KR20160126102A - Method Analysing for Bee Venom Component in Live Stock Using UHPLC - Google Patents
Method Analysing for Bee Venom Component in Live Stock Using UHPLC Download PDFInfo
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- KR20160126102A KR20160126102A KR1020150056234A KR20150056234A KR20160126102A KR 20160126102 A KR20160126102 A KR 20160126102A KR 1020150056234 A KR1020150056234 A KR 1020150056234A KR 20150056234 A KR20150056234 A KR 20150056234A KR 20160126102 A KR20160126102 A KR 20160126102A
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- bee venom
- liquid chromatography
- performance liquid
- high performance
- livestock
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
- G01N33/12—Meat; Fish
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a method for analyzing bee venom components in livestock products using ultra-high performance liauid chromatography (1) a sample preprocessing step for extracting bee venom components from livestock products using distilled water; (2) injecting the extracted sample into ultra high performance liquid chromatography; (3) flowing a mixed solvent comprising a mixture of trifluoroacetic acid, acetonitrile and an aqueous solution as the mobile phase; And (4) By analyzing the components of bee venom through the qualitative and quantitative analysis steps of the bee venom ingredient in the livestock product through the analysis of the absorbance of the ultra high performance liquid chromatography, the bean poison was injected or fed into the livestock, cows, pigs and chickens And to a detection method capable of judging whether or not to remain.
Description
The present invention relates to a method for analyzing bee venom components in livestock products using ultra high performance liquid chromatography (UHPLC), and more particularly, to an ultra high performance liquid chromatography (UHPLC) The present invention relates to a method for detecting the presence or absence of bee venom in livestock products when the bee venom is injected or fed into Korean beef cattle, cows, pigs, and chickens by analyzing the bee venom components in the livestock sample.
The consumption of livestock products continues to increase with the history of human life. In order to supply the increased consumption of livestock products smoothly, various researches and experiments have been continuously carried out for mass production methods and production of fast-growing and high-quality meat products. In livestock farming, in order to facilitate supply to rapidly increasing demand, various types of antimicrobial agents and various growth promoting agents are introduced to induce rapid growth in order to prevent various diseases and side effects caused by mass rearing as well as facilities modernization .
However, due to mass breeding, animals are exposed to various diseases at all times and antimicrobial agents are added to prevent diseases. Such antimicrobial agents must be completely decomposed and discharged from the body at the time of shipment of livestock. Due to rapid up- It remains in the animal's body at the time of shipment, and the human body consuming the meat product has the problem that the immunity is lowered like the animal and causes various diseases.
Several studies have shown that bee venom, a natural substance, can be used as an alternative to antibiotics to solve the problems of overuse of antibiotics in the livestock industry.
Bee venom is known to be involved in antibacterial, antioxidant and anti-inflammatory actions, and is used for the treatment of arthritis, back pain and arteriosclerosis in humans. In addition, bee venom is involved in the activation of immune function and has the effect of treating diseases. Therefore, some farmers have tried to prevent diseases of livestock by use of bio - syringe solution.
Bee venom is composed of more than 40 substances including peptides and proteins (eg enzymes) and active amines. These components are used for anti-inflammatory, anti-inflammatory and antimicrobial actions, as well as promoting the secretion of pituitary hormones and promoting blood circulation. It is known to be effective.
Among them, melitin contains 40 ~ 50% of dry bee venom. In 1954, it was firstly discovered as a hemolytic element by Newman and Harborman. In 1972, Activity properties were observed. It contains 26 amino acids structurally and strongly inhibits macrophage migration and synergizes with phospholipase A2 to increase mutual activity. Stimulates the pituitary gland and adrenal cortex system to promote catecholamine and cortisone secretion, stabilizes the lysosomal membrane, and anti-inflammatory action. In addition, dissolution, enzyme, pain induction, and radiation-resistant action are known.
The phospholipase contains 12% of the bee venom and this enzyme has the cell-tissue destruction and hemolytic action because it makes the BDP-binding fatty acid of glycerin of phospholipid, .
Histamine contains 0.1-1% of bee venom and injected into a rat vein, resulting in blood pressure lowering action, intestinal contraction, and secretion of gastric acid, resulting in redness and swelling, but recovered in a short time It is known as a hook.
