CN109890835B - Method for preparing refined bee venom with removed allergic components and refined bee venom with removed allergic components prepared by the method - Google Patents
Method for preparing refined bee venom with removed allergic components and refined bee venom with removed allergic components prepared by the method Download PDFInfo
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- CN109890835B CN109890835B CN201780038902.6A CN201780038902A CN109890835B CN 109890835 B CN109890835 B CN 109890835B CN 201780038902 A CN201780038902 A CN 201780038902A CN 109890835 B CN109890835 B CN 109890835B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
Abstract
The invention relates to a method for preparing refined bee venom with allergic components removed and refined bee venom with allergic components removed, which is prepared by the method, and is characterized by comprising the following steps of: (a) adding a buffer solution into the bee venom for dilution; (b) adding protease into the diluted diluent in the step (a) to perform enzyme reaction; (c) adding cooled ethanol to the reactant of step (b) to coagulate the protein of the reactant; (d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and (e) taking supernatant liquid in the reactant of the coagulum precipitate in the step (d) and then drying.
Description
Technical Field
The invention relates to a method for preparing refined bee venom with removed allergic components and the refined bee venom with removed allergic components prepared by the method, which is characterized by comprising the following steps: (a) adding a buffer solution into the bee venom for dilution; (b) adding protease into the diluted diluent in the step (a) and then carrying out enzyme reaction; (c) adding cooled ethanol to the reactant of step (b) to coagulate the protein of the reactant; (d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and (e) taking the supernatant liquid in the reaction product of the coagulum precipitation in the step (d) and then drying.
Background
Bee venom refers to the venom of bees, and the medical treatment effect of bee venom has been approved since ancient times. According to the results of recent studies, bee venom has been found to have the cosmetic purpose of improving wrinkles, whitening and the like, in addition to the medical functions such as anticancer action, arthritis alleviation, arteriosclerosis alleviation, lumbago alleviation, skin wound treatment, immunity enhancement action, anti-inflammatory action, blood pressure lowering and increase of the regeneration of lymphocytes and erythrocytes in blood.
The main components of bee venom and its pharmacological functions were observed as shown in the following Table 1.
[ Table 1]
| Composition (I) | Pharmacological function | Composition ratio (%) |
| Peptide (melittin ) | Tranquilizing and immunity enhancing effects | About 50 percent |
| Protein (phospholipase A2) | Cell destruction and allergy | 15％ |
| Amines (histamine) | Lowering blood pressure | 1～2％ |
Bee venom contains peptides, proteins, low-molecular active amines, etc., and is composed of more than 40 kinds of substances, and the main effective components are shown in the table.
In the composition of bee venom, proteins are composed of components such as Phospholipase a2(Phospholipase a2, PLA2), Hyaluronidase (Hyaluronidase), phosphatase (phosphatase), and α -Glucosidase (α -Glucosidase) having a molecular weight of 13kDa or more, and mainly play a role in physiological activities such as disruption of blood cell membranes, blood coagulation, vasodilation and infiltration, promotion of blood circulation, and promotion of hydrolysis of proteins.
In particular, phospholipase and hyaluronidase are bee venom-constituting substances that induce strong allergic reactions, and cause very serious safety problems for users allergic to bee venom. Therefore, in order to use bee venom for therapeutic purposes, it can be said that inactivation or complete removal of the above-mentioned components is necessary.
Accordingly, several techniques for removing the above-mentioned allergy-inducing substances from bee venom are known. For example, korean patent No. 1382404 describes a method of preparing pure bee venom with impurities removed on a large scale by dissolving bee venom to prepare a bee venom solution, adding the bee venom solution to an ethylenediamine-N-Propylsilane (PSA) adsorbent to prepare a mixture, and then subjecting it to microfiltration. Further, Korean patent No. 1364506 discloses a method for removing phospholipase from bee venom using an ultrafiltration membrane having a cut-off (cut-off) molecular weight of 10kDa or more, and making purified bee venom containing 4 wt% or more of melimine and 50 wt% or more of melimine. Further, Korean granted patent No. 1608045 discloses a technique for removing phospholipase from bee venom using a cation exchange resin and isolating only melittin. However, there is no method for preparing purified bee venom without toxicity by removing allergic components using protease as in the present invention.
