**1. Introduction**

Royal jelly is a thick milky-white or yellowish fluid slightly sweet and obviously acidic, which is produced and secreted by nurse honey bees from their hypopharyngeal gland [1]. Due to this kind of food being mainly used to feed to the queen bee, it is called royal jelly and also called bee milk. Royal jelly has been paid more and more attention among researchers because of its rich nutritional value, including protein, lipid, vitamins, trace elements and many other biological activities [2]. For example, 10-hydroxy-decenoic acid is one of the important active components in royal jelly [3]. Royal jelly possesses several pharmacological activities such as anti-oxidation, anti-inflammation, anti-fatigue, antiageing, antineoplastic, and anti-diabetes which can protect neurons and inhibit oxidative stress damage in the brain to effectively improve Alzheimer's disease and Parkinson's disease [4–6]. However, royal jelly is susceptible to the influence of light, temperature, time and other factors, resulting in the loss of components and deterioration of active substances during production, storage, and transportation, which has an impact on the quality of royal jelly. Therefore, to avoid the inactivation of active substances, fresh royal jelly is usually cryopreserved during transportation and storage, which increases the processing cost and difficulty of royal jelly seasonality. The traditional processing method has a serious impact on the quality of royal jelly and destroys its activity. Therefore, vacuum freeze drying (VFD) is widely used as the method to fabricate royal jelly lyophilized powder for convenient transportation and storage.

VFD has been widely used to fabricate products which have high quality and economic value. Freeze-dried foods can maintain almost the same color, flavor, and nutrient value

**Citation:** Li, L.; Wang, P.; Xu, Y.; Wu, X.; Liu, X. Effect of Trehalose on the Physicochemical Properties of Freeze-Dried Powder of Royal Jelly of Northeastern Black Bee. *Coatings* **2022**, *12*, 173. https://doi.org/ 10.3390/coatings12020173

Academic Editor: Jaejoon Han

Received: 2 January 2022 Accepted: 28 January 2022 Published: 29 January 2022

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compared with fresh foods [7]. Recently, the VFD process has increasingly been used to fabricate protein products [8]. However, some proteins could undergo structural changes during the VFD process. For instance, Song et al. reported that the structure of bovine serum albumin changed during the VFD process [9]. A limited number of potentials for biopharmaceuticals might undergo inactivation, denaturation, and other reactions during the fabricating process [10]. Moreover, Maillard reaction and oxidation could also happen during the VFD process, which should be avoided as much as possible during the food production process [11].

It has been reported that the stability of protein can be achieved by the formation of an amorphous glass matrix [12]. The high viscosity and stable structure of the glassy state are the important factors in preventing the protein from unfolding. To enhance the stability of the protein during the VFD process, monosaccharides and disaccharides have been widely used to prevent protein denaturation and protect the stability of the protein crystal structure [13]. Recently, different cryoprotective agents such as sucrose, lactose, mannitol, and trehalose have been used to improve the storage stability and properties of freeze-dried powders [14,15]. For instance, trehalose is a non-reducing disaccharide with a higher glass transition temperature and a lower hygroscopic ability compared with other disaccharides. Trehalose is widely used as a stabilizing agent in the food industries, which can interact intensively with the surface of macromolecules [16,17] and improve the stability of freezedried samples significantly [18,19]. Trehalose has been used to improve the properties of peeled shrimp protein during frozen storage and increase the total polyphenols and antioxidant activity of apple puree [20,21]. However, there are very few studies about the protective effect of trehalose on vacuum freeze drying of northeast black bee royal jelly.

In this study, the protective effect, and physicochemical properties of different trehalose content on vacuum freeze-drying of royal jelly are systemically studied. The effect of trehalose on the physicochemical property, total flavonoid content, solubility and free radical scavenging activity of royal jelly lyophilized powder are investigated. Finally, 0.5% content of trehalose is selected as the best addition content which could reduce the loss of protein and total sugar during fabrication and exhibits the best DPPH radical scavenging ability as well as the lowest hygroscopicity.

