CN110205264B - Saprolegnia antagonist JD03 and application of fermentation liquor and active compound thereof in preparation of products for preventing and treating saprolegniasis - Google Patents
Saprolegnia antagonist JD03 and application of fermentation liquor and active compound thereof in preparation of products for preventing and treating saprolegniasis Download PDFInfo
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Abstract
The invention discloses a saprolegnia antagonistic bacterium JD03, and application of fermentation liquor and active compounds thereof in preparation of products for preventing and treating saprolegnia. The water mold antagonistic bacterium JD03 is Burkholderia ubonensis JD03 with the preservation number: GDMCC NO: 60660. the Saprolegnia antagonist JD03 and the fermentation broth thereof have good, safe, effective and stable Saprolegnia antagonistic effect, can be used as a new biological control product for replacing malachite green, avoids environmental pollution and residues of malachite green and metabolites thereof in aquatic products, meets the requirement of preventing and treating fish saprolegnia, and ensures the quality safety of the aquatic products. The active compound 2-pyrrolidone-5-carboxylic acid is separated from the fermentation liquor of the saprolegnia antagonist strain JD03 for the first time, can obviously inhibit the germination of saprolegnia spores and the growth of hyphae, is environment-friendly and safe to fish, and has great potential for developing novel products for preventing and treating saprolegnia of fish.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to application of Saprolegnia antagonist JD03 and fermentation liquor thereof and an active compound 2-pyrrolidone-5-carboxylic acid in preparation of products for preventing and treating saprolegniasis, in particular to application in preparation of products for preventing and treating fish saprolegniasis.
Background
Saprolegniasis is a common fungal parasitic disease in freshwater aquaculture, has the characteristics of wide epidemic range, long time, no selectivity to hosts and the like, and often causes a large number of dead diseased fishes due to outbreak of saprolegniasis, thereby bringing serious economic loss to the aquaculture industry. The species of Saprolegnia spp (Saprolegnia spp.) is the major parasitic fungal pathogen causing saprolegniasis. Malachite green was once the most effective chemical for preventing and treating saprolegniasis since the last 30 century, but researches found that malachite green is difficult to degrade, has long residual time in water environment and aquatic animals, and has the risks of teratogenesis, carcinogenesis and mutagenesis on mammals and human beings. Therefore, the use of the malachite green as an aquatic medicine is prohibited by governments of various countries in the last 90 th century, and related laws and regulations for forbidding malachite green are also developed in 2002 in China.
Biological control is used as a novel aquatic disease control mode, has the characteristics of safety to human beings and animals and environmental friendliness, and is a hotspot in the current aquatic disease control research field. Therefore, the research and development of a new product for preventing saprolegniasis safely, green and efficiently to replace malachite green is urgent.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide the Saprolegnia antagonist JD03 and the fermentation liquid thereof. The Saprolegnia antagonist JD03 and the fermentation broth thereof have good effect of antagonizing Saprolegnia, can be used as a new biological control product for replacing malachite green, avoids environmental pollution and residues of malachite green and metabolites thereof in aquatic products, meets the requirement of preventing and controlling fish saprolegnia, and ensures the quality safety of the aquatic products.
The invention also aims to provide the application of the Saprolegnia antagonist JD03 and the fermentation liquid thereof.
The invention also aims to provide a method for separating and purifying the active substance 2-pyrrolidone-5-carboxylic acid in the fermentation liquor of the Saprolegnia parasitica JD 03.
Still another object of the present invention is to provide the use of an active compound, 2-pyrrolidone-5-carboxylic acid.
The strain JD03 separated and purified from pond sediment has the effect of remarkably inhibiting spore germination and hypha growth of saprolegnia, and is identified as JD03 in Burkholderia umchenensis (Burkholderia ubonensis) in which an active compound against saprolegnia in the fermentation broth is 2-pyrrolidone-5-carboxylic acid. The method is to discover that the fermentation liquor of the Wenshenburkholderia has the function of resisting the water mildew for the first time, identify the compound with the activity of resisting the water mildew, further complete the control test of the saprolegniasis of the fishes by using the fermentation liquor of the Wenshenburkholderia, and have obvious effect.
The purpose of the invention is realized by the following technical scheme:
the invention provides a water mold antagonistic bacterium JD03, the strain is named as Wen Burkholderia ubonensis JD03, which is obtained by separating and purifying bottom mud of a pond.
The deposited information of the bacterium Wenswerburkholderia (Burkholderia ubonensis) JD 03: the preservation unit: guangdong province culture Collection (GDMCC), preservation date: 5/2019, deposit address: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC NO: 60660.
the liquid culture medium of the Wenshenburg Hold Hall (Burkholderia ubonensis) JD03 strain is a beef extract peptone liquid culture medium, and the beef extract peptone solid culture medium is prepared by adding 15-20 g of agar into every 1000mL of the liquid culture medium and heating and melting the agar.