Disclosure of Invention
Technical problem to be solved
The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a method for producing purified bee venom without toxicity using protease, which can completely retain the active ingredients of bee venom such as melittin and can effectively and completely remove the allergenic ingredients of bee venom, i.e., phospholipase a2 and hyaluronidase.
Technical scheme
In order to solve the above technical problems, the present invention provides a method for preparing purified bee venom with an allergy component removed therefrom, comprising the steps of: (a) adding a buffer solution into the bee venom for dilution; (b) adding protease into the diluted diluent in the step (a) and then carrying out enzyme reaction; (c) adding cooled ethanol to the reactant of step (b) to coagulate the protein of the reactant; (d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and (e) taking the supernatant liquid in the reaction product of the coagulum precipitation in the step (d) and then drying.
In addition, the present invention provides the purified bee venom prepared by the method with the removal of allergic components.
Advantageous effects
The refined bee venom prepared by the method completely removes phospholipase A2 and hyaluronidase which induce strong anaphylactic reaction, thereby being capable of preparing refined bee venom which is safe to anaphylactic reaction fundamentally and well keeps effective components contained in the bee venom, and therefore, the refined bee venom has the advantage of being capable of being used more safely for various pharmacological functions.
Drawings
FIG. 1 shows the results of electrophoretogram analyses of a diluted solution of non-purified bee venom diluted with a phosphoric acid buffer solution and a purified bee venom phosphoric acid buffer solution prepared by the method of preparation example 2.
PLA 2: phospholipase A2
Detailed Description
In order to achieve the object of the present invention, the present invention provides a method for preparing purified bee venom with an allergy component removed therefrom, comprising the steps of:
(a) adding a buffer solution into the bee venom for dilution;
(b) adding protease into the diluted diluent in the step (a) and then carrying out enzyme reaction;
(c) adding cooled ethanol to the reactant of step (b) to coagulate the protein of the reactant;
(d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and
(e) taking the supernatant liquid in the reactant of the coagulum precipitation of the step (d) and then drying.
In the method for preparing refined bee venom of the present invention, the allergenic component may be, but is not limited to, Phospholipase A2(Phospholipase A2) and Hyaluronidase (Hyaluronidase).
In the method for preparing purified bee venom of the present invention, the buffer solution of step (a) may preferably be an EDTA, Tris, phosphate or citrate buffer solution at a pH of 5 to 9 and a concentration of 1 to 150mM, more preferably a phosphate buffer solution at a pH of 6 to 9 and a concentration of 50 to 150mM, and most preferably a phosphate buffer solution at a pH of 7.5 and a concentration of 100 mM. The buffer solution can remove Ca ions which act as a cofactor (cofactor) of phospholipase A2, an allergic component of bee venom. In addition, if the buffer solution contains oxygen, an oxidation reaction occurs during the reaction, resulting in a decrease in the titer of the obtained bee venom. Therefore, in order to remove oxygen in the buffer solution, oxygen can be removed by heating the buffer solution at a temperature of 90 ℃ or higher for 5 to 30 minutes, or by replacing the buffer solution with purified nitrogen gas for 30 minutes or longer.
