*3.1. Chemical Composition and Water Activity of Royal Jelly Powder*

Table 2 showed the chemical composition of lyophilized powder in the control group and TR groups. It shows that TR samples had more proteins and total sugar than the control sample (*p* < 0.05). The reason why the protein contents increased from 33.39% to 35.60% in the lyophilized powder with trehalose was that the trehalose could prevent the loss of active substances such as proteins from drying-related stresses during the VFD process [34]. With the increase of trehalose content of the samples, the total sugar content in the lyophilized powder increased from 33.80% to 36.80%. There were no significant differences in fat, ash, pH value, and 10-HDA between the control group and TR groups (*p* > 0.05). The moisture content and aw of lyophilized powder were 3.32%–4.03%, 0.203–0.235, respectively. The moisture content of all the samples was found to be lower than 5%, which was important for stability. The aw is an important parameter to predict the stability of lyophilized powder. The aw of freeze-dried production is generally between 0.28–0.11 and all aw values of the lyophilized powders in this research were within this range which could help to control non-enzymatic browning and inhibit microbial growth [18]. The aw decreased with the increase of trehalose content, which was due to trehalose having an ability to enhance the hydrogen bond strength. Commonly, the water molecule had the disposition to move to the molecules which were easy to form hydrogen bonds. Due to the existence of trehalose, there was a weaker interaction between water molecules and royal jelly lyophilized powder leading to lower aw [35].


**Table 2.** Proximate composition (%), water activity (av) and acidity of trehalose-royal jelly lyophilized powder (TR).

Notes: Values are given as mean ± standard error. The letter from a to f shows the different value in significant differences from high value to low value (*p* < 0.05).

### *3.2. Evaluation of Royal Jelly Freeze-Dried Powder*

The angle of repose, bulk density and tapped density were used to determine powder fluidity. The flow behavior of the powder is useful to predict its quality characteristics during processing, packaging, and storage [36]. Table 3 shows the parameters of royal jelly lyophilized powder.

**Table 3.** Bulk density, tapped density and angle of repose of control and trehalose-royal jelly lyophilized powder (TR).


Notes: Values are given as mean ± standard error. The letter from a to f shows the different value in significant differences from high value to low value (*p* < 0.05).

### 3.2.1. Angle of Repose

In general, the angles of repose <30◦, 30◦–45◦, 45◦–55◦, and >55◦ indicated good flowability, some cohesiveness, true cohesiveness, and high cohesiveness (very limited flowability), respectively [37]. As shown in Table 3, the control group had the smallest angle of repose but also >50◦ among all the groups, indicating that the lyophilized powder had a high cohesion. The higher angle of repose of the TR groups might be due to the smaller particle size and higher cohesion of the lyophilized powder in TR groups. With the increase of trehalose content, the angle of repose and the fluidity of the powder gradually increased.

### 3.2.2. Bulk Density and Tapped Density

Table 3 shows that the bulk density and tapped density of royal jelly lyophilized powder increased with the increase of the trehalose content. The increased density could be explained by the moisture content and particle shape of the lyophilized powder. First, the moisture content of the powder affects the bulk density and tapped density directly. If the powder contains lower moisture content, the total solid density will increase. Second, the particle shape and size also significantly affect the bulk density and tapped density. For instance, the particles with irregular shapes have low bulk density [18]. According to SEM images, the structure of lyophilized powder with trehalose had a broken glass shape, and the sample in the control group was more complete. Therefore, the royal jelly lyophilized powder with trehalose had a higher bulk density and tapped density than the control sample.

### *3.3. Solubility*

In the food industry, solubility can be regarded as the rate of dissolution to describe powder reconstitution properties [38]. According to the research reported by Jayasundera et al., the low aw value and relatively high moisture content (due to the high water holding capacity of the protein) of powder also contributed to higher solubility [39]. Haque et al. reported that high aw values exacerbate protein denaturation which harmed the solubility [40]. Quek et al. proved that there was a positive correlation between the solubility of spray-dried watermelon powder and water content [41]. As shown in Figure 1, the trehalose could improve the solubility of lyophilized powder and the solubility of powder increased with the increase of trehalose addition level. The control group and TR 9 group were ~70.80% and ~78%, respectively.

**Figure 1.** Solubility and hygroscopicity of control sample and trehalose-royal jelly lyophilized powder (TR).

### *3.4. Hygroscopicity*

Hygroscopicity is the ability of a material to absorb moisture from the environment [42]. Hygroscopic powders will absorb water from the air, increasing their cohesion and decreasing their flowability which adversely affects the quality of the powder. Powders with hygroscopic properties less than 20% are generally considered as not very hygroscopic [43]. It is reported that the powders are considered hygroscopic if the hygroscopicity value of powder is in the range of 15%–20% (determined at 75% of RH) [44]. According to Figure 1, all samples could be considered as not hygroscopic. The hygroscopicity of the powder dropped to the bottom with 0.5 wt.% content of trehalose (TR 5) and then increased slightly.

