Active royal jelly polypeptide freeze-dried powder and preparation method thereof

Abstract

This invention discloses an active royal jelly polypeptide freeze-dried powder and its preparation method. Royal jelly is mixed with water, and then sequentially hydrolyzed using carbon-supported trypsin and carbon-supported papain. Saponin gum is added as an enzyme stabilizer during the hydrolysis. The hydrolysate is then concentrated via microfiltration-ultrafiltration, nanofiltration, freeze-drying, pulverizing, and sieving to obtain the final product. This invention utilizes immobilized enzymes for stepwise hydrolysis, allowing for enzyme recovery and reuse. Saponin gum effectively protects the activity of the immobilized enzymes, improving hydrolysis efficiency. The gentle process throughout avoids damage to heat-sensitive components, effectively preserving the 10-hydroxy-2-decenoic acid content and antioxidant activity. The resulting product exhibits high activity and good solubility, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the technical field of royal jelly preparation, specifically relating to an active royal jelly polypeptide freeze-dried powder and its preparation method. Background Technology

[0002] Royal jelly is rich in proteins, polypeptides, 10-hydroxy-2-decenoic acid (10-HDA), and superoxide dismutase (SOD)-like active substances, possessing various physiological functions such as antioxidation and immune regulation. However, the large protein molecules in natural royal jelly are not easily absorbed directly by the human body, and royal jelly itself has a sour and astringent taste and poor storage stability, limiting its deep processing applications. Enzymatic hydrolysis to convert royal jelly proteins into small polypeptide molecules is a common method to improve its absorption and functional activity.

[0003] Currently, the preparation of royal jelly peptides mostly employs single-enzyme or double-enzyme hydrolysis using free enzymes (such as trypsin and papain). However, the active ingredients in royal jelly are quite sensitive to heat. Traditional enzymatic hydrolysis requires high-temperature enzyme inactivation to terminate the reaction, followed by spray drying or vacuum drying. This process easily leads to 10-HDA degradation and reduced SOD-like activity, thereby weakening the biological efficacy of the final product. Even with freeze-drying, improper control of the pre-freezing rate, sublimation temperature, or vacuum level can easily result in problems such as product collapse, high residual moisture content, and poor solubility, making it unsuitable for long-term storage.

[0004] On the other hand, existing purification methods for royal jelly peptide products mostly employ ultrafiltration or ethanol precipitation. The former has limited ability to remove small molecule impurities, while the latter involves the risk of organic solvent residue and is cumbersome to operate. Although chemical stabilizers commonly used in enzymatic hydrolysis (such as EDTA and DTT) can protect some enzyme activity, their safety is somewhat limited in food applications, necessitating the development of green and efficient enzyme stabilization solutions.

[0005] In summary, how to reduce enzyme usage costs while ensuring enzymatic hydrolysis efficiency, maximize the preservation of active ingredients in royal jelly, and improve the purity and storage stability of the final product are urgent technical problems to be solved in the field of royal jelly deep processing. Summary of the Invention

[0006] This invention discloses an active royal jelly polypeptide freeze-dried powder and its preparation method, aiming to solve the problems of low enzymatic hydrolysis efficiency, difficulty in enzyme recovery, and insufficient retention of product activity in the existing royal jelly polypeptide preparation process.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] (1) Preparation of royal jelly: Take fresh royal jelly and mix it with deionized water at a mass ratio of 1:5-7. Stir well to obtain royal jelly solution;

[0009] (2) First enzymatic hydrolysis: Add carbon-borne trypsin to royal jelly liquid at a solid-liquid mass ratio of 1:25, add 0.02%-0.04% enzyme stabilizer by weight of royal jelly liquid, then heat to 37-40℃ and enzymatically hydrolyze for 2.5h under constant temperature conditions. Adjust the pH to 7.8-8.2 with 0.1mol / L sodium hydroxide solution. After enzymatic hydrolysis, filter carbon-borne trypsin and collect the filtrate for later use.

[0010] Furthermore, the enzyme stabilizer is saponin gum, which has a molecular weight exceeding 400 kDa.

[0011] (3) Second enzymatic hydrolysis: Add carbon-loaded papain to the above filtrate at a solid-liquid mass ratio of 1:20, add 0.02%-0.04% enzyme stabilizer by mass of filtrate, adjust the pH to 6.0-7.0 with 0.1mol / L hydrochloric acid, then heat to 50-54℃ and enzymatically hydrolyze for 3h under constant temperature conditions. After the enzymatic hydrolysis is completed, filter the carbon-loaded papain and collect the filtrate for later use.

[0012] (4) Concentration and freeze-drying: The filtrate after the above-mentioned secondary enzymatic hydrolysis was sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The microfiltration-ultrafiltration was carried out at room temperature and a pressure of 0.05-0.15 MPa to remove high molecular weight saponin gum, and the ultrafiltration permeate was collected. Then, the permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 3 kDa and separated at room temperature and a pressure of 0.5 MPa. The nanofiltration permeate was collected and then concentrated to a solid content of 8%. The concentrate was dispensed into stainless steel freeze-drying trays with a liquid layer thickness of 1.5 cm and pre-frozen at -45℃ for 5 h. After pre-freezing, it was transferred to a freeze dryer. During the sublimation drying stage, the vacuum degree was controlled at 15-25 Pa and the partition temperature at -22℃ for 14 h. During the desorption drying stage, the vacuum degree was controlled at ≤5 Pa and the partition temperature at 20-30℃ for 5 h, so that the residual moisture was ≤2%.

[0013] (5) Crushing and sieving: After freeze-drying, in a clean environment with a relative humidity of 30%, the freeze-dried cake is put into a high-speed crusher and intermittently crushed at a speed of 2000r / min for 10s each time, with an interval of 30s, and repeated 3 times. After crushing, it is passed through a 100-mesh sieve to obtain active royal jelly polypeptide freeze-dried powder.

