A biocontrol bacillus siamese and its metabolites and uses

Abstract

The present invention belongs to the field of microbial secondary metabolites, and specifically relates to a biocontrol Bacillus siamensis and its metabolites and uses. The biocontrol Bacillus siamensis is classified and named as: Bacillus siamensis N‑1, and is preserved in the Guangdong Provincial Microbial Culture Collection Center with a preservation number of: GDMCC 64974 and a preservation date of: August 12, 2024. The preparation method of its metabolites includes strain activation, seed liquid preparation and fermentation. The antimicrobial peptides of the metabolites of the biocontrol Bacillus siamensis have a significant inhibitory effect on common tropical fruit pathogens, and can maintain a higher storage quality and a longer shelf life of post-harvest fruits by reducing the incidence of mango anthrax and wax apple soft rot. In addition, the antimicrobial peptide is a natural fermentation product of microorganisms, which has the characteristics of safety, a wide antibacterial spectrum, and a simple preparation method, which is convenient for industrial production and can achieve large-scale production and application.

Description

Technical Field

[0001] The invention belongs to the field of microbial secondary metabolites, and specifically relates to a biocontrol bacillus siamese and its metabolites and uses. Background Art

[0002] Postharvest plant diseases are a major limiting factor that seriously reduces the economic value of postharvest horticultural products. Excessive fungal spores accumulate in the fruit epidermis, causing irreversible damage to human health. At present, as food safety has become a hot topic of discussion, problems such as low degradation rate, high residues, and environmental pollution of chemical pesticides and chemical fungicides have brought challenges to food safety that cannot be ignored. In recent years, with the country's attention to food safety, natural and safe biological preservatives have increasingly entered the public eye. Microbial secondary metabolites have become a research hotspot due to their sustainable development, eco-friendliness, and good antibacterial and preservation characteristics.

[0003] At present, biocontrol microorganisms mainly include two categories: biocontrol molds and biocontrol bacteria. As another major pillar of biocontrol microorganisms, biocontrol molds occupy an important component in the prevention and control of post-harvest diseases of fruits and vegetables. However, mold culture takes a long time, spores are easily dispersed in the environment, and fermentation liquid is easily contaminated by bacteria, which brings many difficulties in the preparation of antibacterial fermentation liquid and the prevention and control of fruit and vegetable diseases. Biocontrol bacteria have been widely used in the prevention of post-harvest fruit and vegetable diseases due to their fast growth rate, high fermentation yield, and wide control spectrum. Therefore, it is necessary to develop new biocontrol bacteria for the prevention and control of post-harvest plant diseases and insect pests. Summary of the invention

[0004] The purpose of the present invention is to overcome the disadvantages of the prior art and provide a biocontrol bacillus siamese and its metabolites and uses.

[0005] The purpose of the present invention is achieved by the following technical scheme: A biocontrol Bacillus siamensis, classified and named: Bacillus siamensis N-1, preserved in Guangdong Microbiological Culture Collection Center, with a preservation number of: GDMCC 64974, and a preservation date of: August 12, 2024.

[0006] A method for preparing a biocontrol siam bacillus metabolite, comprising the following steps:

[0007] S1. Activation of strains: inoculate the Bacillus siamensis N-1 of claim 1 into LB solid medium and culture in an incubator at 28 to 32°C for 10 to 14 hours;

[0008] S2. Preparation of seed solution: The activated bacteria were inoculated into LB liquid medium and cultured on a shaking table for 22 to 26 hours to obtain seed solution, wherein the culture temperature was 36 to 40°C and the shaking speed was 110 to 130 rpm / min;

[0009] S3. Fermentation: The seed liquid obtained in step S2 is inoculated into a fermentation medium for fermentation. The fermentation temperature is 30 to 34°C, the fermentation speed is 160 to 200 rpm / min, and the fermentation time is 68 to 74 hours. The fermentation liquid is post-treated to obtain antimicrobial peptides.

[0010] Furthermore, the fermentation medium includes the following components: 5 g / L glucose, 10 g / L yeast extract powder, and 10 g / L sodium chloride.

[0011] Furthermore, in step S3, the fermentation temperature is 32° C., the fermentation speed is 180 rpm / min, the fermentation time is 72 h, the inoculation amount of the seed liquid is 3%, and the carrier liquid volume is 150 mL / 500 mL per bottle.

[0012] Furthermore, the specific operation of the post-treatment is: centrifuging the fermentation broth, discarding the precipitate, adding 10wt% hydrochloric acid solution to the supernatant, leaving it at 4°C for 10 to 14 hours, further centrifuging and discarding the supernatant, dissolving the obtained precipitate in methanol solution, adjusting the pH value to 7 with sodium hydroxide solution, and then magnetically stirring for 4.5 to 5.5 hours, centrifuging and discarding the precipitate, and concentrating the supernatant by rotary evaporation and freeze-drying to obtain the antimicrobial peptide, wherein the centrifugal speed is 4500 to 5500 rpm / min, and the centrifugal time is 8 to 12 minutes.

[0013] The biocontrol Bacillus siamense metabolites prepared by the above method are used in preventing and controlling wax apple soft rot and mango anthracnose.

[0014] Furthermore, the metabolites of the biocontrol Bacillus siamea have an inhibitory effect on litchi peronophyte, wax apple microspore pseudodisc multihistosporium and mango anthracnose.

[0015] Furthermore, the optimal concentration of the metabolite antimicrobial peptide was 3.125 mg / mL.

