Bacillus amyloliquefaciens ZH807 and its application

Abstract

The present invention relates to the field of microbial fermentation technology, and specifically discloses a strain of Bacillus amyloliquefaciens ZH807 and its application. The present invention improves the ability of Bacillus amyloliquefaciens to produce active substances during fermentation on the basis of wild-type strains, and solves the problems of fermentation spore production and high viscosity of fermented liquid at the same time, and screens out a strain of Bacillus amyloliquefaciens ZH807 that does not produce spores during liquid fermentation. The strain characteristic is that it does not produce spores during liquid fermentation, and the foam production during fermentation is controllable, and the fermented liquid has low viscosity, which is conducive to the secretion and accumulation of bioactive substances, and is convenient for the subsequent extraction process of the fermented liquid. In addition, the strain is subjected to liquid fermentation to produce alkaline protease, and the alkaline protease activity reaches 14769U / mL, which is 22 times higher than the starting strain, and the enzyme activity has a significant improvement effect.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, in particular to a strain of Bacillus amyloliquefaciens ZH807 and an application thereof, in particular to a strain of Bacillus amyloliquefaciens ZH807 that does not produce spores and an application thereof in liquid fermentation. Background Art

[0002] Bacillus amyloliquefaciens is a type of aerobic, spore-forming Gram-positive bacteria that can produce a variety of bioactive substances including enzymes, nucleosides, amino acids and antibacterial substances. Therefore, it is widely used in agriculture, industry, food and medicine. It is a generally recognized safe microorganism (GRAS) by the U.S. Food and Drug Administration (FDA).

[0003] Early studies reported that the ability of Bacillus amyloliquefaciens isolated from commercial α-amylase concentrate to produce α-amylase is 50-150 times that of Bacillus subtilis, proving that Bacillus amyloliquefaciens has a stronger protein secretion ability than Bacillus subtilis and is a highly efficient potential protein expression host. However, in the fermentation process of using Bacillus amyloliquefaciens as a production strain to secrete bioactive substances, there are problems of spore production and increased concentration of fermentation broth, which directly affects the yield of the target product and increases the difficulty of separation and purification.

[0004] Studies have shown that the formation of biofilm by Bacillus, which causes the fermentation liquid to become sticky, is closely related to the spore formation phenomenon, because both processes depend on the DNA binding protein spo0A. Phosphorylated spo0A can directly activate or inhibit the transcription of target genes. Whether the cell begins to form spores is determined by internal and external environmental conditions. Whether the internal and external environment is suitable for the formation of spores is reflected in the content and phosphorylation level of spo0A. Only when the content and phosphorylation level of spo0A reach a certain threshold, spores begin to form, and the external factors that affect spore formation are mainly cell density and nutritional status.

[0005] In the late stage of Bacillus fermentation, due to the consumption of nutrients such as carbon source and nitrogen source, changes in the cell growth environment and other factors, the genes regulating spore formation lose repression and are activated, thereby forming a large number of spores. The large-scale generation of spores reduces the level of metabolic flow to produce bioactive substances, and increases the cost of removing spores in the post-treatment process of the fermentation broth. In addition, during the growth of Bacillus, a type of substance secreted from the cell wall forms a capsule structure of the bacteria and dissolves in the fermentation broth. When the gas and solid content and the gas and solid particles of the fermentation broth are dispersed to a certain critical value, a mutation of the continuous mobile phase and the gas and solid dispersion phase occurs, which is manifested in the non-Newtonian change of the fermentation broth and the increase in viscosity. The increase in viscosity will first affect the ventilation supply, increase the stirring resistance, and affect the growth of the bacteria; it will also increase the surface tension, affect the foam breakage, and then produce uncontrollable foam during the fermentation process, resulting in fermentation failure or a decrease in the expression of the target protein. Generally, spore formation and reduction of fermentation broth viscosity are prevented by optimizing the culture medium or controlling the fermentation, but such methods have poor repeatability and versatility and need to be optimized separately according to different strains and fermentation conditions.

