CN114262666A - Screening method of leuconostoc mesenteroides and application of leuconostoc mesenteroides in preparation of aquatic bacteriostatic agent - Google Patents
Screening method of leuconostoc mesenteroides and application of leuconostoc mesenteroides in preparation of aquatic bacteriostatic agent Download PDFInfo
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Abstract
The invention discloses a screening method of leuconostoc mesenteroides and application of the leuconostoc mesenteroides in preparing bacteriostatic agent for preventing and treating aquatic bacterial diseases, which comprises the steps of carrying out gradient dilution on pickle juice by using sterile water, coating the diluent on an MRS solid plate added with calcium carbonate, culturing at 37 ℃, selecting a single bacterial colony with a calcium-soluble ring, and carrying out lineation and purification on the MRS solid plate to obtain a pure strain; extracting genome DNA of the screened strain as a template, and carrying out PCR amplification on the 16S rRNA gene; the screened strains after sequencing are inoculated on an MRS solid plate and cultured at the constant temperature of 37 ℃ until colonies are formed. The invention also discloses application of the leuconostoc mesenteroides in preparing a bacteriostatic agent for preventing and treating aquatic bacterial diseases. The leuconostoc mesenteroides screened by the invention has an inhibition effect on various aquatic pathogenic bacteria by producing extracellular bacteriostatic substances, can improve the survival rate of fish under the stress of aeromonas, and can improve the survival rate of bacteria by preparing the viable bacteria agent.
Description
Technical Field
The invention belongs to the technical field of aquatic microorganism application, and particularly relates to a screening method of leuconostoc mesenteroides and application thereof in preparation of a bacteriostatic agent for preventing and treating aquatic bacterial diseases.
Background
Freshwater aquaculture is an important component of inland aquaculture, provides a large amount of high-quality animal protein for human beings, is one of the most main sources for obtaining the high-quality animal protein by the human beings in the future, and has important significance for improving the food structure of people. The freshwater aquaculture product is deeply favored by consumers due to the characteristics of low fat content, fresh and tender meat quality, rich vitamins and the like, the yield and the scale of the freshwater aquaculture industry are increased, and the freshwater aquaculture area and the yield of China are the top in the world. The river system runs through the middle and lower reaches of the yellow river in the south of the Henan, the resources of the beach land of the south of the yellow river are more, the water source is sufficient, the terrain is flat, the water source is tidy and continuous, and the development of aquaculture has the unique advantage. The healthy development of the aquaculture industry in the yellow river basin has important significance for the ecological environment and the economic and civil life of the yellow river.
Aquatic pathogenic bacteria such as Aeromonas veronii, Aeromonas salmonicida, Shewanella, Salmonella, etc. can cause skin ulcer and acute hemorrhagic septicemia of freshwater fish in large scale, and can infect human body to cause diarrhea, food poisoning and secondary infection. Due to unreasonable use of antibacterial drugs and spread of drug-resistant strains and drug-resistant genes, the problem of drug resistance of aquatic pathogenic bacteria is prominent, so that difficulty is brought to prevention and treatment of aquatic bacterial diseases, and meanwhile, a large number of fish infectious diseases caused by pathogenic bacteria bring considerable influence to aquatic breeding industry, and huge economic loss is caused.
The lactobacillus is a probiotic, is an important member forming a normal microbial flora in a specific area, can regulate intestinal flora and improve the intestinal environment, and has an important promotion effect on the health of organisms. Meanwhile, the lactobacillus can also secrete fine protein or peptide (antibacterial peptide) which has a certain inhibition effect on pathogenic bacteria. However, the number of the antibacterial substances of the lactic acid bacteria found at present is not large, and the reports of the application of the antibacterial substances in the prevention and treatment of aquatic bacterial diseases are quite limited. Therefore, the screening of the lactic acid bacteria with the inhibition effect on the growth of the aquatic pathogenic bacteria has important significance and application value for preventing and treating the aquatic bacterial diseases.
