KR20200144800A - Bee venom sterilization process using E-beam and verification of sterilization process using model virus - Google Patents

Abstract

The present invention relates to a method of sterilizing a virus in bee venom by irradiating electron beam (E-beam) to bee venom, and verifying whether the virus is sterilized or not by a sterilization process using a model virus. By using the method of sterilizing viruses in bee venom, including the step of irradiating the E-beam to the bee venom of the present invention, microorganisms present in bee venom can be sterilized. Accordingly, unlike the existing sterilization process, the active ingredient of bee venom is not changed, and it is possible to prepare the bee venom that is safe and sterilized for the human body. In addition, by using the process of confirming sterilization using the model virus of the present invention, it is possible to effectively confirm that all viruses in bee venom are sterilized, thereby preparing safe bee venom with verified sterilization.

Description

Bee venom sterilization process using E-beam and verification of sterilization process using model virus}

The present invention relates to a method for sterilizing viruses in bee venom by irradiating an E-beam to bee venom, and verifying whether or not the virus is sterilized by a sterilization process using a model virus.

One of the animal sources produced by bees, bee venom can contain unwanted and potentially dangerous contaminants such as viruses, bacteria, yeast, fungi, mycoplasma and parasites. Since bee venom can be used for administration to the human body, it is very important to remove contaminants that may exist in bee venom.

Conventionally, methods known as sterilization processes for animal raw materials include heating, filtering, and adding a chemical inert or sensitizer to the product. The heat treatment heats the product to about 60° C. for about 70 hours and can destroy up to about 50% of the activity of the contaminating microorganism, but can cause damage to the sensitive product. The filtration process physically filters the product to remove contaminants. However, this method can remove the active ingredient of bee venom having a high molecular weight, and in some cases, a virus with a small size may remain by filtration. The chemical sterilization process binds to the DNA/RNA of the virus and is UV or ionized to produce free radicals that break the chemical bonds to the DNA/RNA backbone of the virus in such a way that the virus can no longer replicate. It is a process of adding harmful reagents that are activated by radiation. Such a process may cause toxicity, may cause side effects when administered to humans, and problems that may cause modification of the active ingredient of bee venom have been pointed out. Therefore, there has been a need for a virus sterilization process in bee venom that does not cause changes in the active ingredient of bee venom and can ensure the safety of bee venom that can be used as a medicine.

On the other hand, as a conventional inspection method to check whether a contaminant exists in bee venom, a method of individually detecting the contaminating microorganism for suspected contamination of bacteria, viruses, and fungi to check whether or not contamination by individual microorganisms is disclosed. . However, such a process has been pointed out a problem that it is not possible to confirm contamination by an unforeseen contaminant, and a technology capable of confirming the absence of a virus in the bee venom has been required to solve this problem.

Accordingly, the present inventors have studied a safe sterilization process while effectively sterilizing the virus in bee venom, without causing changes in the active ingredient, and studying a method that can effectively confirm that the virus has been sterilized by the sterilization process, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method of sterilizing a virus in bee venom comprising the step of irradiating an E-beam to bee venom, and to confirm whether or not sterilization by using an E-beam to bee venom and a model virus It is to provide a bee venom manufacturing method including the step and sterilized bee venom thereby.

In order to achieve the above object, the present invention provides a method of sterilizing a virus in bee venom comprising the step of irradiating the bee venom with an E-beam (electron beam).

In addition, the present invention (a) the step of irradiating the E-beam to bee venom; And (b) detecting the model virus in the bee venom; provides a method for producing a sterilized bee venom comprising.

In addition, the present invention provides a sterilized bee venom prepared by the method of manufacturing the sterilized bee venom.

By using the method of sterilizing the virus in bee venom including the step of irradiating the E-beam of the present invention to the bee venom, it is possible to sterilize the microorganisms present in the bee venom, through which, unlike the existing sterilization process, bee venom It does not change the active ingredient of, and is safe for the human body and can produce sterilized bee venom. In addition, if the process of confirming sterilization using the model virus of the present invention is used, it is possible to effectively confirm that all viruses in the bee venom are sterilized, thereby producing a safe bee venom whose sterilization has been verified.

