1. A Bacillus amyloliquefaciens (ahsz909) has a preservation number of CGMCC No. 13028.

2. Use of a bacillus amyloliquefaciens comprising the bacillus amyloliquefaciens of claim 1 (ahsz909) in the preparation of a polypeptide.

3. The use according to claim 2, wherein the bacillus amyloliquefaciens uses soybean meal as a substrate to prepare the polypeptide.

4. A method of producing a polypeptide, comprising: inoculating the seed solution of the bacillus amyloliquefaciens into a fermentation culture medium containing soybean meal for culture, wherein the fermentation liquor contains polypeptide after the culture is finished.

5. The preparation method according to claim 4, wherein the mass fraction of the soybean meal in the fermentation medium is 10-15% based on the total mass of the fermentation liquid.

6. The method according to claim 4, wherein the inoculation volume of the seed solution of Bacillus amyloliquefaciens is 1 to 5% of the volume of the fermentation medium.

7. The method according to claim 4, wherein the Bacillus amyloliquefaciens is the Bacillus amyloliquefaciens (ahsz909) of claim 1.

8. The method according to claim 4, wherein the fermentation medium has a pH of 6.5 to 7.5 and/or the temperature of the fermentation medium is 34 to 39 ℃.

9. The method according to claim 4, wherein the shaking table is rotated at 150 to 250r/min during the cultivation in the fermentation medium.

10. The method according to claim 4, wherein the culture is carried out in a fermentation medium for 16 to 24 hours.

Background

The soybean meal is a byproduct obtained by extracting soybean oil from soybeans, can be divided into first-soaked soybean meal and second-soaked soybean meal according to different extraction methods, has advanced production process and is a main variety circulated in the spot market in China. The amount of soybean meal consumed in China at present is about 7000 million tons every year. The content of crude protein in the soybean meal is 45-55%, and the soybean meal has the advantages of wide source, low price, easy storage and more balanced nutrition, and is one of the main plant source proteins.

The conversion of proteins in soybean meal into polypeptides, small peptides and free amino acids is mainly achieved by means of acid, alkali or enzymatic hydrolysis. Although acid-base hydrolysis is simple and convenient, the hydrolysis cannot be determined according to the specified hydrolysis in the production process, and the amino acid is damaged to reduce the nutritional value because the production conditions are harsh, so the method is less adopted at present. The most widely used at present is the enzymatic hydrolysis process. Enzymatic hydrolysis of proteins includes both direct enzymatic hydrolysis and indirect enzymatic hydrolysis. However, the direct enzymolysis method has limited industrial production due to high cost and high price of commercial enzyme preparations.

At present, the strains for fermenting the soybean meal mainly comprise aspergillus, saccharomycetes, bacillus and lactic acid bacteria. Researchers at home and abroad have conducted extensive research on strains and processes, and mainly focus on 3 types of aspergillus, bacillus and mixed strains. Aspergillus has strong enzyme production capability, can degrade anti-nutritional factors and macromolecular proteins, but can only be propagated under aerobic conditions, and has low soybean peptide content (16.68mg/ml) and conversion rate (55.6%). The Bacillus (Bacillus) can produce various digestive enzymes and bacteriostatic substances outside cells, can convert macromolecular protein into small molecular peptide, degrades anti-nutritional factors, is resistant to temperature and acid environment, is convenient to produce and process, and has great development potential. Some patents about protease producing bacteria or fermented soybean meal are reported, but no patent is reported about hydrolysis of soybean meal to produce polypeptide by using protease producing bacteria.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide the use of Bacillus amyloliquefaciens for the preparation of polypeptides, which solves the problems of the prior art.

In order to achieve the above objects and other related objects, the present invention provides a Bacillus amyloliquefaciens ahsz909 with the preservation number of CGMCC No. 13028.

The invention also provides the application of the bacillus amyloliquefaciens in preparing the polypeptide.

Preferably, the bacillus amyloliquefaciens uses bean pulp as a substrate to prepare polypeptide.

The invention also provides a preparation method of the polypeptide, which comprises the following steps: inoculating the seed solution of the bacillus amyloliquefaciens ahsz909 into a fermentation culture medium containing soybean meal for culture, wherein the fermentation liquid contains polypeptide after the culture is finished.

Preferably, the mass fraction of the soybean meal powder in the fermentation medium is 10-15% based on the total mass of the fermentation liquid.

