Use of an enzymatic process in the preparation of a royal jelly preparation having enhanced immune cell activity

Abstract

The application relates to an application of an enzymolysis process in preparation of royal jelly preparation with improved immune cell activity, and belongs to the technical field of enzymolysis. The technical problem to be solved is to provide an enzymolysis process and application thereof which can more optimally improve the immune cell activity of royal jelly preparation. The technical solution is that the enzymolysis process is applied in preparation of royal jelly preparation with improved immune cell activity, the enzymolysis process is that first, a composite enzyme A is used for enzymolysis, and then, a composite enzyme B is used for secondary enzymolysis; the composite enzyme A is a lipase and a flavour protease with a mass ratio of 1:1-5; and the composite enzyme B is a chymotrypsin and a bromelain with a mass ratio of 3-8:1.

Description

Technical Field

[0001] This invention belongs to the field of enzymatic hydrolysis technology, specifically relating to the application of an enzymatic hydrolysis process in the preparation of royal jelly preparations that enhance the activity of immune cells. Background Technology

[0002] Immunity is the core physiological barrier for the human body to resist the invasion of external pathogens and maintain the homeostasis of the internal environment. Its strength directly determines the body's health level and disease resistance. With the continuously accelerating pace of modern life, significantly increased work pressure, irregular work and rest schedules, unbalanced diets, and aggravated environmental pollution, problems such as weakened immune function have become a common sub-health state. These manifest as recurrent colds, persistent fatigue, delayed wound healing, and increased susceptibility to infections. In severe cases, it can significantly increase the incidence of infectious diseases, placing dual pressure on population health and social medical resources. Against this backdrop, the development of safe, efficient functional ingredients with clearly defined immune-enhancing effects has become a real demand in the health consumer market.

[0003] Royal jelly, a natural active substance secreted by bees, is rich in various nutrients such as proteins, polypeptides, amino acids, fatty acids, vitamins, and bioactive factors. Its physiological functions in regulating immunity, anti-oxidation, and delaying aging have been widely confirmed. However, some of the large molecular active substances in natural royal jelly are difficult for the human body to absorb and utilize directly, and its physiological activity is easily lost due to external environmental factors, limiting its practical application in the field of immune enhancement.

[0004] To overcome this technical bottleneck, enzymatic hydrolysis technology has been widely used in the deep processing of royal jelly. Through the directional catalytic action of specific proteases, the large molecular proteins in royal jelly are degraded into small molecular peptides, which can not only significantly improve its bioavailability, but also enrich and enhance its specific physiological activities. It is the core technical means to optimize the efficacy of royal jelly products.

[0005] Patent document CN109198540A discloses a process for comprehensively extracting the effective components of royal jelly, including the following steps: adding royal jelly to purified water at a weight ratio of 1:4-10, mixing, extracting, centrifuging, filtering the supernatant obtained through a 200-300 mesh filter medium, and collecting the royal jelly filter cake; adding trypsin hydrolysate, blueberry juice, and physiological saline to the royal jelly, and enzymatically hydrolyzing at 35-40℃ for 70-80 minutes; boiling the enzymatically hydrolyzed royal jelly liquid in boiling water at 100℃ for 4-5 minutes, and rapidly cooling to 3-5℃; mixing the royal jelly liquid evenly with deionized water, centrifuging to obtain the royal jelly supernatant, filtering the royal jelly liquid using an ultrafiltration membrane, and freeze-drying to obtain the effective components of royal jelly. This invention extracts the non-water-soluble active ingredients in royal jelly by immersing the non-water-soluble components in water and then using enzymatic hydrolysis to recover the non-water-soluble active ingredients from the water. The extraction efficiency is high and the extracted royal jelly contains a large amount of active ingredients. However, this invention does not study the efficacy of its products in enhancing the activity of immune cells.

[0006] Patent document CN113647591A discloses a royal jelly enzymatic hydrolysate, which is obtained by enzymatic hydrolysis of royal jelly with protease M-P001. The enzymatic hydrolysis conditions are as follows: royal jelly is prepared into a 5-20% aqueous solution, and protease M-P001 is added at a ratio of 1500-2500 U / g (based on the raw royal jelly), and enzymatic hydrolysis is carried out at pH 6.0-8.0 and 33-36℃ for 20-25 hours. This royal jelly enzymatic hydrolysate can significantly improve the clearance ability of mouse mononuclear-macrophages and increase the activity of mouse NK cells, thus enhancing immune activity. However, the protease M-P001 in this invention is difficult to obtain and has high production costs, making it difficult to achieve large-scale industrial promotion and application, and failing to meet the actual needs of industrial production.