[0016] The prepared antimicrobial peptide was added to PDB medium and diluted to a concentration of 3.125 mg / mL. It can destroy the hyphae structure of P. microspore and Peronophythora litchi, causing a large accumulation of reactive oxygen (ROS) and cell apoptosis, thereby effectively inhibiting the growth and reproduction of Pestalotiopsis microspore and Peronophythora litchi.

[0017] The prepared antimicrobial peptide was added to PDA solid culture medium and diluted to a concentration of 3.125 mg / mL, which can effectively inhibit the growth and reproduction rate of mango anthracnose (C.gloeosporioides) and wax apple microspore pseudodisc multi-hairy spore (P.microspore). It was verified by in vitro puncture experiments that the spread rate of mango anthracnose and wax apple soft rot was significantly reduced at this concentration.

[0018] The prepared antimicrobial peptide was added to sterile water and diluted to a concentration of 3.125 mg / mL. The harvested mangoes were cleaned and immersed in the antimicrobial peptide solution. The incidence of anthracnose in mango fruits was significantly reduced, and the softening rate of the fruit was significantly reduced. The activity of pectinase and cellulose in the fruit was better maintained, thereby extending the shelf life of post-harvest mangoes.

[0019] The present invention has the following advantages: the reason for the occurrence of post-harvest tropical fruit diseases is that fungal spores lurk in the fruit epidermis and grow and reproduce through the stomata on the surface of the fruit peel, thereby inducing fruit diseases and reducing their commercial value and shelf life. The present invention provides a biocontrol Siamese Bacillus, which is classified and named as: Bacillus siamensis N-1, preserved in the Guangdong Provincial Microbial Culture Collection Center, with a preservation number of: GDMCC 64974, and a preservation date of: August 12, 2024. The antimicrobial peptide metabolite of the biocontrol Siamese Bacillus has a significant inhibitory effect on common tropical fruit pathogens, and can maintain a higher storage quality and a longer shelf life of post-harvest fruits by reducing the incidence of mango anthrax and wax apple soft rot. In addition, the antimicrobial peptide is a natural fermentation product of microorganisms, which has the characteristics of being natural, safe, and having a wide antibacterial spectrum, and the preparation method is simple, which is easy to apply to industrial production, and can achieve large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Figure 4 shows the colony morphology of Bacillus siamensis N-1 and the antibacterial effect of the fermentation liquid of Bacillus siamensis N-1 on 7 different tropical fruit pathogens, including: Figure a1: Thielaviopsis paradoxa, Figure a2: Lasiodiplodia theobromae, Figure a3: Phomopsis caricae-papayae Petrak, Figure a4: Colletotrichum gloeosporioides, Figure a5: Pestalotiopsis microspore, Figure a6: Peronophythora litchi, Figure a7: Fusarium oxysporum, Figure a8: Single colony of Bacillus Siamese (Bacillus Siamese, N-1).

[0021] Figure 2 These are staining diagrams showing the effects of the antimicrobial peptides of the present invention on the hyphae of Peronophythora litchi, wherein Figures a1 and a2: optical microscopy; Figures a3 and a4: scanning electron microscopy; Figures a5 and a6: reactive oxygen (ROS) content in hyphae.

[0022] Figure 3 These are staining diagrams of the effect of the antimicrobial peptide of the present invention on the mycelium of P. microspore, wherein Figures a1 and a2: optical microscope; Figures a3 and a4: scanning electron microscope; Figures a5 and a6: reactive oxygen (ROS) content of mycelium.

[0023] Figure 4 This is a diagram showing the effect of the antimicrobial peptide of the present invention on the extracellular substances of the hyphae of P. microspore. In the diagram, Figure a1: UV nucleic acid, Figure a2: UV protein, Figure a3: total protein, Figure a4: malondialdehyde.

[0024] Figure 5The figures are the effects of the antimicrobial peptides of the present invention on the growth and reproduction of wax apple microspore P. microspore and the control of fruit diseases. In the figures, Figures a1 and a4 are the growth conditions of wax apple microspore P. microspore at 5 days (a1: without antimicrobial peptide, a4: antimicrobial peptide treatment group), Figures a2 and a3 are the incidence of soft rot of wax apples in the control group after 6 days of storage, and Figures a5 and a6 are the incidence of soft rot of wax apples in the lipopeptide treatment group after 6 days of storage.

[0025] Figure 6 These are effect diagrams of the bacterial peptide of the present invention on the prevention and treatment of anthracnose disease in mango fruit. In the diagrams, Figures a1 and a2: the incidence of anthracnose disease in mangoes in the control group at 27 days, and Figures a3 and a4: the incidence of anthracnose disease in mangoes in the lipopeptide-treated group at 27 days. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and examples, and the protection scope of the present invention is not limited to the following: Example 1: A method for preparing a biocontrol siam bacillus metabolite, which comprises the following steps:

[0027] S1. Activation of strains: inoculate Bacillus siamensis N-1 into LB solid medium and culture in an incubator at 28°C for 10 hours; wherein, the Bacillus siamensis N-1 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit number: GDMCC 64974, and the deposit date: August 12, 2024;

[0028] S2. Preparation of seed solution: The activated bacteria were inoculated into LB liquid medium and cultured on a shaking table for 22 h to obtain seed solution. The culture temperature was 36 ° C and the shaking speed was 110 rpm / min;

