Method for separating and purifying active components of wasp venom
The wasp venom solution is centrifuged in sections using ultrafiltration technology, which solves the problems of time-consuming separation and purification and easy inactivation of active components in the existing technology. Components with antifungal and antibacterial activity are obtained, simplifying the operation process and improving efficiency.
Patent Information
- Application Number
- CN202510930827.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-10-03
AI Technical Summary
The existing technology takes a long time to separate and purify the active components of wasp venom, and improper selection of elution solution can easily lead to inactivation of the active components.
Ultrafiltration technology is used to centrifuge the crude venom solution of wasp venom in sections using ultrafiltration tubes with molecular weight cutoffs of 30kDa, 10kDa and 3kDa, to obtain retained concentrates of not less than 30kDa, not less than 10kDa but less than 30kDa, and not less than 3kDa but less than 10kDa, respectively, to avoid inactivation of active components through physical separation methods.
The method achieves efficient separation and purification of active components of wasp venom, obtaining active components with antifungal and antibacterial effects, simplifies the operation process, saves time and avoids the inactivation of active components, and is suitable for process scale-up.
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Figure CN120732897A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of separation and purification, and particularly relates to a method for separating and purifying active components of wasp venom. Background Art
[0002] Wasps are a general term for all insects in the family Vespidae, order Hymenoptera. Over 6,000 species are known worldwide, with over 200 found in China, and 122 documented in Yunnan. The concave-striped wasp (Vespa velutinaauraria Smith) is a major, large-scale species cultivated in Yunnan Province. With mature breeding techniques and high yields, it has become a powerful driver of prosperity for farmers.
[0003] Wasp venom is secreted and stored in a venom sac gland in the tail of a wasp. It is expelled through the stinger during hunting, when threatened, or when stressed. Wasp venom has numerous benefits, including antibacterial, anti-inflammatory, analgesic, and anti-tumor properties. Traditionally, the Jingpo people use wasp medicinal wine to treat rheumatism and rheumatoid arthritis. Modern analytical studies have shown that wasp venom varies among species, but its main components fall into three categories: high-molecular-weight active proteins, including enzymes, toxins, and allergens; low-molecular-weight bioactive amines, including histamine, 5-hydroxytryptamine, and catecholamines; and low-molecular-weight bioactive peptides, including kinins, vesperid venom peptides, and chemotactic peptides. For active proteins and peptides, chromatographic techniques such as size exclusion chromatography, affinity chromatography, and ion exchange chromatography are the preferred methods for isolating active components from wasp venom. However, these methods have drawbacks, including limited sample loading, lengthy extraction times, low yields, and the need for repeated elutions. Protein or peptide components can also be easily inactivated if the elution solvent is not properly selected.
[0004] Therefore, there is an urgent need to provide a new method for isolating wasp venom to solve the problems of time-consuming separation and purification in existing technologies, improper selection of elution solutions, and easy inactivation of active components. Summary of the Invention
[0005] The present invention aims to provide a method for separating and purifying active components of wasp venom. By this method, three active components with antifungal and antibacterial effects can be obtained. At the same time, the method is simple to operate, highly efficient, and saves a lot of purification time. Moreover, the entire process only involves physical separation, avoiding inactivation of the active components.
[0006] The present invention provides a method for separating and purifying active components of wasp venom, comprising the following steps:
[0007] preparing crude wasp venom into a crude wasp venom solution;
[0008] The crude venom solution was centrifuged for the first time using an ultrafiltration tube with a molecular weight cut-off of 30 kDa. The inner tube obtained a concentrate with a molecular weight cut-off of not less than 30 kDa, and the outer tube obtained the first filtrate.
[0009] The first filtrate is subjected to a second centrifugation using an ultrafiltration tube with a molecular weight cut-off of 10 kDa, the inner tube obtains a concentrate with a cut-off of not less than 10 kDa but less than 30 kDa, and the outer tube obtains the second filtrate;
[0010] The second filtrate was centrifuged for the third time using an ultrafiltration tube with a molecular weight cut-off of 3 kDa, and the inner tube obtained a concentrated solution with a cut-off of not less than 3 kDa but less than 10 kDa.
