Multifunctional bacillus velezensis, microbial inoculant and application thereof

Abstract

The application provides a multifunctional bacillus velezensis, a microbial agent and application thereof, and relates to the technical field of microbial development and application. The bacillus velezensis is bacillus velezensis KY950a, which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. 24943. The application screens a multifunctional microbial strain which has strong hair keratin degradation ability, strong animal protein degradation ability, strong gelatin hydrolysis ability and strong cellulose hydrolysis ability. The strain can rapidly and efficiently degrade feather or animal carcass protein into small molecule peptides or amino acids, especially into small molecule peptides with a molecular weight of about 5KD, so that the animal hair or animal carcass can be degraded to form animal-derived biological fertilizer, which is used for promoting plant growth. Meanwhile, the strain has good salt and alkali tolerance, acid tolerance and a wide antibacterial spectrum against plant pathogenic bacteria, and is a multifunctional strain with excellent comprehensive performance and high application value.

Description

Technical Field

[0001] This invention relates to the field of microbial development and application technology, and in particular to a multifunctional Bacillus belye strain, its inoculum, and its applications. Background Technology

[0002] In recent years, news reports of the indiscriminate disposal of animal carcasses have sparked widespread public concern about the proper handling of these carcasses. The safe disposal of animal carcasses is crucial to public health and safety. Implementing harmless treatment of animal carcasses generated during livestock and poultry farming and slaughter is particularly important for disease prevention and food safety. Commonly used methods for harmless treatment of animal carcasses include incineration, burial, chemical hydrolysis, and biodegradation (Ma Juewen et al., 2014). Incineration kills pathogenic microorganisms through oxidation and combustion, turning animal carcasses into ash. While it can eliminate pathogens through high temperatures, incineration produces air pollutants and poses a risk of disease spread (Guo Dongpo, 2013). It also consumes large amounts of fuel, increasing processing costs (Kalbasi A et al., 2005). The burial method involves placing animal carcasses and related animal products into a carcass disposal pit or burial pit, covering and disinfecting them with lime or similar materials to allow fermentation or decomposition. However, it is limited by site constraints, easily causing soil and groundwater pollution and even the re-spread of pathogens. Furthermore, the biosafety after several years is difficult to assess. Therefore, the EU has banned the burial method for treating animal carcasses (Fan Shi, 2012). The rendering method involves using mechanical, heating, or chemical treatments to transform animal carcasses into fertilizer, protein solids, soluble fats or oils, and water. While rendering can inactivate most pathogens and produce valuable byproducts, it requires high temperature and pressure control, specialized equipment, and has high initial costs (Song Jiande, 2013). It also cannot kill prions, thus posing certain biological risks. Chemical hydrolysis refers to the process of hydrolyzing animal carcasses and tissues into bone residue and sterile aqueous solutions under high temperature and alkaline or acidic catalysts for rapid decomposition. Biodegradation refers to the process of using the powerful ability of microorganisms to decompose and transform organic matter into organic fertilizer through bacteria or enzyme preparations. The decomposition products can be processed into organic fertilizer, realizing resource reuse. Due to its simplicity, practicality, economy, environmental protection and ability to produce stable and efficient plant fertilizer, it will become the mainstream method for the harmless treatment of infected animal carcasses.

[0003] The main elements composing organisms are C, H, O, N, P, and S. N is an essential nutrient for plant growth. In animals, N mainly exists in the form of protein, which is the main component of meat and blood. Waste animal carcasses contain a large amount of meat and blood, making them a rich and usable resource that can be degraded into nutrients that plants can absorb and utilize, thus achieving resource reuse. However, animal carcasses have a complex composition, including hair, blood, skin, meat, fat, various gastrointestinal contents, bones, hooves, and horns. Hair is mainly composed of keratin, skin is mainly composed of collagen and fat, gastrointestinal contents are mainly composed of cellulose and fat, and meat and blood are mainly composed of protein. Different enzymes need to be selected for specific degradation based on the different components of the animal carcass. However, it is difficult to match enzyme preparations because the optimal catalytic hydrolysis conditions for each enzyme are different, making it difficult to achieve uniformity. Microbial degradation can effectively solve these problems. The biostimulants produced can promote plant growth and development, alleviate abiotic stress, and improve crop quality (Bai Youlu, 2017). Therefore, screening for multifunctional microbial strains that can degrade animal carcasses is of great significance.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a multifunctional Bacillus belye strain, its inoculum, and its applications. This strain can rapidly and efficiently degrade feather or animal carcass protein into small molecule peptides or amino acids, particularly feather powder / animal carcass protein into small molecule peptides of approximately 5KD. This allows animal hair and carcasses to be degraded into animal-derived bio-fertilizers to promote plant growth.

[0006] The technical solution provided by this invention is as follows: In one aspect, the present invention provides a multifunctional Bacillus belyssus strain, wherein the Bacillus belyssus strain is Bacillus belyssus (… Bacillus velezensis KY950a is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24943.

[0007] This invention employs an enrichment-high-throughput method to screen for multifunctional microorganisms with strong abilities to degrade hair keratin, as well as those with strong abilities to degrade animal proteins and hydrolyze gelatin and cellulose. From soil samples, 207 microorganisms capable of degrading hair keratin were screened, of which 38 exhibited strong, efficient, and rapid keratin degradation, and 8 strains simultaneously possessed relatively strong abilities to degrade proteins and keratin, as well as hydrolyze cellulose and gelatin. One target strain with relatively strong capabilities in all these areas was named KY950a. The study found that this strain significantly increased the water-soluble protein and amino acid content in the fermentation broth under liquid fermentation conditions of feather meal and liquid fermentation (after high-temperature sterilization): compared to the control, the water-soluble protein content increased by 93.7% and the amino acid content increased by 104.1% in feather meal fermentation; and the protein content increased by 117.5% and the amino acid content increased by 256.7% in animal carcass fermentation broth. The results of polyacrylamide gel electrophoresis showed that only strain KY950a could degrade feather powder / animal carcass protein into small peptides or amino acids mainly concentrated in the range of 4.6-10KD.

[0008] Based on the 16S rDNA sequence (SEQ ID No. 1) of strain KY950a, the strain was identified as Bacillus belye (B. belye). Bacillus velezensis ).

[0009] In one aspect, the present invention provides a microbial agent comprising the aforementioned Bacillus belye ( Bacillus velezensis KY950a or its metabolites. In this invention, the microbial agent may also contain excipients or carriers. The excipients or carriers are commonly used excipients or carriers in microbial agents.

