4).