1 is a diagram schematically showing the manufacturing process of bee venom.
2 is a diagram confirming that all of the hepatitis A virus has been killed by irradiating an E-beam on a sample spiked with the hepatitis A virus in bee venom.
3 is a diagram confirming that all of the porcine parvoviruses were killed by irradiating the E-beam to a sample spiked with porcine parvovirus in bee venom.

The present invention provides a method of sterilizing a virus in bee venom, comprising the step of irradiating the bee venom with an E-beam (electron beam).

Hereinafter, the present invention will be described in more detail.

In the present invention,'bee venom' is a venom from the spawning tube of bees.

The bee venom of the present invention may be raw bee venom, dried bee venom, bee venom extract or purified bee venom. Raw bee venom is not dried or purified, has a specific gravity of 1.1 to 1.3, an acidity (pH) of 5.2 to 5.5, and 70 to 80% by weight may be made of a protein or peptide. Bee venom extract can be extracted using various extraction methods known in the art, preferably water, anhydrous or hydrated lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), such lower alcohols and water It may be obtained by using a mixed solvent of and, acetone, ethyl acetate, and butylene glycol as an extraction solvent. Purified bee venom may be obtained through a conventional purification process other than the extract by the above-described extraction solvent, or may be purified by separation using a filtration membrane, separation by various chromatography, or purification methods.

The bee venom of the present invention may be administered to an individual to improve body weight gain, immune function, and pain treatment function.

In the present invention,'sterilization' may be the reduction of biological contaminants or microorganisms, preferably retroviruses, herpes viruses, paramyxoviruses, cytomegaloviruses, A Such as hepatitis virus, hepatitis B virus, hepatitis viruses, pox viruses, toga viruses, Ebstein-Barr virus, and parvoviruses. virus; Bacteria such as Escherichia, Bacillus, Campylobacter, Streptococcus, and Staphalococcus; Parasites such as Trypanosoma; Malaria parasites, including Plasmodium species; leaven; mold; Mycoplasma; And it may be a reduction of any one or more contaminants selected from the group consisting of prion, more preferably, it may be a reduction of any one or more viruses selected from the group consisting of bovine Herpes virus, hepatitis A virus, and porcine parvovirus. .

In the present invention, the'method of sterilization' may be a method using any effective radiation for sterilization of bee venom. Preferably the radiation may be small particles comprising E-beam radiation.

The'E-beam' of the present invention may be an E-beam irradiated with an irradiation amount of 20 to 100 kGy, and preferably an E-beam irradiated with an irradiation amount of 25 to 50 kGy. When the E-beam is irradiated with an irradiation dose of 20 kGy or less, the virus in bee venom may not be sterilized, and when irradiated with an irradiation dose of 100 kGy or more, there is a concern that the color of bee venom is deteriorated due to high power.

The'E-beam' of the present invention does not cause a change in the active ingredient in bee venom, preferably melittin, apamin, and mast cell granulation reducing peptide (MCD-Peptide, Mast Cell Degranulation Peptide, Op), protease inhibitors (Protease Inhibitor), secarpin (Secarpin), touch affine (Tertiapin), does not cause a change of procamine (Procamine), more preferably does not cause a change of melittin.

According to the'method for sterilizing virus in bee venom' of the present invention, microorganisms present in bee venom can be sterilized without changing the active ingredient of bee venom.

In addition, the present invention (a) the step of irradiating the E-beam to bee venom; And (b) detecting the model virus in the bee venom; provides a method for producing a sterilized bee venom comprising.

The E-beam of the step (a) of the present invention may be an E-beam irradiated with an irradiation amount of 20 to 100 kGy, and preferably an E-beam irradiated with an irradiation amount of 25 to 50 kGy. When the E-beam is irradiated with an irradiation dose of 20 kGy or less, the virus in bee venom may not be sterilized, and when irradiated with an irradiation dose of 100 kGy or more, there is a concern that the color of bee venom is deteriorated due to high power.

In the present invention, the detection of step (b) is polymerase chain reaction (PCR), ligase chain reaction (LCR), Gap-LCR (Gap-ligase chain reaction), repair chain reaction, and transcription. -Mediated amplification (TMA), self sustained sequence replication, selective amplification of target polynucleotide sequences, consensus sequence priming polymerase chain reaction (CP-PCR), arbitrary Priming polymerase chain reaction (AP-PCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, ring-mediated constant temperature amplification (LAMP), multiplex polymerase chain reaction (multiplex PCR), double Polymerase chain reaction (nested-PCR), single tube double polymerase chain reaction (single tube nested-PCR), reverse transcription-polymerase chain reaction (RT-PCR), inverse polymerase chain reaction (inverse PCR), real-time polymerase At least one method selected from the group consisting of chain reaction (RT-PCR), real-time polymerase chain reaction quantitative test (RQ-PCR), capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement and phosphorescence measurement It may be performed using, and preferably, reverse transcription-polymerase chain reaction (RT-PCR) method and gel electrophoresis may be used, but is not limited thereto.