[0010] In one aspect, the present invention provides the aforementioned Bacillus belysinus ( Bacillus velezensis The application of KY950a or the aforementioned microbial agents, the application including one or more of the following: (a) degrading keratin or keratin-containing substances into a mixture of polypeptides and amino acids with less than 5KD; (b) degrading animal carcass proteins into a mixture of polypeptides and amino acids with less than 5KD; (c) degrading proteins; (d) hydrolyzing cellulose or cellulose-containing substances; (e) hydrolyzing gelatin or gelatin-containing substances; (f) promoting plant growth or antagonizing plant pathogens.

[0011] Hair degradation is a crucial step in the degradation of animal carcasses. Hair is mainly composed of α-keratin or β-keratin. This invention focuses on hair degradation, first screening for strains with strong keratin-degrading capabilities. The strain of this invention exhibits strong keratin degradation ability, with a keratin hydrolysis zone diameter reaching 9.5 mm, significantly larger than other strains. Furthermore, this strain can grow on protein-degrading solid media, gelatin-containing liquid media, and cellulose-degrading solid media, indicating that it simultaneously possesses protein, gelatin, and cellulose hydrolysis functions. Therefore, this invention provides the application of this strain in the degradation of feather meal or animal carcass proteins to produce amino acids, peptides, and / or soluble peptides.

[0012] Different microorganisms produce different types of enzymes, and their degradation effects on the same target can vary greatly. Peptides are substances between amino acids and proteins, and are one of the functional substances of biostimulants. Small molecule peptides are important intercellular signaling molecules and are important components for the function of amino acid fertilizers. The *Bacillus belyssae* strain screened in this invention... Bacillus velezensis KY950a is a strain that can rapidly and efficiently degrade animal carcass protein into small molecule peptides and amino acids, especially keratin-containing feather powder or animal carcass protein into small molecule peptides and amino acids of about 5KD. It can be used to degrade feather powder or animal carcasses to form animal-derived fertilizer, which can then be used for plant planting or cultivation to promote plant growth or antagonize plant pathogens.

[0013] In one embodiment, the keratin-containing substance comprises animal hair, such as poultry feathers; preferably, the keratin-containing substance is feather powder. The strains of the present invention can utilize untreated animal carcasses or treated animal carcasses (e.g., treated by high temperature or chemical methods).

[0014] In one embodiment, the plant pathogen includes one or more of the following: early blight pathogen of tomato, black spot pathogen of pear, sheath blight pathogen of rice, spot leaf drop pathogen of apple, sheath blight pathogen of wheat, and white wilt pathogen. In one embodiment, promoting plant growth or antagonizing plant pathogens includes promoting the growth of crops such as corn. For example, the fermentation broth of feather meal from strain KY950a or the fermentation broth of animal carcasses treated with strain KY950a (after high-temperature sterilization) can increase dry weight, chlorophyll content, and nitrogen content. The treated corn grows better, with greener leaves and greater leaf spread.

[0015] In one aspect, the present invention provides a method for producing a soluble peptide or amino acid solution by degrading feathers or animal carcass proteins, the method comprising utilizing the aforementioned Bacillus belye ( Bacillus velezensis KY950a bacterial solution is fermented together with feathers or animal carcasses.

[0016] In one aspect, the present invention provides a method for the combined enzymatic and bacterial degradation of feathers or animal carcasses, the method comprising using the aforementioned Bacillus belye (… Bacillus velezensis The bacterial culture of KY950a is used in conjunction with an enzyme preparation to co-ferment feathers or animal carcasses. In one embodiment, the enzyme preparation includes one or more of protease, cellulase, collagen hydrolase, and amylase; preferably, protease.

[0017] In one aspect, the present invention provides a fermented fertilizer containing biostimulants, characterized in that the fermented fertilizer is prepared by any of the foregoing methods. In another aspect, the present invention provides the application of the fermented fertilizer in promoting plant growth and / or preventing plant diseases.

[0018] This invention utilizes microbial degradation to process animal carcasses treated by high-temperature sterilization or other methods (such as chemical methods) to produce amino acid-containing fertilizer. This amino acid fertilizer is a biostimulant product containing a mixture of amino acids, peptides, sugars, etc. Small molecule peptides are crucial components for the effectiveness of amino acid fertilizers. Research in this invention also shows that compared to other strains (including other Bacillus belyssus strains), strain KY950a and the fermentation broth of feather meal / high-temperature sterilized animal carcasses have a stronger growth-promoting effect on plants (such as corn). The fertilizer product is rich in biostimulants, amino acids, small molecule peptides, and proteins.

[0019] Biological sample preservation information: Bacillus belye ( Bacillus velezensis The strain KY950a was deposited on May 23, 2022, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 24943; address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, 100101, China. It was confirmed as a viable strain by the collection center on May 23, 2022.

[0020] Beneficial effects: In addition to efficiently degrading hair keratin and animal protein, the strain of this invention is also a multifunctional microbial strain that can hydrolyze gelatin (the main component of animal carcass skin) and cellulose (the main component of animal carcass intestinal contents). When animal carcass tissue that has been sterilized at high temperature is treated with this multifunctional microbial strain, an ecologically safe amino acid fertilizer product rich in biostimulants, amino acids, small molecule peptides, and proteins can be formed.

[0021] The strain of this invention has a strong ability to degrade animal carcasses, and can degrade and transform animal carcass proteins into small molecule peptides or amino acids that promote healthy plant growth (the fermentation broth samples of treated animal carcasses are all concentrated at around 4.6-10 kDa, and mainly at around 5 kDa), which can simultaneously promote plant growth and prevent plant diseases.

[0022] The feather meal fermentation broth of strain KY950a or the fermentation broth of animal carcasses treated with strain KY950a (after high-temperature sterilization) has a promoting effect on plant growth, which is significantly higher than the effect of treatment with 0.1M NaOH, enzyme preparation 1, commercial strain 92068, DSM7, and Bacillus belyssus KY913 (J-913). Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of screening positive strains using keratin culture medium provided in an embodiment of the present invention; Figure 2 A comparative example of the hydrolysis of strains in keratin / protein / cellulose culture media provided in the embodiments of the present invention (where a and b are keratin solid culture media; c and d are protein solid culture media; e and f are cellulose solid culture media). Figure 3 SDS-PAGE results of animal carcass fermentation broth (after high-temperature sterilization) using different methods and strains provided in the embodiments of the present invention (lanes 1-9 are samples of animal carcass fermentation broth after high-temperature sterilization fermented with water, 0.1M HCl, 0.1M NaOH, enzyme preparation 1 (Kangshengyuan crude enzyme preparation), enzyme preparation 2 (commercially available conventional neutral protease preparation), enzyme preparation 3 (commercially available conventional pepsin preparation), 92068, J-676, and J-962, respectively; lanes 10-18 are samples of animal carcass fermentation broth after high-temperature sterilization fermented with strains J-806, J-955, J-913 (i.e., KY-913), J-552, J-568, J-577, J-662, KY950a, and J-997, respectively). Figure 4 The results of microscopic observation of strain KY950a provided in the embodiments of the present invention; Figure 5Biological results of irrigating corn plants with different feather meal fermentation liquids provided in the embodiments of the present invention; Figure 6 Biological results of irrigating corn plants with fermented animal carcasses treated with different high-temperature sterilization methods provided in the embodiments of the present invention (1). Figure 7 Biological results of irrigating corn plants with fermented animal carcasses treated with different high-temperature sterilization methods provided in the embodiments of the present invention (2); Figure 8 The results of antagonistic tests between strain KY950a provided in this embodiment of the invention and plant pathogenic fungi and bacteria (where a is tomato early blight; b is pear black spot; c is rice sheath blight; d is apple scab; e is wheat sheath blight; f is bacterial bacterial blight). Figure 9 The results of stress resistance determination of strain KY950a provided in the embodiments of the present invention (where a is a medium with pH 4.6; b is a medium with pH 10.5; c is a medium with pH 11.7; d is a medium with 10% KNO3; e is a medium with 15% KNO3; f is a medium with 20% KNO3). Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1. Screening for microbial strains with keratin-degrading capabilities using a high-throughput method. 1.1 Soil Sampling Select representative samples, which can come from different areas such as farmland, grassland, and forest soil. Collect 15-20 g of sample from each point, and indicate the source (province, county), year and month of collection, and the source of the soil (plant or other). Store the sample in a -80℃ refrigerator.

