Preparation method of micromolecule enzymolysis royal jelly capable of improving absorption
By performing multi-step enzymatic hydrolysis on royal jelly, especially by using sea cucumber hydrolase and aminopeptidase followed by pepsin treatment, the problems of insufficient stability and bioavailability caused by the excessively large molecular weight of enzymatically hydrolyzed royal jelly have been solved, resulting in higher absorption and anti-aging effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGXI WANGS BEE GARDEN CO LTD
- Filing Date
- 2026-04-15
- Publication Date
- 2026-05-19
AI Technical Summary
Some peptides in existing enzymatically hydrolyzed royal jelly have molecular weights exceeding 1500 Da, resulting in insufficient stability and bioavailability of royal jelly extracts, making it difficult to meet the requirements for higher absorption and anti-aging effects.
Royal jelly was initially hydrolyzed using sea cucumber hydrolase and aminopeptidase, and then further hydrolyzed using pepsin. The hydrolysis conditions, such as concentration, pH, temperature and time, were controlled to ensure that the molecular weight of the peptides was less than 1000 Da, and in particular, the proportion of peptides less than 500 Da reached more than 80%.
It significantly reduces the molecular weight of enzymatically hydrolyzed royal jelly, improves its stability and bioavailability, enhances its anti-aging effects, and makes it easier to absorb.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of royal jelly preparation technology, specifically relating to a method for preparing small-molecule enzymatically hydrolyzed royal jelly that improves absorption. Background Technology
[0002] Honey is rich in sugars, vitamins, and minerals, while royal jelly contains abundant proteins, royal jelly acid (10-HDA), and vitamins. Propolis contains flavonoids, terpenes, and other active ingredients. Therefore, bee products also have potential applications in cosmetics. The most abundant proteins and peptides in royal jelly are its main active ingredients and are also a research hotspot. Some proteins and peptides have been effectively isolated, and their functions are supported by a large amount of experimental data. However, some proteins and their enzymatic hydrolysis products may still have some undiscovered physiological activities. Furthermore, the physiological effects and toxic side effects of royal jelly have not been fully studied, which limits the scope of its application. Therefore, a better understanding of its biological activities and physiological effects will help promote its application.
[0003] Studies have found that small molecule peptides produced by enzymatic hydrolysis of natural protein royal jelly may have higher biological activity or functions that proteins do not possess. Through enzymatic hydrolysis, separation, and purification, enzymatic hydrolysates with antioxidant activity higher than the original protein extract can be obtained. Furthermore, the high-grade proteins in royal jelly may cause allergies in consumers. Therefore, if the enzymatic hydrolysis process can be optimized to maximize the yield of royal jelly peptides, the utilization value of royal jelly can be improved, and allergic reactions can be eliminated.
[0004] The health benefits of royal jelly have been extensively confirmed by research. As a raw material for health supplements, royal jelly is primarily consumed directly, with limited processing. Increasing the absorption rate and efficacy of royal jelly, and reducing allergic reactions, is crucial for product development. Enzymatic hydrolysis technology breaks down proteins into smaller peptides, which can better address the allergy issues associated with royal jelly and further enhance its bioactivity and added value.
[0005] For example, Chinese patent CN113647591A discloses a royal jelly enzymatic hydrolysate, which is an enzymatic hydrolysate obtained by hydrolyzing royal jelly with protease M-P001; the protease M-P001 is derived from Mycobacterium tuberculosis. The enzymatic hydrolysis conditions are as follows: enzymatic hydrolysis of royal jelly aqueous solution with protease M-P001, the concentration of royal jelly in the aqueous solution is 5-20%; protease M-P001 is added at an amount of 1500-2500 U / g (based on royal jelly raw material), pH 6.0-8.0, and enzymatic hydrolysis is carried out at 33-36°C 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 having the effect of improving immune activity. It can be used as a raw material to prepare health products, medicines, or foods with immune-enhancing effects, which can significantly improve the product value of royal jelly.