### *3.5. Total Flavonoids and Total Phenols Contents (TFC and TPC)*

TFC and TPC of royal jelly have a variety of pharmacological properties, including antibacterial, anticancer, anti-inflammatory, immunomodulatory and antioxidant activities [45]. The TFC and TPC content of royal jelly lyophilized powder are shown in Figure 2. The content of flavonoids and phenols in TR groups were higher than those in the control group (*p* < 0.05) and reached the peak in the TR 5 group of which the TPC and TFC were 2.08 mgGAE·g−<sup>1</sup> and 11.2 mgRE·g−1, respectively. This phenomenon could be due to the lower content of flavonoids and phenols degradation caused by trehalose during the VFD process. Trehalose can interact with bioactive compounds in royal jelly and form complexes [46]. According to the research describing the effect of adding sugars during storage, trehalose has no direct internal hydrogen bonds compared with most other disaccharides. All the four internal hydrogen bonds are connected indirectly via two water molecules which can form part of the natural dihydrate structure. This arrangement makes the molecule unusual flexibility around the disaccharide bond, which may allow trehalose to adhere more closely to the irregular surface of the macromolecule than other disaccharides and protect the bioactive substance [47].

**Figure 2.** TPC and TFC of the control sample and trehalose royal jelly lyophilized powder (TR).

*3.6. The Free Radical Scavenging Activity of Lyophilized Powder*

Figure 3 shows the radical-scavenging effect of samples in control and TR groups upon hydroxyl radicals. All lyophilized powder inhibited the formation of hydroxyl radicals in varying degrees. The DPPH radical-scavenging effect of the control group was 8% and TR samples showed significantly higher free radical scavenging activity than the control group (*p* < 0.05). TR 5 showed the highest radical-scavenging effect compared to other TR samples. However, trehalose had no free radical scavenging activity. It was speculated that the strongest antioxidant capacity of the TR 5 group might be due to the highest TPC and TFC among the TR groups. According to the studies of Guo et al. and Nurcholis et al., TPC of royal jelly showed certain antioxidants and the TFC also had a significant positive correlation with royal jelly antioxidant activity from ethanol [2,48].

**Figure 3.** The free radical scavenging activity of the control sample and trehalose-royal jelly lyophilized powder (TR).

Therefore, lyophilized powder with 0.5% trehalose was then selected for further analysis because of the good particle properties, low moisture, and high antioxidant activity and solubility.

### *3.7. Color*

A higher *L\** value means the brighter color of the lyophilized powder. The negative *a\** value indicates that the color of freeze-dried powder was close to the green. The higher value of *b\** shows lyophilized powder is close to yellow. Table 4 shows that the *L\** values of royal jelly lyophilized powder with different trehalose content were significantly different (*p* < 0.05). The *L\** value of TR groups was slightly decreased and the *b\** value of the TR groups was increased compared with the control sample. The Δ*E\** represented the color difference between the TR groups and the control group. With the addition of trehalose, Δ*E\** of lyophilized powder increased. The addition of trehalose increased the content of phenolic substances in the lyophilized powder, which resulted in the color deepening of lyophilized powder [49]. However, the color of the TR 5 group was closest to the control group. It was speculated that this difference might be due to non-enzymatic browning. Phenolic substances could promote the non-enzymatic browning of royal jelly to a certain extent and form brown substances through their oxidation and condensation which could affect the color of royal jelly lyophilized powder [50].


**Table 4.** Color comparison of royal jelly lyophilized powder (TR) and control group.

Notes: Values are given as mean ± standard error. The letter from a to f shows the different value in significant differences from high value to low value (*p* < 0.05). Control, TR 1, TR 3, TR 5, TR 7, and TR 9 were 0%, 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% addition amount of trehalose, respectively.

### *3.8. Particle Size*

The particle size distribution affects many properties such as bulk behavior and the homogeneity of powder [51]. As shown in Section 3.6, the lyophilized powder with 0.5% trehalose was selected for further analysis. Figure 4 shows that TR 5 appeared approximately as Log-normal distribution. There was a small peak around 10 μm which might be the fractions generation during handling or the sieving process. The median diameter (d50) of lyophilized powder with trehalose (124 μm) was slightly smaller than that of the control group (134 μm). The size of particles could depend on the milling type, operating conditions, and milling time of fabrication [52]. Almost 80% of royal jelly lyophilized powder fell within the range of the opening sizes of the sieve screens.

**Figure 4.** The particle size of the control sample and royal jelly lyophilized powder with 0.5% addition of trehalose (TR).