In the method for preparing purified bee venom of the present invention, the dilution in step (a) may be preferably performed by adding a buffer solution having a pH of 6 to 9 to bee venom and diluting the solution to 8 to 12% (w/v), and more preferably may be performed by adding a buffer solution having a pH of 7.5 to bee venom and diluting the solution to 10% (w/v). The peptide component of bee venom, represented by melittin, is about 50% of the solid component of bee venom, and the rest 50% is protein component such as phospholipase A2 inducing allergy. The total solid content of the two components is 30% of bee venom. Therefore, it is considered that the content of the allergenic protein component in 1mL of bee venom reaches about 150 mg. In the present invention, bee venom is diluted in a buffer solution at a concentration of about 10%, and thus the content of allergy-inducing proteins in 1mL of the reaction mixture is about 15 mg.
In the method for producing purified bee venom of the present invention, the protease in the step (b) may be at least one protease selected from the group consisting of animal-derived proteases such as trypsin and pancreatic enzyme, plant proteases such as bromelain and papain, and proteases derived from microorganisms, and more preferably alkaline protease (Alcalase). The alkaline protease may be Serine endopeptidase (Serine endopeptidase). The alkaline protease is effective for removing the allergenic component from bee venom compared with other proteases, and the concentration of the alkaline protease used is preferably 0.8 to 1.2 units (unit)/mL, more preferably 1 unit/mL. When the bee venom is treated at the above-mentioned concentration, the allergy-inducing components in the bee venom can be effectively removed, but when the alkaline protease is used at a concentration exceeding the above-mentioned range, the effective components of the bee venom, such as melittin, are decomposed, thereby causing a problem of decreasing the content of the effective components.
In addition, in the method for preparing purified bee venom of the present invention, the step (c) may preferably add ethanol cooled to-15 to-25 ℃ to the reactant to coagulate the protein of the reactant, and more preferably may add ethanol cooled to-20 ℃ to the reactant to coagulate the protein of the reactant. For cooling the reaction product, a method of adding ethanol cooled to-20 ℃, adding dry ice, adding liquid nitrogen, or the like may be used, but since the peptide in the bee venom has solubility in ethanol, it is preferable to use ethanol cooled to-20 ℃. The ethanol used in this case is 95% pure ethanol, and the ethanol may be added so that the content of ethanol in the reaction product is 30 to 65%, and more preferably so that the content of ethanol is 50%. When the amount of ethanol added is less than the above range, the protein coagulation effect is low, and even if the amount of ethanol added exceeds the above range, the degree of coagulation of the protein does not change, and therefore, it is preferable to add the ethanol in the above range.
In the method for preparing refined bee venom of the present invention, more specifically, the following steps may be included:
(a) adding a buffer solution with the pH value of 6-9 into the bee venom, and diluting to 8-12% (w/v);
(b) adding 0.8-1.2 units/mL of alkaline protease (Alcalase) into the diluted diluent in the step (a), and then carrying out an enzyme reaction at 34-40 ℃ for 5-20 minutes;
(c) adding ethanol cooled to-15 to-25 ℃ to the reactant of step (b) to coagulate the protein of the reactant;
(d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and
(e) taking the supernatant liquid in the reactant of the coagulum precipitation of the step (d) and then drying.
The method for preparing purified bee venom of the present invention may further comprise a step of heating the reactant subjected to the enzyme reaction of step (b), and more particularly, the method may comprise the steps of:
(a) adding a buffer solution with the pH value of 6-9 into the bee venom, and diluting to 8-12% (w/v);
(b) adding 0.8-1.2 units/mL of alkaline protease (Alcalase) into the diluted diluent in the step (a), and then carrying out an enzyme reaction at 34-40 ℃ for 5-20 minutes;
(c) heating the reactant subjected to the enzyme reaction in the step (b) for 10-20 minutes at 75-85 ℃;
(d) adding ethanol cooled to-15 to-25 ℃ to the heated reactant of step (c) to coagulate the protein of the reactant;
(e) centrifuging the coagulated reactant of step (d) to precipitate a coagulum; and
(f) taking the supernatant liquid in the reactant of the coagulum precipitation of the step (e) and then drying.