[0006] In order to improve the problems of spore production and stickiness of fermentation liquid caused by Bacillus amyloliquefaciens fermentation, early research directions were mainly focused on traditional mutagenesis breeding and fermentation optimization. The frequency of forward mutation induced by this technology was low, and the screening throughput of mutant strains was not high. Genetic engineering breeding methods were gradually developed to transform the genome of Bacillus amyloliquefaciens, but due to the limitations of Bacillus amyloliquefaciens' own modification system, the transformation of Bacillus amyloliquefaciens strains was very difficult, resulting in very limited reports on it as an expression host bacterium. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a strain of Bacillus amyloliquefaciens ZH807 and its application. The present invention screens out a non-spore-forming Bacillus amyloliquefaciens ZH807 based on the wild-type strain. The strain has the characteristics of not producing spores during liquid fermentation, controllable foam production during fermentation, low viscosity of fermentation liquid, which is conducive to the secretion and accumulation of bioactive substances and convenient for the subsequent extraction process of the fermentation liquid.

[0008] To achieve the above purpose, the inventors have finally obtained the following technical solution through a large number of experiments and unremitting exploration:

[0009] The first purpose of the present invention is to provide a strain of Bacillus amyloliquefaciens ZH807, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 14, 2022, with the deposit number GDMCC NO: 62790, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The colony characteristics of Bacillus amyloliquefaciens ZH807 of the present invention are as follows:

[0011] The screened Bacillus amyloliquefaciens ZH807 was cultured in LB medium for 24-48 hours. The colonies were slightly white and opaque, with ridges on the surface and irregular edges. They were Gram-positive and arranged in long rods in a row.

[0012] The Bacillus amyloliquefaciens ZH807 of the present invention is obtained by using Bacillus amyloliquefaciens Y9 as the initial strain and undergoing mutation breeding. The mutant strain is obtained by mutation using a known mutagenesis method (ARTP mutagenesis).

[0013] Specifically, the mutagenesis breeding method of the Bacillus amyloliquefaciens ZH807 strain of the present invention is as follows:

[0014] (1) Mutagenesis: Bacillus amyloliquefaciens Y9 was used as the starting strain and cultured in LB liquid medium. The bacterial liquid was taken in the logarithmic growth phase and induced by a known mutagenesis method (preferably ARTP mutagenesis), and the mutagenesis time was 30-180s (preferably 120s). A single colony induced by ARTP was selected, and the mutant colonies were screened using LB medium plates and DNB medium plates. The mutant strains that grew normally on the LB medium plates but did not grow colonies on the DNB medium plates were selected as the primary screening strains.

[0015] (2) Primary screening: From the primary screening strains, select the Bacillus amyloliquefaciens colonies with large single colonies and good growth, then inoculate the screened strains onto LB solid culture medium plates, culture at 37°C for 72 hours, select the bacteria for spore staining and preparation, and select the strains without spores by microscopic observation as the secondary screening strains;

[0016] (3) Rescreening: After activation, Bacillus amyloliquefaciens Y9 and the re-screened strains were inoculated into the fermentation medium respectively, cultured at 37°C and 220rpm for 72h, centrifuged at 12000g for 10min to remove the bacteria, collected the fermentation broth supernatant, and measured the viscosity of the fermentation broth. Finally, a non-spore-forming Bacillus amyloliquefaciens ZH807 with low fermentation broth viscosity was screened out. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 14, 2022, with the deposit number GDMCC NO: 62790, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0017] The Bacillus amyloliquefaciens ZH807 screened by the present invention is a variant strain obtained by mutagenizing the starting strain using the ARTP mutagenesis method with Bacillus amyloliquefaciens Y9 as the starting bacterium, and the variant strain is screened using a DNB / LB culture medium. A mutagen strain that hardly grows on a DNB culture medium plate after heat treatment and grows normally on an LB culture medium plate is selected as a primary screening strain. The primary screening strain is inoculated into an LB liquid culture medium, and a re-screening strain that does not produce spores in the late culture period is screened out using a spore staining method, and finally the re-screened Bacillus amyloliquefaciens is inoculated into a fermentation culture medium for culture, and the fermentation liquid viscosity is measured, and finally a strain of Bacillus amyloliquefaciens ZH807 that does not produce spores and has a low fermentation liquid concentration is screened out.

