2.3.8. Water Activity

The water activity (aw) of lyophilized powder was determined by a pre-calibrated water activity meter (Rotronic, Bassersdorf, Switzerland) at 26 ± 0.5 ◦C.

### *2.4. Characterization of Royal Jelly Lyophilized Powder*

### 2.4.1. Angle of Repose

Herein, the angle of repose was measured via a fixed funnel method which was similar to the method reported by Alanazi et al. [27]. Firstly, a funnel was fixed on the iron rack vertically above a piece of blank paper. Then, the lyophilized powder was poured into the funnel and slipped on the blank paper until the peak of the powder pile touched the outlet of the funnel. Finally, the angle of repose can be calculated by:

$$\theta = \tan^{-1}(H/R) \tag{1}$$

where *θ* is the angle of repose, *H* is the distance between the blank paper and the funnel outlet, and *R* is the radius at the bottom of the powder cone. The measurement was replicated three times to calculate the average angle.

### 2.4.2. Bulk Density and Tapped Density

The measurements of bulk density and tapped density were carried out with the method reported by Erdem et al. with slight modifications [28]. First, the powder was poured into a clean and dry graduated cylinder (5 mL) which was with a mass of *m*. Tap the cylinder once slightly to remove all the adhering powder on the wall, and then record the volume of the powder *v*<sup>1</sup> and weigh the mass of cylinder and powder *m*1. Then, tap the cylinder until the powder reached a constant volume, record the volume of the powder *v*<sup>2</sup> and weigh the mass of cylinder and powder *m*2. Finally, the bulk density *Db* and the tapped density *Dt* can be calculated by:

$$D\_b = (m\_1 - m) / v\_1 \tag{2}$$

$$D\_t = (m\_2 - m) / v\_2 \tag{3}$$

### *2.5. Solubility*

The solubility of lyophilized powder was carried out with the method reported by Erdem et al. with slight modifications [28]. In total, 2.0 g royal jelly lyophilized powder and 50 mL distilled water were added into a glass beaker and mixed for 5 min at 800 rpm with a stirrer. The obtained suspensions stood at room temperature for 10 min and centrifuged at 500× *g* for 5 min. Finally, the supernatant was dried at 102 ◦C in an incubator oven until obtained a constant weight. The solubility can be calculated by:

$$\text{Solubility} = (m\_s/m\_t) \times 100\% \tag{4}$$

where *ms* is the mass of soluble solid, and *mt* is the mass of the total solid before dissolution.

### *2.6. Hygroscopicity*

Hygroscopicity was determined by the method reported by Tonon et al. [29]. One gram of each powder sample was exposed at 75% RH (saturated sodium chloride solution) for 192 h. The hygroscopic ability was presented as a percentage of the water absorption of the dried powder:

$$\text{Hygroscopicity} = \frac{m\_{\text{wet}} - m\_{\text{dry}}}{m\_{\text{dry}}} \times 100\% \tag{5}$$

where *m*wet is the mass of powder after water absorption, and *m*dry is the mass of the dry powder.

### *2.7. Measurement of Total Flavonoid Content (TFC)*

The TFC of royal jelly lyophilized powder was determined by the aluminium chloride colorimetric method reported by Liu et al. with slight modifications [30]. An amount of 0.5 g lyophilized powder sample was mixed with 15 mL of 95% ethanol and ultra-sonicated for 1 h. Then, the ethanol extract of lyophilized royal jelly powder was obtained by filtration. An amount of 1 mL of ethanol extract, 1 mL of AlCl3 solution (1 M), 1 mL potassium acetate (1 M), and 15 mL ethanol (95 wt.%) were mixed and added distilled water to 50 mL. The solution was kept at room temperature for 40 min and absorbance was recorded at 420 nm wavelength against blank via a UV-visible spectrophotometer (TU-1901L, Beijing Universal Instrument Co., Ltd., Beijing, China). Various concentrations of rutin were prepared in alcohol as a reference flavonoid. Finally, the same process and measurement were repeated and the calibration curve was plotted. The TFC of royal jelly lyophilized powder was expressed as rutin equivalent per gram of royal jelly (mgRE·g<sup>−</sup>1).

### *2.8. Measurement of Total Phenolic Contents (TPC)*

The total phenol in ethanolic extract of royal jelly lyophilized powder was measured via a colorimetric method. The total phenolic contents were measured by the Folin-Ciocalteau method reported by Singleton et al. with slight modifications [31]. In total, 10 mg lyophilized powder sample was mixed with 100 mL of ethanol to prepare the ethanol extract of powder (100 mg·L−1). Moreover, 100 <sup>μ</sup>L of the ethanol extract and 100 <sup>μ</sup>L of 7.5 wt.% Na2CO3 were fully mixed. Then, 50 μL of Folin-Ciocalteu reagent was added and the obtained mixture and kept at room temperature in the dark for 30 min. The absorbance of all the samples was measured at 760 nm via a UV-visible spectrophotometer (TU-1901L, Beijing Universal Instrument Co., Ltd., Beijing, China). Under the same conditions, gallate acid was used as a reference phenolic. Finally, the same process and measurement were repeated and the calibration curve was plotted. TPC of the extract was evaluated based on gallic acid equivalent (mgGAE·g<sup>−</sup>1).

### *2.9. The Free Radical Scavenging Activity Assays*

Antioxidant activities were measured by DPPH radical-scavenging assay based on the method reported by Brand-Williams et al. with slight modification [32]. One gram of lyophilized powder was added to 20 mL of ethanol. The mixture was shocked in the dark for 1 h and filtrated. 3.6 mL of DPPH (0.1 mmol L−<sup>1</sup> in ethanol) and 0.66 mL extract were mixed. The mixture was incubated in the dark for 30 min at room temperature and then the scavenging capacity was measured at 517 nm.