[0027] 1.2 Screening for microbial strains with keratin-degrading capabilities using enrichment methods Step 1 Enrichment: Take 0.5 g of soil sample and shake well in 50 mL of purified water. Take 500 μL of the mixture and put it into a liquid culture medium containing 0.5% keratin (5 g keratin, 1 g dipotassium hydrogen phosphate, 0.5 g potassium dihydrogen phosphate, 0.5 g sodium chloride, 1 L water, sterilized at 121℃ for 20 min). Incubate at 30℃ with shaking at 200 r / min for 2-3 days. Observe and record the changes in color, turbidity, etc. of the liquid culture medium.

[0028] The second step of enrichment: Take 500 μL of the bacterial solution enriched in the previous step into a liquid culture medium containing 0.5% keratin, and incubate at 30℃ with shaking at 200 r / min for 2-3 days. Observe and record the changes in the color and turbidity of the liquid culture medium.

[0029] The third step of enrichment: Take 500 μL of the bacterial solution enriched in the second step and put it into a liquid culture medium containing 0.5% keratin. Incubate at 30℃ and 200 r / min for 2-3 days with shaking. Observe and record the changes in the color and turbidity of the liquid culture medium.

[0030] 1.3 Screening of functional microbial strains capable of degrading keratin using the coating method 100 μL of the last enriched bacterial culture was diluted and spread onto a solid selective medium containing 0.5% keratin (keratin 5 g, dipotassium hydrogen phosphate 1 g, potassium dihydrogen phosphate 0.5 g, sodium chloride 0.5 g, agar powder 15 g, water 1 L, sterilized at 121℃ for 20 min). The growth of the strain and the hydrolysis of keratin on the selective medium containing 0.5% keratin were observed and recorded. Strains with keratin hydrolysis function were selected from the above medium and purified by streaking on R2A medium (peptone 0.5 g, yeast extract 0.5 g, glucose 0.5 g, tryptone 0.5 g, soluble starch 0.5 g, dipotassium hydrogen phosphate 0.3 g, magnesium sulfate 0.05 g, sodium pyruvate 0.3 g, agar powder 15 g, water 1 L, sterilized at 121℃ for 20 min).

[0031] 1.4 Repeat Validation To ensure the keratin-degrading ability of the strain, the target strain that can degrade keratin was streaked again onto keratin solid selective medium and incubated at 30°C to verify whether the selected colonies had the function of degrading keratin and to exclude false positive colonies. Figure 1 This is a schematic diagram of screening positive strains on keratin culture medium.

[0032] 1.5 Results Using the above method, 69 soil samples from 17 different provinces and cities were enriched and screened using a high-throughput method. 207 microbial strains with the ability to degrade keratin were obtained, of which 38 strains had strong, efficient and rapid keratin degradation capabilities.

[0033] Because animal carcasses are complex in composition, containing not only hair, but also skin, fat, meat, bones, gastrointestinal contents, hooves and horns, and because different microbial strains produce different types and amounts of extracellular proteolytic enzymes, their ability to degrade different components will vary. These differences are mainly reflected in the size of peptide molecules, the composition ratio of amino acids, and the composition of biological macromolecules, which will have a differentiated impact on the promotion of plant growth.

[0034] Therefore, this invention aims to screen for multifunctional microbial strains that can rapidly degrade the most abundant and difficult-to-degrade components of animal carcasses (such as hair, skin, and gastrointestinal contents) including animal carcass proteins (after high-temperature sterilization). Thus, the initial step was to screen for microbial strains that degrade hair, followed by strains that specifically degrade keratin. Then, from these 38 relatively strong strains capable of efficiently and rapidly degrading keratin, strains with stronger protein, gelatin, and cellulose hydrolysis functions were selected. Next, the BCA and ninhydrin methods were used to determine the soluble protein and amino acid content of feather meal and (after high-temperature sterilization) animal carcasses under liquid fermentation conditions. Finally, the SDS-PAGE method was used to further screen for target microbial strains that can degrade (after high-temperature sterilization) animal carcass proteins into small molecules of approximately 5 kDa.

[0035] Example 2. Screening for target microbial strains capable of degrading (high-temperature sterilized) animal carcass proteins into small peptides and amino acids of approximately 5 kDa. 2.1 Screening for strains with stronger protein, gelatin, and cellulose hydrolysis functions. 2.1.1 Screening for strains with stronger protein hydrolysis capabilities Thirty-eight microbial strains capable of degrading keratin were cultured on R2A medium for 2 days. They were then needled into solid selective medium containing 0.5% keratin (5 g keratin, 1 g dipotassium hydrogen phosphate, 0.5 g potassium dihydrogen phosphate, 0.5 g sodium chloride, 15 g agar powder, 1 L water, sterilized at 121℃ for 20 min) and solid protein-degrading medium (0.5 g peptone, 0.5 g yeast extract, 0.5 g glucose, 0.5 g tryptone, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate, 0.3 g sodium pyruvate, 5 g skim milk powder, 15 g agar powder, 1000 mL water, sterilized at 121℃ for 20 min) and cultured at room temperature. After the appearance of hydrolysis zones, the diameter of the keratin / protein hydrolysis zones for different strains was measured in mm. Commercial strain 92068 was set as a control (strain 92068 is a commonly used Bacillus subtilis on the market, which is mostly used for plant disease prevention and growth promotion).

