Antagonistic bacterium GHZJ-1 and application thereof

Abstract

The invention discloses an antagonistic bacterium GHZJ-1, the preservation number of the antagonistic bacterium GHZJ-1 is CCTCC (China Center For Type Culture Collection) NO: M2024755, and the antagonistic bacterium GHZJ-1 is classified and named as Bacillus sp. (Bacillus sp.). Antagonistic experiments prove that an extracellular metabolite of the antagonistic bacterium GHZJ-1 classified as bacillus developed by the invention can have an excellent inhibition effect on the metschinia pekinensis, and a new idea can be provided for a metschinia pekinensis inhibitor; and a new direction and a new choice can be provided for preventing and treating the milk disease of biological animals taking the metschinia pekinensis as the pathogeny. A culture medium for obtaining the antagonistic bacterium GHZJ-1 with high inhibition activity on the activity of the metschinia pekinensis is screened and selected through experiments, the fermentation metabolism time is selected, and the result shows that supernate obtained after fermentation metabolism of the antagonistic bacterium GHZJ-1 for more than 48 hours has high inhibition activity.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and particularly to an antagonistic bacterium GHZJ-1 and its application. Background Art

[0002] The Chinese mitten crab (Eriocheir sinensis), also known as the river crab, hairy crab, and Hairy Crab, belongs to the order Decapoda, family Grapsidae, genus Eriocheir, and is the main economically important crab species in freshwater aquaculture in China. The Chinese mitten crab is rich in nutrition and delicious in taste, and is deeply loved by consumers. "Milk disease" is a relatively serious disease that often occurs in the process of culturing Chinese mitten crabs. Its characteristic is that a large amount of milky liquid accumulates in the chest cavity. The diseased crabs move slowly, have reduced vitality, loss of appetite, their walking legs are easily shed, and they are prone to death, usually causing significant losses to crab farmers. It has been confirmed that the pathogen of the "milk disease" of Chinese mitten crabs is Metschnikowia bicuspidata, which is infectious and has a very high mortality rate for infected crabs. Traditional antifungal compounds have very limited effects on inhibiting milk disease and have the risks of causing environmental pollution and reducing food safety. Summary of the Invention

[0003] Therefore, based on the above background, the present invention provides an antagonistic bacterium GHZJ-1 and its application, providing a new direction and more choices for combating Metschnikowia bicuspidata and preventing and controlling aquatic organisms, especially the "milk disease" of Chinese mitten crabs.

[0004] One of the purposes of the present invention is to provide:

[0005] An antagonistic bacterium GHZJ-1, with a preservation number of CCTCC NO: M2024755, and its taxonomic name is Bacillus sp. (bacteria of the genus Bacillus).

[0006] Another purpose of the present invention is the application of the antagonistic bacterium GHZJ-1, and the application at least includes one of the following:

[0007] ① Application in the preparation of an inhibitor with Metschnikowia bicuspidata as the pathogen;

[0008] ② Application in the preparation of a preparation for preventing and controlling the "milk disease" of aquatic animals.

[0009] Furthermore, the pathogen of the "milk disease" is Metschnikowia bicuspidata.

[0010] Furthermore, the aquatic animals include crabs, Macrobrachium rosenbergii, Chinook salmon, Penaeus chinensis, and Oncorhynchus tshawytscha.

[0011] Furthermore, the crabs include Chinese mitten crabs.

[0012] Another purpose of the present invention is to provide:

[0013] A preparation method of an agent against Metschnikowia bicuspidata, which is prepared by using the above-mentioned antagonistic bacterium GHZJ-1 as a fermentation agent.

[0014] Furthermore, it includes the following steps:

[0015] S1: Inoculate the antagonistic bacterium GHZJ-1 into a culture medium for fermentation culture;

[0016] S2: After culturing for at least 48 hours, separate and take the supernatant.

