Preparation method and application of sterile fermentation filtrate of pseudomonas aeruginosa 8-7

Abstract

The invention discloses a preparation method and application of a sterile fermentation filtrate of pseudomonas aeruginosa 8-7, and belongs to the technical field of microorganisms. The invention discloses a preparation method of a Pseudomonas aeruginosa 8-7 sterile fermentation filtrate. The Pseudomonas aeruginosa 8-7 sterile fermentation filtrate is prepared by using an FA fermentation medium. The FA fermentation culture medium comprises the following components: 15g / L of acid hydrolyzed casein 2, 25ml / L of glycerol, 1g / L of MgSO4. 7H2O and 1.5 g / L of K2HPO4, and the pH is natural. The sterile fermentation filtrate of the pseudomonas aeruginosa 8-7 can tolerate strong acid, strong alkali, high temperature, Na2SO3, ultraviolet irradiation and other environments, and is insensitive to metal ions; and the antibacterial agent has a good antibacterial effect on various plant pathogenic bacteria. According to the invention, the fermentation culture condition combination of the antibacterial substances produced by the pseudomonas aeruginosa 8-7 is optimized, and technical support and theoretical guidance are provided for the industrial production of the pseudomonas aeruginosa 8-7 strain and the acquisition of the high-yield antibacterial substances.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and more specifically to a preparation method of Pseudomonas aeruginosa 8-7 sterile fermentation filtrate and application thereof. Background Art

[0002] The rubber tree (Hevea brasiliensis) is a perennial tropical rainforest tree species of the genus Hevea in the family Euphorbiaceae. The natural rubber produced by it is an irreplaceable industrial raw material and is planted on a large scale in Hainan, Yunnan, Guangdong and other places in my country. The rubber planting area in Yunnan is located on the northern edge of the tropics, and the warm and humid climate conditions are conducive to the reproduction and spread of pathogenic microorganisms. Anthracnose of rubber trees caused by anthracnose bacteria occurs frequently, harming rubber seedlings, field saplings and tapped rubber trees, infecting multiple parts such as young leaves, petioles, young shoots and fruits, causing young leaves to fall off, young shoots to wither and fruits to rot, etc. In severe cases, it causes repeated leaf shedding and young shoots to wither, resulting in delayed tapping time and reduced latex production. At present, the prevention and control of anthrax mainly uses chemical pesticides, such as 25% prochloraz emulsifiable concentrate or 2000 times diluted 75% thiophanate-methyl wettable powder or mancozeb, etc. However, chemical control has problems such as pesticide residues, harm to human health, and soil and water pollution.

[0003] In recent years, biological control has become a research hotspot in plant disease control. Among them, Pseudomonas has a strong disease prevention and growth promotion effect and is an important microbial source of biological pesticides. Regarding the use of biocontrol bacteria to control plant fungal diseases, how to effectively improve their ability to produce extracellular antibacterial substances is the key to improving their biocontrol effectiveness. Different strains of the same genus differ in carbon source, nitrogen source utilization and fermentation conditions. The reasons for the differences are closely related to the genetics, physiological characteristics, and isolation sources of the strains themselves. For different strains, different culture medium components and fermentation conditions can have a significant impact on the synthesis and accumulation of antibacterial substances in the fermentation broth. Cheng Liangliang et al. (Screening of antagonistic bacteria HB-10 against wheat sheath blight and optimization of its fermentation conditions) optimized the fermentation medium of Pseudomonas aeruginosa HB-10 and found that the strain grew in the medium with the optimal glucose of 10 g / L, yeast powder of 20 g / L, and KCl of 10 g / L, and the antibacterial effect of the fermentation broth was improved by 5%; Li Shuyan et al. (Optimization of fermentation conditions for iron carrier production by Pseudomonas aeruginosa Gxun-2 and its antibacterial effect) optimized the fermentation medium of Pseudomonas aeruginosa Gxun-2 and found that the best fermentation process of the strain was 2 g / L sodium succinate, 10 g / L glycerol, 1 g / L ammonium sulfate, 10 g / L glucose, initial pH 6.8, temperature 35°C, inoculation amount 1% (v / v), and liquid volume 140 mL / 250 mL; under these conditions, the fermentation broth had no significant effect on the banana wilt pathogen Fusarium oxysporum The antibacterial rate of f.sp.cubense) reached 70.60%±1.87%, which was 52.68%±4.23% higher than that before optimization.

[0004] The stability of antibacterial active substances is the premise of the preparation and production of microbial agents. The stability of fermented bacterial liquid is conducive to industrial mass production and can achieve good control effects when applied to the field. Qiao Tianmin et al. reported (Structural identification and stability detection of antibacterial pigments of Pseudomonas aeruginosa) that the anti-plant pathogen active substances produced by Pseudomonas aeruginosa are more sensitive under acidic conditions. 3+ , Cu 2+ , Fe 3+ , Pb 2+ It has a great impact on active substances, limiting its application in the field.

