Application of rhamnolipid in preparation of functional product for preventing and controlling outbreak of aurelia

Abstract

The invention relates to application of rhamnolipid in preparation of a functional product for preventing and controlling outbreak of aurelia. The invention particularly relates to the application of the rhamnolipid in preparation of an aurelia aurita killing agent. The aurelia comprises at least one of aurelia scyphistoma, discoid and jellyfish. The rhamnolipid has high killing activity on aurelia scyphistoma, ephyra and jellyfish, the aurelia scyphistoma and ephyra can die completely when the concentration of the rhamnolipid is larger than 12 mg / L and the rhamnolipid is exposed for 48 h, and the aurelia jellyfish can die completely when the concentration of the rhamnolipid is larger than 18 mg / L and the rhamnolipid is exposed for 48 h. Meanwhile, compared with a botanical pesticide, the rhamnolipid has higher safety to other aquatic organisms.

Description

Technical Field

[0001] This invention relates to the field of ecological control technology for jellyfish infestations, and in particular to the application of a rhamnose glycolipid in the preparation of functional products for controlling moon jellyfish outbreaks. Background Technology

[0002] Under the combined effects of global climate change, overfishing, and near-shore environmental pollution, the frequency and intensity of jellyfish outbreaks in marine ecosystems have shown a significant upward trend. Moon jellyfish ( Aurelia coerulea Competition for food with commercially important fish species has led to the decline of fishery resources and triggered numerous blockages in the cooling systems of coastal power plants and nuclear power plants, causing significant economic losses. Therefore, effectively controlling the outbreak of moon jellyfish populations has become a major issue that urgently requires in-depth research.

[0003] Currently, patent application number 202511104554.0 discloses the application of tea saponin E3 as a killer of moon jellyfish polyps. The proposed extraction process for tea saponin E3 is simple, and tea saponin E3 exhibits strong killing activity against moon jellyfish polyps at low concentrations, with low application costs. However, tea saponin compounds also exhibit strong toxicity to fish, greatly limiting their application in nearshore moon jellyfish infestation control. In addition, patent application number 202310387878.4 discloses a method for inhibiting the transverse fission reproduction of moon jellyfish polyps using homosalate. This invention controls the moon jellyfish population and reduces the risk of outbreaks by reducing and slowing down the transverse fission proliferation rate of moon jellyfish polyps. However, such compounds are expensive to use and cannot completely kill moon jellyfish polyps.

[0004] In summary, the current efforts to eliminate or control the population of moon jellyfish still face serious secondary risks, high product costs, and incomplete eradication effects. There is an urgent need to develop new active substances that are safe, inexpensive, and highly effective against all stages of the moon jellyfish's life cycle in order to effectively prevent outbreaks of moon jellyfish. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing an application of rhamnolipin in the preparation of functional products for preventing and controlling moon jellyfish outbreaks.

[0006] This invention provides the application of rhamnolipin in the preparation of functional products for preventing and controlling moon jellyfish outbreaks.

[0007] Furthermore, the application of the rhamnose glycolipid in the preparation of a moon jellyfish killer.

[0008] Furthermore, the moon jellyfish includes at least one of the polyp and discoid forms. Furthermore, the concentration of the rhamnolipin is not less than 12 mg / L. Even more further, the concentration of the rhamnolipin is 12-31 mg / L.

[0009] Furthermore, the moon jellyfish includes the moon jellyfish medusa. Furthermore, the concentration of the rhamnolipin is not less than 18 mg / L. Even further, the concentration of the rhamnolipin is 18-31 mg / L.

[0010] This invention also provides a method for killing moon jellyfish with rhamnolipin, the method comprising the following steps: (1) Preparation of rhamnolipin stock solution: Prepare a rhamnolipin stock solution with a concentration of at least 1 g / L using seawater; (2) Dilute the rhamnolipin stock solution in the habitat of the polyps and / or discoids of the moon jellyfish to ensure that the concentration of rhamnolipin is not less than 12 mg / L; or The rhamnolipin stock solution was diluted in the habitat of the moon jellyfish to ensure that the concentration of rhamnolipin was not less than 18 mg / L.

[0011] Furthermore, the preparation method of the rhamnolipin mother liquor is as follows: weigh 1 g of rhamnolipin and place it in a 1L volumetric flask, add 1L of natural seawater that has been sand-filtered and treated with ultraviolet light, and sonicate until completely dissolved to obtain the rhamnolipin mother liquor.

[0012] The present invention also provides a moon jellyfish killer, wherein the active ingredient of the killer comprises rhamnolipid.

