Method for screening, separating and fermenting bacterial strains for producing antibacterial substances from shrimp pond

Abstract

The invention belongs to the technical field of microorganisms, and discloses a method for screening bacterial strains capable of producing antibacterial substances, which comprises the following steps of: activating bacterial strains to be screened and indicating pathogenic bacteria, and respectively culturing at different positions of the same LB flowable double-layer plate, and observing whether the diameter of the pathogenic bacteria indicating the pathogenic bacteria in the LB flowable double-layer plate is reduced or not and whether the middle is hollow or not, thereby screening to obtain the bacterial strains capable of producing the antibacterial substances. The method provided by the invention can be used for simply and rapidly screening strains capable of producing antibacterial substances, and compared with conventional methods (such as paper method screening and turbidimetric method screening), the method provided by the invention is higher in screening effect and better in accuracy. The LB flowable double-layer plate used in the method has relatively strong plate flowability, so that the growth and metabolic activity of microorganisms are promoted, the flowability of antibacterial substances is enhanced, and potential bacterial strains capable of producing the antibacterial substances are rapidly screened.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for screening, separating and fermenting a strain producing antibacterial substances from a shrimp pond. Background Art

[0002] In modern medicine and agriculture, the demand for antimicrobial substances is increasing, especially in the context of the increasingly serious problem of antibiotic resistance, it is particularly important to find new and effective antimicrobial agents. In recent years, the research on antimicrobial substances from microorganisms has gradually become a hot topic. The screening of microorganisms in nature is a key step in discovering new antimicrobial agents, especially bacteria from soil and water bodies, which have become a potential source of new antimicrobial agents due to their rich biodiversity and unique metabolites. Especially in the field of aquaculture, bacterial diseases are one of the main causes of death of fish and shrimp. Shrimp ponds, as a rich source of microorganisms, have shown their potential in screening antimicrobial strains.

[0003] Although the existing technology for screening antibacterial strains from nature has made some progress, there are still some problems and challenges. First, there is a lot of room for optimization in the screening process, and many potential antibacterial strains may be missed due to inappropriate culture conditions or improper screening methods. Secondly, the optimization of fermentation conditions often relies on experience, and the bacteria may be affected by environmental factors during the fermentation process, such as temperature and pH fluctuations, resulting in unstable production and activity of antibacterial substances. Finally, the genetic diversity of strains and the complexity of metabolic pathways make it difficult to achieve consistency and controllability in large-scale production, and better strains need to be continuously explored and paid attention to.

[0004] After screening out strains with good antibacterial effects, optimizing their fermentation conditions is the key to improving the production efficiency of antibacterial substances. The optimization of fermentation conditions includes temperature, pH value, nutrient ratio and other aspects. By systematically studying the effects of different fermentation conditions on the growth of Bacillus and the synthesis of antibacterial substances, the yield and activity of antibacterial substances can be significantly improved. For example, suitable temperature and pH value can promote the growth and metabolism of bacteria, thereby increasing the synthesis rate of antibacterial substances. In addition, the reasonable ratio of nutrients is also crucial. Specific carbon sources and nitrogen sources can significantly enhance the metabolic activity of strains, thereby increasing the yield of antibacterial substances. Summary of the invention

[0005] The first aspect of the present invention aims to provide a method for screening bacterial strains capable of producing antibacterial substances.

[0006] The purpose of the second aspect of the present invention is to provide application of the method of the first aspect of the present invention in screening target strains.

[0007] The third aspect of the present invention aims to provide a use of the fermentation medium in the first aspect of the present invention in improving the ability of Bacillus paralicheniformis to secrete antibacterial substances.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a method for screening a strain capable of producing antibacterial substances, comprising the following steps:

[0010] After the strain to be screened and the indicator pathogen are activated, they are cultured at different positions on the same LB flowable double-layer plate. By observing whether the diameter of the indicator pathogen in the LB flowable double-layer plate is reduced and whether a hollow appears in the middle, the strain that produces antibacterial substances is screened.

[0011] The flowable plate allows the isolated bacteria and pathogens to compete for growth resources. At the same time, due to the presence of a certain physical distance, the pathogens and isolated bacteria are given a certain amount of growth time, which further allows the antibacterial substances secreted by the fermentation broth of the isolated bacteria, such as surfactant and antimicrobial peptides, to penetrate the pathogens.

