Fusarium rubrum GXIMD00551 and application thereof
By screening and identifying the oil and fat components in the new strain Fusariumincarnatum GXIMD00551 and its fermentation products, low-cost antifoulants were prepared, which solved the problems of low content and high production costs of natural sources, and achieved effective inhibition and antifouling effects on marine polluted organisms.
Patent Information
- Application Number
- CN202411807427.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-10
- Publication Date
- 2025-03-25
AI Technical Summary
The existing natural source antifoulants have low content, high production costs, and require complex extraction and purification processes, which limit their application in the field of marine antifouling.
A new strain Fusariumincarnatum GXIMD00551 and its oil and fat components in its fermentation products were screened and identified. Low-cost, easy-to-extract antifouling agents were prepared by extraction of petroleum ether, diethyl ether, dichloromethane or chloroform.
This oil and fat component has a good inhibitory effect on marine defiled bacteria and barnacle larvae, can significantly reduce the adhesion of defiled organisms, and achieve the same antifouling effect as commonly used antifouling agents on the market. At the same time, because fungi are easy to amplify and ferment, their production costs are low, and they have broad application prospects.
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Figure CN119685175A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, in particular to Fusarium erythrocyticum GXIMD00551 and applications thereof. Background Art
[0002] Marine biofouling refers to the phenomenon that fouling organisms in seawater attach and gather on the surface of substrates soaked in seawater, which has an adverse impact on human production activities and the marine ecological environment. General marine fouling organisms can be divided into two categories, one is micro-fouling organisms, such as fouling bacteria and fungi, and the other is large-scale fouling organisms, such as seaweed and fouling animals barnacles, bryozoans, etc. Marine biofouling has a wide impact on human daily production and life. They adsorb, grow, reproduce, metabolize and die on the surface of marine facilities. The final biofouling will cause great damage to marine engineering facilities. In terms of ship shipping, marine fouling organisms attached to the surface of ships will increase their mass and surface friction resistance, leading to higher fuel consumption, causing economic losses, and emitting more greenhouse gases, thereby damaging the environment. Among the many anti-fouling technologies, anti-fouling coatings containing heavy metals are the most economical, effective and widely applicable methods, accounting for about 76% of the market share. However, traditional anti-fouling coatings will release harmful anti-fouling agents that are difficult to decompose, such as organotin, which pollutes the ecological environment. At the same time, it can enter the human food chain through fish, shellfish and other organisms, seriously threatening human health. At present, most countries have restricted the use of traditional antifouling agents. Antifouling agents containing heavy metals such as copper will also harm the marine ecological environment, so the development of green antifouling coatings has become an important direction for marine antifouling.
[0003] With the advancement of science and technology, the extraction of natural ingredients with antifouling activity from marine organisms is becoming a research hotspot for green antifouling coatings. These natural antifouling agents have the advantages of being environmentally friendly, having little harm to marine organisms, and being easily degraded naturally. The structural types include fatty acids, phenols, diphenyl ethers, etc. Bhattarai et al. isolated two organic acids, 2-hydroxytetradecanoic acid and oleic acid, from the chloroform extract of a marine bacterium (Shewanellaoneidensis SCH0402), and both have marine antifouling activity. However, compared with industrially synthesized antifouling agents, natural antifouling agents have less content, high production costs, and require complex extraction and purification processes, which restricts the application of natural antifouling agents. Summary of the invention
[0004] In view of the above, it is necessary to find biological preparations suitable for the preparation of antifouling agents in response to the problems of industrially synthesized antifouling agents, naturally derived antifouling agents with low content, high production costs, and the need for complex extraction and purification processes, and to develop low-cost, easily extractable antifouling agents based on the easy reproduction characteristics of organisms.
[0005] To achieve the above purpose, the present invention screened out a new strain: (Fusariumincarnatum) strain GXIMD00551, which is classified and named: Fusariumincarnatum GXIMD00551, and the preservation number is GDMCCNO.65402; the strain is preserved in Guangdong Provincial Microbiological Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is November 1, 2024.
[0006] Furthermore, the ITS sequence of the strain is shown as SEQ ID NO.1.
[0007] The present invention also includes a bacterial agent containing the Fusarium in carnatum strain GXIMD00551.
[0008] The present invention also includes the oil extract of the Fusarium in carnatum strain GXIMD00551.