[0011] As a preferred embodiment, the method for preparing crude wasp venom comprises the following steps:
[0012] Low-voltage pulses are used to stimulate wasp detoxification, and the crude wasp venom is obtained after the venom is dried; the voltage of the low-voltage pulses is 12V.
[0013] As a preferred solution, the crude wasp venom is dissolved in deionized water to obtain a crude wasp venom solution, wherein the concentration of the crude wasp venom solution is 30 μg / mL to 10 mg / mL.
[0014] As a preferred solution, when preparing the crude wasp venom solution, the process further includes centrifuging after the dissolution to remove the precipitate; the centrifugal force of the centrifugation is 10,000×g, and the centrifugation time is 5 minutes.
[0015] As a preferred solution, the centrifugal force of the first centrifugation is 3000×g, and the time of the first centrifugation is 10 to 30 minutes.
[0016] As a preferred solution, the centrifugal force of the second centrifugation is 3000×g, and the time of the second centrifugation is 15 to 40 minutes.
[0017] As a preferred solution, the centrifugal force of the third centrifugation is 3000×g, and the time of the third centrifugation is 25 to 60 minutes.
[0018] As a preferred embodiment, the wasp includes Vespa concave.
[0019] The present invention also provides a retained concentrate obtained by the separation and purification method described above, wherein the retained concentrate includes a retained concentrate of not less than 30 kDa, a retained concentrate of not less than 10 kDa but less than 30 kDa, and a retained concentrate of not less than 3 kDa but less than 10 kDa.
[0020] The present invention also provides the retained concentrate obtained by the separation and purification method described above, or the use of the retained concentrate in inhibiting fungal and / or bacterial activity, wherein the fungi include Candida albicans, and the bacteria include Escherichia coli and Staphylococcus aureus.
[0021] Beneficial effects: The present invention provides a method for separating and purifying active components of wasp venom, comprising the following steps: preparing crude wasp venom into a crude wasp venom solution; subjecting the crude wasp venom solution to a first centrifugation using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, obtaining a retained concentrate of not less than 30 kDa in the inner tube, and obtaining a first filtrate in the outer tube; subjecting the first filtrate to a second centrifugation using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, obtaining a retained concentrate of not less than 10 kDa but less than 30 kDa in the inner tube, and obtaining a second filtrate in the outer tube; subjecting the second filtrate to a third centrifugation using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, obtaining a retained concentrate of not less than 3 kDa but less than 10 kDa in the inner tube. Utilizing the separation and purification method of the present invention, three active components with antifungal and antibacterial efficacy can be obtained. The method is simple to operate, highly efficient, saves a great deal of purification time, and the entire process involves only physical separation, greatly avoiding the inactivation of the active components, which is very conducive to process scale-up.
[0022] The ultrafiltration of the present invention is a membrane filtration, which separates the feed liquid through a membrane of a specific pore size through pressure or concentration gradient, etc., and can solve the problems of the existing separation and purification technology that is time-consuming, the elution solution is improperly selected, and the active components are easily inactivated.
[0023] In the examples of the present invention, SDS-PAGE experiments were further performed on the ultrafiltration fractions. The results showed that the retained concentrate of no less than 30 kDa contained protein, while the retained concentrates of no less than 10 kDa but less than 30 kDa and no less than 3 kDa but less than 10 kDa contained trace amounts or no protein. This suggests that the overall antibacterial activity of wasp venom may be the result of the combined action of proteins or polypeptides and non-peptide substances. The present invention further clarifies the active sites of wasp venom (based on molecular weight) through ultrafiltration fractionation, providing a theoretical basis and practical guidance for the subsequent diversified utilization of wasp venom, further enhancing the role of wasp farming in increasing farmers' income. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.
