Concentration and purification process and application of royal jelly extract

Abstract

The invention provides a concentration and purification process and application of a royal jelly extract, and belongs to the technical field of royal jelly processing and production. The processing technology comprises the following steps: uniformly mixing fresh royal jelly with distilled water in proportion to obtain a royal jelly diluent; and adding disodium EDTA, potassium alginate, hydroxypropyl-beta-cyclodextrin and sodium lactobionate into the royal jelly diluent to form a mixed solution, performing ultrasonic treatment on the mixed solution, performing centrifugal separation, and performing separation and purification on the collected supernate by using an anion exchange resin chromatographic column to obtain the royal jelly extract. The separation and purification method provided by the invention has the characteristics of high royal jelly major protein extraction rate and purity, convenience, rapidness, good repeatability and the like, and can be used for preserving aquatic products.

Description

Technical Field

[0001] The invention relates to the field of royal jelly processing and production, and in particular to a concentration and purification process for a royal jelly extract and application thereof. Background Art

[0002] Royal jelly, also known as royal jelly, bee milk, royal jelly, etc., is a milky white or light yellow slurry secreted by the tongue glands and maxillary glands of young worker bees in the hive. It looks like cream, tastes sour and slightly spicy, and has a unique aroma. It is mainly used to feed queen bees and larvae. Royal jelly is a complex matrix, which contains a large amount of compounds including water (50%-65%), protein (11%-18%), monosaccharides and disaccharides (10%-25%), fatty acids and lipids (4%-8%), free amino acids and mineral salts (about 1.5%), as well as a small amount of polyphenols, enzymes and vitamins. Royal jelly has been shown to have a variety of functional properties, such as antibacterial activity, anti-inflammatory activity, hypotensive activity, antioxidant effect, anti-hypercholesterolemia activity and anti-tumor effects, so royal jelly has been widely used in medicine, health food, cosmetics and other fields.

[0003] Royal jelly protein is favored by scientists because of its physiological functionality. At present, there are many technologies for extracting royal jelly protein. For example, the patent with application number 201410013192.X discloses a royal jelly extract and its extraction method. The patent first uses an ultrafiltration membrane M1 with a molecular weight cutoff of 100kDa to separate and remove macromolecular impurities in the royal jelly aqueous solution, and then uses an ultrafiltration membrane M2 with a molecular weight cutoff of 49kDa to separate and concentrate the main royal jelly protein in the M1 permeate, and then supplements pure water to allow the small molecule solution to pass through M2. The intercepted liquid obtained is a purified and concentrated royal jelly main protein solution. The paper entitled "Separation, Purification, Biological Activity and Preliminary Structural Characterization of Royal Jelly Active Protein, Liu Hesheng, Huazhong Agricultural University" discloses a study on the extraction, separation, purification and other aspects of highly active royal jelly protein. The paper takes fresh royal jelly protein as the research object, and through the separation and classification of proteins and the determination of the protein content and antioxidant properties of each fraction, albumin is determined as the target protein fraction, and the separation and purification of royal jelly protein is achieved. Although the above technology can also obtain royal jelly protein by separation and purification, the existing technology for separating and purifying royal jelly protein has defects such as the main royal jelly protein is easily inactivated during the purification process at room temperature, resulting in a low extraction rate of the main royal jelly protein. Summary of the invention

[0004] In view of this, the present invention provides a concentration and purification process for royal jelly extract, comprising the following steps:

[0005] (1) mixing fresh royal jelly and distilled water in proportion, adjusting the pH to 6.8-7.5 with alkali solution to obtain a royal jelly dilution;

[0006] (2) adding disodium EDTA to the royal jelly dilution, mixing for 10-15 minutes, and then adding potassium alginate, hydroxypropyl-β-cyclodextrin and sodium lactobionate to form a mixed solution, and placing the mixed solution in an environment of 200-500W for ultrasonic treatment for 60-180 minutes;

[0007] (3) centrifuging the ultrasonically treated mixed solution, collecting the supernatant, and then loading the supernatant onto an anion exchange resin chromatography column equilibrated with a buffer solution, eluting the anion exchange resin chromatography column with a buffer solution to obtain an eluate, and concentrating the eluate to obtain a royal jelly extract.