The reactant subjected to the enzymatic reaction under the conditions as described above is subjected to heat treatment, thereby interrupting the enzymatic activity of the protease contained in the reactant and thermally denaturing the bee venom allergenic proteins phospholipase a2 and hyaluronidase which may be present in the reactant.
In addition, the invention also provides the refined bee venom which is prepared by the method and is used for removing the allergic components. The purified bee venom of the present invention is characterized in that it contains no substances inducing allergy, Phospholipase A2(Phospholipase A2) and Hyaluronidase (Hyaluronidase), and contains melittin of 50% or more and melittin of 1% or more as the active ingredients of bee venom. In addition, in the toxicity evaluation of mice, the purified bee venom of the present invention showed a significantly higher LD50 value, and almost no toxicity, as compared with the LD50 value of bee venom before purification. In addition, in the antibacterial test using Staphylococcus aureus (Staphylococcus aureus) strain and Propionibacterium acnes (Propionibacterium acnes) strain, the purified bee venom of the present invention showed similar antibacterial effect as compared with the bee venom before purification, and as a result of confirming the effect of suppressing allergy of the purified bee venom using guinea pigs, no abnormal change was observed in guinea pigs, and no antigenicity was confirmed.
The following describes the present invention in detail with reference to examples. However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples.
Preparation example 1: preparation method of refined bee venom for removing allergy-inducing substances
(a) To 0.1g of bee venom purchased from the Korean bee-keeping Association and having a solid content of 30% (w/w), 1mL of a phosphate buffer solution having a pH of 7.5 and 100mM was added, and the bee venom was diluted to 10% (w/v).
(b) To the diluted dilution of the step (a), 1 unit/mL of alkaline protease (Alcalase, Novozyme) was added, and then an enzyme reaction was performed at 37 ℃ for 10 minutes.
(c) Adding 1mL of 95% (v/v) ethanol cooled to-20 ℃ to the enzyme-reacted reactant of the step (b), thereby coagulating the protein of the reactant.
(d) Centrifuging the coagulated reactant of step (c) to precipitate a coagulum.
(e) Taking supernatant of the reaction product of the step (d) of the coagulum precipitation, and then performing low-temperature vacuum drying (Speed Vac) to remove ethanol in the solution, thereby obtaining 1mL of purified humus phosphate buffer solution.
Preparation example 2: preparation method of refined bee venom for removing allergy-inducing substances
(a) To 0.1g of bee venom purchased from the Korean bee-keeping Association and having a solid content of 30% (w/w), 1mL of a phosphate buffer solution having a pH of 7.5 and 100mM was added, and the bee venom was diluted to 10% (w/v).
(b) To the diluted dilution of the step (a), 1 unit/mL of alkaline protease (Alcalase, Novozyme) was added, and then an enzyme reaction was performed at 37 ℃ for 10 minutes.
(c) Heating the enzyme-reacted reactant of step (b) at 80 ℃ for 15 minutes.
(d) Adding 1mL of 95% (v/v) ethanol cooled to-20 ℃ to the heated reactant of step (c), thereby coagulating the protein of the reactant.
(e) Centrifuging the coagulated reactant of step (d) to precipitate a coagulum.
(f) Taking supernatant of the reaction product of the step (e) to precipitate the coagulum, and then performing low-temperature vacuum drying (Speed Vac) to remove ethanol in the solution, thereby obtaining 1mL of purified humus phosphate buffer solution.
Example 1: antibacterial ability of refined bee venom
To confirm whether or not the purified bee venom obtained in preparation example 2 maintained physiological activity, antibacterial ability against Staphylococcus aureus (Staphylococcus aureus) strain and Propionibacterium acnes (Propionibacterium acnes) strain was analyzed by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
[ Table 2]
Comparison of antibacterial ability of Pre-refined bee venom and post-refined bee venom
As a result, the purified bee venom of preparation example 2 was found to have increased MIC and MBC compared to the bee venom before purification, but the increase was not so great, and it was confirmed that the original antibacterial effect of the bee venom was well maintained.