[0005] Pseudomonas aeruginosa 8-7 is a strain of biocontrol bacteria isolated by the inventor's research group in the early stage, which has a good antagonistic effect on a variety of pathogens. The strain is now deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 20694, and has an invention patent named "A strain of Pseudomonas aeruginosa 8-7 and its application" with an authorization announcement number of CN112280714B. In order to further increase the content of antibacterial substances in Pseudomonas aeruginosa 8-7 microbial preparations, it is urgent to optimize the fermentation method of Pseudomonas aeruginosa 8-7 to produce antibacterial substances.

[0006] Therefore, providing a method for preparing a sterile fermentation filtrate of Pseudomonas aeruginosa 8-7 and its application is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a preparation method of Pseudomonas aeruginosa 8-7 sterile fermentation filtrate and application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A method for preparing a sterile fermentation filtrate of Pseudomonas aeruginosa 8-7, comprising the following steps:

[0010] The fermentation seed liquid of Pseudomonas aeruginosa 8-7 was inoculated into the FA fermentation medium, the bottling volume of the medium was 50 mL / 500 mL, the shaking flask culture speed was 220 r / min, the inoculation amount was 10%, the culture temperature was 36° C., and the culture time was 5 days;

[0011] The FA fermentation medium comprises: acid hydrolyzed casein② 15 g / L, glycerol 25 ml / L, MgSO4·7H2O 1 g / L, K2HPO4 1.5 g / L, and natural pH.

[0012] Furthermore, the preparation method of the fermentation seed liquid is as follows: Pseudomonas aeruginosa 8-7 is activated in LB solid culture medium, a single colony is picked and inoculated into a triangular flask containing LB liquid culture medium, and cultured at 36°C and 180r / min constant temperature shaking until OD 600 =1.0, and obtain fermentation seed liquid.

[0013] Furthermore, the method described above obtains the aseptic fermentation filtrate of Pseudomonas aeruginosa 8-7.

[0014] Further, the use of the aseptic fermentation filtrate of Pseudomonas aeruginosa 8-7 in the preparation of an agent for inhibiting plant pathogens, wherein the plant pathogens are Colletotrichum gloeosporioides, Corynespora cassiicola, Colletotrichum alienum, Colletotrichum siamense, Alternaria heveae, Fusarium solani, Pestalotiopsis microspora, Calonectria pentaseptata, Peronophythora litchii, Phytophthora melonis Katsura, Phytophthora vignae Purss, Fusarium oxysporum, cubense), Phytophthora capsici, Rhizopus stolonifer, Alternaria solani, Botrytis cinerea, Alternaria alternate, Alternaria alternate or Diaporthe passifloricola.

[0015] The present invention provides a fermentation medium suitable for Pseudomonas aeruginosa 8-7 (see patent 202011210825.8) with a deposit number of CGMCC No. 20694, and further screens and obtains the optimal shake flask fermentation conditions of Pseudomonas aeruginosa 8-7. The present invention solves the problem of low content of antibacterial substances in existing Pseudomonas aeruginosa 8-7 microbial preparations, obtains an optimized fermentation process for high-yield extracellular antibacterial substances of Pseudomonas aeruginosa 8-7, and verifies that the antibacterial substances obtained under the conditions have the characteristics of heat resistance, light resistance, and good storage stability, and the fermentation broth can maintain good antibacterial activity even under acidic conditions. At the same time, the control effect of Pseudomonas aeruginosa 8-7 fermentation broth on 19 plant pathogens is clarified. Compared with the shake flask fermentation conditions before optimization, the antibacterial efficiency of the antibacterial substances produced by Pseudomonas aeruginosa 8-7 on Colletotrichum alienum is improved by adopting a high-yield extracellular antibacterial substance shake flask fermentation process. The invention provides technical support and theoretical guidance for the industrial production of Pseudomonas aeruginosa 8-7 strains and obtaining high-yield antibacterial substances.

[0016] It can be seen from the above technical scheme that compared with the prior art, the present invention discloses a preparation method and application of aseptic fermentation filtrate of Pseudomonas aeruginosa 8-7. The aseptic fermentation filtrate prepared from the fermentation medium of Pseudomonas aeruginosa 8-7 with a preservation number of CGMCC No. 20694 has strong stability. The antibacterial effect is not affected when treated under high temperature (121°C) and high pressure conditions for 30 minutes; the light has little effect on the stability of the antibacterial active substance; the reducing agent has little effect on the stability of the antibacterial active substance; the antibacterial activity of the antibacterial active substance is not affected when treated under pH 1-12 conditions for 30 minutes; after the aseptic fermentation liquid of strain 8-7 is treated with 11 kinds of metal ions, including CuSO4, EDTA-Na, FeCl3, NiSO4, NaCl, MgCl2, KCl, ZnSO4, CaCl2, BaCl2, and Pb(NO3)2, the antibacterial active substance still maintains good antibacterial activity. The invention determines the optimal fermentation medium and fermentation conditions of the strain 8-7, the antibacterial active substance has a broad antibacterial spectrum and good stability, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1The accompanying drawing shows the effect of different inoculation amounts on the antibacterial activity of the aseptic fermentation filtrate of strain 8-7 of the present invention;

[0019] Figure 2 The accompanying drawing shows the effect of different culture temperatures on the antibacterial activity of the aseptic fermentation filtrate of strain 8-7 of the present invention;

