Preparation method of antioxidant royal jelly peptide

Abstract

The invention discloses a preparation method of antioxidant royal jelly peptide, which comprises the following steps: mixing royal jelly with water, regulating the pH value, adding pepsin for enzymolysis, and carrying out gel chromatography and anion exchange chromatography to obtain the antioxidant royal jelly peptide.

Description

Technical Field

[0001] The invention relates to the technical field of royal jelly peptides, and in particular to a preparation method of antioxidant royal jelly peptides. Background Art

[0002] my country is the largest exporter of royal jelly. As a natural health food with a long history, royal jelly has been proven to have a wealth of physiological functions, such as lowering blood pressure, lowering blood sugar, and improving immunity. It is suitable for almost all people, non-toxic and has no side effects. Therefore, it has always been popular in domestic and foreign markets. Among them, the most abundant proteins and peptides are the main active ingredients of dried bee jelly, and are also the research hotspots of royal jelly. Some of the proteins and peptides have been effectively separated, and their functions are also supported by a large amount of experimental data. However, some proteins and their enzymatic hydrolysates may still have some physiological activities that have not been discovered; and the physiological effects and toxic side effects of royal jelly have not been fully studied, which has also led to the limitation of the scope of use of royal jelly. Therefore, knowing more about its biological activity and physiological effects will help promote its application.

[0003] Scholars have found that the small molecule peptides produced after enzymatic hydrolysis of the natural protein royal jelly may have higher biological activity than proteins or have functions that proteins do not have. After enzymatic hydrolysis, separation and purification, the enzymatic hydrolysis products have higher antioxidant activity than the original protein extract. The higher-level 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 polypeptides, the utilization value of royal jelly can be improved and allergic reactions can be eliminated.

[0004] The separation and purification process further improves the biological activity of the sample and provides a preliminary research basis for its mechanism of action and structural analysis. The importance of gel filtration chromatography as a preliminary separation step is that it can quickly and effectively separate macromolecules and small molecules, and can further improve the purification effect by combining with other separation techniques. How to combine and improve the biological activity of the separated and purified royal jelly peptide is the research focus of the present invention. Summary of the invention

[0005] In order to solve the above technical problems, the specific preparation process of the present invention is as follows:

[0006] A method for preparing an antioxidant royal jelly peptide, the preparation method comprising fully mixing 100 parts of royal jelly with distilled water at a ratio of 1:10 to prepare a royal jelly solution, adding an acidic regulator to adjust pH, enzymatically hydrolyzing, centrifugally filtering, and separating and purifying;

[0007] Wherein, the acidity regulator is magnesium DL-aspartate.

[0008] The enzymolysis comprises the following steps: adding an acidic regulator to the royal jelly solution to adjust the pH to 2.3, adding 0.78-0.88 parts of pepsin, and after enzymolysis for 3 hours, adding sodium carbonate to adjust the pH to 7.5, adding 0.3-0.4 parts of papain, and continuing the enzymolysis at 37°C for 2 hours, and then precipitating with saturated ammonium sulfate, collecting the precipitate, and desalting the precipitate through running water dialysis for reuse, boiling the filtered enzymolysis solution in a boiling water bath for 15 minutes, and centrifuging at 10,000 r / min for 10 minutes, filtering the supernatant once with a 0.45 um filter membrane, and setting aside.

[0009] The separation and purification process includes: subjecting the filtered enzymatic hydrolysate to sephadex-LH-20 gel chromatography and anion exchange chromatography from top to bottom, with ultrapure water as the mobile phase and an elution rate of 2.5 ml / min, collecting the eluate, and freeze-drying to obtain antioxidant royal jelly peptides.

[0010] Among them, the anion exchange chromatography was obtained by mixing 5 5 mL ion exchange columns of the ToyoScreen series, namely, GigaCapQ-650M, Q-600CAR, DEAE-650M, SuperQ-650M, and QAE-550C columns from top to bottom.

[0011] The beneficial effects of the present invention are:

[0012] 1. The invention discloses a method for preparing an antioxidant royal jelly peptide, which comprises adjusting the pH value by using an acidic regulator, optimizing the activity of enzymes in the process of protease hydrolysis and the activity of hydrolysis products, minimizing the fishy smell of royal jelly hydrolysis, increasing the yield, activity, and antioxidant properties of small molecule peptides in royal jelly polypeptides, and separating the hydrolyzate by gel-combined anion exchange chromatography to improve the purity and biological activity.