[0025] Figure 1is the activity of each fraction after ultrafiltration against E. coli; where 1 is the retained concentrate of not less than 30 kDa; 2 is the first filtrate; 3 is the retained concentrate of not less than 10 kDa but less than 30 kDa; 4 is the second filtrate; 5 is the retained concentrate of not less than 3 kDa but less than 10 kDa; and 6 is the third filtrate.
[0026] Figure 2 is the activity of each fraction after ultrafiltration against Staphylococcus aureus; where 1 is the retained concentrate of not less than 30 kDa; 2 is the first filtrate; 3 is the retained concentrate of not less than 10 kDa but less than 30 kDa; 4 is the second filtrate; 5 is the retained concentrate of not less than 3 kDa but less than 10 kDa; and 6 is the third filtrate.
[0027] Figure 3 is the activity of each fraction after ultrafiltration against Candida albicans; where 1 is the retained concentrate of not less than 30 kDa; 2 is the first filtrate; 3 is the retained concentrate of not less than 10 kDa but less than 30 kDa; 4 is the second filtrate; 5 is the retained concentrate of not less than 3 kDa but less than 10 kDa; and 6 is the third filtrate.
[0028] Figure 4 The results of 12% SDS-PAGE of each component after ultrafiltration; among them, lane 1 is the retained concentrate of not less than 3KDa but less than 10KDa; lane 2 is the retained concentrate of not less than 10KDa but less than 30KDa; lane 3 is the retained concentrate of not less than 30KDa; lane 4 is the protein marker. DETAILED DESCRIPTION
[0029] The present invention provides a method for separating and purifying active components of wasp venom, comprising the following steps:
[0030] preparing crude wasp venom into a crude wasp venom solution;
[0031] The crude venom solution was centrifuged for the first time using an ultrafiltration tube with a molecular weight cut-off of 30 kDa. The inner tube obtained a concentrate with a molecular weight cut-off of not less than 30 kDa, and the outer tube obtained the first filtrate.
[0032] The first filtrate is subjected to a second centrifugation using an ultrafiltration tube with a molecular weight cut-off of 10 kDa, the inner tube obtains a concentrate with a cut-off of not less than 10 kDa but less than 30 kDa, and the outer tube obtains the second filtrate;
[0033] The second filtrate was centrifuged for the third time using an ultrafiltration tube with a molecular weight cut-off of 3 kDa, and the inner tube obtained a concentrated solution with a cut-off of not less than 3 kDa but less than 10 kDa.
[0034] The method for preparing crude wasp venom of the present invention comprises the following steps: using a low-voltage pulse to stimulate wasp detoxification, drying the venom, and obtaining the crude wasp venom; the voltage of the low-voltage pulse is 12V. The crude wasp venom is dissolved in deionized water to obtain a crude wasp venom solution, wherein the concentration of the crude wasp venom solution is 30 μg / mL to 10 mg / mL. The preparation of the crude wasp venom solution also includes centrifuging after dissolution to remove precipitates; the centrifugation is performed at a centrifugal force of 10,000 × g for 5 minutes. As a specific embodiment, the concentration of the crude venom solution can be any one of 30 μg / mL, 40 μg / mL, 60 μg / mL, 100 μg / mL, 500 μg / mL, 700 μg / mL, 900 μg / mL, 1000 μg / mL, 2000 μg / mL, 5000 μg / mL, 8000 μg / mL, and 10000 μg / mL, as well as any value between the two values. The concentration of the crude venom solution of the present invention can be passed through an ultrafiltration tube with different molecular weight cut-offs of the present invention. When the concentration of the crude venom solution of the present invention is too high, the ultrafiltration tube may be clogged.