[0012] In some embodiments of the present invention, the LB flowable double-layer plate is prepared by comprising an upper layer of LB culture medium containing 0.1% to 0.3% agar and a lower layer of LB culture medium containing 1% to 3% agar.

[0013] In some embodiments of the present invention, the upper layer of the LB flowable double-layer plate is an LB medium containing 0.1% to 0.2% agar, and the lower layer is an LB medium containing 2% to 3% agar.

[0014] In some embodiments of the present invention, the upper layer of the LB flowable double-layer plate is an LB medium containing 0.2% to 0.3% agar, and the lower layer is an LB medium containing 2% to 3% agar.

[0015] In some embodiments of the present invention, during the screening process, the bacterial liquid of the indicator pathogen is dripped into the four corners of the LB fluid double-layer plate, and the bacterial liquid of the strain to be screened is dripped into the middle position of the LB fluid double-layer plate. After culturing, by observing whether the growth of the indicator pathogen on the LB fluid double-layer plate is normal, whether the diameter of the indicator pathogen is reduced, and whether there is a hollow in the middle, it is determined whether the strain to be screened is a strain that produces antibacterial substances; or

[0016] A square is drawn with the bacterial solution of the strain to be screened in an LB fluid double-layer plate, and the bacterial solution of the indicator pathogen is dropped into the square. After cultivation, whether the growth of the indicator pathogen on the LB fluid double-layer plate is normal, whether the diameter of the indicator pathogen is reduced, and whether a hollow appears in the middle is observed to determine whether the strain to be screened is a strain that produces antibacterial substances.

[0017] In some embodiments of the present invention, a control group and an experimental group are set during the screening process, wherein, in the control group, the indicator pathogen is dripped at the four corners and the middle of the LB fluid double-layer plate; in the experimental group, the indicator pathogen is dripped at the four corners of the LB fluid double-layer plate, and the bacterial solution of the strain to be screened is dripped in the middle, cultured, and the growth status of the experimental plate and the control plate is observed, and it is observed whether the indicator bacteria on the experimental plate grow normally, whether the diameter of the indicator bacteria is reduced, and whether a hollow appears in the middle.

[0018] In some embodiments of the present invention, a control group and an experimental group are set during the screening process, wherein, in the control group, an indicator pathogen is added in the middle of the LB fluid double-layer plate; in the experimental group, an indicator pathogen is added in the middle of the LB fluid double-layer plate, and then a square is drawn with the bacterial solution of the strain to be tested with the LB fluid double-layer plate as the center, cultured, and the growth status of the experimental plate and the control plate is observed, and it is observed whether the indicator bacteria on the experimental plate grow normally, whether the diameter of the indicator bacteria is reduced, and whether a hollow appears in the middle.

[0019] In some embodiments of the present invention, the indicator pathogens include at least one of Vibrio, Staphylococcus aureus, Lactococcus garrisonii, Escherichia coli, Streptococcus pneumoniae, Shigella dysenteriae, and Salmonella.

[0020] In some embodiments of the present invention, the culture conditions are 36-37° C. for 8-14 h.

[0021] In some embodiments of the present invention, the culture condition is 37-38° C. for 8-12 h.

[0022] In some embodiments of the present invention, the method further comprises a rescreening step, wherein the rescreening process comprises culturing the screened strains in a fermentation medium, collecting the fermentation supernatant, and evaluating the inhibitory effect of the fermentation supernatant on the indicator pathogens by a punching method.

[0023] In some embodiments of the present invention, the fermentation medium comprises peptone, soluble starch and glucose; or the fermentation medium comprises corn flour, soybean meal, peptone and glucose; or the fermentation medium is BHI medium.

[0024] In some embodiments of the present invention, the fermentation medium is composed of peptone, soluble starch, glucose, KH 2 PO 4 and (NH4 ) 2 SO 4 composition.

[0025] In some embodiments of the present invention, each liter of the fermentation medium contains 10-20 g of peptone, 15-30 g of soluble starch, 1-10 g of glucose, 1-7 g of KH 2 PO 4 and 1-7 g (NH 4 ) 2 SO 4 .