In the present invention, the method of manufacturing the sterilized bee venom may further include, before step (a), spiking the model virus of step (b) to bee venom. If the step of spiking the model virus of step (b) is further included, if the model virus is not detected by detecting the model virus spiked in step (b), bee venom by step (a) I can confirm that my virus is sterile.

In the present invention, the model virus is a virus capable of ensuring sterilization of unknown or unidentified viruses, and may be a virus that may be contaminated with bee venom itself or a virus most similar thereto, preferably bovine Herpis virus, It may be any one or more viruses selected from the group consisting of hepatitis A virus and porcine parvovirus.

According to the'method of manufacturing sterilized bee venom' of the present invention, it is possible to prepare bee venom that has been verified to have sterilized virus in bee venom without changing the active ingredient in bee venom.

In addition, the present invention provides a sterilized bee venom prepared by the method of manufacturing the sterilized bee venom.

The sterilized bee venom of the present invention is a bee venom that has been verified that the virus in bee venom is sterilized, and the active ingredient of bee venom is maintained without change even after the sterilization process.

Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined herein have meanings commonly used in the technical field to which the present invention belongs.

Hereinafter, the present invention will be described in detail by examples.

Example 1. Selection and culture of model virus to verify virus sterilization effect

1.1 Selection of model virus to verify virus sterilization effect

The following criteria were considered to select a model virus to verify the virus sterilization effect of the virus sterilization process in bee venom.

(i) The virus to be used for verification of virus contamination in bee venom must be the virus itself or the most similar virus that may be contaminated with bee venom. In addition, by selecting viruses of various physicochemical properties, it should be possible to indicate that the virus sterilization process to be verified can generally sterilize all types of viruses.

(ii) The selected virus should, as far as possible, be highly resistant to physicochemical treatment to ensure that other unknown or unidentified viruses are sterilized.

(iii) Viruses should be able to be analyzed easily and cultured at high concentrations.

(iv) The selected virus should have a low risk of infection to the experimenter during the testing period being verified.

Considering these factors, bovine herpes virus (BHV), hepatitis A virus (HAV) and porcine parvovirus (PPV) are the most effective model viruses to verify virus in bee venom. Appropriately, it was selected as a model virus to check whether the virus was sterilized in bee venom.

1.2 Cultivation of bovine Herpis virus

MDBK NBL-1 (ATCC CCL-22) cells were placed in a cell culture medium (DMEM medium containing 10% fetal calf serum and L-glutamine) and cultured in an incubator at 37°C and 5% CO ₂ . Monolayer MDBK NBL-1 (ATCC CCL-22) cells cultured in a T-150 flask were infected with bovine Herpes virus (ATCC VR-188), and then virus culture medium (DMEM medium containing 2% fetal calf serum) And then cultured in a 35° C., 5% CO ₂ incubator to periodically observe cytopathic effect (CPE). When the cytopathic effect was clearly observed, the culture medium and cell debris were collected, the cells were crushed through freezing and thawing processes 3 times, and the cell debris was removed by centrifugation. The centrifugal supernatant was filtered through a 0.45 μm filter, subdivided, and stored at -70°C.

1.3 Culture of hepatitis A virus

FRhK-4 (ATCC CRL-1688) cells were put in a cell culture medium (DMEM medium containing 10% fetal calf serum and L-glutamine) and cultured in a 37°C, 5% CO ₂ incubator. Single layer FRhK-4 (ATCC CRL-1688) cells cultured in a T-150 flask were infected with hepatitis A virus (ATCC VR-1402), and then virus culture medium (DMEM medium containing 2% fetal calf serum) And then cultured in a 35° C., 5% CO ₂ incubator to periodically observe cytopathic effect (CPE). When the cytopathic effect was clearly observed, the culture medium and cell debris were collected, the cells were crushed through freezing and thawing processes 3 times, and the cell debris was removed by centrifugation. The centrifugal supernatant was filtered through a 0.45 μm filter, subdivided, and stored at -70°C.

