CN109715176B - Method for preparing refined bee venom with removed allergic components from ovum gallus Domesticus flavus, and refined bee venom with removed allergic components prepared by the method - Google Patents
Method for preparing refined bee venom with removed allergic components from ovum gallus Domesticus flavus, and refined bee venom with removed allergic components prepared by the method Download PDFInfo
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- CN109715176B CN109715176B CN201780038541.5A CN201780038541A CN109715176B CN 109715176 B CN109715176 B CN 109715176B CN 201780038541 A CN201780038541 A CN 201780038541A CN 109715176 B CN109715176 B CN 109715176B
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/304—Foods, ingredients or supplements having a functional effect on health having a modulation effect on allergy and risk of allergy
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/204—Animal extracts
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Insects & Arthropods (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
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) preparing a buffer solution containing egg yolk; (b) adding bee venom into the buffer solution prepared in the step (a) and then stirring; (c) cooling the stirred reactant of step (b) while adding cooled ethanol, thereby coagulating the protein of the reactant; (d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and (e) taking the supernatant liquid of the reaction product of the coagulum precipitation of the step (d), concentrating and 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) preparing a buffer solution containing egg yolk; (b) adding bee venom into the buffer solution prepared in the step (a) and then stirring; (c) cooling the stirred reactant of step (b) while adding cooled ethanol, thereby coagulating 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 precipitated by the coagula of the step (d), concentrating and 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 effect and immunity effect | 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 have 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 components is necessary.
Accordingly, a technique for removing the aforementioned allergy-inducing substances from bee venom has been 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 to purify bee venom containing 4 wt% or more of melimine and 50 wt% or more of melimine. Further, Korean patent laid-open No. 1608045 discloses a technique for removing phospholipase from bee venom using cation exchange resin and isolating only melittin. However, there is no method for preparing purified bee venom from egg yolk to remove allergenic components, 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 it is an object of the present invention to provide a method for preparing purified bee venom which can effectively and completely remove the allergenic components of bee venom, such as phospholipase A2 and hyaluronidase, while keeping the active components of bee venom, such as melittin and melittin, intact.
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) preparing a buffer solution containing egg yolk; (b) adding bee venom into the buffer solution prepared in the step (a) and then stirring; (c) cooling the stirred reactant of step (b) while adding cooled ethanol, thereby coagulating the protein of the reactant; (d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and (e) taking the supernatant liquid of the reaction product of the coagulum precipitation of the step (d), concentrating and drying.
Further, the present invention provides purified bee venom with allergic components removed, which is prepared by the method.
Advantageous effects
The refined bee venom prepared by the method of the present invention completely removes phospholipase A2 and hyaluronidase which induce strong allergic reactions, so that refined bee venom which is safe to allergic reactions fundamentally can be prepared, and the effective components contained in bee venom are well retained, thereby having an advantage of being more confident in use for various pharmacological functions.
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) preparing a buffer solution containing egg yolk;
(b) adding bee venom into the buffer solution prepared in the step (a) and then stirring;
(c) cooling the stirred reactant of step (b) while adding cooled ethanol, thereby coagulating the protein of the reactant;
(d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and
(e) taking the supernatant liquid of the reaction product of the coagulum precipitation of the step (d), concentrating and 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 yolk of the step (a) is raw yolk derived from poultry eggs such as chicken eggs, duck eggs, quail eggs, etc., and yolk obtained by cutting eggs and separating into egg white and yolk may be used.
In the method for producing purified bee venom of the present invention, the buffer solution in the step (a) may preferably be a buffer solution having a pH of 7 to 9 and containing 1 to 10% (w/v) of egg yolk, and more preferably may be a buffer solution having a pH of 8 and containing 5% (w/v) of egg yolk. When yolk is dissolved in a buffer solution, yolk protein, yolk lecithin, neutral lipid, cholesterol, and the like are emulsified with the yolk lecithin to form an emulsified suspension in a colloidal state.
In the method for preparing purified bee venom of the present invention, the step (b) may preferably be performed by adding 0.5 to 1.5% (w/v) of bee venom to a buffer solution and then stirring the mixture at 30 to 50 ℃ for 5 to 20 minutes, and more preferably by adding 1% (w/v) of bee venom to a buffer solution and then stirring the mixture at 35 ℃ for 10 minutes. When bee venom is added to the buffer solution under the above-mentioned conditions and then stirred, the substances inducing allergy contained in bee venom can be effectively bound to the egg yolk lipoprotein.
