Antarctic strain as well as preparation and application thereof

Abstract

The invention belongs to the field of microorganisms, and particularly relates to an Antarctic strain as well as preparation and application thereof. The bacterial strain can reverse fish death caused by pathogenic bacteria and inhibit growth of the pathogenic bacteria, the bacterial strain grows well in a low-temperature environment, an innovative antibiotic replacement prevention and treatment method is provided for prevention and treatment of pathogenic bacteria infection in the aquaculture industry, and the bacterial strain is especially suitable for infection prevention and control in the overwintering period or when the water temperature gets warm in spring. The fish breeding loss is effectively reduced, and the healthy development of the aquatic product breeding industry is promoted.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a novel Antarctic strain CLB_Antarctic_008PLUS which can replace antibiotics, can reduce the mortality of fish infected by A.veronii, and can inhibit the growth of A.veronii. Background Art

[0002] Largemouth bass (Micropterus salmoides), commonly known as black bass or California bass, belongs to the genus Micropterus of the family Micropteridae of the order Perciformes. It is native to North America and has been introduced to other parts of the world. Largemouth bass was introduced to China from the United States in 1983. It has become one of China's main aquaculture species due to its delicious meat, fast growth rate and easy breeding. It has now become a specialty freshwater fish that my country has vigorously developed. In recent years, the scale of breeding has continued to expand, helping the development of aquaculture, one of the key areas of China's economic development.

[0003] However, as the degree of intensive farming of largemouth bass increases, disease problems have also occurred one after another, threatening the healthy development of largemouth bass farming. Largemouth bass aquaculture has suffered serious losses due to infectious diseases caused by bacterial infections. Especially after the wintering period, the immune system function of fish decreases, and they are susceptible to pathogenic bacteria infection when the water temperature rises. In my country, there have been many large-scale deaths of largemouth bass and other fish due to bacterial infections during the spring water temperature rise period. We isolated the pathogenic bacterial strain Aeromonas veronii (in situ temperature is about 15°C) from the large-scale mortality event of largemouth bass after wintering, which can cause 100% mortality of largemouth bass and zebrafish with slightly damaged skin. The largemouth bass farming industry is seriously threatened by pathogens such as Aeromonas veronii, causing significant economic losses.

[0004] Therefore, there is an urgent need to develop an effective prevention and control method at low temperatures that can replace antibiotics to meet the needs of the aquaculture industry and ensure the sustainable development of the aquaculture industry. Summary of the invention

[0005] The present invention discovered an Antarctic strain, which exhibits a good growth rate in a low temperature environment, can reverse fish deaths caused by the pathogenic bacteria strain Aeromonas veronii, and the extract of the fermentation broth of the strain can inhibit the growth of the pathogenic bacteria strain Aeromonas veronii. The Antarctic strain can effectively replace antibiotics for the prevention and treatment of infection by the pathogen Aeromonas veronii at low temperatures, providing a new antibiotic replacement strategy, bringing an innovative prevention and treatment method to the aquaculture industry, and is particularly suitable for infection prevention and control of fish during the winter or when the water temperature warms up in spring for infectious diseases caused by A. veronii.

[0006] The invention provides an Antarctic strain, comprising an Antarctic strain with a deposit number of CCTCC NO: M20241961, and / or a subculture strain of the Antarctic strain with a deposit number of CCTCC NO: M20241961.

[0007] The present invention provides a product of an Antarctic bacterial strain, comprising at least one of the following:

[0008] 1) Cultures of Antarctic strains;

[0009] 2) extracts from cultures of Antarctic strains;

[0010] 3) Fermentation products of Antarctic strains;

[0011] 4) Extracts of fermented Antarctic strains;

[0012] The Antarctic strain comprises the Antarctic strain with a deposit number of CCTCC NO: M20241961, and / or a subculture strain of the Antarctic strain with a deposit number of CCTCC NO: M20241961.

[0013] The extract of the Antarctic strain culture is an ethyl acetate extract of the Antarctic strain culture, and the extract of the Antarctic strain fermentation product is an ethyl acetate extract of the Antarctic strain fermentation product.