The beef extract peptone liquid culture medium: 10g/L of peptone, 3g/L of beef extract, 5g/L of sodium chloride and 15-20 g/L of agar, wherein the pH value is 7.0-7.2, and the high-temperature sterilization treatment is carried out at 121 ℃ for 20 min; the beef extract peptone liquid culture medium does not need to add agar.
The fermentation culture medium of the Wenswenburkholderia ubonensis JD03 strain is a potato glucose liquid culture medium (PDB) which comprises the following components: peeling potato 200g/L, glucose 20.0g/L, and autoclaving at 121 deg.C for 20 min.
A biological agent, the active ingredient of which is fermentation broth of Burkholderia umchenensis (Burkholderia ubonensis) JD03 and/or JD 03.
At least one of the Wenswenburkholderia ubonensis JD03 strain, fermentation liquor and biological agent is applied to the preparation of products for preventing and treating saprolegniasis, and further applied to the preparation of products for preventing and treating fish saprolegniasis. Specifically, the JD03 strain and the fermentation liquid thereof can obviously inhibit the growth of saprolegnia hyphae and the germination of saprolegnia spores; and can obviously reduce the saprolegnia infection rate of the fish.
Preferably, the fish is freshwater fish, and grass carp is a model species, but not limited thereto.
Further, the pathogenic fungus causing saprolegniasis is a species of Saprolegnia (Saprolegnia), but is not limited thereto.
The active compound in the fermentation liquor of the Burkholderia monocytogenes JD03 strain is 2-pyrrolidone-5-carboxylic acid, and the separation and purification method comprises the following steps:
(1) and (2) mixing JD03 strain fermentation liquor and an organic reagent according to a volume ratio of 1:1, repeatedly shaking and uniformly mixing, and repeating for 2-3 times to ensure that effective substances are extracted by an organic reagent as much as possible; extracting the supernatant with organic reagent, evaporating to dryness, dissolving with methanol, and oven drying to obtain extract as active substance extract;
further, the organic reagent is n-butanol, but is not limited thereto;
further, the preparation method of the JD03 strain fermentation broth comprises the following steps:
inoculating the activated JD03 strain into a beef extract peptone liquid culture medium, and culturing to obtain a seed solution; then adding the seed solution into a potato dextrose liquid medium (PDB) according to the inoculation amount of 2% (v/v), and carrying out fermentation culture to obtain JD03 strain fermentation liquor.
Preferably, the culture condition is constant temperature shaking culture at 160-180 rpm for 8-12 h at 25-28 ℃;
more preferably, the culture conditions are constant temperature shaking culture at 160rpm for 10h at 28 ℃;
preferably, the fermentation culture conditions are constant temperature shaking culture at 160rpm for 48h at 28 ℃.
(2) Dissolving the active matter extract with methanol, uniformly mixing the active matter extract with the methanol by ultrasonic, and then mixing the active matter extract with the methanol according to a mass ratio of 1:1, fully mixing with 200-300 meshes of silica gel, drying, fully grinding, filtering with four layers of gauze to obtain sample powder, and sealing and storing; filling the silica gel into a column by a wet method, namely soaking the 100-200-mesh silica gel by petroleum ether to fully expand the silica gel and remove air, then continuously pouring the silica gel into a glass chromatographic column (10 multiplied by 120cm), and simultaneously discharging redundant petroleum ether to slowly precipitate silica gel particles to a required height; finally, adopting a dry method for loading, slowly adding sample powder into the chromatographic column, and ensuring that the contact surface is neat; eluting by using a mobile phase with the polarity from small to large, wherein the mobile phase is ethyl acetate: petroleum ether 1:2, ethyl acetate: 1:1 of petroleum ether and ethyl acetate; collecting the components eluted from the three mobile phases, combining, and evaporating to dryness to obtain an active ingredient SY 1;
(3) the SY1 active ingredient is firstly analyzed by an analytical high performance liquid chromatography, the mobile phase comprises 20% methanol and 80% deionized water, the flow rate is 1mL/min, the sample amount is 10 mu L, the ultraviolet detection wavelength is 210nm, and the active ingredient can be sequentially divided into I-1, I-2 and I-3 wave peaks; and respectively preparing the wave crests I-1, I-2 and I-3 by preparative high performance liquid chromatography, wherein the mobile phase is methanol: the flow rate of water is 1:4, the sample volume is 1mL, the ultraviolet detection wavelength is 210nm, and the active peak is mainly concentrated on the I-3 wave peak; then carrying out second preparative high performance liquid chromatography separation on a sample corresponding to the I-3 wave peak, wherein the mobile phase is methanol: water is 1:4, the flow rate is 8mL/min, the sample volume is 1mL, the ultraviolet detection wavelength is 210nm, II-1 and II-2 wave peaks can be obtained in sequence, and the active site is positioned in the II-2 wave peak; and then carrying out third preparative high performance liquid chromatography separation on the sample corresponding to the II-2 wave peak, wherein the mobile phase is methanol: water is 1:4, the flow rate is 8mL/min, the sample volume is 1mL, the ultraviolet detection wavelength is 210nm, III-1 and III-2 wave peaks can be obtained in sequence, and the active site is positioned in the III-2 wave peak; drying the sample corresponding to the III-2 wave peak, and determining the purity of the dried sample by adopting a high performance liquid chromatography, wherein the purity is higher than 95%; to obtain the active compound 2-pyrrolidone-5-carboxylic acid with the molecular formula C5H7O3N, the structural formula is shown as follows:
further, the purity was determined using conditions such that the mobile phase was methanol: water 2: 3, the ultraviolet detection wavelength is 210nm, the sample injection amount is 10 mu L, and the time is 15 min.