In the method for preparing purified bee venom of the present invention, the step (c) may preferably include cooling the stirred reactant to-5 to-15 ℃ and adding ethanol cooled to-10 to-20 ℃ to coagulate the protein of the reactant, and more preferably may cool the stirred reactant to-10 ℃ and adding ethanol cooled to-15 ℃ to coagulate the protein of the reactant. As described above, when the yolk lipoprotein to which the allergy-inducing substance is bound is coagulated, the coagulation product can be precipitated by centrifugation, and the allergy-inducing substance can be easily removed.
As for the method for producing purified bee venom of the present invention, more specifically, it may comprise the steps of:
(a) preparing a buffer solution containing 1-10% (w/v) of yolk and having a pH of 7-9;
(b) adding 0.5-1.5% (w/v) of bee venom into the buffer solution prepared in the step (a), and then stirring for 5-20 minutes at 30-50 ℃;
(c) cooling the stirred reactant of the step (b) to-5 to-15 ℃, and simultaneously adding ethanol cooled to-10 to-20 ℃, thereby coagulating the protein of the reactant;
(d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and
(e) taking the supernatant liquid of the reaction product of the coagulum precipitation of the step (d), concentrating and drying.
More specifically, the following steps may be included:
(a) preparing a buffer solution of pH 8 containing 5% (w/v) egg yolk;
(b) adding 1% (w/v) of bee venom to the buffer solution prepared in the step (a), followed by stirring at 35 ℃ for 10 minutes;
(c) cooling the stirred reactant of step (b) to-10 ℃, and adding ethanol cooled to-15 ℃ 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 of the reaction product of the coagulum precipitation of the step (d), concentrating and drying.
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 does not detect the substances Phospholipase A2(Phospholipase A2) and Hyaluronidase (Hyaluronidase) which induce allergy, and contains melittin of 60% or more and melittin of 2% or more as the active ingredients of bee venom. 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, it was confirmed that no abnormal change was observed in the guinea pigs and no antigenicity was observed.
The following describes embodiments of the present invention in detail. 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) 100mL of 10mM EDTA buffer solution of pH 8, in which 5% (w/v) of yolk isolated from raw eggs was dissolved, was prepared.
(b) Adding 1% (w/v) of bee venom available from the Korean bee-keeping Association to the buffer solution prepared in the step (a), and then stirring at 35 ℃ for 10 minutes.
(c) Moving the stirred reactant of the step (b) to an ice bag at-10 ℃, cooling, and slowly adding 95% (v/v) ethanol cooled to-15 ℃, thereby coagulating the protein of the reactant.
(d) Centrifuging the coagulated-10 ℃ reactant of step (c) to precipitate a coagulum.
(e) Collecting supernatant of the reaction product of the step (d) of coagulating product precipitation, vacuum concentrating to remove ethanol, and freeze drying to obtain 0.75g refined bee venom.
Example 1: antibacterial ability of refined bee venom
To confirm whether or not the purified bee venom obtained in preparation example 1 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, it was confirmed that the purified bee venom of preparation example 1 well maintained the antibacterial effect as compared with the bee venom before purification.
Example 2: changes in the composition of refined bee venom
The components of melittin, phospholipase A2, and hyaluronidase of the bee venom before purification and the bee venom purified by the method of preparation example 1 were analyzed.
The analytical equipment used a liquid chromatograph, and the columns were Sephadex TM 75, Source 15RPC ST, C18, RP-amide, and Peptide ES-C18, 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]
Changes in the composition of refined bee venom
| Content of ingredients (%) | Melittin | Bemisin peptide | Phospholipase A2 | Hyaluronidase |
| Bee venom before refining | 65 | 2.5 | 11 | 1.8 |
| Preparation example 1 | 63 | 2.7 | 0 | 0 |
As a result, it was confirmed that the purified bee venom of the present invention did not detect the substances Phospholipase A2(Phospholipase A2) and Hyaluronidase (Hyaluronidase) which induce allergy, and further contained melittin in an amount of 60% or more and melittin in an amount of 2% or more as active ingredients of bee venom.
Example 3: test of Effect of purified bee venom on suppressing allergy
In order to confirm the effect of suppressing allergy of the purified bee venom of the present invention, the following Passive Cutaneous Anaphylaxis (PCA) test was performed.