[0008] Preferably, the volume ratio of royal jelly to distilled water in step (1) is 1:6-10.

[0009] Preferably, the amount of disodium EDTA added to the mixed solution in step (2) is 5.5-8wt%.

[0010] Preferably, the amount of potassium alginate added to the mixed solution in step (2) is 0.1-3wt%, the amount of hydroxypropyl-β-cyclodextrin added to the mixed solution is 0.1-5wt%, and the amount of sodium lactobionate added to the mixed solution is 1-5.5wt%.

[0011] Preferably, the centrifugal speed in step (3) is 3000-5000 r / min.

[0012] Preferably, the buffer in step (3) is 28M dithioerythritol, 35mM Na2HPO4, 12mM NaH2PO4, and the volume ratio of the supernatant to the buffer is 1:2-3.

[0013] Preferably, the anion exchange resin in step (3) is 719 anion exchange resin.

[0014] Preferably, the flow rate of the buffer solution during the elution in step (3) is 1.5-5 ml / min.

[0015] The present invention also provides a royal jelly extract prepared according to the method, and application of the royal jelly extract in the field of aquatic product preservation.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The invention adopts disodium EDTA, potassium alginate, hydroxypropyl-β-cyclodextrin and sodium lactobionate to pretreat the royal jelly dilution, wherein the disodium EDTA can effectively inhibit the activity of protease in the royal jelly dilution and improve the extraction rate of the main protein of the royal jelly.

[0018] The present invention maintains the activity of the main protein in the royal jelly by adopting a specific buffer solution to avoid the inactivation of the main protein; potassium alginate, hydroxypropyl-β-cyclodextrin and sodium lactobionate are used in combination as a protective agent for the main royal jelly protein in the present invention, which can improve the stability of the main royal jelly protein at room temperature and ensure the extraction rate.

[0019] The invention realizes the purification of the main royal jelly protein by adopting the ion exchange chromatography method, so that the main royal jelly protein obtained has higher purity.

[0020] The separation and purification method provided by the invention has the characteristics of high extraction rate and purity of the main royal jelly protein, convenience, speed, good repeatability and the like. DETAILED DESCRIPTION

[0021] The present invention provides a concentration and purification process for a royal jelly extract, comprising the following steps:

[0022] (1) mixing fresh royal jelly and distilled water in proportion, adjusting the pH to 6.8-7.5 with alkali solution to obtain a royal jelly dilution;

[0023] (2) adding disodium EDTA to the royal jelly dilution, mixing for 10-15 minutes, and then adding potassium alginate, hydroxypropyl-β-cyclodextrin and sodium lactobionate to form a mixed solution, and placing the mixed solution in an environment of 200-500W for ultrasonic treatment for 60-180 minutes;

[0024] (3) centrifuging the ultrasonically treated mixed solution, collecting the supernatant, and then loading the supernatant onto an anion exchange resin chromatography column equilibrated with a buffer solution, eluting the anion exchange resin chromatography column with a buffer solution to obtain an eluate, and concentrating the eluate to obtain a royal jelly extract.

[0025] In some embodiments of the present invention, the volume ratio of the royal jelly to distilled water in step (1) is 1:6-10, preferably, the volume ratio of the royal jelly to distilled water is 1:8.

[0026] In some embodiments of the present invention, the amount of disodium EDTA added to the mixed solution in step (2) is 5.5-8 wt %. Preferably, the amount of disodium EDTA added to the mixed solution is 6 wt %.