[0013] Furthermore, the killing agent is used to kill at least one of the moon jellyfish polyps, discoids, and medusa bodies. Beneficial effects

[0014] The rhamnolipin of this invention exhibits strong killing activity against the polyps, discoids, and medulla of the moon jellyfish. Exposure to rhamnolipin concentrations greater than 12 mg / L for 48 hours leads to complete death of the polyps and discoids of the moon jellyfish, while exposure to concentrations greater than 18 mg / L for 48 hours leads to complete death of the medulla. Furthermore, compared to existing jellyfish outbreak control agents such as tea saponin E3 and homosalate, rhamnolipin is inexpensive, readily degrades under natural environmental conditions, and has high safety for other aquatic organisms. Attached Figure Description

[0015] Figure 1 These are images showing the state of the polyps, discoids, and medulla of the moon jellyfish after 48 h and 96 h of treatment with 20 mg / L rhamnolipid. Figure 2The graph shows the statistical results of mortality of moon jellyfish polyps after treatment with different concentrations of rhamnolipin, as well as the toxicity regression curves after 48h and 96h of exposure. Figure 3 The graph shows the statistical results of mortality of moon jellyfish discoids after treatment with different concentrations of rhamnolipin, as well as the toxicity regression curves after 48h and 96h of exposure. Figure 4 The graph shows the statistical results of mortality rates of moon jellyfish after treatment with different concentrations of rhamnolipin, as well as the toxicity regression curves after 48 hours and 96 hours of exposure. Detailed Implementation

[0016] This invention relates to the application of rhamnolipin in the preparation of functional products for preventing and controlling moon jellyfish outbreaks.

[0017] The moon jellyfish used in this invention ( A. coerulea The polyps, discoids, and medulla were provided by the Muping Integrated Environmental Experiment Station Laboratory of the Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences.

[0018] Rhamnolipids are a class of glycolipid biosurfactants obtained by fermentation of *Pseudomonas aeruginosa*. Rhamnolipids can rapidly disrupt microbial cell membranes, thereby effectively inhibiting the growth and reproduction of pathogenic microorganisms such as bacteria, fungi, and nematodes. They have been widely used as plant protection additives for crop disease control. Simultaneously, rhamnolipids can ultimately be utilized as nutrients by environmental microorganisms, plankton, and aquatic plants, exhibiting advantages such as high safety and environmental friendliness. The rhamnolipids used in this invention were purchased from Sigma-Aldrich.

[0019] The moon jellyfish referred to in this invention includes at least one of the following: polyp, discoid body, and medusa body. The state of the moon jellyfish polyps, discoid bodies, and medusa bodies after rhamnolipin treatment was observed: Healthy moon jellyfish polyps, discoid bodies, and medusa bodies were selected and cultured in a 19°C constant temperature incubator. Rhamnolipin stock solution was added to the culture water, controlling the final concentration of rhamnolipin to be 20 mg / L. The exposure experiment lasted for 96 hours. Observations were made before the start of the rhamnolipin exposure experiment, and at 48 hours and 96 hours of exposure to determine the state of the moon jellyfish polyps, discoid bodies, and medusa bodies, and photographs were taken and recorded.

[0020] The states of the polyp, discoid body, and medulla of the moon jellyfish before the start of the rhamnolipin exposure experiment, and 48 and 96 hours after the exposure experiment are shown in the figure. Figure 1 .

[0021] Before the rhamnolipin exposure experiment, the polyps of the moon jellyfish were in good condition, with extended tentacles and active predation behavior. After 48 hours of exposure to 20 mg / L rhamnolipin, the tentacles of the moon jellyfish polyps contracted, showed no obvious response to needle pricking, and a small amount of ulceration appeared at the tissue edges. After 96 hours of exposure to 20 mg / L rhamnolipin, the ulceration of the moon jellyfish polyps worsened, and a large amount of fragmented tissue appeared.

[0022] Before the rhamnolipin exposure experiment, the moon jellyfish discoids were in a relaxed state with normal contraction frequency and active feeding behavior. After 48 hours of exposure to 20 mg / L rhamnolipin, the moon jellyfish discoids lay still at the bottom of the six-well plate, and the tentacles retracted and the edges became blurred. After 96 hours of exposure to 20 mg / L rhamnolipin, the moon jellyfish discoids further contracted and showed no response to needle pricking.

[0023] Before the rhamnolipin exposure experiment, the moon jellyfish was in good growth condition with intact tissue structure, and its oral arms and tentacles were naturally extended, with normal umbrella contraction frequency. After 48 hours of exposure to 20 mg / L rhamnolipin, the moon jellyfish's umbrella became thinner and stopped contracting, and a small amount of fragmented tissue appeared around the individual. After 96 hours of exposure to 20 mg / L rhamnolipin, the edges of the moon jellyfish's umbrella ulcerated, the tentacles fell off, and a large amount of fragmented tissue appeared around the individual.