Disclosure of Invention
The invention aims to provide a screening method of leuconostoc mesenteroides, and the leuconostoc mesenteroides screened by the method can be used for effectively preventing and treating aquatic bacterial diseases.
The invention adopts the following technical scheme for solving the technical problems, and the screening method of leuconostoc mesenteroides is characterized by comprising the following specific steps of: subjecting kimchi juice to sterile water treatment 10-1-10-5Gradient dilution, namely coating the diluents with different gradients on an MRS solid plate added with calcium carbonate, culturing for 48h at 37 ℃, selecting a single colony with a calcium-dissolving ring, streaking and purifying on the MRS solid plate, repeatedly operating for 2-3 times to obtain a pure strain, and preserving the pure strain with glycerol at-80 ℃; extracting genome DNA of the screened strain as a template, and carrying out PCR amplification on the 16S rRNA gene under the conditions that: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 45s, 30 cycles, extension at 72 ℃ for 2min, sequencing after purifying PCR products, performing BLAST comparison on sequencing results and NCBI, and selecting seeds or genera with high similarity to the sequencing results; and (3) inoculating the screened strain after sequencing on an MRS solid plate, and culturing at the constant temperature of 37 ℃ for 24-48h until a colony is formed.
The leuconostoc mesenteroides can inhibit the formation of bacterial biofilms and the secretion of bacterial extracellular proteases.
Further defined, the aquatic bacteria is one or more of salmonella, escherichia coli, pseudomonas aeruginosa, aeromonas salmonicida, shewanella or aeromonas veronii.
Further limited, the bacteriostatic agent for preventing and treating the aquatic bacterial diseases is a leuconostoc mesenteroides ethyl acetate extract, and the specific preparation process of the leuconostoc mesenteroides ethyl acetate extract is as follows: activating leuconostoc mesenteroides in an MRS liquid culture medium, culturing at 37 ℃ overnight, centrifuging fermentation liquor of the leuconostoc mesenteroides for 3min at 12000rpm, taking supernatant, adding ethyl acetate with the same volume, shaking and uniformly mixing for 3 times, 2h each time, mixing ethyl acetate extract obtained each time, decompressing and evaporating by using a rotary evaporator, and adding dimethyl sulfoxide to dissolve to obtain a crude extract, namely the leuconostoc mesenteroides ethyl acetate extract.
Further limiting, the bacteriostatic agent for preventing and treating the aquatic bacterial diseases is a leuconostoc mesenteroides freeze-dried viable bacterial agent, the freeze-dried viable bacterial agent takes skimmed milk powder, trehalose, sodium glutamate and xylo-oligosaccharide as freeze-dried protective agents, and the survival rate of the leuconostoc mesenteroides in the freeze-dried viable bacterial agent can reach 82 percent to the maximum; the preparation process of the freeze-dried viable bacteria agent comprises the following steps: activating Leuconostoc mesenteroides in MRS liquid culture medium, culturing at 37 deg.C overnight, centrifuging the fermented liquid of Leuconostoc mesenteroides at 12000rpm for 3min, and collecting thallus; sterilizing trehalose, sodium glutamate and xylooligosaccharide with a bacteria filter with a membrane pore size of 0.22 μm, and refrigerating for later use; keeping the skimmed milk powder at 110 deg.C for 10min for sterilization; mixing the bacteria with skimmed milk powder, trehalose, sodium glutamate and xylo-oligosaccharide, uniformly mixing on a vortex mixer to prepare a suspension, pre-freezing the suspension at-70 ℃ for 2h, taking out and freeze-drying in a vacuum freeze dryer to prepare the live bacteria agent of leuconostoc mesenteroides, wherein the freeze-drying condition is that the vacuum degree is 5Pa, the heating temperature of a clapboard is 20 ℃, the cold trap temperature is-55 ℃, and the freeze-drying time is 30 h.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the leuconostoc mesenteroides strain has wide tolerance range on pH, is suitable for growth in aquaculture water, has high acid production capacity, and is convenient for relieving the phenomenon of pH rise of the water caused by algae outbreak.