Preferably, the pH value of the fermentation medium is 6.5-7.5.

Preferably, the temperature for culturing in the fermentation medium is 34-39 ℃.

Preferably, the rotating speed of the shaking table is 150-250 r/min when the fermentation medium is cultured.

Preferably, the culture is carried out in a fermentation medium for 16-24 h.

The seed solution of the bacillus amyloliquefaciens ahsz909 is obtained by the following method: activating the frozen bacillus amyloliquefaciens ahsz909, inoculating the activated bacillus amyloliquefaciens into a seed culture medium for culture, and obtaining a seed solution after the culture is finished.

Preferably, the inoculation volume of the seed solution of the bacillus amyloliquefaciens ahsz909 is 1-5% of the volume of the fermentation medium.

As mentioned above, the application and method of the Bacillus amyloliquefaciens in the preparation of the polypeptide have the following beneficial effects: the utilization of protein in soybean meal by a microbial fermentation method is the mainstream of the current research. The screened bacillus amyloliquefaciens ahsz909 has the property of producing protease, is used for hydrolyzing bean pulp to produce polypeptide, and can ensure that the polypeptide content is between 60 and 80mg/ml after fermentation culture for 16 to 24 hours. The strain is used for hydrolyzing the soybean meal and has the advantages of short fermentation period, easily obtained raw materials, low cost and easy control of fermentation products. The fermentation product has no pollution and can be used for producing microbial fertilizer. The invention reduces the production cost by microbial fermentation of an indirect enzymolysis method and utilizing the enzyme-producing strain to directly act on the substrate bean pulp, and the fermentation process is controllable, simple and convenient. The fermentation liquor has the effect of promoting the root growth of the potted cucumber.

Drawings

FIG. 1 shows a photograph of a transparent circle of the Bacillus amyloliquefaciens ahsz909 according to the present invention formed on a casein medium plate.

FIG. 2 shows the observation of the results of the first rescreening (plate well experiment) of the Bacillus amyloliquefaciens ahsz909 of the present invention.

FIG. 3 shows the colony morphology of the Bacillus amyloliquefaciens ahsz909 of the present invention on nutrient agar plates.

Detailed Description

The invention provides a bacillus amyloliquefaciens ahsz909, wherein the bacillus amyloliquefaciens ahsz909 is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms in 2016, 09 and 21, and is classified and named as bacillus amyloliquefaciens, the preservation number is CGMCC No.13028, and the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

The bacillus amyloliquefaciens ahsz909 is a gram-positive bacterium and rod-shaped.

The polypeptide yield of the bacillus amyloliquefaciens ahsz909 in 16 hours is more than 64 mg/ml. The polypeptide yield of 16h is obtained by the following method:

1) inoculating seed liquid of bacillus amyloliquefaciens ahsz909 into a fermentation medium according to the inoculation amount of 3% of the volume of the fermentation medium, culturing for 18h at the temperature of 37 ℃ and the rotating speed of a shaking table of 200r/min, and obtaining fermentation liquid after the culture is finished;

2) separation of fermentation liquor: the fermentation broth is frozen and centrifuged at 10000r/min for 10min at 4 ℃. Filtering the supernatant with 0.45 μm microporous membrane to obtain filtrate as polypeptide liquid mixture;

3) the content of the polypeptide was measured by the biuret method, a standard curve was prepared using casein, and the regression equation Y ═ aX + b was obtained with the concentration of the polypeptide as the abscissa X (mg/ml) and the OD value as the ordinate Y. The polypeptide content is calculated by acid soluble polypeptide, the polypeptide liquid mixed solution and 10% trichloroacetic acid (TCA) aqueous solution are mixed uniformly in equal volume, the mixture is kept stand for 30min, and then the mixture is centrifuged at 10000r/min for 10 min. Taking the supernatant to dilute by a proper time, and adding the following components in percentage by weight: biuret reagent ═ 3: 2(V: V), standing for 15min, measuring OD at 540nm, and calculating polypeptide content by using formula (1).

Polypeptide content (mg/ml) ═ a-b/a × N (1)

In formula (1), A is OD540, and N is the dilution factor of the sample.