[0013] 2. The role of magnesium DL-aspartate as an acidity regulator is one of its most important functions. During the enzymatic hydrolysis process, the activity of pepsin usually depends on the pH of the solution. By adjusting the pH value, magnesium DL-aspartate helps to create an environment more suitable for the activity of pepsin, optimize the enzymatic hydrolysis reaction, and obtain peptides with strong antioxidant activity. In the enzymatic hydrolysis of royal jelly, the addition of magnesium DL-aspartate helps to improve the enzymatic hydrolysis effect of pepsin, thereby increasing the yield of royal jelly polypeptides. Magnesium DL-aspartate plays a positive role in the biological activity of the enzymatic hydrolysis products. In the enzymatic hydrolysis products, magnesium DL-aspartate helps to improve the activity of polypeptides by optimizing the reaction conditions and obtain more small molecule peptides. The enzymatic hydrolysis products not only have high DPPH free radical scavenging activity, but also have significantly improved inhibitory activity against lipase and β-glucosidase. These biological activities help to improve the functionality of royal jelly and increase its health benefits.

[0014] 3. During the enzymatic hydrolysis process, pepsin, papain, etc. may cause the product to have an unpleasant smell or odor, which will have a negative impact on the product's acceptance. DL-Aspartate magnesium can help reduce the generation of these unpleasant flavors by adjusting the pH value or neutralizing the odorous substances. It can effectively improve the flavor, reduce the odor, improve the sensory quality of the product, and increase its market acceptance.

[0015] 4. The polypeptide components with high α-glucosidase inhibitory activity are separated by combining gel filtration chromatography and anion exchange chromatography. After separation and purification, the inhibitory activity of the target product is significantly improved compared with that before purification. After impurities or irrelevant components are removed, the purity and biological activity are enhanced. And the separated and purified products generally have better digestion stability. Its bioavailability in the body may be high and it can maintain activity in the digestive tract. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to the embodiments.

[0017] All raw materials used in the embodiments of the present invention are from Jiangxi Wang's Bee Farm. The molecular weight of the pepsin used below is 35000a, the product specification is 3000-10000NF, the papain specification is 600000-240000 μ / g, and the implementation standard is GB1886.174. They are purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., and the raw materials or chemical reagents, unless otherwise specified, are obtained through conventional commercial channels.

[0018] Example 1

[0019] 100 parts of royal jelly and distilled water were fully mixed in a ratio of 1:10 to prepare a royal jelly solution, DL-aspartate magnesium was added to the royal jelly solution to adjust the pH to 2.3, 0.82 parts of pepsin were added, and after enzymatic hydrolysis for 3 hours, sodium carbonate was added to adjust the pH to 7.5, 0.35 parts of papain were added, and enzymatic hydrolysis was continued at 37°C for 2 hours, and then saturated ammonium sulfate was used for precipitation, and the precipitate was collected. The precipitate can be reused by desalting through running water dialysis, and the filtered enzymatic hydrolyzate was boiled in a boiling water bath for 15 minutes, centrifuged at 10000r / min for 10 minutes, and the supernatant was filtered once with a 0.45um filter membrane for standby use.

[0020] The filtered enzymatic hydrolysate is separated from top to bottom by sephadex-LH-20 gel chromatography and anion exchange chromatography. The anion exchange chromatography is obtained by mixing 5 5 mL ion exchange columns of the ToyoScreen series, namely GigaCapQ-650M, Q-600CAR, DEAE-650M, SuperQ-650M, and QAE-550C columns from top to bottom; the mobile phase is ultrapure water, the elution rate is 2.5 ml / min, the eluate is collected, and lyophilized to obtain the antioxidant royal jelly peptide.