[0027] In some embodiments of the present invention, the amount of potassium alginate added to the mixed solution in step (2) is 0.1-3wt%, preferably, the amount of potassium alginate added is 1wt%, the amount of hydroxypropyl-β-cyclodextrin added to the mixed solution is 0.1-5wt%, preferably, the amount of hydroxypropyl-β-cyclodextrin added to the mixed solution is 3wt%, and the amount of sodium lactobionate added to the mixed solution is 1-5.5wt%, preferably, the amount of sodium lactobionate added is 1.5wt%.

[0028] In some embodiments of the present invention, the centrifugal speed in step (3) is 3000-5000 r / min, preferably, the centrifugal speed is 4000 r / min.

[0029] In some embodiments of the present invention, the composition of the buffer in step (3) is 28M dithioerythritol, 35mMNa2HPO4, and 12mM NaH2PO4, and the volume ratio of the supernatant to the buffer is 1:2-3, preferably, the volume ratio is 1:2.

[0030] In some embodiments of the present invention, the anion exchange resin in step (3) is 719 anion exchange resin.

[0031] The royal jelly protein carries a negative charge at pH 6-8, so the present invention selects anion exchange resin for purification treatment. Through the positive and negative charge interaction between the royal jelly protein and the anion exchange resin, and elution is performed at a specific flow rate in combination with a specific eluent, the purified royal jelly protein is obtained.

[0032] In some embodiments of the present invention, the flow rate of the buffer solution during elution in step (3) is 1.5-5 ml / min, and preferably, the flow rate of the buffer solution during elution is 2 ml / min.

[0033] The present invention also provides a royal jelly extract prepared according to the method, and application of the royal jelly extract in the field of aquatic product preservation.

[0034] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0035] The experimental methods or test methods described in the following examples are conventional methods unless otherwise specified; the raw materials and auxiliary agents are obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.

[0036] Example 1

[0037] A concentration and purification process for royal jelly extract, the specific steps are as follows:

[0038] (1) fresh royal jelly and distilled water were mixed uniformly in a volume ratio of 1:8, the pH value was adjusted to 7.0 with a 1 mol / L sodium hydroxide solution, and the mixture was filtered through a 0.2 μm microporous filter membrane to obtain a royal jelly dilution;

[0039] (2) adding 6 wt % disodium EDTA to the royal jelly dilution, mixing for 10 min, and then adding 1 wt % potassium alginate, 3 wt % hydroxypropyl-β-cyclodextrin and 1.5 wt % sodium lactobionate to form a mixed solution, and placing the mixed solution in a 300 W room temperature environment for ultrasonic treatment for 80 min;

[0040] (3) The ultrasonically treated mixed solution is centrifuged at a speed of 4000 r / min, and the supernatant is collected. The supernatant is then loaded onto a 719 anion exchange resin chromatography column equilibrated with a buffer solution (35 mM Na2HPO4, 12 mM NaH2PO4, 28 M dithioerythritol), and the 719 anion exchange resin chromatography column is eluted with a buffer solution (35 mM Na2HPO4, 12 mM NaH2PO4, 28 M dithioerythritol). The volume ratio of the supernatant to the buffer solution is 1:2, and the flow rate of the buffer solution during the elution is 2 ml / min to obtain an eluate, and the eluate is concentrated to one third of the original volume to obtain a royal jelly extract.

[0041] Example 2

[0042] A concentration and purification process for royal jelly extract, the specific steps are as follows:

[0043] (1) mixing fresh royal jelly and distilled water in a volume ratio of 1:6, adjusting the pH to 7.3 with a 1 mol / L sodium hydroxide solution, and filtering through a 0.2 μm microporous filter membrane to obtain a royal jelly dilution;

[0044] (2) adding 5.5 wt % disodium EDTA to the royal jelly dilution, mixing for 15 min, and then adding 0.1 wt % potassium alginate, 0.5 wt % hydroxypropyl-β-cyclodextrin and 2.5 wt % sodium lactobionate to form a mixed solution, and placing the mixed solution in a 500 W room temperature environment for ultrasonic treatment for 70 min;

[0045] (3) The ultrasonically treated mixed solution was centrifuged at a speed of 3000 r / min, and the supernatant was collected. The supernatant was then loaded onto a 719 anion exchange resin chromatography column equilibrated with a buffer solution (35 mM Na2HPO4, 12 mM NaH2PO4, 28 M dithioerythritol), and the 719 anion exchange resin chromatography column was eluted with a buffer solution (35 mM Na2HPO4, 12 mM NaH2PO4, 28 M dithioerythritol). The volume ratio of the supernatant to the buffer solution was 1:3, and the flow rate of the buffer solution during the elution was 5 ml / min to obtain an eluate. The eluate was concentrated to one third of the original volume to obtain a royal jelly extract.