[0024] This invention also provides a method for killing moon jellyfish with rhamnolipin, comprising the following steps: (1) Preparation of rhamnolipin mother liquor: Weigh 1 g of rhamnolipin and place it in a 1L volumetric flask. Add 1L of natural seawater that has been sand filtered and treated with ultraviolet light. Shake until completely dissolved to obtain rhamnolipin mother liquor. (2) Dilute the rhamnolipin stock solution in the living environment of the polyps and / or discs of the moon jellyfish so that the concentration of rhamnolipin is not less than 12 mg / L; or dilute the rhamnolipin stock solution in the living environment of the moon jellyfish so that the concentration of rhamnolipin is not less than 18 mg / L.

[0025] Experimental Example 1: Test of the bactericidal activity of rhamnolipin against the polyps of moon jellyfish Healthy moon jellyfish polyps were selected and transferred to 12-well plates, with 12 polyps per well. After one week of temporary rearing, a simulated exposure experiment was conducted in the laboratory. One control group and five rhamnolipin concentration levels were set up, with each treatment group containing three wells as parallels, for a total of 36 polyps. The exposure experiment lasted for 96 hours, during which the seawater temperature was 18.5±0.3℃, pH was 8.12±0.03, salinity was 31.4±0.4, and the light conditions were set to light:dark = 12 h:12 h. After 48 h and 96 h of exposure, the status of the moon jellyfish polyps was observed and the number of dead individuals was recorded. Probability analysis was used to calculate the toxicity regression curve and LC50 value of rhamnolipin to moon jellyfish polyps.

[0026] The mortality rates of moon jellyfish polyps after treatment with different concentrations of rhamnolipin for 48 h and 96 h are shown in the table below. Figure 2 A. By Figure 2 A indicates that after 48 hours of exposure to 10 mg / L rhamnolipin, 86.7% of the polyps of the moon jellyfish died, and after 96 hours of continued exposure, 96.7% of the polyps died; while 48 hours of exposure to 12 mg / L rhamnolipin resulted in the death of 100% of the polyps.

[0027] The toxicity regression curves of moon jellyfish polyps treated with rhamnolipin for 48 h and 96 h are shown in the figure. Figure 2 B, C. Calculations show that after treatment with rhamnolipin for 48 h, the median lethal concentration (LC50) of *Lycopodium clavatum* was 6.74 mg / L, with a 95% confidence interval of [6.13, 7.32]; after treatment for 96 h, the median lethal concentration was 6.20 mg / L, with a 95% confidence interval of [5.67, 6.71].

[0028] Experimental Example 2: Test of the bactericidal activity of rhamnolipin against the discoid body of the moon jellyfish Twelve moon jellyfish discoids in good condition were selected from 6-well plates and cultured for one week before a simulated exposure experiment was conducted in the laboratory. One control group and five rhamnolipin concentration levels were set up, with each treatment group containing three wells as parallels, for a total of 36 discoids. The exposure experiment lasted for 96 hours, during which the seawater temperature was 18.5±0.4 ℃, pH was 8.10±0.02, salinity was 31.5±0.4, and the light conditions were set at a light:dark ratio of 12 h:12 h. After 48 h and 96 h of exposure, the condition of the moon jellyfish discoids was observed and the number of dead individuals was recorded. Probability analysis was used to determine the toxicity regression curve and LC50 value of rhamnolipin to moon jellyfish discoids.

[0029] The mortality rates of moon jellyfish discoid bodies treated with different concentrations of rhamnolipin for 48 h and 96 h are shown in the table below. Figure 3 A. By Figure 3 A indicates that after 48 hours of exposure to 10.5 mg / L rhamnolipin, 70.83% of the moon jellyfish discoids died, and after 96 hours of continued exposure, 95.8% of the moon jellyfish discoids died; while 48 hours of exposure to 12 mg / L rhamnolipin resulted in the death of 100% of the moon jellyfish discoids.

[0030] The toxicity regression curves of *Gnaphalium affine* discoids treated with rhamnolipin for 48 h and 96 h are shown in the figure. Figure 3 B, C. Calculations show that after treatment with rhamnolipin for 48 h, the median lethal concentration (LC50) of *Syngonium spp.* was 8.58 mg / L, with a 95% confidence interval of [7.79, 8.86]; after treatment for 96 h, the median lethal concentration was 7.97 mg / L, with a 95% confidence interval of [7.67, 8.25].