2. The leuconostoc mesenteroides bacteriostatic substance is a high-temperature-resistant substance, has an inhibiting effect on various aquatic pathogenic bacteria, and has high application potential.
3. The Leuconostoc mesenteroides freeze-dried viable bacteria agent has high survival rate of bacteria, and can be used for effectively improving the survival rate of bacteria, particularly fish under the stress of aeromonas.
In conclusion, the leuconostoc mesenteroides screened by the invention has an inhibiting effect on various aquatic pathogenic bacteria by producing extracellular bacteriostatic substances, can improve the survival rate of fish stressed by aeromonas, improves the survival rate of bacteria by preparing the viable bacteria agent, and has a higher application prospect and a higher practical value.
Drawings
FIG. 1 is a phylogenetic tree diagram (A) of Leuconostoc mesenteroides constructed based on 16S rRNA gene sequence and its colony morphology diagram (B) and simple staining diagram (C) of cells.
FIG. 2 is a graph of Leuconostoc mesenteroides growth at different pH (A) and its acid production potential (B).
FIG. 3 shows the bacteriostatic ability of Leuconostoc mesenteroides, wherein A is Salmonella, B is Escherichia coli, C is Pseudomonas aeruginosa, D is Aeromonas salmonicida Z2, E is Shewanella Z3, and F is Aeromonas veronii Z12.
Fig. 4 is a study of the bacteriostatic effect and thermal stability of the ethyl acetate extract of leuconostoc mesenteroides.
FIG. 5 shows the effect of Leuconostoc mesenteroides on the biofilm-forming ability (A) and protease production (B) of pathogenic bacteria.
FIG. 6 shows the survival rate of bacteria in the freeze dried live Leuconostoc mesenteroides bacterial agent.
FIG. 7 is a graph of the effect of Leuconostoc mesenteroides on fish survival.
Detailed Description
The following detailed description of the embodiments of the present invention will be described in detail with reference to the accompanying drawings and examples, which are provided for illustration of the present invention and are not intended to limit the scope of the present invention, and the parameters, proportions, etc. of the examples may be selected according to circumstances without substantially affecting the results. Unless otherwise specified, the methods described in the examples are all conventional methods, and the reagents used are all conventional reagents or reagents formulated in a conventional manner.
Example 1
Separation, screening and identification of leuconostoc mesenteroides
(1) Subjecting kimchi juice to sterile water treatment 10-1-10-5Gradient dilution, applying different gradient dilutions to MRS solid plate added with calcium carbonateCulturing at 37 deg.C for 48h, selecting single colony with calcium-dissolving ring, streaking and purifying on MRS solid plate, repeating the operation for 2-3 times to obtain pure strain, storing the pure strain with-80 deg.C glycerol, and performing subsequent experiments.
(2) Extracting the genome DNA of the selected strain as a template, and carrying out PCR amplification on the 16S rRNA gene. The PCR amplification conditions were: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 45s, 30 cycles, extension at 72 ℃ for 2min, purification of PCR products and sequencing. And performing BLAST comparison on the sequencing result on NCBI, and selecting the species or the genus with high similarity to the sequencing result. Phylogenetic trees were constructed using MEGA 5 biological software (a in fig. 1). The results show that the strain and the lactic acid bacteria leuconostoc mesenteroides (B)Leuconostoc mesenteroidesLm) had the highest homology and was designated asLeuconostoc mesenteroides DH（MW821358）。
(3) The selected strain is inoculated on an MRS solid plate, and is cultured for 24-48h at the constant temperature of 37 ℃ until a colony is formed, and the colony morphology is observed. And their cell morphology was observed under an oil lens by simple staining (B and C in FIG. 1). After the strain grows for 24 hours on an MRS solid culture medium, the strain is observed to be milky, round, neat in edge and opaque; after simple staining of the bacterial cells, the bacterial cells were observed under an optical microscope to be spherical, short rod-shaped, about 2 μm long and about 1 μm wide.
