A method for preparing anti-fatigue royal jelly peptides

Abstract

This invention provides a method for preparing anti-fatigue royal jelly peptides. Through a two-stage immobilized enzyme membrane reactor, continuous enzymatic hydrolysis of royal jelly protein and online product separation are coupled, ultimately yielding royal jelly peptide powder with high peptide content and a molecular weight primarily less than 3000 Da. This invention achieves deep coupling of dual-enzyme synergistic hydrolysis and membrane separation, significantly improving substrate conversion efficiency and product purity, and providing a technical solution for the safe, efficient, and quality-controllable industrial production of anti-fatigue royal jelly peptides.

Description

Technical Field

[0001] This invention belongs to the technical field of royal jelly peptide preparation, specifically relating to a method for preparing anti-fatigue royal jelly peptides. Background Technology

[0002] In the field of food biotechnology, royal jelly, a natural mixture secreted by the pharyngeal and mandibular glands of worker bees, is widely used in the research and development of health foods and functional foods due to its rich protein content and diverse bioactivities. Protein is a crucial component of royal jelly, accounting for approximately 9% to 18% of its dry matter, with the main royal jelly protein accounting for as much as 82% to 90%. This protein possesses various bioactivities, including immunomodulatory, hypotensive, anti-inflammatory, and antibacterial effects. The preparation of bioactive peptides from royal jelly proteins through proteolytic hydrolysis has become an important technical pathway to enhance its nutritional value and functional activity. Studies have shown that royal jelly proteins themselves have relatively weak antioxidant activity, but this activity is significantly enhanced after enzymatic hydrolysis. Furthermore, peptides with molecular weights less than 3 kDa, especially less than 1 kDa, exhibit the strongest free radical scavenging ability and total antioxidant capacity. Therefore, developing efficient, controllable, and safe royal jelly peptide preparation technologies is of great significance for realizing the high-value utilization of royal jelly resources.

[0003] However, existing royal jelly peptide preparation technologies still face several pressing technical challenges in practical applications. Firstly, regarding enzymatic hydrolysis, current methods primarily employ single proteases in batch reactors for hydrolysis. Commonly used proteases include protease N, pepsin, trypsin, and papain. The limited specificity of cleavage sites in single-enzyme systems makes it difficult to fully hydrolyze the diverse peptide bonds in royal jelly proteins, resulting in a wide molecular weight distribution in the product and low enrichment of the target active peptides. Furthermore, batch hydrolysis presents difficulties in separating the enzyme from the product. After hydrolysis, proteases must be removed through heating inactivation or centrifugation and filtration. These operations not only increase production steps but may also damage heat-sensitive active peptides. More importantly, during batch reactions, hydrolysis products continuously accumulate in the reaction system, creating feedback inhibition of the protease and significantly reducing hydrolysis efficiency in the later stages, making it difficult to further increase the degree of hydrolysis.

[0004] Secondly, regarding food safety, to address the difficulty of separating enzymes from products during batch enzymatic hydrolysis, some technical solutions attempt to immobilize proteases on the surface of a carrier to achieve enzyme reuse and continuous operation. However, when existing immobilized enzyme technologies are applied to the preparation of food-grade products, they generally only use water or buffer solutions to wash away unreacted glutaraldehyde, lacking systematic safety verification and deep purification treatment for food-grade applications. This limits the widespread application of immobilized enzyme membrane reactors in the health food field.

[0005] Furthermore, regarding the operational stability of enzyme membrane reactors, membrane fouling and enzyme leakage constrain the long-term stable operation of the system when enzymatic hydrolysis is coupled with membrane separation. Regarding membrane fouling, royal jelly feedstock contains a large amount of insoluble lipid complexes, which easily adsorb onto the membrane surface and clog the pores, leading to a rapid decline in membrane flux, shortening the single-cycle operation, and increasing the cleaning frequency. Existing continuous feeding methods cannot effectively alleviate the accumulation of concentration polarization layers and contaminants on the membrane surface. During operation, membrane flux continues to decline, affecting the continuous collection efficiency and production stability of the product. Regarding enzyme leakage, studies have shown that while increasing the operating pressure in enzyme membrane reactors helps improve protein recovery, it also causes a large amount of immobilized enzyme to leak into the product, causing severe membrane fouling. The leaked protease not only results in the loss of catalytic enzymes, but more seriously, the free protease continues to hydrolyze product peptides that have already permeated the membrane, leading to an uncontrollable shift in the molecular weight distribution of the target product, making it difficult to guarantee batch-to-batch consistency.

[0006] Furthermore, regarding post-processing of the product, the enzymatic hydrolysis product contains a large amount of salt and free amino acids. Traditional processes typically employ heating concentration or direct freeze-drying for this purpose. Heating concentration can easily lead to the inactivation of heat-sensitive active peptides, while direct freeze-drying is unsuitable for industrial production due to the large volume of materials and high energy consumption. Moreover, neither of these post-processing methods can effectively remove any remaining trace amounts of cross-linking agent fragments or small molecule impurities, posing a challenge to improving product purity.