[0018] The present invention utilizes an ARTP mutagenesis method to screen and obtain a non-spore-producing Bacillus amyloliquefaciens ZH807. The method has a high positive mutation rate and a large throughput for screening mutants. The strain characteristics are that almost no spores are produced during liquid fermentation, foam production during fermentation is controllable, and the fermentation liquid has low viscosity, which is beneficial to the secretion and accumulation of bioactive substances and convenient for subsequent extraction of the fermentation liquid.

[0019] In some embodiments, the formula of the DNB medium in step (1) above is: 3 g beef extract, 5 g peptone, 15 g agar, pH 6.6-7.0.

[0020] In some embodiments, the formula of the fermentation medium in step (3) above is: 20 g sucrose, 20 g yeast powder, 20 g trypsin, and 1 g magnesium sulfate.

[0021] In some embodiments, the passage stability test of Bacillus amyloliquefaciens ZH807:

[0022] Bacillus amyloliquefaciens ZH807 was inoculated into LB solid medium and cultured at 37°C for 24 hours. Then, it was transferred to LB solid medium and cultured at 37°C for 24 hours. The different generations of strains were obtained by analogy. The subcultured strain was inoculated into DNB solid medium and cultured for 72 hours to determine whether spores were formed. The selected mutagenic strain was continuously streaked and subcultured on LB solid medium until the 10th generation.

[0023] The results showed that the non-spore-forming characteristics of each generation of strains inoculated in DNB solid medium remained stable, indicating that Bacillus amyloliquefaciens ZH807 has good generation stability and is suitable for industrial application.

[0024] The second purpose of the present invention is to provide the use of the above-mentioned Bacillus amyloliquefaciens ZH807 in the preparation of fermentation products.

[0025] The screened Bacillus amyloliquefaciens ZH807 simultaneously solves the problems of fermentation spore production and high viscosity of the fermentation liquid, thereby improving the ability of Bacillus amyloliquefaciens to produce active substances during the fermentation process.

[0026] As a preferred embodiment of the application of the present invention, the fermentation product includes a fermentation product that does not contain spores.

[0027] As a preferred embodiment of the application of the present invention, the application of Bacillus amyloliquefaciens ZH807 to increase the content of alkaline protease during fermentation.

[0028] The application of Bacillus amyloliquefaciens ZH807 in fermentation can produce a higher content of alkaline protease, controllable foam production, significantly improve enzyme activity, and low viscosity of the fermentation liquid during the fermentation process.

[0029] The third purpose of the present invention is to provide a use of the above-mentioned Bacillus amyloliquefaciens ZH807 in reducing the viscosity of the fermentation liquid during the fermentation process.

[0030] The fourth purpose of the present invention is to provide a method for increasing the content of alkaline protease in a fermentation process, comprising adding the above-mentioned Bacillus amyloliquefaciens ZH807 for fermentation.

[0031] Bacillus amyloliquefaciens ZH807 was fermented in liquid form to produce alkaline protease. During the fermentation process, the viscosity of the fermentation liquid was low, and the alkaline protease activity in the fermentation tank reached 14769U / mL, which was 22 times higher than that of the starting strain.

[0032] The fifth purpose of the present invention is to provide a fermentation broth, wherein the fermentation broth contains the above-mentioned Bacillus amyloliquefaciens ZH807.

[0033] The sixth object of the present invention is to provide a method for preparing the fermentation broth, comprising the following steps:

[0034] (1) Seed solution preparation: Bacillus amyloliquefaciens ZH807 was inoculated into a seed culture medium and cultured at 30° C. to 40° C. for 12 to 16 hours to obtain a seed solution;

[0035] (2) Preparation of fermentation broth: The seed liquid of step (1) is inoculated into a liquid fermentation medium at an inoculation amount of 6%, a culture temperature of 30° C. to 40° C., and a fermentation time of 12 to 16 hours to obtain the fermentation broth.

[0036] The sixth purpose of the present invention is to provide an alkaline protease prepared by using the above-mentioned Bacillus amyloliquefaciens ZH807.