[0006] For example, a master's thesis from Zhejiang University, titled "Study on the Anti-Photoaging Activity of Royal Jelly and its Enzymatic Hydrolysates," authored by Qian Jiale and published on January 1, 2021, investigated the molecular weight distribution of royal jelly and its enzymatic hydrolysates. The study found that enzymatic hydrolysis significantly reduced the molecular weight of the samples. The molecular weight of the freeze-dried royal jelly powder was concentrated in the range of greater than 6500 Da (47%) and less than 700 Da (45.1%), indicating that the protein content of the freeze-dried royal jelly powder was approximately 47%, while the content of its main components, such as fatty acids and amino acids, was approximately 45.1%. The molecular weights of the products after enzymatic hydrolysis were all less than 6500 Da. The 180-700 Da and 700-1500 Da fractions had relatively high yields (65.15% and 23.44%, respectively), indicating that most of the proteins in the royal jelly hydrolysate obtained by the enzymatic hydrolysis process were hydrolyzed and converted into specific peptide fragments and amino acids. Most of the peptides were concentrated in the 180-1500 Da range, while a small number of peptides had molecular weights exceeding 1500 Da.
[0007] However, the molecular weight of existing enzymatically hydrolyzed royal jelly still has a small number of peptides with molecular weights exceeding 1500 Da, which means that the stability and bioavailability of royal jelly extracts cannot better meet the requirements. Therefore, there is a need to develop a small-molecule enzymatically hydrolyzed royal jelly with good stability and peptides with molecular weights less than 1000 Da that are easy to absorb, as well as its preparation process. Summary of the Invention
[0008] To achieve the above objectives, this invention selects the type of enzyme and the enzymatic hydrolysis conditions to prepare a small-molecule enzymatically hydrolyzed royal jelly with peptide molecular weights all less than 1000 Da.
[0009] To achieve the above objectives, the present invention adopts the following technical solution:
[0010] A method for preparing small-molecule enzymatically hydrolyzed royal jelly with improved absorption includes the following steps: (1) Preparation of royal jelly aqueous solution: Dissolve royal jelly raw material in deionized water to obtain royal jelly aqueous solution; (2) Take royal jelly aqueous solution and adjust the pH value to 7-8, then add sea cucumber hydrolase and aminopeptidase, carry out enzymatic hydrolysis, sterilize, and obtain enzymatic hydrolysate 1; (3) Take the enzymatic hydrolysate 1, adjust the pH to 1-2, add pepsin, carry out enzymatic hydrolysis, sterilize and freeze dry to obtain the small molecule enzymatic hydrolysed royal jelly.
[0011] in, The concentration of the royal jelly aqueous solution mentioned in step (1) is 10-25%, preferably 15-20%; The concentration of the royal jelly aqueous solution is any value within the range of 10-25%, for example, any of the following values or any range between two: 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%; Preferably, the concentration of the royal jelly aqueous solution is any value within the range of 15-20%, for example, any of the following values or any range between the two: 15%, 16%, 17%, 18%, 19%, 20%; During the implementation of this invention, it was discovered that the concentration of the royal jelly aqueous solution has a certain impact on the enzymatic hydrolysis efficiency. When the concentration is too low, the probability of the substrate and enzyme specifically binding decreases, resulting in a decrease in enzymatic hydrolysis efficiency. When the concentration is too high, the enzyme reaction rate reaches saturation, and the enzymatic hydrolysis effect no longer increases. This invention controls the concentration of the royal jelly aqueous solution to 10-25%, preferably 15-20%, which results in a moderate enzymatic hydrolysis rate that meets the requirements of enzymatic hydrolysis.
[0012] The concentration of the royal jelly aqueous solution is the mass-volume ratio of royal jelly to water, expressed in g / mL.
[0013] The mass ratio of sea cucumber hydrolase and aminopeptidase in step (2) above is 1-3:1-2; preferably 3:2.
[0014] The mass ratio of sea cucumber hydrolase and aminopeptidase in step (2) is any value in the range of 1-3:1-2, such as any of the following values or any range between the two: 1:1, 1:2, 2:1, 2.5:1, 3:1, 3:2; Preferably, the mass ratio of sea cucumber hydrolase and aminopeptidase in step (2) is any value in the range of 1-3:1-2, such as any of the following values or any range between the two: 2:1, 2.5:1, 3:1, 3:2; More preferably, the mass ratio of sea cucumber hydrolase and aminopeptidase in step (2) is 3:2.