Example 2
Research on growth condition and acid production capacity of leuconostoc mesenteroides under different pH values
(1) Leuconostoc mesenteroides after activation in MRS was inoculated into MRS liquid media of pH =2, pH =4, pH =6, pH =8, respectively, mixed well, shake-cultured at a constant temperature of 37 ℃, and sampled after intervals to determine its absorbance value at 600nm (a in fig. 2). The results show that in the environment with pH =8, the strain enters the logarithmic growth phase after growing for 12h and enters the stationary phase after 20 h; in a growth environment with pH =4, the strain has a short lag phase, rapidly enters a logarithmic growth phase, and enters a stationary phase after 10 h; the growth condition at pH =6 is approximately similar to that at pH =4, and in contrast, the growth of lactic acid bacteria is more suitable in an environment with pH =6, and lactic acid bacteria hardly grow in an environment with pH = 2.
(2) The leuconostoc mesenteroides is activated in MRS and then inoculated into an MRS liquid culture medium, is subjected to constant temperature shaking culture at 37 ℃, and the pH value of the leuconostoc mesenteroides is measured by using a pH value meter at 0h, 2h, 4h, 6h, 8h, 10h, 24h, 36h and 48h (B in figure 2). The result shows that the pH value of the strain after inoculation for 4-24h is in a descending trend, which indicates that the cell metabolism is vigorous at the moment and is the main period of acid production, and the pH value after 24h is stable and is as low as 4.06.
Example 3
Bacteriostatic ability of leuconostoc mesenteroides
Pathogenic bacteria Escherichia coli, pseudomonas aeruginosa, salmonella, Aeromonas salmonicida Z2, Shewanella Z3 and Aeromonas veronii Z12 are activated in LB liquid, Leuconostoc mesenteroides is activated in MRS liquid culture medium and cultured overnight at 37 ℃, the fermentation broth of Leuconostoc mesenteroides is taken and centrifuged for 3min under the condition of 12000rpm, and the supernatant is taken. Absorbing 100 mu L of the pathogenic bacteria fermentation liquid, coating the pathogenic bacteria fermentation liquid on different LB solid culture media, and uniformly coating the bacterial liquid by using a coating rod. Sterile filter paper sheets are placed on each plate, 10 mu L of lactic acid sterile supernatant is dripped to serve as a treatment group, 50 mu L of MRS is dripped to serve as a blank control, and three parallel control groups are arranged for treatment of each pathogenic bacterium. The plate was incubated overnight in an incubator at 37 ℃ and the zone size was observed (FIG. 3). The inhibition effect of the strain culture supernatant on the indicator strain is shown in figure 3, and the strain metabolite has a certain inhibition effect on aeromonas, escherichia coli, salmonella and pseudomonas aeruginosa.
Example 4
Research on bacteriostatic effect and thermal stability of leuconostoc mesenteroides ethyl acetate extract
(1) Activating Leuconostoc mesenteroides in MRS liquid culture medium, culturing at 37 deg.C overnight, centrifuging the fermentation liquid of Leuconostoc mesenteroides at 12000rpm for 3min, and collecting the supernatant. Adding equal volume of ethyl acetate, shaking and mixing for 3 times, each time for 2 h. Mixing the ethyl acetate extracts obtained each time, evaporating to dryness in a rotary evaporator under reduced pressure, and adding a small amount of dimethyl sulfoxide (DMSO) to dissolve to obtain a crude extract. The bacteriostatic test was carried out according to the above method to test the inhibitory effect of the ethyl acetate extract on Aeromonas veronii Z12.
(2) Respectively heating a certain amount of ethyl acetate extract at 60 ℃, 80 ℃ and 121 ℃, cooling, and then performing a bacteriostatic experiment according to the method, and verifying the influence of temperature on bacteriostatic substances. As shown in figure 4, the ethyl acetate extract (positive control) has an obvious inhibition effect on Aeromonas Z12, and meanwhile, the bacteriostatic substance treated at the high temperature of 60 ℃, 80 ℃ and 121 ℃ still has an inhibition effect on Aeromonas Z12, which indicates that the bacteriostatic substance leuconostoc mesenteroides ethyl acetate extract has better thermal stability.