[0029] S3. Fermentation: The seed liquid obtained in step S2 is inoculated into a fermentation medium for fermentation, wherein the fermentation medium comprises the following components: 5 g / L glucose, 10 g / L yeast extract powder, and 10 g / L sodium chloride. The fermentation temperature is 30°C, the fermentation speed is 160 rpm / min, the fermentation time is 68 h, the inoculation amount of the seed liquid is 3%, the carrier liquid volume is 150 mL / 500 mL per bottle, the fermentation liquid is centrifuged at 4500 rpm / min for 8 min, the precipitate is discarded, a 10 wt % hydrochloric acid solution is added to the supernatant, the mixture is placed at 4°C for 10 h, and the supernatant is further centrifuged at 4500 rpm / min for 8 min to discard the supernatant, the obtained precipitate is dissolved in a methanol solution, the pH value is adjusted to 7 with a 1 mol / L sodium hydroxide solution, and then the mixture is magnetically stirred for 4.5 h, centrifuged at 4500 rpm / min for 8 min to discard the precipitate, and the supernatant is concentrated by rotary evaporation and freeze-dried to obtain an antimicrobial peptide.

[0030] Embodiment 2: A method for preparing a biocontrol Bacillus siamese metabolite, comprising the following steps:

[0031] S1. Activation of strains: inoculate Bacillus siamensis N-1 into LB solid medium and culture in an incubator at 32°C for 14 hours; wherein the Bacillus siamensis N-1 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit number: GDMCC 64974, and the deposit date: August 12, 2024;

[0032] S2. Preparation of seed solution: The activated bacteria were inoculated into LB liquid medium and cultured on a shaking table for 26 h to obtain seed solution. The culture temperature was 40 ° C and the shaking speed was 130 rpm / min;

[0033] S3. Fermentation: The seed liquid obtained in step S2 is inoculated into a fermentation medium for fermentation, wherein the fermentation medium comprises the following components: 5 g / L glucose, 10 g / L yeast extract powder, and 10 g / L sodium chloride. The fermentation temperature is 34°C, the fermentation speed is 200 rpm / min, the fermentation time is 74 h, the inoculation amount of the seed liquid is 3%, the carrier liquid volume is 150 mL / 500 mL per bottle, the fermentation liquid is centrifuged at 5500 rpm / min for 12 min, the precipitate is discarded, a 10 wt % hydrochloric acid solution is added to the supernatant, the mixture is placed at 4°C for 14 h, and the supernatant is further centrifuged at 5500 rpm / min for 12 min to discard the supernatant, the obtained precipitate is dissolved in a methanol solution, the pH value is adjusted to 7 with a 1 mol / L sodium hydroxide solution, and then the mixture is magnetically stirred for 5.5 h, centrifuged at 5500 rpm / min for 12 min to discard the precipitate, and the supernatant is concentrated by rotary evaporation and freeze-dried to obtain an antimicrobial peptide.

[0034] Embodiment 3: A method for preparing a biocontrol Bacillus siamese metabolite, comprising the following steps:

[0035] S1. Activation of strains: inoculate Bacillus siamensis N-1 into LB solid medium and culture in an incubator at 30°C for 12 hours; wherein, the Bacillus siamensis N-1 is deposited in Guangdong Microbiological Culture Collection Center, with the deposit number: GDMCC 64974, and the deposit date: August 12, 2024;

[0036] S2. Preparation of seed solution: The activated bacteria were inoculated into LB liquid medium and cultured on a shaking table for 24 h to obtain seed solution. The culture temperature was 38 ° C and the shaking speed was 120 rpm / min;

[0037] S3. Fermentation: The seed liquid obtained in step S2 is inoculated into a fermentation medium for fermentation, wherein the fermentation medium comprises the following components: 5 g / L glucose, 10 g / L yeast extract powder, and 10 g / L sodium chloride. The fermentation temperature is 32°C, the fermentation speed is 180 rpm / min, the fermentation time is 72 h, the inoculation amount of the seed liquid is 3%, the carrier liquid volume is 150 mL / 500 mL per bottle, the fermentation liquid is centrifuged at 5000 rpm / min for 10 min, the precipitate is discarded, a 10 wt% hydrochloric acid solution is added to the supernatant, the mixture is placed at 4°C for 12 h, and the supernatant is further centrifuged at 5000 rpm / min for 10 min to discard the supernatant, the obtained precipitate is dissolved in a methanol solution, the pH value is adjusted to 7 with a 1 mol / L sodium hydroxide solution, and then the mixture is magnetically stirred for 5 h, centrifuged at 5000 rpm / min for 10 min to discard the precipitate, and the supernatant is concentrated by rotary evaporation and freeze-dried to obtain an antimicrobial peptide.

[0038] Example 4: Isolation and identification of Bacillus siamensis N-1

[0039] The soil from the root system of fruit trees in the wax apple orchard in Qionghai, Hainan Province was collected and placed in a fresh-keeping bag and immediately brought back to the laboratory of the School of Life Sciences and Health of Hainan University for the isolation of antagonistic bacteria. 10g of wax apple orchard soil sample was added to 90mL of sterile water and mixed on a shaker at 28℃ and 150rpm / min for about 2h. The mixed sample solution was diluted in a gradient manner with a concentration range of 10 -3 , 10 -4 , 10 -5 , 10 -6 , take 100 μL of each dilution sample and spread it on NA medium and culture it for 48 hours, pick colonies of different morphologies and separate them by streaking, and obtain single colonies, which will be stored in the refrigerator for later use. The purified antagonistic strains were preliminarily screened by plate confrontation, and the strains with the best antagonistic effect were saved. 16S rDNA sequencing was performed to determine the strains.