[0036] Keratin / protein removal capacity = Keratin / protein ring diameter in millimeters + X; X is a weighting coefficient, which is 0, 1, or 2 depending on the transparency of the protein dissolution zone of the strain; (Note: X is a weighting coefficient, which is 0, 1, or 2 depending on the transparency of the hydrolysis zone of the strain. The number 2 represents a completely transparent hydrolysis zone; the number 1 represents a semi-transparent dissolution zone; and 0 represents an opaque dissolution zone.) 2.1.2 Screening for strains with gelatin hydrolysis function Thirty-eight microbial strains capable of degrading keratin were screened and cultured on R2A medium for 2 days, with OD... 600nm =0.05g of the culture medium was inoculated into a liquid medium containing 1% gelatin (10g gelatin, 1g glucose, 0.25g peptone, 0.3g dipotassium hydrogen phosphate, 0.05g magnesium sulfate, 1L water, sterilized at 121℃ for 20 min). After setting up a blank control, the medium was cultured overnight at 30℃ on a shaker at 200 rpm / min. After the liquid became turbid, it was placed at 4℃ and the liquid was observed to see if it solidified. If it solidified, it indicated that the strain did not have the ability to hydrolyze gelatin. If the liquid was still flowing, it indicated that the strain had the ability to hydrolyze gelatin.

[0037] 2.1.3 Screening for strains with stronger cellulose hydrolysis function Thirty-eight microbial strains capable of degrading keratin were screened and cultured on R2A medium for 2 days. They were then needled into cellulose-degrading solid medium (2 g sodium carboxymethyl cellulose, 2 g yeast extract, 1 g dipotassium hydrogen phosphate, 0.25 g magnesium sulfate, 15 g agar powder, 1000 mL water, sterilized at 121℃ for 20 min). Commercial Bacillus subtilis (strain 92068) was used as a control. The cultures were incubated at 30℃ for 1-2 days, followed by fumigation with iodine solution. The diameter of the CMC degradation zone was measured in mm.

[0038] CMC solving capability = number of millimeters of CMC loop diameter solved + X; X is a weighting coefficient, which is 0, 1, or 2 depending on the transparency of the hydrolysis zone of the strain. (Note: X is a weighting coefficient, which is 0, 1, or 2 depending on the transparency of the hydrolysis zone of the strain. The number 2 represents a completely transparent hydrolysis zone; the number 1 represents a semi-transparent dissolution zone; and 0 represents an opaque dissolution zone.) 2.1.4 Results of the determination of keratin / protein / gelatin / cellulose hydrolysis function of microbial strains The keratin / protein / gelatin / cellulose hydrolysis functions of the 38 screened microbial strains were determined. The results showed that 8 of the 38 microbial strains (J-577, J-662, J-997, J-552, J-568, J-955, J-676 and KY950a) simultaneously possessed relatively strong abilities to degrade proteins and keratin, as well as hydrolyze cellulose and gelatin.

[0039] Figure 2 The hydrolysis performance of strains on keratin / protein / cellulose media is shown. Table 1 shows the hydrolytic ability of some strains on keratin, protein, cellulose, and gelatin.

[0040] Table 1. Results of the hydrolytic ability of some strains on keratin, protein, cellulose and gelatin.

[0041] from Figure 2 As shown in Table 1, some microbial strains possess both strong keratin and protein degradation abilities, and also hydrolyze cellulose and gelatin, such as strains KY950a, J-997, J-955, and J-577. However, this also reflects that the ability of microbial strains to degrade keratin is not necessarily positively correlated with their ability to degrade proteins. Strains with weak keratin degradation abilities can also have strong protein degradation abilities, such as strains J-456, J-806, and J-913; conversely, strains with strong keratin degradation abilities can have very weak protein degradation abilities, such as strain J-568. Among these 38 strains, strain J-913 (also referred to as KY-913 in this invention) is a Bacillus belesii strain, but its keratin degradation ability is very weak.

[0042] Therefore, the BCA method was used to determine whether these 8 microbial strains had the expected ability to degrade feather keratin and (high-temperature sterilized) animal carcasses in liquid fermentation. Then, the SDS-PAGE method was used to screen out target microbial strains that could degrade feather keratin and (high-temperature sterilized) animal carcasses into small molecules of about 5 kDa.

[0043] 2.2 Determination of the ability of microbial strains to degrade feather meal and (high-temperature sterilized) animal carcass proteins under liquid fermentation conditions using the BCA method. 2.2.1 Liquid fermentation Eight selected bacterial strains were inoculated into 100 mL of liquid culture medium (1 g soybean meal, 1 g glucose, 0.30 g dipotassium hydrogen phosphate, 0.30 g sodium pyruvate, 0.05 g magnesium sulfate, 1000 mL water, sterilized at 121℃ for 20 min) and cultured overnight at 30℃ and 200 rpm in shake flasks. Then, 10 mL of each bacterial culture was added to an Erlenmeyer flask containing 10 g feather meal / (high-temperature sterilized) animal carcass (mixed pig and cattle carcasses, treated at ≥140℃ and ≥0.5 MPa for ≥4 hours) for liquid fermentation. An equal volume of water and an equal volume of commercial strain 92068 bacterial culture were used as controls for the liquid fermentation of feather meal / (high-temperature sterilized) animal carcass. After fermentation, the supernatant was taken and analyzed using the BCA method and ninhydrin method to determine the content of soluble protein and amino acids in the supernatant.

[0044] 2.2.2 Determination of soluble protein content by BCA method Take 0.1 mL of each dilution of protein standard and the protein sample to be tested, and add them to the labeled test tubes; Add 2.0 mL of working solution to each test tube and mix thoroughly. Seal the test tubes and incubate at 37°C for 30 min, then cool the test tubes to room temperature. Set the spectrophotometer to 562 nm, zero it with water as a control, and measure the absorbance of the samples sequentially over 10 minutes. Subtract the average absorbance of the blank standard at 562 nm from the absorbance of each standard and the test sample at 562 nm. Plot the absorbance of the BSA standard at 562 nm after blank correction against its concentration (μg / mL) to create a standard curve. Use this standard curve to determine the protein concentration of each test sample.

[0045] 2.2.3 Determination of amino acid content in fermentation broth using the ninhydrin method 1. Take 50 mg of tryptophan standard, add water to make up to 100 mL, to obtain a 0.5 mg / mL tryptophan standard solution. Take the standard solution at certain gradients (0, 50, 100, 150, 200, 250, 300 μg / mL) and react with ninhydrin reagent for color development. Measure the OD using a spectrophotometer. 570nm . with OD 570nm Plot a standard curve for the ordinate.

