*2.6. Antibacterial Assays*

The antibacterial activity of scotta hydrolysates was tested against six bacterial strains (see Table 1) belonging to three different species, namely, *Listeria monocytogenes*(four strains), *Staphylococcus aureus* and *Salmonella bongori*. The strains, stored at −80 ◦C, were thawed and precultured in Brain Heart infusion broth medium (BHI, WVR, Milano, Italy) for 24 h at 37 ◦C. Overnight cultures were then used to prepare microbial inoculation used for the test. One milliliter of overnight culture was centrifuged at 14,000× *g* for 2 min, then the pellets were resuspended in saline solution until reaching 0.2 optical density (OD) (~8 log10 CFU mL−1).

**Table 1.** List of microorganisms, medium and culture condition for testing the antimicrobial activity of enzymatic hydrolysate of Scotta 1.


1 DSMZ, Deutsche SammLung von Mikroorganismen und Zellkulturen, German Collection of Microorganism of Cell Cultures; DAFES, Collection of Microorganisms of Dipartimento di Agraria of the University of Sassari, Section of Food and Environmental Science.

The lyophilized hydrolysates (BSPH, PSPH) and non-hydrolysate (CTRL) were weighed and dissolved in Brain Heart Infusion broth (BHI), giving a final concentration of 100 mg mL−1.The solutions were then filter-sterilized on a 0.22 μm filter (Sartorius). Aliquots of 100 μL of filtered BSPH, PSPH, CTRL, and BHI without hydrolysates (BHI-WH) as positive control were dispensed on 96-wells microtiter plates and inoculated with 5 μL of the bacterial suspension as previous prepared. Four wells for each strain and for each solution (BSPH, PSPH, CTRL and BHI-WH) were set up. The antibacterial assay was performed separately on separate microtiter plates for each sample and for each batch.

As blank samples, 100 μL BHI-hydrolysate and BHI-WH solutions before incubation were used. The microtiter plates were then incubated at 37 ◦C for 24 h and growth was measured automatically every 30 min at OD600 using a SPECTROstar nano microplate spectrophotometer reader (BMG Labtech, Ortenberg, Germany). Each growth curve was fitted by the primary model of Baranyi and Roberts [39] wrapped in DMFit Excel add-in [40], that was utilized also to evaluate the maximum specific growth rate (μ), the duration of lag phase (λ) according to Petretto et al. [38]. The 1000XOD absorbance values were log transformed to calculate the growth parameters with DMFIT add-in. Analysis of variance (ANOVA) was performed separately for each bacterial strain tested, using as factor the four treatments: BSPH, PSPH, CTRL, and BHI-WH to evaluate the influence of the two hydrolysates on the values of maximum specific growth rate (μmax) and lag phase (λ). When a significant effect was observed (*p* < 0.05), the differences between means were separated using the Tukey-Kramer multiple comparisons test. SPSS software, version 22, was used to conduct the statistical analyses.