[0014] Furthermore, the coconut shell activated carbon also includes pretreatment: coconut shell activated carbon with a pore size of 5-30 nm, a specific surface area ≥800 m² / g, and a particle size of 100-200 mesh is boiled with 1 mol / L nitric acid for 2 h, washed with deionized water until neutral, and dried for later use; then KH-550 silanization modification is performed: 0.5%-2% KH-550 relative to the mass of activated carbon is added, the pH is adjusted to 4-5 with glacial acetic acid, and mechanically stirred and refluxed in a 60℃ water bath for 6 h. After the reaction is completed, it is filtered, washed three times each with anhydrous ethanol and deionized water, and dried under vacuum at 80℃ to constant weight.

[0015] Further, the preparation of the carbon-supported trypsin includes: mixing coconut shell activated carbon and trypsin solution at a mass ratio of 15:1; the trypsin solution is prepared with phosphate buffer at pH 7.5 and the enzyme concentration is 0.5 mg / mL; the mixture is shaken and adsorbed at 25℃ and 150 r / min for 3 h; during the adsorption process, 0.05%-0.1% Tween-80 is added as a dispersant; after the adsorption is completed, the mixture is washed 3 times with buffer containing 0.05% Tween-80, then washed once with deionized water, and then centrifuged at 4℃ at 4000 r / min for 10 min, and then refrigerated at 4℃ under humid conditions for later use.

[0016] Further, the preparation of the carbon-supported papain: coconut shell activated carbon and papain solution were mixed at a mass ratio of 12:1. The papain solution was prepared with citrate buffer at pH 7.0, with an enzyme concentration of 0.8 mg / mL. 0.004 mol / L EDTA and 0.5% food-grade Tween-80 were added as a dispersant. The mixture was shaken and adsorbed at 25℃ and 150 r / min for 4 h. After adsorption, it was washed 3 times with buffer containing 0.05% Tween-80, then washed once with deionized water, and then centrifuged at 4000 r / min for 10 min at 4℃. It was then refrigerated at 4℃ under humid conditions for later use.

[0017] The advantages and beneficial effects of this invention are as follows:

[0018] The present invention provides an active royal jelly polypeptide freeze-dried powder, which has significant advantages in improving enzymatic hydrolysis efficiency, preserving product activity, improving product stability, and achieving green production through immobilized enzyme stepwise hydrolysis combined with a mild freeze-drying process.

[0019] Free enzymes are difficult to recover after reaction and require high-temperature inactivation to terminate the reaction, leading to the loss of heat-sensitive active ingredients in royal jelly. Immobilized enzymes, on the other hand, can be gently separated through simple filtration, reducing the need for high-temperature inactivation. Stepwise enzymatic hydrolysis is performed using carbon-supported trypsin and carbon-supported papain, allowing both enzymes to function sequentially under their optimal conditions, synergistically hydrolyzing royal jelly proteins. Immobilized enzymes can be quickly recovered and reused through simple filtration, reducing production costs. Gum saponin is a neutral / weakly anionic heteropolysaccharide containing galactose and mannose backbones. Its long-chain structure, exceeding 400 kDa, forms a physical barrier (steric hindrance effect) around the immobilized enzyme particles, effectively preventing enzyme molecules from desorbing from the carrier surface. Simultaneously, its abundant hydroxyl groups (-OH) can form a hydrogen bond network with the hydrophilic regions of the enzyme surface, maintaining the hydration layer structure of the enzyme in the aqueous phase, thereby slowing down conformational unfolding under high-temperature (50-54℃) and acidic (pH 6.0) conditions. In the first enzymatic hydrolysis step, adding saponin gum can inhibit its spontaneous degradation, maintain the conformational stability of the immobilized trypsin, and improve its reusability. In the second enzymatic hydrolysis step, the hydrolysis temperature needs to be increased. Adding saponin gum can further protect the active site of papain, reduce the impact of high temperature and acidic environment on enzyme inactivation, and thus ensure the continuous and efficient execution of the second enzymatic hydrolysis.

[0020] The subsequent processes employed low-temperature pre-freezing, low-temperature sublimation drying, and room-temperature nanofiltration concentration to avoid the destruction of heat-sensitive active substances in royal jelly (such as 10-hydroxy-2-decenoic acid and SOD-like substances) caused by prolonged high-temperature processing, thus ensuring the bioactivity of the final freeze-dried powder. The molecular weight of saponin gum ranges from tens of thousands to millions of Da. Microfiltration-ultrafiltration effectively retains residual saponin gum, avoiding the impact of high-molecular-weight polysaccharides on the purity of the final product, while not affecting the enrichment of target royal jelly peptides with a molecular weight ≤3kDa.

[0021] Subsequent separation and concentration using a nanofiltration membrane with a molecular weight cutoff of 3 kDa effectively enriches the active peptides in royal jelly, resulting in a concentrated distribution of peptide molecular weight. Through optimized freeze-drying parameters, the product has low residual moisture and can be stored for a long time at room temperature or under refrigeration without the need for preservatives.

[0022] In summary, this invention achieves a good balance between process economy, product activity retention, quality stability, and environmental friendliness, and has significant practical value and broad application prospects. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments.

[0024] I. Materials and Pretreatment

[0025] In the following embodiments, the coconut shell activated carbon was purchased from Zhengzhou Yongkun Environmental Protection Technology Co., Ltd., and underwent the following treatment: Coconut shell activated carbon with a pore size of 5-30 nm, a specific surface area ≥800 m² / g, and a particle size of 100 mesh was boiled with 1 mol / L nitric acid for 2 hours, washed with deionized water until neutral, and dried for later use; then, KH-550 silanization modification was performed: 1% KH-550 relative to the mass of activated carbon was added, the pH was adjusted to 4.5 with glacial acetic acid, and the mixture was mechanically stirred and refluxed in a 60℃ water bath for 6 hours. After the reaction was completed, the mixture was filtered, washed three times each with anhydrous ethanol and deionized water, and vacuum dried at 80℃ to constant weight for later use.