[0014] Cultures and fermentations refer to products prepared with the help of the life activities of microorganisms, including but not limited to the microorganisms themselves, direct metabolites, or secondary metabolites.

[0015] The invention provides a culture method or fermentation method of an Antarctic strain, comprising the steps of: placing the Antarctic strain in a culture medium for culture or fermentation, wherein the Antarctic strain comprises an Antarctic strain with a preservation number of CCTCC NO: M20241961 and / or a subculture strain of the Antarctic strain with a preservation number of CCTCC NO: M20241961.

[0016] The culture medium is a mixture of nutrients required for the life activity of microorganisms, used for co-cultivation or fermentation, and can be in the form of liquid, solid or semi-solid.

[0017] The culture medium is 2216E culture medium, which can be in the form of liquid, solid or semi-solid.

[0018] The culture or fermentation temperature is 4°C-35°C, preferably 10°C-28°C, more preferably 15°C-25°C.

[0019] The invention provides a method for preparing an extract from a culture or fermentation product of an Antarctic strain, the steps comprising: extracting the culture or fermentation product of the Antarctic strain with ethyl acetate, wherein the Antarctic strain comprises an Antarctic strain with a preservation number of CCTCC NO: M20241961 and / or a subculture strain of the Antarctic strain with a preservation number of CCTCC NO: M20241961.

[0020] The culture or fermentation product of the Antarctic strain is obtained by placing the Antarctic strain in a culture medium for culturing or fermentation.

[0021] The culture medium is 2216E culture medium, which can be in the form of liquid, solid or semi-solid.

[0022] The culture or fermentation temperature is 4°C-35°C, preferably 10°C-28°C, more preferably 15°C-25°C.

[0023] The volume ratio of ethyl acetate to the culture or fermentation product of the Antarctic bacterial strain is 1-2:1, preferably 1:1.

[0024] The cultured bacterial solution is extracted with ethyl acetate and then purified and concentrated by vacuum distillation, for example, vacuum distillation at 41° C., 210 kPa, and a rotation speed of 50 rpm.

[0025] The extract of the Antarctic strain culture (fermentation) provided by the present invention inhibits the growth of the pathogenic bacteria A.veronii. The culture (fermentation) of the Antarctic strain provided by the present invention can reverse the fish death caused by the pathogenic bacteria A.veronii and improve the survival rate of the fish. Therefore, the Antarctic strain, the Antarctic strain culture (fermentation) and the extract of the Antarctic strain culture (fermentation) provided by the present invention can be used to prepare products for the prevention and treatment of pathogenic bacteria, and are used for the prevention, control or treatment of fish pathogenic bacteria infection.

[0026] The present invention provides a product for preventing and treating fish pathogenic bacteria, comprising the Antarctic strain and / or the product of the Antarctic strain provided by the present invention.

[0027] The product for preventing and treating fish pathogenic bacteria provided by the present invention can be a medicine or reagent, can be a feed or additive, can be a water quality improver, or can be a bottom quality improver, and its form can be solid, semi-solid, liquid or gas.

[0028] The present invention provides a fish breeding method, comprising the following steps: the fish breeding environment contains the Antarctic strain and / or the product of the Antarctic strain provided by the present invention.

[0029] The crude extract of the fermentation liquid of the Antarctic strain CLB_Antarctic_008PLUS of the present invention can significantly inhibit the growth of A.veronii. In addition, after the fermentation liquid of CLB_Antarctic_008PLUS was added to the water where the largemouth bass and zebrafish with slightly damaged skin were located, the mortality rate of the largemouth bass related to A.veronii infection was reduced to 10%, and the mortality rate of the zebrafish was reduced to 20%.

[0030] The present invention provides an innovative, green and efficient biological control method for the prevention and control of pathogenic bacteria A. veronii infection in aquaculture, especially under low temperature conditions (such as 15-20°C). This method can effectively reduce the breeding losses of largemouth bass and other economic fish and promote the healthy development of aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a picture of the cultured CLB_Antarctic_008PLUS strain.

[0032] Figure 2 This is the Gram staining image of CLB_Antarctic_008PLUS strain.