[0017] Furthermore, the culture medium used in step S1 can be prepared by dissolving 8 - 15 g of peptone, 15 - 25 g of glucose, and 3 - 8 g of yeast extract in 1 - 3 L of deionized water;

[0018] Or it can be prepared by dissolving 8 - 15 g of yeast extract, 15 - 25 g of tryptone, and 3 - 8 g of NaCl powder in 1 - 3 L of deionized water;

[0019] Or it can be prepared by dissolving 8 - 15 g of yeast extract and 15 - 25 g of tryptone in 1 - 3 L of deionized water;

[0020] Or it can be prepared by dissolving 15 - 30 g of glucose, 0.5 - 1.5 g of yeast extract, 3 - 10 g of L-glutamic acid, 1 - 5 mg of L-phenylalanine, 0.5 - 1.5 g of L-tryptophan 1.0 g, 0.2 - 1.2 g of potassium chloride, 0.5 - 1.5 g of potassium dihydrogen phosphate, 0.2 - 1.0 g of magnesium sulfate heptahydrate, 2 - 8 mg of manganese sulfate tetrahydrate, 0.05 - 0.5 mg of copper sulfate pentahydrate, and 0.08 - 0.3 mg of ferrous sulfate heptahydrate in 1 - 3 L of deionized water.

[0021] The fourth object of the present invention is to use the above-mentioned agent against Metschnikowia bicuspidata alone or in combination with other drugs for preparing an inhibitor against Metschnikowia bicuspidata, or a preparation for preventing and treating "milk disease" of aquatic animals.

[0022] Furthermore, the aquatic animals include crabs, Macrobrachium rosenbergii, Chinook salmon, Palaemonetes sinensis, and Oncorhynchus tshawytscha.

[0023] The effective effects achieved by the present invention are as follows:

[0024] Proved by the antagonistic experiment, the extracellular metabolites of the antagonistic bacterium GHZJ-1 classified as Bacillus in the present invention can have excellent inhibitory effects on Metschnikowia bicuspidata, which can provide new ideas for the preparation of inhibitors against Metschnikowia bicuspidata, and can provide new directions and choices for the prevention and treatment of "milk disease" of aquatic animals with Metschnikowia bicuspidata as the pathogen.

[0025] The medium for the antagonistic bacterium GHZJ-1 with high inhibitory activity against Metschnikowia bicuspidata was screened through experiments, and the fermentation and metabolism time was also selected. The results showed that the supernatant of the antagonistic bacterium GHZJ-1 after fermentation and metabolism for more than 48 hours had high inhibitory activity.

[0026] Explanatory drawings of the specification

[0027] Appendix Figure 1 This shows the biofilm formation of Bacillus sp. GHZJ-1 in the improved Landy medium after 48 hours of growth in the embodiment of the present invention;

[0028] Appendix Figure 2 This shows the antagonistic effect of Bacillus sp. GHZJ-1 in the YPD agar medium after 48 hours of growth on the indicator bacterium Metschnikowia bicuspidata in the embodiment of the present invention;

[0029] Among them Figure 2 At the arrow pointing upward in the upper left figure is the hole into which the bacterial liquid of this spore strain was injected; the arrow below is the biofilm formed by this spore bacillus around the hole; the right figure is the negative reference, that is, the YPD agar medium without inoculating Bacillus sp. GHZJ-1 bacteria.

[0030] Appendix Figure 3 This shows the inhibitory effect of the culture of Bacillus sp. GHZJ-1 strain (YPD broth for 72 hours) on Metschnikowia bicuspidata in the embodiment of the present invention:

[0031] Among them, (A) is the supernatant of the bacterial broth culture; (B) is the lysate of the broken bacterial cells; the black line segment represents a length of 10 mm; the results shown in the figure are representatives of three biological replicate experiments.

[0032] Appendix Figure 4 This shows the inhibitory effect of the supernatant samples obtained by fermenting and culturing Bacillus sp. GHZJ-1 with different media on Metschnikowia bicuspidata in the embodiment of the present invention:

[0033] Among them, (A) is the supernatant of the bacterial YTNaCl broth culture (72 hours); (B) is the supernatant of the bacterial YT broth culture (72 hours); (C) is the supernatant of the bacterial YT broth culture (24 hours); (D) is the supernatant of the bacterial YT broth culture (48 hours); the black line segment represents a length of 10 mm; the results shown in the figure are representatives of three biological replicate experiments.