[0021] Example 2

[0022] 100 parts of royal jelly and distilled water were fully mixed in a ratio of 1:10 to prepare a royal jelly solution, DL-aspartate magnesium was added to the royal jelly solution to adjust the pH to 2.3, 0.78 parts of pepsin were added, and after enzymatic hydrolysis for 3 hours, sodium carbonate was added to adjust the pH to 7.5, 0.3 parts of papain were added, and enzymatic hydrolysis was continued at 37°C for 2 hours, and then precipitated with saturated ammonium sulfate, and the precipitate was collected. The precipitate can be reused by desalting through running water dialysis, and the filtered enzymatic hydrolyzate was boiled in a boiling water bath for 15 minutes, centrifuged at 10000r / min for 10 minutes, and the supernatant was filtered once with a 0.45um filter membrane for standby use.

[0023] The filtered enzymatic hydrolysate is separated from top to bottom by sephadex-LH-20 gel chromatography and anion exchange chromatography. The anion exchange chromatography is obtained by mixing 5 5 mL ion exchange columns of the ToyoScreen series, namely GigaCapQ-650M, Q-600CAR, DEAE-650M, SuperQ-650M, and QAE-550C columns from top to bottom; the mobile phase is ultrapure water, the elution rate is 2.5 ml / min, the eluate is collected, and lyophilized to obtain the antioxidant royal jelly peptide.

[0024] Example 3

[0025] 100 parts of royal jelly and distilled water were fully mixed in a ratio of 1:10 to prepare a royal jelly solution. DL-aspartate magnesium was added to the royal jelly solution to adjust the pH to 2.3. 0.88 parts of pepsin were added. After enzymolysis for 3 hours, sodium carbonate was added to adjust the pH to 7.5. 0.4 parts of papain were added. The enzymolysis was continued at 37°C for 2 hours. Saturated ammonium sulfate was used for precipitation, and the precipitate was collected. The precipitate can be reused by desalting through running water dialysis. The filtered enzymolysis solution was boiled in a boiling water bath for 15 minutes, centrifuged at 10000 r / min for 10 minutes, and the supernatant was filtered once with a 0.45um filter membrane for standby use.

[0026] The filtered enzymatic hydrolysate is separated from top to bottom by sephadex-LH-20 gel chromatography and anion exchange chromatography. The anion exchange chromatography is obtained by mixing 5 5 mL ion exchange columns of the ToyoScreen series, namely GigaCapQ-650M, Q-600CAR, DEAE-650M, SuperQ-650M, and QAE-550C columns from top to bottom; the mobile phase is ultrapure water, the elution rate is 2.5 ml / min, the eluate is collected, and lyophilized to obtain the antioxidant royal jelly peptide.

[0027] Comparative Example 1

[0028] In this comparative example, magnesium DL-aspartate is tartaric acid, and the rest is the same as in Example 1.

[0029] Comparative Example 2

[0030] The proteases used in this comparative example are all papain, specifically: 100 parts of royal jelly and distilled water are fully mixed in a ratio of 1:10 to prepare a royal jelly solution, sodium carbonate is added to the royal jelly solution to adjust the pH to 7.5, 0.35 parts of papain are added, and enzymolysis is carried out at 37°C for 5 hours, and then precipitated with saturated ammonium sulfate, the precipitate is collected, and the precipitate can be reused by desalting through running water dialysis, the filtered enzymolysis solution is boiled in a boiling water bath for 15 minutes, and centrifuged at 10000r / min for 10 minutes, and the supernatant is filtered once with a 0.45um filter membrane for standby use; the rest is the same as Example 1.

[0031] Comparative Example 3

[0032] The difference between this comparative example and comparative example 1 is that the pH value adjusted by DL magnesium aspartate is different, specifically: 100 parts of royal jelly and distilled water are fully mixed in a ratio of 1:10 to prepare a royal jelly solution, DL magnesium aspartate is added to the royal jelly solution to adjust the pH to 2.8, 0.82 parts of pepsin are added, and after enzymolysis for 3 hours, sodium carbonate is added to adjust the pH to 7.5, 0.35 parts of papain are added, and enzymolysis is continued at 37°C for 2 hours, and then saturated ammonium sulfate is used for precipitation, and the precipitate is collected. The precipitate can be reused by desalting through flowing water dialysis, and the filtered enzymolysis solution is boiled in a boiling water bath for 15 minutes, centrifuged at 10000r / min for 10 minutes, and the supernatant is filtered once with a 0.45um filter membrane for standby use, and the rest is the same as in Example 1.