Other components of bee venom include eupamines, MCD peptides, hyaluronidase, adolapine, dopamine, protease inhibitors, secapine, deltapine, progamines, and alpha-glucosidase.
Recently, bee venom has been shown to be effective not only in the prevention and treatment of diseases of human body, but also in the improvement of productivity of livestock products and prevention and treatment of diseases.
Korean Registered Patent No. 10-1391911 entitled " Bee-to-Toe Composition with Improved Growth Rate and Immune Function ", Korean Registered Patent No. 10-1156082, "Composition for Treatment of Cow Mastitis ", Korean Registered Patent No. 10-1055689, ≪ / RTI > Korean Patent No. 10-1318787 entitled " Antimicrobial Functional Natural Feed Additive ", Korean Patent Laid-Open Publication No. 19990-37904, "Method for Treating Anemia in Sows Using Live Bee Venom ", Korean Registered Patent No. 10-1288389" Feed Composition for Poultry "Shows that the bee venom ingredient improves the productivity of livestock products and has the effect of preventing and treating diseases.
However, even though bee venom is a natural substance that can act as a substitute for antibiotics, bee venom itself is used to prevent or treat diseases of livestock products. If bee venom remains in the livestock product, it can be indirectly ingested, which can cause problems in the stability of the livestock product. There has been no report on the method of pretreatment of livestock products and the method of optimal analysis of residual bee venom components in livestock products in order to determine the presence of bee venom components in livestock products when bee venom is developed into natural antibiotics or animal medicines and feedstuffs and injected or fed into livestock products .
The present invention provides a method of pretreatment of animal products and an optimal method of analyzing residual bee venom, which can determine whether bee venom remains in livestock products, which is increasingly used in livestock products, so that it can be used as stability data for future use of bee venom in livestock products Do.
It is an object of the present invention to provide a method for detecting the presence or absence of beverage in livestock products when the bee venom is injected or fed for improvement of productivity of dairy cattle, cows, pigs and chickens and prevention and treatment of diseases.
It is another object of the present invention to provide a method for pretreating animal products to detect bee venom components in an animal product.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.
In order to achieve the above object, the present invention is characterized by including a method for analyzing bee venom components in an animal product using ultra high performance liquid chromatography.
(1) Preparation of livestock products, livestock products are lyophilized, diluted with distilled water to a concentration of 10mg / ml or more and less than 60mg / ml, and extracted using ultrasound A sample pretreatment step including a step of taking only the supernatant by centrifugation followed by a filtering step;
(2) injecting the extracted sample into ultra high performance liquid chromatography;
(3) flowing a mixed solvent comprising trifluoroacetic acid, acetonitrile and water as the mobile phase; And
(4) qualitative and quantitative analysis of the bee venom ingredient in the livestock product through the analysis of the absorbance of ultra high performance liquid chromatography.
The bee keeping component is characterized in that at least one of melatin, histamine and phospholipase is selected.
In the step (1), the ultrasonic extraction is performed at a frequency of 20 kHz or more and less than 40 kHz in the sample preprocessing step.
When the livestock products are selected from the group consisting of Korean, Korean, Korean, and Chinese pigs, dilution with distilled water is performed at a concentration of 30 mg / ml or more and 50 mg / ml or less in the step (1) Minute.
When the livestock product is milk, dilution with distilled water is performed at a concentration of 40 mg / ml or more and 60 mg / ml or less in the sample preparation step of step (1), and ultrasonic extraction is performed for 20 minutes to 40 minutes.
When the livestock product is an egg, dilution with distilled water is performed at a concentration of 10 mg / ml or more and 30 mg / ml or less in the pretreatment step of the step (1), and ultrasonic extraction is performed for 20 minutes to 40 minutes.
Is a reversed phase non-polar column packed with octasilane (C8) as a column used for ultrahigh performance liquid chromatography in the step (2).
The column has a column length of 8 cm or more and 12 cm or less, a column inner diameter of 4 mm or more and 5 mm or less, and a column particle size of 2 占 퐉 or more to 3 占 퐉 or less.
The column temperature is characterized by a temperature of 40 ° C or more and 60 ° C or less
The mixed solvent in step (3) is a mixture of solution A and solution B, wherein solution A is a mixture of 20 mM trifluoroacetic acid and acetonitrile, and solution B is a mixture of 20 mM trifluoroacetic acid and water.