The application of an active compound 2-pyrrolidone-5-carboxylic acid in fermentation liquor of a Wenshenburg Holeria (Burkholderia ubonensis) JD03 strain in the preparation of a product for preventing and treating saprolegniasis; in particular to the application in preparing products for preventing and treating fish saprolegniasis.
Further, the fish is freshwater fish, and grass carp is used as a model species, but not limited thereto.
Further, the pathogenic fungus causing saprolegniasis is a species of Saprolegnia (Saprolegnia), but is not limited thereto.
Furthermore, when the effective concentration of the 2-pyrrolidone-5-carboxylic acid is more than or equal to 0.125mg/mL, the germination of the saprolegnia spores can be completely inhibited; preferably 0.125mg/mL to 1 mg/mL.
The dosage form of the product is liquid medicine or powder;
the product contains one or more pharmaceutically acceptable carriers or excipients.
Compared with the prior art, the invention has the following advantages and effects:
(1) according to the invention, the saprolegnia antagonist strain JD03 and the fermentation broth thereof are applied to the prevention and treatment of fish saprolegnia for the first time, so that the morbidity of the grass carp saprolegnia can be effectively reduced, and the effect of obviously preventing and treating the fish saprolegnia is achieved;
(2) the water mold antagonistic strain JD03 disclosed by the invention has strong antagonistic capability on the water molds of fishes, is safe, effective and stable, and has a wide application prospect;
(3) the active compound 2-pyrrolidone-5-carboxylic acid is separated from the fermentation liquor of the saprolegnia antagonist strain JD03 for the first time, can obviously inhibit the germination of saprolegnia spores and the growth of hyphae, is environment-friendly and safe to fish, and has great potential for developing novel products for preventing and treating saprolegnia of fish.
Drawings
FIG. 1 shows the inhibitory effect of JD03 strain fermentation broth on the growth of saprolegnia hyphae; note: a: CK is a control group; b: experimental group.
FIG. 2 is a graph of the inhibition of Saprolegnia spore germination by sterile fermentation broths of varying concentrations; note: 1.2, 3, 4, 5, 6, 7 and 8 represent dilutions of JD03 at 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5625% and 0.78125% of sterile fermentation broth concentration, respectively; EG is an experimental group; CK is a control group.
FIG. 3 shows the effect of JD03 aseptic fermentation 10 times the concentration on the growth of saprolegnia hypha; note: a: CK is a control group; b: experimental group.
FIG. 4 is a graph showing the effect of a high temperature of 100 ℃ on the inhibition of the growth of saprolegnia by a 10-fold concentrate of JD03 sterile fermentation broth; note: a: CK is a control group; b: experimental group.
FIG. 5 shows the effect of fermentation broth of antagonistic bacteria JD03 strain in preventing grass carp from being infected by saprolegnia spores; note: the different capital letters in the time points of the control group indicate significant differences within the group (p < 0.05); the different lower case letters in each time point of the experimental group indicate significant intra-group differences (p < 0.05); indicates that the difference between the control group and the experimental group is very significant (p < 0.01).
FIG. 6 is a graph of the inhibitory effect of different mobile phase components on the germination of Saprolegnia spores; wherein, a, b and c in the figure respectively represent different components which are extracted from the same mobile phase.
FIG. 7 shows the purity of the active peak III-2.