[0007] In summary, there is an urgent need to develop a royal jelly enzymatic hydrolysate that is safe, has a simple and controllable process, and can effectively enhance the activity of immune cells, so as to promote the industrial application of royal jelly deep processing technology in the health field. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides an application of an enzymatic hydrolysis process in the preparation of royal jelly preparations that enhance the activity of immune cells.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides: an enzymatic hydrolysis process for preparing royal jelly preparations that enhance the activity of immune cells, comprising the following steps: (1) Mix royal jelly and water and homogenize to obtain a royal jelly solution; (2) Add compound enzyme A to the royal jelly solution for enzymatic hydrolysis to obtain the hydrolysate; (3) Add compound enzyme B to the enzymatic hydrolysate for secondary enzymatic hydrolysis, inactivate the enzyme, separate the solid and liquid, obtain a clear liquid, and dry it. The complex enzyme A mentioned in step (2) is a lipase and a flavor protease; The complex enzyme B mentioned in step (3) is chymotrypsin and bromelain.

[0010] In some implementations, the mass ratio of royal jelly to water in step (1) is 1:10-15; preferably 1:10-13; and more preferably 1:11-12.

[0011] In some implementations, the homogenization in step (1) is at least once at 5-15 MPa, for 3-5 min each time; preferably, the homogenization is 2-4 times at 5-12 MPa, for 3-5 min each time; more preferably, the homogenization is 2-3 times at 5-10 MPa, for 3-5 min each time.

[0012] In some embodiments, the mass ratio of lipase to flavor protease in step (2) is 1:1-5; preferably 1:2-4; and more preferably 1:3.

[0013] In some embodiments, the amount of compound enzyme A added in step (2) is 0.2%-0.8% of the mass of the royal jelly solution; preferably 0.4%-0.6%; and more preferably 0.5%.

[0014] In some implementations, the enzymatic hydrolysis conditions in step (2) are: 40-45℃ for 5-12 hours and pH 7.0-7.5.

[0015] In some embodiments, the mass ratio of chymotrypsin and bromelain in step (3) is 3-8:1; preferably 5-7:1; and more preferably 6:1.

[0016] In some embodiments, the amount of compound enzyme B added in step (3) is 0.3%-0.8% of the mass of the royal jelly solution; preferably 0.4%-0.6%; and more preferably 0.5%.

[0017] In some implementations, the conditions for the secondary enzymatic hydrolysis in step (3) are: enzymatic hydrolysis at 45-55℃ for 5-12 hours and pH 7.5-8.0.

[0018] In some implementations, the enzyme inactivation in step (3) is performed by inactivating the enzyme at 100°C for 5-10 minutes.

[0019] In some implementations, the solid-liquid separation in step (3) is centrifugation, specifically centrifugation at 5000-10000 rpm for 15-30 min; preferably centrifugation at 6000-8000 rpm for 15-25 min.

[0020] In some implementations, the drying in step (3) is vacuum drying, specifically: temperature ≤ 50°C, vacuum degree -0.08 to -0.1 MPa.

[0021] Secondly, the present invention provides the application of the above-mentioned enzymatic hydrolysis process in the preparation of royal jelly preparations that enhance the activity of immune cells.

[0022] The royal jelly preparation described in this invention is a medicine.

[0023] The beneficial effects of this invention are as follows: 1. This invention uses a compound enzyme A (lipase + flavor protease) to degrade lipid components and initially hydrolyze short peptides, and then uses a compound enzyme B (chymotrypsin + bromelain) to specifically hydrolyze large molecular proteins, which significantly improves the enzymatic hydrolysis efficiency and the release of active ingredients in the product.

[0024] 2. Experiments have shown that the enzymatically hydrolyzed royal jelly prepared by this invention can significantly improve the cell viability and phagocytic capacity of macrophages. Compared with conventional enzymatically hydrolyzed or unhydrolyzed royal jelly, this invention has a greater advantage in enhancing the activity of immune cells.

[0025] 3. The four enzymes used in this invention are made from readily available raw materials and have simple preparation methods, which meet the requirements for industrialization.