The step (3) is characterized in that the volume percentage of the solution A is increased with time.
The solution A is characterized by having a volume ratio of 40% by volume to 40% by volume for 4.5 minutes and 4.5% by volume for 9 minutes.
The absorbance analysis in the step (4) is characterized by using an ultraviolet detector of 200 nm-280 nm as a detector used in ultrahigh performance liquid chromatography.
The present invention can be used as a future stability data to determine whether bee venom remains in livestock products when bean sprouts are injected or fed into livestock products with natural antibiotics or animal drug feeds, by providing a pretreatment method for livestock samples and an optimal analysis method of bee venom components in livestock samples Do.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view showing a method for analyzing bee venom components in animal products using ultra high performance liquid chromatography. FIG.
FIG. 2 is a view showing a pretreatment process of a livestock sample for analysis of bee venom components in an animal product.
FIG. 3A is a view showing a pretreatment process of a livestock sample for bee venom analysis when a livestock sample is a cattle, a cow, a chicken, or a pig.
FIG. 3B is a view showing a pretreatment process of a livestock sample for bee venom analysis when the livestock sample is milk.
FIG. 3C is a view showing a pretreatment process of a livestock sample for bee venom analysis when the livestock sample is an egg.
Fig. 4 is an analysis of melittin at a concentration of 5 ㎍ / ml of bee venom.
Fig. 5 is a view showing the detection of the bee venom component remaining amount in the livestock product on the first day after the bee venom was injected or fed into the livestock product.
Fig. 6 is a chart showing the remaining amount of bee-venom components in each livestock sample containing the tablet bee venom before lyophilization. Fig.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.
The present invention provides a pretreatment method for an animal material sample necessary for determining whether or not to retain the bee venom.
In addition, the present invention provides an optimal method for analyzing bee venom components in an animal product sample to determine whether bee venom components remain in livestock products including a pretreatment method of a livestock sample.
The present invention provides a method for quantitatively and qualitatively analyzing at least one of histamine and phospholipase including melittin, which is a main component of bee venom, and a method for analyzing the optimum bee venom component using ultra high performance liquid chromatography (UHPLC) do.
The bee venom components were analyzed using an ultrahigh performance liquid chromatography (UHPLC) -DAD 1290 infinity model of Agilent (Santa Clara, CA, USA). The principle of UHPLC is to separate phases according to the degree of adsorption to the packed material inside the column by using the polar molecular weight structure which is inherent property of each sample.
The bee venom components in the livestock sample are differentiated by the difference in affinity for the filler in the column. The difference in polarity, which is a unique characteristic of each component, appears depending on the chemical characteristics. Since the polarity difference is a factor determining the affinity for the filler, the time taken to pass through the column, that is, the retention time, .
The bee venom components that have passed through the column are detected in succession according to elution time through peaks which are the increase in absorbance on an ultraviolet absorptiometer (UV). The detected bee venom component is identified by qualitative comparison with elution time with a reference material (qualitative analysis), and each type of bee poison contained in the sample is calculated by the height or area of the peak (quantitative analysis).
1, a method for analyzing bee venom components in animal products using ultra high performance liquid chromatography comprises the steps of (1) a sample preprocessing step of extracting bee venom components from livestock products using distilled water; (2) injecting the extracted sample into ultra high performance liquid chromatography; (3) flowing a mixed solvent comprising trifluoroacetic acid, acetonitrile and water as the mobile phase; And (4) qualitative and quantitative analysis of bee venom components in the livestock product through the analysis of the absorbance of ultra high performance liquid chromatography.
The step (1) is a sample preprocessing step for extracting the bee venom component, and FIG. 2 shows a sample preprocessing step. The sample pretreatment step comprises (A) preparing an animal product to be tested; (B) lyophilizing livestock products and diluting them with distilled water; (C) extracting using ultrasonic waves; (D) centrifuging and taking only the supernatant; And (E) filtering with a membrane filter.