[0037] The Bacillus amyloliquefaciens ZH807 of the present invention can produce a relatively high content of alkaline protease during the fermentation process. The alkaline protease activity of the purified Bacillus amyloliquefaciens ZH807 is 28710 U / mL, and the total recovery rate is 64%.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention is to improve the ability of Bacillus amyloliquefaciens to produce active substances during fermentation on the basis of wild-type strains, and to solve the problem of fermentation spore production and high viscosity of fermented liquid at the same time, and screen out a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZH807 that does not produce spores, and the strain characteristic is that it does not produce spores during liquid fermentation, and the foam production during fermentation is controllable, and the fermented liquid viscosity is low, which is conducive to the secretion accumulation of bioactive substances, and facilitates the subsequent extraction process of the fermented liquid. In addition, the strain is subjected to liquid fermentation to produce alkaline protease, and the alkaline protease activity reaches 14769U / mL, which is 22 times higher than the starting strain, and the enzyme activity has a significant improvement effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The results of lethality at different mutagenesis times are shown;

[0041] Figure 2 The colony morphology of Bacillus amyloliquefaciens ZH807 is shown;

[0042] Figure 3 This is a microscope picture of spore staining of Bacillus amyloliquefaciens Y9 in the late growth stage on LB solid medium plate;

[0043] Figure 4 This is a microscope picture of spore staining of Bacillus amyloliquefaciens ZH807 at the late growth stage on LB solid culture medium plate. DETAILED DESCRIPTION

[0044] In order to promote understanding of the present invention, some embodiments will be referred to below, and specific language will be used to describe the present invention. However, it should be understood that these specific embodiments are not intended to limit the scope of the present invention. Any changes and further modifications in the described embodiments, as well as any further applications of the present invention, are generally expected by those skilled in the art.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0047] In the following examples, the percentages are by mass unless otherwise specified.

[0048] Example 1. Mutagenesis of Bacillus amyloliquefaciens

[0049] Mutagenesis of bacteria:

[0050] Bacillus amyloliquefaciens Y9 was used as the starting strain, cultured in LB liquid medium, and the bacterial solution was taken for mutagenesis during the logarithmic growth phase. The strain was cultured in a shake flask until the OD600 value was 0.6, 1 mL of fresh bacterial solution was centrifuged to collect cells, and the cells were washed with physiological saline containing 5% glycerol and resuspended 3 times, and the bacterial concentration absorbance value was adjusted to 0.6-0.8, 10 μL of the resuspended solution was coated on the surface of the metal slide, and the instrument was set to a mutagenesis power of 120 W and a helium flow rate of 10 SL / min on the ARTP mutagenesis breeding operation station, the irradiation spacing was 2 mm, and different treatment times were set to 30 / 60 / 90 / 120 / 150 / 180 s. After the mutagenesis was completed, the slide was removed with tweezers, and then the slide was transferred to a centrifuge tube containing 1 mL of PBS buffer, and vortexed for 2 minutes to wash all the bacteria on the slide. The eluted bacterial solution was appropriately diluted in gradients, and 100 μL was spread on LB solid plate culture medium, cultured at 37°C for 24 h, and counted. The lethality at different mutagenesis times was calculated (as shown in Table 1) to determine the optimal mutagenesis conditions.

[0051] Lethality rate = (number of colonies without mutagenesis treatment - number of colonies with mutagenesis treatment) / number of colonies without mutagenesis treatment × 100%.

[0052] Table 1

[0053]

[0054]

[0055] Results: When the lethality rate was about 90%, the strain had a high positive mutation rate, so the appropriate irradiation treatment time for the strain was set at 120s (e.g. Figure 1 ).

[0056] Example 2: Screening of Bacillus amyloliquefaciens

[0057] Pick a single colony induced by ARTP and transfer it to a 96-well plate containing 50 μL of physiological saline, shake and mix, inoculate it on the LB medium plate with a 96-well plate spotter, heat treat the 96-well plate at 80°C for 15 minutes, and then inoculate it on the DNB medium (3g beef extract, 5g peptone, 15g agar, pH6.6-7.0) plate with a 96-well plate spotter, and count it at 37°C for 24 hours. The number of the colonies that grow normally on the LB medium but not on the DNB medium is used as the primary screening strain.