[0035] The centrifugal force of the first centrifugation of the present invention is 3000 × g, and the time of the first centrifugation is 10 to 30 min. As a specific embodiment, the time of the first centrifugation can be any one of 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min and 30 min, and any value between two numerical values. When the centrifugal force is 3000 × g, within the time range of the present invention, the centrifugal force (3000 × g) and the time work together to efficiently enrich the retained concentrate of not less than 30 kDa, ensuring sufficient recovery. When the centrifugation time is too short (<10 min), the insufficient centrifugation time will cause the target substance (not less than 30 kDa retained concentrate) to fail to be fully concentrated, resulting in a too low recovery rate of the retained concentrate, which may not meet the needs of subsequent experiments or applications. When the centrifugation time is too long (>30 minutes), the amount of target material collected may no longer increase significantly after a certain period of time, but the experimental period is extended, reducing overall efficiency. In the embodiment of the present invention, the centrifugal force is 3000×g and the centrifugation time is 25 minutes, which can reduce the volume of the concentrate with a cutoff of not less than 30 kDa to approximately 300 μL.
[0036] The centrifugal force of the second centrifugation of the present invention is 3000 × g, and the time of the second centrifugation is 15 to 40 min. As a specific embodiment, the time of the first centrifugation can be any one of 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min and 40 min, and any value between two numerical values. When the centrifugal force is 3000 × g, within the time range of the present invention, the centrifugal force (3000 × g) and the time act synergistically to make not less than 10 kDa but less than 30 kDa trapped concentrate efficiently enriched, ensuring sufficient recovery. When the centrifugation time is too short (<15min), the insufficient centrifugation time will cause the target substance (not less than 10kDa but less than 30KDa retained concentrate) to fail to be fully concentrated, resulting in too low a recovery rate of the retained concentrate, which may not meet the needs of subsequent experiments or applications. When the centrifugation time is too long (>40min), after a certain period of time, the collection amount of the target substance may no longer increase significantly, but the experimental cycle is extended, reducing the overall efficiency. In an embodiment of the present invention, the centrifugal force is 3000×g and the centrifugation time is 18min, which can make the volume of the retained concentrate of not less than 10kDa but less than 30KDa reach about 300μL.
[0037] The centrifugal force of the third centrifugation of the present invention is 3000×g, and the time of the third centrifugation is 25 to 60 min. As a specific embodiment, the time of the first centrifugation can be any one of 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min and 60 min, and any value between the two values. When the centrifugal force is 3000×g, within the time range of the present invention, the centrifugal force (3000×g) and time work together to efficiently enrich the retained concentrate of not less than 3kDa but less than 10KDa, ensuring a sufficient recovery rate. When the centrifugation time is too short (<25min), insufficient centrifugation time will cause the target substance (not less than 3kDa but less than 10KDa retained concentrate) to fail to be fully concentrated, resulting in a too low recovery rate of the retained concentrate, which may not meet the needs of subsequent experiments or applications. When the centrifugation time is too long (>60min), after a certain period of time, the collection amount of the target substance may no longer increase significantly, but the experimental cycle is extended, reducing the overall efficiency. In an embodiment of the present invention, the centrifugal force is 3000×g and the centrifugation time is 35min, which can make the volume of the retained concentrate of not less than 3kDa but less than 10KDa reach about 300μL.
[0038] As a specific embodiment, the wasp includes Vespa concave.
[0039] The present invention also provides a retained concentrate obtained by the separation and purification method as described above, wherein the retained concentrate includes a retained concentrate of not less than 30kDa, a retained concentrate of not less than 10kDa but less than 30KDa, and a retained concentrate of not less than 3kDa but less than 10KDa. As a specific embodiment, the retained concentrate obtained by the present invention can be preserved by freeze-drying. In an embodiment of the present invention, an SDS-PAGE experiment was further performed on each component obtained by ultrafiltration. The results showed that the retained concentrate of not less than 30kDa contained protein, while the retained concentrate of not less than 10kDa but less than 30KDa and not less than 3kDa but less than 10KDa contained trace amounts or no protein, indicating that the overall antibacterial activity of wasp venom may be the result of the combined action of proteins or polypeptides and non-peptide substances. The present invention further clarifies the active parts of the wasp venom (based on molecular weight) through ultrafiltration segmentation, provides a theoretical basis and practical guidance for the subsequent diversified utilization of wasp venom, and further enhances the status of wasp breeding in increasing farmers' income.