[0026] 4. The enzymatically hydrolyzed royal jelly prepared by this invention has a good effect on enhancing the activity of immune cells and can be used as a raw material for drug preparation, promoting the industrial application of royal jelly deep processing technology in related health fields. Detailed Implementation

[0027] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0028] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms, the definition provided in this chapter shall prevail.

[0029] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0030] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0031] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0032] The term "solid-liquid separation" as used in this article refers to the process of separating solid and liquid phases through physical means, including but not limited to separation methods such as filtration, centrifugation, sedimentation, vacuum filtration, and pressure filtration.

[0033] The term "enzyme inactivation" as used in this article refers to the process of eliminating the catalytic activity of endogenous enzymes in raw materials through heating, pH adjustment, or other physical or chemical methods, thereby terminating enzymatic reactions and preventing the degradation of active ingredients. This includes, but is not limited to, inactivation methods such as high-temperature inactivation, boiling water bath, steam heating, and microwave treatment.

[0034] The term "drying" as used in this article refers to the process of removing moisture or other volatile solvents from materials through physical or physicochemical means to bring the materials to a specified moisture content, including but not limited to freeze drying, vacuum drying, hot air drying, spray drying, natural drying, and other drying methods.

[0035] All numerical values ​​or expressions relating to component amounts, process conditions, etc., used herein shall, in all cases, be understood to be modified by the word “about.” When referring to a quantity or range of values, the quantity or range is an approximation within experimental variability (or within statistical experimental error). In this invention, the term “about” shall have the meaning of being within 10%, preferably within 5%, of the specified value or range.

[0036] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0037] The enzymes used in this example all comply with the requirements for use of food additives in GB2760-2024. These include chymotrypsin (CAS: 9004-07-3, enzyme activity approximately 100,000 U / g), lipase (CAS: 9001-62-1, enzyme activity approximately 100,000 U / g), flavor protease (Nanning Dongheng Huadao Biotechnology Co., Ltd., enzyme activity approximately 30,000 U / g), and bromelain (Nanning Dongheng Huadao Biotechnology Co., Ltd., enzyme activity approximately 100,000 U / g). Royal jelly was sourced from Jiangxi Wangshi Bee Garden Co., Ltd., batch number 20250729. The preparation of unhydrolyzed royal jelly involved vacuum drying (50℃, -0.08 MPa).

[0038] In this invention, sodium hydroxide and citric acid are used to adjust the pH value.

[0039] Example 1 An enzymatic hydrolysis process for preparing royal jelly preparations that enhance the activity of immune cells. The steps are as follows: (1) Mix royal jelly and water at a mass ratio of 1:12, homogenize twice at 8 MPa for 4 min each time to obtain royal jelly solution.

[0040] (2) Add compound enzyme A to the royal jelly solution for enzymatic hydrolysis to obtain the hydrolysate; in, The amount of the compound enzyme A added is 0.5% of the mass of the royal jelly solution; The complex enzyme A is a lipase and a flavor protease in a mass ratio of 1:3. The enzymatic hydrolysis conditions are: 42℃ for 8 hours, pH 7.0-7.5.

[0041] (3) Add compound enzyme B to the enzymatic hydrolysate for secondary enzymatic hydrolysis. After the secondary enzymatic hydrolysis, inactivate the enzyme at 100℃ for 5 min, centrifuge at 7000 rpm for 20 min, and dry the clear liquid under vacuum (50℃, -0.08MPa). in, The amount of compound enzyme B added is 0.5% of the mass of the royal jelly solution; The compound enzyme B is composed of chymotrypsin and bromelain in a mass ratio of 6:1. The conditions for the secondary enzymatic hydrolysis are: enzymatic hydrolysis at 50℃ for 8 hours, pH 7.5-8.

[0042] Example 2 An enzymatic hydrolysis process for preparing royal jelly preparations that enhance the activity of immune cells. The steps are as follows: (1) Mix royal jelly and water at a mass ratio of 1:10, homogenize at 5 MPa for 3 times, 3 minutes each time, to obtain a royal jelly solution.

[0043] (2) Add compound enzyme A to the royal jelly solution for enzymatic hydrolysis to obtain the hydrolysate; The amount of the compound enzyme A added is 0.2% of the mass of the royal jelly solution; The complex enzyme A is a lipase and a flavor protease in a mass ratio of 1:1. The enzymatic hydrolysis conditions are: 42℃ for 12 hours, pH 7.0-7.5.

