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

Abstract

The present invention discloses a preparation method and application of a sterile fermentation filtrate of Pseudomonas aeruginosa 8-7, and belongs to the field of microbial technology. The present invention discloses a preparation method of a sterile fermentation filtrate of Pseudomonas aeruginosa 8-7, which is prepared using FA fermentation medium. The components of the FA fermentation medium are: acid hydrolyzed casein ②15g / L, glycerol 25ml / L, MgSO4·7H2O 1g / L, K2HPO41.5g / L, and natural pH. The sterile fermentation filtrate of Pseudomonas aeruginosa 8-7 can withstand environments such as strong acid, strong alkali, high temperature, Na2SO3 and ultraviolet irradiation, and is insensitive to metal ions; and has a good antibacterial effect on a variety of plant pathogens. The present invention optimizes the combination of fermentation culture conditions for the production of antibacterial substances by Pseudomonas aeruginosa 8-7, and provides technical support and theoretical guidance for the industrial production of Pseudomonas aeruginosa 8-7 strains and the acquisition of high-yield antibacterial substances.

Description

Technical Field

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

[0002] The rubber tree (Hevea brasiliensis) is a perennial tropical rainforest tree in the genus Hevea, Euphorbiaceae. The natural rubber it produces is an irreplaceable industrial raw material and is cultivated extensively in Hainan, Yunnan, and Guangdong provinces of my country. Yunnan's rubber-growing region, located on the northern edge of the tropics, enjoys a warm, humid climate conducive to the reproduction and spread of pathogenic microorganisms. Anthracnose, caused by Colletotrichum, is a frequent disease of rubber trees, harming seedlings, field saplings, and tapped rubber trees. It infects young leaves, petioles, shoots, and fruits, causing leaf drop, shoot dieback, and fruit rot. In severe cases, it can lead to repeated leaf drop and shoot dieback, delaying the start of harvest and reducing latex production. Currently, the main chemical pesticides used to control anthrax are chemical pesticides, such as 25% prochloraz emulsifiable concentrate or 2000 times diluted 75% chlorothalonil 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 for plant disease prevention and control. Pseudomonas aeruginosa, with its potent disease prevention and growth-promoting properties, is an important microbial source for biopesticides. When using biocontrol bacteria to control plant fungal diseases, effectively enhancing their ability to produce extracellular antimicrobial substances is key to improving their effectiveness. Different strains of the same genus differ in their utilization of carbon and nitrogen sources, as well as in fermentation conditions. These differences are closely related to the strain's genetic and physiological characteristics, as well as its isolation source. For different strains, differences in culture medium composition and fermentation conditions can significantly influence the synthesis and accumulation of antimicrobial 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 optimal medium of 10 g / L glucose, 20 g / L yeast powder, and 10 g / L KCl, and the antibacterial effect of the fermentation liquid increased 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 optimal 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 size 1% (v / v), and liquid volume 140 mL / 250 mL; under these conditions, the fermentation liquid had no effect on the banana wilt pathogen Fusarium oxysporum The antibacterial rate of f.sp.cubense was 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, a biocontrol bacterium isolated by the inventors' research team, exhibits potent antagonistic effects against a variety of pathogens. This strain is currently deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection under the deposit number CGMCC No. 20694. It is also covered by a patent entitled "A strain of Pseudomonas aeruginosa 8-7 and its use," with the grant number CN112280714B. To further increase the content of antibacterial substances in microbial preparations derived from Pseudomonas aeruginosa 8-7, it is imperative to optimize the fermentation method for producing these substances.

[0006] Therefore, providing a preparation method of Pseudomonas aeruginosa 8-7 sterile fermentation filtrate and its application is a problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a preparation method of a 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 solutions:

[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 FA fermentation medium, the medium bottling volume was 50 mL / 500 mL, the shake 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: 15 g / L of acid hydrolyzed casein ②, 25 ml / L of glycerol, 1 g / L of MgSO4·7H2O, and 1.5 g / L of K2HPO4, with a natural pH.

