*2.6. Statistical Analysis*

All the data was analyzed using the Kruskal–Wallis test, with an initial analysis of the distribution goodness of fit using the Kolmogorov–Smirnov test. All analysis was carried out with Statgraphics Centurion XVI Software (Statpoint Technologies Inc., Warrenton, VA, USA). The significant differences were validated with a probability < 0.05.

#### **3. Results and Discussion**

One of the most important aspects in the study of AMPs is to determine its antifungal action mechanism. In relation to antimicrobial peptides to biocontrol contaminant microorganisms in winemaking, Enrique et al. (2008) [9] studied the antifungal effect of the synthetic peptide LfcinB17-31 on *B. bruxellensis*, determining that its action mechanism is related to the penetration of the peptides into the cell cytoplasm. Additionally, by fluorescence microscopy, Branco et al. (2017) [12] have described that saccharomycin (antifungal peptides produced by *S. cerevisiae* CCMI885 strain) produce cell membrane disruption and internalization of the peptides in *Hanseniaspora guilliermondii* and *B. bruxellensis*. Our previous results have demonstrated that *C. intermedia* LAMAP1790 releases peptides in the culture medium with masses under 4.6 kDa, which show selective antifungal activity on *B. bruxellensis* strains [16,17]. With the purpose of defining the cell damaged produced by the antifungal supernatant of *C. intermedia* LAMAP1790 (which contains these peptides) on *B. bruxellensis* LAMAP2480, different assays were carried out using calcofluor white (CW), propidium iodide (PI), or 6-carboxy-2,7-dichlorodihydrofluorescein diacetate (C400) [12,22,28] (Figure 1).

Under optimum growth condition, the yeas<sup>t</sup> wall stains bright blue-white by the CW assembly [28], being impermeable to PI [12] and the C400 cannot be oxidized to its fluorescence form [22]. As a negative control, cells were inoculated in buffer HEPES saline pH 7.0 (Figure 1A–C), it can be observed that neither the wall cellular nor the impermeability of the membrane was affected. Only 29.91 ± 7.29% of the observed cells in the medium show green fluorescence (Figure 1C) and none show red color. This increase of green fluorescence would be related to the lack of nutrients that *B. bruxellensis* had during the 24 h trail, due has been reported that such periods may activate an autophagy process [29,30]. Autophagy is a non-selective degradation of organelles or intracellular macromolecules, a recycling process that allows the amino acid supply and survival. *S. cerevisiae* can do mitophagy (removal of damaged mitochondria), therefore, releasing mitochondrial ROS into the cytoplasm [29,30]. On the other hand, when the cells have damage in the membrane, this is no longer impermeable to PI, dying cells in red [23].

Thus, as a positive control of both processes, we carried out an induction to the oxidative stress and membrane damage by zymolyase and H2O2 treatment (Figure 1D–F). As observed in this figure the treatment produced a 63.39 ± 6.92% permeabilization to cell surface membrane, allowing the penetration of PI into the cell (compared with control sample 1B). Besides, a 63.61 ± 8.17% of cells show a rise of intracellular ROS, which allowed the observation of green fluorescence derived from C400 oxidation (Figure 1F). Additionally, when yeasts were exposed to *C. intermedia* supernatant at 12h, it was observed a rise in the number of cells which oxide C400 (Figure 1I) which is sustained at 24 h of incubation (Figure 1L), while it is observed a little rise of permeable cells of PI to 24 h of incubation (Figure 1H,K). When the *C. intermedia* supernatant is treated with protease, a decrease decrease in the number of cells that oxidize C400 and the permeable cells of PI (Figure 1N,O) was observed, confirming that antifungal compounds have protein nature [16].

By comparing the percentage of fluorescent yeas<sup>t</sup> in different conditions (Figure 2), it can be observed that the incubation of *B. bruxellensis* with the antifungal supernatant produce a sustaining little rise in the number of permeable cells to PI at incubation time, is not statistically different from the negative control (Figure 2A).