1.4 Culture of porcine parvovirus

MPK (ATCC CCL-166) cells were placed in a cell culture medium (DMEM medium containing 10% fetal calf serum and L-glutamine) and cultured in a 37°C, 5% CO ₂ incubator. After infecting porcine parvovirus (ATCC VR-742) into monolayer MPK (ATCC CCL-166) cells cultured in a T-150 flask, virus culture medium (DMEM medium containing 2% fetal calf serum) was added. Cytopathic effect (CPE) was observed periodically while culturing in a 35°C, 5% CO ₂ incubator. When the cytopathic effect was clearly observed, the culture medium and cell debris were collected, the cells were crushed through freezing and thawing processes 3 times, and the cell debris was removed by centrifugation. The centrifugal supernatant was filtered through a 0.45 μm filter, subdivided, and stored at -70°C.

Example 2. Determination of E-beam dose for virus inactivation process in bee venom

2.1 Confirmation of the sterilization effect according to the E-beam irradiation dose on the sample spiking bovine Herpes virus in bee venom

In the bee venom manufacturing process, the E-beam irradiation process is a sterilization process in which 25 kGy of E-beam is irradiated on the freeze-dried bee venom powder, and it is the final process to inactivate infectious agents. For the E-beam, thermal company E-beam was prepared, and for bee venom, purified bee venom sold in Korea was purchased and used. A scale-down process of the E-beam irradiation process was designed to verify the virus inactivation effect in the E-beam irradiation process. Bee Venom An experiment was performed to confirm the optimal E-beam irradiation dose that can indicate virus inactivation in the reduction scale process. As a control, a purified bee venom sample that was not treated with any treatment and bovine Herpes virus cultured in Example 1.2 were prepared. As an experimental group, a freeze-dried sample of purified bee venom spiked with bovine Herpis virus, a model virus cultured in Example 1.2 at a concentration of 1 x 10 ⁷ cfu/ml, was prepared, and purified bee venom spiked with the virus was prepared. Freeze-dried, 1kGy, 5kGy, 10kGy, 25kGy irradiated E-beams were used to prepare samples subjected to a sterilization process. The presence of bovine Herpes virus in the sample was confirmed by performing RT-PCR using Bovine herpesvirus 1 putative fibronectin binding protein genesig® Advanced Kit (Primerdesign TM Ltd). Table 1 shows the reduction ratio in the designed scale-down process, and Table 2 shows the results of detection of bovine Herpes virus in each experimental group and control group.

lane Processing conditions Bovine Herpis virus detection One Bovine herpes virus sample detection 2 Freeze-dried sample of purified bee venom spiked with bovine Herpes virus that did not undergo E-beam sterilization detection 3 Purified bee venom freeze-dried sample spiked with bovine Herpes virus through 1kGy E-beam sterilization process detection 4 Freeze-dried sample of purified bee venom spiked with bovine Herpis virus through 5kGy E-beam sterilization process detection 5 Purified bee venom freeze-dried sample spiked with bovine Herpes virus that went through 10kGy E-beam sterilization process detection 6 Freeze-dried sample of purified bee venom spiked with bovine Herpes virus through 25kGy E-beam sterilization process Not detected 7 Untreated purified bee venom freeze-dried sample Not detected

2.2 Confirmation of the sterilization effect according to the dose of E-beam irradiation on the sample that spiked the hepatitis A virus in bee venom

In order to confirm the optimal E-beam irradiation dose that can indicate virus inactivation in the reduction scale process, an experiment was additionally performed on the hepatitis A virus. As a control, a purified bee venom sample without any treatment and a hepatitis A virus cultured in Example 1.3 were prepared. As an experimental group, a freeze-dried sample of purified bee venom spiked with hepatitis A virus, a model virus cultured in Example 1.3 at a concentration of 1 x 10 ⁷ cfu/ml, was prepared, and purified bee venom spiked with the virus Were freeze-dried, and samples subjected to a sterilization process were prepared using E-beams of irradiation doses of 1kGy, 5kGy, 10kGy, and 25kGy. The following experiment was performed to detect the hepatitis A virus in the sample.