[0009] Furthermore, the extract contains the following components: methyl palmitate with a relative content of 2.73%, palmitic acid with a relative content of 28.35%, 9-octadecenoic acid methyl ester with a relative content of 1.38%, 2,5-di-tert-butylphenol with a relative content of 1.2%, ethyl palmitate with a relative content of 0.6%, methyl linoleate with a relative content of 1.7%, methyl stearate with a relative content of 1.43%, dodecyl-9-ynyl chloroacetate with a relative content of 34.8%, cis-13-octadecenoic acid with a relative content of 15.11%, stearic acid with a relative content of 8.96%, butyl hexadecanoate with a relative content of 1.5%, 2-chloroethyl linoleate with a relative content of 0.72%, trifluoroacetic acid oleyl alcohol with a relative content of 0.52% and / or butyl octadecanoate with a relative content of 0.54%.
[0010] The present invention also includes a method for preparing the oil extract, which comprises: fermenting the Fusarium incarnatum strain GXIMD00551, and then extracting the oil components in the fermentation product with petroleum ether, ether, dichloromethane or chloroform.
[0011] The present invention also includes the use of the Fusarium in carnatum strain GXIMD00551, the bacterial agent or the lipid extract in the preparation of an antifouling agent.
[0012] The present invention also includes the use of the Fusarium in carnatum strain GXIMD00551, the bacterial agent or the lipid extract in inhibiting the growth of fouling bacteria and / or barnacle larvae.
[0013] Furthermore, the fouling bacteria is Vibrio Pelagius; and the barnacle larvae is Amphibalanusamphitrite.
[0014] The present invention also includes a method for applying the oil extract to prevent fouling, which comprises applying the oil extract to the surface of an object to be prevented from fouling.
[0015] The present invention has the following beneficial effects: the strain GXIMD00551 of the present invention is obtained by screening and separation by the applicant from the salt fields of Beihai, Guangxi. After activity detection, the strain, especially the oil and fat extract components after fermentation, have a good inhibitory effect on marine fouling bacteria, fouling animal barnacle larvae, etc., and can be used as an antifouling agent. Experiments have proved that after the oil and fat extract components of the strain GXIMD00551 of the present application are used to coat the PVC board, the attachment of fouling organisms is significantly reduced, and can reach the same level as the commonly used antifouling agents and antifouling means on the market. At the same time, since the oil and fat extract components of the present application are mainly obtained from the fungal fermentation liquid, the fungus has the characteristics of easy to scale up fermentation and low production cost. In addition, the oil and fat components in the strain account for a high proportion, which is easy to obtain and develop and utilize, and is an antifouling agent with broad prospects in the future. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is the colony morphology of strain GXIMD00551.
[0017] Figure 2 This is an antibacterial plate experiment of the fermentation product of strain GXIMD00551 against the fouling bacteria VibrioPelagius.
[0018] Figure 3 This is a picture of the offshore antifouling experiment on the oil composition of strain GXIMD00551.
[0019] Figure 4 It is the biological attachment rate of the oil component of strain GXIMD00551 in the offshore hanging plate experiment.
[0020] Figure 5 It is the positive control of biological attachment rate in offshore hanging board experiment.
[0021] Figure 6 It is the biological attachment rate of the blank control offshore hanging board experiment.
[0022] Biomaterial Deposit Information
[0023] The strain information deposited in this application is: Fusarium incarnatum strain GXIMD00551, its classification name is: Fusarium incarnatum GXIMD00551, its Chinese classification name is: Fusarium incarnatum GXIMD00551, and the preservation number is GDMCC NO.65402; the strain is deposited in Guangdong Provincial Microbiological Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is November 1, 2024. DETAILED DESCRIPTION
[0024] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.
[0025] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.
[0026] Example 1
[0027] This example is the separation and identification of Fusarium erythrocyticum GXIMD00551.
[0028] 1. Isolation and purification of strain GXIMD00551: The strain of the present application was isolated from saturated brine in Zhulin Salt Field, Beihai, Guangxi. The specific isolation method is as follows: salt field water is directly used as the sample stock solution, and the sample stock solution is diluted with sterile seawater into a sample suspension of 1:10 and 1:100. Take 100 μL of each of the two concentrations of sample suspension and apply it to a red tiger medium and a Czapek medium containing 10% sea salt, and place it in a constant temperature incubator at 25°C for 12 days. Use a sterilized bamboo stick to pick up single colonies with different morphologies and inoculate them on a PDA medium to purify the strain.
[0029] 2. Identification of strain GXIMD00551: ① Morphological identification The specific operation is as follows: inoculate the strain GXIMD00551 on PDA medium containing 10% sea salt, place it in a constant temperature incubator for 24 hours, and observe the appearance of round colonies on the plate. Figure 1 As shown, Figure 1② The genomic DNA of the strain was extracted, and the ITS rRNA sequence was amplified and sequenced using universal primers for fungi to obtain the ITS sequence. The specific sequence is shown in the sequence table SEQ ID NO.1. After comparison with the NCBI database, the strain GXIMD00551 was finally identified as the species Fusarium incarnatum.