[0020] Figure 3 The accompanying drawing shows the effect of different culture speeds on the antibacterial activity of the aseptic fermentation filtrate of strain 8-7 of the present invention;

[0021] Figure 4 The accompanying drawing shows the effect of different culture times on the antibacterial activity of the aseptic fermentation filtrate of strain 8-7 of the present invention;

[0022] Figure 5 The accompanying drawing shows the effect of different liquid loading amounts on the antibacterial activity of the aseptic fermentation filtrate of strain 8-7 of the present invention. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Colletotrichum gloeosporioides, Corynespora cassiicola, Colletotrichum alienum, Colletotrichum siamense, Alternaria heveae, Fusarium solani, Pestalotiopsis microspora, Calonectria pentaseptata, Peronophythora litchii, Phytophthora melonis Katsura, Phytophthora vignae Purss, Fusarium oxysporumf.sp.cubense, Phytophthora capsici, Rhizopus stolonifer), Alternaria solani, Botrytis cinerea, Alternaria alternate, Alternaria alternate, and Diaporthe passifloricola were all preserved by the Plant Protection and Microbial Utilization Research Center of Yunnan Tropical Crops Science Research Institute. Glucose, yeast extract, tryptone and other analytical reagents were from Sangon Biotechnology Co., Ltd.; acid hydrolyzed casein ① (C8221): Beijing Solebold Technology Co., Ltd.; acid hydrolyzed casein ② (C822594): Shanghai MacLean Biochemical Technology Co., Ltd.; constant temperature incubator, Minnesota Mining and Manufacturing Company; constant temperature shaking incubator, Shanghai Boxun Industrial Co., Ltd.

[0025] Example 1

[0026] Pseudomonas aeruginosa 8-7, with the deposit number: CGMCC No.20694 (see patent 202011210825.8), is preserved by the General Microbiology Center of China National Microbiological Culture Collection Administration.

[0027] This embodiment involves 8 kinds of liquid culture media: LB, PDA, soybean powder, pigment growth-promoting medium, beef extract peptone medium, sucrose medium, TB medium and FB medium, and the specific formulas are as follows;

[0028] LB liquid medium: 5 g yeast powder, 10 g NaCl, 10 g tryptone, and distilled water to 1000 mL.

[0029] PDA medium: 200 g potatoes, 20 g glucose, distilled water to 1000 mL.

[0030] Soybean powder culture medium: 65g soybean powder, 16g corn steep liquor, 12g glucose, 22mL anhydrous ethanol, and distilled water to 1000mL.

[0031] Pigment-promoting medium: 22 g tryptone, 20 g glucose, 5 g potassium nitrate, pH 7.5, distilled water to 1000 mL.

[0032] Beef extract peptone medium: NaCl 5g, peptone 10g, beef extract 3g, pH 7.2-7.4, distilled water to 1000mL.

[0033] Sucrose medium: sucrose 3 g, NaCl 2.5 g, peptone 10 g, pH 7.5, distilled water to 1000 mL.

[0034] TB medium: 12 g tryptone, 24 g yeast extract, 9.4 g K2HPO4, 2.2 g KH2PO4, 4 mL glycerol, and distilled water to 1000 mL.

[0035] FB culture medium: MgSO4·7H2O 1g, K2HPO4 2.5g, acid hydrolyzed casein① 5g, glycerol 8mL, distilled water to 1000mL, natural pH.

[0036] Preparation of fermentation seed liquid: Pseudomonas aeruginosa 8-7 was activated in LB solid medium and cultured in a 37°C incubator for 24 h. A single colony was picked and inoculated into a 300 mL flask containing LB liquid medium (100 mL). The culture was kept at 36°C and 180 rpm for 12 h (OD 600 =1.0), and obtain the fermentation seed liquid.

[0037] Screening of culture media producing antibacterial substances: 100 mL of fermentation culture media producing antibacterial substances of the above 8 strains 8-7 to be screened were respectively placed in 500 mL Erlenmeyer flasks, and the cultured seed liquid was inoculated in the Erlenmeyer flask at an inoculation rate of 15%. After culturing in a 30°C constant temperature shaking incubator at 180 r / min for 4 days, the fermentation liquid was centrifuged at 8000 r / min for 4 min, the supernatant was aspirated into another sterilized centrifuge tube, and filtered through a 0.22 μm microporous filter membrane to obtain sterile fermentation filtrates of 8 screening culture media.

[0038] Determination of antibacterial activity: The mycelium growth rate method was used, and Colletotrichum heteromorphum was used as the pathogen target bacteria. After Colletotrichum heteromorphum was activated on a PDA plate for 4 days, a bacterial cake was made using a 5mm hole puncher and inoculated into the center of a PDA plate (6 cm in diameter) coated with 100 μL of sterile fermentation filtrate, and repeated 3 times; after culturing in a 28°C incubator for 4 days, the colony diameters of each treatment were measured using the cross method, and the mycelium growth inhibition rate was calculated: inhibition rate (%) = [(control pathogen colony diameter - treatment pathogen colony diameter) / (control pathogen colony diameter - bacterial cake diameter)] × 100. The PDA plate not coated with sterile fermentation filtrate was used as the control.