[0058] Bacillus amyloliquefaciens colonies with large single colonies and good growth were selected, and the streaking purification was repeated twice, and a total of 610 strains were initially screened. The 610 strains were streaked on LB plates, and the morphology of single colonies was observed. The strains with consistent colony morphology, no film production, smooth surface, and drier colonies than the starting strains were selected, and 15 strains were screened.

[0059] The 15 strains were stained and rescreened. The strains were inoculated on LB solid culture medium plates and cultured at 37°C for 72 hours. The bacteria were picked for spore staining and slide preparation. The rescreened strains without spores were screened by microscopic observation, and 5 strains of non-spore-forming Bacillus amyloliquefaciens were obtained, numbered LF03, LS301, LS302, LF78, and LF81.

[0060] Spore quantity detection method: Take 100 μL of the homogenous bacterial solution cultured in a shake flask and dilute it 10 times in series to 10 with sterile saline. -6 The diluted bacterial solution was placed in an 85°C water bath for 30 minutes. After cooling to room temperature, 100uL of the diluted bacterial solution was taken and spread on LB solid medium. Incubate at 37°C for 24 hours. Select the plate for colony count and calculate the number of spores in the original bacterial solution. The spore count of Bacillus amyloliquefaciens Y9 is 4.3×106CFU / mL, and the spore count of Bacillus amyloliquefaciens ZH807 is 0.

[0061] Then, the initial strain Bacillus amyloliquefaciens Y9 and the above-mentioned 5 strains LF03, LS301, LS302, LF78, and LF81 were activated and inoculated into fermentation medium (20 g sucrose, 20 g yeast powder, 20 g tryptone, and 1 g magnesium sulfate) respectively, and cultured at 37°C with constant temperature shaking at 220 rpm for 72 h. The cells were removed by centrifugation at 12000 g for 10 min, and the fermentation supernatant was collected. The viscosity of the fermentation broth was measured by a viscometer, and finally a strain of Bacillus amyloliquefaciens LF03, which did not produce spores and had low fermentation broth viscosity, was selected. The strain was named ZH807 and entrusted to the Guangdong Institute of Microbiology for preservation. The preservation information is: Bacillus amyloliquefaciens ZH807 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 14, 2022, with the preservation number GDMCC NO: 62790, and the preservation address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0062] The colony characteristics of Bacillus amyloliquefaciens ZH807 are as follows: Bacillus amyloliquefaciens ZH807 is cultured in LB medium for 24-48 hours, and the colonies are slightly white and opaque, with ridges on the surface, irregular edges, positive Gram staining, and long rods arranged continuously. Figure 2 The spore staining micrograph of Bacillus amyloliquefaciens Y9 in the late growth stage on LB solid medium plate is shown in the figure. Figure 3As shown, the spore staining micrograph of Bacillus amyloliquefaciens ZH807 in the late growth stage on LB solid medium plate is shown in Figure 4 shown.

[0063] Example 3: Test on the stability of Bacillus amyloliquefaciens ZH807

[0064] Bacillus amyloliquefaciens ZH807 was inoculated into LB solid medium and cultured at 37°C for 24 hours, then transferred to LB solid medium and cultured at 37°C for 24 hours, and so on to obtain strains of different generations. The subcultured strain was inoculated into DNB solid medium and cultured for 72 hours to determine whether spores were formed. The screened mutagenic strain was continuously streaked and subcultured on LB solid medium plates to the 10th generation. The results showed that the non-spore-forming characteristics of each generation of strains inoculated into DNB solid medium remained stable, indicating that Bacillus amyloliquefaciens ZH807 has good subculture stability and is suitable for industrial applications.

[0065] Example 4: A fermentation liquid and a method for preparing the same

[0066] A method for preparing a fermentation broth comprises the following steps:

[0067] Preparation of primary seed solution: Inoculate activated Bacillus amyloliquefaciens ZH807 into LB liquid culture medium and culture at 37° C. for 12-16 hours to obtain primary seed solution.