FIG. 8 shows the effect of 2-pyrrolidone-5-carboxylic acid, an active compound, on the inhibition of the germination of Saprolegnia diclina spores, in fermentation broth of strain JD 03; note: 1.2, 3, 4, 5, 6, 7, 8 represent concentrations of 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL, 0.015625mg/mL, respectively; a is the experimental group and B is the control group.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
The host model fish used in the examples was grass carp (Ctenophagogondon idellus) from the Wuxincun fish fry farm in the Kwangzhou city Fangdu area.
The saprolegnia described in the examples is disclosed in the literature "screening of saprolegnia antagonistic bacteria and preliminary analysis of antagonistic properties [ J ]. ecology science, 2013,32(2): 189-.
Example 1: identification of antagonistic Strain JD03
(1) Activation of JD03 strain: JD03 strain was isolated from a pond sediment sample and was kept in the laboratory. Activating the preserved JD03 strain on a beef extract peptone solid culture medium by adopting a plate-streaking method, culturing for 16h at the constant temperature of 28 ℃, selecting a monoclonal to inoculate the monoclonal to a beef extract peptone liquid culture medium, and performing constant-temperature shaking culture for 10h at the constant temperature of 160rpm at the temperature of 28 ℃. The bacterial liquid can be used as seed liquid for fermentation culture and can also be used as bacterial liquid for bacteriostasis test.
(2) Identification of strain JD 03:
the morphological and physiological and biochemical indexes of JD03 strain were determined by reference to Berger's Manual of identification of bacteria (ninth edition).
The results show that: the JD03 strain was a gram-negative bacterium with flagellar extremes clustered. JD03 colonies were smooth and bright, milky in color, small round spheres and regular in edge.
JD03 strain can utilize glucose, fructose, galactose, trehalose, arabinose, xylose, mannose, ribose, maltose, arabitol, mannitol, sorbitol, citrate, histamine, and does not utilize sorbitol, melibiose, lactulose, or galactitol. JD03 strain can hydrolyze gelatin, and is positive for urease and arginine double hydrolase.
The morphological and physiobiochemical characteristics of JD03 were consistent with those of Burkholderia ubonensis, recorded in Berger's Manual of identification of bacteria (ninth edition), English, and therefore JD03 strain isolated from pond sediment samples was identified as Burkholderia ubonensis, Wenshole (Burkholderia ubonensis) JD 03.
The deposited information of the bacterium Wenswerburkholderia (Burkholderia ubonensis) JD 03: the preservation unit: guangdong province culture Collection (GDMCC), preservation date: 5/2019, deposit address: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC NO: 60660.
example 2: inhibitory effect of antagonistic strain JD03 on Saprolegnia parasitica
(1) Preparation of saprolegnia hypha and saprolegnia spore suspension: collecting the preserved Saprolegnia parasitica plate, perforating with sterile perforator with diameter of 8mm to obtain Saprolegnia parasitica block, and uniformly inoculating 4 Saprolegnia parasitica blocks to potato dextroseAdding 10-12 Saprolegnia parasitica blocks into a triangular flask filled with 150mL sterile water by using sterile tweezers after punching holes on a fresh Saprolegnia parasitica plate by using an aseptic puncher to obtain Saprolegnia parasitica blocks, adding the 10-12 Saprolegnia parasitica blocks into the triangular flask filled with 150mL sterile water, culturing for 2d at the constant temperature of 25 ℃, filtering the water in the triangular flask by using 8 layers of sterile gauze to remove hyphae, finally counting the number of Saprolegnia parasitica spores in the water by using a dilution coating method, and adjusting the concentration of the spores to 5 × 10 by using the sterile water3piece/mL, stored in a refrigerator at 4 ℃ for subsequent experiments.
(2) Preparation of fermentation liquor and 10-fold concentrated solution of JD03 strain: activating a preserved JD03 strain on a beef extract peptone solid culture medium by adopting a plate-streaking method, culturing for 16h at the constant temperature of 28 ℃, selecting a monoclonal to inoculate the monoclonal to a beef extract peptone liquid culture medium, and performing constant-temperature shaking culture at the constant speed of 160rpm for 10h at the temperature of 28 ℃, wherein the bacterial liquid can be used as a seed liquid for fermentation culture and can also be used as a bacterial liquid for a bacteriostatic test. Adding the seed solution of JD03 strain into Potato Dextrose Broth (PDB) according to the inoculum size of 2% (v/v), and performing constant temperature shaking culture at 160rpm at 28 ℃ for 48h to obtain JD03 strain fermentation liquor. Centrifuging the fermentation broth at 4 deg.C and 7500rpm for 15min, removing thallus, collecting supernatant, and filtering with sterile filter membrane with pore diameter of 0.22 μm to obtain sterile fermentation broth. Taking sterile fermentation liquor of JD03 strain, carrying out vacuum freeze drying, dissolving with sterile distilled water to obtain 1/10 of the original fermentation liquor volume, and filtering with a sterile filter membrane with the pore diameter of 0.22 mu m to obtain 10 times of concentrated solution of the sterile fermentation liquor.