[0015] The total amount of sea cucumber hydrolase and aminopeptidase added is 2-5% of the weight of royal jelly, preferably 2.5-4.5%; The total amount of sea cucumber hydrolase and aminopeptidase added in step (2) is any value within the range of 2-5% of the royal jelly mass, for example, any of the following values or any range between the two: 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.5%, 3.8%, 4.0%, 4.2%, 4.5%, 4.8%, 5.0%; Preferably, the total amount of sea cucumber hydrolase and aminopeptidase added in step (2) is any value within the range of 2-5% of the royal jelly mass, for example, any of the following values or any range between the two: 2.8%, 3.0%, 3.2%, 3.5%, 3.8%, 4.0%, 4.2%.
[0016] More preferably, the total amount of sea cucumber hydrolase and aminopeptidase added in step (2) is 3.5% of the weight of royal jelly.
[0017] The temperature of enzymatic hydrolysis in step (2) above is 50-55℃; preferably 50℃.
[0018] The enzymatic hydrolysis time described in step (2) above is 1-3 hours; preferably 2-3 hours. The enzymatic hydrolysis time in step (2) is any value within the range of 1-3 hours, such as any of the following values or any range between the two: 1h, 1.5h, 2h, 2.5h, 3h; Preferably, the enzymatic hydrolysis time in step (2) is any value within the range of 1-3 hours, such as any of the following values or any range between the two: 1.5h, 2h, 2.5h.
[0019] More preferably, the enzymatic hydrolysis time in step (2) is 2 hours.
[0020] The amount of pepsin added in step (3) above is 1-3% of the weight of royal jelly, preferably 1.5-2.5%; The amount of pepsin added in step (3) is any value within the range of 1-3% of the royal jelly mass, for example, any of the following values or any range between the two: 1%, 1.2%, 1.5%, 1.8%, 2.0%, 2.2%, 2.5%, 3.0%; Preferably, the amount of pepsin added in step (3) is any value in the range of 1-3% of the royal jelly mass, for example, any of the following values or any range between the two: 1.5%, 1.8%, 2.0%.
[0021] More preferably, the amount of pepsin added in step (3) is 2.0% of the weight of royal jelly.
[0022] The temperature for enzymatic hydrolysis in step (3) above is 35-40℃; preferably 37℃.
[0023] The enzymatic hydrolysis time in step (3) above is 2-4 hours; preferably 3-4 hours. The enzymatic hydrolysis time in step (3) is any value within the range of 2-4 hours, for example, any of the following values or any range between the two: 2h, 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h, 3.8h, 4.0h; Preferably, the enzymatic hydrolysis time in step (3) is any value within the range of 3-4 hours, such as any of the following values or any range between the two: 3h, 3.2h, 3.5h, 3.8h, 4.0h.
[0024] More preferably, the enzymatic hydrolysis time in step (3) is 3.5 hours.
[0025] A small-molecule enzymatically hydrolyzed royal jelly prepared by the above preparation method, wherein the molecular weight of the peptides in the small-molecule enzymatically hydrolyzed royal jelly is less than or equal to 1000 Da, and the proportion of peptides with a molecular weight of less than or equal to 500 Da is greater than or equal to 80%.
[0026] During the implementation of this invention, it was unexpectedly discovered that firstly, sea cucumber hydrolase and aminopeptidase were used to enzymatically hydrolyze royal jelly, and then pepsin was used to further enzymatically hydrolyze the hydrolysate. The resulting enzymatically hydrolyzed royal jelly had a significantly lower molecular weight, with peptides having a molecular weight of 500 Da or less accounting for more than 80%, and all peptides having a molecular weight of 1000 Da or less, which is more conducive to absorption and has relatively high stability. This small-molecule enzymatically hydrolyzed royal jelly has high bioavailability and therefore has a better anti-aging effect.