[0033] Figure 3 Gel electrophoresis of the 16S rDNA amplification product (sample) used for BLAST confirmation of the species of the CLB_Antarctic_008PLUS strain.

[0034] Figure 4 The results of the antibacterial experiment of the crude extract of CLB_Antarctic_008PLUS against Aeromonas veronii are shown in the figure. The upper left is the CLB_Antarctic_008PLUS group, the upper right is the negative control ddH2O group, and the bottom is the neomycin group.

[0035] Figure 5 This is a picture of a sick sea bass.

[0036] Figure 6 Image of zebrafish that died during the A. veronii bacterial immersion experiment. DETAILED DESCRIPTION

[0037] This study isolated and identified pathogenic bacteria from largemouth bass, used secondary metabolites of Antarctic bacteria to conduct antibacterial experiments, and verified them through in vivo rescue experiments, providing a theoretical basis for further exploring prevention and control measures for diseases caused by bacteria. This study helps to further understand the pathological characteristics of pathogenic microorganisms of diseased largemouth bass, and provides a theoretical basis for early diagnosis of bacterial diseases in bass and timely and accurate formulation of reasonable treatment methods.

[0038] Specifically, we isolated the pathogenic bacterial strain Aeromonas veronii from a mass mortality event after overwintering of largemouth bass (in situ temperature approximately 15°C), which was able to cause 100% mortality in largemouth bass and zebrafish with mild skin damage.

[0039] The wild strain CLB_Antarctic_008PLUS was collected from the intestinal contents of Antarctic fish (Dissostichus mawsoni). After a series of scientific steps, including nutrient broth (NB) medium enrichment, plate separation, purification, bacterial genome extraction, 16S ribosomal DNA amplification and sequence alignment, it was finally determined to be a new species of Vitreosilla. CLB_Antarctic_008 can grow in the range of 4°C to 35°C, and shows a certain growth ability in both marine and freshwater culture media. It can grow stably at 4°C. After continuous subculture, a low-temperature acclimated strain, namely CLB_Antarctic_008PLUS, was obtained.

[0040] The fermentation broth of CLB_Antarctic_008PLUS and its secondary metabolites showed significant inhibitory effects on the fish pathogen A.veronii. In addition, the fermentation broth can significantly reduce the mortality of zebrafish and largemouth bass caused by A.veronii infection.

[0041] The technical solution of the present invention is described below in conjunction with specific embodiments.

[0042] 1. Extraction of secondary metabolites of Antarctic strains of the present invention

[0043] 1. Isolation and identification of Antarctic strains

[0044] Dissostichus mawsoni was taken out from -80℃, stored at -20℃ and 4℃ for 12h, and then dissected after thawing. The morphology, body length and weight of the fish were recorded before dissection, and the species molecular identification was performed. The surface of the fish was wiped with 75% alcohol for disinfection, and the fish was dissected with pre-sterilized scissors and forceps. The intestinal contents, stomach contents, body fluids and various tissues were taken out in turn, and the tissues were placed in 1.5ml EP tubes, and 1ml TRIzol was added for storage. The intestinal contents, stomach contents and body fluids were placed in sterile EP tubes.

[0045] Isolation and purification of strains: Take 100 μl of the intestinal contents obtained by dissection and put them in EP tubes, dilute them 10 times with seawater filtered with a 0.22 μm filter membrane and sterilized by high pressure, and evenly spread 50 μl of the diluted samples on nutrient broth (NB) and 2216E solid culture medium plates, culture them at 18°C ​​for 2-3 days, select single colonies with large differences in morphology and size from the plates, and culture them in a shaker at 18°C ​​and 200 rpm with the corresponding liquid culture medium. After constant temperature culture for 2-3 days, streak culture on solid culture medium, and purify repeatedly until a single colony is obtained for subsequent experiments. At the same time, the obtained strains are stored in 50% glycerol and placed in a -80°C refrigerator.