[0026] II. Preparation of Immobilized Enzymes

[0027] The carbon-carrying enzymes used in the following embodiments were prepared according to the following method:

[0028] (1) Preparation of carbon-supported trypsin: The coconut shell activated carbon treated above was mixed with trypsin solution at a mass ratio of 15:1. The trypsin solution was prepared with phosphate buffer at pH 7.5 and the enzyme concentration was 0.5 mg / mL. The mixture was shaken and adsorbed at 25℃ and 150 r / min for 3 h. During the adsorption process, 0.08% Tween-80 was added as a dispersant. After the adsorption was completed, the mixture was washed 3 times with buffer containing 0.05% Tween-80 and then washed once with deionized water. Then it was centrifuged at 4℃ at 4000 r / min for 10 min and stored in a humid state at 4℃ for later use.

[0029] (2) Preparation of carbon-supported papain: The coconut shell activated carbon treated above was mixed with papain solution at a mass ratio of 12:1. The papain solution was prepared with citrate buffer at pH 7.0 with an enzyme concentration of 0.8 mg / mL. 0.004 mol / L EDTA was added, and 0.5% food-grade Tween-80 was added as a dispersant. The mixture was shaken and adsorbed at 25℃ and 150 r / min for 4 h. After adsorption, it was washed 3 times with buffer containing 0.05% Tween-80, and then washed once with deionized water. Then it was centrifuged at 4℃ at 4000 r / min for 10 min and stored in a humid state at 4℃ for later use.

[0030] III. Preparation of Royal Jelly

[0031] In the following embodiments, the royal jelly was taken from the Wang Family Bee Garden in Jiangxi Province. The royal jelly was mixed with deionized water at a mass ratio of 1:6 and stirred evenly to obtain royal jelly liquid for later use.

[0032] Example 1

[0033] (1) First enzymatic hydrolysis: Carbon-borne trypsin and royal jelly liquid solid-liquid mass ratio of 1:25 were added, along with 0.03% of saponin gum by weight of royal jelly liquid. The temperature was then raised to 38℃ and enzymatically hydrolyzed for 2.5 h under constant temperature conditions. The pH was adjusted to 8.0 with 0.1 mol / L sodium hydroxide solution. After the enzymatic hydrolysis was completed, the carbon-borne trypsin was filtered out and the filtrate was collected for later use.

[0034] (2) Second enzymatic hydrolysis: Add carbon-carrying papain to the above filtrate at a solid-liquid mass ratio of 1:20, add 0.03% of the filtrate mass of saponin gum, adjust the pH to 6.5 with 0.1 mol / L hydrochloric acid, then heat to 52℃ and enzymatically hydrolyze for 3 hours under constant temperature conditions. After the enzymatic hydrolysis is completed, filter the carbon-carrying papain and collect the filtrate for later use.

[0035] (3) Concentration and freeze-drying: The filtrate after the above-mentioned secondary enzymatic hydrolysis was sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The microfiltration-ultrafiltration was carried out at room temperature and 0.1 MPa pressure to remove high molecular weight saponin gum and collect the ultrafiltration permeate. Then, the permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 3 kDa and separated at room temperature and 0.5 MPa pressure. The nanofiltration permeate was collected and then concentrated to a solid content of 8%. The concentrate was dispensed into stainless steel freeze-drying trays with a liquid layer thickness of 1.5 cm and pre-frozen at -45℃ for 5 h. After pre-freezing, it was transferred to a freeze dryer. During the sublimation drying stage, the vacuum degree was controlled at 20 Pa and the partition temperature at -22℃ for 14 h. During the desorption drying stage, the vacuum degree was controlled at 4 Pa ​​and the partition temperature at 25℃ for 5 h to ensure that the residual moisture was ≤2%.

[0036] (4) Crushing and sieving: After freeze-drying, in a clean environment with a relative humidity of 30%, the freeze-dried cake is put into a high-speed crusher and intermittently crushed at a speed of 2000r / min for 10s each time, with an interval of 30s, and repeated 3 times. After crushing, it is passed through a 100-mesh sieve to obtain active royal jelly polypeptide freeze-dried powder.

[0037] Example 2

[0038] (1) First enzymatic hydrolysis: Carbon-borne trypsin and royal jelly liquid solid-liquid mass ratio of 1:25 were added, along with 0.02% of saponin gum by weight of royal jelly liquid. The temperature was then raised to 40℃ and enzymatically hydrolyzed for 2.5 h under constant temperature conditions. The pH was adjusted to 7.8 with 0.1 mol / L sodium hydroxide solution. After the enzymatic hydrolysis was completed, the carbon-borne trypsin was filtered and the filtrate was collected for later use.

[0039] (2) Second enzymatic hydrolysis: Add carbon-carrying papain to the above filtrate at a solid-liquid mass ratio of 1:20, add 0.02% saponin gum by mass of filtrate, adjust the pH to 7.0 with 0.1 mol / L hydrochloric acid, then heat to 50℃ and enzymatically hydrolyze for 3 hours under constant temperature conditions. After the enzymatic hydrolysis is completed, filter the carbon-carrying papain and collect the filtrate for later use.

[0040] (3) Concentration and freeze-drying: The filtrate after the above-mentioned secondary enzymatic hydrolysis was sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The microfiltration-ultrafiltration was carried out at room temperature and 0.15 MPa pressure to remove high molecular weight saponin gum and collect the ultrafiltration permeate. Then, the permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 3 kDa and separated at room temperature and 0.5 MPa pressure. The nanofiltration permeate was collected and then concentrated to a solid content of 8%. The concentrate was dispensed into stainless steel freeze-drying trays with a liquid layer thickness of 1.5 cm and pre-frozen at -45℃ for 5 h. After pre-freezing, it was transferred to a freeze dryer. During the sublimation drying stage, the vacuum degree was controlled at 25 Pa and the partition temperature at -22℃ for 14 h. During the desorption drying stage, the vacuum degree was controlled at 5 Pa and the partition temperature at 20℃ for 5 h to ensure that the residual moisture was ≤2%.