1g untreated purified bee venom sample for RNA isolation, 0.5mg of purified bee venom freeze-dried sample spiked with hepatitis A virus without E-beam sterilization, spiked hepatitis A virus through E-beam sterilization process 0.5 mg of purified bee venom freeze-dried sample was added to 5 mL, 1 mL, and 1 mL of TRIzol Reagent (Thermo Fisher Scientific, CA, USA), homogenized, reacted at room temperature for at least 15 minutes, and then added to 800 μL of supernatant in chloroform (Sigma-aldrich, MI, USA) ) 200 μL was mixed and reacted at room temperature for 5 minutes, followed by centrifugation (12000 g, 15 minutes, 4°C). The supernatant was transferred to a new tube and reacted with 500 μL of isopropanol (Sigma-aldrich) for 10 minutes at room temperature to precipitate RNA, followed by centrifugation (12000 g, 10 minutes, 4°C), and 80% ethanol was added after removing the supernatant. Washed by using and centrifuged (12000 g, 5 minutes, 4°C), the pellet was dissolved in DEPC treated water (WelGENE, Korea) to a concentration of about 1 μg/μL and used in the experiment. After 1 μg of RNA isolated using AmfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Korea) was synthesized with cDNA, 2 μL of cDNA and 10 pmoles of primer were added in a 20 μL reaction using AccuPower PCR Premix of Bioneer (Korea). RT-PCR (TP3300, Takara, Japan) was performed to detect RNA of hepatitis A virus. The reaction conditions were denatured at 95°C for 5 minutes, followed by 40 cycles of 95°C for 20 seconds, 60°C for 30 seconds, and 72°C for 1 minute each, followed by treatment at 72°C for 5 minutes to amplify. The amplified product was electrophoresed at 100V on a 1.2% Agarose gel using 0.5 × TAE solution, and expression was confirmed using Safe-Pinky DNA Gel Staining solution (GenDEPOT). The primer sets used for RT-PCR are shown in Table 3, and the detection results are shown in FIGS. 2 and 4.

lane Processing conditions Hepatitis A virus detection One Hepatitis A virus sample detection 2 Freeze-dried sample of purified bee venom spiked with hepatitis A virus without E-beam sterilization detection 3 Freeze-dried sample of purified bee venom spiked with hepatitis A virus through 1kGy E-beam sterilization process detection 4 Freeze-dried sample of purified bee venom spiked with hepatitis A virus through 5kGy E-beam sterilization process detection 5 Purified bee venom freeze-dried sample spiked with hepatitis A virus through 10kGy E-beam sterilization process detection 6 Freeze-dried sample of purified bee venom spiked with hepatitis A virus through 25kGy E-beam sterilization process Not detected 7 Untreated purified bee venom freeze-dried sample Not detected

2.3 Confirmation of the sterilization effect according to the dose of E-beam irradiation for samples spiked with porcine parvovirus in bee venom

In order to confirm the optimal E-beam irradiation dose that can indicate virus inactivation in the reduced scale process, an experiment was additionally performed on porcine parvovirus. As a control, a purified bee venom sample without any treatment and a porcine parvovirus cultured in Example 1.4 were prepared. As an experimental group, a freeze-dried sample of purified bee venom spiked with porcine parvovirus, a model virus cultured in Example 1.4 at a concentration of 1 x 10 ⁷ cfu/ml, was prepared, and purified bee venom spiked with the virus was prepared. Freeze-dried, 1kGy, 5kGy, 10kGy, 25kGy irradiated E-beams were used to prepare samples subjected to a sterilization process. The following experiment was performed to detect porcine parvovirus in the sample.