[0012] Furthermore, the preparation method of the fermentation seed liquid is as follows: Pseudomonas aeruginosa 8-7 is activated in LB solid medium, a single colony is picked and inoculated into a triangular flask containing LB liquid 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 sterile fermentation filtrate of Pseudomonas aeruginosa 8-7.

[0014] Furthermore, the use of the Pseudomonas aeruginosa 8-7 sterile fermentation filtrate 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, and Phytophthora raphis. 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 for 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 these 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 against Colletotrichum alienum is improved by the high-yield extracellular antibacterial substance shake flask fermentation process. The present invention provides technical support and theoretical guidance for the industrial production of Pseudomonas aeruginosa 8-7 strains and the acquisition of high-yield antibacterial substances.

[0016] It can be seen from the above technical solution that compared with the prior art, the present invention discloses a preparation method and application of a sterile fermentation filtrate of Pseudomonas aeruginosa 8-7. The sterile 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; 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 sterile fermentation broth of strain 8-7 is treated with 11 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

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

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

[0021] Figure 4 The accompanying figure 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 figure shows the effect of different liquid volumes on the antibacterial activity of the aseptic fermentation filtrate of strain 8-7 of the present invention. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts 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 oxysporum f.sp.cubense, Phytophthora capsici, Rhizopus stolonifer), Alternaria solani, Botrytis cinerea, Alternaria alternate, Alternaria alternate, and Diaporthe passifloricola are all maintained by the Plant Protection and Microbial Utilization Research Center of the Yunnan Institute of Tropical Crops. Analytical-grade reagents such as glucose, yeast extract, and tryptone were obtained from Sangon Biotechnology Co., Ltd.; acid-hydrolyzed casein ① (C8221) was obtained from Beijing Solebold Technology Co., Ltd.; acid-hydrolyzed casein ② (C822594) was obtained from Shanghai MacLean Biochemical Technology Co., Ltd.; a constant-temperature incubator was obtained from Minnesota Mining and Manufacturing Company; and a constant-temperature shaking incubator was obtained from Shanghai Boxun Industrial Co., Ltd.

[0025] Example 1

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

[0027] This example involves 8 liquid culture media: LB, PDA, soybean powder, pigment-promoting medium, beef extract peptone medium, sucrose medium, TB medium, and FB medium. 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: 65 g soybean powder, 16 g corn steep liquor, 12 g glucose, 22 mL anhydrous ethanol, and distilled water to 1000 mL.

[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, dilute to 1000mL with distilled water.

[0033] Sucrose culture 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 1 g, K2HPO4 2.5 g, acid hydrolyzed casein ① 5 g, glycerol 8 mL, distilled water to 1000 mL, 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 100 mL LB liquid medium and cultured at 36°C and 180 rpm for 12 h (OD 600 =1.0), and obtain fermentation seed liquid.

[0037] Screening of culture media producing antibacterial substances: 100 mL of the fermentation culture medium producing antibacterial substances of the above-mentioned 8 strains 8-7 to be screened was respectively placed in a 500 mL Erlenmeyer flask, and the cultured seed liquid was inoculated into 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 minutes, and 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 the 8 screening culture media.

[0038] Antibacterial activity was determined using the hyphal growth rate method, using Colletotrichum heteromorphum as the target pathogen. After activation on PDA plates for 4 days, a bacterial cake was prepared using a 5 mm borer and inoculated three times onto the center of a PDA plate (6 cm diameter) coated with 100 μL of sterile fermentation filtrate. The plates were incubated in a 28°C incubator for 4 days and then removed. The diameters of the colonies from each treatment were measured using the cross-hatch method, and the hyphal growth inhibition rate was calculated as follows: Inhibition rate (%) = [(control pathogen colony diameter - treatment pathogen colony diameter) / (control pathogen colony diameter - bacterial cake diameter)] × 100. A PDA plate not coated with sterile fermentation filtrate served as a control.