[0040] Bacillus siamensis N-1 strain is a biocontrol bacterium isolated from the soil of wax apple orchard in 2019. The bacterium is a Gram-positive bacterium with white wrinkled colonies. It was identified as Bacillus siamensis N-1 based on physiological characteristics and molecular identification. It is classified and named as Bacillus siamensis N-1 and preserved in Guangdong Microbial Culture Collection Center with the preservation number of GDMCC 64974 and the preservation date of August 12, 2024. The preservation address is: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0041] The physiological and biochemical characteristics and molecular identification results of Bacillus siamensis provided in this experiment are as follows:

[0042] The single colony of N-1 is white with white wrinkled edges. After streaking on a plate, it is milky white with a raised center and uneven wrinkled edges. N-1 can grow under sterile conditions, has the ability to hydrolyze starch, tolerate salt, and utilize carbon and nitrogen sources such as glucose. The catalase and VP assays are positive, which is consistent with the characteristics of Bacillus. After amplification and sequencing, the sequence is as follows:

[0043]

[0044] The results showed that the genome of N-1 was highly similar to that of Bacillus siamensis (GenBank: KU597567.1), and the homology similarity of 16S rDNA comparison was 100%. Therefore, it was further confirmed to be Bacillus siamensis.

[0045] Example 5: Effects of different fermentation medium components and fermentation conditions on the content of antimicrobial peptides

[0046] Comparative Example 1:

[0047] The N-1 seed liquid was inoculated into LB medium (5 g of yeast extract, 10 g of tryptone and 10 g of sodium chloride, 2%, 200 mL / bottle), placed in a 28°C incubator, and cultured on a shaking table at 120 rpm / min for 24 hours. The optimal carbon source, nitrogen source and inorganic salt were screened respectively based on the antibacterial rate of the culture medium against T. paradoxa, L. theobromae, Phomopsis caricae-papayae Petrak, C. gloeosporioides, P. microspore, Peronophythora litchi and F. oxysporum, and the content of antibacterial active peptides in the fermentation broth was extracted and determined.

[0048] Comparative Example 2:

[0049] The different components of the culture medium - carbon source: glucose / yeast powder / soluble starch were used as carbon sources from different sources, 5 g each, the nitrogen source was tryptone, and the inorganic salt was sodium chloride. The carbon source of Bacillus siamese was screened, and the inhibition rate of the carbon source against T.paradoxa, L.theobromae, Phomopsis caricae-papayae Petrak, C.gloeosporioides, P.microspore, Peronophythora litchi, and F.oxysporum was used as the standard.

[0050] Comparative Example 3:

[0051] The different components of the culture medium - nitrogen source: yeast extract powder / beef extract / tryptone as nitrogen sources from different sources, 10g each, the carbon source is glucose, and the inorganic salt is sodium chloride. The nitrogen source of Bacillus siamese was screened, and the antibacterial rate of the nitrogen source against T.paradoxa, L.theobromae, Phomopsis caricae-papayae Petrak, C.gloeosporioides, P.microspore, Peronophythora litchi, and F.oxysporum was used as the standard to screen the optimal nitrogen source, and the content of antibacterial active peptides in the fermentation broth was extracted and determined.

[0052] Comparative Example 4:

[0053] For different components of the culture medium - inorganic salts: sodium chloride / potassium chloride / magnesium chloride are used as inorganic salts from different sources, 10g each, the carbon source is glucose, and the nitrogen source is yeast extract powder. The inorganic salts of Bacillus siamea are screened, and the antibacterial rates of the inorganic salts against T.paradoxa, L.theobromae, Phomopsis caricae-papayae Petrak, C.gloeosporioides, P.microspore, Peronophythora litchi and F.oxysporum are used as standards to screen the optimal inorganic salts, and the content of antibacterial active peptides in the fermentation broth is extracted and determined.

[0054] Comparative Example 5:

[0055] Optimize the fermentation conditions - carrier volume: Taking 500mL triangular flasks as an example, each bottle was filled with 100mL, 150mL, and 200mL of optimized LB culture medium (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride) of different contents, and the fermentation conditions of Bacillus siamense - carrier volume were screened. The antibacterial rates of T.paradoxa, L.theobromae, Phomopsis caricae-papayaePetrak, C.gloeosporioides, P.microspore, Peronophythora litchi, and F.oxysporum were used as standards to screen the optimal carrier volume, and the antibacterial active peptide content of the fermentation broth was extracted and determined.

[0056] Comparative Example 6:

[0057] Optimize the fermentation conditions - inoculation amount: Taking 500mL triangular flasks as an example, each bottle is filled with 150mL of optimized LB medium (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride), and different amounts of seed liquid (2%, 3%, and 4%) are inoculated to screen the fermentation conditions of Bacillus siamense - inoculation amount. The antibacterial rate of T.paradoxa, L.theobromae, Phomopsiscaricae-papayae Petrak, C.gloeosporioides, P.microspore, Peronophythora litchi, and F.oxysporum is used as the standard, and the antibacterial active peptide content of the fermentation liquid is extracted and determined, and the optimal inoculation amount is screened respectively.

