## 3 Results and Discussion  
### Characterization of the Grape Pomace Dietary Supplement  
Diet plays a fundamental role in providing strong and healthy honeybee colonies [41]. In fact, there is evidence that the capacity of the colony to face both biotic and abiotic stressors can be enhanced by providing strong bees through a convenient supply of nutrients [42], activating the most important protective physiological system of all organisms, the immune system. The workflow presented in this study follows the scheme depicted in Figure 1.  
Grape pomace [43] and GPP obtained by spray drying were chromatographically analyzed (Figure S2). It is important to point out that the percentage of wall material used (20% maltodextrin) had been previously studied, with satisfactory results [26]. Different phenolic compounds presented a high content, such as syringic acid (153 and 75 mg 100 g\({}^{-1}\) DW for GP and GPP, respectively), and catechin (67 and 43 mg 100 g\({}^{-1}\) DW for GP and GPP, respectively) (Table 2). However, the greater phenolic content was found in the anthocyanin group, reaching a total of 1873 mg 100 g\({}^{-1}\) DW for GP and 1102 mg 100 g\({}^{-1}\) DW for GPP. Additionally, ten anthocyanins, reaching high content, were identified by HPLC-DAD, and the resulting concentration agreed with those reported by [PERSON] et al. [43]. The main anthocyanins quantified from GP raw material and GPP were malvidin 3-\(O\)-hexoside and petunidin 3-\(O\)-hexoside, while content losses of the compounds ranged between 36 and 53%, with an average of 41% of the GPP with respect to the GP raw material. It is important to point out that despite the loss of phenolic compounds due to the drying process (see GPP), the final content was still high, and the phenolic compound and anthocyanin compound concentrations were found to be within the range reported for dry grape pomace (1598.57 mg 100 g\({}^{-1}\) gallic acid equivalents and between 730.7-1850.3 mg 100 g\({}^{-1}\) malvidin 3-glucoside equivalents, respectively) by [PERSON] et al. [44]. Furthermore, it \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Primers** & & **Sequences** & **Reference** \\ \hline \multirow{3}{*}{[PERSON]} & F & AGCATGATGATGATGATGATGATGATGATG & [38] \\  & R & TGAAGGATGATGATGATG & \\ \multirow{3}{*}{[PERSON]} & F & CTCATCGGAAGACATG & [38] \\  & R & TGAATTCAAAGCCAGTTCGAAAA & \\ \multirow{3}{*}{[PERSON]} & F & CAACTCCAGAATTGGAAATAGCA & [39] \\  & R & TTTGAAATAGGATTG & [39] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: \(Cont.\)is important to mention their high anthocyanin content, particularly if they are compared with food with great value of phenolic compounds such as blueberry (around 840 mg 100 g\({}^{-1}\) DW) [45] or elderberry (range 800-1000 mg 100 g\({}^{-1}\) DW) [46], making, therefore, this ingredient a source of bioactive compounds to supplement the diet of bees. It is demonstrated that spray-drying treatments would be adequate for the treatment of waste with a high content of bioactive compounds, such as the case of grape pomace, since it is easy to handle, cheap, scalable, and the ingredient obtained would remain enriched in compounds of interest. Furthermore, it is worthy of note that this research was based only on the polyphenolic composition of grape pomace; however, grape skins represent a more complex matrix. In fact, other compounds or nutrients such as vitamins can contribute substantially to the biological activity [47]. Figure 1: Workflow of the production of GPP as a dietary supplement to strengthen the honeybee immune system. The antioxidant activity was determined by using two assays, 2,2-diphenyl-1-picrylhydrazyl (DPPH*) and oxygen radical absorbance capacity [28]. Assays were carried out and measured for the raw material, GP, and for the ingredients obtained after the