In step (A), livestock products are collectively referred to as products produced by livestock, as well as livestock such as cows, cows, chickens, and pigs. First, prepare the part to be examined of the livestock products to be inspected. Preferably, when the specimen is in a lump state, the test site is finely divided to facilitate lyophilization. If the specimen is egg, remove egg shell and collect only the contents and freeze dry. Freeze-drying in step (B) is carried out at -20 캜 or lower. When inserting the inspecting body into the freeze dryer, be sure to use a container with a lid, drill a fine hole, and freeze dry until the water is completely removed. After lyophilization, the lyophilized material is diluted with distilled water. As shown in FIG. 3A, when the livestock sample is a cattle, a cow, a chicken, and a pig, the dilution is preferably performed at a concentration of 30 mg / ml or more and 50 mg / ml or less. More preferably at a concentration of 40 mg / ml. If the concentration is less than 30 mg / ml, the component to be analyzed may not be detected in ultra high performance liquid chromatography. If the concentration is more than 50 mg / ml, the component separation may be difficult in ultra high performance liquid chromatography.
As shown in FIG. 3B, when the livestock sample is milk, the dilution with distilled water is preferably diluted to a concentration of 40 mg / ml or more and 60 mg / ml or less of dry livestock product weight. More preferably at a concentration of 50 mg / ml. If the concentration is less than 40 mg / ml, the component to be detected in ultra high performance liquid chromatography may not be detected. If the concentration is higher than 60 mg / ml, the component separation may be difficult in ultra high performance liquid chromatography.
In Fig. 3C, when the livestock sample is an egg, dilution with distilled water is preferably carried out by diluting the dried livestock product to a concentration of 10 mg / ml or more and 30 mg / ml or less. More preferably 20 mg / ml.
If the concentration is less than 10 mg / ml, it may not be detected in the ultra high performance liquid chromatography. If the concentration is more than 30 mg / ml, it may be difficult to separate the components in ultra high performance liquid chromatography.
The reason for the different concentration of dilution is that the dilution rate of distilled water varies depending on the meat quality of the livestock products.
After dilution with distilled water, ultrasonic extraction is performed using ultrasonic waves of 20 to 40 kHz in step (C). Ultrasonic extraction is a type in which ultrasonic waves are converted into heat energy from the inside, instead of directly heating the heat. Compared with the conventional extraction method, it prevents destruction of the components and has excellent extraction yield. If the frequency of the ultrasonic wave extraction is lower than the above condition, the extraction of the desired component is not performed well, or the frequency of the frequency is larger than the above condition, the object component may be destroyed or denatured.
If the specimen is either Hanwoo, cow, chicken or pig, ultrasonically extract for 40 minutes to 80 minutes. The extraction is preferably carried out for 60 minutes. If the sample is milk or egg, ultrasonically extract for 20 minutes to 40 minutes. Preferably 30 minutes (Figs. 3A, 3B, 3C). If the extraction time of the ultrasonic extraction is shorter than the above conditions, the desired component may not be extracted well or the extraction time may be longer than the above condition, thereby causing the problem that the target component is destroyed or denatured.
After extracting the supernatant, the supernatant is centrifuged at 10000 rpm to 15,000 rpm at 4 ° C in step (D), and the supernatant is filtered with a 0.45-μm membrane filter in step E. More preferably, in step (D), the extract is centrifuged at 4,000 rpm at 13,000 rpm to remove only the supernatant, followed by filtration through a 0.45-um membrane filter.
If the centrifugation in step (D) is less than 10,000 rpm or more than 15,000 rpm, the desired components may not be separated from other components.
The column used in the ultra high performance liquid chromatography in the step (2) is preferably a reversed phase nonpolar column packed with octasilane (C8) dodecylsilane (C12) or octadecylsilane (C18) Lt; / RTI > This is because columns filled with silica, columns packed with amino group (NH 2) or packed with nitrile (CN) groups have high polarity, so that bee venom components are not adsorbed and eluted. However, when dodecylsilane (C12) or octasilane (C8) is used, components having similar chemical properties may overlap with each other to reduce the separation. Therefore, for accurate analysis, An absilane (C18) column is most preferred. The octadecylsilane (C18) column is packed with 18 chains of non-polar carbon in the column. The polar component does not bond with the non-polar column functionalities and dissolves rapidly, and the non-polar component binds to the non- .