[0058] Comparative Example 7:

[0059] Optimize the fermentation conditions - speed: take 500mL triangular flask as an example, each bottle is filled with 150mL of optimized LB medium (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride), and inoculated with 3% seed liquid, and different shaking table speeds are used as screening conditions 120rpm / min, 150rpm / min, 180rpm / min to screen the fermentation conditions of Bacillus siamense - shaking table speed, and the antibacterial rate of pineapple paradoxa (T.paradoxa), longan cocoa hairy two spores (L.theobromae), papaya brown stem rot fungus (Phomopsis caricae-papayae Petrak), mango anthracnose (C.gloeosporioides), wax apple microspore pseudo-disk multi-hairy spore (P.microspore), litchi downy mildew (Peronophythora litchi), dragon fruit Fusarium (F.oxysporum) is used as the standard, the antibacterial active peptide content of the fermentation liquid is extracted and measured, and the optimal shaking table speed is screened respectively.

[0060] Comparative Example 8:

[0061] Optimize the fermentation conditions-fermentation temperature: take 500mL triangular flask as an example, each bottle is filled with 150mL of optimized LB medium (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride), and inoculated with 3% seed liquid, the shaking table speed is 150rpm / min, and different fermentation temperatures (27°C, 32°C, 38°C) are used as the screening basis to screen the fermentation conditions-fermentation temperature of siam Bacillus, and the antibacterial rate of pineapple paradoxa (T.paradoxa), longan cocoa hairy two spores (L.theobromae), papaya brown stem rot fungus (Phomopsis caricae-papayae Petrak), mango anthracnose (C.gloeosporioides), wax apple microspore pseudo-disk multi-hairy spore (P.microspore), litchi downy mildew (Peronophythora litchi), dragon fruit Fusarium (F.oxysporum) is used as the standard, the antibacterial active peptide content of the fermentation liquid is extracted and measured, and the optimal fermentation temperature is screened respectively.

[0062] Comparative Example 9:

[0063] Fermentation conditions are optimized-fermentation time: taking 500mL triangular flask as an example, each bottle is filled with 150mL of optimized LB medium (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride), and 3% seed liquid is inoculated, the shaking table speed is 150rpm / min, the fermentation temperature is 32°C, and different fermentation times (24h, 48h, 72h) are used as the screening basis to screen the fermentation conditions-fermentation temperature of siam Bacillus, and the antibacterial rate of pineapple miraculous root tremella (T.paradoxa), longan cocoa hairy two spore bacteria (L.theobromae), papaya brown stem rot fungus (Phomopsis caricae-papayae Petrak), mango anthracnose (C.gloeosporioides), wax apple microspore spore (P.microspore), litchi downy mildew (Peronophythora litchi), dragon fruit Fusarium (F.oxysporum) is used as the standard, and the antibacterial active peptide content of the fermentation liquid is extracted and determined.

[0064] The experiments and calculations of the antibacterial rate involved in the above experiments are as follows:

[0065] A cross was drawn in the center of a PDA plate about 2.0 mm thick, and six different tropical fruit pathogen cakes with a diameter of 0.8 mm were placed in the center. 10 μL of differently treated Siamese Bacillus broth and LB liquid culture medium were inoculated on filter paper pieces 2.5 cm away from the center. The mycelial radius of the pathogen in the group treated with LB liquid culture medium and the mycelial radius of the pathogen in the groups treated with different Siamese Bacillus fermentation broths were measured respectively, and the antibacterial rates of different groups were calculated according to the following formula. The antibacterial rates of Siamese Bacillus fermentation broths in different treatment groups on different tropical fruit pathogens were measured respectively, and the antibacterial rates of different treatment groups were compared. The antibacterial rate of the treatment group with the highest antibacterial rate was selected as the standard, and the active peptides of the fermentation broth of this group were extracted.

[0066] Bacterial inhibition rate (%) = (mycelium of LB liquid medium treatment group - mycelium of fermentation liquid treatment group) / mycelium of LB liquid medium treatment group

[0067] The experimental results are shown in Tables 1 and 2. It can be seen from Tables 1 and 2 that the optimal fermentation conditions are determined to be carbon source (5 g glucose), nitrogen source (10 g yeast extract powder), inorganic salt (10 g sodium chloride) and carrier liquid volume (150 mL), inoculation amount (3%), rotation speed (180 rpm / min), fermentation temperature (32°C), and fermentation time (72 h).

[0068] Table 1 Effects of different culture medium components and fermentation conditions on the content of antimicrobial peptides

[0069]

[0070] Table 2 Effect of different fermentation conditions on the content of antimicrobial peptides

[0071]

[0072]

[0073] Implementation group: The fermentation medium composition and fermentation conditions were: carbon source (glucose 5g), nitrogen source (yeast extract powder 10g), inorganic salt (sodium chloride 10g) and carrier volume (150mL), inoculation amount (3%), rotation speed (180rpm / min), fermentation temperature (32°C), fermentation time (72h), and the antibacterial rate of pineapple paradoxa, longan cocoa hair color two spores (L.theobromae), papaya brown stem rot fungus (Phomopsis caricae-papayae Petrak), mango anthracnose (C.gloeosporioides), wax apple microspore pseudo-disk multi-hairy spore (P.microspore), litchi downy mildew (Peronophythora litchi), dragon fruit Fusarium (F.oxysporum) was used as the standard to extract the antibacterial active peptides from the fermentation liquid. The experimental results are shown in Table 3 and Figure 1 As shown, through optimization, 1 g of active peptide can be extracted from each liter of N-1 fermentation broth, and the fermentation supernatant has a good inhibitory effect on 7 different tropical fruit pathogens.