[0007] In summary, existing royal jelly peptide preparation technologies have varying degrees of shortcomings in areas such as enzymatic hydrolysis efficiency and precise control of product molecular weight distribution, food safety of immobilized enzyme processes, inhibition of membrane fouling and enzyme leakage, purification effects of post-processing, and membrane module lifespan. Developing a royal jelly peptide preparation method that can achieve efficient continuous enzymatic hydrolysis, ensure food-grade safety, precisely control product molecular weight distribution, effectively inhibit membrane fouling and enzyme leakage, and balance post-processing purification and module lifespan is of significant technological value and practical importance for promoting the development of the royal jelly deep processing industry. Summary of the Invention

[0008] This invention discloses a method for preparing anti-fatigue royal jelly peptides to solve any of the above-mentioned or potential problems in the prior art. To solve the above-mentioned technical problems, this invention specifically includes the following steps:

[0009] (1) Prepare a 0.5% sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer and circulate it through the membrane cavity for 2 hours at 4°C for cross-linking treatment. Then rinse with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Immobilize the thermophilic protease on the inner surface of the first-stage hollow fiber enzyme membrane reactor. Prepare an enzyme solution with a concentration of 5-8 g / L. Use 0.1 mol / L phosphate buffer as solvent, add 2 mmol / L CaCl2 and adjust the pH to 8.0 as buffer. Circulate it through the membrane cavity at 4°C for 12 hours. After immobilization, rinse with buffer until no protein is detected. Then perform a rinsing treatment: circulate the membrane cavity with 0.1 mol / L glycine-phosphate buffer at pH 7.0 at 25°C at 30 mL / min for 30 minutes. Then rinse with 10 times the membrane volume of sterile deionized water until the residual sodium tripolyphosphate is ≤0.5 mg / L to obtain the first-stage immobilized enzyme membrane reactor.

[0010] (2) Take fresh royal jelly, freeze dry and then pulverize it, pass it through an 80-mesh sieve to obtain royal jelly freeze-dried powder. Weigh the freeze-dried powder and prepare a substrate solution of 50-60 g / L with 0.1 mol / L phosphate buffer. After 0.45 μm pre-microfiltration, pump it into the tube side of the first-stage immobilized enzyme membrane reactor using a pulse feeding method. The pulse mode is 3 seconds of feeding and 1 second of pause, with an average feed flow rate of 60-80 mL / min.

[0011] (3) The first stage of continuous enzymatic hydrolysis and membrane separation coupling is carried out: the reactor water bath is controlled at 58-64℃, the transmembrane pressure difference is 0.1-0.15MPa, the pH is maintained at 8.0 by 0.5mol / L NaOH, the circulation flow rate is 200-260mL / min, and the permeate with a molecular weight of less than 3kDa is collected.

[0012] (4) The collected permeate was used as the substrate for the second-stage reaction and enzymatically hydrolyzed in the second-stage immobilized enzyme membrane reactor. The reaction temperature was 50℃, the transmembrane pressure difference was 0.1-0.15MPa, the pH was maintained at 7.5-8.0 with 0.5mol / L NaOH, the circulation flow rate was 200-240mL / min, and the permeate was separated again by an ultrafiltration membrane with a molecular weight cutoff of 3kDa to obtain the second-stage permeate.

[0013] (5) The solution is then concentrated and desalted by passing it through a nanofiltration membrane with a molecular weight cutoff of 1 kDa at room temperature and 0.5 MPa pressure. The nanofiltration retentate is then freeze-dried to obtain anti-fatigue royal jelly peptide powder.

[0014] Furthermore, the preparation of the second-stage immobilized enzyme membrane reactor includes: preparing a 0.5% volume concentration sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer, circulating it through the membrane cavity at 4°C for 2 hours for cross-linking treatment, then rinsing with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L, immobilizing Bacillus subtilis protease on the inner surface of the second-stage hollow fiber enzyme membrane reactor, preparing an enzyme solution with a concentration of 5-8 g / L, using 0.1 mol / L phosphate buffer at pH 8.0 as the solvent, circulating it through the membrane cavity at 4°C for 12 hours, rinsing with buffer until no protein is detected after immobilization, and then performing the same rinsing treatment as in step (1) to obtain the second-stage immobilized enzyme membrane reactor;

[0015] Cleaning and regeneration of membrane modules: After each operating cycle, the first and second stage immobilized enzyme membrane reactors were rinsed with deionized water at 100 mL / min for 30 minutes, followed by sequential cleaning with 0.1 mol / L sodium hydroxide solution and 0.1 mol / L hydrochloric acid solution at 40°C for 60 minutes each, and then rinsed with deionized water until neutral, and equilibrated with phosphate buffer. After every 5 cycles, each cycle lasting 24 hours, after cleaning, the first stage membrane module was circulated with 1 g / L thermophilic protease solution and the second stage membrane module was circulated with 1 g / L Bacillus subtilis protease solution at 4°C for 2 hours to replenish enzyme activity.

[0016] The advantages and beneficial effects of this invention are as follows:

[0017] This invention provides a method for preparing anti-fatigue royal jelly peptides, which constructs a two-stage tandem immobilized enzyme membrane reactor system. Thermophilic bacterial protease and subtilisin protease are sequentially immobilized on the inner surface of a hollow fiber ultrafiltration membrane, achieving a high degree of coupling between continuous enzymatic hydrolysis of royal jelly proteins and product separation. Compared with existing single-enzyme batch hydrolysis techniques, this method allows hydrolysis products to immediately permeate through the membrane and exit the reaction zone after generation, avoiding feedback inhibition of the enzyme by the product and significantly improving hydrolysis efficiency and substrate conversion rate. Simultaneously, the sequential action of the two enzyme stages fully leverages the preferential cleavage of hydrophobic amino acid residues by thermophilic bacterial protease and the broad-spectrum hydrolytic ability of subtilisin protease, significantly enhancing the functional activity of the product.