[0027] Furthermore, this invention provides the application of the above-mentioned small molecule enzymatic hydrolysis of royal jelly in the preparation of products with anti-aging effects.
[0028] The products mentioned include cosmetics.
[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a method for preparing small molecule enzymatically hydrolyzed royal jelly with improved absorption. The small molecule enzymatically hydrolyzed royal jelly peptides prepared by this method have a molecular weight of less than or equal to 1000 Da, of which the proportion of peptides with a molecular weight of less than or equal to 500 Da is greater than or equal to 80%. Since the proportion of peptides with a molecular weight of less than or equal to 500 Da is relatively high, they are easier to be absorbed and utilized, and the bioavailability is significantly improved.
[0030] (2) During the implementation of this invention, it was unexpectedly discovered that royal jelly was first enzymatically hydrolyzed with sea cucumber hydrolase and aminopeptidase, and then the hydrolysate was enzymatically hydrolyzed again with pepsin. The resulting enzymatically hydrolyzed royal jelly not only had a significantly lower molecular weight, but also significantly improved stability. In addition, because the molecular weight of the enzymatically hydrolyzed royal jelly was relatively low, its bioavailability was improved, which significantly improved its anti-aging effect. Detailed Implementation
[0031] 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.
[0032] 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.
[0033] The sea cucumber hydrolase, aminopeptidase, and pepsin used in the following examples and comparative examples were all purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.
[0034] The royal jelly used in the following examples and comparative examples was fresh royal jelly, and the mass of royal jelly used was 200g.
[0035] Example 1: A method for improving the absorption of small-molecule enzymatic hydrolysis of royal jelly and its preparation process (1) Preparation of royal jelly aqueous solution: Dissolve royal jelly raw material in deionized water to obtain a royal jelly aqueous solution with a concentration of 20%; (2) Take a royal jelly aqueous solution and adjust the pH value to 7.5. Then add sea cucumber hydrolase and aminopeptidase at a mass ratio of 2% of royal jelly and 1:1. Enzymatically hydrolyze for 3 hours at 50℃, sterilize, and obtain hydrolysate 1. (3) Take the enzymatic hydrolysate 1, adjust the pH to 1.5, add pepsin at 3% of the weight of royal jelly, enzymatically hydrolyze at 37°C for 2 hours, sterilize and freeze dry to obtain the small molecule enzymatically hydrolyzed royal jelly.
[0036] Example 2: A method for improving the absorption of small-molecule enzymatic hydrolysis of royal jelly and its preparation process (1) Preparation of royal jelly aqueous solution: Dissolve royal jelly raw material in deionized water to obtain a royal jelly aqueous solution with a concentration of 15%; (2) Take a royal jelly aqueous solution and adjust the pH value to 7.5. Then add sea cucumber hydrolase and aminopeptidase at a mass ratio of 3:1 to 5% of the royal jelly mass. Enzymatically hydrolyze for 1 hour at 50°C and sterilize to obtain hydrolysate 1. (3) Take the enzymatic hydrolysate 1, adjust the pH to 1.5, add pepsin at 1% of the weight of royal jelly, enzymatically hydrolyze at 37°C for 4 hours, sterilize and freeze dry to obtain the small molecule enzymatically hydrolyzed royal jelly.
[0037] Example 3: A method for improving the absorption of small-molecule enzymatic hydrolysis of royal jelly and its preparation process. (1) Preparation of royal jelly aqueous solution: Dissolve royal jelly raw material in deionized water to obtain a royal jelly aqueous solution with a concentration of 25%; (2) Take a royal jelly aqueous solution and adjust the pH value to 7.5. Then add sea cucumber hydrolase and aminopeptidase at a mass ratio of 2:1 to 5% of the royal jelly mass. Enzymatically hydrolyze for 3 hours at 50°C and sterilize to obtain hydrolysate 1. (3) Take the enzymatic hydrolysate 1, adjust the pH to 1.5, add pepsin at 2% of the weight of royal jelly, enzymatically hydrolyze at 37°C for 3 hours, sterilize and freeze dry to obtain the small molecule enzymatically hydrolyzed royal jelly.