[0040] The present invention also provides a retained concentrate obtained by the separation and purification method described above, or the use of the retained concentrate in inhibiting fungal and / or bacterial activity, wherein the fungi include Candida albicans, and the bacteria include Escherichia coli and Staphylococcus aureus. In the Examples of the present invention, using Escherichia coli, Staphylococcus aureus, and Candida albicans as indicator strains, the antibacterial activity of three ultrafiltration concentrates (a retained concentrate of not less than 30 kDa, a retained concentrate of not less than 10 kDa but less than 30 kDa, and a retained concentrate of not less than 3 kDa but less than 10 kDa) was investigated using the inhibition zone method. The results showed that all three concentrates showed significant inhibitory effects on the indicator strains.
[0041] To further illustrate the present invention, the method for separating and purifying the active components of wasp venom provided by the present invention is described in detail below with reference to the following examples, but they should not be construed as limiting the scope of protection of the present invention.
[0042] Unless otherwise specified, the present invention has no special requirements for the raw materials, and commercially available products known to those skilled in the art can be used.
[0043] Example 1
[0044] (1) Collection of venom from the concave-striped wasp: Use low voltage pulses (12V) to stimulate the wasp to excrete venom on the collector. After drying under natural conditions, scrape it with a venom scraper to obtain the crude venom from the concave-striped wasp.
[0045] (2) Pretreatment of Crude Bee Venom: Weigh crude bee venom and dilute it with deionized water to a final concentration of 1.6 mg / mL. Centrifuge at room temperature for 5 min (10,000 × g) and discard the precipitate. If any floating or suspended debris remains in the supernatant, filter it with a 0.45 μm aqueous membrane.
[0046] (3) Ultrafiltration of crude bee venom - First, the crude venom solution of Vespa contagiosum was centrifuged for 25 minutes (room temperature, 3000×g) using an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The operation ended when the volume of the retained concentrate of 15 mL of the crude venom solution was about 300 μL. The inner tube obtained a retained concentrate of not less than 30 kDa, and the outer tube obtained the first filtrate. The filtrate was further centrifuged for 18 minutes using an ultrafiltration tube with a molecular weight cutoff of 10 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was about 300 μL. The inner tube obtained a retained concentrate of not less than 10 kDa but less than 30 kDa, and the outer tube obtained the second filtrate. The filtrate was further centrifuged for 35 minutes using an ultrafiltration tube with a molecular weight cutoff of 3 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was about 300 μL. The inner tube obtained a retained concentrate of not less than 3 kDa but less than 10 kDa, and the outer tube obtained the third filtrate. 10 μL of each concentrate was taken for activity detection experiment, and the remaining concentrate was freeze-dried.
[0047] (4) Antifungal and antibacterial activity test - Escherichia coli, Staphylococcus aureus and Candida albicans were used as indicator strains. The antibacterial activity of the three ultrafiltration concentrates was tested by the inhibition zone method. The results showed that all three concentrates showed significant inhibitory effects on the indicator strains. Figures 1 to 3 .
[0048] Antibacterial activity assay - filter paper method was used. Escherichia coli, Staphylococcus aureus and Candida albicans were cultured on agar slants overnight and sterilized saline was adjusted to 0.5 McFarland turbidimetric (1×10 8 CFU / mL), take 350 μL of each and add it to a plate (9 cm in diameter) filled with culture medium, stick a sterilized filter paper, add 3-5 μL of the sample solution to each filter paper, culture bacteria inverted at 36°C for 18-24 hours, and culture fungi at 28°C for 36 hours, measure the size of the inhibition zone, repeat 3 times for each sample, and take the average value.
