Sea urchin tubulopod extract with high antibacterial activity and preparation method and application of sea urchin tubulopod extract
By extracting antibacterial active ingredients from the red tube foot of the middle ball sea urchin to prepare sea urchin duct foot extract, the problem of Vibrio infection in sea urchin breeding was solved, effective inhibition of Vibrio and improved the immunity and survival rate of sea urchin.
Patent Information
- Application Number
- CN202411988358.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-04
AI Technical Summary
During sea urchin breeding, sea urchin is susceptible to Vibrio infection and causes diseases. The existing technology lacks effective antibacterial measures, which affects the immunity and survival rate of sea urchin.
The ingredients with high antibacterial activity were extracted from the red tube foot of the middle ball sea urchin, and the extract with significant antibacterial effect was obtained by mixing with acidic ethanol solution, centrifuging, adsorption resin treatment and other steps.
The chuckling duct foot extract has a significant inhibitory effect on the pathogenic bacteria of Vibrio, which can effectively prevent and control bacterial invasion in aquaculture, reduce the incidence and mortality rate, and increase the profits of aquatic products.
Smart Images
Figure CN119745928A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of aquaculture, and in particular to a sea urchin tube foot extract with high antibacterial activity, and a preparation method and application thereof. Background Art
[0002] The intermediate ball sea urchin (Strongylocentrotus intermedius), also known as the Yezo sea urchin, belongs to the class Echinoides in the phylum Echinodermata. It is one of the important marine echinoderm resources and has extremely high nutritional and medicinal value. As a high-end seafood ingredient, sea urchins are fully recognized and loved by consumers, and their breeding prospects are broad. In the process of sea urchin breeding, they are faced with the infection of Vibrio in seawater and cause diseases. Sea urchins with strong antibacterial ability have better immunity and survival rate during the breeding process. The tube feet are important movement and sensory organs of sea urchins. In the living state, the tube feet of sea urchins extend out of the body, which is easy to observe and collect. After collecting the tube feet, the sea urchins can continue to be cultured in seawater. Therefore, the tube feet of sea urchins have the potential to become a source of natural bioactive substances. However, there is currently no research on the extraction and preparation of bioactive substances in sea urchin tube feet, especially antibacterial substances, and there is also a lack of application research on sea urchin tube foot extracts. Summary of the invention
[0003] The purpose of the present invention is to provide a sea urchin tube foot extract with high antibacterial activity and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. The present invention extracts a component with high antibacterial activity from the red tube foot of the sea urchin. It has been verified that the red tube foot extract has a significant inhibitory effect on Vibrio pathogenic bacteria, and can be used to prevent and control pathogenic bacteria in aquaculture, reduce the incidence and mortality rate in aquaculture, and increase aquatic product income.
[0004] To achieve the above object, the present invention provides the following solutions:
[0005] The invention provides an application of a sea urchin tube foot extract in preparing an antibacterial agent. The sea urchin tube foot extract is extracted from the red tube foot of the intermedia ball sea urchin.
[0006] Strongylocentrotus intermedius, also known as the Yezo sea urchin, has tube feet that can be roughly divided into red and white due to different pigment cell contents. The present invention has found that the red tube foot extract of the intermediate ball sea urchin has a significant antibacterial effect, which is significantly higher than the white tube foot extract. Therefore, the present invention uses the red tube foot of the intermediate ball sea urchin as a raw material to prepare a sea urchin tube foot extract with high antibacterial activity.
[0007] Furthermore, the antibacterial agent has the function of inhibiting the activity of pathogenic bacteria of the genus Vibrio.
[0008] Optionally, the Vibrio pathogenic bacteria include Vibrio alginolyticus, Vibrio Parahaemolyticus, Vibrio vulnificus, Vibrio anguillarum and / or Vibrio harveyi.
[0009] The present invention also provides a method for preparing the sea urchin tube foot extract in the above application, comprising the following steps:
[0010] (1) grinding the red tube foot tissue of the intermedius sea urchin into a slurry, mixing the obtained slurry with an acidic ethanol solution for reaction, centrifuging, and collecting the supernatant;
[0011] (2) The supernatant is adsorbed and eluted by a macroporous adsorption resin, and then dried to obtain the sea urchin tube foot extract.