Example 5
Influence of Leuconostoc mesenteroides on biofilm forming capability and protease yield of pathogenic bacteria
(1) Capability of leuconostoc mesenteroides in forming pathogenic bacteria biofilm
The biofilm-forming ability of the above strains was measured by crystal violet staining. Activating the strains Z2, Z3 and Z12 for multiple times, inoculating the activated strains into LB liquid culture medium with the inoculation amount of 1wt%, and diluting the strain fermentation liquor which is cultured overnight to OD by using fresh LB culture medium600And =0.85, inoculating the mixture into a 24-well plate containing 1mL of LB liquid culture medium in each well according to the inoculation amount of 2wt%, adding 50 μ L of sterile supernatant of lactic acid bacteria D01 to the treatment group, setting and adding 50 μ L of fresh MRS as a control group, setting 3 groups in each group in parallel, and culturing at the constant temperature of 37 ℃ for 72 hours. The culture medium was aspirated from each well with a pipette, washed with distilled water 3 times, dried at room temperature for more than 30min, and fixed at 65 ℃ for 15 min. Adding 1mL of crystal violet staining solution into each of the treatment group and the control group for staining for 30min, removing the crystal violet staining solution by suction, washing the crystal violet with deionized water for 3-4 times until the crystal violet is colorless, and drying at room temperature for 20 min. Adding 1mL ethanol solution with volume fraction of 95% for decolorizing for 30min, and measuring OD with enzyme-labeling instrument595And observing the film forming condition by using a digital microscope system. As shown in a of fig. 5, the level of pathogenic biofilm formation with the addition of the sterile supernatant of the strain was significantly lower than the control, indicating that leuconostoc mesenteroides was able to significantly inhibit the formation of pathogenic biofilm.
(2) Influence of Leuconostoc mesenteroides on yield of pathogenic bacteria protease
Inoculating strains Z2, Z3 and Z12 in a culture medium containing 1wt% of skimmed milk, respectively taking 1mL of 1wt% of skimmed milk powder LB culture solution of Z2, Z3 and Z12 after culturing for 24 hours, placing the culture solution in a centrifuge tube, centrifuging for 2min at 12000rpm, taking 500 mu L of Z2 sterile supernatant in the centrifuge tube, taking three centrifuge tubes, respectively subpackaging 100 mu L of each centrifuge tube, arranging three centrifuge tubes in parallel, and carrying out the same operation on Z3 and Z12.
② 100 μ L of azocasein solution (prepared with 50mM Tris-HCl buffer solution with pH 8.0) is added to each 100 μ L of sterile supernatant and placed in a 37 ℃ constant temperature incubator for reaction for 2 h.
And thirdly, adding 500 mu L10% (w/v) trichloroacetic acid solution into each tube of reaction solution, and standing for 30min at room temperature to terminate the reaction.
And fourthly, placing the reaction solution in a centrifuge, centrifuging for 10min at 12000rpm, taking 500 muL of supernatant, adding 500 muL of 1M NaOH solution into the supernatant, and mixing uniformly.
Fifthly, adding 500 mu L of Tris-HCl buffer solution into the centrifuge tube filled with 500 mu L of sterile supernatant, mixing uniformly, and taking the mixture as a blank control. Determination of OD Using an ultraviolet Spectrophotometer440In OD440The change 0.001 is defined as one enzyme activity unit (1U).