[0026] In some embodiments of the present invention, each liter of the fermentation medium contains 14-18 g of peptone, 18-22 g of soluble starch, 4-6 g of glucose, 2-6 g of KH 2 PO 4 and 2-5 g (NH 4 ) 2 SO 4 .

[0027] The second aspect of the present invention provides the use of the method of the first aspect of the present invention in screening target strains.

[0028] In some embodiments of the present invention, the target strain is a strain that produces antibacterial substances.

[0029] The third aspect of the present invention provides use of the fermentation culture in the first aspect of the present invention in improving the ability of Bacillus paralicheniformis to secrete antibacterial substances.

[0030] Compared with other culture media, the use of the fermentation culture medium can significantly improve the inhibitory effect of the fermentation broth of Bacillus paralicheniformis on pathogens (such as Vibrio), and can increase the diameter of the inhibition zone from the initial 11 mm to 20 mm, and the antibacterial effect is improved by 42%.

[0031] The beneficial effects of the present invention are:

[0032] The method provided by the present invention can be used to simply and quickly screen strains that produce antibacterial substances, and has a higher screening effect and better accuracy than conventional methods (such as paper disc screening and turbidimetric screening). The LB flow double-layer plate used in the method has strong plate flowability, thereby promoting the growth and metabolic activity of microorganisms, enhancing the flowability of antibacterial substances, and enabling rapid screening of potential antibacterial substance-producing strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of a flowing flat bilayer.

[0034] Figure 2 Schematic diagram of flow plate screening.

[0035] Figure 3 The results of the flowability plate screening are shown in Figure 1. A is the pathogen control plate of the flowability plate screening method 1), B is the strain test plate of the flowability plate screening method 1), C is the pathogen control plate of the flowability plate screening method 2), and D is the strain test plate of the flowability plate screening method 2).

[0036] Figure 4 The antibacterial results of rescreening AES1.

[0037] Figure 5 The identification results of AES1 selected strains, A is the streaking morphology, B is the Gram staining microscopy, and C is the 16S sequencing colony PCR electrophoresis. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below through specific examples.

[0039] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0040] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0041] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0042] Example 1 Screening of strains with antibacterial activity from isolated strains

[0043] A method for screening strains producing antimicrobial substances from shrimp ponds comprises the following steps:

[0044] (1) Sample collection

[0045] Multiple sampling points were selected in different areas of the shrimp pond, including the edge of the shrimp pond, the central area, the deep water area, and the shallow water area.

[0046] Use sampling tools, such as sterile spoons, to collect soil samples at different depths at each sampling point. Collect soil samples from the surface (0-5 cm), middle (5-15 cm), and deep (15-25 cm). Collect about 50 grams of soil at each depth and place the soil samples collected at different depths in sterile sample tubes.

[0047] Open the sample tube in the clean bench. When performing microbial separation, first mix the collected soil sample with sterile water at a ratio of 1:10 to obtain a preliminary dilution. Then, take 1 mL from the preliminary dilution and mix it with 9 mL of sterile water to obtain a 100-fold dilution; take 1 mL from the 100-fold dilution and continue to mix it with 9 mL of sterile water to obtain a 1000-fold dilution. Repeat this step for 10 -3 , 10 -4 , 10 -5 Diluent. Take 0.2mL of 10 -3 , 10 -4 , 10 -5 Add the dilution to the LB solid plate and spread it with coating beads to ensure uniform coating. Place it in a 37°C incubator and culture overnight.

[0048] According to the colony morphology, select representative single colonies to ensure selection diversity, streak on a new LB solid plate, place in a 37°C incubator, and culture overnight. Repeat the streaking step three times, and observe the streaked strains under a microscope to ensure pure culture. A total of 15 isolated bacteria were obtained, numbered from 01 to 15.

[0049] (2) Strain screening

[0050] The flowable plate allows the isolated bacteria and pathogens to compete for growth resources. At the same time, due to the presence of a certain physical distance, the pathogens and isolated bacteria are given a certain amount of growth time, which further allows the antibacterial substances secreted by the fermentation broth of the isolated bacteria, such as surfactant and antimicrobial peptides, to penetrate the pathogens.