The specification of the column is possible within the range normally used. However, since the length, the inner diameter and the particle size of the column are related to the analysis time and the separation, it is preferable to use the column in an appropriate category in consideration of promptness and selectivity. As one example, when considering the separation efficiency, the standard of the column can be selected within a range of 8 cm to 12 cm and a column inner diameter within a range of 3 mm to 6 mm. Particularly, in the case of an octacetylsilane (C18) column, the column length is 10 cm, the column inner diameter is 4.5 mm, and the column particle size is more preferably 2.7 m.
In the step (3), various types of organic solvents such as ethanol, methanol, or acetonitrile, which are conventionally used as the mobile phase of liquid chromatography, can be used. However, when only the above-mentioned organic solvents were used, some components having similar polarities were not separated.
In order to solve such a problem, in one embodiment, a mixed solvent in which an organic solvent is mixed with an aqueous solution at a predetermined ratio is used as the mobile phase, and the organic solvent may be acetonitrile having a relatively low polarity.
However, when the mixed solvent of 40% or more and 45% or less of acetonitrile is used as the mobile phase as described above, the peak may be nonpolar or peak separation may be observed when water is used as a water-soluble solvent. These problems can be solved by using 20 mM ammonium formate.
In another preferred embodiment, in order to selectively move the main components of the bee venom without overlapping, the mobile phase in step (3) is mixed with 20 mM trifluoroacetic acid and 20% acetonitrile in an organic solvent, and 20 mM trifluoroacetic acid and water is changed from 0 to 4.5 minutes with time, the mixture of 20 mM trifluoroacetic acid and acetonitrile is made to have a volume volume of 40 vol% and when it is 4.5 minutes to 9 minutes, it is more than 40% and less than 45% The bee venom component can be separated optimally with the volume ratio composition.
In the detection of absorbance analysis in the step (4), the ultraviolet detector can be used in a range of 200 nm or more and 280 nm or less, and most preferably 220 nm. At wavelengths below 200 nm and above 280 nm, absorption of light is small and can not be detected.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
<Example 1> A pretreatment method of a sample of animal products of the present invention
1) Livestock products: Hanwoo, cow, pig, chicken
2) Conditions and methods
① Collect the parts (liver and flesh) to be examined and divide them for easy freeze drying.
② Freeze dry and measure the weight of the specimen that has completely removed the water. Dilute it with distilled water so that the concentration of test substance is 40 mg / ml.
③ In order to extract bee venom components from the specimens, ultrasonic waves are extracted for 1 hour by using ultrasonic waves.
④ Ultrasonically inspected specimens should be centrifuged at 13,000 rpm for 5 minutes at 4 ° C and only supernatant is taken.
(5) The supernatant is filtered using a 0.45 ㎛ membrane filter to obtain a specimen.
≪ Example 2 > A pretreatment method for a sample of animal products
1) Livestock products: Milk
2) Conditions and methods
① Collected milk is freeze-dried to completely remove moisture.
② Weigh the powdered milk and dilute with distilled water to a concentration of 50 mg / ml.
③ To extract bee venom components from milk, ultrasonic extraction is performed for 30 minutes using ultrasonic waves.
④ Ultrasonic milk is centrifuged at 13,000 rpm for 30 minutes at 4 ℃, and only supernatant is taken.
(5) The supernatant is filtered using a 0.45 ㎛ membrane filter to obtain a specimen.
≪ Example 3 > A pretreatment method for a sample of animal products
1) Livestock products: Eggs
2) Conditions and methods
① The eggs are crushed, the skin is removed and only the contents are lyophilized to completely remove water.
② Measure the weight of the test material and dilute it with distilled water so that the concentration of test substance is 20 mg / ml.
③ To extract the bee venom components from the specimens, ultrasonic waves are extracted for 30 minutes using ultrasonic waves.
④ Ultrasonically examined specimens should be centrifuged at 13,000 rpm for 30 minutes at 4 ° C, and then only the supernatant is taken.
(4) The supernatant is filtered using a 0.45 ㎛ membrane filter to obtain a specimen.