[0041] (4) Crushing and sieving: After freeze-drying, in a clean environment with a relative humidity of 30%, the freeze-dried cake is put into a high-speed crusher and intermittently crushed at a speed of 2000r / min for 10s each time, with an interval of 30s, and repeated 3 times. After crushing, it is passed through a 100-mesh sieve to obtain active royal jelly polypeptide freeze-dried powder.

[0042] Example 3

[0043] (1) First enzymatic hydrolysis: Carbon-borne trypsin and royal jelly liquid solid-liquid mass ratio of 1:25 were added, along with 0.04% of saponin gum by weight of royal jelly liquid. The temperature was then raised to 37°C and enzymatically hydrolyzed for 2.5 h under constant temperature conditions. The pH was adjusted to 8.2 with 0.1 mol / L sodium hydroxide solution. After the enzymatic hydrolysis was completed, the carbon-borne trypsin was filtered out and the filtrate was collected for later use.

[0044] (2) Second enzymatic hydrolysis: Add carbon-carrying papain to the above filtrate at a solid-liquid mass ratio of 1:20, add 0.04% of the filtrate mass of saponin gum, adjust the pH to 6.0 with 0.1 mol / L hydrochloric acid, then heat to 54℃ and enzymatically hydrolyze for 3 hours under constant temperature conditions. After the enzymatic hydrolysis is completed, filter the carbon-carrying papain and collect the filtrate for later use.

[0045] (3) Concentration and freeze-drying: The filtrate after the above-mentioned secondary enzymatic hydrolysis was sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The microfiltration-ultrafiltration was carried out at room temperature and 0.05 MPa pressure to remove high molecular weight saponin gum and collect the ultrafiltration permeate. Then, the permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 3 kDa and separated at room temperature and 0.5 MPa pressure. The nanofiltration permeate was collected and then concentrated to a solid content of 8%. The concentrate was dispensed into stainless steel freeze-drying trays with a liquid layer thickness of 1.5 cm and pre-frozen at -45℃ for 5 h. After pre-freezing, it was transferred to a freeze dryer. During the sublimation drying stage, the vacuum degree was controlled at 15 Pa and the partition temperature at -22℃ for 14 h. During the desorption drying stage, the vacuum degree was controlled at 5 Pa and the partition temperature at 30℃ for 5 h to ensure that the residual moisture was ≤2%.

[0046] (4) Crushing and sieving: After freeze-drying, in a clean environment with a relative humidity of 30%, the freeze-dried cake is put into a high-speed crusher and intermittently crushed at a speed of 2000r / min for 10s each time, with an interval of 30s, and repeated 3 times. After crushing, it is passed through a 100-mesh sieve to obtain active royal jelly polypeptide freeze-dried powder.

[0047] Comparative Example 1

[0048] (1) Enzymatic hydrolysis: Take royal jelly, add trypsin, the amount of trypsin added is 0.4% of the weight of royal jelly, add saponin gum 0.06% of the weight of royal jelly, heat to 38℃, adjust the pH to 8.0 with 0.1mol / L NaOH, and hydrolyze at a constant temperature for 2.5h. Then add papain, the amount of papain added is 0.4% of the weight of royal jelly, adjust the pH to 6.5 with 0.1mol / L HCl, heat to 52℃, and hydrolyze at a constant temperature for 3h. After the hydrolysis is completed, heat to 85℃ and keep for 15min to inactivate the enzyme. After cooling, filter and use the filtrate for later use.

[0049] (2) Concentration and freeze-drying: The filtrate after enzymatic hydrolysis was sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The microfiltration-ultrafiltration was carried out at room temperature and 0.1 MPa pressure to remove high molecular weight saponin gum and collect the ultrafiltration permeate. Then, the permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 3 kDa and separated at room temperature and 0.5 MPa pressure. The nanofiltration permeate was collected and then concentrated to a solid content of 8%. The concentrate was dispensed into stainless steel freeze-drying trays with a liquid layer thickness of 1.5 cm and pre-frozen at -45℃ for 5 h. After pre-freezing, it was transferred to a freeze dryer. During the sublimation drying stage, the vacuum degree was controlled at 20 Pa and the partition temperature at -22℃ for 14 h. During the desorption drying stage, the vacuum degree was controlled at 4 Pa ​​and the partition temperature at 25℃ for 5 h to ensure that the residual moisture was ≤2%.

[0050] (3) Crushing and sieving: After freeze-drying, in a clean environment with a relative humidity of 30%, the freeze-dried cake is put into a high-speed crusher and intermittently crushed at a speed of 2000r / min for 10s each time, with an interval of 30s, and repeated 3 times. After crushing, it is passed through a 100-mesh sieve to obtain active royal jelly polypeptide freeze-dried powder.

[0051] Comparative Example 2

[0052] (1) Enzymatic hydrolysis: Add carbon-borne trypsin to royal jelly liquid at a solid-liquid mass ratio of 1:25, add 0.03% of saponin gum by weight of royal jelly liquid, then heat to 38℃ and enzymatically hydrolyze for 5.5h under constant temperature conditions. Adjust the pH to 8.0 with 0.1mol / L sodium hydroxide solution. After the enzymatic hydrolysis is completed, filter the carbon-borne trypsin and collect the filtrate for later use.