[0046] Example 3

[0047] A concentration and purification process for royal jelly extract, the specific steps are as follows:

[0048] (1) fresh royal jelly and distilled water were mixed uniformly in a volume ratio of 1:9, the pH value was adjusted to 7.2 with a 1 mol / L sodium hydroxide solution, and the mixture was filtered through a 0.2 μm microporous filter membrane to obtain a royal jelly dilution;

[0049] (2) adding 6.5 wt % disodium EDTA to the royal jelly dilution, mixing for 12 min, and then adding 2 wt % potassium alginate, 5 wt % hydroxypropyl-β-cyclodextrin and 3.5 wt % sodium lactobionate to form a mixed solution, and placing the mixed solution in a 500 W room temperature environment for ultrasonic treatment for 75 min;

[0050] (3) The ultrasonically treated mixed solution was centrifuged at a speed of 5000 r / min, and the supernatant was collected. The supernatant was then loaded onto a 719 anion exchange resin chromatography column equilibrated with a buffer solution (35 mM Na2HPO4, 12 mM NaH2PO4, 28 M dithioerythritol), and the 719 anion exchange resin chromatography column was eluted with a buffer solution (35 mM Na2HPO4, 12 mM NaH2PO4, 28 M dithioerythritol). The volume ratio of the supernatant to the buffer solution was 1:3, and the flow rate of the buffer solution during the elution was 4 ml / min to obtain an eluate. The eluate was concentrated to one third of the original volume to obtain a royal jelly extract.

[0051] Comparative Example 1

[0052] The difference from Example 1 is that potassium alginate is not added in step (2), and the remaining steps are consistent with Example 1.

[0053] Comparative Example 2

[0054] The difference from Example 1 is that hydroxypropyl-β-cyclodextrin is not added in step (2), and the remaining steps are consistent with Example 1.

[0055] Comparative Example 3

[0056] The difference from Example 1 is that sodium lactobionate is not added in step (2), and the remaining steps are consistent with Example 1.

[0057] Comparative Example 4

[0058] The difference from Example 1 is that no disodium EDTA is added in step (2), and the remaining steps are consistent with Example 1.

[0059] Comparative Example 5

[0060] The difference from Example 1 is that in step (1), the volume ratio of royal jelly to distilled water is 1:15, and the other steps are consistent with Example 1.

[0061] Comparative Example 6

[0062] The difference from Example 1 is that the ultrasonic power in step (2) is 200 W, and the other steps are consistent with Example 1.

[0063] The royal jelly extracts of Examples 1-3 and Comparative Examples 1-6 were freeze-dried using conventional processes to obtain freeze-dried powders. The purity of the royal jelly main protein in the freeze-dried powders was measured. The results are shown in Table 1.

[0064] Extraction rate (%) = (main protein content of royal jelly / total protein content of fresh royal jelly) × 100%; wherein, the MRJPs content is determined by the Bradford method, and the total protein content is determined by the Kjeldahl method.