[0044] (3) Add compound enzyme B to the enzymatic hydrolysate for secondary enzymatic hydrolysis. After the secondary enzymatic hydrolysis, inactivate the enzyme at 100℃ for 5 min, centrifuge at 7000 rpm for 15-25 min, and dry the clear liquid under vacuum (50℃, -0.08MPa). The amount of compound enzyme B added is 0.8% of the mass of the royal jelly solution; The complex enzyme B is composed of chymotrypsin and bromelain in a mass ratio of 3:1. The conditions for the secondary enzymatic hydrolysis are: 55℃ for 12 hours, pH 7.5-8.

[0045] Example 3 An enzymatic hydrolysis process for preparing royal jelly preparations that enhance the activity of immune cells. The steps are as follows: (1) Mix royal jelly and water at a mass ratio of 1:15, homogenize once at 10 MPa for 5 min each time to obtain royal jelly solution.

[0046] (2) Add compound enzyme A to the royal jelly solution for enzymatic hydrolysis to obtain the hydrolysate; The amount of the compound enzyme A added is 0.8% of the mass of the royal jelly solution; The complex enzyme A is a lipase and a flavor protease in a mass ratio of 1:5. The enzymatic hydrolysis conditions are: 40℃ for 5 hours, pH 7.0-7.5.

[0047] (3) Add compound enzyme B to the enzymatic hydrolysate for secondary enzymatic hydrolysis. After the secondary enzymatic hydrolysis, inactivate the enzyme at 100℃ for 5 min, centrifuge at 7000 rpm for 15-25 min, and dry the clear liquid under vacuum (50℃, -0.08MPa). The amount of compound enzyme B added is 0.3% of the mass of the royal jelly solution; The compound enzyme B is composed of chymotrypsin and bromelain in a mass ratio of 8:1. The conditions for the secondary enzymatic hydrolysis are: enzymatic hydrolysis at 45℃ for 12 hours, pH 7.5-8.

[0048] Comparative Example 1 The only difference between this comparative example and Example 1 is the order of complex enzyme A and complex enzyme B.

[0049] Specifically, it is an enzymatic hydrolysis process for preparing royal jelly preparations that enhance the activity of immune cells. The steps are as follows: (1) Mix royal jelly and water at a mass ratio of 1:12, homogenize twice at 8 MPa for 4 min each time to obtain royal jelly solution.

[0050] (2) Add compound enzyme B to the royal jelly solution for enzymatic hydrolysis to obtain the hydrolysate; in, The amount of compound enzyme B added is 0.5% of the mass of the royal jelly solution; The compound enzyme B is composed of chymotrypsin and bromelain in a mass ratio of 6:1. The enzymatic hydrolysis conditions are: 50℃ for 8 hours, pH 7.5-8.

[0051] (3) Add compound enzyme A to the enzymatic hydrolysate for secondary enzymatic hydrolysis. After the secondary enzymatic hydrolysis, inactivate the enzyme at 100℃ for 5 min, centrifuge at 7000 rpm for 20 min, and dry the clear liquid under vacuum (50℃, -0.08MPa). in, The amount of the compound enzyme A added is 0.5% of the mass of the royal jelly solution; The complex enzyme A is a lipase and a flavor protease in a mass ratio of 1:3. The enzymatic hydrolysis conditions are: 42℃ for 8 hours, pH 7.0-7.5.

[0052] Comparative Example 2 The only difference between this comparative example and Example 1 is that it does not contain compound enzyme B, and the enzymatic hydrolysis process is slightly different.

[0053] Specifically, an enzymatic hydrolysis process for preparing royal jelly preparations that enhance the activity of immune cells. The steps are as follows: (1) Mix royal jelly and water at a mass ratio of 1:12, homogenize twice at 8 MPa for 4 min each time to obtain royal jelly solution.

[0054] (2) Add compound enzyme A to the royal jelly solution for enzymatic hydrolysis to obtain the hydrolysate; in, The amount of the compound enzyme A added is 1% of the mass of the royal jelly solution; The complex enzyme A is a lipase and a flavor protease in a mass ratio of 1:3. The enzymatic hydrolysis conditions are: 42℃ for 16 hours, pH 7-7.5.

[0055] (3) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 100℃ for 5 min, centrifuged at 7000 rpm for 20 min, and the clear liquid is dried under vacuum (50℃, -0.08MPa). Comparative Example 3 The only difference between this comparative example and Example 1 is that it does not contain complex enzyme A, and the enzymatic hydrolysis process is slightly different.