[0039] Screening of culture medium producing antibacterial substances: strain 8-7 was cultured in 8 liquid culture media including LB, PDA, soybean powder, pigment growth-promoting medium, beef extract peptone medium, sucrose medium, TB and FB, and the inhibition of the obtained 100 μL sterile fermentation filtrate on the growth of Colletotrichum heteromorpha mycelium was detected, and the inhibition rate of each treatment group was compared. The results are shown in Table 1.

[0040] Table 1 Inhibitory effect of sterile fermentation filtrate of screening medium of strain 8-7 on Colletotrichum heteromorphum

[0041]

[0042]

[0043] Note: Different lowercase letters after the data in the same column indicate significant differences (P<0.05).

[0044] The results in Table 1 show that FB medium has the best antibacterial effect, which is significantly higher than the other 7 screening mediums. Therefore, the components of FB medium were used as the basis for the next step of fermentation condition optimization.

[0045] Example 2 Screening of the best fermentation factor

[0046] 1) Screening of the best fermentation carbon source

[0047] FB medium was used as the basic fermentation medium, and glycerol, mineral oil, soybean oil, corn oil, peanut oil, rapeseed oil, olive oil, palm oil, sucrose, xylose, mannitol, glucose, soluble starch, maltose, lactose, galactose, and fructose were selected as carbon sources (replacing 8 mL of glycerol) at a ratio of 1% (mass volume ratio) for further optimization experiments. The mycelium growth rate method was used, and Colletotrichum heteromorphum was used as the pathogenic target bacteria to determine the antibacterial activity (operation method as above) of 100 μL of sterile fermentation filtrate (preparation method as above) in each treatment group, and each treatment was repeated 3 times. The results are shown in Table 2.

[0048] Table 2 Effects of different carbon source culture media on the antibacterial activity of aseptic fermentation filtrate of strain 8-7

[0049]

[0050]

[0051] The results in Table 2 show that among the 17 carbon sources used to obtain the aseptic fermentation filtrates of strain 8-7, the aseptic fermentation filtrates of 9 carbon sources have a certain inhibitory effect on Colletotrichum heteromorphum, among which the fermentation filtrate of the strain with glycerol as the carbon source has the highest antibacterial rate, which is significantly higher than other carbon sources. The aseptic fermentation filtrate also has an antibacterial rate when soybean oil, corn oil, peanut oil, rapeseed oil, olive oil, and palm oil are used as carbon sources. The aseptic fermentation filtrate of the strain has no antibacterial activity when mineral oil, xylose, sucrose, galactose, soluble starch, lactose, glucose, and maltose are used as carbon sources; the antibacterial rate of the aseptic fermentation filtrate is extremely low when fructose is used as a carbon source; the antibacterial rate of the fermentation filtrate of the strain when mannitol is used as a carbon source is equivalent to that of glycerol. For cost considerations, glycerol is selected as the best carbon source for liquid fermentation medium.

[0052] 2) Screening of the best nitrogen source for fermentation

[0053] FB was used as the basic fermentation medium, glycerol was the best carbon source (added at 1%), and acid hydrolyzed casein ① (Solebo C8221), acid hydrolyzed casein ② (McLean C822594), casein hydrolyzate, beef extract, tryptone, and yeast powder were selected as nitrogen sources (nitrogen source was added at a ratio of 2%, replacing acid hydrolyzed casein ① 5g), and further optimization experiments were performed. The mycelial growth rate method was used, and the heteromorphic anthracnose bacteria were used as the pathogenic target bacteria to determine the antibacterial activity of 100μL sterile fermentation filtrate (preparation method as above) of each treatment group (operation method as above), and each treatment was repeated 3 times. The results are shown in Table 3.

[0054] Table 3 Effects of different nitrogen source culture media on the antibacterial activity of the aseptic fermentation filtrate of strain 8-7

[0055]

[0056] The results in Table 3 show that the aseptic fermentation filtrates of the strains obtained using 6 nitrogen sources all have inhibitory effects on Colletotrichum heteromorphum. Among them, the fermentation broth using acid hydrolyzed casein ② and casein hydrolyzate as nitrogen sources has the highest antibacterial rate, which is significantly higher than other nitrogen sources. For cost considerations, acid hydrolyzed casein ② was selected as the optimal nitrogen source for liquid fermentation medium.

[0057] 3) Orthogonal test

[0058] According to the test results of carbon source and nitrogen source, four factors, namely, acid hydrolyzed casein②, K2HPO4, MgSO4·7H2O, and glycerol, were selected to conduct a 4-factor 4-level orthogonal test (Table 4). The mycelial growth rate method was used, and Colletotrichum heteromorphum was used as the pathogenic target bacteria to determine the antibacterial activity of 100 μL of sterile fermentation filtrate (preparation method as above) in each treatment group (operation method as above), and each treatment was repeated 3 times.

[0059] Table 4 Factors and levels design of orthogonal experiment

[0060]

[0061] The orthogonal experiment results are shown in Table 5.