[0074] Table 3 Antimicrobial peptide content and its inhibition rate against different pathogens

[0075]

[0076] Example 6: Experiment on the effect of antimicrobial active peptides on litchi peronophyte

[0077] The fermentation liquid obtained by the above-mentioned optimal conditioned medium composition (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride) and culture conditions (fermentation time: 72h, fermentation temperature: 32°C, shaker speed: 150rpm / min, inoculation amount: 3%, carrier volume: 150mL) was centrifuged at 5000rpm / min for 10min, and the precipitate was discarded. The supernatant was added with 10% hydrochloric acid solution and precipitated in a refrigerator at 4°C overnight. The precipitate was further centrifuged at 5000rpm / min and the supernatant was discarded. The precipitate was dissolved in methanol solution, the pH was adjusted to 7.0 with 1mol / L NaOH solution, and then placed on a magnetic stirrer for stirring for 5h, centrifuged and the precipitate was discarded, and finally rotary evaporation was concentrated for freeze-drying.

[0078] Implementation group: The antimicrobial peptide obtained above was diluted with PDB liquid medium to a concentration of 3.125 mg / mL. Preparation of pathogenic bacteria: After culturing Peronophythora litchi for 5 days, a sufficient amount of bacterial cake was taken with a 1 mL pipette tip (8 mm) and placed in sterile water, and diluted to (10 6 CFU / mL), take 2mL of spore liquid and add it to PDB medium (100mL / bottle), and culture it in a shaker at 28℃ and 120rpm / min for 72d. Filter the mycelium with gauze, discard the culture liquid, add the mycelium to PDB medium containing 3.125mg / mL antimicrobial peptide, and culture it in a shaker for 24h. Collect the mycelium and observe the mycelium structure under optical microscope and scanning electron microscope. Add DCFH2-DA, stain it in the dark at 28℃ for 30min, wash it with PBS buffer and observe it under fluorescence microscope. The ROS excitation wavelength is 428nm and the emission wavelength is 535nm.

[0079] Comparative Example 10:

[0080] PDB liquid medium was used to culture Peronophythora litchi. The culture was shaken at 120 rpm / min and 28°C for 24 h. The hyphal structure, reactive oxygen (ROS) content and cell apoptosis of Peronophythora litchi were determined.

[0081] Experimental results: Figure 2 As shown, 3.125 mg / mL of antimicrobial peptide can significantly destroy the hyphae structure of Litchi Downy Phytophthora, causing mycelial apoptosis, and the mycelium exhibits strong green fluorescence.

[0082] Example 7: Experimental study on the effect of antimicrobial peptides on Pseudomonas syringae

[0083] The fermentation liquid obtained by the above-mentioned optimal conditioned medium composition (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride) and culture conditions (fermentation time: 72h, fermentation temperature: 32°C, shaker speed: 150rpm / min, inoculation amount: 3%, carrier volume: 150mL) was centrifuged at 5000rpm / min for 10min, and the precipitate was discarded. The supernatant was added with 10% hydrochloric acid solution and precipitated in a refrigerator at 4°C overnight. The precipitate was further centrifuged at 5000rpm / min and the supernatant was discarded. The precipitate was dissolved in methanol solution, the pH was adjusted to 7.0 with 1mol / L NaOH solution, and then placed on a magnetic stirrer for stirring for 5h, centrifuged and the precipitate was discarded, and finally rotary evaporation was concentrated for freeze-drying.

[0084] Implementation group: The antimicrobial peptide obtained above was diluted with PDB liquid medium to a concentration of 3.125 mg / mL. Preparation of pathogenic bacteria: After culturing P. microspore for 5 days, a sufficient amount of bacterial cake was taken with a 1 mL pipette tip (8 mm) and placed in sterile water, and diluted to (10 6 CFU / mL), take 2mL of spore liquid and add it to PDB medium (100mL / bottle), and culture it in a shaker at 28℃ and 120rpm / min for 72d. Filter the mycelium with gauze, discard the culture liquid, add the mycelium to PDB medium containing 3.125mg / mL antimicrobial peptide, and culture it in a shaker for 24h. Collect the mycelium and observe the mycelium structure under optical microscope and scanning electron microscope. Add DCFH2-DA, stain it in the dark at 28℃ for 30min, wash it with PBS buffer and observe it under fluorescence microscope. The ROS excitation wavelength is 428nm and the emission wavelength is 535nm.

[0085] Comparative Example 11:

[0086] P. microspore was cultured in PDB liquid medium at 120 rpm / min and 28°C for 24 h, and the hyphal structure, reactive oxygen (ROS) content and cell apoptosis of P. microspore were determined.

[0087] Experimental results: Figure 3 As shown, 3.125 mg / mL of antimicrobial peptide can significantly destroy the hyphae structure of the wax apple pathogen Pseudomonas microsporus, causing mycelial apoptosis and the mycelium showing green fluorescence.

[0088] Example 8: Experiment on the effect of antimicrobial active peptides on extracellular substances of Pseudomonas syringae

[0089] Implementation group: The fermentation liquid obtained by the above-mentioned optimal conditioned medium composition (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride) and culture conditions (fermentation time: 72h, fermentation temperature: 32°C, shaker speed: 150rpm / min, inoculation amount: 3%, carrier volume: 150mL) was centrifuged at 5000rpm / min for 10min, and the precipitate was discarded. The supernatant was added with 10% hydrochloric acid solution and precipitated in a refrigerator at 4°C overnight. The precipitate was further centrifuged at 5000rpm / min and the supernatant was discarded. The precipitate was dissolved in methanol solution, the pH was adjusted to 7.0 with 1mol / L NaOH solution, and then placed on a magnetic stirrer for stirring for 5h, centrifuged and the precipitate was discarded, and finally concentrated by rotary evaporation for freeze-drying. The antimicrobial active peptide obtained above was diluted with PDB liquid culture medium to a concentration of 3.125 mg / mL, and P. microspore was cultured, and cultured in a shaking table at 120 rpm / min and 28°C for 24 hours, and the extracellular substance content of mycelium of P. microspore was measured at 0h, 6h, 12h, 18h, and 24h. The measured extracellular substance refers to the supernatant taken for measurement after the liquid mycelium of P. microspore was centrifuged.