[0051] Activate the pathogen indicator bacteria (wild Vibrio parahaemolyticus) and the strains to be screened overnight in advance, that is, inoculate the pathogen indicator bacteria and the strains to be screened 01-15 into LB liquid culture medium (10.0 g of tryptone, 5.0 g of yeast extract and 10.0 g of NaCl are dissolved in 1 L of distilled water, the pH value is adjusted to 7.0±0.2, and sterilized at 0.11 MPa and 121°C for 30 min), place at 37°C, 220 rpm shaking, and culture overnight.

[0052] Prepare LB double-layer plates with 0.2% agar concentration plus 2% agar concentration: take 5 mL of 2% agar LB (weigh 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl and 20.0 g agar and dissolve them in 1 L of distilled water, adjust the pH value to 7.0 ± 0.2, and sterilize at 0.11 MPa, 121 ° C for 30 min) and pour it into the plate. After it hardens, take 8 mL of 0.2% agar LB (weigh 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl and 2.0 g agar and dissolve them in 1 L of distilled water, adjust the pH value to 7.0 ± 0.2, and sterilize at 0.11 MPa, 121 ° C for 30 min to prepare 0.2% agar LB medium) and pour it into the plate. After it hardens, you can get a LB double-layer plate.

[0053] Liquidity Tablet Screening:

[0054] Screening method 1): Control plate: Take 1 μL of freshly cultured pathogenic bacteria solution with a bacterial concentration of 10 8 cfu / mL, drip at the four corners and the middle of the plate ( Figure 2 ); Experimental plate: Similarly, take 1 μL of freshly cultured pathogenic bacteria solution and drop it on the four corners of the plate, and add 1 μL of freshly cultured isolated bacteria solution in the middle ( Figure 2 ). Move horizontally, do not shake, and place in a 37℃ incubator for overnight culture; or

[0055] Screening method 2): Control plate: 100 μL pathogen indicator solution ( Figure 2 ); Experimental plate: Similarly, take 100 μL of pathogen indicator solution and drop it in the middle of the plate, then take 1 μL of freshly cultured isolated bacterial solution and draw a square with the plate as the center ( Figure 2 ). Move horizontally without shaking, place in a 37℃ incubator, and culture overnight.

[0056] Screening result determination: For screening method 1), observe the growth status of the experimental plate and the control plate to see whether the pathogen growth on the experimental plate is normal, whether the diameter of the pathogen is reduced, and whether there is a hollow in the middle. It can be observed that the diameter of the pathogen growth on the test plate with the antibacterial effect strain is smaller than that on the control plate, and a hollow transparent ( Figure 3 ), that is, the strain is judged to have antibacterial activity. For screening method 2), observe the growth status of the experimental plate and the control plate, and observe whether the pathogen growth on the experimental plate is normal and whether there is a hollow in the middle. It can be observed that the pathogen growth on the test plate with antibacterial effect strains is hollow and transparent compared with the control plate ( Figure 3 ), that is, the strain was judged to have antibacterial activity.

[0057] Example 2

[0058] The agar concentration of the flowable plate in Example 1 was optimized, and the agar concentration of the upper plate was screened by fixing the agar concentration of the lower plate to ensure that the strain to be screened and the pathogenic bacteria had a certain contact after overnight culture. The spores with antibacterial effects preserved in the laboratory were selected as experimental strains, and the indicator bacteria were wild Vibrio parahaemolyticus. The specific steps were:

[0059] Activate pathogen indicator bacteria and antibacterial spores overnight in advance: inoculate into LB liquid medium (10.0 g tryptone, 5.0 g yeast extract and 10.0 g NaCl dissolved in 1 L distilled water, adjust pH to 7.0 ± 0.2, sterilize at 0.11 MPa, 121 ° C for 30 min), place at 37 ° C, 220 rpm shaker, and culture overnight;

[0060] Prepare a double-layer LB plate with an upper layer of 0.1% to 1% agar and a lower layer of 2% agar: Pour 5 mL of 2% agar LB into the plate, wait for it to harden, then pour 8 mL of 0.1% to 1% agar LB into the plate ( Figure 1 ); 20 μL of pathogenic bacteria and 20 μL of fermentation liquid of antibacterial spores were added dropwise on a double-layer plate with a spacing of 1 cm; the plates were moved horizontally and placed in a 37°C incubator for overnight culture.