[0053] (2) Concentration and freeze-drying: The filtrate after enzymatic hydrolysis was sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The microfiltration-ultrafiltration was carried out at room temperature and 0.1 MPa pressure to remove high molecular weight saponin gum and collect the ultrafiltration permeate. Then, the permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 3 kDa and separated at room temperature and 0.5 MPa pressure. The nanofiltration permeate was collected and then concentrated to a solid content of 8%. The concentrate was dispensed into stainless steel freeze-drying trays with a liquid layer thickness of 1.5 cm and pre-frozen at -45℃ for 5 h. After pre-freezing, it was transferred to a freeze dryer. During the sublimation drying stage, the vacuum degree was controlled at 20 Pa and the partition temperature at -22℃ for 14 h. During the desorption drying stage, the vacuum degree was controlled at 4 Pa ​​and the partition temperature at 25℃ for 5 h to ensure that the residual moisture was ≤2%.

[0054] (3) Crushing and sieving: After freeze-drying, in a clean environment with a relative humidity of 30%, the freeze-dried cake is put into a high-speed crusher and intermittently crushed at a speed of 2000r / min for 10s each time, with an interval of 30s, and repeated 3 times. After crushing, it is passed through a 100-mesh sieve to obtain active royal jelly polypeptide freeze-dried powder.

[0055] Comparative Example 3

[0056] (1) Enzymatic hydrolysis: Add carbon-carried papain to royal jelly liquid at a solid-liquid mass ratio of 1:20, add 0.03% of saponin gum by weight of royal jelly liquid, adjust the pH to 6.5 with 0.1 mol / L hydrochloric acid, then heat to 52℃ and enzymatically hydrolyze for 5.5 h under constant temperature conditions. After the enzymatic hydrolysis is completed, filter carbon-carried papain and collect the filtrate for later use.

[0057] (2) Concentration and freeze-drying: The filtrate after enzymatic hydrolysis was sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The microfiltration-ultrafiltration was carried out at room temperature and 0.1 MPa pressure to remove high molecular weight saponin gum and collect the ultrafiltration permeate. Then, the permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 3 kDa and separated at room temperature and 0.5 MPa pressure. The nanofiltration permeate was collected and then concentrated to a solid content of 8%. The concentrate was dispensed into stainless steel freeze-drying trays with a liquid layer thickness of 1.5 cm and pre-frozen at -45℃ for 5 h. After pre-freezing, it was transferred to a freeze dryer. During the sublimation drying stage, the vacuum degree was controlled at 20 Pa and the partition temperature at -22℃ for 14 h. During the desorption drying stage, the vacuum degree was controlled at 4 Pa ​​and the partition temperature at 25℃ for 5 h to ensure that the residual moisture was ≤2%.

[0058] (3) Crushing and sieving: After freeze-drying, in a clean environment with a relative humidity of 30%, the freeze-dried cake is put into a high-speed crusher and intermittently crushed at a speed of 2000r / min for 10s each time, with an interval of 30s, and repeated 3 times. After crushing, it is passed through a 100-mesh sieve to obtain active royal jelly polypeptide freeze-dried powder.

[0059] Comparative Example 4

[0060] (1) First enzymatic hydrolysis: Add carbon-borne trypsin to royal jelly liquid at a solid-liquid mass ratio of 1:25, then heat to 38℃ and enzymatically hydrolyze for 2.5h under constant temperature conditions. Adjust the pH to 8.0 with 0.1mol / L sodium hydroxide solution. After the enzymatic hydrolysis is completed, filter the carbon-borne trypsin and collect the filtrate for later use.

[0061] (2) Second enzymatic hydrolysis: Add carbon-carrying papain to the above filtrate at a solid-liquid mass ratio of 1:20, adjust the pH to 6.5 with 0.1 mol / L hydrochloric acid, then raise the temperature to 52℃ and enzymatically hydrolyze for 3 hours under constant temperature conditions. After the enzymatic hydrolysis is completed, filter the carbon-carrying papain and collect the filtrate for later use.

[0062] (3) Concentration and freeze drying: The filtrate after the above secondary enzymatic hydrolysis is passed through a nanofiltration membrane with a molecular weight cutoff of 3 kDa and separated at room temperature and 0.5 MPa pressure. The nanofiltration permeate is collected and concentrated to a solid content of 8%. The concentrate is dispensed into stainless steel freeze-drying trays with a liquid layer thickness of 1.5 cm and pre-frozen at -45℃ for 5 h. After pre-freezing, it is transferred to a freeze dryer. During the sublimation drying stage, the vacuum degree is controlled at 20 Pa and the partition temperature is -22℃ for 14 h. During the desorption drying stage, the vacuum degree is controlled at 4 Pa ​​and the partition temperature is 25℃ for 5 h to ensure that the residual moisture is ≤2%.

[0063] (4) Crushing and sieving: After freeze-drying, in a clean environment with a relative humidity of 30%, the freeze-dried cake is put into a high-speed crusher and intermittently crushed at a speed of 2000r / min for 10s each time, with an interval of 30s, and repeated 3 times. After crushing, it is passed through a 100-mesh sieve to obtain active royal jelly polypeptide freeze-dried powder.

[0064] Comparative Example 5

[0065] The difference between this comparative example and Example 1 is that the saponin gum is replaced with trehalose in this comparative example; otherwise, it is the same as Example 1.

[0066] Experiment 1: 10-Hydroxy-2-decenoic acid content

[0067] Weigh 0.2 g of the final royal jelly peptide powder sample and place it in a 50 mL volumetric flask. Add approximately 30 mL of a methanol-water-phosphoric acid (volume ratio 55:45:2) mixed solvent, and extract ultrasonically for 15 minutes. After cooling, dilute to the mark with the same mixed solvent, shake well, and filter through a 0.45 μm microporous membrane to obtain the test solution. The chromatographic conditions were as follows: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol-water-phosphoric acid (55:45:2, volume ratio), flow rate: 1.0 mL / min, detection wavelength: 212 nm, column temperature: 30 ℃, injection volume: 20 μL. Accurately weigh 10-hydroxy-2-decenoic acid reference standard, dissolve and dilute with methanol to prepare a reference solution containing 0.1 mg per mL, and determine using the same method. Calculate the content of 10-hydroxy-2-decenoic acid in the sample using the external standard method based on the peak area of ​​the reference solution.