[0030] Example 2
[0031] This example is about the fermentation products of Fusarium incarnatum strain GXIMD00551 (hereinafter referred to as: F. incarnatum GXIMD00551) and their functional identification.
[0032] 1. Preparation of fermentation product of strain GXIMD00551: 80 g of rice, 0.4 g of yeast extract, 0.4 g of glucose, 3.6 g of sea salt, and 120 ml of water are placed in a 1-liter conical flask and sterilized in an autoclave at 121°C for 20 minutes. F.incarnatum GXIMD00551 is activated on a suitable plate culture medium. Use a sterile bamboo stick to pick the activated mycelium into about 100 bottles of culture medium prepared above, and culture them at a constant temperature of 25°C for 30 days to obtain a fermentation product. The fermentation product is extracted three times with petroleum ether, and the extract is concentrated under reduced pressure to obtain the oil component (the fermentation product can also be extracted with ether, dichloromethane or chloroform).
[0033] 2. GC-MS analysis of oil components:
[0034] Pipette 20 μL of the oil component sample prepared in step 1 into a 2 mL centrifuge tube and dissolve the sample in 980 μL of hexane. Add 50 μL of 2 mol / L potassium hydroxide methanol solution (11.2 g potassium hydroxide dissolved in 100 mL methanol). Seal the centrifuge tube, sonicate for 10 min, and centrifuge. Transfer 1 mL of the transparent supernatant to a sample bottle.
[0035] Chromatographic conditions: The chromatographic column is a HP-5MS quartz capillary column (30m×250μm×0.25μm); the carrier gas is He. The flow rate is 1ml / min. No splitting, the injection volume is 1μL, and the injection port temperature is 250℃. The heating program: the initial temperature is 100℃, maintained for 2min, increased to 180℃ at 3℃ / min and maintained for 1min, and finally increased to 250℃ at 10℃ / min and maintained for 5min.
[0036] Mass spectrometry conditions: EI ion source, MS ion source temperature 230°C, MS quadrupole temperature 150°C, electron energy 70 eV, solvent delay time 3 min; detection range m / z 50 to 550. The results of oil component analysis are shown in Table 1.
[0037] Table 1 Oil components and relative contents of F. incarnatum GXIMD00551
[0038]
[0039] As shown in Table 1, the present invention identified 14 compounds from the oil components, accounting for 99.46% of the total volatile components. Dodecyl-9-alkynyl chloroacetate (34.8%), palmitic acid (28.35%), cis-13-octadecenoic acid (15.11%) and stearic acid (8.96%) are the main components.
[0040] 3. Anti-fouling bacteria activity test: The filter paper method was used to determine the inhibitory activity of oil components against fouling bacteria. The fouling bacteria Vibrio Pelagius (VP) was isolated from polyvinyl chloride plates (PVC plates) attached by marine fouling organisms. The activated indicator bacteria were inoculated into LB medium and cultured at 30°C and 180r / min with shaking until the logarithmic growth phase. Pour the bacterial solution into the sterilized LB solid medium and complete the plate pouring. Select a filter paper with a diameter of 6mm and sterilize it in a culture dish before use. Dissolve the oil components with DMSO and prepare a 10mg / mL sample. Pipette 5μL of the above solution onto the sterilized filter paper, and then transfer the filter paper to the bacterial culture medium. Set up 3 parallel samples for each sample. The culture medium was placed in a constant temperature incubator at 37°C for 24 hours, and the diameter of the inhibition zone in each culture dish was measured. The inhibition zone of the oil component is 6.82±0.1mm (specifically, Figure 2 shown).
[0041] 4. Anti-fouling biological barnacle Venus larvae attachment activity: The adult barnacles required for the experiment were collected from Fangchenggang, Guangxi, and identified as Amphibalanus amphitrite by Dr. Liu Xinming from the Institute of Marine Drugs of Guangxi University of Chinese Medicine. The adult barnacles were cultured in aged seawater (salinity: 2.8%), and the barnacle larvae were collected. The barnacle larvae were cultured in seawater (salinity: 2.8%), and some stage IV nauplii metamorphosed into Venus larvae after 4 days, with Phaeodactylumtricornutum Bohlin as bait. After 4-5 days of culture, the barnacle larvae metamorphosed from nauplii to Venus larvae, and the Venus larvae were collected. The experiment set up a blank group, a sample group and a solvent control group, and the samples were set with a final concentration gradient. An equal volume of seawater was used as the blank control group, an equal volume of DMSO was used as the solvent control group, and 4,5-dichloro-2-octyl-isothiazolone (DCOIT) was used as the positive control group. Set up three parallel wells and place them in a dark constant temperature room for 48 hours. Observe the attachment and metamorphosis of Venus larvae in each well and calculate the attachment rate of Venus larvae. 50 The median lethal concentration (LC) is 0.51 μg / mL. 50 Greater than 50μg / mL, activity index (LD50 / EC50) greater than 98, a highly effective and low-toxic inhibitor.