[0046] 2. Take 0.1 mL of sample solution, add 2 mL of pH 5.5, 0.2 mol / L acetate buffer and 2 mL of ninhydrin colorimetric solution, mix well, and boil in a 100℃ water bath for 30 min. Cool to room temperature with tap water. After standing for 5 min, dilute with 6 mL of 50% ethanol, shake well, and then measure the OD. 570nm (The generated color is stable within 60 min). The OD of the sample was measured. 570nm By comparing with the standard curve, the amino acid content (μg / ml) in the sample can be determined.

[0047] 2.2.4 Results The bacterial culture of the selected strains was subjected to liquid fermentation with feather meal / (high-temperature sterilized) animal carcass, followed by centrifugation. The supernatant was then analyzed using the BCA method and the ninhydrin method to determine the content of soluble protein and amino acids in the supernatant. The results are shown in Tables 2 and 3 below: Table 2. Results of BCA determination of soluble protein after fermentation of strains and feather meal / (sterilized animal carcass protein)

[0048] Table 3. Results of amino acid content determination of ninhydrin after fermentation of some strains with feather meal / (high-temperature sterilized) animal carcass protein.

[0049] As shown in Tables 2 and 3, under liquid fermentation conditions, compared with the control (water-treated feather meal / animal carcass fermentation broth), the content of water-soluble proteins in the feather meal / (high-temperature sterilized) animal carcass fermentation broth obtained by the eight selected strains was increased to varying degrees. Furthermore, different strains showed differences in their hydrolytic ability towards feather meal / (high-temperature sterilized) animal carcass. Among them, strain KY950a significantly increased the content of water-soluble proteins and amino acids in the fermentation broth under both liquid fermentation conditions of feather meal and (high-temperature sterilized) animal carcass: compared with the control, the water-soluble protein content increased by 93.7% and the amino acid content increased by 104.1% in feather meal fermentation; and in the (high-temperature sterilized) animal carcass fermentation broth, the protein content increased by 117.5% and the amino acid content increased by 256.7%.

[0050] Therefore, KY950a can be preliminarily identified as the target strain for this study to conduct subsequent experiments. Next, polyacrylamide gel electrophoresis (SDS-PAGE) was used to further screen the selected strain, ultimately identifying the target strain capable of degrading (high-temperature sterilized) animal carcasses to approximately 5 kDa small peptides and amino acids.

[0051] 2.3 Further screening of target strains capable of degrading feather meal / (high-temperature sterilized) animal carcass proteins to approximately 5 kDa using SDS-PAGE was conducted. 2.3.1 The ability of the target strain to degrade (high-temperature sterilized) animal carcass proteins was determined by polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE: separating gel 16.5%; stacking gel 5%.

[0052] Composition of 16.5% separating gel (10 mL): 1.8 mL distilled water; 5.5 mL 30% gel stock solution (Acry); 2.5 mL pH 8.8 Tris-HCl buffer; 0.1 mL 10% SDS; 0.1 mL 10% AP; 4 μL TEMED; 1.0 mL glycerol.

[0053] Composition of 5% stacking gel (5 mL): 3.40 mL distilled water; 0.83 mL 30% gel stock solution (Acry); 0.63 mL pH 6.8 Tris-HCl buffer; 0.05 mL 10% SDS; 0.05 mL 10% AP; 2 μL TEMED.

[0054] 1. Using a pipette, pour the prepared separating gel solution into the prepared glass plate until it is 2 / 3 full. Immediately seal the gel with distilled water or anhydrous ethanol. Let it stand at room temperature for about 60 minutes until a clear boundary is visible. Pour off the distilled water or anhydrous ethanol and blot away any remaining liquid with filter paper. Prepare the stacking gel as described above, pour it between the glass plates, immediately add a comb, and fix for about 40 minutes.

[0055] 2. Based on the protein concentration measured by BCA, the sample loading amount for each well is 60 μg. Calculate the protein sample volume, mix it with 5× loading buffer, mix well, and boil at 100℃ for 5 min to denature.

[0056] 3. After the stacking gel solidifies, assemble the electrophoresis apparatus, add SDS electrophoresis buffer, remove the comb, and load the samples (using Thermo Spectra™ multicolor low molecular weight protein molecular weight standards as markers). Use water, acid, alkali, three different enzyme preparations, and fermented animal carcass broth treated with 92068 (after high-temperature sterilization) as controls for electrophoresis. The electrophoresis conditions are: 80 V constant voltage for the stacking gel and 120 V constant voltage for the separating gel. Stop electrophoresis when the bromophenol blue reaches approximately the bottom of the gel.

[0057] 2.3.2 Results After electrophoresis, the gel was stained with staining solution and then destained with destaining solution until the bands were clearly visible. The gel was then placed in an electrophoresis imaging system for imaging. The results are shown below. Figure 3 .

[0058] Compared to the animal carcass fermentation broth samples treated with water (after high-temperature sterilization) in lanes 2-6 and lane 1 (blank control), under the same loading conditions, 0.1M HCl, 0.1M NaOH, and three different enzyme preparations all showed degradation activity against the animal carcasses (after high-temperature sterilization). The gel running results showed that acid, alkali, and enzyme preparation 2 had some degradation ability against the animal carcasses (after high-temperature sterilization), but the strongest degradation ability was observed with enzyme preparations 1 and 3, corresponding to lanes 4 and 6. Both enzyme preparations degraded the products into amino acid molecules, but since the molecular weight of amino acids is 128 Da, no bands were observed on the gel, which is normal. Lanes 7-18 show the gel running results of animal carcass fermentation broth samples treated with different strains (after high-temperature sterilization). It can be seen that under the same loading conditions, different strains showed some differences in their hydrolytic ability against the animal carcasses (after high-temperature sterilization). Compared to lanes 2-3 (0.1M HCl, 0.1M NaOH ... Compared to the gel running results of lanes 4 and 6 (NaOH), lanes 7-9, 10-12, and 14 (enzyme preparations 1 and 3), the microbial strains corresponding to lanes 7-9, 10-12, and 14 showed relatively weaker degradation ability of animal carcasses (after high-temperature sterilization), as reflected in the gel: each lane showed a large number of bands of varying depths. The gel running results of the fermentation broth of animal carcasses (after high-temperature sterilization) treated with the strains corresponding to lanes 13 and 15-18 showed that, compared to the microbial strains corresponding to lanes 7-9, 10-12, and 14, these... The animal carcass fermentation broth samples treated with this strain (after high-temperature sterilization) showed fewer molecular weight bands on the gel and were relatively lighter in color, indicating a stronger degradation ability for the animal carcasses (after high-temperature sterilization). Among them, the animal carcass fermentation broth samples treated with lane 17 (KY950a) all had molecular weights concentrated in the range of 4.6-10 kDa, mainly around 5 kDa. This strain also showed stronger degradation ability for animal carcasses (after high-temperature sterilization) than enzyme preparation 2. Therefore, this strain was selected as the research subject for subsequent experiments.

