# The Possibility of Using Bee Drone Brood to Design Novel Dietary Supplements for Apitherapy
## 3 Results and Discussion
### Protein  
In vitro protein digestibility was tested on soluble protein content, protein degradation profiles (sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)) and HPTLC profiles of free amino acids. The analysis of protein digestibility in the prepared capsules showed that the ES capsule itself contained a small amount of soluble protein, probably derived from the cuticle (Table 4). Drone brood is the main source of soluble protein in the capsules analyzed. Among all the prepared preparations, the highest protein content was found in the DB capsule with the highest share of drone brood (300 mg of drone brood/capsule), compared to DB + ES or DB + CC (300 mg of drone brood/capsule of 550 mg). In the gastric phase, it was found that the protein is best digested in the DBand DB + ES capsules (84.09 and 83.13%, respectively). A significantly lower percentage of protein digestion was also observed in the DB + CC (46.03%) capsule. In the intestinal fractions after in vitro digestion was found, a high level of protein digestion in the DB + ES capsule at a level of 60.76% was present. Significantly lower results were shown for the DB + CC capsule, which was 25.15% lower compared to the DB + ES capsules (Figure 2a).  
SDS-PAGE protein profiling was performed to understand changes in protein content during in vitro digestion of the supplement capsules. Most of the proteins came from the drone brood (Figure 3). The undigested DB sample showed a rich protein profile of 10, 12, 25, 26, 40, 42, 61, 70, 74, 80, 115, 130, and 175 kDa. In all the samples, further digestion in the stomach resulted in the elimination of a large amount of protein and the generation of fuzzy bands (10, 42, and 72 kDa). This indicates that the organized protein network began to disintegrate in the gastric phase, and most of the protein macromolecules disintegrated rapidly. In the intestinal fractions of the preparations, mainly bands with a mass of 10, 26, 42, 74, and 130 were identified, and their intensity was higher compared to the non-digested fractions. This may indicate the participation of digestive enzymes (lipase, amylase, and protease) in the protein profile of the intestinal fraction, whose molecular weights are 48, 56, and 22 kDa, respectively [42].  
Similar observations were made by other researchers [43]. The authors tested the protein content by electrophoresis in soy milk. They showed that after digestion in the oral cavity, the protein content did not change, which could be influenced by the lack of enzymes responsible for protein digestion in the oral cavity. After 5 and 60 min of digestion in the stomach, the number of bands and their intensity decreased significantly compared to the undigested sample, with a significant decrease in protein content after 5 min. However, after 30 and 120 min of digestion in the intestines, they observed an intensification of some bands with a mass of 25 and 35 kDa, compared to the gastric fraction.  
Figure 3: Protein profiles of fractions obtained by SDS-PAGE. M—molecular weight marker; 1—DB (undigested); 2—DB (gastric phase); 3—DB (intestinal phase); 4—DB + ES (undigested); 5—DB + ES (gastric phase); 6—DB + ES (intestinal phase); 7—DB + CC (undigested); 8—(gastric phase); 9—DB + CC (intestinal phase).