[0018] Secondly, regarding the preparation process of the immobilized enzyme, this invention adds a rinsing pretreatment step after sodium tripolyphosphate cross-linking and immobilization. The enzyme is first treated with glycine-phosphate buffer, and the final detection shows that the residual sodium tripolyphosphate content is less than 0.5 mg / L, significantly improving the safety and stability of the royal jelly peptide powder.

[0019] Third, this invention adopts a combination of pulsed feeding and substrate pre-microfiltration. By pre-microfiltration of the polyethersulfone membrane at room temperature, insoluble lipid complexes in royal jelly are effectively removed, reducing the sources of contaminants on the membrane surface. At the same time, the pulse mode utilizes instantaneous pressure fluctuations to disturb the concentration polarization layer on the membrane surface, which slows down the membrane flux decay, significantly extends the effective working time of a single operation, and reduces the frequency of membrane cleaning and operating costs.

[0020] Fourth, in the post-processing stage, this invention directly concentrates and desaltes the permeate from the second-stage reactor through a nanofiltration membrane. This nanofiltration step removes small molecule salts and free amino acids, further improving the purity of the peptide powder. Compared with conventional heating concentration or spray drying, nanofiltration operates at room temperature, effectively protecting the bioactivity of heat-sensitive active peptides. Compared with direct freeze drying, nanofiltration concentration significantly reduces freeze-drying energy consumption and lowers production costs.

[0021] Fifth, regarding the cleaning and regeneration of membrane modules, compared with the existing technology where membrane modules need to be re-immobilized or replaced after the enzyme activity of the membrane module declines, the "patch-style" regeneration method of the present invention significantly reduces the replacement frequency and operating cost of immobilized enzymes, achieving a balance between economy and continuity.

[0022] In summary, this invention, through the systematic integration of multiple technologies such as dual-enzyme sequential immobilization, pretreatment, pulsed feeding, nanofiltration cascade concentration, and enzyme activity compensation and repair, solves the problems of insufficient safety and uneven molecular weight distribution in existing royal jelly peptide preparation technologies. The prepared anti-fatigue royal jelly peptide powder has the characteristics of high purity, high activity, low residue, and batch stability. Moreover, the entire process is continuous, controllable, and scalable, providing a safe, efficient, and economical solution for the industrial production of high-value-added royal jelly products. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments. The membrane material of the hollow fiber enzyme membrane reactors used below is polyethersulfone, with a membrane area of ​​0.5 square meters and a pore size cutoff of 3 kDa. The thermophilic proteases used below have a specific activity of 30-350 units / mg protein, a molecular weight of 34.6 kDa, CAS number 9073-78-3, and were purchased from Shanghai Xuanya Biotechnology Co., Ltd. The subtilisin is Alcalase 2.4 L, purchased from Novozymes (China) Biotechnology Co., Ltd., its component is subtilisin A, CAS number 9014-01-1, and its enzyme activity is 2.4 AU-A / g. The phosphate buffers used below are a mixed salt of disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0024] The specific rinsing process for preparing the enzyme membrane reactor is as follows: 0.1 mol / L glycine-phosphate buffer solution at pH 7.0 is circulated through the membrane lumen at 30 mL / min for 30 minutes at 25°C, and then rinsed with 10 times the membrane volume of sterile deionized water until the residual sodium tripolyphosphate is ≤0.5 mg / L.

[0025] Example 1

[0026] (1) Prepare a 0.5% sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer and circulate it through the membrane cavity for 2 hours at 4°C for cross-linking treatment. Then wash with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Immobilize the thermophilic protease on the inner surface of the first-stage hollow fiber enzyme membrane reactor. Prepare an enzyme solution with a concentration of 6 g / L. Use 0.1 mol / L phosphate buffer as solvent, add 2 mmol / L CaCl2 and adjust the pH to 8.0 as buffer. Circulate it through the membrane cavity at 4°C for 12 hours. After immobilization, wash with buffer until no protein is detected. Then perform rinsing treatment to obtain the first-stage immobilized enzyme membrane reactor.

[0027] (2) Take fresh royal jelly, freeze dry and then pulverize it, pass it through an 80-mesh sieve to obtain royal jelly freeze-dried powder. Weigh 200g of the freeze-dried powder and prepare a substrate solution of 55g / L with 0.1mol / L phosphate buffer. After 0.45μm pre-microfiltration, pump it into the tube side of the first-stage immobilized enzyme membrane reactor using a pulse feeding method. The pulse mode is 3 seconds of feeding and 1 second of pause, with an average feed flow rate of 70mL / min.

[0028] (3) The first stage of continuous enzymatic hydrolysis and membrane separation coupling is carried out: the reactor water bath is controlled at 61℃, the transmembrane pressure difference is 0.12MPa, the pH is maintained at 8.0 by 0.5mol / L NaOH, the circulation flow rate is 230mL / min, and the permeate with a molecular weight of less than 3kDa is collected.