[0056] Specifically, an enzymatic hydrolysis process for preparing royal jelly preparations that enhance the activity of immune cells. The steps are as follows: (1) Mix royal jelly and water at a mass ratio of 1:12, homogenize twice at 8 MPa for 4 min each time to obtain royal jelly solution.

[0057] (2) Add compound enzyme B to the royal jelly solution for enzymatic hydrolysis to obtain the hydrolysate; in, The amount of compound enzyme B added is 1.0% of the mass of the royal jelly solution; The compound enzyme B is composed of chymotrypsin and bromelain in a mass ratio of 6:1. The enzymatic hydrolysis conditions are: 50℃ for 16 hours, pH 7.5-8.

[0058] (3) Inactivate the enzyme in the enzyme hydrolysate at 100℃ for 5 min, centrifuge at 7000 rpm for 20 min, and dry the clear liquid under vacuum (50℃, -0.08MPa). Comparative Example 4 The only difference between this comparative example and Example 1 is that: complex enzyme A is replaced with lipase and bromelain in a mass ratio of 1:3, and complex enzyme B is replaced with chymotrypsin and flavor protease in a mass ratio of 6:1.

[0059] The other steps are the same as in Example 1.

[0060] Detection Example 1 Effects of royal jelly preparations (enzymatically hydrolyzed royal jelly) on macrophage activity RAW 264.7 mouse macrophages were cultured in DMEM complete medium at a concentration of 1×10⁻⁶. 5 Inoculate at a density of 100 cells / mL into 96-well plates and incubate overnight, then discard the culture medium. Perform experiments according to the following groups, with 3 replicates per group; Blank control group: Add 100 μL of DMEM complete culture medium; Examples 1-3 and Comparative Examples 1-4: 100 μL of DMEM complete medium containing 200 μg / mL enzymatically hydrolyzed royal jelly was added; Positive control group: Add 100 μL of DMEM complete medium containing 1 μg / mL LPS; Unhydrolyzed royal jelly group: Add 100 μL of DMEM complete medium containing 200 μg / mL unhydrolyzed royal jelly; Incubate at 37℃ and 5% CO2 for 24 h. Add 10 μL of CCK-8 reagent to each well and continue incubating at 37℃ for 1 h. Mix well and measure the absorbance of each well at 450 nm using a microplate reader.

[0061] The cell viability relative to the blank control group is calculated using the following formula: Cell viability (%) = OD of experimental group 450 / Blank control group OD 450 ×100%.

[0062] The results are shown in Table 1.

[0063] Table 1

[0064] Note: Compared to the blank control group, P < 0.05.

[0065] The results showed that, compared with the blank control group, the enzymatically hydrolyzed royal jelly prepared in Examples 1-3 of the present invention could significantly enhance the activity of RAW 264.7 macrophages (P<0.05); the enzymatically hydrolyzed royal jelly and unenzymatically hydrolyzed royal jelly prepared in Comparative Examples 1-4 had no significant effect on enhancing the activity of RAW264.7 macrophages (P>0.05).

[0066] Furthermore, comparing Example 1 and Comparative Examples 1-4, it can be seen that the stepwise enzymatic hydrolysis process of the present invention, which uses specific complex enzyme A and specific complex enzyme B in combination, can significantly enhance the efficacy of royal jelly in improving the activity of immune cells.

[0067] Detection Example 2 Effects of royal jelly preparations (enzymatically hydrolyzed royal jelly) on the phagocytic capacity of macrophages RAW 264.7 mouse macrophages were cultured in DMEM complete medium at a concentration of 1×10⁻⁶. 5 Inoculate at a density of 100 cells / mL into 96-well plates and incubate overnight, then discard the culture medium. Perform experiments according to the following groups, with 3 replicates per group; Blank control group: Add 100 μL of DMEM complete culture medium; Examples 1-3 and Comparative Examples 1-4: 100 μL of DMEM complete medium containing 200 μg / mL enzymatically hydrolyzed royal jelly was added; Positive control group: Add 100 μL of DMEM complete medium containing 1 μg / mL LPS; Unhydrolyzed royal jelly group: Add 100 μL of DMEM complete medium containing 200 μg / mL unhydrolyzed royal jelly; After culturing at 37℃ and 5% CO2 for 24 h, the culture medium was discarded, and 100 μL of 0.075% neutral red staining solution was added to each well. Incubation continued for 1 h. The neutral red staining solution was discarded, and the cells were gently washed twice with PBS to remove any unphagocytosed neutral red dye. 100 μL of cell lysis buffer (anhydrous ethanol: glacial acetic acid = 1:1, v / v) was added to each well, and the cells were incubated at room temperature in the dark for 30 min, followed by low-speed shaking until complete cell lysis. The absorbance of each well was measured at 540 nm using a microplate reader.