[0062] Table 5 Results of orthogonal experiments on nutrients in the fermentation medium of the strain

[0063]

[0064]

[0065] The orthogonal experimental results in Table 5 show that the order of influence of the four factors on the inhibition rate of Colletotrichum heteromorphum is acid hydrolyzed casein ②> glycerol> K2HPO4> MgSO4·7H2O, and the best level combination is A3B3C1D2, that is, the combination scheme number 11. In the variance analysis of the 16 groups of antibacterial effects, number 11 has the highest inhibition rate on Colletotrichum heteromorphum. The optimized FB fermentation medium was determined to be the FA fermentation medium, and the components of the FA fermentation medium were: acid hydrolyzed casein ② 15g / L, glycerol 25ml / L, MgSO4·7H2O 1g / L, K2HPO41.5g / L, and natural pH.

[0066] 4) Screening of the optimal inoculum size

[0067] Place 100 mL of the FA fermentation medium obtained by the above screening in a 500 mL Erlenmeyer flask, and inoculate 5%, 10%, 15%, 20%, and 25% of the cultured fermentation seed liquid in the Erlenmeyer flask respectively; place it in a 36°C constant temperature shaking incubator at 180 r / min for 4 days, centrifuge the fermentation liquid at 8000 r / min for 4 minutes, pipette the supernatant into another sterilized centrifuge tube, and filter it through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate; use the tested Colletotrichum heteromorphum as the pathogenic target bacteria, and determine the antibacterial activity of each treatment group, with each treatment repeated 3 times. Results are shown in Figure 1 When the inoculation amount was 10%, the antibacterial activity of the sterile fermentation filtrate reached its peak value, and the antibacterial rate was 71.56%; therefore, the optimal inoculation amount of strain 8-7 was selected as 10% for the next experiment.

[0068] 5) Screening of optimal culture temperature

[0069] Place 100 mL of the FA fermentation medium obtained by the above screening in a 500 mL Erlenmeyer flask, and inoculate 10 mL of the cultured fermentation seed liquid in the Erlenmeyer flask; set the shaking incubator culture temperature to 30, 33, 36, and 39 ° C respectively; after culturing in a constant temperature shaking incubator at 180 r / min for 4 days, centrifuge the fermentation liquid at 8000 r / min for 4 minutes, pipette the supernatant into another sterilized centrifuge tube, and filter through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate; use the tested Colletotrichum heteromorphum as the pathogenic target bacteria to determine the antibacterial activity of each treatment group, and repeat each treatment 3 times. Results are shown in Figure 2 When the culture temperature was 36℃, the antibacterial activity of the sterile fermentation filtrate reached its peak value, and the antibacterial rate was 72.24%; therefore, the optimal fermentation temperature of strain 8-7 was selected as 36℃ for the next experiment.

[0070] 6) Screening of the optimal culture speed

[0071] 100 mL of the selected FA fermentation medium was placed in a 500 mL Erlenmeyer flask, and 10 mL of the cultured fermentation seed liquid was added to the flask; the shaking incubator temperature was set to 36°C; the shaking speed was set to 120, 140, 160, 180 and 220 r / min, respectively. After 4 days of incubation in an oscillating incubator, the fermentation liquid was centrifuged at 8000 r / min for 4 minutes, and the supernatant was transferred to another sterilized centrifuge tube and filtered through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate; the tested Colletotrichum heteromorphum was used as the pathogenic target bacteria to determine the antibacterial activity of each treatment group, with 3 replicates for each treatment. Results are shown in Figure 3 When the culture speed was 220r / min, the antibacterial activity of the sterile fermentation filtrate reached its peak value, and the antibacterial rate was 81.45%; therefore, the culture speed of strain 8-7 was selected as 220r / min for the next experiment.

[0072] 7) Screening of the optimal culture time

[0073] 100 mL of the FA fermentation medium obtained by the above screening was placed in a 500 mL conical flask, and 10 mL of the cultured fermentation seed liquid was added to the conical flask; the shaking incubator temperature was set to 36°C; the shaking speed was 220 r / min, and the incubator was cultured for 4, 5, 6, 7, and 8 days respectively. The optimal culture time was determined, and the fermentation liquid was centrifuged at 8000 r / min for 4 minutes. The supernatant was aspirated into another sterilized centrifuge tube and filtered through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate; the heteromorphic anthracnose fungus was used as the pathogenic target bacteria, and the antibacterial activity of each treatment group was determined, and each treatment was repeated 3 times. The results are shown in Figure 4 When the culture time was 5-7 days, the antibacterial activity of the sterile fermentation filtrate was 85.56%, 84.96% and 82.71% respectively; the culture time of strain 8-7 was 5 days for the next experiment.

[0074] 8) Screening of the optimal fermentation liquid volume

[0075] The FA fermentation medium obtained by screening was injected with a liquid volume gradient of 50mL / 500mL, 100mL / 500mL, 150mL / 500mL and 200mL / 500mL, and the cultured fermentation seed liquid was inoculated in a triangular flask with an inoculation amount of 10%, the fermentation temperature was 36°C, and the shaker speed was 220r / min in an oscillating incubator for 5 days. The fermentation liquid was centrifuged at 8000r / min for 4min, and the supernatant was aspirated into another sterilized centrifuge tube and filtered through a 0.22μm microporous filter membrane to obtain a sterile fermentation filtrate; the test anthracnose fungus was used as the pathogenic target bacteria to determine the antibacterial activity of each treatment group, and each treatment was repeated 3 times. The results are shown in Figure 5 When the liquid volume of the sterile fermentation filtrate was 50mL / 500mL, the antibacterial rate reached a peak of 89.12%. Then, with the increase of the liquid volume, the antibacterial activity decreased significantly, indicating that the liquid volume had a great influence on the accumulation of antibacterial substances in strain 8-7. Therefore, the liquid volume of 50mL / 500mL was selected for subsequent experiments.