[0012] Furthermore, the mass ratio of the slurry to the acidic ethanol solution is 1:(2-4).
[0013] Optionally, the acidic ethanol solution includes a hydrochloric acid ethanol solution or a sulfuric acid ethanol solution.
[0014] Furthermore, the mixing reaction time is 1.5-3h.
[0015] Furthermore, the adsorption time is 2-3h.
[0016] Furthermore, the eluent during the elution is anhydrous ethanol, and the elution time is 0.5-2h.
[0017] The invention also provides a sea urchin tube foot extract prepared by the preparation method.
[0018] The present invention also provides an antibacterial agent, which comprises the sea urchin tube foot extract and auxiliary materials.
[0019] The present invention discloses the following technical effects:
[0020] The present invention proves through experiments that the red tube foot extract of the intermedia ball sea urchin has good anti-Vibrio ability and has higher antibacterial activity than the white tube foot extract. It can be used to inhibit pathogenic bacteria of the genus Vibrio and prevent and treat Vibrio-related diseases, improve the antibacterial ability of the intermedia ball sea urchin, accelerate the breeding process of sea urchins, effectively prevent sea urchin diseases caused by Vibrio during the breeding process, and improve the economic benefits of breeding sea urchins and other aquatic organisms. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0022] Figure 1 The appearance and morphological characteristics of the red-tube-footed intermediate sea urchin and the white-tube-footed intermediate sea urchin;
[0023] Figure 2 The results of drug sensitivity test of sea urchin red tube foot extract and sea urchin white tube foot extract;
[0024] Figure 3 The results of drug sensitivity test of sea urchin red tube foot extract, oxytetracycline and sterile water;
[0025] Figure 4 The test results of the minimum inhibitory concentration test of sea urchin red tube foot extract;
[0026] Figure 5 These are the results of in vivo antibacterial experiments of sea urchin red tube foot extract, sea urchin red tube foot extract, positive control and negative control; * indicates significant difference from the negative control (P<0.05). DETAILED DESCRIPTION
[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0032] The intermediate ball sea urchins used in the embodiments of the present invention were purchased from Laizhou breeding farm, including intermediate ball sea urchins with red tube feet and intermediate ball sea urchins with white tube feet, and their morphology is as follows: Figure 1 shown.
[0033] Example 1 Preparation of sea urchin red tube foot extract
[0034] 1. Place the middle ball sea urchin with red tube feet into sea water, cut off the spines on its back, and after the tube feet are extended, quickly cut off the tube foot tissue to obtain the red tube foot tissue.
[0035] 2. Quickly collect the cut tube foot tissue with a 1mL pipette, place it in a 1.5mL EP tube, centrifuge to remove excess seawater, and wash with 1×PBS.
[0036] 3. Grind the podia tissues into a homogenate, add hydrochloric acid ethanol solution (30% HCl + 70% ethanol) at a mass ratio of 1:3, and mix thoroughly on a rotary mixer at room temperature for 2 h.
[0037] 4. Centrifuge the mixture produced in the previous step in a refrigerated centrifuge at 4°C and 12,000 rpm for 10 minutes, and aspirate the supernatant.
[0038] 5. Place the supernatant into NKA-9 macroporous adsorption resin (S25839-250g) and fully adsorb on a rotary mixer at room temperature for 2 hours.
[0039] 6. Clean the macroporous adsorption resin with deionized water, then soak it in anhydrous ethanol for 1 hour to elute the tube foot extract from the adsorption resin.
[0040] 7. The eluted solution was freeze-dried to obtain red tube foot extract powder, which was frozen at -20°C and stored away from light.
[0041] Comparative Example 1 Preparation of Sea Urchin White Tube Foot Extract
[0042] 1. Place the middle ball sea urchin with white tube feet into sea water, cut off the spines on its back, and after the tube feet are extended, quickly cut off the tube foot tissue to obtain the white tube foot tissue.
[0043] 2. Quickly collect the cut tube foot tissue with a 1mL pipette, place it in a 1.5mL EP tube, centrifuge to remove excess seawater, and wash with 1×PBS.