1) Test substance: preparation example 1 of purified bee venom
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) Determination of the composition of the test groups, the amount applied, isolation and application of the groups
[ Table 4]
Determination of the composition and application rates of the test groups
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 of the test sera, 2 animals used for priming were used per serum from different individuals to perform the PCA reaction. The test serum was diluted from 10-fold to 5120-fold with a physiological saline injection, and serially diluted 2-fold each time, and then administered into dorsal skin of guinea pigs in an amount of 100. mu.l each. 4 hours after intradermal administration, a priming antigen solution containing 2% Evans'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 PCA reaction was that after 30 minutes from initiation of PCA reaction, the test animals were sacrificed by ether anesthesia, and the dorsal skin of guinea pigs was peeled off and observed for the presence of bluish-white spots. 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.
10) Test results
General symptoms: no special symptoms and dead animals caused by the bee venom of the present invention were observed.
-change in body weight: no weight change caused by bee venom of the present invention was observed.
-determination of PCA: the antigenicity of the bee venom of the present invention was measured using the PCA reaction-inducing ability of guinea pigs as an index, and the results are shown in table 5 below.
[ Table 5]
PCA results (number of positive animals/total number of test animals)
| Dilution factor | G1 | G2 | G3 | G4 |
| 10 | 0/10 | 0/10 | 0/10 | 10/10 |
| 20 | 0/10 | 0/10 | 0/10 | 10/10 |
| 40 | 0/10 | 0/10 | 0/10 | 10/10 |
| 80 | 0/10 | 0/10 | 0/10 | 10/10 |
| 160 | 0/10 | 0/10 | 0/10 | 10/10 |
| 320 | 0/10 | 0/10 | 0/10 | 10/10 |
| 640 | 0/10 | 0/10 | 0/10 | 8/10 |
| 1280 | 0/10 | 0/10 | 0/10 | 6/10 |
| 2560 | 0/10 | 0/10 | 0/10 | 5/10 |
| 5120 | 0/10 | 0/10 | 0/10 | 3/10 |
As a result of the PCA reaction, no bluish-white spots were observed in neither the bee venom low dose group G2 nor the bee venom high dose group G3, including the negative control group G1. On the other hand, in the positive control group G4, the antibody titer was 5120-fold, and a significant PCA reaction occurred. From this result, it was judged that the purified bee venom prepared according to the present invention was not antigenic.
Claims (3)
1. A method for preparing refined bee venom with an allergic component removed, comprising the steps of:
(a) preparing a buffer solution containing yolk;
(b) adding bee venom into the buffer solution prepared in the step (a) and then stirring;
(c) cooling the stirred reactant of step (b) while adding cooled ethanol, thereby coagulating 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 precipitation of the step (d), concentrating and drying,
wherein the allergy component comprises phospholipase A2 and hyaluronidase.
2. The method for producing purified bee venom with removed allergenic component according to claim 1, wherein the buffer solution of step (a) is a buffer solution of pH 7 to 9 containing yolk in an amount of 1 to 10% by mass/volume.
3. The method for preparing purified bee venom with removed allergenic component according to claim 1, wherein said method comprises the steps of:
(a) preparing a buffer solution with pH of 7-9, wherein the buffer solution contains 1-10% by mass of yolk in volume;
(b) adding bee venom with the mass volume ratio of 0.5-1.5% into the buffer solution prepared in the step (a), and then stirring for 5-20 minutes at the temperature of 30-50 ℃;
(c) cooling the stirred reactant of the step (b) to-5 to-15 ℃, and simultaneously adding ethanol cooled to-10 to-20 ℃, thereby coagulating the protein of the reactant;
(d) centrifuging the coagulated reactant of step (c) to precipitate a coagulum; and
(e) taking the supernatant liquid of the reaction product of the coagulum precipitation of the step (d), concentrating and drying.
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| KR1020160079370A KR101667995B1 (en) | 2016-06-24 | 2016-06-24 | Method for producing nontoxic purified bee venom not causing allergy using yolk |
| KR10-2016-0079370 | 2016-06-24 | ||
| PCT/KR2017/006540 WO2017222305A1 (en) | 2016-06-24 | 2017-06-21 | Method for producing allergenic component-free purified bee venom using yolk, and allergenic component-free purified bee venom produced thereby |
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