(3) Inhibition of growth of Saprolegnia by JD03 strain fermentation broth: testing the inhibition effect of JD03 strain fermentation liquor and 10-fold concentrate of sterile fermentation liquor on the growth of saprolegnia hypha by adopting an agar diffusion method, adding 200 microliter JD03 bacterial liquid prepared in the central hole of a PDA plate, culturing at the constant temperature of 25 ℃ for 72 hours, and measuring the diameter of a bacteriostatic zone; sterile water was added to the control group, three in parallel per group (fig. 1, 3). The inhibition effect of JD03 strain sterile fermentation liquid on the germination of the saprolegnia spores is tested by a micropore equal-time dilution method, the sterile fermentation liquid is diluted by an equal-time dilution method and then is respectively added into a 96-pore plate, 10 mu L of saprolegnia spore liquid (about 50 spores) is respectively added into each pore, after the mixture is cultured for 48 hours at a constant temperature of 25 ℃, microscopic examination is carried out by an optical microscope, and the concentration corresponding to the complete non-germination of the spores is taken as the minimum inhibition concentration; sterile water was added to the control group, three in parallel per group (fig. 2).
(4) The effect of the high temperature of 100 ℃ on the inhibition of the growth of the saprolegnia by the JD03 sterile fermentation liquid 10 times concentrated solution is as follows: the experimental group of JD03 sterile fermentation broth 10 times concentrated solution was treated at 100 ℃ for 10min, while the control group was added with JD03 sterile fermentation broth 10 times concentrated solution without 100 ℃ treatment, and the diameter of the zone of inhibition was measured for three parallel groups (FIG. 4).
The results show that: in the PDA plate, the fermentation liquor of JD03 strain can obviously inhibit the germination of saprolegnia spores and the growth of saprolegnia hyphae, and a bacteriostatic zone with the diameter of 23.04 +/-0.99 mm is formed in the center of the plate, while the saprolegnia hyphae of the control group grows over the plate, as shown in figure 1.
When the sterile fermentation broth of JD03 strain was diluted twice, it could not completely inhibit spore germination, and when diluted twice, no spore germination was observed under microscope, which indicates that when the fermentation broth was diluted twice, it could completely inhibit spore germination, while the spores in the control group all germinated completely to grow hyphae, as shown in FIG. 2.
In the PDA plate, the JD03 10-fold concentrated solution of the sterile fermentation liquid can obviously inhibit the germination of the saprolegnia spores and the growth of saprolegnia hyphae, and a bacteriostasis zone with the diameter of 19.54 +/-0.84 mm is formed in the center of the plate, while the saprolegnia hyphae of the control group grows over the plate, as shown in figure 3.
The JD03 sterile fermentation liquid 10 times concentrated solution is heated at 100 ℃ for 10min, the liquid color is changed from light yellow to dark brown red, the diameter of the inhibition zone is 22.14 +/-1.12 mm, and the inhibition zone is larger than that of the control group which is not heated, which shows that the inhibition activity is enhanced, and is shown in figure 4.
Example 3: prevention effect of antagonistic strain JD03 fermentation liquor on saprolegniasis of artificial infection of grass carp
(1) Preparation of JD03 strain fermentation broth and water mold spore suspension: the same as in example 2.
(2) Taking healthy grass carp, placing the grass carp close to the tail behind dorsal fin, wiping off mucus on body surface with gauze, then scratching the body surface with rough object, drawing a wound with length of about 1.5cm and depth of about 1mm on two sides of the back respectively with syringe needle, dripping prepared water mould spore suspension on the wound, and putting into water containing water mould spores, wherein the experiment is divided into two groups, namely JD03 experiment group and control group, three parallel groups are respectively arranged, each barrel is filled with 25L of water, the treated 15-tail grass carp is placed, and 500mL of prepared water mould spore suspension (2 × 10) is added into each barrel4one/mL) to make the final concentration of the saprolegnia spores in the water body about 400/mL, 50mL of JD03 strain fermentation liquid (with the bacterial concentration of 5.9 × 10) was added to each barrel of the experimental group8cfu/mL), adding 50mL PDB culture solution into a bucket of a control group, continuously inflating in the experimental process, not feeding bait, replacing 1/2 water every other day, supplementing 250mL saprolegnia spore suspension into each bucket after water replacement, supplementing 30mL JD03 strain fermentation liquor into each bucket of the experimental group, wherein the concentration of the bacterial liquid is 5.9 × 108cfu/mL, control supplemented 30mL PDB medium per barrel. Observing and recording the condition of the Chinese herbal fish in each group every day, recording the number of diseased fish with saprolegnia hypha visible to naked eyes, fishing out in time if dead fish exists, and recording the number of dead fish.