[0076] The present invention optimizes the fermentation conditions based on the optimal fermentation medium formula determined by orthogonal test. Considering the antibacterial rate as the only indicator, the optimal fermentation conditions of strain 8-7 are clarified: the medium bottling volume is 50mL / 500mL, the shaking flask culture speed is 220r / min, the inoculation amount is 10%, the culture temperature is 36°C, and the culture time is 5d, at which the antibacterial activity is the best.

[0077] Example 3 Inhibitory effect of aseptic fermentation filtrate of strain 8-7 on 19 different plant pathogens

[0078] The pathogens include Colletotrichum gloeosporioides, Corynespora cassiicola, Colletotrichum alienum, Colletotrichum siamense, Alternaria heveae, and Fusarium solani from rubber trees; Pestalotiopsis microspora and Calonectria pentaseptata from nut trees; Peronophythora litchii from litchi; Phytophthora melonis Katsura from melon; Phytophthora purss from cowpea; and Fusarium oxysporum from banana. cubensis); Phytophthora capsici from pepper; Rhizopus stolonifer from peach; Alternaria solani from tomato; Botrytis cinerea from grape; Alternaria alternate from tobacco; Alternaria alternate from winter jujube and Diaporthe passifloricola from citrus were the pathogens; the pathogens and sterile fermentation filtrate (100 μL, prepared by optimal fermentation conditions using FA fermentation medium) were inoculated on PDA plates at the same time, and the treatment inoculated with pathogens alone was used as the control. The experiment was repeated three times and cultured in a constant temperature box at 28°C for 5 days. The diameter of the inhibition zone was measured, the inhibition was observed and photographed. The broad-spectrum antibacterial activity of the antagonistic bacteria was evaluated, and the results are shown in Table 6.

[0079] Table 6 Inhibitory effect of aseptic fermentation liquid of strain 8-7 on 19 plant pathogenic fungi

[0080]

[0081]

[0082] Note: Different lowercase letters indicate significant differences in the same column of data at the P<0.05 level.

[0083] The results in Table 6 show that the aseptic fermentation filtrate of strain 8-7 has inhibitory effects on 19 tested pathogenic fungi, among which the best inhibitory effect is on Phytophthora melonii from melon and Phytophthora downyii from litchi, with the inhibition rates of the two Phytophthora being 93.87±1.69 and 94.56±1.38 mm, respectively; followed by citrus black spot pathogen and Colletotrichum heteromorphum, with inhibition rates of 88.93±0.54 and 89.81±0.44 mm, respectively. The inhibition rate of eight plant pathogens, including Corynespora leaf fall pathogen, Fusarium solani, Macrospora pentaphyllosporin, Alternaria solani, Alternaria tobacco, Phytophthora capsici, Rhizopus stolonifer, and Fusarium oxysporum, is less than 50%.

[0084] Example 4 Study on the stability of antimicrobial substances in the aseptic fermentation filtrate of strain 8-7

[0085] Sterile fermentation filtrate: prepared using FA fermentation medium under optimal fermentation conditions.

[0086] (1) Effect of light on the stability of antimicrobial substances

[0087] Place the sterile fermentation filtrate under natural light, set 1, 2, 3, 4, 5 days, 5 treatments, and measure its activity every 1 day; place the sterile fermentation filtrate under a 30W ultraviolet lamp, irradiate with ultraviolet light at a distance of 15cm from the lamp, set 30, 60, 90, 120min for a total of 4 treatments, use the untreated sterile fermentation filtrate as the control (CK), and repeat three times for each treatment to take the average value. Pick a 5mm heteromorphic anthracnose fungus cake and inoculate it on a PDA plate containing 100μL of sterile fermentation filtrate from each experimental group, invert and culture at a constant temperature of 28℃ for 4 days, observe the changes in the inhibition zone, and measure the diameter of the inhibition zone. The results are shown in Table 7.

[0088] Table 7 Effect of light on the stability of antibacterial substances

[0089]

[0090] The results in Table 7 show that the sterile fermentation filtrate still has high antibacterial activity after being irradiated with ultraviolet light at different time gradients, and even after 120 minutes of treatment, there is still an 82.82% antibacterial rate. After the sterile fermentation filtrate was placed under natural light for different time periods, it was found that the antibacterial substances had little effect within 5 days, indicating that the sterile fermentation filtrate placed in the natural environment will not reduce its activity or change its properties.