[0033] Comparative Example 4

[0034] The difference between this comparative example and comparative example 1 is that the pH value adjusted by DL magnesium aspartate is different, specifically: 100 parts of royal jelly and distilled water are fully mixed in a ratio of 1:10 to prepare a royal jelly solution, DL magnesium aspartate is added to the royal jelly solution to adjust the pH to 2.0, 0.82 parts of pepsin are added, and after enzymolysis for 3 hours, sodium carbonate is added to adjust the pH to 7.5, 0.35 parts of papain are added, and enzymolysis is continued at 37°C for 2 hours, and then saturated ammonium sulfate is used for precipitation, and the precipitate is collected. The precipitate can be reused by desalting through running water dialysis, and the filtered enzymolysis solution is boiled in a boiling water bath for 15 minutes, centrifuged at 10000r / min for 10 minutes, and the supernatant is filtered once with a 0.45um filter membrane for standby use, and the rest is the same as in Example 1.

[0035] Comparative Example 5

[0036] The separation and purification process in this comparative example is sephadex-LH-20 gel chromatography separation, the mobile phase is ultrapure water, the elution rate is 2.5 ml / min, the eluate is collected and freeze-dried to obtain the antioxidant royal jelly peptide; the rest is the same as Example 1.

[0037] Comparative Example 6

[0038] The separation and purification process in this comparative example is anion exchange chromatography, which is obtained by mixing 5 5 mL ion exchange columns of the ToyoScreen series, namely GigaCapQ-650M, Q-600CAR, DEAE-650M, SuperQ-650M, and QAE-550C columns from top to bottom, the mobile phase is ultrapure water, the elution rate is 2.5 ml / min, the eluate is collected and freeze-dried to obtain the antioxidant royal jelly peptide; the rest is the same as Example 1.

[0039] Experiment 1: Yield and relative molecular weight distribution of peptide freeze-dried powder

[0040] The yield and relative molecular weight distribution of the obtained royal jelly peptide freeze-dried powder were determined, and the results are shown in Table 1.

[0041] Table 1

[0042]

[0043] Test 2: Antioxidant Determination

[0044] (1) DPPH free radical scavenging rate: prepare 79 mg / L DPPH-ethanol solution, dissolve the royal jelly peptide freeze-dried powder sample in anhydrous ethanol to obtain a sample solution with a mass concentration of 1000 ug / mL, take 0.5 mL of the sample solution, add 5.0 mL of DPPH-ethanol solution and mix well, react at 37°C for 1 h, use anhydrous ethanol as blank, and compare the color at a wavelength of 517 nm to calculate the DPPH free radical scavenging rate, DPPH free radical scavenging rate = (blank absorbance - sample absorbance) / blank absorbance x 100;

[0045] (2) ABTS free radical scavenging rate: 25 mL of 7.4 mol / L ABTS solution and 25 mL of 2.6 mmol / L potassium persulfate solution were measured and mixed in equal volumes, and reacted at room temperature in the dark for 12 to 16 hours, then diluted 40 to 50 times with anhydrous ethanol, and placed at room temperature in the dark for 30 minutes to prepare ABTS working solution: the royal jelly peptide freeze-dried powder sample was dissolved in anhydrous ethanol to prepare a sample solution with a concentration of 20 mg / ml, 20 L of sample solution 280 W ABTS working solution was taken to a 96-well ELISA plate, and the absorbance value A1 was measured at 734 nm. The absorbance value of the sample solution replaced by deionized water was A2, and vitamin C was used as a positive control. The ABTS free radical scavenging rate was calculated as follows: Scavenging rate (%) = (2-A1) / A2x100:

[0046] (3) Superoxide anion free radical scavenging rate: Take 4.5 mL of 50 mmol / L tris(hydroxymethyl)aminomethane hydrochloric acid buffer in a test tube, take 2.0 mL of distilled water in a test tube, then take 1.0 mL of royal jelly peptide freeze-dried powder sample solution (microcapsules dissolved in ethanol, concentration 20 mg / mL) in the test tube, mix thoroughly, place in a 25°C water bath, place for 20 min, then take 0.5 ml of 3.0 mol / L pyrogallol solution in the test tube, shake quickly, adjust to zero with distilled water, and use an ultraviolet spectrophotometer to measure the absorbance A1 at a wavelength of 325 nm. The absorbance value of the microcapsule sample solution replaced by deionized water is A2. Vitamin C is used as a positive control. The superoxide anion free radical scavenging rate is calculated as follows: scavenging rate (%) = (A2-A1) / A2x100.