The results show that: on the 5 th day after the experiment, the control group had developed macroscopic saprolegnia hyphae at the wound of the grass carp, and the hyphae were taken to confirm that saprolegnia is saprolegnia under a microscope, indicating that the injured fish is infected with saprolegnia. The infection rate of the grass carps in the control group is increased along with the prolonging of the experimental time in the period of 5-15 days. Compared with the control group, the infection rate of the grass carp in the experimental group is significantly lower than that of the control group at each time point (figure 5), and at the end of the experiment, the infection rate of the control group is 84.4% + -0.04, and is significantly higher than that of the experimental group by 31.1% + -1.28. Therefore, the JD03 fermentation liquor reduces the infection rate of the saprolegnia infected grass carp by 53.3 percent, and the incidence rate of the saprolegnia is remarkably reduced.
Example 4: separation and purification of anti-mildew active compound in JD03 strain fermentation liquor
(1) Preparation of fermentation broth of JD03 strain: the same as in example 2.
(2) Mixing JD03 strain fermentation liquor and n-butanol according to a volume ratio of 1:1, repeatedly shaking and uniformly mixing for 30s, repeating for 2-3 times, and extracting effective substances by the organic reagent as far as possible. Extracting the supernatant with organic reagent, evaporating to dryness with rotary evaporator, dissolving with methanol, placing in beaker, oven drying to obtain extract as active substance extract.
Dissolving 270g of active matter extract obtained by repeated extraction with n-butanol with methanol, uniformly mixing the active matter extract with the methanol by ultrasound, and then mixing the active matter extract with the methanol according to a mass ratio of 1:1 and 270g of silica gel (200-300 meshes) and mixing the two by continuous stirring. Drying in a 50 deg.C oven, grinding, filtering with four layers of gauze to obtain sample powder, and sealing for storage. And (3) filling the silica gel into a column by a wet method, namely soaking the silica gel (100-200 meshes) in petroleum ether to fully expand the silica gel and remove air, then continuously pouring the silica gel into a glass chromatographic column (10 multiplied by 120cm), and discharging the redundant petroleum ether to slowly precipitate the silica gel particles to the required height. And finally, adopting a dry method for loading, and slowly adding the sample powder into the chromatographic column to ensure that the contact surface is neat. Eluting by using a mobile phase with the polarity from small to large, wherein the mobile phase is ethyl acetate: petroleum ether 1:2, ethyl acetate: 1:1, ethyl acetate: methanol-8: 1, ethyl acetate: methanol 4:1, ethyl acetate: methanol 2:1, ethyl acetate: methanol 1:1, ethyl acetate: methanol 1:2, ethyl acetate: methanol-1: 4, methanol.
Weighing active parts obtained by eluting each mobile phase and evaporating to dryness by adopting a micropore equal-time dilution method, dissolving the active parts by using distilled water, filtering the active parts by using a filter membrane with the aperture of 0.22 mu m, obtaining active solutions with various concentrations by adopting the equal-time dilution method, adding the active solutions into a 96-pore plate respectively, adding 10 mu L of saprolegnia spore suspension (about 50 spores) into each pore, culturing the mixture at the constant temperature of 28 ℃ for 48 hours, and determining the minimum inhibitory concentration for inhibiting spore germination by using an optical microscope.
Activity detection, wherein the ratio of mobile phase ethyl acetate: petroleum ether 1:2, ethyl acetate: the petroleum ether 1:1 and ethyl acetate fractions may be combined, and the active ingredients of the fractions may be separated and purified by high performance liquid chromatography. The specific chromatographic conditions were as follows:
a chromatographic column: agilent TC-C18column, 5 μm, 4.6mm × 250 mm; welch UltimateXB-C18, 5 μm, 21.2mm × 250 mm;
mobile phase (V/V): methanol: 1:4 of water; methanol: water 2: 3;
flow rate: 1 mL/min; 8 mL/min;
wavelength: 210 nm;
sample introduction amount: 10 mu L of the solution; 1 mL.
Identifying chemical structure of the separated and purified anti-mildew active compound by using mass spectrum (EI-MS) and proton nuclear magnetic resonance hydrogen spectrum (f: (a))1H-NMR)、13C nuclear magnetic resonance carbon spectrum (C13C-NMR) to determine the structure of the compound active against water mold.