[0038] Example 4: A method for improving the absorption of small-molecule enzymatic hydrolysis of royal jelly and its preparation process (1) Preparation of royal jelly aqueous solution: Dissolve royal jelly raw material in deionized water to obtain a royal jelly aqueous solution with a concentration of 20%; (2) Take a royal jelly aqueous solution and adjust the pH value to 7.5. Then add sea cucumber hydrolase and aminopeptidase at a mass ratio of 3:2 to 3.5% of the royal jelly. Enzymatically hydrolyze for 2 hours at 50°C and sterilize to obtain hydrolysate 1. (3) Take the enzymatic hydrolysate 1, adjust the pH to 1.5, add pepsin at 2.0% of the weight of royal jelly, enzymatically hydrolyze at 37°C for 3.5 h, sterilize and freeze dry to obtain the small molecule enzymatically hydrolyzed royal jelly.
[0039] Comparative Example 1: The difference from Example 4 is that only sea cucumber hydrolytic enzyme is used in step (2), while the rest is the same as in Example 4.
[0040] Comparative Example 2: The difference from Example 4 is that the mass ratio of sea cucumber hydrolase and aminopeptidase in step (2) is 1:3, while the rest is the same as in Example 4.
[0041] Comparative Example 3: The difference from Example 4 is that the order of steps (2) and (3) is changed, that is: (2) Take a royal jelly aqueous solution, adjust the pH to 1.5, add pepsin at 2.0% of the royal jelly mass, enzymatically hydrolyze at 37℃ for 3.5h, sterilize, and obtain enzymatic hydrolysate 1; (3) Take the enzymatic hydrolysate 1 and adjust the pH value to 7.5. Then add sea cucumber hydrolase and aminopeptidase at a mass ratio of 3:2 to 3.5% of the royal jelly. Enzymatically hydrolyze at 50°C for 2 hours, sterilize and freeze dry to obtain the small molecule enzymatically hydrolyzed royal jelly.
[0042] Everything else is the same as in Example 4.
[0043] Comparative Example 4: The preparation method for unenzymatically hydrolyzed royal jelly freeze-dried powder is as follows: dissolve royal jelly raw material in deionized water to obtain a 25% royal jelly aqueous solution, and then freeze-dry it directly.
[0044] Effect test: 1. Anti-aging effect test Hydroxyl radicals are among the most reactive and toxic free radicals. They can react with any molecule in the living cells of an organism, causing lesions in the body's tissues and cells, leading to disease and aging. Moreover, the reaction is very rapid. Therefore, research on hydroxyl radicals is of great significance in terms of biological damage and aging.
[0045] Experimental Method: Take 9 test tubes and add 2 mL of 6 mmol / L salicylic acid solution, 1 mL of 2 mmol / L FeSO4 solution, and 1 mL of 6 mmol / L H2O2 to each test tube. Shake well and incubate at 37℃ for 15 min. Then, add 3 mg / mL of the small-molecule enzymatically hydrolyzed royal jelly solution prepared in Examples 1-4 and Comparative Examples 1-4 to 8 test tubes, respectively, as experimental groups 1-8. Add 2.0 mmol / mL vitamin C to the remaining test tube as the control group. Incubate the test tubes at 37℃ for 60 min and measure the absorbance at 536 nm using a spectrophotometer. 样品 Then, using distilled water as a blank control, the absorbance was measured as A. 空白 The clearance rate is calculated using the formula: Clearance rate (%) = (1-A) 样品 / A 空白 )×100. The experimental results are shown in Table 1 below.
[0046] Table 1
[0047] As can be seen from the test results in Table 1 above, the small molecule enzymatically hydrolyzed royal jelly provided by the present invention has a better effect in scavenging hydroxyl free radicals, and therefore has a better anti-aging effect. The reason is that the small molecule enzymatically hydrolyzed royal jelly provided by the present invention has a better absorption effect and bioavailability, which leads to its improved anti-aging effect. On the other hand, changing the molecular weight of royal jelly or not performing enzymatic hydrolysis significantly reduces its effect in scavenging hydroxyl free radicals, thereby reducing its anti-aging effect.