[0039] Screening of culture media producing antibacterial substances: Strain 8-7 was cultured in eight different liquid media: LB, PDA, soybean powder, pigment-promoting medium, beef extract peptone medium, sucrose medium, TB, and FB. 100 μL of the resulting sterile fermentation filtrate was tested for its inhibitory effect on the growth of Colletotrichum heteromorpha mycelium. The inhibition rates of the various treatment groups were 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 seven screening media. 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 base fermentation medium. 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 at a 1% (mass-volume) ratio as carbon sources (replacing 8 mL of glycerol). Further optimization experiments were conducted. Using the mycelial growth rate method, the antibacterial activity of 100 μL of sterile fermentation filtrate (prepared as above) from each treatment group was determined using the same procedure as above, with three replicates per treatment. The results are shown in Table 2.

[0048] Table 2 Effects of different carbon source media on the antibacterial activity of the 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 sterile fermentation filtrates of strain 8-7, 9 of the 17 carbon sources had a certain inhibitory effect on Colletotrichum heteromorphum. Among them, the fermentation filtrate of the strain using glycerol as the carbon source had the highest antibacterial rate, which was significantly higher than that of other carbon sources. The sterile fermentation filtrates using soybean oil, corn oil, peanut oil, rapeseed oil, olive oil, and palm oil as carbon sources also had an antibacterial rate. The sterile fermentation filtrates of the strain using mineral oil, xylose, sucrose, galactose, soluble starch, lactose, glucose, and maltose as carbon sources had no antibacterial activity; the sterile fermentation filtrate using fructose as the carbon source had an extremely low antibacterial rate; and the fermentation filtrate using mannitol as the carbon source had an antibacterial rate comparable to that of glycerol. For cost considerations, glycerol was selected as the optimal carbon source for the liquid fermentation medium.

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

[0053] Using FB as the base fermentation medium and glycerol as the optimal carbon source (added at 1%), further optimization experiments were conducted using acid-hydrolyzed casein ① (Solaibo C8221), acid-hydrolyzed casein ② (McLean C822594), casein hydrolyzate, beef extract, tryptone, and yeast extract as nitrogen sources (nitrogen source added at a 2% ratio, replacing 5g of acid-hydrolyzed casein ①). The antibacterial activity of 100 μL of sterile fermentation filtrate (prepared as above) from each treatment group was determined using the hyphal growth rate method with Colletotrichum spp. as the target pathogen (operation method as above). Three replicates were performed for each treatment. 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 the six nitrogen sources all had inhibitory effects on Colletotrichum heteromorphum. Among them, the fermentation broth using acid-hydrolyzed casein ② and casein hydrolyzate as nitrogen sources had the highest inhibition rate, which was significantly higher than that of 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] Based on the experimental results of carbon and nitrogen sources, four factors (acid hydrolyzed casein②, K₂HPO₄, MgSO₄·7H₂O, and glycerol) were selected for a four-factor, four-level orthogonal experiment (Table 4). The antibacterial activity of 100 μL of sterile fermentation filtrate (prepared as above) from each treatment group was determined using the mycelial growth rate method with Colletotrichum heteromorphum as the target pathogen (operation method as above), with three replicates per treatment.

[0059] Table 4 Factor and level design of orthogonal experiment

[0060]

[0061] The results of the orthogonal experiment 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. The optimal combination of levels is A3B3C1D2, i.e., combination number 11. In the analysis of variance of the 16 inhibitory effects, combination number 11 had the highest inhibition rate against Colletotrichum heteromorphum. The optimized FB fermentation medium was determined to be the FA fermentation medium. The FA fermentation medium composition was: acid hydrolyzed casein ② 15 g / L, glycerol 25 ml / L, MgSO₄·7H₂O 1 g / L, K₂HPO₄ 1.5 g / L, and a natural pH.