[0046] Microbial identification: 20 μl PCR reaction system, 27F primer (5'-AGAGTTTGATCCTGGCTCAG-3') 1 μl, 1492R primer (5'-GGTTACCTTGTTACGACTT-3') 1 μl, dNTP Mix solution 10 μl, bacterial liquid template 1 μl, double distilled water 7 μl, centrifuge and mix, then place 20 μl of the mixture in a PCR instrument, and amplify through the PCR amplification program: 95℃10min pre-denaturation, then 95℃30s, 55℃30s, 72℃1min 30s, 29 cycles, and finally extend at 72℃ for 10min. After the reaction is completed, the PCR product is taken and subjected to 1% agarose gel electrophoresis, and the product with a single bright band is selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Its species is determined by BLAST comparison, and it is a new species of Vitreosilla.

[0047] CLB_Antarctic_008PLUS was deposited in the China Center for Type Culture Collection (Wuhan University, Wuhan, China, Postal Code 430072) on September 13, 2024, with the deposit number of CCTCC NO:M20241961, the culture name of Vitreoscilla sp.CLB_ANTARCTIC_008Plus, and the deposit result was survival.

[0048] 2. Extraction of Crude Extracts of Antarctic Bacteria

[0049] According to the inoculation amount of 10%, the bacterial solution of CLB_Antarctic_008PLUS obtained after separation and purification was added with 300ml2216E liquid culture solution in a conical flask, and the culture was shaken at 18°C ​​and 200rpm for 60h, and 60L of CLB_Antarctic_008PLUS culture solution was fermented and cultured cumulatively. An equal volume of ethyl acetate was added, and the extraction was carried out according to the ratio of bacterial solution to ethyl acetate of 1:1. After sufficient stirring, it was allowed to stand, and it was stirred once every five minutes and then allowed to stand, and repeated three times. The supernatant was taken and rotary evaporated, and the temperature was set to 41°C, the pressure was 210kPa, and the speed was 50rpm using a rotary evaporator (RV 3pro V Package, IKA, Germany). The light yellow concentrate left at the end of the rotary evaporation was the crude extract of bacterial metabolites, which was dissolved in 1ml of sterilized H2O.

[0050] 2. Antimicrobial activity experiment of Antarctic bacteria metabolites

[0051] In order to avoid bacterial contamination, this experiment was carried out in a biological safety cabinet, and all reagents and consumables used in the operation were strictly sterilized to avoid foreign bacterial contamination during the operation.

[0052] The activity of the secondary metabolite (crude extract) of Antarctic bacteria CLB_Antarctic_008PLUS in inhibiting the growth of A.veronii was tested by the filter paper method. The pathogenic bacteria A.veronii was made into a bacterial suspension, 50μl was taken and dropped on the NB solid plate, and evenly coated with a glass coating rod to make a plate containing indicator bacteria. The metabolite concentrate of Antarctic bacteria CLB_Antarctic_008PLUS was dissolved with 1ml of sterile seawater, and the secondary metabolites were fully dipped with a blank drug-sensitive paper (6mm) and placed on the plate. Sterile seawater was used as a negative control, and 100μg / ml neomycin was used as a positive control. The plate was placed in a 28℃ incubator for 20h, and the diameter data of the transparent sterile aperture around the filter paper was observed and recorded. It was found that the metabolite of Antarctic bacteria CLB_Antarctic_008PLUS can inhibit the growth of A.veronii, no colonies grew around the filter paper, and there was a transparent aperture. The positive control neomycin also had an inhibition zone, while the negative control sterile water had no inhibition zone, indicating that the secondary metabolites of the Antarctic bacteria CLB_Antarctic_008PLUS had an inhibitory effect on the growth of the pathogenic bacteria A.veronii.

[0053] 3. Bacterial challenge experiment

[0054] The A. veronii bacteria isolated from the wounds of diseased largemouth bass were cultured on a shaker at 18°C, and the OD value of 600 was measured every 4 hours using a spectrophotometer (BioSpectrometer, Eppendorf, Germany) until the OD value of the bacterial solution reached 3. At the same time, the number of bacteria in the bacterial solution was counted using a bacterial counting plate, and a certain volume of the sample bacterial suspension was placed in the counting cell of the bacterial counting plate, and the count was observed under a microscope. According to the number of bacteria in the counting chamber scale, the number of bacteria in the sample was calculated according to the number of bacteria in the counting chamber scale. A standard curve between the number of bacteria and the absorbance of the bacterial culture was established. When the OD value reached 3, the bacterial concentration was 3-4×10 9 CFU / ml.