1g untreated purified bee venom sample for RNA isolation, purified bee venom spiked with porcine parvovirus without E-beam sterilization, 0.5mg freeze-dried sample, purified bee venom spiked with porcine parvovirus through E-beam sterilization process 0.5 mg of freeze-dried sample was added to 5 mL, 1 mL, and 1 mL of TRIzol Reagent (Thermo Fisher Scientific, CA, USA), homogenized, reacted at room temperature for at least 15 minutes, and then 200 μL of chloroform (Sigma-aldrich, MI, USA) in 800 μL of the supernatant. The mixture was reacted for 5 minutes at room temperature and centrifuged (12000 g, 15 minutes, 4°C). The supernatant was transferred to a new tube and reacted with 500 μL of isopropanol (Sigma-aldrich) for 10 minutes at room temperature to precipitate RNA, followed by centrifugation (12000 g, 10 minutes, 4°C), and 80% ethanol was added after removing the supernatant. The pellet was washed and centrifuged (12000 g, 5 min, 4°C) in DEPC treated water (WelGENE, Korea) and used in the experiment at a concentration of about 1 μg/μL. After 1 μg of RNA isolated using AmfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Korea) was synthesized with cDNA, 2 μL of cDNA and 10 pmoles of primer were added in a 20 μL reaction using AccuPower PCR Premix of Bioneer (Korea). RT-PCR (TP3300, Takara, Japan) was performed so as to be. The reaction conditions were denatured at 95°C for 5 minutes, followed by 40 cycles of 95°C for 20 seconds, 60°C for 30 seconds, and 72°C for 1 minute each, followed by treatment at 72°C for 5 minutes to amplify. The amplification product was electrophoresed at 100 V on a 1.2% Agarose gel using 0.5 × TAE solution, and the detection of porcine parvovirus was confirmed using Safe-Pinky DNA Gel Staining solution (GenDEPOT). The primer sets used for RT-PCR are shown in Table 5, and the detection results are shown in Figs. 3 and 6.

Lane Processing conditions Whether porcine parvovirus is detected One Porcine parvovirus sample detection 2 Freeze-dried sample of purified bee venom spiked with porcine parvovirus without E-beam sterilization detection 3 Freeze-dried sample of purified bee venom spiked with porcine parvovirus that went through 1kGy E-beam sterilization process detection 4 Freeze-dried sample of purified bee venom spiked with swine parvovirus that went through 5kGy E-beam sterilization process detection 5 Freeze-dried sample of purified bee venom spiked with porcine parvovirus that went through 10kGy E-beam sterilization process detection 6 Freeze-dried sample of purified bee venom spiked with porcine parvovirus through 25kGy E-beam sterilization process Not detected 7 Untreated purified bee venom freeze-dried sample Not detected

As shown in Table 2, Table 4, and Table 6, it was confirmed that no spiked bovine Herpes virus, hepatitis A virus, or porcine parvovirus were detected in purified bee venom subjected to the E-beam sterilization process with an irradiation dose of 25 kGy. I did.

Therefore, it was confirmed that all viruses that can contaminate bee venom, such as bovine Herpes virus, hepatitis A virus, and porcine parvovirus, are sterilized when the bee venom undergoes an E-beam sterilization process.

Example 3. Changes in melittin content in bee venom according to virus inactivation process in bee venom

Unlike other virus inactivation processes, the sterilization process of the present invention does not cause degeneration of the main active ingredients in bee venom, and an experiment was conducted to confirm this. Melittin was selected as the main active ingredient, and autoclave sterilization and alcohol sterilization were selected as virus inactivation processes for comparison.

3.1 Measurement of changes in melittin content in bee venom by autoclaving process

As a control group, purified bee venom on the market that has not undergone a sterilization process was prepared without treatment. As an experimental group, purified bee venom was dissolved in 100% distilled water to make 1 mg/mL, and then heated samples at 121° C. for 5 minutes, 10 minutes, 15 minutes, and 20 minutes using a sterilizer were set as experimental groups. The melittin content analysis for each experimental group was measured using a UPLC I class model equipped with a diode array detector from Waters (Minneapolis, MN, USA), and the column was Halo ES- from Advanced Material Technology (Wilmington, DE, USA). C18 (2.1×100 mm, 2.7 μm,) was used. UPLC analysis conditions were flow rate 0.8 mL/min, injection volume 2 μL, detection wavelength 220 nm, column temperature 50°C, and mobile phase was (A) 20 mM TFA/MeCN, (B) 20 mM TFA/H2O solvent. 31%; 0-3 min, (A) 31-40%; 3-5 minutes, (A) 40-45%; Set to 5-10 minutes. Table 7 shows a table measuring the melittin content of each experimental group and control group.

Processing conditions Melittin content (%(w/w)) Untreated purified bee venom sample 64.1 Purified bee venom sample sterilized at 121℃ for 5 minutes 51.8 Purified bee venom sample sterilized at 121℃ for 10 minutes 51.0 Purified bee venom sample sterilized at 121℃ for 15 minutes 50.7 Purified bee venom sample sterilized at 121℃ for 20 minutes 46.0

As shown in Table 7, when the purified bee venom sample was sterilized by the autoclave process, it was confirmed that the content of melittin, the main active ingredient of bee venom, was significantly reduced by 12% or more even after 5 minutes of the autoclaving process.