[0029] (4) The preparation of the second-stage immobilized enzyme membrane reactor includes: preparing a 0.5% volume concentration sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer, circulating it through the membrane cavity for 2 hours at 4°C for cross-linking treatment, and then rinsing with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Immobilizing Bacillus subtilis protease on the inner surface of the second-stage hollow fiber enzyme membrane reactor, preparing an enzyme solution with a concentration of 6 g / L, using 0.1 mol / L phosphate buffer with pH 8.0 as the solvent, circulating it through the membrane cavity at 4°C for 12 hours, and rinsing with buffer until no protein is detected after immobilization, followed by rinsing treatment to obtain the second-stage immobilized enzyme membrane reactor;

[0030] (5) The collected permeate was used as the substrate for the second-stage reaction and enzymatically hydrolyzed in the second-stage immobilized enzyme membrane reactor. The reaction temperature was 50℃, the transmembrane pressure difference was 0.12MPa, the pH was maintained at 7.8 by 0.5mol / L NaOH, the circulation flow rate was 220mL / min, and it was separated again by an ultrafiltration membrane with a molecular weight cutoff of 3kDa to obtain the second-stage permeate.

[0031] (6) The solution is then concentrated and desalted by passing it through a nanofiltration membrane with a molecular weight cutoff of 1 kDa at room temperature and 0.5 MPa pressure. The nanofiltration retentate is then freeze-dried to obtain anti-fatigue royal jelly peptide powder.

[0032] Example 2

[0033] (1) Prepare a 0.5% sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer and circulate it through the membrane cavity for 2 hours at 4°C for cross-linking treatment. Then wash with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Immobilize the thermophilic protease on the inner surface of the first-stage hollow fiber enzyme membrane reactor. Prepare an enzyme solution with a concentration of 5 g / L. Use 0.1 mol / L phosphate buffer as solvent, add 2 mmol / L CaCl2 and adjust the pH to 8.0 as buffer. Circulate it through the membrane cavity at 4°C for 12 hours. After immobilization, wash with buffer until no protein is detected. Then perform rinsing treatment to obtain the first-stage immobilized enzyme membrane reactor.

[0034] (2) Take fresh royal jelly, freeze dry and then pulverize it, pass it through an 80-mesh sieve to obtain royal jelly freeze-dried powder. Weigh 200g of the freeze-dried powder and prepare a 60g / L substrate solution with 0.1mol / L phosphate buffer. After 0.45μm pre-microfiltration, pump it into the tube side of the first-stage immobilized enzyme membrane reactor using a pulse feeding method. The pulse mode is 3 seconds of feeding and 1 second of pause, with an average feed flow rate of 60mL / min.

[0035] (3) The first stage of continuous enzymatic hydrolysis and membrane separation coupling is carried out: the reactor water bath is controlled at 64℃, the transmembrane pressure difference is 0.15MPa, the pH is maintained at 8.0 by 0.5mol / L NaOH, the circulation flow rate is 200mL / min, and the permeate with a molecular weight of less than 3kDa is collected;

[0036] (4) The preparation of the second-stage immobilized enzyme membrane reactor includes: preparing a 0.5% volume concentration sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer, circulating it through the membrane cavity for 2 hours at 4°C for cross-linking treatment, and then rinsing with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Immobilizing Bacillus subtilis protease on the inner surface of the second-stage hollow fiber enzyme membrane reactor, preparing an enzyme solution with a concentration of 8 g / L, using 0.1 mol / L phosphate buffer with pH 8.0 as the solvent, circulating it through the membrane cavity at 4°C for 12 hours, and rinsing with buffer until no protein is detected after immobilization, followed by rinsing treatment to obtain the second-stage immobilized enzyme membrane reactor;

[0037] (5) The collected permeate was used as the substrate for the second-stage reaction and enzymatically hydrolyzed in the second-stage immobilized enzyme membrane reactor. The reaction temperature was 50℃, the transmembrane pressure difference was 0.1MPa, the pH was maintained at 8.0 by 0.5mol / L NaOH, the circulation flow rate was 240mL / min, and the permeate was separated again by an ultrafiltration membrane with a molecular weight cutoff of 3kDa to obtain the second-stage permeate.

[0038] (6) The solution is then concentrated and desalted by passing it through a nanofiltration membrane with a molecular weight cutoff of 1 kDa at room temperature and 0.5 MPa pressure. The nanofiltration retentate is then freeze-dried to obtain anti-fatigue royal jelly peptide powder.

[0039] Example 3

[0040] (1) Prepare a 0.5% sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer and circulate it through the membrane cavity for 2 hours at 4°C for cross-linking treatment. Then wash with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Immobilize the thermophilic protease on the inner surface of the first-stage hollow fiber enzyme membrane reactor. Prepare an enzyme solution with a concentration of 8 g / L. Use 0.1 mol / L phosphate buffer as solvent, add 2 mmol / L CaCl2 and adjust the pH to 8.0 as buffer. Circulate it through the membrane cavity at 4°C for 12 hours. After immobilization, wash with buffer until no protein is detected. Then perform rinsing treatment to obtain the first-stage immobilized enzyme membrane reactor.

[0041] (2) Take fresh royal jelly, freeze dry and then pulverize it, pass it through an 80-mesh sieve to obtain royal jelly freeze-dried powder. Weigh 200g of the freeze-dried powder and prepare a 50g / L substrate solution with 0.1mol / L phosphate buffer. After 0.45μm pre-microfiltration, pump it into the tube side of the first-stage immobilized enzyme membrane reactor using a pulse feeding method. The pulse mode is 3 seconds of feeding and 1 second of pause, with an average feed flow rate of 80mL / min.