As shown in B in FIG. 5, the extracellular protease activity of Aeromonas Z2 was high, and the inhibitory effect of Leuconostoc mesenteroides on the protease production of Z2 was significant (B)P<0.001), the activity of Shewanella Z3 and its monad Z12 extracellular protease is lower, and the leuconostoc mesenteroides metabolite has no significant difference to the yield of Aeromonas Z12 protease compared with the control group (P >0.999）。
Example 6
Survival rate of bacteria in leuconostoc mesenteroides freeze-dried viable bacteria agent
Activating Leuconostoc mesenteroides in MRS liquid culture medium, culturing at 37 deg.C overnight, diluting and coating Leuconostoc mesenteroides fermentation liquid on MRS plate, and counting bacteria. Centrifuging the rest Leuconostoc mesenteroides fermentation liquor for 3min at 12000rpm, and collecting thallus. Sterilizing trehalose, sodium glutamate and xylooligosaccharide with a bacteria filter with a membrane pore size of 0.22 μm, and refrigerating for later use; keeping the skimmed milk powder at 110 deg.C for 10min, and sterilizing. Mixing thallus with skimmed milk powder, trehalose, sodium glutamate and xylooligosaccharide, and mixing on vortex mixer to obtain suspension. Pre-freezing at-70 deg.C for 2h, taking out, and freeze-drying in vacuum freeze-drying machine under vacuum degree of 5Pa, heating temperature of the baffle plate of 20 deg.C, cold trap temperature of-55 deg.C, and freeze-drying time of about 30 h. And taking the freeze-dried thallus, resuspending the freeze-dried thallus with sterile PBS, diluting, coating and counting. And calculating the survival rate of the bacteria by using the ratio of the number of the bacteria after freeze-drying to the number of the bacteria before freeze-drying. As shown in FIG. 6, the survival rate of the strain added with the lyoprotectant is significantly higher than that of the strain without the lyoprotectant, and the survival rate is as high as 82%.
Example 7
Action of leuconostoc mesenteroides on attacking toxin of aeromonas by bathing
The experimental fish species were zebra fish, which were purchased and then normally fed for 5 days, and randomly divided into 4 groups. Wherein 2 groups are fed with feed, and the other 2 groups are fed with Leuconostoc mesenteroides (1.5 × 10) with different concentrations7CFU/mL and 4.5X 107CFU/mL) of equal quality feed, no fish died in each group for 10 days, indicating that leuconostoc mesenteroides had no effect on fish growth. Activating Aeromonas veronii Z12 in LB liquid culture medium, inoculating to LB liquid culture medium, shake culturing at 37 deg.C, centrifuging the fermentation liquid at 12000rpm for 10min, removing supernatant, and collecting thallus. After the cells were resuspended and washed several times with sterile PBS, the cell concentration was adjusted to 5X 108CFU/mL final concentration was added to the above-mentioned 2 treatment groups with addition of Leuconostoc mesenteroides and 1 other group of zebrafish, and the remaining 1 group was added with an equal volume of PBS as a control. Survival of groups of zebrafish was observed and recorded daily. The results are shown in fig. 7, the survival rate of the zebrafish in the control group without bacteria and the prevention group with high-concentration leuconostoc mesenteroides reaches 100% in the observation process of 10d, and the phenomenon of fish body death does not occur. The low-concentration prevention group and the aeromonas immersion-bath counteracting group have the phenomenon that fish die in the 2 nd day, the survival rate of the fish is reduced to 90 percent, the survival rate of the low-concentration prevention group is kept at 90 percent in the rest observation time,the phenomenon of fish death does not occur any more. However, the aeromonas bathing and attacking group suffered from massive death at 3d, and the survival rate was reduced to 40% until the final survival rate was only 20%. The experiments of air monad bathing and counteracting poison show that the feeding of leuconostoc mesenteroides can enhance the resistance of zebra fish to air monad and greatly improve the survival rate of fish bodies, so the leuconostoc mesenteroides has certain application value in the prevention and treatment of aquatic pathogenic bacteria.
Finally, it is to be noted that: it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention, and it is intended that all such modifications and variations be considered as within the scope of the invention.
Claims (5)
1. A screening method of leuconostoc mesenteroides is characterized by comprising the following specific steps: subjecting kimchi juice to sterile water treatment 10-1-10-5Gradient dilution, namely coating the diluents with different gradients on an MRS solid plate added with calcium carbonate, culturing for 48h at 37 ℃, selecting a single colony with a calcium-dissolving ring, streaking and purifying on the MRS solid plate, repeatedly operating for 2-3 times to obtain a pure strain, and preserving the pure strain with glycerol at-80 ℃; extracting genome DNA of the screened strain as a template, and carrying out PCR amplification on the 16S rRNA gene under the conditions that: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 45s, 30 cycles, extension at 72 ℃ for 2min, sequencing after purifying PCR products, performing BLAST comparison on sequencing results and NCBI, and selecting seeds or genera with high similarity to the sequencing results; and (3) inoculating the screened strain after sequencing on an MRS solid plate, and culturing at the constant temperature of 37 ℃ for 24-48h until a colony is formed.