[0044] 3. Grind the tube feet tissues into a homogenate, add hydrochloric acid ethanol solution (30% HCl + 70% ethanol) at a mass ratio of 1:3, and mix thoroughly on a rotary mixer at room temperature for 2 hours.
[0045] 4. Centrifuge the mixture produced in the previous step in a refrigerated centrifuge at 4°C and 12,000 rpm for 10 minutes, and aspirate the supernatant.
[0046] 5. Place the supernatant into NKA-9 macroporous adsorption resin (S25839-250g) and fully adsorb on a rotary mixer at room temperature for 2 hours.
[0047] 6. Clean the macroporous adsorption resin with deionized water, then soak it in anhydrous ethanol for 1 hour to elute the tube foot extract from the adsorption resin.
[0048] 7. The eluted solution was freeze-dried to obtain the white tube foot extract powder, which was frozen at -20°C and stored away from light.
[0049] Test Example 1
[0050] The antibacterial function of the sea urchin tube foot extract prepared in Example 1 and Comparative Example 1 was verified, and the verification test was as follows:
[0051] 1. Drug sensitivity test
[0052] (1) Prepare 2216E agar medium: weigh 5.24 g of 2216E agar, dissolve it in 100 mL of deionized water, sterilize it by autoclaving at 121°C for 15 min, cool it to about 50°C, pour it into a sterile culture dish, and wait for it to solidify before use.
[0053] 200 μL of Vibrio alginolyticus bacterial solution (concentration of bacterial solution was 0.8×10 8 CFU / mL) was evenly spread on 2216E agar medium using a spreading rod.
[0054] (2) Dissolve the sea urchin red tube foot extract and the sea urchin white tube foot extract in sterile ultrapure water to a concentration of 1000 μg / mL. Use sterile tweezers to pick up a blank drug sensitivity paper (6 mm in diameter) and fully immerse it in the sea urchin extract solution.
[0055] (3) Place the drug-sensitive paper strips soaked with the drug solution in the culture medium coated with Vibrio, use sterile ultrapure water as the negative control group, and use 1000 μg / mL oxytetracycline solution as the positive control group to conduct the Vibrio inhibition experiment. Set up two replicate plates for each drug solution, culture at 28°C for 22 hours, and observe whether a transparent antibacterial zone appears around the paper strips.
[0056] The antibacterial results of different sea urchin tube foot extracts are as follows Figure 2 As shown in the figure, the red tube foot extract showed an obvious inhibition zone around the paper in the culture medium, and the arrow indicated a transparent inhibition zone, but no obvious inhibition zone appeared in the white tube foot extract, indicating that the red tube foot extract had a significant inhibitory effect on the growth of Vibrio, and the red tube foot sea urchin had a higher antibacterial activity than the white tube foot sea urchin. The size of the inhibition zone of the two sea urchin tube foot extracts was counted, and the average size of the inhibition zone of each liquid was taken, and the inhibition rate was calculated according to the following formula. The results are shown in Table 1.
[0057] Formula: Inhibition rate (%) = (colony radius of control group - colony radius of treatment group) / colony radius of control group × 100%.
[0058] Table 1 Antibacterial rate of different sea urchin tube foot extracts
[0059] Liquid Antibacterial rate% Sea Urchin Red Tube Podia Extract 85.0±0.4 Sea Urchin White Tube Podia Extract 10.0±0.3
[0060] The full picture of the culture medium for the drug sensitivity test of sea urchin red tube foot extract is as follows Figure 3 As shown, it can be seen that obvious transparent inhibition zones appeared around the drug-susceptibility paper strips soaked in sea urchin red tube foot extract and oxytetracycline, and the growth of Vibrio alginolyticus was inhibited, while the growth of Vibrio in other parts of the culture medium was normal. However, no inhibition zones appeared around the drug-susceptibility paper strips soaked in sterile ultrapure water in the negative control group.