[0066] 4) Screening of the optimal inoculum size

[0067] 100 mL of the FA fermentation medium obtained by the above screening was placed in a 500 mL triangular flask, and 5%, 10%, 15%, 20%, and 25% of the cultured fermentation seed liquid were respectively inoculated into the triangular flask; after being cultured in a 36°C constant temperature shaking incubator at 180 r / min for 4 days, the fermentation liquid was centrifuged at 8000 r / min for 4 minutes, and 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 antibacterial activity of each treatment group was determined using the tested Colletotrichum heteromorphum as the pathogenic target bacteria, and each treatment was repeated 3 times. The results are shown in FIG. Figure 1 When the inoculation amount was 10%, the antibacterial activity of the sterile fermentation filtrate reached its peak, 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] 100 mL of the FA fermentation medium obtained by the above screening was placed in a 500 mL triangular flask, and 10 mL of the cultured fermentation seed liquid was added to the triangular flask; the shaking incubator temperature was set to 30, 33, 36, and 39 ° C respectively; after culturing in a constant temperature shaking incubator at 180 r / min for 4 days, 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 antibacterial activity of each treatment group was determined using the tested Colletotrichum heteromorphum as the pathogenic target bacteria, and each treatment was repeated 3 times. The results are shown in FIG. Figure 2 The antibacterial activity of the sterile fermentation filtrate reached its peak when the culture temperature was 36°C, and the antibacterial rate was 72.24%; therefore, the optimal fermentation temperature of strain 8-7 was selected as 36°C 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 triangular flask, and 10 mL of the cultured fermentation seed liquid was added to the triangular 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 culturing in an oscillating incubator for 4 days, 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 antibacterial activity of each treatment group was determined using the tested anthracnose fungus as the pathogenic target bacteria, and each treatment was repeated 3 times. The results are shown in FIG. Figure 3 When the culture speed was 220 r / 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 at 220 r / 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 triangular flask, and 10 mL of the cultured fermentation seed liquid was added to the triangular 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 to determine the optimal culture time. 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 heteromorphic 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 FIG. 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 accessed in a triangular flask with an inoculation amount of 10%. The fermentation temperature was 36℃, and the shaker speed was 220r / min. The shaking incubator was cultured 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 antibacterial activity of each treatment group was determined with Colletotrichum heteromorphum as the pathogenic target bacteria, and each treatment was repeated 3 times. The results are shown in Figure 5 When the volume of the sterile fermentation filtrate was 50mL / 500mL, the antibacterial rate reached a peak of 89.12%. Then, with the increase of the volume, the antibacterial activity decreased significantly, indicating that the volume of the liquid had a great influence on the accumulation of antibacterial substances in strain 8-7. Therefore, the volume of 50mL / 500mL was selected for subsequent experiments.

[0076] Based on the optimal fermentation medium formula determined through orthogonal experiments, the present invention optimized fermentation conditions. Taking the antibacterial rate as the sole indicator, the optimal fermentation conditions for strain 8-7 were determined: a medium filling volume of 50 mL / 500 mL, a shake flask culture speed of 220 r / min, an inoculum size of 10%, a culture temperature of 36°C, and a culture time of 5 days, at which the antibacterial activity was optimal.

[0077] Example 3 Inhibitory Effects of Sterile 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. oxysporumf.sp.cubense); 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 used as pathogens. PDA plates were inoculated with the pathogens and sterile fermentation filtrate (100 μL, prepared under optimal fermentation conditions using FA fermentation medium) at the same time. The treatment inoculated with the pathogen alone was used as the control. The test was repeated three times and cultured in a constant temperature chamber at 28°C for 5 days. The diameters of the inhibition zones were measured, the inhibition conditions were observed, and photos were taken. The broad-spectrum antibacterial activity of the antagonistic bacteria was evaluated. The results are shown in Table 6.