The seed culture medium comprises the following components in percentage by mass: beef extract powder 0.5%, bone peptone 1%, sodium chloride 0.5%, and pH 7.0. The fermentation medium comprises the following components in percentage by mass: 10-15% of soybean meal, 1-2.5% of soluble starch, 1-7% of corn flour, 0.03-1% of magnesium sulfate, 0.05-0.3% of calcium chloride, 0.4% of disodium hydrogen phosphate, 0.03% of dipotassium hydrogen phosphate, 0.05% of manganese sulfate and the balance of water. The pH value is 6.5-7.5.

The invention provides an application of bacillus amyloliquefaciens in preparing polypeptide.

The polypeptide is a product after proteolysis.

The bacillus amyloliquefaciens comprises bacillus amyloliquefaciens ahsz 909.

The bacillus amyloliquefaciens ahsz909 belongs to a protease producing bacterium, and the produced protease can hydrolyze protein into polypeptide, small peptide or free amino acid.

Specifically, the bacillus amyloliquefaciens ahsz909 utilizes soybean meal as a substrate to prepare polypeptide.

The invention also provides a preparation method of the polypeptide, which comprises the following steps: inoculating the seed solution of the bacillus amyloliquefaciens ahsz909 into a fermentation culture medium containing soybean meal for culture, wherein the fermentation liquid contains polypeptide after the culture is finished.

Taking the total mass of the fermentation liquid as a reference, the mass fraction of the soybean meal powder in the fermentation medium is 10-15%.

Taking the total mass of fermentation liquor as a reference, the fermentation medium comprises the following substances in percentage by mass: 10-15% of soybean meal, 1-2.5% of soluble starch, 1-7% of corn flour, 0.03-1% of magnesium sulfate, 0.05-0.3% of calcium chloride, 0.3-0.6% of disodium hydrogen phosphate, 0.03-0.05% of dipotassium hydrogen phosphate, 0.03-0.05% of manganese sulfate and the balance of water.

The pH value of the fermentation medium is 6.5-7.5.

The temperature for culturing in the fermentation medium is 34-39 ℃.

The rotating speed of a shaking table is 150-250 r/min when the culture is carried out in a fermentation culture medium.

Culturing in a fermentation culture medium for 16-24 h.

The seed solution of the bacillus amyloliquefaciens ahsz909 is obtained by the following method: activating the frozen bacillus amyloliquefaciens ahsz909, inoculating the activated bacillus amyloliquefaciens into a seed culture medium for culture, and obtaining a seed solution after the culture is finished.

The temperature for culturing in the seed culture medium is 34-39 ℃.

The rotating speed of a shaking table is 150-250 r/min when the seeds are cultured in a seed culture medium.

Culturing in a seed culture medium for 12-16 h.

In one embodiment, the seed medium comprises the following: beef extract powder, bone peptone and sodium chloride.

In one embodiment, the seed solution of Bacillus amyloliquefaciens ahsz909 is inoculated in a volume of 1-5% of the fermentation medium volume.

The content of the polypeptide obtained by the method is 6-8%.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Example 1

Screening strains of protease producing bacteria producing polypeptide from bean pulp, and screening from a company strain bank.

Primary screening: after 15 strains of the spore bacteria preserved in a strain bank of the company are activated on an operating agar culture medium plate, the strains are respectively spotted on casein culture medium plates and cultured at 28-37 ℃. 12 strains with clear transparent circles were selected, wherein the strain with the largest HC (transparent circle diameter/colony diameter) ratio was Bacillus amyloliquefaciens ahsz 909. The casein culture medium comprises the following components in percentage by mass: 0.5% of beef extract powder, 0.5% of casein, 0.2% of ammonium sulfate, 0.5% of dipotassium phosphate, 0.02% of magnesium sulfate, 0.01% of anhydrous calcium chloride, 0.5% of sodium chloride, 2% of agar powder and about 7.0% of pH.

Re-screening:

preparing a crude enzyme solution:

and (3) selecting 2 rings of the primarily screened strains, respectively inoculating the strains into a seed culture medium, performing overnight culture at 28-37 ℃ and 180-200 r/min for 16-17 h, inoculating the strains into a fermentation culture medium according to the inoculum size of 3%, and performing overnight culture at 28-37 ℃ and 180-200 r/min for 16-24 h. Taking 10mL of fermentation liquor, centrifuging at 4 ℃ and 10000r/min for 10min, taking supernatant, and centrifuging twice under the condition to take supernatant, namely the crude enzyme solution. The seed culture medium comprises the following components in percentage by mass: beef extract powder 0.5%, bone peptone 1%, sodium chloride 0.5%, and pH 7.0. The fermentation medium comprises the following components in percentage by mass: 10-15% of soybean meal, 1-2.5% of soluble starch, 1-7% of corn flour, 0.03-1% of magnesium sulfate, 0.05-0.3% of calcium chloride, 0.4% of disodium hydrogen phosphate, 0.03% of dipotassium hydrogen phosphate, 0.05% of manganese sulfate and the balance of water. The pH value is 6.5-7.5.