3.2 Measurement of change of melittin content in bee venom by alcohol sterilization process

Purified bee venom samples were added to 100% distilled water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, and 100% ethanol to make 1 mg/mL, vortexed for 1 minute, and then filtered through a 0.2 μm membrane filter as a control. And experimental groups, and melittin content analysis was performed in the same manner as in Example 3.1. Table 8 shows the melittin content of each control and experimental group.

Processing conditions Melittin content (%(w/w)) 100% distilled water treated purified bee venom sample 60.7 20% ethanol-treated purified bee venom sample 63.7 40% ethanol-treated purified bee venom sample 61.8 60% ethanol-treated purified bee venom sample 57.4 80% ethanol-treated purified bee venom sample 3.1 100% ethanol-treated purified bee venom sample 0

As shown in Table 8, when passing through the alcohol sterilization process of 20% to 40%, the melittin content was found to increase in a small amount, but this means that other components in the bee venom were eluted and the melittin was relatively increased, The alcohol sterilization process is a result showing that the composition of bee venom changes. In addition, when sterilized by an alcohol sterilization process of 60% or more, it was confirmed that the content of melittin, the main active ingredient of bee venom, was reduced.

3.3 Measurement of changes in melittin content in bee venom by E-beam sterilization process

A purified bee venom sample subjected to a 25kGy E-beam sterilization process and an untreated purified bee venom sample were prepared, and each melittin content was measured and compared in the same manner as in Example 3.1. The measured values of the melittin content of each sample are shown in Table 9.

Processing conditions Melittin content (%(w/w)) Untreated purified bee venom sample 62.1 25kGy E-beam sterilization process purified bee venom sample 62.2

As shown in Table 9, in the case of sterilization by the 25kGy E-beam sterilization process, it was confirmed that the content of melittin, the main active ingredient of bee venom, was constant before and after sterilization. Therefore, it was confirmed that the sterilization process using the E-beam did not cause a change in the active ingredient content of bee venom compared to the alcohol sterilization process or the high pressure sterilization process.

Hereinafter, a formulation example of a composition containing bee venom of the present invention will be described, but it is not intended to limit the present invention, but to explain it in detail.

Formulation Example 1. Preparation of powder

100 mg of bee venom extract of the present invention

1 g lactose

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2. Preparation of tablet

100 mg of bee venom extract of the present invention

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet preparation method.

Formulation Example 3. Preparation of Capsule

100 mg of bee venom extract of the present invention

Corn starch 100 mg

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, a gelatin capsule was filled according to a conventional capsule preparation method to prepare a capsule.

Formulation Example 4. Preparation of Pill

100 mg of bee venom extract of the present invention

1.5 g lactose

1 g glycerin

0.5 g xylitol

After mixing the above components, it was prepared so as to be 4 g per ring according to a conventional method.

Formulation Example 5. Preparation of granules

100 mg of bee venom extract of the present invention

Soybean extract 50 mg

200 mg of glucose

Starch 600 mg

After mixing the above ingredients, 100 mg of 30% ethanol was added and dried at 60°C to form granules, and then filled into a cloth.

Claims (10)

A method for sterilizing a virus in bee venom, comprising: irradiating an E-beam (electron beam) to the bee venom.
The method of claim 1, wherein the E-beam is irradiated with an irradiation amount of 20 to 100 kGy.
The method of claim 1, wherein the E-beam does not cause changes in melittin in bee venom.
The method of claim 1, wherein the virus is a bovine Herpes virus.
(a) irradiating the bee venom with an E-beam; And
(b) detecting a model virus in bee venom; a method of producing a sterilized bee venom comprising.

The method of claim 5, further comprising: spiking the model virus of step (b) to bee venom before step (a).
The method of claim 5, wherein the model virus is a bovine Herpes virus.
The method of claim 5, wherein the E-beam is irradiated with an irradiation amount of 20 to 100 kGy.
The method of claim 5, wherein the E-beam does not cause a change in melittin in the bee venom.
The sterilized bee venom manufactured by the method of manufacturing the sterilized bee venom of claim 5.

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