[0061] The results are shown in Table 1, and 0.2% upper layer concentration was finally selected as the optimal concentration.

[0062] Table 1 Analysis of the concentration of agar in the upper layer of the flow plate

[0063]

[0064]

[0065] Example 3

[0066] This example uses the method of Example 1 as well as the paper disc method and the turbidimetric method to screen strains with antibacterial activity, and compares the screening effects of the three methods, as follows:

[0067] Screening by paper strip method: Activate the pathogen indicator bacteria and the colonies to be screened 01-15 (step (1) of Example 1) overnight in advance, inoculate them into LB liquid culture medium, place them in a shaker at 37°C, 220rpm, and culture them overnight; take 200μL of the cultured pathogens and add them to 20mL LB to prepare a bacterial plate; take 30μL of the supernatant of the culture of the bacteria to be screened 01-15 and add them to the sterilized filter paper. Use sterile tweezers to take the drug-sensitive paper strip, stick it on the surface of the plate, and press the paper strip lightly with the tweezers to make it flat. The spacing between each paper strip is not less than 24mm, and the center of the paper strip is not less than 15mm from the edge of the plate. After the plate with the paper strip is placed in a 37°C incubator and left to stand overnight, use a vernier caliper to measure the diameter of the inhibition zone.

[0068] Turbidimetric screening: Activate the pathogen indicator bacteria and strains 01 to 15 to be screened (step (1) of Example 1) overnight in advance, inoculate them into LB liquid culture medium, place them in a shaker at 37°C, 220 rpm, and culture them overnight; take 1 mL of the culture medium of the strain to be screened and 1 mL of the culture medium of the pathogenic bacteria and mix them in a test tube, add the culture medium to the mixed culture medium, and adjust the final volume to 10 mL. Use a spectrophotometer to measure the OD of the mixed culture medium. 600 The value was taken as the initial turbidity value (T0). The test tube was placed in an incubator at 37°C and 220 rpm for overnight culture. The OD of the mixed culture solution was measured again. 600 The final turbidity value (Tf) was used to calculate the inhibition rate of each candidate strain.

[0069]

[0070] Fluidity plate screening: Same as Example 1.

[0071] The results of screening by the three methods are shown in Table 2. The paper disc method and the turbidimetric method did not screen out strains with antibacterial activity, while the method of Example 1 screened out two strains with antibacterial activity. The two strains were further screened by the punching method. The results showed that the two strains had good antibacterial effects on Vibrio parahaemolyticus. It is suggested that the method of Example 1 can effectively screen out strains with antibacterial effects from unknown strains, and its screening effect is better than that of the traditional paper disc method and turbidimetric method.

[0072] Table 2 Comparison of screening methods

[0073]

[0074]

[0075] Example 4

[0076] The two strains with antibacterial effects obtained in Example 3 (named AES strains for the convenience of description) were cultured using different fermentation media, and their antibacterial effects were measured using the punching method to screen the best fermentation medium, as follows:

[0077] (1) Culture medium screening

[0078] Medium preparation: Prepare liquid fermentation medium according to Table 3 below, mix well, and sterilize.

[0079] Table 3 Culture medium formula

[0080]

[0081] (2) Preparation of bacterial solution

[0082] AES was inoculated into 5 mL of LB liquid seed liquid, placed in a 37°C, 220 rpm shaker overnight (16 h) culture, transferred to 50 mL of fermentation medium (Table 3) at 1:100, placed in a 37°C, 220 rpm shaker culture for 24 h until the bacterial liquid reached the logarithmic growth phase; at the same time, the commercial subtilis spore strain BS168 (Beina Bio, BNCC371816) was selected as a negative control. After the fermentation was completed, the fermentation liquid in the shake bottle was taken out, and the fermentation liquid was centrifuged at 10000 rpm and 4°C for 10 min, and the supernatant (i.e., the crude liquid containing antibacterial substances) was collected and stored at 4°C for the time being.

[0083] (3) Preparation of Oxford cup antibacterial plates

[0084] A. Inoculate the pathogen into 10 mL of LB liquid seed solution and culture it in a shaking incubator at 37°C and 220 rpm overnight.