[0091] (2) Effect of temperature on the stability of antibacterial substances

[0092] The sterile fermentation filtrate was placed in a water bath at 80℃ for 1h, a water bath at 100℃ for 1h, and a high temperature (121℃) high pressure condition for 30min. After cooling to room temperature, 100μL of the sterile fermentation filtrate was applied to a PDA solid plate. A 5mm heteromorphic anthracnose cake was picked and inoculated on a PDA plate containing the sterile fermentation filtrate of each experimental group. The plate was inverted and cultured at 28℃ for 4d. The changes in the inhibition zone were observed and the diameter of the inhibition zone was measured. The sterile fermentation filtrate without water bath treatment was used as the control (CK). Each treatment was repeated three times and the average value was taken. The results are shown in Table 8.

[0093] Table 8 Effect of temperature on the stability of antibacterial substances

[0094]

[0095] The results in Table 8 show that temperature has little effect on the activity of antibacterial substances. Even under the conditions of 100°C boiling water treatment for 1 h and 121°C high pressure sterilization treatment for 30 min, the antibacterial activity is still very high, indicating that the antibacterial substances have strong thermal stability.

[0096] (3) Effect of pH on the stability of antimicrobial substances

[0097] 0.1mol / L HCl and 0.1mol / L NaOH were used to prepare sterile fermentation filtrates with final pH values ​​of 1, 3, 4, 5, 7, 9, 11, and 12, respectively. After standing for 30 minutes, the pH value was adjusted to the untreated pH value (7.2). 100μL of the treated sterile fermentation filtrate was spread on a PDA solid plate, and a 5mm anthracnose cake was picked and inoculated on the PDA medium containing the pH-treated sterile fermentation filtrate. The culture was inverted at 28°C for 4 days, and the changes in the inhibition zone were observed and the diameter of the inhibition zone was measured. The untreated sterile fermentation filtrate was used as the control (CK), and the average value was taken for each treatment repeated three times. The results are shown in Table 9.

[0098] Table 9 Effect of pH on the stability of antibacterial substances

[0099]

[0100] The results in Table 9 show that under slightly acidic conditions (pH 1-4), pH changes have little effect on the activity of antibacterial substances. When the pH is 12, the sterile fermentation filtrate still has 81.49% antibacterial activity, indicating that the stability of the antibacterial substances is relatively strong.

[0101] (4) Effect of reducing agents on the stability of antibacterial substances

[0102] The final concentration was 0.4×10 -4 mol / L、5×10 -4 mol / L、2×10 -3 mol / L、5×10-3 mol / L Na2SO3 sterile fermentation filtrate, after standing for 30 minutes, take 100μL of the treated sterile fermentation filtrate and spread it on the PDA solid plate, pick 5mm heteromorphic anthrax cake and inoculate it on the treated sterile fermentation liquid PDA medium, invert and culture at 28℃ constant temperature for 4 days, observe the changes in the inhibition zone, and measure the diameter of the inhibition zone of each treatment; and use the corresponding concentration of Na2SO3 solution as the control to measure the colony diameter of each control pathogen; calculate the mycelium growth inhibition rate: inhibition rate (%) = [(control pathogen colony diameter-treatment pathogen colony diameter) / (control pathogen colony diameter-cake diameter)]×100. Each treatment was repeated three times and the average value was taken. The results are shown in Table 10.

[0103] Table 10 Effect of reducing agent on the stability of antibacterial substances

[0104]

[0105] Note: CK(0) was treated with sterile fermentation filtrate without adding Na2SO3, and the PDA plate without sterile fermentation filtrate was used as the control to calculate the inhibition rate.

[0106] The results in Table 10 show that the higher the Na2SO3 content in the salt solution, the lower the antibacterial rate of the antibacterial substance; when the concentration of the Na2SO3 salt solution is less than 5×10 -4 The inhibition rate was not significantly affected.

[0107] (5) Effect of metal ions on the stability of antimicrobial substances

[0108] Prepare CuSO4 and EDTA-Na with a concentration of 0.1 mol / L 2、 , FeCl3, NiSO4, NaCl, MgCl2, KCl, ZnSO4, CaCl2, BaCl2, Pb(NO3)2 salt solutions. Take 11 EP tubes, add 10 μL of the freshly prepared salt solution to each tube, then add 190 μL of sterile fermentation filtrate to each of the 11 test tubes to make the final concentration of metal ions 0.005 mol / L. After standing for 5 hours, take 100 μL of the treated sterile fermentation filtrate and apply it on the PDA solid plate, pick 5 mm heteromorphic anthrax cake and inoculate it on the treated sterile fermentation filtrate PDA medium, invert and culture at 28°C for 4 days, observe the changes in the inhibition zone, and determine the diameter of the inhibition zone of each treatment; and use the corresponding salt solution as a control to determine the colony diameter of each control pathogen; calculate the mycelium growth inhibition rate: inhibition rate (%) = [(control pathogen colony diameter-treatment pathogen colony diameter) / (control pathogen colony diameter-cake diameter)] × 100. Each treatment was repeated three times and the average value was taken. The results are shown in Table 11.

[0109] Table 11 Effect of metal ions on the activity of antibacterial substances

[0110]

[0111]

[0112] Note: CK(0) refers to the treatment with sterile fermentation filtrate without adding salt solution, and the PDA plate without sterile fermentation filtrate was used as the control to calculate the inhibition rate.