[0061] 2. Minimum inhibitory concentration determination experiment
[0062] The minimum inhibitory concentration (MIC) of the sea urchin tube foot extract prepared in Example 1 was determined by the two-fold dilution method. Vibrio alginolyticus was selected as the test bacteria, and the sea urchin tube foot extract solution was diluted in a gradient manner by the two-fold dilution method. The alginolytic Vibrio bacterial solution was treated respectively, and the culture was kept at 28°C for 24 hours. The growth of the bacterial solution was observed, and the lowest concentration at which no bacteria grew was taken as the MIC of the sea urchin tube foot extract. The results are shown in FIG. Figure 4 As shown in Table 2. It can be seen that the drug-sensitive paper pieces soaked in 500μg / mL and 100μg / mL sea urchin red tube foot extracts have obvious inhibition zones around them, and the inhibition rates are all above 60%; while the drug-sensitive paper pieces soaked in 50μg / mL sea urchin red tube foot extracts have no obvious inhibition zones around them, and the inhibition rates are less than 10%; and the drug-sensitive paper pieces soaked in 25μg / mL sea urchin red tube foot extracts have no inhibition zones around them. Therefore, the minimum inhibitory concentration of red tube foot extract is 50μg / mL.
[0063] Table 2 Antibacterial rate of sea urchin red tube foot extracts at different concentrations
[0064] Concentration of sea urchin red tube foot extract μg / mL Antibacterial rate% 500 79.8±0.8 100 62.0±0.9 50 9.0±0.3
[0065] 3. Animal experiments
[0066] The cultured intermediate sea urchins were treated with Vibrio alginolyticus as the test bacteria, and then the sea urchin tube foot extract prepared in Example 1 and Comparative Example 1 was used to conduct an in vivo antibacterial experiment. 240 intermediate sea urchins with a diameter of 2-3 cm were randomly placed into 8 breeding tanks, 30 in each tank, and they were set up into 4 groups: an experimental group of sea urchin red tube foot extract, an experimental group of sea urchin white tube foot extract, a positive control group of oxytetracycline, and a negative control group of sterilized seawater. First, 50 μL of 1000 CFU / mL of Vibrio alginolyticus was injected into each intermediate sea urchin, and then 50 μL of 1000 μg / mL of sea urchin red tube foot extract and sea urchin white tube foot extract were injected into the intermediate sea urchins in the experimental group; 50 μL of sterilized seawater was injected into the negative control group; and 50 μL of 1000 μg / mL of oxytetracycline solution was injected into the positive control group. The survival rate of the intermediate sea urchins was counted, and the results are as follows Figure 5 It can be seen that the sea urchin red tube foot extract can reduce the death of sea urchins caused by Vibrio and improve the survival rate.
[0067] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. Use of a sea urchin tube foot extract in the preparation of an antibacterial agent, characterized in that: The sea urchin tube foot extract is extracted from the red tube foot of the intermedius sea urchin.
2. The use according to claim 1, characterized in that: The antibacterial agent has the function of inhibiting the activity of pathogenic bacteria of the genus Vibrio.
3. The use according to claim 2, characterized in that: The pathogenic bacteria of the genus Vibrio include Vibrio alginolyticus, Vibrio Parahaemolyticus, Vibrio vulnificus, Vibrio anguillarum and / or Vibrio harveyi.
4. A method for preparing a sea urchin tube foot extract for use according to any one of claims 1 to 3, characterized in that: The steps include: (1) grinding the red tube foot tissue of the intermedius sea urchin into a slurry, mixing the obtained slurry with an acidic ethanol solution for reaction, centrifuging, and collecting the supernatant; (2) The supernatant is adsorbed and eluted by a macroporous adsorption resin, and then dried to obtain the sea urchin tube foot extract.
5. The preparation method according to claim 4, characterized in that: The mass ratio of the slurry to the acidic ethanol solution is 1:(2-4).
6. The preparation method according to claim 4, characterized in that: The mixing reaction time is 1.5-3h.
7. The preparation method according to claim 4, characterized in that: The adsorption time is 2-3h.
8. The preparation method according to claim 4, characterized in that: The eluent during the elution is anhydrous ethanol, and the elution time is 0.5-2h.
9. A sea urchin tube foot extract obtained by the preparation method according to any one of claims 4 to 8.
10. An antibacterial agent, characterized in that The antibacterial agent comprises the sea urchin tube foot extract according to claim 9 and auxiliary materials.