[0042] (3) The first stage of continuous enzymatic hydrolysis and membrane separation coupling is carried out: the reactor water bath is controlled at 58℃, the transmembrane pressure difference is 0.1MPa, the pH is maintained at 8.0 by 0.5mol / L NaOH, the circulation flow rate is 260mL / min, and the permeate with a molecular weight of less than 3kDa is collected.

[0043] (4) The preparation of the second-stage immobilized enzyme membrane reactor includes: preparing a 0.5% volume concentration sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer, circulating it through the membrane cavity for 2 hours at 4°C for cross-linking treatment, and then rinsing with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Immobilizing Bacillus subtilis protease on the inner surface of the second-stage hollow fiber enzyme membrane reactor, preparing an enzyme solution with a concentration of 5 g / L, using 0.1 mol / L phosphate buffer with pH 8.0 as the solvent, circulating it through the membrane cavity at 4°C for 12 hours, and rinsing with buffer until no protein is detected after immobilization, followed by rinsing treatment to obtain the second-stage immobilized enzyme membrane reactor;

[0044] (5) The collected permeate was used as the substrate for the second-stage reaction and enzymatically hydrolyzed in the second-stage immobilized enzyme membrane reactor. The reaction temperature was 50℃, the transmembrane pressure difference was 0.15MPa, the pH was maintained at 7.5 by 0.5mol / L NaOH, the circulation flow rate was 200mL / min, and the permeate was separated again by an ultrafiltration membrane with a molecular weight cutoff of 3kDa to obtain the second-stage permeate.

[0045] (6) The solution is then concentrated and desalted by passing it through a nanofiltration membrane with a molecular weight cutoff of 1 kDa at room temperature and 0.5 MPa pressure. The nanofiltration retentate is then freeze-dried to obtain anti-fatigue royal jelly peptide powder.

[0046] Comparative Example 1

[0047] Fresh royal jelly was freeze-dried and then pulverized through an 80-mesh sieve to obtain freeze-dried royal jelly powder. 200g of the freeze-dried powder was weighed and mixed with deionized water at a ratio of 1:4. The initial pH was adjusted to 5.0, and Aspergillus niger acidic protease was added at a dosage of 9000 U / g substrate. The mixture was enzymatically hydrolyzed for 5 hours in a constant temperature water bath at 45℃. During the enzymatic hydrolysis, the pH was maintained at 5.0 using 0.5mol / L sodium hydroxide solution. After the enzymatic hydrolysis was completed, the hydrolysate was heated to 90℃ and held for 15 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged at 4000 r / min for 20 minutes. The supernatant was filtered through a 0.45μm microporous membrane to remove insoluble residues. The filtrate was collected and then separated through an ultrafiltration membrane with a molecular weight cutoff of 3kDa at a pressure of 0.15MPa. The permeate was collected, concentrated by rotary evaporation, and then freeze-dried to obtain anti-fatigue royal jelly peptide powder.

[0048] Comparative Example 2

[0049] (1) Prepare a 0.5% sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer and circulate it through the membrane cavity for 2 hours at 4°C for cross-linking treatment. Then wash with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Immobilize the thermophilic protease on the inner surface of the hollow fiber enzyme membrane reactor. Prepare an enzyme solution with a concentration of 6 g / L. Use 0.1 mol / L phosphate buffer as solvent, add 2 mmol / L CaCl2 and adjust the pH to 8.0 as buffer. Circulate it through the membrane cavity at 4°C for 12 hours. After immobilization, wash with buffer until no protein is detected. Then perform rinsing treatment to obtain the immobilized enzyme membrane reactor.

[0050] (2) Take fresh royal jelly, freeze dry and then pulverize it, pass it through an 80-mesh sieve to obtain royal jelly freeze-dried powder. Weigh 200g of the freeze-dried powder and prepare a substrate solution of 55g / L with 0.1mol / L phosphate buffer. After 0.45μm pre-microfiltration, pump it into the tube side of the immobilized enzyme membrane reactor using a pulse feeding method. The pulse mode is 3 seconds of feeding and 1 second of pause, with an average feed flow rate of 70mL / min.

[0051] (3) Coupled continuous enzymatic hydrolysis and membrane separation: The reactor water bath is controlled at 61℃, the transmembrane pressure difference is 0.12MPa, the pH is maintained at 8.0 by 0.5mol / L NaOH, the circulation flow rate is 230mL / min, and the permeate with a molecular weight of less than 3kDa is collected;

[0052] (4) The solution is then concentrated and desalted by passing it through a nanofiltration membrane with a molecular weight cutoff of 1 kDa at room temperature and 0.5 MPa pressure. The nanofiltration retentate is then freeze-dried to obtain anti-fatigue royal jelly peptide powder.

[0053] Comparative Example 3

[0054] (1) Prepare a 0.5% sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer and circulate it through the membrane cavity for 2 hours at 4°C for cross-linking treatment. Then wash with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Immobilize Bacillus subtilis protease on the inner surface of the hollow fiber enzyme membrane reactor. Prepare an enzyme solution with a concentration of 6 g / L, use 0.1 mol / L phosphate buffer as solvent, and adjust the pH to 8.0 as buffer. Circulate it through the membrane cavity at 4°C for 12 hours. After immobilization, wash with buffer until no protein is detected. Then perform rinsing treatment to obtain the immobilized enzyme membrane reactor.