≪ Example 4 > Optimum condition for analysis of bee venom component
In the Examples and Experimental Examples according to the present invention, UHPLC was performed using Agilent's UHPLC-DAD 1290 infinity model and Halo peptide ES-C18 (particle size: 2.7 μm, inner diameter: (A) 20 mM trifluoroacetic acid (TFA) / acetonitrile (ACN) and (B) 20 mM trifluoroacetic acid (TFA) / water (H2O) Mixed solvent was used. The volume ratio of the mixture (A) to the mixture (B) was adjusted to 40%: 60% when the volume ratio was from 0 to 4.5 minutes, and from 40% to 45%: 60 to 55% when the volume ratio was from 4.5 minutes to 9 minutes. The flow rate of the mobile phase was 2.0 mL / min. The sample injection amount was 2 μL. The wavelength of the ultraviolet absorptiometer was set at 220 nm. The temperature of the column was adjusted to 50 ° C. during the analysis.
≪ Experimental Example 1 >
1) Experimental method
Melulin was analyzed at an ultra-high performance liquid chromatography optimum condition of the above Example 4 for bee venom at a concentration of 5 / / ml.
2) Experimental results
As shown in FIG. 4, the melittin peak was confirmed by only 5 μg / ml bee venom. Therefore, it is possible to analyze the bee venom component in the test substance if only the amount of 2 ㎍ / ml is converted into the amount of melitin.
<Experimental Example 2> Analysis of bee venom components in livestock samples
1) Experimental method
Generally, bean stocks used in beef farm were 0.02mg / kg for dairy cows and 0.05mg / kg for pigs, respectively. Milk was also milked from the cow one day later. Chickens were mixed with water and fed at a concentration of 0.02ppm for 3 days and chickens and eggs were collected.
The bee venom components were extracted from the livestock samples prepared in Examples 1, 2 and 3 according to the types of livestock products, and analyzed for melittin using the bee venom component optimum analysis method according to Example 4.
The positive control group was
2) Experimental results
The results are shown in Fig. Melitin was detected in the positive control group but not in the Hanwoo, cow, pig, chicken, milk and egg samples.
The bee venom component is not detected in all the sample groups, and the bee venom ingredient remains in the livestock product can be relieved.
≪ Experimental Example 3 > Analysis of bee venom components in
1) Experimental method
The pre-treatment according to Example 1, Example 2 and Example 3 was carried out in the same manner as in the freeze-drying method, except that the concentration of 50 ㎍ / ml of purified bee venom was directly added to each livestock product sample, .
After the pretreatment, bee venom components were analyzed using the bee venom component optimum analysis method according to Example 4 above.
2) Experimental results
As shown in FIG. 6, histamine and phospholipase, including melittin, a major component of bee venom, were detected in all the livestock sample groups.
Therefore, it can be confirmed that the bee venom component in the livestock product pretreatment process is lost and not detected.
It was also confirmed that bee venom was not injected into the meat, milk and eggs after 1 day of feeding.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be possible. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.
Claims (13)
(2) injecting the extracted sample into ultra high performance liquid chromatography;
(3) flowing a mixed solvent comprising trifluoroacetic acid, acetonitrile and water as the mobile phase; And
(4) qualitative and quantitative analysis of the bee venom ingredient in the livestock product through analysis of the absorbance of the ultrahigh performance liquid chromatography; and (4) analyzing the bee venom ingredient in the livestock product using the ultra high performance liquid chromatography.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101896938B1 (en) * | 2017-03-16 | 2018-09-12 | 대한민국 | Separation method of phospholipase A2 in bee venom using chromatography |
| CN113109466A (en) * | 2021-03-27 | 2021-07-13 | 广东互信生物科技有限公司 | Fingerprint spectrum detection method for wasp venom of black peltate wasp |
| KR20210154415A (en) | 2020-06-12 | 2021-12-21 | 대한민국(농촌진흥청장) | Detection method of putrescine from bee venom |
| KR102342400B1 (en) | 2020-06-16 | 2021-12-23 | 대한민국 | Detection method of cadaverine from bee venom |
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2015
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101896938B1 (en) * | 2017-03-16 | 2018-09-12 | 대한민국 | Separation method of phospholipase A2 in bee venom using chromatography |
| KR20210154415A (en) | 2020-06-12 | 2021-12-21 | 대한민국(농촌진흥청장) | Detection method of putrescine from bee venom |
| KR102342400B1 (en) | 2020-06-16 | 2021-12-23 | 대한민국 | Detection method of cadaverine from bee venom |
| CN113109466A (en) * | 2021-03-27 | 2021-07-13 | 广东互信生物科技有限公司 | Fingerprint spectrum detection method for wasp venom of black peltate wasp |
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