[0068] Preparation of secondary seed solution: The primary seed solution was inoculated into LB liquid culture medium at an inoculation volume of 6%, and cultured at 37° C. for 12-16 h to obtain the secondary seed solution of Bacillus amyloliquefaciens ZH807.

[0069] Fermentation test of Bacillus amyloliquefaciens ZH807:

[0070] The secondary seed liquid of Bacillus amyloliquefaciens ZH807 was inoculated into a 15L fermenter for batch fermentation. The initial pH was adjusted to 7.0 with ammonia water. The fermentation temperature was controlled at 37°C, the rotation speed was 200-500 rpm, and the ventilation volume was 0.72 m 3 / h, and ferment continuously for 55h before being put into the tank. The viscosity of the fermentation liquid was tested, and the activity of alkaline protease in the fermentation liquid was determined.

[0071] Results: Compared with the initial strain Y9 fermentation test, the fermentation broth OD600 of Bacillus amyloliquefaciens Y9 reached 10.3, the fermentation broth viscosity was 43.64mPas, and the alkaline protease activity was 663U / mL; the fermentation broth OD600 of Bacillus amyloliquefaciens ZH807 reached 45.9, the fermentation broth viscosity was 19.59mPas, and the alkaline protease activity was 14769U / mL. Compared with the initial strain Y9, the fermentation broth viscosity of Bacillus amyloliquefaciens ZH807 decreased by 55%, and the alkaline protease production increased by 22 times. The fermentation broth of the initial strain was too viscous and difficult to post-process, so the alkaline protease activity detected was low. Bacillus amyloliquefaciens ZH807 is suitable for large-scale production of enzyme preparations and other secondary metabolites. The fermentation broth of Bacillus amyloliquefaciens ZH807 has low viscosity, which is conducive to the separation and purification of the fermentation broth to obtain alkaline protease.

[0072] Example 5: Isolation and purification of alkaline protease

[0073] The fermented liquid of the lower tank in Example 4 was centrifuged at 4000rpm for 10min to take the supernatant, and 5% diatomaceous earth was added to stir for 30min to mix. Then the plate-and-frame filter press process was carried out to obtain the clear liquid, and the ultrafiltration concentration column with a molecular weight of 20kDa was used to concentrate, and the waste liquid was discarded, and the concentration multiple was 3-5 times. The alkaline protease after purification was obtained, and the alkaline protease activity was measured by sampling. The starting strain Y9 was too large in fermented liquid viscosity, and centrifugal and plate-and-frame filter press processes were difficult to obtain clarified fermented liquid, and finally the alkaline protease could not be obtained by separation and purification smoothly, and the alkaline protease activity after purification of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZH807 was 28710U / mL, and the total recovery rate was 64%.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens ZH807, characterized in that: The Bacillus amyloliquefaciens ZH807 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 14, 2022, with the collection number GDMCC NO: 62790, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of Bacillus amyloliquefaciens ZH807 as claimed in claim 1 in preparing fermentation products.

3. The use according to claim 2, characterized in that The fermentation product includes a fermentation product that does not contain spores.

4. The use according to claim 2, characterized in that The invention discloses an application of Bacillus amyloliquefaciens ZH807 in increasing the content of alkaline protease during fermentation.

5. Use of Bacillus amyloliquefaciens ZH807 as claimed in claim 1 in reducing the viscosity of fermentation liquid during fermentation.

6. A method for increasing the content of alkaline protease in a fermentation process, characterized in that: The method comprises adding the Bacillus amyloliquefaciens ZH807 described in claim 1 for fermentation.

7. A fermentation broth, characterized in that: The fermentation broth contains the Bacillus amyloliquefaciens ZH807 according to claim 1.

8. The method for preparing a fermentation liquid according to claim 7, characterized in that: The following steps are involved: (1) Seed solution preparation: Bacillus amyloliquefaciens ZH807 was inoculated into a seed culture medium and cultured at 30° C. to 40° C. for 12 to 16 hours to obtain a seed solution; (2) Preparation of fermentation broth: The seed liquid of step (1) is inoculated into a liquid fermentation medium at an inoculation amount of 6%, a culture temperature of 30° C. to 40° C., and a fermentation time of 12 to 16 hours to obtain the fermentation broth.

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