[0055] (2) Take fresh royal jelly, freeze dry and then pulverize it, pass it through an 80-mesh sieve to obtain royal jelly freeze-dried powder. Weigh 200g of the freeze-dried powder and prepare a substrate solution of 55g / L with 0.1mol / L phosphate buffer. After 0.45μm pre-microfiltration, pump it into the tube side of the immobilized enzyme membrane reactor using a pulse feeding method. The pulse mode is 3 seconds of feeding and 1 second of pause, with an average feed flow rate of 70mL / min.

[0056] (3) Coupled continuous enzymatic hydrolysis and membrane separation: The reactor water bath is controlled at 61℃, the transmembrane pressure difference is 0.12MPa, the pH is maintained at 8.0 by 0.5mol / L NaOH, the circulation flow rate is 230mL / min, and the permeate with a molecular weight of less than 3kDa is collected;

[0057] (4) The solution is then concentrated and desalted by passing it through a nanofiltration membrane with a molecular weight cutoff of 1 kDa at room temperature and 0.5 MPa pressure. The nanofiltration retentate is then freeze-dried to obtain anti-fatigue royal jelly peptide powder.

[0058] Comparative Example 4

[0059] (1) Prepare a 0.5% sodium tripolyphosphate solution with 0.1 mol / L phosphate buffer and circulate it through the membrane cavity for 2 hours at 4°C for cross-linking treatment. Then wash with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L. Mix thermophilic protease and Bacillus subtilis protease at a 1:1 enzyme activity ratio and fix them together on the inner surface of the same hollow fiber enzyme membrane reactor. Prepare an enzyme solution with a concentration of 6 g / L. Use 0.1 mol / L phosphate buffer as solvent, add 2 mmol / L CaCl2 and adjust the pH to 8.0 as buffer. Circulate it through the membrane cavity at 4°C for 12 hours for immobilization. After immobilization, wash with buffer until no protein is detected. Then perform rinsing treatment to obtain the immobilized enzyme membrane reactor.

[0060] (2) Take fresh royal jelly, freeze dry and then pulverize it, pass it through an 80-mesh sieve to obtain royal jelly freeze-dried powder. Weigh 200g of the freeze-dried powder and prepare a substrate solution of 55g / L with 0.1mol / L phosphate buffer. After 0.45μm pre-microfiltration, pump it into the tube side of the immobilized enzyme membrane reactor using a pulse feeding method. The pulse mode is 3 seconds of feeding and 1 second of pause, with an average feed flow rate of 70mL / min.

[0061] (3) Coupled continuous enzymatic hydrolysis and membrane separation: The reactor water bath is controlled at 61℃, the transmembrane pressure difference is 0.12MPa, the pH is maintained at 8.0 by 0.5mol / L NaOH, the circulation flow rate is 230mL / min, and the permeate with a molecular weight of less than 3kDa is collected;

[0062] (4) The solution is then concentrated and desalted by passing it through a nanofiltration membrane with a molecular weight cutoff of 1 kDa at room temperature and 0.5 MPa pressure. The nanofiltration retentate is then freeze-dried to obtain anti-fatigue royal jelly peptide powder.

[0063] Experiment 1: Product yield and molecular weight distribution

[0064] The final lyophilized peptide powder obtained after nanofiltration concentration and freeze-drying was weighed and recorded in grams. The yield was calculated by dividing the mass of the lyophilized peptide powder by the amount of royal jelly lyophilized powder used and then multiplying by 100%. Simultaneously, high-performance gel filtration chromatography was used to determine the peptide distribution in different molecular weight ranges of the product, and the percentages of components with molecular weights greater than 3 kDa, less than or equal to 3 kDa, and less than or equal to 1 kDa were calculated.

[0065] The results are shown in Table 1 below:

[0066] Table 1

[0067]

[0068] As can be seen from the data in Table 1, the peptide yields (15.62%-16.53%) and product molecular weight distributions (≤3kDa 93.72%-96.87%, ≤1kDa 64.56%-68.41%) of Examples 1-3 were significantly better than all comparative examples. Comparative Example 1 (traditional single-enzyme batch hydrolysis) had the lowest yield (8.56%), with the highest residual high molecular weight peptides (>3kDa) (28.59%), and only 35.24% ≤1kDa. This is because the batch reaction resulted in significant product feedback inhibition, incomplete hydrolysis, and potential destruction of bioactive peptides by heat inactivation. Comparative Example 2 (thermophilic protease single-enzyme continuous hydrolysis only) and Comparative Example 3 (subtilisin single-enzyme continuous hydrolysis only) showed significantly better results. The yields (11.45% and 10.29%) and ≤1kDa percentages (50.96% and 47.72%) of the single enzyme were better than those of Comparative Example 1, but still significantly lower than those of the Example. This indicates that the single enzyme has limited cleavage sites and cannot fully hydrolyze the diverse peptide bonds in royal jelly proteins. The yields (12.81%) and ≤1kDa percentages (56.85%) of Comparative Example 4 (both enzymes were co-immobilized in the same reactor) were better than those of the single enzyme comparative example, but still lower than those of the Example. This is mainly because the optimal temperatures (thermophilic protease 58-64℃, Bacillus subtilis protease 50℃) and reaction conditions of the two enzymes are different. They cannot achieve optimal catalytic efficiency at the same time in the same reactor, and co-immobilization may produce steric hindrance or competitive substrate binding. In contrast, the embodiments of the present invention employ a two-stage tandem immobilized enzyme membrane reactor, sequentially immobilizing thermophilic bacterial protease and Bacillus subtilis protease in independent reactors. This not only achieves immediate membrane separation and removal of the product after generation, completely eliminating feedback inhibition, but also allows for independent optimization of reaction temperature, pH, and other conditions for each enzyme, fully leveraging the synergistic hydrolysis effect of the two enzymes. This results in higher product yield, more thorough substrate conversion, and a more concentrated concentration of small molecular weight active peptides (≤3kDa >93%, 500-3000 Da ≥70%). Simultaneously, combined with rinsing treatment, it ensures that sodium tripolyphosphate residue is ≤0.5mg / L and free protease is undetectable, demonstrating the comprehensive advantages of the present invention in terms of enzymatic hydrolysis efficiency, product purity, and food safety.