Example 2: composition change of refined bee venom for changing bee venom species
The change in the components of phospholipase A2 and hyaluronidase, which were substances inducing allergy in purified bee venom, was analyzed using a diluent obtained by diluting non-purified bee venom in a phosphate buffer solution and purified bee venom prepared by preparing purified bee venom according to the method of preparation example 1 while changing the place of purchase of bee venom (preparation examples 1-2) and purified bee venom prepared by changing the place of purchase of alkaline protease (preparation examples 1-3).
Whether or not the allergic components were removed was analyzed by liquid chromatography using Sephadex TM 75, Source 15RPC ST, C18, RP-amide, and Peptide ES-C18 as columns, and the contents of melittin, phospholipase A2, and hyaluronidase in bee venom were calculated by the following formulas, and the results are shown in Table 3 below.
Amount of the taken standard (mg) × (purity of standard/100) × (peak area of desired component in bee venom)/(peak area of standard) × (100/amount of bee venom)
[ Table 3]
Composition change of refined bee venom (%)
| Content of ingredients (%) | Melittin | Bemisin peptide | Phospholipase A2 | Hyaluronidase |
| Bee venom before refining | 65 | 2.5 | 11 | 1.8 |
| Refined bee venom (preparation example 1) | 61 | 2.0 | 0 | 0 |
| Refined bee venom (preparation examples 1-2) | 60 | 2.1 | 0 | 0 |
| Refined bee venom (preparation examples 1-3) | 61 | 2.0 | 0 | 0 |
Preparation example 1: purified bee venom using pre-purified bee venom from the Dahan bee-keeping Association and alkaline protease from Novoxin Co
Preparation examples 1 to 2: purified bee venom Using Pre-purified bee venom available from IBEE Agricultural Union Corporation and alkaline protease available from Novitin (Novo Co.)
Preparation examples 1 to 3: purified bee venom prepared from pre-purified bee venom obtained from the Korean bee-keeping Association and alkaline protease obtained from Sigma
As a result of confirming whether or not the substances phospholipase A2 and hyaluronidase which induce allergy in the purified bee venom were removed, as shown in Table 3, it was confirmed that the phospholipase A2 and hyaluronidase components were not detected in the purified bee venom of preparation examples 1-2 prepared by changing the kind of bee venom, and the allergic components were not detected in the purified bee venom of preparation examples 1-3 prepared by changing the kind of alkaline protease. It was confirmed that the purified bee venom prepared by the method of preparation example 1 of the present invention contained not only Phospholipase A2(Phospholipase A2) and Hyaluronidase (Hyaluronidase) which were substances inducing allergy but also melittin 60% or more and melittin 2% or more as active ingredients of bee venom.
Example 3: changes of components of refined bee venom by changing protease
The results of the electropherogram analysis of the non-purified bee venom diluted solution and the purified bee venom phosphate buffer solution prepared by the method of preparation example 2 were shown in FIG. 1. In the electrophoretogram, phospholipase A2 in the refined bee venom of the present invention was completely removed compared to non-refined bee venom, and the concentration of melittin did not show much change compared to non-refined bee venom.
Further, the contents of melittin, phospholipase a2, and hyaluronidase were analyzed using lyophilized powders of the purified bee venom phosphate buffer solution prepared by the method of preparation example 2 and the purified bee venom phosphate buffer solution prepared by the method of preparation example 2, but varying the kind of protease in step (b), respectively.
[ Table 4]
Composition change of refined bee venom (%)
As a result, it was confirmed that the purified bee venom of preparation example 2 did not detect the allergy-inducing substances Phospholipase A2(Phospholipase A2) and Hyaluronidase (Hyaluronidase), and further contained 50% or more of melittin and 1% or more of melittin as the active ingredients of the bee venom. On the other hand, it was confirmed that in the case of purified bee venom prepared using neutral protease or complex protease without using alkaline protease, the content of the active ingredient of bee venom was significantly reduced and the substances inducing allergy of bee venom were not completely removed, as compared with preparation example 2.