[0059] Example 3. 16S DNA sequencing of target strain KY950a and physiological and morphological analysis of the strain. 3.1 16S DNA sequencing of strain KY950a 3.1.1 Extraction of bacterial DNA using the CTAB method 1. Inoculate a single colony into 5 mL of R2A and incubate overnight at 30°C; 2. Take 1 mL of seed culture medium and inoculate it into 100 mL of R2A liquid, and incubate at 37℃ and 220 r / min for 16 hours; 3. Centrifuge at 5000 r / min for 10 minutes and discard the supernatant.

[0060] 4. After centrifugation and washing with 10 mL TE, dissolve the bacterial cells with 10 mL TE, mix well, and store at -20℃ for later use. 5. Take 3.5 mL of bacterial suspension, add 184 μL of 10% SDS, mix well, add 37 μL of 10 mg / mL proteinase K, mix well, and incubate at 37℃ for 1 hour. 6. Add 740 μL of 5 mol / L NaCl, then add 512 μL of CTAB / NaCl, mix well, and incubate at 65℃ for 10 minutes; 7. Add an equal volume of chloroform / isoamyl alcohol, mix well, centrifuge at 10000 r / min for 5 minutes, and retain the supernatant; 8. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) to the supernatant, mix well, centrifuge at 10000 r / min for 5 minutes, and retain the supernatant; 9. Add 0.6 times the amount of isopropanol, mix well, centrifuge at 10000 r / min for 5 minutes, collect the DNA precipitate, and wash the DNA precipitate with 70% ethanol by centrifugation. 10. Dissolve the DNA in 1 mL TE, add RNase A to a final concentration of 20 μg / mL, and store at 4℃.

[0061] 3.1.2 Amplification and Sequencing PCR amplification of 16S rDNA was performed using universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 2) and 1492r (5'-GGTTACCTTGTTACGACTT-3', SEQ ID No. 3). PCR reaction conditions were: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 60 s, for 35 cycles. PCR products were subjected to 1.5% agarose gel electrophoresis, and the PCR products were recovered, purified, and sequenced after agarose gel electrophoresis.

[0062] The 16S DNA sequencing results of strain KY950a are shown in Sequence 1 (SEQ ID No. 1):

[0063] Based on the obtained 16S rDNA sequence of KY950a (Sequence 1), homologous sequences were searched in GenBank and compared with each other. Simultaneously, sequence alignment was performed with the 16S RNA database (Chun's Lab) recognized by the International Committee for Bacteriology, combined with literature analysis, to determine the taxonomic position of the target microorganism (Yoon, SH, Ha, SM, Kwon, S., Lim, J., Kim, Y., Seo, H. and Chun, J. (2017). Introducing EzBioCloud: Ataxonomically united database of 16S rRNA and whole genome assemblies. Int JSyst Evol Microbiol. 67:1613-1617). The results showed that the 1456-base sequence of this strain is similar to that of the strain *Bacillus belyssae* (…). Bacillus velezensis The two structures have a high degree of homology with a similarity of 99.22, confirming KY950a as a related species. Bacillus velezensis .

[0064] 3.2 KY950a ( Bacillus velezensis Observation of strain morphology The selected strains were inoculated onto R2A plates and cultured at room temperature for 24 hours. The size, shape, color, gloss, viscosity, raised shape, transparency, and edge characteristics of the colonies were observed.

[0065] 3.2.1 Results of Observation of Strains' Morphology Observations showed that strain KY950a grew on R2A medium for 2 days. Under a microscope, strain KY950A was rod-shaped, and the colonies on the medium were round, white, with relatively neat edges, large, opaque, and viscous. Microscopic measurements showed that the colony diameter was about 4-8 μm and the spore diameter was about 2-3 μm.

[0066] Example 4. Biological assay of strain KY950a + feather meal / high-temperature sterilized animal carcass fermentation broth To verify the ability of strain KY950a to hydrolyze and ferment feather meal / (high-temperature sterilized) animal carcasses, a widely used commercial strain, 92068 (strain 92068 is a commonly used Bacillus subtilis strain in the market, mainly used for plant disease prevention and growth promotion), will be used as a control to compare their effects on plant growth. Simultaneously, a synergistic approach involving bacteria and enzymes will be further utilized to optimize the combination of strain KY950a with enzyme preparations, hydrolyzing animal carcasses into peptides and amino acids as much as possible. These peptides and amino acids will then be combined with the bacterial biostimulants produced by KY950a itself to form a "biostimulant-amino acid fertilizer" product.

[0067] 4.1 Biological determination of the effect of feather meal fermentation broth of strain KY950a on maize plant growth Take uniformly sized, plump corn seeds and sow them in pots filled with vermiculite. Observe the moisture level of the vermiculite in the pot and water as needed (generally water every 2 days) until the corn germinates. Control strains: Select commercial strain 92068, strain DSM7 (a commonly used Bacillus amyloliquefaciens, often used for plant disease prevention and growth promotion), and another strain, Bacillus velezensis KY913, as control strains. OD... 600 The strains 92068, DSM7, KY913, and KY950a (with a concentration of 0.05) were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of liquid culture medium and cultured at 30°C and 200 r / min for 48 hours. The bacterial suspensions were then added in equal volumes to beakers containing 100 g of feather meal and mixed thoroughly, allowing to ferment at room temperature for 24 hours. Commercial strains 92068-feather meal fermentation broth, DSM7-feather meal fermentation broth, KY913-feather meal fermentation broth, and KY950a-feather meal fermentation broth were diluted by the same factor and applied to corn pots at appropriate times. Water was used as a blank control, water-feather meal fermentation broth as a negative control, and feather meal fermentation broth treated with enzyme preparation 1 and 0.1 M NaOH as positive controls. Corn growth was observed and recorded daily, and watering was replenished regularly. As the corn plants grew, the chlorophyll and nitrogen content of the leaves of different groups of corn plants was measured and recorded every two days using a chlorophyll meter. Thirty days after the corn plants grew, the stems and leaves of different groups of corn were cut off, placed in dried petri dishes, and dried in an oven (105℃ for blanching, 85℃ for drying to constant weight). The dry weight of the corn plants in different groups was recorded.

[0068] 4.1.1 Results of biological assays on the effects of feather meal fermentation broth of strain KY950a on maize plant growth Thirty days after the corn plants matured, chlorophyll and nitrogen content were measured in different groups of corn (6 plants per group). The differences between the corn plants in different groups were also photographed. After completion, the corn stems and leaves were dried. The results are as follows: Table 4. Dry weight of corn after drying in different groups

[0069] Table 5. Results of chlorophyll and nitrogen content in maize leaves of different groups

[0070] As shown in Table 4: ① The dry weight of corn irrigated with feather meal fermentation broth treated with different strains and water was higher than that of corn irrigated with water alone; ② The dry weight of corn irrigated with feather meal fermentation broth of strain KY950a was 95.7% and 40.6% higher than that of corn treated with water and water + feather meal, respectively, and was significantly higher than that of corn irrigated with feather meal fermentation broth treated with 0.1M NaOH, enzyme preparation 1, commercial strain 92068, DSM7, and Bacillus belyssus KY913.