(1) Re-screening for the first time in a flat plate hole experiment:

punching holes on casein plates by using a puncher, uniformly punching 3 holes on each casein plate, and enabling the hole diameter to be 0.80 cm. And (3) injecting 25 mu L of crude enzyme solution into a plate hole (three holes are parallel), culturing the plate at the constant temperature of 28-37 ℃ for 16-24 h, dropwise adding 2.5% TCA solution, just paving the plate in a dish, and measuring the diameter of the hydrolysis transparent ring. Meanwhile, a centrifugal liquid injection flat plate hole without inoculating a bacterial culture medium is arranged as a blank control so as to eliminate the interference of the culture medium on the experiment. And selecting the bacterial strain corresponding to the enzyme solution with larger diameter of the formed transparent ring as a second secondary screening.

And (3) forming transparent rings by the crude enzyme liquid of all the strains through 1 st secondary screening, wherein the diameter of each transparent ring is 0.4-2.9 cm. Wherein the diameter D of the transparent ring is more than or equal to 2.0cm, and 2 strains are shown in figure 1-2, wherein the diameter of the transparent ring formed by the bacillus amyloliquefaciens ahsz909 is larger and is 2.8 +/-0.05 cm. Thus, the strain was selected for a second rescreening.

(2) Secondary rescreening for protease activity determination

Selecting a strain with the hydrolysis ring diameter D being more than or equal to 2.0cm selected by a plate hole experimental method, and taking the corresponding crude enzyme liquid to detect the enzyme activity of the crude enzyme liquid according to an ultraviolet spectrophotometry in QB/1803-1993 universal test method for industrial enzyme preparations. The method comprises the following specific steps:

drawing a standard curve: drying L-tyrosine (purity is more than or equal to 95%) at 105 ℃ to constant weight, weighing 0.1000g, dissolving with 20mL of 1mol/L HCl, and fixing the volume to 100mL to obtain 1mg/mL of L-tyrosine standard stock solution. 10mL of 1mg/mL L-tyrosine was taken and made up to 100mL with 0.1mol/L HCl to give 100. mu.g/mL L-tyrosine standard solution. Finally, 100. mu.g/mL of the L-tyrosine standard solution was diluted with distilled water to concentrations of 0, 20, 40, 60, 80, 100 and 120. mu.g/mL of L-tyrosine, respectively, and the absorbance thereof was measured at a wavelength of 275. Establishing a standard curve equation of L-tyrosine concentration C (mu g/mL) and absorbance A: C133.9A +0.995, r 0.9970. When a is 1, the absorption constant K is 134.895. K is more than or equal to 130 and less than or equal to 135.

And (3) enzyme activity determination:

crude enzyme solution: centrifuging the fermentation liquor at room temperature of 10000r/min for 10min, and taking supernatant fluid, namely the enzyme solution to be detected. Taking 1mL of enzyme solution to be tested, dissolving the enzyme solution with a buffer solution with the pH value of 7.5, and diluting by proper times for testing (diluting until the absorbance value of the liquid to be tested is in the range of 0.25-0.40). Firstly, 1% casein solution is put into a constant temperature water bath kettle with the temperature of 40 +/-0.2 ℃, and is preheated for 5 min.

(1) Control group:

taking a test tube, marking 2mL of enzyme solution diluted by A, adding 4mL of trichloroacetic acid in water bath at 40 +/-0.2 ℃ for 2min, shaking up, adding 2mL of 1% casein solution in water bath at 40 +/-0.2 ℃ for 10min, shaking up, continuously heating for 10min, and measuring the light absorption value of the filtrate obtained by centrifugal filtration at 275nm by using an ultraviolet spectrophotometer.