[0085] B. Double-layer agar diffusion-perforated bottom layer: Pour less than 10 mL of sterilized LB solid culture medium (agar content 2%) into a culture dish, place it horizontally, and the bottom layer will be formed after solidification.

[0086] C. Punching: Place a sterilized 8mm diameter Oxford cup with smooth ends on the bottom layer of the culture medium without damaging the bottom layer to form a circular hole with a diameter of 8mm on the culture medium.

[0087] D. Bacterial layer: Take 20 mL of LB solid medium (1% agar content) cooled to about 40°C and pour it into a centrifuge tube. Add indicator bacteria solution (wild Vibrio parahaemolyticus, bacterial concentration is 1*10 6 CFU / mL) and mix it upside down, then pour it horizontally onto the bottom culture medium. Be careful not to pour it into the Oxford cup. This layer, after solidification, is the bacterial layer.

[0088] E. Sample addition: After the plate solidifies, add the fermentation liquid of the bacteria to be tested into the round wells, adding 100 μL of fermentation supernatant to each well.

[0089] F. Observation: After culturing in a 37°C incubator for 16 hours, observe the formation of the inhibition zone and measure the diameter of the inhibition zone.

[0090] AES was cultured in different fermentation mediums. The results showed that after AES was cultured in medium No. 6, the diameter of the inhibition zone was the largest, among which AES1 could reach 20mm. The degree of transparency of the inhibition zone was high, indicating that it had a significant effect on Vibrio. At the same time, it was observed that the fermentation supernatant of the negative control BS168 had no inhibition zone ( Figure 4 and Table 4). AES1 using medium No. 6 can increase the diameter of the inhibition zone from the lowest 11 mm (using LB medium fermentation culture) to 20 mm, an increase of 42%.

[0091] Table 4 The fermentation supernatants of two AES strains in different culture media inhibited the diameter of Vibrio

[0092]

[0093] Example 5 Identification of species classification of AES strains

[0094] Since AES1 has a better antibacterial effect than AES2, AES1 was selected for identification.

[0095] 1. Observe the morphology by streaking on a plate: streak the AES strain on an LB plate and place it in a 37°C incubator. After one day of incubation, observe the colony characteristics, including the size, shape, edge, color, surface, and texture of the colonies.

[0096] like Figure 5 As shown in A, based on the observed morphological characteristics, the AES1 strain is white, opaque, has single colonies on the streaks, has irregular edges, and a rough surface. It is preliminarily judged that the AES strain may belong to the genus Bacteria.

[0097] 2. Gram staining:

[0098] (1) Preparation of smear: Take a small amount of pure culture solution of AES1 strain with a sterile pipette and drop it on a clean sterile slide. Use sterile tweezers to gently flatten the solution to form a uniform thin layer and wait for the smear to dry.

[0099] (2) Fixation: Soak the dried smear in a fixative (95% ethanol) for about 1 minute to fix the bacteria. Remove the smear and remove the excess fixative with a paper towel.

[0100] (3) Primary staining: Immerse the smear in crystal violet staining solution and stain for about 1 minute. Remove the smear and gently absorb the excess stain with a paper towel.

[0101] (4) Mordanting: Immerse the smear in iodine solution for about 1 minute, remove the smear and absorb the excess iodine solution with a paper towel.

[0102] (5) Decolorization: Place the smear in 95% ethanol and shake gently to decolorize until the smear changes from purple to colorless, which takes about 30 seconds to 1 minute. Remove the smear and absorb the excess ethanol with a paper towel.

[0103] (6) Counterstaining: Immerse the smear in the counterstain solution (safranin) for about 30 seconds to 1 minute. Remove the smear and absorb the excess counterstain with a paper towel.

[0104] (7) Observation: Use a microscope to observe the smear and observe the staining characteristics of the bacteria.

[0105] According to the staining results, purple, spherical or rod-shaped bacteria were observed and arranged in pairs or chains ( Figure 5 B), strain AES1 was identified as a Gram-positive bacterium.