[0071] Table 5 shows that: ① The average chlorophyll content and average nitrogen content of corn irrigated with feather meal fermentation broth treated with different strains and water were higher than the dry weight of corn irrigated with water alone; ② The average chlorophyll content and average nitrogen content of corn irrigated with feather meal fermentation broth treated with KY950a were significantly higher than those of corn irrigated with water, 0.1M NaOH, commercial strain 92068, DSM7, and feather meal fermentation broth treated with Bacillus belye KY913; The average chlorophyll content and average nitrogen content of corn irrigated with feather meal fermentation broth treated with KY950a were not significantly different from those of corn irrigated with feather meal fermentation broth treated with enzyme preparation 1.

[0072] from Figure 5 As can be seen from Figures A and B, the corn irrigated with KY950a-treated feather meal fermented liquid grew better than the corn irrigated with water alone and the corn irrigated with water-treated feather meal fermented liquid. Furthermore, the top view shows that the leaves of the corn irrigated with KY950a-treated feather meal fermented liquid were greener and broader. Figure 5 As can be seen from the CH, compared with maize plants irrigated with feather meal fermentation broth treated with 0.1M NaOH, commercial strain 92068, and DSM7, maize plants irrigated with feather meal fermentation broth treated with KY950a showed superior leaf expansion, color, and leaf size; from Figure 5 The results showed that, compared with the corn plants irrigated with feather meal fermentation liquid treated with enzyme preparation 1 and strain KY913 (also a Bacillus belye), the corn plants irrigated with feather meal fermentation liquid treated with KY950a were taller overall, with greener leaves and greater spread.

[0073] In summary, regardless of the dry weight of the corn plants, the average chlorophyll content, the average nitrogen content, or the width of the corn leaves, the corn irrigated with the feather meal fermentation liquid treated with strain KY950a showed better growth. This indicates that strain KY950a itself has a stronger degradation effect on feather meal and a stronger promoting effect on plant growth.

[0074] 4.2 Biological assay of the effect of fermentation broth from animal carcasses treated with strain KY950a (after high-temperature sterilization) on maize plant growth. (1) Take corn seeds of uniform size and full grains and sow them in a pot containing vermiculite. Observe the moisture of the vermiculite in the pot and water it in a timely manner (generally water it once every 2 days) until the corn germinates. (2) Control strains: Commercial strain 92068, strain DSM7 (commonly used Bacillus amyloliquefaciens in the market, mostly used for plant disease prevention and growth promotion) and another strain Bacillus velezensis KY913 were selected as control strains. (3) OD 600 Strains 92068, DSM7, KY913, and KY950a with a concentration of 0.05 were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of liquid culture medium and cultured at 30 °C and 200 r / min for 48 hours. (4) Add the above bacterial suspension in equal volumes to beakers containing 100 grams of animal carcasses that have been sterilized at high temperature (hereinafter referred to as "animal carcasses"), mix well, and let ferment at room temperature for 24 hours. (5) Commercial strains 92068-animal carcass fermentation broth, DSM7-animal carcass fermentation broth, KY913-animal carcass fermentation broth, enzyme preparation 1-animal carcass fermentation broth, enzyme preparation 2-animal carcass fermentation broth, KY950a-animal carcass fermentation broth, and KY950a-enzyme preparation 1-animal carcass fermentation broth were diluted by the same multiple and then applied to corn pots at appropriate times. Water was set as a blank control, water-animal carcass fermentation broth was set as a negative control, and feather meal fermentation broth treated with enzyme preparation 1, enzyme preparation 2, and 0.1M NaOH was set as a positive control. The growth of corn was observed and recorded every day, and watering was replenished regularly.

[0075] (6) As the corn plants grow, use a chlorophyll meter to measure and record the chlorophyll content and nitrogen content of the leaves of different groups of corn plants every 2 days. (7) After the corn has grown for 30 days, the corn stems and leaves of different groups were cut off, placed in dried petri dishes, and dried in an oven (105℃ for blanching, 85℃ for drying to constant weight). The dry weight of the corn plants of different groups was recorded.

[0076] 4.2.1 Biological assay results of the effect of fermentation broth from animal carcasses treated with strain KY950a (after high-temperature sterilization) on maize plant growth. Thirty days after the corn plants matured, chlorophyll and nitrogen content were measured in different groups of corn (6 plants per group). The differences between the corn plants in different groups were also photographed. After completion, the corn stems and leaves were dried. The results are as follows: Table 6. Dry weight of corn after drying in different groups

[0077] Table 7. Results of chlorophyll and nitrogen content in maize leaves of different groups

[0078] As shown in Table 6: ① The dry weight of corn irrigated with animal carcass fermentation broth treated with different strains and water (after high-temperature sterilization) was higher than that of corn irrigated with water alone; ② The dry weight of corn irrigated with animal carcass fermentation broth of strain KY950a (after high-temperature sterilization) was 104.3% and 51.6% higher than that of corn irrigated with water alone and water-treated animal carcass fermentation broth (after high-temperature sterilization), respectively, and was significantly higher than that of corn irrigated with fermentation broth of animal carcass fermentation broth treated with 0.1M NaOH, enzyme preparation 1, commercial strain 92068, DSM7, and Bacillus belyssus KY913 (after high-temperature sterilization); ③ The dry weight of corn irrigated with animal carcass fermentation broth treated with KY950a strain compound enzyme preparation 1 (after high-temperature sterilization) was the highest.

[0079] Table 7 shows that: ① The average chlorophyll content and average nitrogen content of corn irrigated with animal carcass fermentation liquid treated with different strains and water (after high-temperature sterilization) were higher than the dry weight of corn irrigated with water alone; ② The average chlorophyll content and average nitrogen content of corn irrigated with animal carcass fermentation liquid treated with KY950a (after high-temperature sterilization) were the highest, and significantly higher than those of corn irrigated with water, 0.1M NaOH, enzyme preparation 1, enzyme preparation 2, commercial strain 92068, DSM7, and animal carcass fermentation liquid treated with Bacillus belye KY913 (after high-temperature sterilization).