(2) Test groups:

taking a test tube, marking B (parallel for 3 times), adding 2mL of diluted enzyme solution into the test tube B, adding 2mL of 1% casein solution into water bath at 40 +/-0.2 ℃ for 2min, shaking up, adding 4mL of trichloroacetic acid into water bath at 40 +/-0.2 ℃ for 10min, shaking up, continuously heating for 10min, and measuring the light absorption value of the filtrate obtained by centrifugal filtration at 275nm by using an ultraviolet spectrophotometer.

(3) The hydrolysis of casein at 40 ℃ per ml enzyme solution per minute was calculated to yield 1. mu.g of tyrosine, defined as 1 protein activity.

X＝A×K×8/2×1/10×n×E＝2/5×A×K×n×E

Wherein X is the enzyme activity (mu/g or mu/mL) of the sample; a is the average absorbance of the sample solution; k: a light absorption constant; 8, the total volume of the reaction reagent is mL; 2: sucking 2mL of enzyme solution, wherein the amount is 1 mL; 1/10: the reaction time is 10min, counted as 1 min; n: dilution times; e: the conversion factor between the UV method and the Fulin method (neutral and alkaline protease factor of 0.5; and acid protease factor of 0.77). The results obtained are expressed as integers. The relative error of the parallel test should not exceed 3%.

The enzyme activity of the fermentation liquor obtained by secondary re-screening is more than 190U/ml, and the protease activity of the bacillus amyloliquefaciens ahsz909 is the highest and is 214U/ml.

And (3) combining the primary screening result and the secondary screening result, and finally selecting the protease producing strain which utilizes the bean pulp to produce the polypeptide as the bacillus amyloliquefaciens ahsz 909.

Example 2:

the use method of the protease producing strain for producing the polypeptide by using the bean pulp comprises the following steps: taking strain ahsz909 preserved in glycerin tube to carry out nutrient agar plate activation (as shown in figure 3), taking 2 rings to be inoculated in a seed culture medium, culturing for 12h at the temperature of 37 ℃ and the rotating speed of a shaking table of 200r/min to obtain seed liquid, inoculating the seed liquid into the fermentation culture medium according to the inoculation amount of 3 percent, culturing for 18h at the temperature of 37 ℃ and the rotating speed of the shaking table of 200r/min, and obtaining polypeptide-containing fermentation liquid after the culture is finished. The content of the polypeptide in the fermentation liquor is detected by the following method:

(1) separation of polypeptide liquid:

the fermentation broth is frozen and centrifuged at 10000r/min for 10min at 4 ℃. Filtering the supernatant with microporous membrane (0.45 μm) to obtain filtrate, which is polypeptide liquid mixture.

(2) Preparation of Standard Curve

Accurately weighing 12g of casein standard substance, and preparing into mother liquor of 12mg/ml with distilled water for later use. Preparing the solution into 0, 2, 4, 6, 8, 10 and 12mg/ml solution by adopting a gradient dilution method, then respectively taking 6.0ml of standard solution, adding 4.0ml of biuret reagent, and uniformly mixing on a vortex mixer. Standing for 15min, measuring OD value at 540nm (with the first tube as blank control), and making standard curve with polypeptide concentration as abscissa X (mg/ml) and OD value as ordinate Y to obtain regression equation Y of 0.0657X +0.0269 and R of 0.997

(3) Determination of polypeptide content

The polypeptide content is calculated by acid soluble polypeptide, the polypeptide liquid mixed solution is mixed with 10% trichloroacetic acid (TCA) aqueous solution in equal volume, and is uniformly mixed on a vortex instrument, the mixture is kept stand for 30min, and then the mixture is centrifuged at 10000r/min for 10 min. Taking the supernatant to dilute by a proper time, and adding the following components in percentage by weight: biuret reagent ═ 3: 2 (V), mixing uniformly on a vortex oscillator, standing for 15min, and measuring the OD value at 540 nm. The content of the polypeptide in the sample solution was calculated according to the formula (1) with reference to the standard curve.

Polypeptide content (mg/ml) ═ a-0.0269)/0.0657 XN (1)

In the formula (1), A is OD₅₄₀And N is the dilution factor of the sample.

The seed culture medium comprises the following components in percentage by mass: beef extract powder 0.5%, bone peptone 1%, sodium chloride 0.5%, and pH 7.0. The fermentation medium comprises the following components in percentage by mass: 10-15% of soybean meal, 1-2.5% of soluble starch, 1-7% of corn flour, 0.03-1% of magnesium sulfate, 0.05-0.3% of calcium chloride, 0.4% of disodium hydrogen phosphate, 0.03% of dipotassium hydrogen phosphate, 0.05% of manganese sulfate and the balance of water. The pH value is 6.5-7.5.

