## 2 Materials and Methods
### In Vitro Digestion Protocol  
The protocol was initiated by adding a 5 mL sample (aqueous suspension the Vlp and Zg propolis complexes or standard mix complex with HP-\(\beta\)-CD) to a 50 mL amber centrifuge tube and was mixed with 4.0 mL of SSF. After that, 0.5 mL of \(\alpha\)-amylase aqueous solution (1500 U/mL in SSF), 25 mL of 0.3 mol/L CaCl\({}_{2}\) and 225 mL of distilled water were added to the mixture to make the ratio of sample to SSF 1:1 (\(v/v\)). The mixture was incubated in a water bath at 37 \({}^{\circ}\)C for 5 min with a uniform shake. To simulate the gastric phase, 8.0 mL of SGF, 0.667 mL of pepsin solution (2000 U/mL in SGF), 5 mL of 0.3 mol/L CaCl\({}_{2}\), and 0.448 mL of distilled water were added to the mixture from an oral phase. The mixture was incubated in a water bath at 37 \({}^{\circ}\)C for 2 h with stirring.  
Two separate protocols were performed to simulate the intestinal phase:  
* Centrifugation model: The volume of 8.0 mL SIF was added to gastric phase, followed by adding 5.0 mL of pancreatin solution (800 U/mL in SIF), 3.0 mL of 160 mmol/L bile salts, 40 mL of 0.3 mol/L CaCl\({}_{2}\), 0.15 mL of 1 mol/L NaOH, and 3.16 mL of distilled water. A sodium hydroxide solution was added to the mixture to neutralize the mixture to pH 7.0. The reaction mixture was again incubated in a water bath at 37 \({}^{\circ}\)C for 2 h.  
Figure 1: Illustration of the in vitro gastrointestinal digestion protocol. Created with BioRender.com (accessed on 20 July 2023). Centrifugation tube marked with: “OF” is oral fraction; “GF” is gastric fraction; “IF-C” is intestinal fraction obtained by centrifugation model; “Dout” is fraction outside the membrane; and “Din” is dialyzable fraction.  
* Dialysis model: Enzymes and reagents were added to the gastric phase in the same concentrations as in the 1 st model. However, a semi-permeable membrane for dialysis, which simulated the wall of the small intestine, was filled with NaHCO\({}_{3}\) and NaCl solution and immersed in the test tube with the gastric phase. Simulated intestinal fluid, pancretain, and bile salts were then added in concentrations as already described in the IF-C model. The reaction tube with membrane was placed in a water bath at 37 \({}^{\circ}\)C for 2 h with agitation. The solution outside and inside the dialysis tubing (referred to as \"Dout samples\" and \"Din samples\", respectively) were taken to represent the material that remained within the gastrointestinal tract and the fraction that was accessible for absorption, respectively. At the end of each phase, the oral (OF), gastric (GF), intestinal fraction with centrifugation (IF-C), and the fractions obtained by dialysis model Din and Dout were placed in an ice bath for 10 min and centrifuged at 4472\(\times\)\(g\) for 20 min at 4 \({}^{\circ}\)C to separate soluble (bioaccessible) from insoluble compounds, and then aliquots were stored at \(-\)80 \({}^{\circ}\)C until analysis. The concentration of individual standards in the ethanolic extract of the mixture of standards and HP-\(\beta\)-CD complex during the in vitro digestion was determined, taking into account the obtained average concentrations of polyphenic compounds in the EEP complex with HP-\(\beta\)-CD. Blank sample contained water, enzymes, and digestive fluids. Before spectrophotometric analysis, aliquots of the samples were diluted or directly analyzed without dilution, depending on the analyte concentration in the individual phase. Bioaccessibility Index Bioaccessibility (%) was described as the content of phenolic compounds released in the in vitro digestion process compared to the content of phenolic compounds in the tested samples, and the percentage of bioaccessibility was calculated according to the following equation: Bioaccessibility (%) = \frac{\text{content of compounds released after simulated intestinal digestion}}{\text{content of compounds in the undigested sample}}\times 100\] (1)