[0080] from Figure 6As can be seen from the data, corn irrigated with KY950a (high-temperature sterilized) animal carcass fermentation liquid showed better growth than corn irrigated with water alone, water-treated corn, corn irrigated with 0.1M NaOH, commercial strain 92068, DSM7, and strain KY913 (both Bacillus species, high-temperature sterilized) animal carcass fermentation liquid. Furthermore, from the top view, the leaves of corn irrigated with KY950a fermentation liquid were more vibrant green and broader. Figure 7 As can be seen from the data, compared with the corn plants irrigated with the fermentation liquid treated with enzyme preparation 1 and enzyme preparation 2, the corn plants irrigated with the fermentation liquid of animal carcasses treated with KY950a (after high-temperature sterilization) were taller overall, with greener leaves and greater spread.

[0081] In summary, animal carcasses (after high-temperature sterilization) can provide the nutrients needed for plant growth after being treated with microbial strains, and the growth-promoting effect of animal carcass fermentation broth (after high-temperature sterilization) varies among different strains. Compared with commercial strains 92068, DSM7, and Bacillus kyleseus strain KY913, corn plants treated with feather meal or irrigated with animal carcass fermentation broth (after high-temperature sterilization) showed better growth in terms of dry weight, average chlorophyll content, average nitrogen content, and leaf width. This was also superior to animal carcass fermentation broth treated with chemical methods or single enzyme preparations (after high-temperature sterilization). This indicates that KY950a has a stronger growth-promoting effect on plants, and that strain KY950a produces substances that promote plant growth during its growth and reproduction. The feather meal fermentation broth and animal carcass fermentation broth (after high-temperature sterilization) amplify this effect, achieving a synergistic effect ("1+1>2").

[0082] Example 5. Multifunctional assay for KY950a strain Since the animal carcass fermentation broth product treated with strain KY950a contains the microbial strain KY950a, which has been reported to have highly efficient broad-spectrum antibacterial activity and plant growth-promoting ability, the purpose of this experiment is to determine the specific antifungal and antibacterial effects of this strain and to determine other functions of this strain, so as to ensure the resistance of the subsequent animal-derived amino acid fermentation broth product to fungi and improve the product efficacy.

[0083] 5.1 Antagonistic test between strains and plant pathogenic fungi and bacteria Antagonistic experiment against plant pathogenic fungi: Strains KY950a and existing plant pathogenic fungi in the laboratory were inoculated onto PDA medium for a plate confrontation experiment. The strains were cultured at temperatures suitable for different pathogenic fungi, and the results were observed to determine their resistance to the pathogenic fungi. Bacillus subtilis strain 92068 was used as a control.

[0084] Plant pathogenic bacteria antagonism experiment: Plant pathogenic bacteria were inoculated into R2A liquid medium and cultured at 30℃ for 24 h. OD was then measured. 600 Then according to the final concentration OD 600 =0.05% diluted in R2A solid medium, poured into a petri dish, and inoculated strain KY950a onto the dish to determine the strain's resistance to the pathogenic bacteria. Bacillus subtilis strain 92068 was used as a control.

[0085]

[0086] 5.1.1 Results of the test on the strain's resistance to plant pathogens Results determination ( Figure 8 The results showed that, compared with the control strain 92068, strain KY950a had stronger or similar antagonistic effects against early blight of tomato, black spot of pear, sheath blight of rice, leaf spot of apple, and sheath blight of wheat, and had a stronger antagonistic effect against white blight of plant pathogens.

[0087] 5.2 Determination of strain resistance Strain strain KY950a was inoculated onto R2A medium with pH 4.6, pH 10.5, pH 11.7, 10% KNO3, 15% KNO3, and 20% KNO3, respectively. The growth of the strain was observed, as well as whether its growth was inhibited and the rate of growth. Bacillus subtilis strain 92068 was used as a control.

[0088] 5.2.1 Results of stress resistance test for strain KY950a Results determination ( Figure 9 The results showed that strain KY950a had similar salt and alkali tolerance to the control strain 92068; however, strain KY950a had stronger acid tolerance than strain 92068.

[0089] Conclusion: This invention isolated a strain of Bacillus belye KY950a from soil collected in Daguan Village, Gaoqing County, Zibo City, Shandong Province, using a high-throughput screening method. This strain can degrade animal carcass protein (after high-temperature sterilization) into small peptides and amino acids of approximately 5KD, promoting plant growth while also enhancing crop biocontrol and stress resistance. The 1456-base sequence of this strain is consistent with that of the strain. Bacillus velezensis With a high degree of homology of 99.22%, KY950a was identified as... Bacillus velezensisThis invention found that compared with the commercial Bacillus subtilis strain 92068, KY950a has a stronger ability to hydrolyze animal carcass proteins and keratin (after high-temperature sterilization), with the hydrolysed animal carcass protein products concentrated at around 5 kDa. Compared with commercial strain 92068, feather meal fermentation broth treated with commercial enzymes, and animal carcass fermentation broth (after high-temperature sterilization), the feather meal fermentation broth treated with KY950a and the animal carcass fermentation broth (after high-temperature sterilization) have a stronger ability to promote plant growth. The study also found that strain KY950a also has strong cellulose degradation and gelatin hydrolysis capabilities, and the bacterium also has good salt, alkali, and acid tolerance, as well as a broad spectrum of inhibition against plant pathogens. It is evident that KY950a is a strain of microorganisms that has a strong ability to degrade various complex components of animal carcasses (after high-temperature sterilization). It can convert animal carcasses (after high-temperature sterilization) into small molecule peptides and amino acids, promote plant growth, prevent and control plant diseases, and is tolerant to salt and acid / alkali.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multifunctional Bacillus belye strain, characterized in that, The *Bacillus belesii* is *Bacillus belesii* (… Bacillus velezensis KY950a is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24943.

2. A microbial agent, characterized in that, The bacterial agent contains Bacillus berberis as described in claim 1. Bacillus velezensis )KY950a.

3. The Bacillus belesiensis as described in claim 1 ( Bacillus velezensis The application of KY950a or the microbial agent according to claim 2 is characterized in that, The application is one or more of the following: (a) Degrading keratin or keratin-containing substances into a mixture of peptides and amino acids with less than 5 kDa; (b) Degrading animal carcass proteins into a mixture of peptides and amino acids with less than 5 kDa; (c) Hydrolyzed gelatin or substances containing gelatin; (d) Antagonism of plant pathogens; the plant pathogen is *Early Blight of Tomato*. Alternaria solani Black spot fungus of pear Alternaria alternata Rice sheath blight fungus Thanatephorus cucumeris wheat sheath blight fungus Rhizoctonia cerealis White blight fungus Xanthomonas oryzae One or more of them.

4. The application according to claim 3, characterized in that, The keratin-containing substance is feather powder.

5. A method for producing soluble peptides or amino acid solutions by degrading feathers or animal carcass proteins, characterized in that, The method includes using Bacillus belesiensis as described in claim 1 (… Bacillus velezensis KY950a bacterial solution is used for liquid fermentation with feathers or animal carcasses.

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