[0049] (5) The whole venom of wasps is usually quite complex. In addition to proteins or peptides, there are other small molecules such as histamine. The three components (not less than 30kDa retained concentrate, not less than 10kDa but less than 30KDa retained concentrate and not less than 3kDa but less than 10KDa retained concentrate) shown by the antibacterial experiment in step (4) all have the activity of inhibiting fungi and bacteria. The present invention further performs SDS-PAGE experiments on each component obtained by ultrafiltration. It can be seen from the figure that the retained concentrates of not less than 3kDa but less than 10kDa and not less than 10kDa but less than 30kDa basically do not contain proteins or polypeptides at an SDS-PAGE resolution of 12%. The results show that the retained concentrate of not less than 30kDa contains protein, while the retained concentrates of not less than 10kDa but less than 30kDa and not less than 3kDa but less than 10kDa contain trace amounts of or no protein, see Figure 4 In other words, the overall antibacterial activity of wasp venom may be the result of the combined action of proteins or polypeptides and non-peptide substances. This invention further clarifies the active sites of wasp venom (based on molecular weight) through ultrafiltration segmentation, providing a theoretical basis and practical guidance for the subsequent diversified utilization of wasp venom, further enhancing the role of wasp farming in increasing farmers' income.
[0050] Example 2
[0051] (1) Collection of venom from the concave-striped wasp: Use low voltage pulses (12V) to stimulate the wasp to excrete venom on the collector. After drying under natural conditions, scrape it with a venom scraper to obtain the crude venom from the concave-striped wasp.
[0052] (2) Pretreatment of Crude Bee Venom: Weigh crude bee venom and dilute it with deionized water to a final concentration of 5.00 mg / mL. Centrifuge at room temperature for 5 min (10,000 × g) and discard the precipitate. If any floating or suspended debris remains in the supernatant, filter it with a 0.45 μm aqueous membrane.
[0053] (3) Ultrafiltration of Crude Bee Venom - First, the crude venom solution of Vespa contagiosum was centrifuged for 27 minutes (room temperature, 3000×g) using an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The operation was terminated when the volume of the retained concentrate of 15 mL of the crude venom solution was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 30 kDa, and the outer tube obtained the first filtrate. The filtrate was further centrifuged for 17 minutes using an ultrafiltration tube with a molecular weight cutoff of 10 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 10 kDa but less than 30 kDa, and the outer tube obtained the second filtrate. The filtrate was further centrifuged for 35 minutes using an ultrafiltration tube with a molecular weight cutoff of 3 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 3 kDa but less than 10 kDa, and the outer tube obtained the third filtrate. 10 μL of each concentrate was taken for activity detection experiment, and the remaining concentrate was freeze-dried.
[0054] (4) Antifungal and antibacterial activity assay - Using Escherichia coli, Staphylococcus aureus, and Candida albicans as indicator strains, the antibacterial activity of the three ultrafiltration concentrates was evaluated using the inhibition zone assay. All three concentrates exhibited significant inhibitory effects against the indicator strains.
[0055] Example 3
[0056] (1) Collection of venom from the concave-striped wasp: Use low voltage pulses (12V) to stimulate the wasp to excrete venom on the collector. After drying under natural conditions, scrape it with a venom scraper to obtain the crude venom from the concave-striped wasp.
[0057] (2) Pretreatment of Crude Bee Venom: Weigh crude bee venom and dilute it with deionized water to a final concentration of 2.00 mg / mL. Centrifuge at room temperature for 5 min (10,000 × g) and discard the precipitate. If any floating or suspended debris remains in the supernatant, filter it with a 0.45 μm aqueous membrane.