[0034] The antagonistic bacterium GHZJ-1, with the preservation number of CCTCC NO: M2024755; its Latin taxonomic name is Bacillus sp.; the preservation date is April 24, 2024; the preservation institution is the China Center for Type Culture Collection; the preservation location is Wuhan University, China Detailed implementation manners

[0035] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] Sources of the materials used in the following experiments:

[0037] Yeast extract: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 119-44-8;

[0038] Glucose: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 50-99-7;

[0039] L-Glutamic acid: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 56-86-0;

[0040] L-Phenylalanine: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 63-91-2;

[0041] L-Tryptophan: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 73-22-3;

[0042] Potassium chloride: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 7447-40-7;

[0043] Potassium dihydrogen phosphate: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 7778-77-0;

[0044] Magnesium sulfate heptahydrate: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 10034-99-8;

[0045] Manganese sulfate tetrahydrate: Thermo Fisher Scientific, CAS number: 10101-68-5;

[0046] Copper sulfate pentahydrate: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 7758-99-8;

[0047] Ferrous sulfate heptahydrate: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 7720-78-7;

[0048] Peptone: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 73049-73-7;

[0049] Agar powder: Beijing Solarbio Science & Technology Co., Ltd., CAS number: 9002-18-0.

[0050] Example 1: Screening and cultivation of antagonistic bacteria GHZJ-1, comprising the following steps:

[0051] (1) Bacillus strain isolation steps:

[0052] Collect water samples from the tailwater ecological treatment pool of Panjin Photosynthetic Crab Co., Ltd. (the water in the ecological treatment pool is non-flowing, and its water mainly comes from the tailwater discharged from a main pipeline. However, before the tailwater is discharged, the sewage will be filtered at the outlet of the pipeline. The treatment pool will be regularly aerated to stimulate the growth of aerobic microorganisms and algae, so as to further treat the sewage ecologically more efficiently.), dilute the water sample with physiological saline, and then culture the treated sample at high temperature (80°C) for about fifteen minutes, and finally spread the sample after high temperature culture on the nutrient agar plate to isolate the single colony of Bacillus strain;

[0053] (2) Steps for culturing Bacillus strains:

[0054] The Bacillus strain isolated in the previous step was inoculated into a modified Landy liquid medium (formula: 20.0 g of glucose, 1.0 g of yeast extract, 5.0 g of L-glutamic acid, 2 mg of L-phenylalanine, 1.0 g of L-tryptophan, 0.5 g of potassium chloride, 1.0 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 5 mg of manganese sulfate tetrahydrate, 0.14 mg of copper sulfate pentahydrate, and 0.15 mg of ferrous sulfate heptahydrate were dissolved in 1 L of deionized water, and the pH value of the culture medium was controlled within the range of 7.0-7.2), and cultured at room temperature for 48-72 hours, and the production of biofilm by the strain in the culture medium was observed, and then the strain with strong ability to produce biofilm was screened out, and the screened strain was the antagonistic bacterium GHZJ-1 of the present invention, and then the antagonistic bacterium GHZJ-1 was sent to the China Center for Type Culture Collection for preservation, and the preservation number was CCTCCNO: M2024755; the Latin classification name was Bacillus sp.

[0055] The antagonistic bacteria GHZJ-1 was inoculated into the modified Landy liquid medium for cultivation, and it showed a strong ability to synthesize biofilms, such as Figure 1 shown.

[0056] Specifically, the GHZJ-1 colony was inoculated into a conical flask containing 50 mL of culture medium (the capacity of the conical flask was 250 mL), and cultured in an air shaker at 30°C with a shaker speed of 200 rpm and no light. The air in the shaker incubator was normal air.

[0057] In this example, for the diluted water sample, the sample is first treated at high temperature to kill most of the non-spore-forming microorganisms. Since Bacillus can produce spores, it can survive in the form of spores after high-temperature treatment and can be revived into the vegetative form in a nutrient agar medium (a common medium for enriching bacterial strains). Therefore, this example adopts the methods of high-temperature treatment and nutrient agar enrichment to isolate Bacillus strains. The modified Landy liquid medium is an oligotrophic medium, and components such as glutamate in it can promote Bacillus to produce various secondary metabolites with potential antifungal activities. The accumulation of these secondary metabolites can promote the formation of biofilms. Therefore, the ability of the strain to produce biofilms in the Landy liquid medium is used as an indicator phenotype for its production of antifungal active substances, so as to conduct a preliminary evaluation and screening of the isolated Bacillus strains.