[0069] Experiment 2: Fatigue resistance test

[0070] SPF-grade male Kunming mice, weighing 18-22g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. After 3 days of acclimatization, they were randomly divided into 9 groups of 10 mice each: a blank control group (physiological saline), Examples 1-3, Comparative Examples 1-4, and a positive control group (rhodioloside, 50mg / kg). Samples for the examples and comparative examples were prepared with physiological saline, and the gavage dose was 200mg / kg body weight (based on the dry weight of the peptide powder). The blank control group received an equal volume of physiological saline. Administration was once daily for 30 consecutive days.

[0071] Weighted swimming experiment: 30 minutes after the last administration, mice were placed in a swimming tank with a lead weight of 5% of their body weight on the base of their tails and in water at a depth of 30 cm and a temperature of (25±1) ℃. The time it took for the mice to be unable to float to the surface within 10 seconds after entering the water until their heads were completely submerged was recorded as the weighted swimming time.

[0072] Serum urea nitrogen determination: 30 minutes after the last administration, mice were placed in 30°C water and swam without load for 90 minutes. After resting for 60 minutes, blood was collected by enucleation of the eyeballs, and serum was separated. Serum urea nitrogen content was determined by diacetyl oxime colorimetric method.

[0073] Blood lactate assay: Thirty minutes after the last administration, 20 μL of blood was collected from the tail of the mice to measure the baseline blood lactate level at rest. Subsequently, the mice swam in 30°C water without load for 10 minutes. Blood samples were collected again immediately after swimming and 20 minutes after rest. The blood lactate content was measured using the lactate oxidase method (blood lactate test kit), and the area under the blood lactate curve was calculated.

[0074] Liver glycogen assay: Mice were sacrificed 30 minutes after the last administration, and livers were harvested. The liver glycogen content was determined using the anthrone colorimetric method (liver glycogen test kit).

[0075] All data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed using SPSS 22.0 software. LSD test was used for comparisons between groups, and P < 0.05 was considered statistically significant. The results are shown in Table 2.

[0076] Table 2

[0077]

[0078] Note: Compared with the blank control group * P<0.05, ** P<0.01; compared with the positive control group, # P<0.05, ## P<0.01.

[0079] As shown in Table 2, the weight-bearing swimming time (284.6–312.7 s) of mice in Examples 1–3 of this invention was significantly longer than that of all comparative groups (178.2–252.8 s) and the blank control group (126.4 s). Simultaneously, serum urea nitrogen (6.98–7.68 mmol / L) and the area under the curve of blood lactate (186.5–210.4 mmol / L·min) were significantly reduced, while liver glycogen reserves (26.38–29.52 mg / g) were significantly increased. Example 1 achieved the best results in all of the above indicators, even slightly better than the positive control group. Small molecule active peptides are more easily absorbed by the intestines, can rapidly replenish amino acids and inhibit the accumulation of blood lactate and urea nitrogen after exercise, thereby synergistically enhancing the overall efficacy. In comparison, Comparative Example 1 (traditional single-enzyme batch hydrolysis) had the worst anti-fatigue effect due to the destruction of heat-sensitive active ingredients by heating inactivation and the wide molecular weight distribution of the product (only 35.24% ≤1kDa). Comparative Examples 2 and 3 (single-enzyme continuous hydrolysis) avoided thermal destruction, but the single enzyme cleavage sites were limited, and the proportion of small molecule peptides was insufficient (50.96% and 47.72% ≤1kDa, respectively), resulting in limited improvement in effect. Comparative Example 4 (dual-enzyme co-immobilization) had insufficient hydrolysis due to the conflict between the optimal temperatures of the two enzymes, and its proportion of small molecule peptides (56.85%) was still lower than that of the Example group, and its anti-fatigue index was also inferior to that of the Example group. This invention employs a two-stage tandem immobilized enzyme membrane reactor, providing independently optimized reaction conditions for thermophilic bacterial protease and Bacillus subtilis protease (61°C for the first stage and 50°C for the second stage), achieving full synergistic hydrolysis of the two enzymes. The product exhibits the highest enrichment of ≤1kDa active peptides, while avoiding the heating inactivation step and maximizing the preservation of heat-sensitive components, thereby endowing royal jelly peptide powder with significant and controllable anti-fatigue function.