Example 4: passive skin allergy (PCA) reaction in order to confirm the effect of suppressing allergy of the purified bee venom prepared by the methods of preparation examples 1 and 2 of the present invention, the following Passive skin allergy (PCA) test was performed, and the experimental method was as follows.
1) Test substance: purified bee venom of production example 1 or production example 2
2) Excipient: physiological saline injection
3) Positive control: ovalbumin (Ovalbumin: from egg)
4) Positive control substance excipient: physiological saline injection
5) Test animals: 300-380 g of Specific Pathogen Free (SPF) guinea pig (guineapig)
6) Composition of test groups, determination of the amount of application, isolation and application of groups
[ Table 5]
Determination of the composition and application rates of the test groups
| Group of | Number of animals | Amount of liquid to be applied (ml/kg) | Amount administered (mg/kg) |
| G1 (vehicle control group) | 5 | 1 | 0 |
| G2 (Low dose group) | 5 | 1 | 0.025 |
| G3 (high dose group) | 5 | 1 | 0.05 |
| G4(OVA + adjuvant group) | 5 | 1 | 2.5 |
7) Determination of the application amount: sensitization and PCA were applied at the same rate.
8) Application: sensitization is applied to the dorsum neck of guinea pigs and the PCA reaction is initiated using serum from blood collected after sensitization. For the day of PCA priming, 2 animals used for priming were used per serum from different individuals to perform the PCA reaction. The test serum was diluted from 10 to 5120 times with a physiological saline injection, serially diluted 2 times each time, and then applied to the dorsal skin of guinea pigs in 100. mu.l each. 4 hours after intradermal administration, a priming antigen solution containing 2% evans blue (Evan's blue) was administered intravenously to the hind limb to prime the PCA response.
9) And (3) observation and inspection: general symptoms and body weight were examined. The judgment of the PCA reaction was made by sacrificing the test animals by ether anesthesia 30 minutes after the PCA reaction was initiated, then peeling the back skin of the guinea pigs, and observing whether there was a bluish-white spot. When a bluish white spot appears, the major axis and the minor axis are measured, and the serum is determined to be positive when the average value calculated by (major axis + minor axis)/2 is 5mm or more, and the final serum dilution factor (maximum dilution factor) showing positive is determined as the final titer (antibody titer) of the serum.
[ Table 6]
PCA results (number of positive reaction animals/total number of test animals) of refined bee venom of preparation examples 1 and 2
As a result of confirming the effect of suppressing allergy of the purified bee venom prepared by the methods of preparation examples 1 and 2, as shown in table 6, it was confirmed that no specific symptoms and dead animals were observed due to the purified bee venom of the present invention, and as a result of PCA reaction, no bluish white spots were observed in both the low dose application group G2 and the high dose application group G3 of the bee venom including the negative control group G1, but in the positive control group G4, the antibody titer was 5120 times, and a significant PCA reaction was observed. From the above results, it was finally confirmed that the purified bee venom prepared according to the present invention was not antigenic.
Claims (4)
1. A method for preparing refined bee venom with an allergic component removed, comprising the steps of:
(a) adding a buffer solution into the bee venom for dilution;
(b) adding protease into the diluted diluent in the step (a) and then carrying out enzyme reaction;
(c) adding cooled ethanol to the reactant of step (b) to coagulate the protein of the reactant;
(d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and
(e) taking supernatant liquid in the reactant precipitated by the coagulum in the step (d) and then drying;
wherein the allergic components are phospholipase A2 and hyaluronidase,
wherein the protease of step (b) is an alkaline protease.
2. The method for preparing purified bee venom for removing allergenic components according to claim 1, further comprising a step of heating the enzymatically reacted reactant of step (b).