### **2. Materials and Methods**

### *2.1. Materials*

The frozen northeast black bee royal jelly (Jilin Hanfeng Agricultural Science and Technology Development Co., Ltd., Changchun, China) was stored at −20 ◦C and used to prepare the lyophilized powder. All the frozen royal jelly was transported by a cold chain and maintained for −20 ◦C in the whole process. Trehalose, Folin-Ciocalteu reagent, gallate acid, 2,2-diphenyl-2-picrylhydrazyl (DPPH), and 10-Hydroxy-2-decanoic acid (10-HDA) were purchased from Meilun Co., Ltd. (Dalian, China). Phenol, vitriolic acid, methanol, phosphoric acid, aluminium chloride, sodium carbonate, and potassium acetate were purchased from Beijing Beihua Co., Ltd. (Beijing, China). All chemicals and solvents were of analytical grade and used as received.

### *2.2. Fabrication of Lyophilized Powder*

Different contents of trehalose, including 0 wt.% (Control), 0.1 wt.% (TR 1), 0.3 wt.% (TR 3), 0.5 wt.% (TR 5), 0.7 wt.% (TR 7), and 0.9 wt.% (TR 9), were added into fresh royal jelly and made into lyophilized powder via a vacuum freeze drier (Beijing Boyikang Instrument Co., Ltd., Beijing, China) (Table 1). The group with trehalose addition of 0% was the control group. First, 10 g royal jelly was defrosted at room temperature for 10 min. Then, the trehalose particles were fully dissolved in distilled water, and the visible impurities were removed from fresh royal jelly. Finally, the trehalose solution and royal jelly were poured into a centrifugal tube, mixed with a vortex oscillator for 60 s and pre-freezed at −40 ◦C for 6 h. During VFD process, the freezing time, the vacuum degree, the temperature of the heating plate, and the temperature of the cold trap were 48 h, 40 Pa, −18 ◦C, and −85 ◦C, respectively. Each formulation group was replicated three times for repeatability.

**Table 1.** Formulations of lyophilized powder with royal jelly add trehalose (TR).


Note: 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.

### *2.3. Characterization of Compositional of Royal Jelly Powder*

### 2.3.1. Protein

The protein content was measured according to the Association of Official Analytical Chemists (AOAC) (2006) method [22].

### 2.3.2. Total Sugars

The total sugars were evaluated according to the Phenol-Sulfuric Acid Assay method [23].

### 2.3.3. Fat

The fat content was determined by a fat analyser (SOX500, Hanon, Jinan, China) based on the Soxhlet extractor method.

### 2.3.4. 10-Hydroxy-2-Decanoic Acid (10-HDA)

The 10-HDA analysis was performed by high-performance liquid chromatography (HPLC) based on published protocols with minor modifications [24]. Prepare 100 mL of 100 μg mL−<sup>1</sup> 10-HDA and standard substance 0, 5, 10, 20, 30, and 40 Cg mL−<sup>1</sup> reserve solution in methanol: water (50:50, V/V).

Approximate 50 mg lyophilized powder was dissolved in 25 mL methanol: water (50:50, V/V) solvent and then treated with 35 kHz ultrasound in an ultrasonic cleaner (Barker, Shanghai, China) for 30 min. After ultrasonic treatment, the sample solution was filtered (0.45 μm filter) and transferred to 2 mL autosampler vials prior for injection.

HPLC separation was performed on a 100 mm × 4.6 mm × 3.5 μm C18 column (Meilun Co., Ltd., Dalian, China) at 30 ◦C with a mobile phase flow rate of 1 mL min−<sup>1</sup> (using isometric conditions). The mobile phase consists of methanol: water: phosphoric acid (50:50:0.3, V/V/V). The maximum absorbance of 10-HDA is 210 nm and the injection volume was 10 μL.

### 2.3.5. Moisture

The moisture content was determined by a rapid moisture meter (HB43-S, Mettler Toledo, Greifensee, Switzerland).