[0048] 2. Stability testing The samples were stored at 25℃ and 60% relative humidity for 5 months and at 40℃ and 75% relative humidity for 3 months, respectively. The content of active ingredients at the initial and final stages was determined. The retention rate was calculated as (final stage content / initial content) × 100%. The test results are shown in Table 2 below.
[0049] The active ingredient is 10-hydroxy-2-decenoic acid; the content of 10-hydroxy-2-decenoic acid is detected according to the method described in the national standard GB9697-2008.
[0050] Table 2
[0051] According to the test results in Table 2 above, the small-molecule enzymatically hydrolyzed royal jelly provided by this invention can achieve an active ingredient retention rate of over 85% after 5 months of storage at 25℃ and 60% relative humidity; and still achieves an active ingredient retention rate of over 80% after 3 months of storage at 40℃ and 75% relative humidity. This indicates that the small-molecule enzymatically hydrolyzed royal jelly of this invention has excellent room temperature and high temperature stability and is easy to preserve. However, in Comparative Examples 1-3, changing the type or ratio of enzymes or changing the enzymatic hydrolysis steps all affected the stability of the products. This may be because changes in the type, ratio, or enzymatic hydrolysis steps affect the enzymatic hydrolysis effect of royal jelly, resulting in significant differences in the degree of protein decomposition, thus affecting the stability of the products. In Comparative Example 4, no enzymatic hydrolysis was performed, and the stability of the freeze-dried royal jelly powder was significantly reduced.
[0052] 3. Bioavailability testing: Nitrogen is a unique marker distinguishing proteins from other macronutrients, and its metabolic fate in an animal's body directly reflects the efficiency of protein digestion, absorption, and utilization. This test follows the internationally recognized classical principle of "intake-excretion" nitrogen balance, which states that under controlled conditions, if a protein is completely digested and absorbed, its nitrogen content should be retained and utilized by the body. Therefore, by accurately measuring the total nitrogen content before and after digestion, the true digestibility and absorption rate of the protein can be calculated.
[0053] Methods for detecting gastrointestinal absorption rate: (1) Preparation of artificial gastrointestinal fluid: According to the Chinese Pharmacopoeia, artificial gastrointestinal fluid and artificial blank gastrointestinal fluid were prepared using the following specific methods: Preparation of artificial gastric juice: Take 16.4 mL of dilute hydrochloric acid, then add 800 mL of water and 10 g of pepsin. After dissolving completely, adjust the pH to 1.3 with 3.65 g / L hydrochloric acid solution, then dilute with water and bring the volume to 1000 mL to obtain artificial gastric juice.
[0054] Preparation of artificial intestinal fluid: Dissolve 6.8g of potassium dihydrogen phosphate in 500mL of water, and adjust the pH to 6.8 with 4g / L sodium hydroxide solution; weigh 10g of trypsin and dissolve it in an appropriate amount of water; mix the two solutions and dilute with water to a final volume of 1000mL to obtain artificial intestinal fluid. (2) Protein absorption efficiency test: Take 1 mg of the microcapsules prepared in Examples 1-4 and Comparative Examples 1-5 respectively and put them into 15 mL centrifuge tubes. Add 3 mL of artificial gastric fluid and incubate at 37 °C on a shaker for 2 h. Then add 6 mL of artificial intestinal fluid to the centrifuge tubes and incubate at 37 °C on a shaker for 2 h. After digestion, centrifuge (10000 g, 30 min, 4 °C), take the supernatant, and use liquid chromatography to detect the nitrogen content of the sample before and after digestion.
[0055] Protein absorption rate (%) = nitrogen content of supernatant / nitrogen content of sample before digestion × 100%.
[0056] The test results are shown in Table 3 below.
[0057] Table 3
[0058] According to the test results in Table 3 above, the small-molecule enzymatically hydrolyzed royal jelly provided in Examples 1-4 of this invention can be absorbed more effectively, with a protein absorption rate of over 95%. However, in Comparative Examples 1-3, changing the type or ratio of enzymes or altering the enzymatic hydrolysis steps all affected the absorption effect of the enzymatically hydrolyzed royal jelly, reducing its bioavailability. This may be because changes in the type, ratio, or enzymatic hydrolysis steps affect the enzymatic hydrolysis effect of royal jelly, resulting in significant differences in the degree of protein decomposition, i.e., large differences in molecular weight, thus affecting the product's bioavailability. In Comparative Example 4, no enzymatic hydrolysis was performed, and the absorption effect of the freeze-dried royal jelly powder was significantly reduced.