[0058] (3) Ultrafiltration of Crude Bee Venom - First, the crude venom solution of Vespa contagiosum was centrifuged for 30 minutes (room temperature, 3000×g) using an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The operation was terminated when the volume of the retained concentrate of 15 mL of the crude venom solution was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 30 kDa, and the outer tube obtained the first filtrate. The filtrate was further centrifuged for 17 minutes using an ultrafiltration tube with a molecular weight cutoff of 10 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 10 kDa but less than 30 kDa, and the outer tube obtained the second filtrate. The filtrate was further centrifuged for 35 minutes using an ultrafiltration tube with a molecular weight cutoff of 3 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 3 kDa but less than 10 kDa, and the outer tube obtained the third filtrate. 10 μL of each concentrate was taken for activity detection experiment, and the remaining concentrate was freeze-dried.
[0059] (4) Antifungal and antibacterial activity assay - Using Escherichia coli, Staphylococcus aureus, and Candida albicans as indicator strains, the antibacterial activity of the three ultrafiltration concentrates was evaluated using the inhibition zone assay. All three concentrates exhibited significant inhibitory effects against the indicator strains.
[0060] Example 4
[0061] (1) Collection of venom from the concave-striped wasp: Use low voltage pulses (12V) to stimulate the wasp to excrete venom on the collector. After drying under natural conditions, scrape it with a venom scraper to obtain the crude venom from the concave-striped wasp.
[0062] (2) Pretreatment of Crude Bee Venom: Weigh crude bee venom and dilute it with deionized water to a final concentration of 2.00 mg / mL. Centrifuge at room temperature for 5 min (10,000 × g) and discard the precipitate. If any floating or suspended debris remains in the supernatant, filter it with a 0.45 μm aqueous membrane.
[0063] (3) Ultrafiltration of crude bee venom - The crude venom solution of Vespa contagiosum was first centrifuged for 27 minutes (room temperature, 3000×g) using an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The operation was terminated when the volume of the retained concentrate of 15 mL of the crude venom solution was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 30 kDa, and the outer tube obtained the first filtrate. The filtrate was further centrifuged for 24 minutes using an ultrafiltration tube with a molecular weight cutoff of 10 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 10 kDa but less than 30 kDa, and the outer tube obtained the second filtrate. The filtrate was then centrifuged for 35 minutes using an ultrafiltration tube with a molecular weight cutoff of 3 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 3 kDa but less than 10 kDa, and the outer tube obtained the third filtrate. 10 μL of each concentrate was taken for activity detection experiment, and the remaining concentrate was freeze-dried.
[0064] (4) Antifungal and antibacterial activity assay - Using Escherichia coli, Staphylococcus aureus, and Candida albicans as indicator strains, the antibacterial activity of the three concentrates was evaluated using the inhibition zone assay. Results: The three concentrates exhibited significant inhibitory effects against the indicator strains.
[0065] Example 5
[0066] (1) Collection of venom from the concave-striped wasp: Use low voltage pulses (12V) to stimulate the wasp to excrete venom on the collector. After drying under natural conditions, scrape it with a venom scraper to obtain the crude venom from the concave-striped wasp.
[0067] (2) Pretreatment of Crude Bee Venom: Weigh crude bee venom and dilute it with deionized water to a final concentration of 2.00 mg / mL. Centrifuge at room temperature for 5 min (10,000 × g) and discard the precipitate. If any floating or suspended debris remains in the supernatant, filter it with a 0.45 μm aqueous membrane.
[0068] (3) Ultrafiltration of Crude Bee Venom - First, the crude venom solution of Vespa contagiosum was centrifuged for 27 minutes (room temperature, 3000×g) using an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The operation was terminated when the volume of the retained concentrate of 15 mL of the crude venom solution was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 30 kDa, and the outer tube obtained the first filtrate. The filtrate was further centrifuged for 32 minutes using an ultrafiltration tube with a molecular weight cutoff of 10 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 10 kDa but less than 30 kDa, and the outer tube obtained the second filtrate. The filtrate was further centrifuged for 40 minutes using an ultrafiltration tube with a molecular weight cutoff of 3 kDa (room temperature, 3000×g). At this time, the volume of the retained concentrate was approximately 300 μL. The inner tube obtained a retained concentrate of not less than 3 kDa but less than 10 kDa, and the outer tube obtained the third filtrate. 10 μL of each concentrate was taken for activity detection experiment, and the remaining concentrate was freeze-dried.