[0058] Example 2: Experiment on the antagonistic ability of the antagonistic bacterium GHZJ-1 against Metschnikowia bicuspidata

[0059] Using YPD solid medium (10 g peptone, 20 g glucose, 5 g yeast extract, 15 g agar powder dissolved in 1 L deionized water) as the medium for the antagonistic experiment, the activity of the antagonistic bacterium GHZJ-1 against Metschnikowia bicuspidata (indicator bacterium) was tested by the punching method.

[0060] The specific operation is as follows: First, melt the YPD solid medium, pour Metschnikowia bicuspidata into the petri dish to make a plate, and punch a hole in the middle.

[0061] After injecting the antagonistic bacterium GHZJ-1 cultured in the modified Landy liquid medium in Example 1 above and its fermentation metabolite supernatant into the punched hole, after culturing for 48 h, the results can be seen as follows. Figure 2 As shown, in the left figure below, obvious boundaries can be seen and biofilms are produced. The right figure is the colony of Metschnikowia bicuspidata without injecting the antagonistic bacterium GHZJ-1 and its supernatant, indicating that the antagonistic bacterium GHZJ-1 and its fermentation metabolite supernatant have a significant inhibitory and antagonistic effect on Metschnikowia bicuspidata. Figure 2 As shown in the left figure below, obvious boundaries can be seen and biofilms are produced. The right figure is the colony of Metschnikowia bicuspidata without injecting the antagonistic bacterium GHZJ-1 and its supernatant, indicating that the antagonistic bacterium GHZJ-1 and its fermentation metabolite supernatant have a significant inhibitory and antagonistic effect on Metschnikowia bicuspidata.

[0062] Example 3:

[0063] In this example, the antagonistic bacterium GHZJ-1 was cultured in YPD broth medium (10 g peptone, 20 g glucose, 5 g yeast extract dissolved in 1 L deionized water), and the inhibitory effect of the fermentation metabolite (supernatant) or the lysate of the broken cells on Metschnikowia bicuspidata was explored.

[0064] The specific operation is as follows:

[0065] The antagonistic bacterium GHZJ-1 was inoculated into YPD broth medium and cultured for 72 h. After that, the bacterial cells and the supernatant were centrifuged and separated to obtain the bacterial cell liquid and the supernatant respectively.

[0066] Preparation steps of the bacterial cell lysate:

[0067] 1. 50 mL of the above bacterial cell liquid was treated with a centrifuge (8000 rpm, 5 min, 25 °C), and the bacterial cells settled. The supernatant was discarded.

[0068] 2. The bacterial cells collected in the previous step were resuspended with 5 mL of PBS solution, and the bacterial suspension was placed under an ultrasonic cell disruptor for lysis. After the bacterial suspension became completely clear after treatment, the obtained liquid was the bacterial cell lysate.

[0069] The YPD solid medium was melted, and Metschnikowia bicuspidata was poured into a petri dish to make a plate, and holes were punched in the middle (two groups).

[0070] After the bacterial cell lysate and the supernatant were respectively injected into the punched holes and cultured for 48 h, the results are shown in the appendix Figure 3 , it can be seen that the culture supernatant of this bacterium showed an obvious inhibitory effect on Metschnikowia bicuspidata in the agar well diffusion assay for antibacterial activity, and the diameter of the inhibition zone was about 19 mm ( Figure 3 A). However, the bacterial cell lysate did not show inhibitory activity against Metschnikowia bicuspidata ( Figure 3 B). It is speculated that the antagonistic activity of Bacillus sp. GHZJ-1 against Metschnikowia bicuspidata mainly comes from the extracellular (secondary metabolite) products of the bacteria.

[0071] Example 4: Effects of different media on the inhibitory effect of the fermentation metabolites of the antagonistic bacterium GHZJ-1 on Metschnikowia bicuspidata

[0072] In this example, three media were selected for the fermentation culture of the antagonistic bacterium GHZJ-1, namely:

[0073] YPD broth medium: 10 g of peptone, 20 g of glucose, and 5 g of yeast extract were dissolved in 1 L of deionized water ( Figure 3 A);

[0074] YTNaCl broth medium: 10 g of yeast extract, 20 g of tryptone, and 5 g of NaCl powder were dissolved in 1 L of deionized water ( Figure 4 A);

[0075] YT broth medium: 10 g of yeast extract and 20 g of tryptone were dissolved in 1 L of deionized water;