[0068] The results are shown in Table 1 below.

[0069] Table 1

[0070] Example 1 2.06±0.04 Example 2 1.97±0.05 Example 3 1.91±0.06 Comparative Example 1 1.42±0.07 Comparative Example 2 1.63±0.05 Comparative Example 3 1.58±0.06 Comparative Example 4 1.74±0.05 Comparative Example 5 1.82±0.04

[0071] Note: The data are the results of three independent and repeated experiments (n=3), and the content is calculated on a dry basis.

[0072] As shown in Table 1, the 10-hydroxy-2-decenoic acid (10-HDA) contents of Examples 1-3 of the present invention were 2.06%, 1.97%, and 1.91%, respectively, significantly higher than all comparative examples. Comparative Example 1, which used a mixture of free enzymes for enzymatic hydrolysis followed by high-temperature inactivation at 85°C, had the lowest 10-HDA content (only 1.42%). This is because 10-HDA is heat-sensitive, and high-temperature treatment led to its significant degradation. The 10-HDA contents of Comparative Example 2 (trypsin hydrolysis only) and Comparative Example 3 (papain hydrolysis only) were 1.63% and 1.58%, respectively, slightly higher than Comparative Example 1 but significantly lower than the examples. This is because the depth of single-enzymatic hydrolysis was insufficient, and after hydrolysis, a large number of large protein molecules still encapsulated or adsorbed 10-HDA, which was then further degraded in subsequent processes. Losses occurred during filtration and concentration. Comparative Example 4, without added saponin, had a 10-HDA content of 1.74%, higher than the single-enzyme hydrolysis group but still lower than the example. This indicates that saponin, as an enzyme stabilizer, not only protects the activity of the immobilized enzyme but also helps maintain mild conditions in the hydrolysis system, thereby indirectly reducing the destruction of active ingredients. Comparative Example 5, with saponin replaced by trehalose, had a 10-HDA content of 1.82%, slightly lower than Example 1 (2.06%), indicating that saponin has a better stabilizing effect on the immobilized enzyme system than trehalose. These results demonstrate that the present invention, through stepwise enzymatic hydrolysis of immobilized enzymes and the addition of saponin, avoids the high-temperature enzyme inactivation step, maximally preserving the characteristic active ingredient 10-HDA in royal jelly.

[0073] Experiment 2: Antioxidant Properties Determination

[0074] (1) DPPH free radical scavenging rate: Prepare a 79 mg / L DPPH-ethanol solution, dissolve the royal jelly peptide lyophilized powder sample in anhydrous ethanol to obtain a sample solution with a mass concentration of 1000 μg / mL, take 0.5 mL of the sample solution, add 5.0 mL of DPPH-ethanol solution and mix well, react at 37℃ for 1 h, use anhydrous ethanol as blank, measure the color at a wavelength of 517 nm, and calculate the DPPH free radical scavenging rate. DPPH free radical scavenging rate = (blank absorbance - sample absorbance) / blank absorbance x 100;

[0075] (2) ABTS free radical scavenging rate: 25 mL of 7.4 mmol / L ABTS solution and 25 mL of 2.6 mmol / L potassium persulfate solution were measured and mixed in equal volumes. The mixture was reacted at room temperature in the dark for 12-16 h. Then, it was diluted 40-50 times with anhydrous ethanol and placed at room temperature in the dark for 30 min to prepare ABTS working solution. The royal jelly peptide lyophilized powder sample was dissolved in anhydrous ethanol to prepare a sample solution with a concentration of 20 mg / mL. 20 μL of the sample solution was taken and 280 μL of ABTS working solution was added to a 96-well microplate. ABTS free radical scavenging rate (%) = (A2-A1) / A2×100, where A1 is the absorbance value of the sample and A2 is the absorbance value of the blank control (deionized water instead of sample solution).

[0076] (3) Superoxide anion free radical scavenging rate: Take 4.5 mL of 50 mmol / L tris(hydroxymethyl)aminomethane hydrochloride buffer into a test tube, then take 2.0 mL of distilled water into a test tube, and then take 1.0 mL of royal jelly peptide lyophilized powder sample solution (sample dissolved in ethanol, concentration 20 mg / mL) into the test tube. After mixing thoroughly, place it in a 25℃ water bath for 20 min. Then take 0.5 mL of 3.0 mmol / L pyrogallol solution into the test tube, shake it quickly, zero the tube with distilled water, and measure the absorbance A1 of the sample at a wavelength of 325 nm using an ultraviolet spectrophotometer. The absorbance value of the sample solution is A2, which is replaced by deionized water. The superoxide anion free radical scavenging rate is calculated as follows: Scavenging rate (%) = (A2-A1) / A2x100.

[0077] The results are shown in Table 2.

[0078] Table 2

[0079] Example 1 93.34±0.82 94.01±0.76 90.58±0.91 Example 2 93.21±0.79 93.87±0.81 90.21±0.88 Example 3 92.85±0.88 93.56±0.84 89.90±0.95 Comparative Example 1 82.72±1.23 81.26±1.35 79.43±1.41 Comparative Example 2 86.49±1.05 85.47±1.12 83.57±1.18 Comparative Example 3 87.74±1.01 87.48±1.09 85.41±1.14 Comparative Example 4 90.64±0.96 87.92±1.18 85.95±1.07 Comparative Example 5 91.58±0.89 90.15±1.03 87.69±1.02

[0080] Note: Each sample was measured in triplicate (n=3), and the results are expressed as mean ± standard deviation. All clearance rates were calculated using the corresponding blank as a control.