[0055] In the zebrafish challenge experiment, 60 healthy adult zebrafish were randomly selected and observed at 27±1℃ for two days. After the state stabilized, the bacterial challenge experiment was performed. The zebrafish were randomly divided into 2 groups, 1 control group (3 breeding tanks) and 1 experimental group (3 breeding tanks). Each group had 30 zebrafish, and every 10 zebrafish were placed in a 3L water breeding tank. The final bacterial concentration in the experimental group tank was 1×10 7 CFU / ml, and an equal volume of PBS was added to the control group. During the experiment, the water temperature, pH, dissolved oxygen and other breeding environments of each culture tank were kept consistent.

[0056] Infection experiment: zebrafish were anesthetized with eugenol, surgical blades were wiped and disinfected with ethanol, and a 5mm incision was made obliquely on the abdomen of the fish with a scalpel, and then the fish was returned to the fish tank. The fish were observed for signs of infection and death every 12 hours. Finally, the bacterial community in the wounds of dead zebrafish was detected. The results showed that A. veronii was present in large quantities in the intestines and gills of zebrafish in the infection group, which may be the main cause of death of zebrafish. After 72 hours, the mortality rate of zebrafish was 100%, and there was no death in the control group.

[0057] 4. Rescue Experiment

[0058] The strain CLB_Antarctic_008PLUS isolated from the intestinal contents of Antarctic fish was placed in a liquid culture medium in a constant temperature shaker at 10°C for expansion culture, and the strain A.veronii isolated from diseased fish in the Central Ocean was placed in a constant temperature shaker at 18°C ​​for expansion culture.

[0059] Zebrafish experiment: 120 healthy zebrafish were taken from the laboratory breeding system and observed for 48 hours to ensure that there was no death and stress before conducting subsequent experiments. They were randomly divided into 4 groups: control group (3 breeding tanks), challenge group (3 breeding tanks), Antarctic bacteria group (3 breeding tanks) and rescue group (3 breeding tanks). Each group contained 30 zebrafish, divided into 3 breeding tanks with 3 liters of water, with 10 zebrafish placed in each tank. After anesthesia, a wound of about 5 mm was scratched on the abdomen of the zebrafish. After the zebrafish regained consciousness in the breeding water, they were placed in the corresponding breeding tanks. The control group was soaked in PBS, and the pathogenic bacteria A.veronii was added to the challenge group at a concentration of 1×10 7 CFU / ml, Antarctic bacteria CLB_Antarctic_008PLUS was added to the Antarctic bacteria group at a concentration of 1×10 7 CFU / ml, pathogenic bacteria A.veronii and Antarctic bacteria CLB_Antarctic_008PLUS were added to the rescue group to make the concentration of A.veronii and CLB_Antarctic_008PLUS 1×10 7 CFU / ml. During the experiment, the water temperature, pH, dissolved oxygen and other breeding environments of each breeding tank were kept consistent.

[0060] Largemouth black bass experiment: 120 healthy largemouth black bass were randomly selected, with a body length of about 15 cm and a body weight of about 100 g. They were observed at 15±2℃ for two days. No mortality was observed for the next experiment. They were randomly divided into 4 groups, 1 control group (3 breeding tanks), 1 challenge group (3 breeding tanks), 1 Antarctic bacteria group (3 breeding tanks), and 1 rescue group (3 breeding tanks). There were 30 bass in each group, and every 10 were placed in a breeding tank with 10L water. If there was no mortality, the challenge and rescue experiments were carried out again. After anesthesia, a wound of about 1 cm was scratched on the abdomen. After the fish regained consciousness in clean water, subsequent treatment was carried out, and the subsequent steps were the same as those for the zebrafish challenge and rescue experiments. The final bacterial concentrations of the Antarctic strain CLB_Antarctic_008PLUS and the pathogenic bacterium A.veronii were 3×10 7 CFU / mL. During the experiment, the water temperature, pH, dissolved oxygen and other aquaculture environments of each aquaculture tank were kept consistent.