[0079] Table 6 Inhibitory effects of sterile fermentation broth of strain 8-7 on 19 plant pathogenic fungi

[0080]

[0081]

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

[0083] The results in Table 6 show that the sterile fermentation filtrate of strain 8-7 has inhibitory effects on 19 test pathogenic fungi, among which the best inhibitory effect is on melon-derived Phytophthora melonis and litchi-derived Phytophthora downyon, with the inhibition rates of the two Phytophthora fungi being 93.87±1.69 and 94.56±1.38 mm, respectively; followed by citrus black spot pathogen and anthracnose pathogen, with inhibition rates of 88.93±0.54 and 89.81±0.44 mm, respectively. The inhibition rate on eight plant pathogens, including Corynespora leaf fall pathogen, Fusarium solani, Macrospora pentaphyllosporium, 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 sterile 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 antibacterial substances

[0087] Sterile fermentation filtrates were placed under natural light for five treatments: 1, 2, 3, 4, and 5 days. Activity was measured every other day. The sterile fermentation filtrates were irradiated under a 30W UV lamp at a distance of 15 cm for 30, 60, 90, and 120 minutes. Untreated sterile fermentation filtrate served as the control (CK). Three replicates were used for each treatment, and the average value was calculated. A 5mm C. heteromorpha cake was inoculated onto a PDA plate containing 100 μL of sterile fermentation filtrate from each experimental group. The plates were incubated inverted at 28°C for 4 days. The inhibition zone was observed and its diameter was measured. 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 maintained high antibacterial activity after exposure to UV light for varying durations, with an inhibition rate of 82.82% even after 120 minutes. Exposure to natural light for varying durations revealed minimal effects on antimicrobial substances within 5 days, indicating that exposure to natural light does not reduce the activity or alter the properties of the sterile fermentation filtrate.

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

[0092] The sterile fermentation filtrate was placed in a water bath at 80°C for 1 hour, a water bath at 100°C for 1 hour, and a high-temperature (121°C) high-pressure treatment for 30 minutes. After cooling to room temperature, 100 μL of the sterile fermentation filtrate was spread on a PDA solid plate. A 5 mm C. anthracis cake was selected and inoculated on the PDA plate containing the sterile fermentation filtrate of each experimental group. The plate was incubated at 28°C for 4 days. The changes in the inhibition zone were observed and the diameter of the inhibition zone was measured. The sterile fermentation filtrate not treated in the water bath was used as the control (CK). Three replicates for each treatment were repeated and the average value was calculated. 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℃ boiling water treatment for 1h and 121℃ high pressure sterilization for 30min, 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 antibacterial substances

[0097] Sterile fermentation filtrates were prepared with 0.1 mol / L HCl and 0.1 mol / L NaOH to final pH values of 1, 3, 4, 5, 7, 9, 11, and 12, respectively. After standing for 30 minutes, the pH was adjusted to the untreated pH value (7.2). 100 μL of the treated sterile fermentation filtrate was spread on a PDA solid plate. A 5 mm C. anthracis cake was inoculated onto the PDA medium containing the pH-treated sterile fermentation filtrate. The plate was incubated inverted at 28°C for 4 days. The inhibition zone was observed and its diameter was measured. The untreated sterile fermentation filtrate was used as the control (CK). Three replicates for each treatment were used and the average value was calculated. 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), changes in pH have little effect on the activity of the antimicrobial substances. When the pH is 12, the sterile fermentation filtrate still has 81.49% antibacterial activity, indicating that the antimicrobial substances are relatively stable.

[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 After 30 minutes of filtration with a mol / L Na2SO3 solution, 100 μL of the treated sterile fermentation filtrate was spread onto a PDA solid plate. A 5 mm Colletotrichum spp. cake was then inoculated onto the treated sterile fermentation broth PDA medium. The culture was inverted and incubated at 28°C for 4 days. The inhibition zone was observed and the diameter of the inhibition zone for each treatment was measured. The colony diameter of each control pathogen was measured using a Na2SO3 solution of the corresponding concentration as a control. The mycelial growth inhibition rate was calculated as follows: Inhibition rate (%) = [(control pathogen colony diameter - treatment pathogen colony diameter) / (control pathogen colony diameter - cake diameter)] × 100. Three replicates were performed for each treatment, and the average value was calculated. 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) refers to the treatment 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 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 antibacterial 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 solution. Eleven EP tubes were prepared and 10 μL of the freshly prepared salt solution was added to each tube. Subsequently, 190 μL of the sterile fermentation filtrate was added to each of the eleven test tubes to a final metal ion concentration of 0.005 mol / L. After 5 h of stagnation, 100 μL of the treated sterile fermentation filtrate was spread onto a PDA plate. A 5 mm cake of Colletotrichum heteromorphum was inoculated onto the treated PDA plate. The plate was incubated inverted at 28°C for 4 days. The inhibition zone was observed and the diameter of the inhibition zone for each treatment was measured. The colony diameter of each control pathogen was measured using the corresponding salt solution as a control. The mycelial growth inhibition rate was calculated as: Inhibition rate (%) = [(control pathogen colony diameter - treatment pathogen colony diameter) / (control pathogen colony diameter - cake diameter)] × 100. Three replicates were performed for each treatment, and the average value was calculated. 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 effect 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 components of sterile fermentation broth of strain 8-7