[0047] The results are shown in Table 2.

[0048] Table 2

[0049]

[0050] Test 3: α-Glucosidase enzyme inhibition activity assay

[0051] Preparation of crude mouse intestinal α-glucosidase enzyme solution: 0.1g of dry powder of acetone extract of house mouse intestine, add 10ml of pH6.9, 0.1mol / L phosphate buffer, mix with a vortex mixer for 3min, let stand in a 4℃ refrigerator for 1h, and filter in the refrigerator. Dilute the filtrate by half to obtain the required enzyme solution, and place it in a -20℃ refrigerator for later use.

[0052] The method for detecting the inhibitory effect of α-glucosidase is to dissolve 0.1g of royal jelly peptide freeze-dried powder obtained in Examples 1-3 and Comparative Examples 1-6 in 10ml of water to obtain a sample solution, take 50μL of sample solution and 100μL of enzyme solution and incubate at 37°C for 20 minutes with a 96-well plate, and then add 50μL2.5mMpNPG pre-incubated. The reaction mixture was incubated at 37°C for 5 minutes, and the absorbance was measured at 405nm with an ELISA reader, measured once every 5min, measured 13 times in total, and timed for 1h. Buffer was used instead of the sample extract as a blank control, and the α-glucosidase inhibitory activity was expressed as inhibition%. Because pNPG can be hydrolyzed by a-glucosidase to produce p-nitrophenol (pNP), and the absorbance value corresponding to its concentration at 405nm has good linearity, the increase in the generated pNP can be represented by the increase in absorbance value. △A1 represents the increase in absorbance value after adding the inhibitor, and △A2 represents the increase in absorbance value of the blank control. The inhibition rate calculation formula is: Inhibition rate % = (△A2-△A1) / △A2x100%

[0053] The results are shown in Table 3.

[0054] Table 3

[0055] Group α-Glucosidase inhibition rate (%) Example 1 42.58 Example 2 41.79 Example 3 42.15 Comparative Example 1 28.54 Comparative Example 2 32.18 Comparative Example 3 36.54 Comparative Example 4 37.07 Comparative Example 5 37.54 Comparative Example 6 38.06

Claims

1. A method for preparing an antioxidant royal jelly peptide, characterized in that: The preparation method comprises the following steps: fully mixing 100 parts of royal jelly with distilled water at a ratio of 1:10 to prepare a royal jelly solution, adding an acidic regulator to adjust pH, performing enzymatic hydrolysis, centrifugal filtration, and separation and purification; The acidity regulator is magnesium DL-aspartate.

2. The method for preparing an antioxidant royal jelly peptide according to claim 1, characterized in that: The enzymolysis comprises the following steps: adding an acidic regulator to the royal jelly solution to adjust the pH to 2.3, adding 0.78-0.88 parts of pepsin, adjusting the temperature in a water bath to 35° C., adding sodium carbonate to adjust the pH to 7.5 after enzymolysis for 3 hours, adding 0.3-0.4 parts of papain, continuing the enzymolysis at 37° C. for 2 hours, and then precipitating with saturated ammonium sulfate, collecting the precipitate, and desalting the precipitate through running water dialysis for reuse, boiling the filtered enzymolysis solution in a boiling water bath for 15 minutes, centrifuging at 10000 r / min for 10 minutes, filtering the supernatant once with a 0.45 um filter membrane, and setting aside.

3. The method for preparing an antioxidant royal jelly peptide according to claim 1, characterized in that: The separation and purification comprises: subjecting the filtered enzymatic hydrolysate to sephadex-LH-20 gel chromatography and anion exchange chromatography from top to bottom, with ultrapure water as the mobile phase and an elution rate of 2.5 ml / min, collecting the eluate, and freeze-drying to obtain the antioxidant royal jelly peptide.

4. The method for preparing an antioxidant royal jelly peptide according to claim 3, characterized in that: The anion exchange chromatography is obtained by mixing five 5 mL ion exchange columns of the ToyoScreen series, namely, GigaCapQ-650M, Q-600CAR, DEAE-650M, SuperQ-650M, and QAE-550C columns from top to bottom.

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