3. The method for preparing purified bee venom with removed allergenic component according to claim 1, wherein said method comprises the steps of:
(a) adding a buffer solution with the pH value of 6-9 into the bee venom, and diluting to 8-12% w/v;
(b) adding 0.8-1.2 units/mL of alkaline protease into the diluted diluent in the step (a), and then carrying out an enzymatic reaction at 34-40 ℃ for 5-20 minutes;
(c) adding ethanol cooled to minus 15 to minus 25 ℃ into the reactant in the step (b) so as to coagulate the protein of the reactant;
(d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and
(e) taking the supernatant liquid in the reactant of the coagulum precipitation of the step (d) and then drying.
4. The method for preparing purified bee venom with removed allergenic component according to claim 2, wherein said method comprises the steps of:
(a) adding a buffer solution with the pH value of 6-9 into the bee venom, and diluting to 8-12% w/v;
(b) adding 0.8-1.2 units/mL of alkaline protease into the diluted diluent in the step (a), and then carrying out an enzymatic reaction at 34-40 ℃ for 5-20 minutes;
(c) heating the reactant subjected to the enzyme reaction in the step (b) for 10-20 minutes at 75-85 ℃;
(d) adding ethanol cooled to minus 15 to minus 25 ℃ into the heated reactant in the step (c) so as to coagulate the protein of the reactant;
(e) centrifuging the coagulated reactant of step (d) to precipitate a coagulum; and
(f) taking the supernatant liquid in the reactant of the coagulum precipitation of the step (e) and then drying.
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| KR10-2016-0079374 | 2016-06-24 | ||
| KR10-2016-0079375 | 2016-06-24 | ||
| KR1020160079374A KR101659894B1 (en) | 2016-06-24 | 2016-06-24 | Method for producing nontoxic bee venom peptide having excellent thermal stability and removing allergy |
| KR20160079375 | 2016-06-24 | ||
| KR10-2017-0037709 | 2017-03-24 | ||
| KR1020170037709A KR101774197B1 (en) | 2016-06-24 | 2017-03-24 | Method for producing nontoxic bee venom peptide |
| PCT/KR2017/006527 WO2017222300A1 (en) | 2016-06-24 | 2017-06-21 | Method for producing allergenic component-free purified bee venom, and allergenic component-free purified bee venom produced thereby |
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| KR101659894B1 (en) * | 2016-06-24 | 2016-09-26 | 김용수 | Method for producing nontoxic bee venom peptide having excellent thermal stability and removing allergy |
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2017
- 2017-06-21 WO PCT/KR2017/006527 patent/WO2017222300A1/en active Application Filing
- 2017-06-21 CN CN201780038902.6A patent/CN109890835B/en active Active
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| JP2002112715A (en) * | 2000-10-03 | 2002-04-16 | Api Co Ltd | Low-allergenized royal jelly and method for producing the same |
| CN103006530A (en) * | 2012-12-20 | 2013-04-03 | 中国科学院南海海洋研究所 | Bee venom compound with sun-proof effect |
| KR101418067B1 (en) * | 2012-12-20 | 2014-07-14 | 주식회사 삼양제넥스 | Preparation method of hypoallergenic royal jelly by enzyme treatment |
| KR101364506B1 (en) * | 2013-06-07 | 2014-02-21 | 주식회사 청진바이오텍 | Preparation of bee venom removed allergic ingredients |
| KR20150036856A (en) * | 2013-09-30 | 2015-04-08 | 주식회사 청진바이오텍 | Method for manufacturing functional cosmetic composite using no allergic bee venom |
| KR20160057662A (en) * | 2014-11-14 | 2016-05-24 | 대한민국(농촌진흥청장) | Method for seperating non-allergenic bee venom |
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| WO2017222300A1 (en) | 2017-12-28 |
| CN109890835A (en) | 2019-06-14 |
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