[0068] The phagocytic rate relative to the blank control group was calculated using the following formula: Phagocytic rate (%) = OD of experimental group 540 / Blank control group OD 540 ×100%.

[0069] The results are shown in Table 2.

[0070] Table 2

[0071] Note: Compared to the blank control group, P < 0.05.

[0072] The results showed that, compared with the blank control group, the enzymatically hydrolyzed royal jelly prepared in Examples 1-3 of this invention could significantly improve the phagocytic capacity of RAW 264.7 macrophages (P<0.05); the enzymatically hydrolyzed royal jelly and unenzymatically hydrolyzed royal jelly prepared in Comparative Examples 1-4 had no significant effect on improving the phagocytic capacity of RAW 264.7 macrophages (P>0.05).

[0073] Furthermore, comparing Example 1 and Comparative Examples 1-4, it can be seen that the stepwise enzymatic hydrolysis process of the present invention, which uses specific complex enzyme A and specific complex enzyme B in combination, can effectively activate macrophage function and significantly enhance its phagocytic ability.

[0074] In summary, the royal jelly preparation (enzymatically hydrolyzed royal jelly) prepared by this invention can enhance the activity of immune cells, and its effect is significantly better than that of unenzymatically hydrolyzed royal jelly.

[0075] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of an enzymatic hydrolysis process in the preparation of royal jelly preparations that enhance the activity of immune cells, characterized in that, The enzymatic hydrolysis process involves first performing enzymatic hydrolysis using compound enzyme A, followed by a secondary enzymatic hydrolysis using compound enzyme B. Compound enzyme A consists of lipase and flavor protease in a mass ratio of 1:1-5; compound enzyme B consists of chymotrypsin and bromelain in a mass ratio of 3-8:

1.

2. The application according to claim 1, characterized in that, The enzymatic hydrolysis process includes the following steps: (1) Mix royal jelly and water and homogenize to obtain a royal jelly solution; (2) Add compound enzyme A to the royal jelly solution for enzymatic hydrolysis to obtain the hydrolysate; (3) Add compound enzyme B to the enzymatic hydrolysate for secondary enzymatic hydrolysis, inactivate the enzyme, separate the solid and liquid, obtain a clear liquid, and dry it.

3. The application according to claim 2, characterized in that, The mass ratio of royal jelly to water in step (1) is 1:10-15; The homogenization described in step (1) is performed at least once at 5-15 MPa, for 3-5 minutes each time.

4. The application according to claim 2, characterized in that, The mass ratio of lipase and flavor protease in step (2) is 1:2-4.

5. The application according to claim 2, characterized in that, The amount of compound enzyme A added in step (2) is 0.2%-0.8% of the mass of the royal jelly solution.

6. The application according to claim 2, characterized in that, The conditions for enzymatic hydrolysis in step (2) are: 40-45℃ for 5-12 hours, pH 7.0-7.

5.

7. The application according to claim 2, characterized in that, The mass ratio of chymotrypsin and bromelain in step (3) is 5-7:

1.

8. The application according to claim 2, characterized in that, The amount of compound enzyme B added in step (3) is 0.3%-0.8% of the mass of the royal jelly solution.

9. The application according to claim 2, characterized in that, The conditions for the secondary enzymatic hydrolysis in step (3) are: enzymatic hydrolysis at 45-55℃ for 5-12 hours and pH 7.5-8.

0.

10. An enzymatic hydrolysis process for preparing royal jelly preparations that enhance the activity of immune cells, characterized in that, Includes the following steps: (1) Mix royal jelly and water and homogenize to obtain a royal jelly solution; (2) Add compound enzyme A to the royal jelly solution for enzymatic hydrolysis to obtain the hydrolysate; (3) Add compound enzyme B to the enzymatic hydrolysate for secondary enzymatic hydrolysis, inactivate the enzyme, separate the solid and liquid, obtain a clear liquid, and dry it. The complex enzyme A is a lipase and flavor protease in a mass ratio of 1:1-5; the complex enzyme B is a chymotrypsin and bromelain in a mass ratio of 3-8:1.

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