Claims

1. A method for preparing an anti-fatigue royal jelly peptide, characterized in that, The process includes the following steps: Fresh royal jelly is taken, freeze-dried, pulverized, and passed through an 80-mesh sieve to obtain freeze-dried royal jelly powder. The freeze-dried powder is weighed and prepared into a substrate solution of 50-60 g / L with 0.1 mol / L phosphate buffer. After 0.45 μm pre-microfiltration, a two-stage immobilized enzyme membrane reaction is performed, followed by concentration and desalting at room temperature and 0.5 MPa pressure using a nanofiltration membrane with a molecular weight cutoff of 1 kDa. The nanofiltration retentate is then freeze-dried to obtain anti-fatigue royal jelly peptide powder. In the two-stage immobilized enzyme membrane, the first-stage immobilized enzyme is thermophilic protease, and the second-stage immobilized enzyme is subtilisin.

2. The method according to claim 1, characterized in that, The two-stage immobilized enzyme membrane reaction is specifically described as follows: (1) The pre-microfiltered substrate solution is pumped into the tube side of the first-stage immobilized enzyme membrane reactor using a pulse feeding method. The pulse mode is 3 seconds of feeding followed by 1 second of pause, with an average feed flow rate of 60-80 mL / min. (2) The first stage of continuous enzymatic hydrolysis and membrane separation coupling is carried out: the reactor water bath is controlled at 58-64℃, the transmembrane pressure difference is 0.1-0.15MPa, the pH is maintained at 8.0 by 0.5mol / L NaOH, the circulation flow rate is 200-260mL / min, and the permeate with a molecular weight of less than 3kDa is collected. (3) The collected permeate was used as the substrate for the second-stage reaction and enzymatically hydrolyzed in the second-stage immobilized enzyme membrane reactor at a reaction temperature of 50°C and a transmembrane pressure difference of 0.1-0.15 MPa. The pH was maintained at 7.5-8.0 by using 0.5 mol / L NaOH and the circulation flow rate was 200-240 mL / min. The permeate was then separated again by an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain the second-stage permeate. The permeate was then passed through a nanofiltration membrane with a molecular weight cutoff of 1 kDa and concentrated and desalted at room temperature and a pressure of 0.5 MPa. The nanofiltration retentate was freeze-dried to obtain anti-fatigue royal jelly peptide powder.

3. The method according to claim 2, characterized in that, The preparation of the first-stage immobilized enzyme membrane reactor includes: preparing a 0.5% (v / v) sodium tripolyphosphate solution using 0.1 mol / L phosphate buffer, circulating it through the membrane cavity at 4°C for 2 hours for cross-linking treatment, then rinsing with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L, immobilizing the thermophilic protease on the inner surface of the first-stage hollow fiber enzyme membrane reactor, preparing an enzyme solution with a concentration of 5-8 g / L, using 0.1 mol / L phosphate buffer as the solvent, and adding 2 mmol / L of... L of CaCl2 was used as a buffer solution, and the pH was adjusted to 8.

0. The buffer solution was circulated through the membrane lumen at 4°C for 12 hours. After immobilization, the membrane was washed with buffer solution until no protein was detected. Then, a rinsing process was performed: the membrane lumen was circulated with 0.1 mol / L glycine-phosphate buffer solution at pH 7.0 at 25°C at 30 mL / min for 30 minutes. Then, the membrane was washed with 10 times the membrane volume of sterile deionized water until the sodium tripolyphosphate residue was ≤0.5 mg / L, thus obtaining the first-stage immobilized enzyme membrane reactor.

4. The method according to claim 2, characterized in that, The preparation of the second-stage immobilized enzyme membrane reactor includes: preparing a 0.5% (v / v) sodium tripolyphosphate solution using 0.1 mol / L phosphate buffer, circulating it through the membrane cavity at 4°C for 2 hours for cross-linking treatment, then rinsing with phosphate buffer until the residual sodium tripolyphosphate is ≤0.5 mg / L; immobilizing Bacillus subtilis protease on the inner surface of the second-stage hollow fiber enzyme membrane reactor; preparing an enzyme solution with a concentration of 5-8 g / L, using 0.1 mol / L phosphate buffer at pH 8.0 as the solvent, circulating it through the membrane cavity at 4°C for 12 hours for immobilization; after immobilization, rinsing with buffer until no protein is detected, followed by rinsing treatment: circulating the solution through the membrane cavity at 30 mL / min using 0.1 mol / L glycine-phosphate buffer at pH 7.0 at 25°C for 30 minutes, then rinsing with 10 times the membrane volume of sterile deionized water until the residual sodium tripolyphosphate is ≤0.5 mg / L, thus obtaining the second-stage immobilized enzyme membrane reactor.

5. The method according to any one of claims 1, 3, and 4, characterized in that, The phosphate buffer solutions described are all mixed salts of disodium hydrogen phosphate and sodium dihydrogen phosphate.

6. The method according to claim 3 or 4, characterized in that, The membrane material of both the first-stage hollow fiber enzyme membrane reactor and the second-stage hollow fiber enzyme membrane reactor is polyethersulfone, the membrane area is 0.5 square meters, and the molecular weight cutoff of the membrane pore size is 3 kDa.

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