2. The application of leuconostoc mesenteroides screened according to the method of claim 1 in preparing bacteriostatic agents for preventing and treating aquatic bacterial diseases, wherein the leuconostoc mesenteroides can inhibit the formation of bacterial biofilms and the secretion of bacterial extracellular proteases.
3. Use according to claim 2, characterized in that: the aquatic bacteria is one or more of salmonella, escherichia coli, pseudomonas aeruginosa, aeromonas salmonicida, shewanella or aeromonas veronii.
4. Use according to claim 2, characterized in that: the bacteriostatic agent for preventing and treating the aquatic bacterial diseases is a leuconostoc mesenteroides ethyl acetate extract, and the specific preparation process of the leuconostoc mesenteroides ethyl acetate extract is as follows: activating leuconostoc mesenteroides in an MRS liquid culture medium, culturing at 37 ℃ overnight, centrifuging fermentation liquor of the leuconostoc mesenteroides for 3min at 12000rpm, taking supernatant, adding ethyl acetate with the same volume, shaking and uniformly mixing for 3 times, 2h each time, mixing ethyl acetate extract obtained each time, decompressing and evaporating by using a rotary evaporator, and adding dimethyl sulfoxide to dissolve to obtain a crude extract, namely the leuconostoc mesenteroides ethyl acetate extract.
5. Use according to claim 2, characterized in that: the bacteriostatic agent for preventing and treating aquatic bacterial diseases is a leuconostoc mesenteroides freeze-dried viable bacterium agent, the freeze-dried viable bacterium agent takes skim milk powder, trehalose, sodium glutamate and xylo-oligosaccharide as freeze-dried protective agents, and the survival rate of the leuconostoc mesenteroides in the freeze-dried viable bacterium agent can reach 82 percent to the maximum; the preparation process of the freeze-dried viable bacteria agent comprises the following steps: activating Leuconostoc mesenteroides in MRS liquid culture medium, culturing at 37 deg.C overnight, centrifuging the fermented liquid of Leuconostoc mesenteroides at 12000rpm for 3min, and collecting thallus; sterilizing trehalose, sodium glutamate and xylooligosaccharide with a bacteria filter with a membrane pore size of 0.22 μm, and refrigerating for later use; keeping the skimmed milk powder at 110 deg.C for 10min for sterilization; mixing the bacteria with skimmed milk powder, trehalose, sodium glutamate and xylo-oligosaccharide, uniformly mixing on a vortex mixer to prepare a suspension, pre-freezing the suspension at-70 ℃ for 2h, taking out and freeze-drying in a vacuum freeze dryer to prepare the live bacteria agent of leuconostoc mesenteroides, wherein the freeze-drying condition is that the vacuum degree is 5Pa, the heating temperature of a clapboard is 20 ℃, the cold trap temperature is-55 ℃, and the freeze-drying time is 30 h.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974450A (en) * | 2010-09-13 | 2011-02-16 | 郑州大学 | Leuconostoc mesenteroides and application thereof |
| CN113308408A (en) * | 2021-07-06 | 2021-08-27 | 广东省科学院生物工程研究所 | Leuconostoc mesenteroides for producing bacteriocin and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974450A (en) * | 2010-09-13 | 2011-02-16 | 郑州大学 | Leuconostoc mesenteroides and application thereof |
| CN113308408A (en) * | 2021-07-06 | 2021-08-27 | 广东省科学院生物工程研究所 | Leuconostoc mesenteroides for producing bacteriocin and application thereof |
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| Title |
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| 李祎等: "一株肠膜明串珠菌的分离鉴定及其抑菌特性" * |
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