[0106] 3. Molecular cloning identification: The 16S rRNA gene identification primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1) and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 2) of bacteria were synthesized by entrusting Shanghai Sangon Biotechnology Co., Ltd., and colony PCR was performed using 2×UTaq PCR MasterMix (Zhuangmeng Biotechnology). The reaction solution was prepared according to Table 5 in a sterile and enzyme-free PCR tube, and the PCR reaction was performed using a PCR instrument set to the program in Table 5. The colony PCR product was run on a gel, 1% agarose gel was prepared, 5 μL of sample and marker were spotted, and the setting was 120V for 30 min. After the gel run was completed, the results were observed on a gel imaging instrument.

[0107] Table 5 PCR colony amplification reaction system and reaction procedure

[0108]

[0109]

[0110] The results are as follows Figure 5 As shown in C, the band of the gel running result is consistent with the size of bacterial 16S, which is about 1600bp. The PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Finally, the 16S rRNA gene sequence obtained by sequencing was compared with the NCBI database, and the BLAST bioinformatics tool was used for homology analysis. According to the sequence alignment results, it was determined that the AES1 strain had the highest similarity with the known species as Bacillus paralicheniformis, and the sequence homology percentage reached 99% (Table 6). By Gram staining, it was observed that the cells of the AES1 strain were purple. Combined with the 16S rRNA gene sequence analysis and plate streak morphology observation, it was finally determined that AES1 belonged to the genus Bacillus paralicheniformis. It is further proved that the method of Example 1 can effectively screen strains with antibacterial effects, and the screening accuracy is high.

[0111] Table 6 16S alignment results

[0112]

[0113] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for screening strains capable of producing antibacterial substances, comprising the following steps: After the strain to be screened and the indicator pathogen are activated, they are cultured at different positions on the same LB flowable double-layer plate. By observing whether the diameter of the indicator pathogen in the LB flowable double-layer plate is reduced and whether a hollow appears in the middle, the strain that produces antibacterial substances is screened.

2. The method according to claim 1, characterized in that The LB fluid double-layer plate is prepared by comprising an upper layer of LB culture medium containing 0.1% to 0.3% agar and a lower layer of LB culture medium containing 1% to 3% agar.

3. The method according to claim 1, characterized in that During the screening process, the bacterial solution of the indicator pathogen is added dropwise to the four corners of the LB fluid double-layer plate, and the bacterial solution of the strain to be screened is added dropwise to the middle position of the LB fluid double-layer plate. After culturing, by observing whether the growth of the indicator pathogen on the LB fluid double-layer plate is normal, whether the diameter of the indicator pathogen is reduced, and whether there is a hollow in the middle, it is determined whether the strain to be screened is a strain that produces antibacterial substances; or A square is drawn with the bacterial solution of the strain to be screened in an LB fluid double-layer plate, and the bacterial solution of the indicator pathogen is dropped into the square. After cultivation, whether the growth of the indicator pathogen on the LB fluid double-layer plate is normal, whether the diameter of the indicator pathogen is reduced, and whether a hollow appears in the middle is observed to determine whether the strain to be screened is a strain that produces antibacterial substances.

4. The method according to any one of claims 1 to 3, characterized in that The indicator pathogenic bacteria include at least one of Vibrio, Staphylococcus aureus, Lactococcus garrisonii, Escherichia coli, Streptococcus pneumoniae, Shigella dysenteriae, and Salmonella.

5. The method according to claim 4, characterized in that The culture condition is 36-38° C. for 8-14 hours.

6. The method according to claim 5, characterized in that The method further comprises a rescreening step, wherein the rescreening process comprises culturing the screened strains in a fermentation medium, collecting the fermentation supernatant, and evaluating the inhibitory effect of the fermentation supernatant on the indicator pathogens by a punching method.

7. The method according to claim 6, characterized in that The fermentation medium comprises peptone, soluble starch and glucose; or The fermentation medium comprises corn meal, soybean meal, peptone and glucose; or The fermentation medium is BHI medium.

8. The method according to claim 6, characterized in that The fermentation medium consists of peptone, soluble starch, glucose, KH2PO4 and (NH4)2SO4.

9. Use of the method according to any one of claims 1 to 8 in screening target strains.

10. Use of the fermentation culture according to claim 8 for improving the ability of Bacillus paralicheniformis to secrete antimicrobial substances.

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