[0076] After the antagonistic bacterium GHZJ-1 was fermented and cultured for 72 h using different media respectively, the obtained supernatant was injected into the holes of the YPD solid medium inoculated with Metschnikowia bicuspidata, and after culturing for 48 h, the results are shown in Figure 3 A and Figure 4 . It can be seen that the supernatant obtained from the YTNaCl broth medium ( Figure 4 A) had significantly lower antibacterial effect than the supernatant obtained from the YT broth medium ( Figure 4 B), and the latter could reach the antibacterial effect of the bacterial supernatant in the YPD broth medium ( Figure 3 A). The YPD broth medium is a commonly used yeast medium containing 2% glucose, while the common media for bacteria are nutrient broth medium or LB broth medium, both containing 0.5% NaCl to maintain the cell osmotic pressure. Therefore, in order to explore whether glucose and salinity affect the anti-Metschnikowia bicuspidata activity of Bacillus sp. GHZJ-1, this example compared the ability of the antagonistic bacterium GHZJ-1 to produce yeast-inhibiting substances under the conditions of YPD, YT and YTNaCl broth media. The results showed that the antagonistic bacterium GHZJ-1 could produce substances with strong inhibitory effects ( Figure 3 and Figure 4 ) in the absence of salt and glucose.

[0077] Example 5: Effect of fermentation culture time on the inhibitory effect of the fermentation metabolites of the antagonistic bacterium GHZJ-1 on Metschnikowia bicuspidata

[0078] In this example, the yeast-inhibiting activities of the supernatant samples obtained by splitting the YT broth medium at different culture durations were detected, and it was found that short-term culture (24 hours) was not sufficient for Bacillus sp. GHZJ-1 to accumulate active substances against yeast, while the yeast-inhibiting activity of the supernatant after 48-hour culture could reach the same level as that of the 72-hour supernatant ( Figure 4 B-D).

[0079] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Antagonistic bacteria GHZJ-1, characterized in that Its deposit number is CCTCCNO: M2024755, and its classification name is Bacillus sp.

2. The use of the antagonistic bacterium GHZJ-1 according to claim 1, characterized in that: The application includes at least one of the following: ① Application in the preparation of inhibitors of the pathogenic yeast Metschnikowia bicuspidatum; ②Application in the preparation of preparations for preventing and treating "milk disease" in aquatic animals.

3. The use according to claim 2, characterized in that: The pathogen of the "milk disease" is the yeast Metschnikowia bicuspidatum.

4. The use according to claim 3, characterized in that: The aquatic animals include crabs, Macrobrachium rosenbergii, Chinook salmon, Chinese grass shrimp and Chinook salmon.

5. The use according to claim 4, characterized in that: The crabs include Chinese mitten crab.

6. A method for preparing a preparation for resisting Metschnikova bicuspidatum, characterized in that: The fermentation agent is prepared by using the antagonistic bacteria GHZJ-1 described in any one of claims 1 to 4 as a fermentation agent.

7. The method for preparing an anti-Metschnikowia bicuspidatum preparation according to claim 6, characterized in that: It includes the following steps: S1: The antagonistic bacteria GHZJ-1 was inoculated into the culture medium for fermentation; S2: After culturing for at least 48 hours, separate and take the supernatant.

8. The method for preparing a preparation against the yeast Mycobacterium bicuspidatum according to claim 7, characterized in that: The culture medium used in step S1 can be prepared by dissolving peptone, glucose, and yeast extract in deionized water; or prepared by dissolving yeast extract, tryptone, and NaCl powder in deionized water; or prepared by dissolving yeast extract and tryptone in deionized water; Or it is prepared by dissolving glucose, yeast extract, L-glutamic acid, L-phenylalanine, L-tryptophan, potassium chloride, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, copper sulfate pentahydrate, and ferrous sulfate heptahydrate in deionized water.

9. Use of the preparation against Metschnikova bicuspidatum according to any one of claims 6 to 8 alone or in combination with other drugs in the preparation of inhibitors of Metschnikova bicuspidatum pathogenic agent or preparations for preventing and treating "milk disease" of aquatic animals.

10. The use according to claim 9, characterized in that: The aquatic animals include crabs, Macrobrachium rosenbergii, Chinook salmon, Chinese grass shrimp and Chinook salmon.

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