[0069] (4) Antifungal and antibacterial activity assay - Using Escherichia coli, Staphylococcus aureus, and Candida albicans as indicator strains, the antibacterial activity of the three concentrates was evaluated using the inhibition zone assay. Results: The three concentrates exhibited significant inhibitory effects against the indicator strains.
[0070] Thus, the present invention utilizes only ultrafiltration membranes, operating in reverse order from largest to smallest, using 30kDa, 10kDa, and 3kDa filters to sequentially intercept and freeze-dry the Vespa venom solution, thereby obtaining three active components with both antifungal (Candida albicans) and antibacterial (Escherichia coli and Staphylococcus aureus) properties. This method is simple to operate, highly efficient, and significantly reduces purification time. Furthermore, the entire process involves only physical separation, significantly avoiding inactivation of active components and significantly facilitating process scale-up.
[0071] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A method for separating and purifying active components of wasp venom, characterized in that: The following steps are involved: preparing crude wasp venom into a crude wasp venom solution; The crude venom solution was centrifuged for the first time using an ultrafiltration tube with a molecular weight cut-off of 30 kDa. The inner tube obtained a concentrate with a molecular weight cut-off of not less than 30 kDa, and the outer tube obtained the first filtrate. The first filtrate is subjected to a second centrifugation using an ultrafiltration tube with a molecular weight cut-off of 10 kDa, the inner tube obtains a concentrate with a cut-off of not less than 10 kDa but less than 30 kDa, and the outer tube obtains the second filtrate; The second filtrate was centrifuged for the third time using an ultrafiltration tube with a molecular weight cut-off of 3 kDa, and the inner tube obtained a concentrated solution with a cut-off of not less than 3 kDa but less than 10 kDa.
2. The separation and purification method according to claim 1, wherein The preparation method of the crude wasp venom comprises the following steps: Low-voltage pulses are used to stimulate wasp detoxification, and the crude wasp venom is obtained after the venom is dried; the voltage of the low-voltage pulses is 12V.
3. The separation and purification method according to claim 1 or 2, wherein The crude wasp venom is dissolved in deionized water to obtain a crude wasp venom solution. The concentration of the crude wasp venom solution is 30 μg / mL to 10 mg / mL.
4. The separation and purification method according to claim 3, wherein When preparing the crude wasp venom solution, the method further comprises centrifuging after dissolving to remove the precipitate; the centrifugal force of the centrifugation is 10,000×g, and the centrifugation time is 5 minutes.
5. The separation and purification method according to claim 1, wherein The centrifugal force of the first centrifugation is 3000×g, and the time of the first centrifugation is 10 to 30 minutes.
6. The separation and purification method according to claim 1, wherein The centrifugal force of the second centrifugation is 3000×g, and the time of the second centrifugation is 15 to 40 minutes.
7. The separation and purification method according to claim 1, wherein The centrifugal force of the third centrifugation is 3000×g, and the time of the third centrifugation is 25 to 60 minutes.
8. The separation and purification method according to any one of claims 1 to 7, characterized in that: The wasps include Vespa concave.
9. The retentate concentrate obtained by the separation and purification method according to any one of claims 1 to 8, characterized in that: The retained concentrate includes a retained concentrate of not less than 30 kDa, a retained concentrate of not less than 10 kDa but less than 30 kDa, and a retained concentrate of not less than 3 kDa but less than 10 kDa.
10. Use of the trapped concentrate obtained by the separation and purification method according to any one of claims 1 to 8 or the trapped concentrate according to claim 9 in inhibiting fungal and / or bacterial activity, characterized in that: The fungi include Candida albicans, and the bacteria include Escherichia coli and Staphylococcus aureus.