**Figure 1.** Evaluation of permeability and reactive oxygen species (ROS) accumulation in *B. bruxellensis* LAMAP2480 cells exposed to *C. intermedia* LAMAP1790 antifungal supernatant at different times, using epifluorescence microscopy. Graphics at the right side of each line represents a percentage of fluorescent cells per field counted in each treatment. (**A**–**C**): untreated yeasts (Negative control). (**D**–**F**): yeasts exposed to H2O2 30% *v*/*v* for 30 min, after treatment with zymolyase (600 μg/mL) for 2 h at 37 ◦C (Positive control). (**G**–**I**): yeasts exposed to *C. intermedia* antifungal supernatant for 12 h. (**J**–**L**): yeasts exposed to *C. intermedia* antifungal supernatant for 24 h. (**M**–**O**): yeasts exposed to *C. intermedia* antifungal supernatant for 24 h after a proteolytic treatment to the supernatant for 4 h at 37 ◦C with 2 mg/mL of *Streptomyces griseus* protease (Sigma®). CW: calcofluor white staining, PI: propidium iodide staining, C400: 6-carboxy-2,7-dichlorodihydrofluorescein diacetate staining.

**Figure 2.** Evaluation of damage produced in *B. bruxellensis* cells after 12 h and 24 h of exposition to antifungal supernatant from *C. intermedia* LAMAP1790. (**A**): Membrane damage observed by cell permeability to propidium iodide staining, (**B**): Oxidative damage derived of ROS accumulation measured with 6-carboxy-2,7-dichlorodihydrofluorescein diacetate staining. The evaluation was performed comparing the percentage of stained fluorescent cells of *B. bruxellensis* after each treatment. White bars correspond to control treatments, black bars correspond to yeasts exposed to antifungal supernatant at labeled times and bars in striped lines (labeled 24 h + P) correspond to 24 h of yeasts exposition to antifungal supernatant treated previously with 2 mg/mL of *Streptomyces griseus* protease (Sigma®). Different letter above each column represents a significative difference (*p* < 0.05).

Nevertheless, by evaluating the percentage of cells which oxidize C400, they increase significantly as incubation time increases, even achieving similar values to the obtained due to the introduction to oxidative stress with H2O2 (Figure 2B). These results allow to demonstrate that the sustaining increase of ROS in *B. bruxellensis* is related to the presence of peptides of mass under 10 kDa in the antifungal supernatant of *C. intermedia* LAMAP1790 and propose that its antifungal action mechanism would be related to the oxidative damage that the exposed cell suffers to the supernatant. This should be proved by the non-significant rise of the observed permeability to PI in *B. bruxellensis* between 12 h and 24 h of exposure to antifungal supernatant, because it has been demonstrated that the induction of ROS in yeasts such as *H. guilliermondii* produces the permeabilization of its cellular membrane [11]. Similar effects have been reported to synthetic peptide PAF26 and other similar peptides in which have been demonstrated that they can penetrate the cytoplasm of *S. cerevisiae*, without affecting firstly the integrity of the cellular membrane [13,14,31]. Thus, it determined that the synthetic peptide PAF26 would have a multistep mechanism of action, where it first interacts with the wall or cellular membrane, then it would be endocytosed and accumulated in the vacuoles, and finally, it would be transported to the

cytoplasm and perform its antifungal action [14]. This mechanism would be like the observations made for *B. bruxellensis* by means of fluorescence microscopy.

