*3.5. Determination of of DPPH*• *Scavenging Potential*

The radical *2,2-diphenyl-1-picrylhydrazyl* (DPPH•) is a stable free radical which is reduced in the presence of hydrogen-donating antioxidants, promoting a color change that can be measured by spectrophotometry [54]. The DPPH• scavenging activity of propolis was determined by mixing 50 µL of samples at concentrations of 1 to 50 µg/mL (in absolute ethanol) and 100 µL of 0.004% (*w*/*v*) DPPH•, followed by 20 min incubation at RT, in the dark, and an absorbance measurement at 517 nm. The percentage reduction for each tested concentration was calculated using the following equation:

$$\text{Inhibition (\%)} = \times 100\tag{1}$$

where ASample is the absorbance of the extract with DPPH• and AControl the absorbance of the control (DPPH• and ethanol). EC<sup>50</sup> (µg/mL), which defines the propolis concentration needed to scavenge 50% of the free radical, was calculated by interpolation from those values. Gallic acid was used as a standard.

#### *3.6. Evaluation of Antimicrobial Activity*

To evaluate propolis antimicrobial activity, a panel of microorganisms was selected, taking into consideration their susceptibility and their clinical and pharmaceutical importance. Gram-negative (*Escherichia coli* CECT 423) and Gram-positive bacteria (*Bacillus subtilis* 48886, *Bacillus cereus* ATCC 7064, *Bacillus megaterium*, *Propionibacterium acnes* H60803, *Staphylococcus aureus* ATCC 6538 and *Methicillin-resistant Staphylococcus aureus* M746665 (MRSA)), as well as the yeast *Candida albicans* 53B and *Saccharomyces cerevisiae* BY4741, were all provided by the culture collection of the Department of Biology of the University of Minho. Bacteria were cultured in LB medium (Difco) for 24 h at 37 ◦C and 200 rpm and yeast in YPD medium (Difco) for 48 h at 30 ◦C and 200 rpm. Agar 2% (*w*/*v*) was added to each recipe to prepare solid media (LBA and YPDA).

An adaptation of the agar dilution method was used to determine the antibacterial and antifungal activities of all of the propolis mixtures. Each mixture was incorporated in LBA and YPDA media at various concentrations (25, 50, 100, 200, 500, 750, 1000, 1500 or 2000 µg/ ml) depending on the strain under study. Subsequently, 5 µL drops of exponential phase microbial cultures (OD<sup>600</sup> = 0.4–0.6) were transferred to the Petri dishes, in triplicate, with incubation for 24 h at 37 ºC in the case of bacteria, or 48 h at 30 ºC in the case of yeast. Minimum inhibitory concentration (MIC) values were obtained upon observation of the presence/absence of microbial growth. Experiments were repeated three times.

#### *3.7. Statistical Analysis*

All of the assays for chemical characterization and antioxidant potential were analyzed and the results were presented as mean ± standard deviation from a variable number of assays, always equal to or greater than three (*n* ≥ 3). GraphPad Prism for Windows (version 8.0.1, GraphPad Software, San Diego, California USA, www.graphpad.com, accessed on 2 December 2021 was used in the statistical analysis of the results. The results were analyzed using analysis of variance (ANOVA) followed by the Tukey test. Differences considered statistically significant (*p* ≤ 0.05) were expressed with the alphabetical notation system, using different letters (lowercase when comparing mixtures and individual extracts, uppercase when comparing mixtures).

#### **4. Conclusions**

This study is a first approach to mixing propolis samples from different years and regions, and consequently with different bio and chemical profiles. We found that when mixing propolis from the same apiary but collected over different years, or when combining propolis samples collected from different regions and years, the antimicrobial and antioxidant activities of the most active of the individual extracts were either preserved or enhanced. Considering the results regarding the chemical characterization (TPC and TFC) and bioactivities (antioxidant and antimicrobial activities) of the mixtures, we can also conclude that the differences between individual propolis samples can be attenuated and a reduction in heterogeneity was obtained, thereby contributing to propolis standardization. These findings support the great potential of propolis and add even more value to this hive resource. Such valorization is also related to a greater use of the product, since samples from different years can be used without any loss of bioactivity. This efficiency in combining different propolis extracts/ samples can contribute to increasing beekeepers' interest in this product and enable them to face larger demands for this natural product [55]. Together, these main outcomes are two important starting points for the valorization and standardization of propolis.

Characterization and quantification of propolis bioactive molecules, such as polyphenols, work as a fingerprint of propolis samples, being of interest in medicine and nutraceuticals [56] and several other applications. With the possible standardization of propolis, its application in combating several health problems, such as obesity and associated metabolic disorders becomes easier [57], or as an antioxidant and anti-inflammatory agent in the prevention and care of various diseases [58,59].

**Author Contributions:** Conceptualization, C.A.-A. and R.O.; methodology, M.P. and A.S.F.; formal analysis, M.P., A.S.F., A.C., R.O. and C.A.-A.; investigation, M.P. and A.S.F.; resources, A.C., R.O. and C.A.-A.; writing—original draft preparation, M.P. writing—review and editing, M.P., A.S.F., A.C., R.O. and C.A.-A.; supervision, R.O. and C.A.-A.; project administration, C.A.-A.; funding acquisition, A.C., R.O. and C.A.-A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by European Investment Funds by FEDER/COMPETE/POCI— Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145- FEDER-006958 and National Funds by FCT—Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2019. Ana Freitas acknowledges the financial support provided by national funds through FCT—Portuguese Foundation for Science and Technology (PD/BD/128276/2017), under the Doctoral Programme "Agricultural Production Chains—from fork to farm" (PD/00122/2012) and from the European Social Funds and the Regional Operational Programme Norte 2020. This study was also supported by CITAB research unit (UIDB/04033/2020) and by CBMA research unit (UIDB/04050/2020) funded by national funds through the FCT I.Pg.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank beekeepers Amadeu Fortunas (Casa do Couto; Montalegre, Portugal and Pedro Fernandes (Encostas do Côa; Pinhel, Quinta Nova, Portugal for the propolis samples used in this work.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