The results show that: active extract obtained by extracting fermentation liquor of JD03 strain with n-butanol is subjected to silica gel column chromatography, components eluted from different mobile phases are collected, evaporated to dryness and redissolved, and the activity of inhibiting germination of saprolegnia spores is determined, and the result is shown in figure 6. Wherein the mobile phase is mixed with ethyl acetate: petroleum ether 1:2, ethyl acetate: the petroleum ether 1:1 and ethyl acetate fractions were the most active, with the lowest inhibitory concentration for spore germination inhibition (fig. 6). The three are combined together, named as SY1 active ingredient, the dry weight is about 5g, and the next high performance liquid chromatography separation is carried out.
The active ingredient SY1 is analyzed by analytical high performance liquid chromatography, the mobile phase is 20% methanol and 80% deionized water, the flow rate is 1mL/min, the sample amount is 10 μ L, the ultraviolet detection wavelength is 210nm, and the active ingredient SY1 can be divided into I-1, I-2 and I-3 wave peaks. And preparing the wave crests of I-1, I-2 and I-3 by preparative high performance liquid chromatography, wherein the mobile phase is methanol: the flow rate of water is 1:4, the sample injection amount is 1mL/min, and the ultraviolet detection wavelength is 210 nm. The bacteriostatic activity of the reagent on the saprolegnia spore is determined by adopting a 96-well plate-micropore equal-fold dilution method, the activity peak is mainly concentrated on an I-3 wave peak, and the minimum concentration for inhibiting the saprolegnia spore germination is 3mg/mL, so that the reagent can be further separated and purified.
And (3) analyzing and preparing a sample corresponding to the active peak I-3 by using a second preparative high performance liquid chromatography. The mobile phase for the preparation of the liquid phase was methanol: the flow rate of water is 1:4, the sample injection amount is 1mL/min, and the ultraviolet detection wavelength is 210 nm. The active peak I-3 can also obtain two peaks II-1 and II-2, the activity of the two peaks for inhibiting the germination of the saprolegnia spores is detected by a 96-pore plate-micropore equal-time dilution method, the active part is positioned in the peak II-2, and the minimum concentration for inhibiting the germination of the saprolegnia spores is 0.75 mg/mL. Therefore, a sample corresponding to the II-2 peak is further separated to obtain two peaks III-1 and III-2, the activity of the two peaks for inhibiting the germination of the saprolegnia spores is detected by a 96-well plate-micropore equal-fold dilution method, the active part is positioned in the peak III-2, the minimum active concentration of the peak III-2 is 0.25mg/mL, and the peak III-2 is separated for further activity determination and purity detection.
Drying the active peak III-2, and determining the purity of the active peak by high performance liquid chromatography (figure 7), wherein the mobile phase is methanol: water 2: 3, the ultraviolet detection wavelength is 210nm, the sample injection amount is 10 mu L, and the time is 15 min. Referring to FIG. 7, the peak appeared at 3.6min and the purity was greater than 95%.
Mass spectrometry (EI-MS) gave the molecular weight of active compound III-2 as 129. At the same time, the nuclear magnetic displacement is13C-NMR(125MHz,DMSO)C1 177.43,C2 174.06,C3 55.46,C4 30.06,C5 25.47；1H-NMR (500MHz, DMSO)12.75(s,1H),7.70(s,1H),3.92(s,1H),2.24(d, J ═ 41.4Hz,1H),2.08(s,2H),1.95(s, 1H). Comparing the data, the active compound was identified as 2-pyrrolidone-5-carboxylic acid, formula C5H7O3N, the structural formula is shown as follows:
example 5: 2-pyrrolidone-5-carboxylic acid activity for inhibiting germination of saprolegnia spores
(1) Preparation of a saprolegnia spore suspension: the same as in example 2.
(2) The test method is a micropore equal-fold dilution method, the concentration of the 2-pyrrolidone-5-carboxylic acid in a 96-well plate is respectively 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL and 0.015625mg/mL, and 10 mu L of saprolegnia spore suspension (about 50 spores) is added in each well; sterile water was added to the control group, three in parallel per group. Under microscopic examination, the concentration corresponding to the complete non-germination of the saprolegnia spores is taken as the Minimum Inhibitory Concentration (MIC) for inhibiting the spore germination. The same as in example 2.
The results show that: the inhibitory effect of the active compound 2-pyrrolidone-5-carboxylic acid of JD03 strain fermentation broth on Saprolegnia spores is shown in FIG. 8. When the concentration of the active compound is more than or equal to 0.125mg/mL, the germination of the saprolegnia spores can be completely inhibited.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A water mould antagonistic bacterium JD03, characterized in that: the microbial strain preservation center is named as Burkholderia ubonensis JD03 and is preserved in 5.5.2019 at the Guangdong province microbial strain preservation center of No. 59 building No. 5 building Guangdong province microbial research institute of Miehuo 100, Guangzhou city, with the preservation number: GDMCC NO: 60660.