[0081] From the antioxidant activity data in Table 2, the DPPH radical scavenging rate (92.85%-93.34%), ABTS radical scavenging rate (93.56%-94.01%), and superoxide anion radical scavenging rate (89.90%-90.58%) of Examples 1-3 were significantly better than those of the comparative examples. Comparative Example 1 had the lowest scavenging rates (82.72%, 81.26%, and 79.43%, respectively). High-temperature enzyme inactivation not only destroyed 10-HDA but also damaged the antioxidant groups of the peptides themselves. Furthermore, the free enzymatic hydrolysis produced peptides with a wider molecular weight distribution and a lower proportion of small-molecule active peptides. The antioxidant activities of the single-enzyme hydrolysis products of Comparative Examples 2 and 3 were between those of Comparative Examples 1 and 4, indicating that stepwise hydrolysis with two enzymes is more effective than single-enzyme hydrolysis in producing small-molecule peptides with high antioxidant activity. The DPPH scavenging rate of Comparative Example 4 (without saponin gum) (90.64%) was significantly lower than that of Example 1 (93.34%). While the differences are relatively small, the ABTS and superoxide anion scavenging rates (87.92%, 85.95%) are significantly lower than those in Example 1 (94.01%, 90.58%), indicating that the addition of saponin gum is crucial for maintaining the stability of the immobilized enzyme and ensuring complete hydrolysis during the enzymatic hydrolysis process. It is particularly beneficial for producing peptide components with stronger scavenging capabilities against ABTS and superoxide anion radicals. The three scavenging rates (91.58%, 90.15%, 87.69%) in Comparative Example 5 (trehalose replacement) are all lower than those in Example 1, further confirming the superiority of saponin gum in the specific system of this invention. In summary, the lyophilized powder obtained in the embodiments of this invention exhibits excellent antioxidant activity, which is attributed to the high-quality active peptides produced by the stepwise hydrolysis of the immobilized enzyme and the synergistic protection of saponin gum, as well as the effective preservation of peptide activity by the mild process throughout.

[0082] Experiment 3: Detection of residual process additives in the final product

[0083] (1) Detection of saponin residue

[0084] The total polysaccharide content was determined using the phenol-sulfuric acid method, with galactose as the standard to construct a standard curve. Accurately weigh 0.5 g of the lyophilized powder sample, dissolve it in 10 mL of deionized water, add 1.0 mL of 5% phenol solution, and quickly add 5.0 mL of concentrated sulfuric acid. Shake well, incubate at room temperature for 20 min, and measure the absorbance at 490 nm. Simultaneously, using a blank royal jelly polypeptide lyophilized powder without added saponin as the matrix, a series of saponin reference solutions (0.5–50 μg / mL) were prepared to construct the standard curve. The saponin content in the sample was calculated using the external standard method, and the detection limit was 0.5 μg / g (based on sample dry weight).

[0085] (2) Detection of Tween-80 residues

[0086] HPLC-evaporative light scattering (HPLC-ELSD) was used to determine the content of polysorbate 80 (Tween-80) in pharmaceutical preparations. 1.0 g of the lyophilized powder sample was accurately weighed, dissolved in 10 mL of a methanol-water (50:50) mixture by sonication, and filtered through a 0.22 μm filter. Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-water (85:15); flow rate: 1.0 mL / min; column temperature: 35 ℃; drift tube temperature of the evaporative light scattering detector: 80 ℃; carrier gas flow rate: 2.0 L / min. A series of standard solutions (0.1–20 μg / mL) were prepared using Tween-80 reference standard (purity ≥99%) to construct a standard curve. Quantification was performed using the external standard method, with a detection limit of 0.1 μg / g (based on sample dry weight).

[0087] (3) EDTA residue detection

[0088] Referring to GB 5009.278-2016, "National Food Safety Standard - Determination of ethylenediaminetetraacetic acid (EDTA) in Food," high-performance liquid chromatography (HPLC) was used. 0.5 g of the lyophilized powder sample was accurately weighed, dissolved in 10 mL of deionized water, purified using a mixed-type strong anion exchange solid-phase extraction column, derivatized with ferric chloride solution, and determined by HPLC (equipped with a UV detector or diode array detector). Quantification was performed using the external standard method, and the detection limit was 0.02 μg / g. This standard requires a mobile phase of methanol and sodium acetate buffer solution, and detection at a wavelength of 254 nm.

[0089] (4) Detection of KH-550 (γ-aminopropyltriethoxysilane) residue

[0090] Since KH-550 is bonded to the activated carbon surface in the form of siloxanes after silanization modification, trace amounts of free KH-550 can be estimated by determining the total silicon (Si) content. The total silicon content in the sample was determined by inductively coupled plasma mass spectrometry (ICP-MS), with background silicon subtracted (using a blank sample without modified activated carbon as a control). 0.2 g of the lyophilized powder sample was accurately weighed, digested with nitric acid-hydrogen peroxide (5:1) using microwave, and then diluted to 25 mL before analysis. A standard curve was plotted using silicon standard solutions (0–100 μg / L). The limit of detection (LOD) for free KH-550 was 0.05 μg / g, and the limit of quantitation (LOQ) was 0.15 μg / g.

[0091] The test results of the samples in each embodiment are shown in Table 3.