[0059] 4. Molecular weight detection Protein molecular weight distribution was determined using Appendix A of GB / T22492-2008, GPC / UV, and Novozymes' proprietary detection method, which was modified based on the national standard.
[0060] The test results are shown in Table 4 below.
[0061] Table 4
[0062] Note: "-" indicates no.
[0063] According to the test results in Table 4 above, the molecular weight of the enzymatically hydrolyzed royal jelly prepared by the method provided by this invention is all below 1000 Da, and the proportion of molecular weight less than or equal to 500 Da is all above 80%. In Comparative Examples 1-3, changing the type or ratio of enzymes or changing the enzymatic hydrolysis steps will affect the molecular weight distribution of the enzymatically hydrolyzed royal jelly, increasing the proportion of molecular weight greater than 1000 Da, thus affecting the absorption effect of the enzymatically hydrolyzed royal jelly and reducing its bioavailability, which corresponds to the test results in Table 3. In Comparative Example 4, no enzymatic hydrolysis was performed, and the content of royal jelly freeze-dried powder with a molecular weight greater than 1000 Da increased significantly, thus significantly reducing the absorption effect.
[0064] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. It should be noted that those skilled in the art can make various improvements and modifications without departing from the technical principles of the present invention, and these modifications should all be considered within the scope of protection of the present invention.
Claims
1. A method for preparing small-molecule enzymatically hydrolyzed royal jelly to improve absorption, characterized in that: Includes the following steps: (1) Preparation of royal jelly aqueous solution: Dissolve royal jelly raw material in deionized water to obtain royal jelly aqueous solution; (2) Take royal jelly aqueous solution and adjust the pH value to 7-8, then add sea cucumber hydrolase and aminopeptidase, carry out enzymatic hydrolysis, sterilize, and obtain enzymatic hydrolysate 1; (3) Take the enzymatic hydrolysate 1, adjust the pH to 1-2, add pepsin, carry out enzymatic hydrolysis, sterilize and freeze dry to obtain the small molecule enzymatic hydrolysed royal jelly.
2. The preparation method according to claim 1, characterized in that: The mass ratio of sea cucumber hydrolase and aminopeptidase in step (2) is 1-3:1-2.
3. The preparation method according to claim 2, characterized in that: The mass ratio of sea cucumber hydrolase and aminopeptidase mentioned in step (2) is 3:
2.
4. The preparation method according to claim 1, characterized in that: The total amount of sea cucumber hydrolase and aminopeptidase added in step (2) is 2-5% of the weight of royal jelly.
5. The preparation method according to claim 1, characterized in that: The temperature for enzymatic hydrolysis in step (2) is 50-55℃; the time for enzymatic hydrolysis is 1-3 hours.
6. The preparation method according to claim 1, characterized in that: The amount of pepsin added in step (3) is 1-3% of the weight of royal jelly.
7. The preparation method according to claim 1, characterized in that: The temperature for enzymatic hydrolysis in step (3) is 35-40℃; the time for enzymatic hydrolysis is 2-4 hours.
8. A small-molecule enzymatically hydrolyzed royal jelly prepared by the preparation method according to any one of claims 1-7, characterized in that: The peptides in the small-molecule enzymatically hydrolyzed royal jelly have a molecular weight of less than or equal to 1000 Da, and the proportion of peptides with a molecular weight of less than or equal to 500 Da is greater than or equal to 80%.
9. The application of the small molecule enzymatically hydrolyzed royal jelly prepared by the preparation method according to any one of claims 1-7 in the preparation of products with anti-aging effects.
10. A product with anti-aging effects, characterized in that, This includes small-molecule enzymatically hydrolyzed royal jelly prepared by the preparation method according to any one of claims 1-7.