[0090] Comparative Example 12:

[0091] PDB liquid culture medium was used to culture P. microspore. The culture was shaken at 120 rpm / min and 28°C for 24 hours to determine the effects of P. microspore on mycelial oxidase and energy levels.

[0092] Experimental results: Figure 4 As shown in the data, 3.125 mg / mL of antimicrobial peptides can significantly destroy the hyphae structure of the wax apple pathogen Pseudomonas microsporus, causing damage to its cell membrane, extravasation of intracellular substances such as ultraviolet nucleic acids and proteins, a decrease in total protein content, and a large accumulation of oxidation product malondialdehyde, leading to cell apoptosis.

[0093] Example 9: Antibacterial test of antimicrobial active peptides on freshly picked wax apple fruits

[0094] Implementation group: The fermentation broth obtained by the above-mentioned optimal conditioned medium composition (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride) and culture conditions (fermentation time: 72h, fermentation temperature: 32°C, shaker speed: 150rpm / min, inoculation amount: 3%, carrier volume: 150mL) was centrifuged at 5000rpm / min for 10min, and the precipitate was discarded. The supernatant was added with 10% hydrochloric acid solution and precipitated in a refrigerator at 4°C overnight. The precipitate was further centrifuged at 5000rpm / min and the supernatant was discarded. The precipitate was dissolved in methanol solution, adjusted to pH 7.0 with 1mol / L NaOH solution, and then placed on a magnetic stirrer for 5h, centrifuged and discarded, and finally concentrated by rotary evaporation for freeze-drying. The antimicrobial active peptide obtained above was diluted with sterile water as the medium to a concentration of 3.125mg / mL. Freshly picked wax apple fruits were rinsed with tap water, soaked in sodium hypochlorite solution, and inoculated with 20 μL spores of P. microspore on the surface of the fruits (10 6 CFU / mL) and 20 μL of antimicrobial peptide solution were used to determine the disease status of wax apple fruits after 6 days.

[0095] Comparative Example 13:

[0096] The fruit surface was inoculated with 20 μL of spores of P. microspore (10 6 CFU / mL) and 20 μL sterile water were used to measure the disease of wax apple fruits after 6 days. The freshly picked wax apples were cleaned with tap water, soaked in sterile water for 10 min, taken out and dried, and the storage of the fruits was observed.

[0097] Experimental results: Figure 5 As shown, 3.125 mg / mL of antimicrobial peptides can significantly delay the incidence of diseases in fresh wax apple fruits after harvest and maintain their good storage quality after harvest.

[0098] Example 10: Experiment on the inhibition of diseases of freshly picked mango fruits by antimicrobial active peptides

[0099] Implementation group: The fermentation liquid obtained by the above-mentioned optimal conditioned medium composition (carbon source is glucose, nitrogen source is yeast extract powder, and inorganic salt is sodium chloride) and culture conditions (fermentation time: 72h, fermentation temperature: 32℃, shaker speed: 150rpm / min, inoculation amount: 3%, carrier volume: 150mL) was centrifuged at 5000rpm / min for 10min, and the precipitate was discarded. The supernatant was added with 10% hydrochloric acid solution and precipitated in a refrigerator at 4℃ overnight. The precipitate was further centrifuged at 5000rpm / min to discard the supernatant, dissolved in methanol solution, adjusted to pH 7.0 with 1mol / L NaOH solution, and then placed on a magnetic stirrer for 5h, centrifuged and discarded, and finally concentrated by rotary evaporation for freeze-drying. The antimicrobial active peptide obtained above was diluted with sterile water to a concentration of 3.125mg / mL. After soaking the freshly picked mango in sodium hypochlorite solution, wash it with sterile water and dry the fruit. The fruit surface was inoculated with 20 μL of spores of C. gloeosporioides (10 6 CFU / mL) and 20 μL antimicrobial peptide solution were used to measure the disease status of mango fruits after 27 days. Freshly picked mangoes were washed with tap water, soaked in antimicrobial peptide solution for 10 min, taken out and dried, and the storage status of the fruits was observed.

[0100] Comparative Example 14:

[0101] The fruit surface was inoculated with 20 μL of spores of C. gloeosporioides (10 6 CFU / mL) and 20 μL sterile water, and the disease status of mango fruits was determined after 27 days. Freshly picked mangoes were washed with tap water, soaked in sterile water for 10 min, fished out and dried, and the storage status of the fruits was observed.

[0102] Experimental results: Figure 6 As shown, 3.125 mg / mL of antimicrobial peptides can significantly delay the spread rate of anthracnose in fresh mangoes after harvest and reduce the incidence of anthracnose on the fruit skin.