It was carried out a qualitative antifungal test with *S. cerevisiae*, *B. bruxellensis*, and two strains of *P. guilliermondii* in solid MBA agar plates to determine the formation of inhibition halos produced by *C. intermedia* LAMAP1790. The two strains of *P. guilliermondii* selected was previously studied by Sangorrín et al. (2013) [20]. In that work, from a pool of 15 strains, it was possible to conclude that strains LAMAP3202 and LAMAP3203 (labeled by Sangorrín as P7 and P8) have the highest transformation efficiencies of *p*-coumaric acid in 4-vinylphenol (more aggressive wine-spoilage phenomena). For these reasons, we considered these strains as the best model to our study. As shown in Table 1, *S. cerevisiae* EC1118 strain does not show growth inhibition, while the *B. bruxellensis* LAMAP2480 strain shows a clear inhibition halo surrounding culture of *C. intermedia*, whose diameter reached 19.00 ± 0.62 mm, as it was described by Peña et al. [16,17]. By analyzing the behavior of *P. guilliermondii*, LAMAP3202 and LAMAP3203 strains can be observed that an inhibition halo appears, whose diameters reached 15.33 ± 0.82 mm and 16.17 ± 0.75 mm, respectively (Table 1). Then, *B. bruxellensis* shows a greater sensitivity to the presence of *C. intermedia* than *P. guilliermondii*. Similar studies carried out by Lopes and Sangorrín (2010) [32] have demonstrated that *P. guillermondii* sensitivity depends on the yeas<sup>t</sup> strains to which it is exposed. On the other hand, Villalba et al. (2016) [23] demonstrated that the production of antifungal compounds of protein nature produced by *Torulaspora delbrueckii*, which has a molecular mass above 30 kDa, shows glucanase and chitinase activity. Therefore, the authors conclude that this would be a killer toxin rather than an antimicrobial peptide (AMP). Thus, this work would constitute the first qualitative evidence which shows the sensitivity of *P. guilliermondii* strains to antimicrobial peptides produced by non-*Saccharomyces* yeasts.


**Table 1.** Inhibition halos obtained after the exposure of *C. intermedia* LAMAP1790 against strains of *S. cerevisiae*, *B. bruxellensis* and *Pichia guilliermondii* in MBA medium.

Values with the same superscript letter are not significantly different (*p* < 0.05). † ND: Non-Detected.

With the purpose of determining whether the antifungal e ffect of *C. intermedia* LAMAP1790 is similar in winemaking conditions, it was carried out assays on synthetic must [12]. We decided to use this media to avoid the antimicrobial influence on yeas<sup>t</sup> described to the hydroxycinnamic acids present in the natural grape must (mainly *p*-coumaric and ferulic acid) [33–35]. Thus, the viability of the spoilage strains *B. bruxellensis* LAMAP2480 and *P. guilliermondii* LAMAP3202 were assessed in mixed culture with *S. cerevisiae* EC1118 for 21 days (Figure 3).

The synthetic must was supplemented with 1 μg of low-mass protein fraction obtained from *C. intermedia* supernatant, and then was inoculated using the spoilage yeasts. Posteriorly, it was inoculated with *S. cerevisiae* EC1118 (fermentation starter). As can be seen in Figure 3 (A, B), the growth of *S. cerevisiae* is not a ffected, demonstrating the harmlessness of the antifungal peptides against this yeast. In the case of the e ffect on *B. bruxellensis*, it was observed that its growth decreases in one magnitude order of di fference compared to the control (3A), while in the case of *P. guilliermondii* L3202, minimal changes between the treatment and control were observed (Figure 3B). Despite having growth inhibition of *P. guilliermondii* in solid medium (Table 1), this e ffect was not seen in synthetic must, which can be related to a greater concentration of an antifungal compound, possibly requiring a greater concentration for this specie compared to *B. bruxellensis*. To date, there are no previous studies that assess the antifungal capacity of a compound of protein nature (AMP or killer toxin) on the growth of *P. guilliermondii* in mixed cultures with *S. cerevisiae* in synthetic wine must. Thus, it would be necessary to further study the action of *C. intermedia* peptides in winemaking conditions.

**Figure 3.** Antifungal activity of 1 μg of low mass protein fraction (under 10 kDa) concentrated from *C. intermedia* L1790 supernatant (solid lines) used synthetic wine must. (**A**) *S. cerevisiae* with *B. bruxellensis* L2480 (**B**) *S. cerevisiae* with *P. guilliermondii* 3202. The controls (stripped lines) corresponds to the concentrated supernatant of *S. cerevisiae* BY4741 (not antifungal activity). All assays were performed in triplicate.