[0042] 5. Field hanging board attachment experiment: Use PVC board to determine the anti-fouling activity at sea. Use a disposable pipette to absorb about 5mL of primer, add it to a 10cm×10cm PVC board, add the sample (400mg), mix well, evenly apply the board, and dry it. Set up an anti-fouling paint positive control (cuprous oxide) and a primer blank control. Check the adhesion of the PVC board at 15d, 30d, 45d, 60d and 90d, and measure the area covered by biological fouling. The specific results are as follows Figure 3 As shown, compared with the blank control and positive control PVC plates ( Figure 3-Figure 6 ), oil and fat components showed significant antifouling activity ( Figure 3 ). A small amount of Hydroides ezoensir and algae had attached to the blank control PVC board at 15 days, and the fouling coverage rate of the PVC board reached 100% at 90 days, while the fouling coverage rate of the positive control PVC board was only 14%. The coverage rate of biological fouling attached to the PVC board coated with the oil component of F. incarnatum GXIMD00551 was greatly reduced, basically equivalent to the positive control, but the toxicity was lower and the threat to the marine environment was less.
[0043] In summary, the red Fusarium erythrocytes GXIMD00551 and its fermentation products screened by the applicant of the present invention, and the oil components in the fermentation products have clear inhibitory activity against fouling bacteria and barnacle larvae attachment, and still have excellent anti-fouling effect after 90 days of offshore hanging board experiment, indicating that the oil extract of the strain of the present application has a good anti-fouling effect, and the fungus can be reproduced in large quantities, with a very low production cost, which is convenient for subsequent development and utilization.
[0044] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.
Claims
1. Fusarium in carnatum strain GXIMD00551, whose deposit number is GDMCC NO.65402.
2. The Fusarium in carnatum strain GXIMD00551 according to claim 1, characterized in that The ITS sequence of the strain is shown in SEQ ID NO.
1.
3. A bacterial agent comprising the Fusarium in carnatum strain GXIMD00551 according to claim 1 or 2.
4. The oil extract of Fusarium in carnatum strain GXIMD00551 as claimed in claim 1 or 2.
5. The oil extract according to claim 4, characterized in that The extract contains the following components: methyl palmitate with a relative content of 2.73%, palmitic acid with a relative content of 28.35%, 9-octadecenoic acid methyl ester with a relative content of 1.38%, 2,5-di-tert-butylphenol with a relative content of 1.2%, ethyl palmitate with a relative content of 0.6%, methyl linoleate with a relative content of 1.7%, methyl stearate with a relative content of 1.43%, dodecyl-9-ynyl chloroacetate with a relative content of 34.8%, cis-13-octadecenoic acid with a relative content of 15.11%, stearic acid with a relative content of 8.96%, butyl hexadecanoate with a relative content of 1.5%, 2-chloroethyl linoleate with a relative content of 0.72%, trifluoroacetic acid oleyl alcohol with a relative content of 0.52% and / or butyl octadecanoate with a relative content of 0.54%.
6. A method for preparing the oil extract according to any one of claims 4 or 5, characterized in that: The method comprises the following steps: fermenting the Fusarium incarnatum strain GXIMD00551, and then extracting the oil components in the fermentation product by using petroleum ether, ethyl ether, dichloromethane or chloroform.
7. Use of the Fusarium in carnatum strain GXIMD00551 according to claim 1 or 2, the bacterial agent according to claim 3 or the lipid extract according to any one of claims 4 or 5 in the preparation of an antifouling agent.
8. Use of the Fusarium in carnatum strain GXIMD00551 according to claim 1 or 2, the bacterial agent according to claim 3 or the lipid extract according to any one of claims 4 or 5 in inhibiting the growth of fouling bacteria and / or barnacle larvae.
9. The use according to claim 8, characterized in that The fouling bacteria is Vibrio Pelagius; the barnacle larvae is Amphibalanus amphitrite.
10. A method for antifouling using the oil extract according to any one of claims 4 or 5, characterized in that: The method comprises the following steps: applying the oil extract to the surface of the object to be antifouled.