[0061] The experimental results show that:

[0062] The zebrafish blank control group did not die, with a mortality rate of 0%, the mortality rate of the perch in the pathogenic bacteria A.veronii group was 100%, the mortality rate of the Antarctic bacteria CLB_Antarctic_008PLUS group was 0%, and the mortality rate of the group soaked in both Antarctic bacteria CLB_Antarctic_008PLUS and pathogenic bacteria A.veronii was 20%. The experimental results showed that Antarctic bacteria CLB_Antarctic_008PLUS significantly inhibited the pathogenic bacteria A.veronii and increased the survival rate by 80%.

[0063] In the blank control group of largemouth bass, there was no death of largemouth bass, with a mortality rate of 0%, the mortality rate of the challenge group soaked in A. veronii was 100%, the mortality rate of the bass soaked in Antarctic bacteria CLB_Antarctic_008PLUS group was 10%, and the mortality rate of the bass soaked in both Antarctic bacteria CLB_Antarctic_008PLUS and pathogenic bacteria A. veronii was 10%. The experimental results showed that the mortality rate of bass was reduced by 90% when Antarctic bacteria CLB_Antarctic_008PLUS and A. veronii were added simultaneously.

[0064] This study helps to further understand the pathological characteristics of pathogenic microorganisms of diseased largemouth bass and provide a theoretical basis for the early diagnosis of bacterial (A. veronii) diseases of bass and the timely and accurate formulation of reasonable treatment methods.

[0065] Bacteria immersion experiment death statistics:

[0066]

[0067] It should be pointed out that the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology of this project to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An Antarctic bacterial strain, characterized in that It comprises the Antarctic strain with the deposit number of CCTCC NO:M20241961, and / or the subculture strain of the Antarctic strain with the deposit number of CCTCC NO:M20241961.

2. A product of an Antarctic bacterial strain, characterized in that Include at least one of the following: 1) Cultures of Antarctic strains; 2) extracts from cultures of Antarctic strains; 3) Fermentation products of Antarctic strains; 4) Extracts of fermented Antarctic strains; The Antarctic strain comprises the Antarctic strain with a deposit number of CCTCC NO: M20241961, and / or a subculture strain of the Antarctic strain with a deposit number of CCTCC NO: M20241961.

3. A method for culturing or fermenting an Antarctic bacterial strain, characterized in that: The Antarctic strain is placed in a culture medium for culture or fermentation, wherein the Antarctic strain comprises the Antarctic strain with a deposit number of CCTCC NO: M20241961, and / or a subculture strain of the Antarctic strain with a deposit number of CCTCC NO: M20241961.

4. The culture method or fermentation method according to claim 3, characterized in that: The culture or fermentation temperature is 4°C-35°C.

5. A method for preparing an extract from a culture or fermentation product of an Antarctic bacterial strain, comprising: The culture or fermentation product of the Antarctic strain is extracted with ethyl acetate, wherein the Antarctic strain comprises the Antarctic strain with a deposit number of CCTCC NO: M20241961 and / or a subculture strain of the Antarctic strain with a deposit number of CCTCC NO: M20241961.

6. The method according to claim 5, characterized in that The volume ratio of ethyl acetate to culture or fermentation product is 1-2:

1.

7. Use of the Antarctic strain described in claim 1 and / or the product described in claim 2 in preparing products for preventing and treating fish pathogens.

8. The use according to claim 7, characterized in that: The product includes at least one of the following: 1) Drugs or reagents; 2) Feed or additives; 3) Water quality improver; 4) Soil conditioner.

9. A product for preventing and treating fish pathogens prepared from the Antarctic strain according to claim 1 and / or the product according to claim 2, comprising at least one of the following: 1) Drugs or reagents; 2) Feed or additives; 3) Water quality improver; 4) Soil conditioner.

10. A method for breeding fish, characterized in that: The steps include: The fish breeding environment contains the Antarctic strain described in claim 1 and / or the product described in claim 2.

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