[0113] The results in Table 11 show that 11 different metal salt solutions have different effects on the activity of antibacterial substances. The eight salt solutions of EDTA-Na2, FeCl3, NaCl, MgCl2, CaCl2, BaCl2, Pb(NO3)2, and NiSO4 have no significant effect on the activity of antibacterial substances; CuSO4 has the greatest impact on the activity of antibacterial substances; but even if the sterile fermentation filtrate contains 0.005 mol / L CuSO4, the inhibition rate against Colletotrichum heteromorphum remains at 81.28%.

[0114] Example 5 Identification of metabolite composition of sterile fermentation broth of strain 8-7

[0115] A single colony of the activated strain 8-7 was picked and inoculated into LB medium and cultured at 180 r / min and 36°C for 12 h (OD 600 =1.15), and used it as seed solution, inoculated into FA fermentation medium at 10% inoculation amount, and cultured at 220r / min, 36℃ for 120h (OD 600=2.00), each treatment was repeated three times, sampling, 8000r / min, centrifugation for 10min, the supernatant was filtered and sterilized with a 0.22μm microporous filter to obtain a sterile fermentation broth, and 10mL of the sterile fermentation broth was stored in a -80°C refrigerator for later use. The sterile fermentation broth was subjected to non-targeted metabolomics determination, and the FA fermentation medium without inoculation of strain 8-7 was used as a control, and each treatment was repeated three times. T3 chromatographic conditions: the chromatographic column was Waters ACQUITY UPLC HSS T3 Column 1.8μm, 2.1mm×100mm, the mobile phase A was ultrapure water containing 0.1% formic acid, the mobile phase B was acetonitrile containing 0.1% formic acid, the column temperature was 40°C, the flow rate was 0.4mL / min, and the injection volume was 4μL. The mobile phase elution gradient is shown in Table 12; mass spectrometry conditions: electrospray ionization (ESI); scanning mode is positive and negative ion mode, capillary voltage +5.5kV; capillary temperature 550℃; resolution: 7500; collision energy: -30~30eV; scanning range m / 25~1250; nebulizer pressure 50psi. Data processing: The original data was converted into mzXML format by ProteoWizard, and the XCMS program was used for peak extraction, alignment, and retention time correction. Peaks with a missing rate greater than 50% in the sample were filtered, and the blank values ​​were filled with KNN. The peak area was corrected by the SVR method. After correction and screening, the Myvi metabolic database, public library, prediction library and metDNA method were searched for metabolite identification.

[0116] Table 12T3 chromatographic column mobile phase gradient conditions

[0117]

[0118] The screening criteria for differential metabolites were Fold change ≥ 2; fold change ≤ 0.5 and P-value < 0.05; FA fermentation medium was used as the control and 120 h was used as the experimental group. After correction and screening, 2990 differential metabolites were obtained from the sterile fermentation broth, including 2086 upregulated substances and 904 downregulated substances. Based on the antibacterial and anti-inflammatory substances reported in the references, the upregulated metabolites were screened, and a total of 45 target metabolites with antibacterial activity were obtained (Table 13); including 15 flavonoids, 7 phenolic acids, 15 alkaloids, 6 organic acids, and 2 coumarins.

[0119] Table 13 Antibacterial metabolite information

[0120]

[0121]

[0122] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a sterile fermentation filtrate of Pseudomonas aeruginosa 8-7, characterized in that: The specific steps are as follows: The fermentation seed liquid of Pseudomonas aeruginosa 8-7 was inoculated into the FA fermentation medium, the bottling volume of the medium was 50 mL / 500 mL, the shaking flask culture speed was 220 r / min, the inoculation amount was 10%, the culture temperature was 36° C., and the culture time was 5 days; The FA fermentation medium comprises: acid hydrolyzed casein② 15 g / L, glycerol 25 ml / L, MgSO4·7H2O 1 g / L, K2HPO4 1.5 g / L, and natural pH.

2. The method for preparing a Pseudomonas aeruginosa 8-7 sterile fermentation filtrate according to claim 1, characterized in that: The preparation method of the fermentation seed liquid is as follows: Pseudomonas aeruginosa 8-7 is activated in LB solid culture medium, a single colony is picked and inoculated into a triangular flask filled with LB liquid culture medium, and cultured at 36°C and 180 rpm at constant temperature until OD 600 =1.0, and obtain fermentation seed liquid.

3. The aseptic fermentation filtrate of Pseudomonas aeruginosa 8-7 obtained by the method according to claim 1 or 2.

4. Use of the aseptic fermentation filtrate of Pseudomonas aeruginosa 8-7 according to claim 3 in the preparation of an agent for inhibiting plant pathogens, characterized in that: The plant pathogens are Colletotrichum gloeosporioides, Corynespora cassiicola, Colletotrichum alienum, Colletotrichum siamense, Alternaria heveae, Fusarium solani, Pestalotiopsis microspora, Calonectria pentaseptata, Peronophythora litchii, Phytophthora melonis Katsura, Phytophthora vignae Purss, Fusarium oxysporumf.sp.cubense, Phytophthora capsici, Rhizopus stolonifera, and Phytophthora stolonifera. stolonifer), Alternaria solani, Botrytis cinerea, Alternaria alternate, Alternaria alternate, or Diaporthe passifloricola.

Images