[0031] Experimental Example 3: Test of the bactericidal activity of rhamnolipin against moon jellyfish. Healthy moon jellyfish were selected and placed in 10L glass tanks, with 12 jellyfish per tank. A simulated exposure experiment was conducted in the laboratory, with one control group and five rhamnolipin concentration levels. Each treatment group consisted of three tanks as parallels, totaling 36 jellyfish. The exposure experiment lasted for 96 hours, during which the seawater temperature was 18.1±0.3 ℃, pH was 8.09±0.02, and salinity was 31.2±0.1. The light conditions were set at a light:dark ratio of 12 h:12 h. After 48 h and 96 h of exposure, the condition of the moon jellyfish was observed and the number of dead individuals was recorded. The toxicity regression curve and LC50 value of rhamnolipin were obtained using probability analysis.

[0032] The mortality rates of moon jellyfish treated with different concentrations of rhamnolipin for 48 hours and 96 hours are shown in the table below. Figure 4 A. By Figure 4 A indicates that after 48 hours of exposure to 16 mg / L rhamnolipin, 64.55% of the moon jellyfish died; with continued exposure up to 96 hours, 83.3% of the moon jellyfish died. However, 48 hours of exposure to 18 mg / L rhamnolipin resulted in 100% mortality of the moon jellyfish. The toxicity regression curves for moon jellyfish treated with rhamnolipin after 48 hours and 96 hours are shown in [the table / incomplete]. Figure 4B, C. Calculations show that after 48 h of treatment with rhamnolipin, the median lethal concentration (LC50) of the moon jellyfish was 14.72 mg / L, with a 95% confidence interval of [14.29, 15.15]; after 96 h of treatment, the median lethal concentration was 13.92 mg / L, with a 95% confidence interval of [13.51, 14.35].

[0033] The results show that rhamnolipin has strong killing activity against the polyps, discoids, and medulla of moon jellyfish, and can cause tissue ulceration. It can be applied to the subsequent product development in the field of moon jellyfish outbreak prevention and control, such as the application of rhamnolipin in the preparation of functional products for the prevention and control of moon jellyfish outbreaks, and the preparation of moon jellyfish killers for killing the polyps, discoids, and medulla of moon jellyfish.

[0034] This invention can be summarized in other specific forms that do not depart from the spirit or essential features of the invention. Therefore, in all respects, the above embodiments of the invention should be considered illustrative only and not limiting, while the claims define the scope of the invention. The foregoing description does not define the scope of the invention; therefore, any changes within the meaning and scope equivalent to the claims should be considered to be included within the scope of the claims.

Claims

1. Application of a rhamnolipin in the preparation of functional products for preventing and controlling moon jellyfish outbreaks.

2. The application of rhamnolipin as described in claim 1 in the preparation of functional products for preventing and controlling moon jellyfish outbreaks, characterized in that, The application of rhamnolipin in the preparation of moon jellyfish killer.

3. The application of rhamnolipin as described in claim 2 in the preparation of functional products for preventing and controlling moon jellyfish outbreaks, characterized in that, The moon jellyfish includes at least one of the polyp and discoid forms.

4. The application of the rhamnose glycolipid as described in claim 3 in the preparation of functional products for preventing and controlling moon jellyfish outbreaks, characterized in that, The concentration of the rhamnolipin is not less than 12 mg / L.

5. The application of the rhamnose glycolipid as described in claim 2 in the preparation of functional products for preventing and controlling moon jellyfish outbreaks, characterized in that, The moon jellyfish mentioned includes the moon jellyfish medullaris.

6. The application of the rhamnose glycolipid as described in claim 5 in the preparation of functional products for preventing and controlling moon jellyfish outbreaks, characterized in that, The concentration of the rhamnolipin is not less than 18 mg / L.

7. A method for killing moon jellyfish with rhamnolipin, characterized in that, The method includes the following steps: (1) Preparation of rhamnolipin stock solution: Prepare a rhamnolipin stock solution with a concentration of at least 1 g / L using seawater; (2) Dilute the rhamnolipin stock solution in the habitat of the polyps and / or discoids of the moon jellyfish to ensure that the concentration of rhamnolipin is not less than 12 mg / L; or The rhamnolipin stock solution was diluted in the habitat of the moon jellyfish to ensure that the concentration of rhamnolipin was not less than 18 mg / L.

8. The method for killing moon jellyfish with rhamnolipin as described in claim 7, characterized in that, The preparation method of the rhamnolipin mother liquor is as follows: weigh 1 g of rhamnolipin and place it in a 1L volumetric flask, add 1L of natural seawater that has been sand-filtered and treated with ultraviolet light, and sonicate until completely dissolved to obtain the rhamnolipin mother liquor.

9. A jellyfish killer, characterized in that, The active ingredient in the insecticide includes rhamnolipin.

10. The jellyfish killer as described in claim 9, characterized in that, The killing agent is used to kill at least one of the polyps, discoids, and medulla of the moon jellyfish.

Images