2. the application of the Saprolegnia antagonist JD03 in preparing products for preventing and treating saprolegniasis as claimed in claim 1, wherein: the pathogenic fungus causing saprolegniasis is a species of the genus Saprolegnia (Saprolegnia).
3. Use according to claim 2, characterized in that: the application of the Saprolegnia antagonist JD03 in preparing products for preventing and treating fish saprolegniasis.
4. Use according to claim 3, characterized in that:
the fish is freshwater fish.
5. Use according to claim 2, 3 or 4, characterized in that:
the active compound in the fermentation liquor of the Saprolegnia parasitica antagonist JD03 is 2-pyrrolidone-5-carboxylic acid.
6. A preparation method of 2-pyrrolidone-5-carboxylic acid is characterized by comprising the following steps: the method comprises the following steps:
(1) mixing the fermentation liquor of Saprolegnia antagonist JD03 of claim 1 and an organic reagent according to a volume ratio of 1:1, repeatedly shaking and uniformly mixing, and repeating for 2-3 times; extracting the supernatant with organic reagent, evaporating to dryness, dissolving with methanol, and oven drying to obtain extract as active substance extract;
wherein the organic reagent is n-butanol;
(2) dissolving the active matter extract with methanol, uniformly mixing the active matter extract with the methanol by ultrasonic, and then mixing the active matter extract with the methanol according to a mass ratio of 1:1, fully mixing with 200-300 meshes of silica gel, drying, fully grinding, filtering with four layers of gauze to obtain sample powder, and sealing and storing; filling the silica gel into a column by a wet method, namely soaking the 100-200-mesh silica gel by petroleum ether to fully expand the silica gel and remove air, then continuously pouring the silica gel into a glass chromatographic column, and simultaneously discharging redundant petroleum ether to slowly precipitate silica gel particles to a required height; finally, adopting a dry method for loading, slowly adding sample powder into the chromatographic column, and ensuring that the contact surface is neat; eluting by using a mobile phase with the polarity from small to large, wherein the mobile phase is ethyl acetate: petroleum ether 1:2, ethyl acetate: 1:1 of petroleum ether and ethyl acetate; collecting the components eluted from the three mobile phases, combining, and evaporating to dryness to obtain an active ingredient SY 1;
(3) the active ingredient SY1 is analyzed by an analytical high performance liquid chromatography, the mobile phase is 20% methanol and 80% deionized water, the flow rate is 1mL/min, the sample amount is 10 mu L, the ultraviolet detection wavelength is 210nm, and the active ingredient SY1 is sequentially divided into I-1, I-2 and I-3 wave peaks; and respectively preparing the wave crests I-1, I-2 and I-3 by preparative high performance liquid chromatography, wherein the mobile phase is methanol: the flow rate of water is 1:4, the sample volume is 1mL, the ultraviolet detection wavelength is 210nm, and the active peak is mainly concentrated on the I-3 wave peak; then the I-3 wave crest pairsAnd carrying out second preparative high performance liquid chromatography on the corresponding sample, wherein the mobile phase is methanol: water is 1:4, the flow rate is 8mL/min, the sample volume is 1mL, the ultraviolet detection wavelength is 210nm, II-1 and II-2 wave peaks are obtained in sequence, and the active site is positioned in the II-2 wave peak; and then carrying out third preparative high performance liquid chromatography separation on the sample corresponding to the II-2 wave peak, wherein the mobile phase is methanol: water is 1:4, the flow rate is 8mL/min, the sample volume is 1mL, the ultraviolet detection wavelength is 210nm, III-1 and III-2 wave peaks are obtained in sequence, and the active site is located in the III-2 wave peak; drying the sample corresponding to the III-2 wave peak, and determining the purity of the dried sample by adopting a high performance liquid chromatography, wherein the purity is more than 95%; to obtain the active compound 2-pyrrolidone-5-carboxylic acid with the molecular formula C5H7O3N, the structural formula is shown as follows:
the application of 2-pyrrolidone-5-carboxylic acid in preparing products for preventing and treating saprolegniasis is characterized in that:
the pathogenic fungus causing saprolegniasis is a species of the genus Saprolegnia (Saprolegnia).
8. Use according to claim 7, characterized in that: the 2-pyrrolidone-5-carboxylic acid is applied to the preparation of products for preventing and treating fish saprolegniasis.
9. Use according to claim 8, characterized in that:
the fish is freshwater fish.
10. Use according to claim 7, 8 or 9, characterized in that:
the effective concentration of the 2-pyrrolidone-5-carboxylic acid is more than or equal to 0.125 mg/mL;
the dosage form of the product is liquid medicine or powder;
the product contains one or more pharmaceutically acceptable carriers or excipients.
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