[0092] Table 3

[0093] Example 1 Not detected (<0.5) Not detected (<0.1) Not detected (<0.02) Not detected (<0.05) Example 2 Not detected (<0.5) Not detected (<0.1) Not detected (<0.02) Not detected (<0.05) Example 3 Not detected (<0.5) Not detected (<0.1) Not detected (<0.02) Not detected (<0.05)

[0094] As shown in Table 3, the contents of saponin, Tween-80, EDTA, and KH-550 in the freeze-dried powders obtained in Examples 1-3 were all below the corresponding detection limits, and were all determined to be undetectable. This indicates that the microfiltration-ultrafiltration combined with the present invention (molecular weight cutoff of 100 kDa) can effectively remove residual high molecular weight saponin (molecular weight > 400 kDa) and Tween-80 micelles in the enzymatic hydrolysis system; subsequent 3 kDa nanofiltration membrane separation and concentration further retained any possible EDTA metal complexes and a small amount of free silane hydrolysis products. Simultaneously, the KH-550 on the immobilized enzyme carrier is firmly bound to the activated carbon surface in a chemical bond form, and does not detach during the enzymatic hydrolysis filtration process, ensuring the purity and safety of the final product, meeting the safety requirements for food and health food raw materials. These residue detection results further verify that the process of the present invention is green and efficient, the product has high purity, and it has good application safety.

Claims

1. A lyophilized powder of active royal jelly polypeptides, characterized in that, Includes the following steps: (1) Preparation of royal jelly: Take fresh royal jelly and mix it with deionized water at a mass ratio of 1:5-7. Stir well to obtain royal jelly solution; (2) First enzymatic hydrolysis: Add carbon-borne trypsin to royal jelly liquid at a solid-liquid mass ratio of 1:25, add 0.02%-0.04% enzyme stabilizer by weight of royal jelly liquid, then heat to 37-40℃ and enzymatically hydrolyze for 2.5h under constant temperature conditions. Adjust the pH to 7.8-8.2 with 0.1mol / L sodium hydroxide solution. After enzymatic hydrolysis, filter carbon-borne trypsin and collect the filtrate for later use. (3) Second enzymatic hydrolysis: Add carbon-loaded papain to the above filtrate at a solid-liquid mass ratio of 1:20, add 0.02%-0.04% enzyme stabilizer by mass of filtrate, adjust the pH to 6.0-7.0 with 0.1mol / L hydrochloric acid, then heat to 50-54℃ and enzymatically hydrolyze for 3h under constant temperature conditions. After the enzymatic hydrolysis is completed, filter the carbon-loaded papain and collect the filtrate for later use. (4) Concentration and freeze-drying: The filtrate after the above-mentioned secondary enzymatic hydrolysis was sequentially passed through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The microfiltration-ultrafiltration was carried out at room temperature and a pressure of 0.05-0.15 MPa to remove high molecular weight impurities, and the ultrafiltration permeate was collected. Then, the permeate was passed through a nanofiltration membrane with a molecular weight cutoff of 3 kDa and separated at room temperature and a pressure of 0.5 MPa. The nanofiltration permeate was collected and then concentrated to a solid content of 8%. The concentrate was dispensed into stainless steel freeze-drying trays with a liquid layer thickness of 1.5 cm and pre-frozen at -45℃ for 5 h. After pre-freezing, it was transferred to a freeze dryer. During the sublimation drying stage, the vacuum degree was controlled at 15-25 Pa and the partition temperature at -22℃ for 14 h. During the desorption drying stage, the vacuum degree was controlled at ≤5 Pa and the partition temperature at 20-30℃ for 5 h, so that the residual moisture was ≤2%. (5) Crushing and sieving: After freeze-drying, in a clean environment with a relative humidity of 30%, the freeze-dried cake is put into a high-speed crusher and intermittently crushed at a speed of 2000r / min for 10s each time, with an interval of 30s, and repeated 3 times. After crushing, it is passed through a 100-mesh sieve to obtain active royal jelly polypeptide freeze-dried powder. The enzyme stabilizer is saponin gum.

2. The freeze-dried active royal jelly polypeptide powder as described in claim 1, characterized in that, The preparation of the carbon-supported trypsin includes: mixing coconut shell activated carbon and trypsin solution at a mass ratio of 15:

1. The trypsin solution is prepared with phosphate buffer at pH 7.5 and the enzyme concentration is 0.5 mg / mL. The mixture is shaken and adsorbed at 25℃ and 150 r / min for 3 h. During the adsorption process, 0.05%-0.1% Tween-80 is added as a dispersant. After the adsorption is completed, the mixture is washed 3 times with buffer containing 0.05% Tween-80, then washed once with deionized water, and then centrifuged at 4000 r / min for 10 min at 4℃. The mixture is then humidified at 4℃ for later use.

3. The freeze-dried active royal jelly polypeptide powder as described in claim 1, characterized in that, Preparation of carbon-supported papain: Coconut shell activated carbon and papain solution were mixed at a mass ratio of 12:

1. The papain solution was prepared with citrate buffer at pH 7.0, with an enzyme concentration of 0.8 mg / mL. 0.004 mol / L EDTA and 0.5% food-grade Tween-80 were added as a dispersant. The mixture was shaken and adsorbed at 25℃ and 150 r / min for 4 h. After adsorption, it was washed 3 times with buffer containing 0.05% Tween-80, then washed once with deionized water, and then centrifuged at 4000 r / min for 10 min at 4℃. It was then refrigerated at 4℃ under humid conditions for later use.

4. The freeze-dried active royal jelly polypeptide powder as described in claim 2 or 3, characterized in that, The coconut shell activated carbon further includes pretreatment: coconut shell activated carbon with a pore size of 5-30 nm, a specific surface area ≥800 m² / g, and a particle size of 100-200 mesh is boiled with 1 mol / L nitric acid for 2 hours, washed with deionized water until neutral, and dried for later use; then, KH-550 silanization modification is performed: 0.5%-2% KH-550 relative to the mass of activated carbon is added, the pH is adjusted to 4-5 with glacial acetic acid, and the mixture is mechanically stirred and refluxed in a 60℃ water bath for 6 hours. After the reaction is completed, the mixture is filtered, washed three times each with anhydrous ethanol and deionized water, and dried under vacuum at 80℃ to constant weight.