[0103] Experimental results analysis:

[0104] 1. Effects of different culture medium components and fermentation conditions on the content of antimicrobial peptides

[0105] When the culture medium components were: carbon source (glucose), nitrogen source (yeast extract powder), inorganic salt (sodium chloride), fermentation time (72h), fermentation temperature (32℃), rotation speed (180rpm / min), and carrier liquid volume (150mL / 500mL per bottle), the maximum antimicrobial peptide extraction amount was 1g / L. The fermentation supernatant had the highest inhibition rate against pathogens of 6 different tropical fruits, which was significantly different compared with the control group (Table 1, Table 2 and Table 3). In addition, the optimal fermentation liquid can significantly inhibit the growth and reproduction of seven different tropical fruit pathogens, including Pestalotiopsis microspore, Fusarium oxysporum, Peronophythora litchi, Fusarium oxysporum, Lasiodiplodia theobromae, Phomopsis caricae-papayae Petrak, and Thielaviopsis paradoxa. Figure 1 ).

[0106] 2. Inhibition rate of antimicrobial peptides against Peronophythora litchi

[0107] After treatment with 3.125 mg / mL of antimicrobial peptide, the hyphae of Peronophythora litchi appeared vesicular, the hyphae became significantly thicker, and the surface was broken and not smooth. After staining with ROS dye, the hyphae showed obvious green fluorescence and obvious apoptosis. The hyphae of the control group were not damaged and no green fluorescence appeared after fluorescence staining. Figure 2 .

[0108] 3. Inhibition rate of antimicrobial peptides against P. microspore

[0109] After treatment with 3.125 mg / mL of antimicrobial peptide, the hyphae of P. microspore in wax apple were shorter than those in the control group, and contained bubble-like structures in the middle of the hyphae. The hyphae in the control group were longer, smoother, and had a complete structure. After staining with ROS dye, the hyphae showed bright green fluorescence, and obvious apoptosis occurred in the hyphae, while no green fluorescence appeared in the control group. After treatment with antimicrobial peptide, the cell wall structure of the hyphae of Microspore was broken, and the contents such as ultraviolet nucleic acids and proteins were extravasated, and the oxidizing substance malondialdehyde accumulated in large quantities, leading to cell apoptosis. In addition, the results of live stabbing showed that the antimicrobial peptide can inhibit the spread rate of wax apple soft rot ( Figure 3-5 ).

[0110] 4. The effect of antimicrobial peptides on the prevention and treatment of mango anthracnose

[0111] After treatment with 3.125 mg / mL of antimicrobial peptides, the diameter of the anthracnose lesions on the surface of mango fruit was 2.2 cm, which was significantly smaller than that of the control group (4.0 cm), with a significant difference (P<0.01). In addition, when freshly picked mangoes were soaked in the diluted antimicrobial peptide solution, no obvious lesions appeared on the mango fruit skin, and the skin remained obviously green. Compared with the control group, antimicrobial peptide treatment can better maintain the storage quality of mango fruit, and the storage period of mango fruit at room temperature can be extended to 27 days ( Figure 6 ).

[0112] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A biocontrol bacillus siamese, characterized in that: The classification name is: Bacillus siamensis N-1, deposited in Guangdong Provincial Microbiological Culture Collection Center, the deposit number is: GDMCC 64974, and the deposit date is: August 12, 2024.

2. A method for preparing a biocontrol Bacillus siamensis metabolite, characterized in that: It includes the following steps: S1. Activation of strains: inoculate the Bacillus siamensis N-1 of claim 1 into LB solid medium and culture in an incubator at 28 to 32°C for 10 to 14 hours; S2. Preparation of seed solution: The activated bacteria were inoculated into LB liquid medium and cultured on a shaking table for 22 to 26 hours to obtain seed solution, wherein the culture temperature was 36 to 40°C and the shaking speed was 110 to 130 rpm / min; S3. Fermentation: The seed liquid obtained in step S2 is inoculated into a fermentation medium for fermentation. The fermentation temperature is 30 to 34°C, the fermentation speed is 160 to 200 rpm / min, and the fermentation time is 68 to 74 hours. The fermentation liquid is post-treated to obtain antimicrobial peptides.

3. The method for preparing a biocontrol siam bacillus metabolite according to claim 2, characterized in that: The fermentation medium includes the following components: 5 g / L glucose, 10 g / L yeast extract powder, and 10 g / L sodium chloride.

4. The method for preparing a biocontrol Bacillus siamensis metabolite according to claim 2, characterized in that: In step S3, the fermentation temperature is 32° C., the fermentation speed is 180 rpm / min, the fermentation time is 72 h, the inoculation amount of the seed liquid is 3%, and the carrier liquid volume is 150 mL / 500 mL per bottle.

5. The method for preparing a biocontrol Bacillus siamensis metabolite according to claim 2, characterized in that: The specific operation of the post-treatment is: centrifuging the fermentation liquid, discarding the precipitate, adding a 10wt% hydrochloric acid solution to the supernatant, placing it at 4°C for 10 to 14 hours, further centrifuging and discarding the supernatant, dissolving the obtained precipitate in a methanol solution, adjusting the pH value to 7 with a sodium hydroxide solution, and then magnetically stirring for 4.5 to 5.5 hours, centrifuging and discarding the precipitate, and concentrating the supernatant by rotary evaporation and freeze-drying to obtain the antimicrobial peptide, wherein the centrifugal speed is 4500 to 5500 rpm / min, and the centrifugal time is 8 to 12 minutes.

6. Use of the biocontrol Bacillus siamense metabolites prepared according to the method described in any one of claims 2 to 5 in preventing and controlling wax apple soft rot and mango anthracnose.

7. The use according to claim 6, characterized in that: The biocontrol Bacillus siamense metabolites have an inhibitory effect on litchi peronophyte, wax apple microsporum and mango anthracnose.

8. The use according to claim 6, characterized in that: The concentration of the metabolite antimicrobial peptide was 3.125 mg / mL.

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