[0065] Table 1 Purity and extraction rate of royal jelly protein in Examples 1-3 and Comparative Examples 1-6

[0066] Group purity(%) Extraction rate (%) Example 1 82.5 85.6 Example 2 81.3 81.7 Example 3 81.6 82.3 Comparative Example 1 81.7 50.5 Comparative Example 2 80.2 48.9 Comparative Example 3 81.0 53.1 Comparative Example 4 81.4 55.6 Comparative Example 5 79.5 84.3 Comparative Example 6 78.6 80.4

[0067] It can be seen from Table 1 that the purity and extraction rate of the main royal jelly protein in the royal jelly extracts obtained in Examples 1-3 of the present application are relatively high. At the same time, due to the differences in the solid-liquid ratio, ultrasonic conditions, the dosage of potassium alginate, hydroxypropyl-β-cyclodextrin, sodium lactobionate and disodium EDTA, and process parameters during the purification process in Examples 1-3, the purity and extraction rate of the main royal jelly protein in Example 1 are the best.

[0068] However, due to the lack of potassium alginate, hydroxypropyl-β-cyclodextrin, sodium lactobionate or disodium EDTA in comparative examples 1-4, a large amount of royal jelly protein was degraded and inactivated during the extraction process, resulting in a low extraction rate of royal jelly protein, but it had little effect on the purity of the product.

[0069] Comparative Example 5 increases the solid-liquid ratio, and although the extraction rate of the main royal jelly protein is increased, the increase in the solid-liquid ratio increases the production cost and the amount of waste liquid discharged. In addition, the increase in the solid-liquid ratio causes impurities to be extracted, resulting in a certain degree of reduction in the purity of the main royal jelly protein.

[0070] Comparative Example 6 reduced the ultrasonic power, which reduced the dissolution of impurities, but also reduced the dissolution rate of the effective ingredients. Finally, the purity and extraction rate of the royal jelly protein were reduced.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A concentration and purification process for royal jelly extract, characterized in that: The following steps are involved: (1) mixing fresh royal jelly and distilled water in proportion, adjusting the pH to 6.8-7.5 with alkali solution to obtain a royal jelly dilution; (2) adding disodium EDTA to the royal jelly dilution, mixing for 10-15 minutes, and then adding potassium alginate, hydroxypropyl-β-cyclodextrin and sodium lactobionate to form a mixed solution, and placing the mixed solution in an environment of 200-500W for ultrasonic treatment for 60-180 minutes; (3) centrifuging the ultrasonically treated mixed solution, collecting the supernatant, and then loading the supernatant onto an anion exchange resin chromatography column equilibrated with a buffer solution, eluting the anion exchange resin chromatography column with a buffer solution to obtain an eluate, and concentrating the eluate to obtain a royal jelly extract.

2. The concentration and purification process of the royal jelly extract according to claim 1, characterized in that: The volume ratio of the royal jelly to distilled water in step (1) is 1:6-10.

3. The concentration and purification process of the royal jelly extract according to claim 1, characterized in that: The amount of disodium EDTA added to the mixed solution in step (2) is 5.5-8wt%.

4. The concentration and purification process of the royal jelly extract according to claim 1, characterized in that: In step (2), the amount of potassium alginate added to the mixed solution is 0.1-3wt%, the amount of hydroxypropyl-β-cyclodextrin added to the mixed solution is 0.1-5wt%, and the amount of sodium lactobionate added to the mixed solution is 1-5.5wt%.

5. The concentration and purification process of the royal jelly extract according to claim 1, characterized in that: The centrifugal speed in step (3) is 3000-5000r / min.

6. The concentration and purification process of the royal jelly extract according to claim 1, characterized in that: The buffer in step (3) is 28M dithioerythritol, 35mM Na2HPO4, 12mM NaH2PO4, and the volume ratio of the supernatant to the buffer is 1:2-3.

7. The concentration and purification process of royal jelly extract according to claim 1, characterized in that: The anion exchange resin in step (3) is 719 anion exchange resin.

8. The concentration and purification process of royal jelly extract according to claim 1, characterized in that: The flow rate of the buffer solution during the elution in step (3) is 1.5-5 ml / min.

9. The royal jelly extract prepared according to the method according to any one of claims 1 to 8.

10. Use of the royal jelly extract prepared according to the method according to any one of claims 1 to 8, or the royal jelly extract according to claim 9 in the field of aquatic product preservation.