[0115] After activation, a single colony of 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 this as the seed solution, inoculated into FA fermentation medium at 10% inoculum, and cultured at 220 r / min and 36 ° C for 120 h (OD 600=2.00), with each treatment repeated three times. Samples were collected and centrifuged at 8000 rpm for 10 min. The supernatant was sterilized by filtration through a 0.22 μm microporous filter to obtain a sterile fermentation broth. 10 mL of the sterile fermentation broth was stored in a -80°C freezer until further use. The sterile fermentation broth was subjected to non-targeted metabolomics analysis, using FA fermentation medium without inoculation of strain 8-7 as a control. Each treatment was repeated three times. T3 chromatographic conditions: Column: Waters ACQUITY UPLC HSS T3 Column 1.8 μm, 2.1 mm × 100 mm; Mobile phase A: Ultrapure water containing 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% formic acid; Column temperature: 40°C; Flow rate: 0.4 mL / min; Injection volume: 4 μL. The mobile phase gradient is shown in Table 12. Mass spectrometry conditions: electrospray ionization (ESI); scanning mode: positive and negative ion mode, capillary voltage +5.5 kV; capillary temperature: 550°C; resolution: 7500; collision energy: -30 to 30 eV; scan range: m / 25 to 1250; nebulizer pressure: 50 psi. Data processing: Raw data were converted to mzXML format using ProteoWizard, and peak extraction, alignment, and retention time correction were performed using the XCMS program. Peaks with a missing rate greater than 50% in the samples were filtered, blank values were filled using KNN, and peak areas were corrected using the SVR method. After correction and screening, metabolite identification was performed using the Myvi Metabolism Database, public libraries, predicted libraries, and metDNA methods.

[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. Using FA fermentation medium as the control and 120 h as the experimental group, 2990 differential metabolites were obtained from the sterile fermentation broth after calibration and screening, including 2086 upregulated substances and 904 downregulated substances. Based on the antibacterial and anti-inflammatory substances reported in the reference literature, the upregulated metabolites were screened and a total of 45 target metabolites with antibacterial activity were obtained (Table 13); these included 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 is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily 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 is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of the sterile fermentation filtrate of Pseudomonas aeruginosa 8-7 in the preparation of an agent for inhibiting plant pathogens, characterized in that: The plant pathogen is Peronophythora litchii, Phytophthora melonis Katsura, Phytophthora vignae Purss or Diaporthe passifloricola; The preparation method of the Pseudomonas aeruginosa 8-7 sterile fermentation filtrate is as follows: The fermentation seed liquid of Pseudomonas aeruginosa 8-7 was inoculated into FA fermentation medium, the medium bottling volume was 50 mL / 500 mL, the shake 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: 15 g / L of C822594 acid hydrolyzed casein, 25 ml / L of glycerol, 1 g / L of MgSO4·7H2O, and 1.5 g / L of K2HPO4, with a natural pH.

2. The use 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 medium, a single colony is picked and inoculated into a triangular flask containing LB liquid medium, and cultured at 36°C and 180 rpm to an OD of 600 =1.0, and obtain fermentation seed liquid.

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