A first group: the application method of the protease producing strain for producing polypeptide by using bean pulp comprises the steps of taking a strain ahsz909 preserved in a glycerin pipe to carry out nutrient agar plate activation, taking 2 rings to be connected in a seed culture medium, culturing for 12 hours at the temperature of 37 ℃ and the rotating speed of a shaking table of 200r/min to obtain a seed solution, inoculating the seed solution into the fermentation culture medium according to the inoculation amount of 3 percent, and culturing for 18 hours at the temperature of 37 ℃ and the rotating speed of the shaking table of 200 r/min. The polypeptide content in the fermentation liquor reaches 78.2 mg/ml.

The seed culture medium comprises the following components in percentage by mass: beef extract powder 0.5%, bone peptone 1%, sodium chloride 0.5%, and pH 7.0. The fermentation medium comprises the following components in percentage by mass: 15% of soybean meal, 2% of soluble starch, 3% of corn flour, 0.01% of magnesium sulfate, 0.3% of calcium chloride, 0.4% of disodium hydrogen phosphate, 0.03% of dipotassium hydrogen phosphate, 0.05% of manganese sulfate and the balance of water, wherein the pH value is 6.7.

Second group: the application method of the protease producing strain for producing polypeptide by using bean pulp comprises the steps of taking a strain ahsz909 preserved in a glycerin pipe to carry out nutrient agar plate activation, taking 2 rings to be connected in a seed culture medium, culturing for 12 hours at the temperature of 37 ℃ and the rotating speed of a shaking table of 200r/min to obtain a seed solution, inoculating the seed solution into the fermentation culture medium according to the inoculation amount of 3 percent, and culturing for 16 hours at the temperature of 37 ℃ and the rotating speed of the shaking table of 200 r/min. The polypeptide content in the fermentation liquor reaches 64 mg/ml.

The seed culture medium comprises the following components in percentage by mass: beef extract powder 0.5%, bone peptone 1%, sodium chloride 0.5%, and pH 7.0. The fermentation medium comprises the following components in percentage by mass: 10% of soybean meal, 2% of soluble starch, 7% of corn flour, 0.05% of magnesium sulfate, 0.3% of calcium chloride, 0.4% of disodium hydrogen phosphate, 0.03% of dipotassium hydrogen phosphate, 0.05% of manganese sulfate and the balance of water, wherein the pH value is 7.0.

Third group: the application method of the protease producing strain for producing polypeptide by using bean pulp comprises the steps of taking a strain ahsz909 preserved in a glycerin pipe to carry out nutrient agar plate activation, taking 2 rings to be connected in a seed culture medium, culturing for 12 hours at the temperature of 37 ℃ and the rotating speed of a shaking table of 200r/min to obtain a seed solution, inoculating the seed solution into the fermentation culture medium according to the inoculation amount of 3 percent, and culturing for 16 hours at the temperature of 37 ℃ and the rotating speed of the shaking table of 200 r/min. The polypeptide content in the fermentation liquor reaches 79.3 mg/ml.

The seed culture medium comprises the following components in percentage by mass: beef extract powder 0.5%, bone peptone 1%, sodium chloride 0.5%, and pH 7.0. The fermentation medium comprises the following components in percentage by mass: 15% of soybean meal, 2.5% of soluble starch, 3% of corn flour, 0.03% of magnesium sulfate, 0.3% of calcium chloride, 0.4% of disodium hydrogen phosphate, 0.03% of dipotassium hydrogen phosphate, 0.05% of manganese sulfate and the balance of water, wherein the pH value is 7.0.

Example 3 experiment for promoting growth of potted cucumber by using 6-8% polypeptide fermentation liquid

Culturing cucumber seeds (autumn cucumber in Tangshan) in a greenhouse, selecting healthy plants with consistent growth vigor and fixedly planting the plants into pots when the cucumber grows to have two leaves and one heart, wherein 2kg of nutrient soil is filled in each pot, and repeating the treatment for 3 times. The first 3d of watering the seedling-reviving water, and measuring basic data (plant height, leaf length, leaf width and stem thickness). Then fertilizing cucumber seedlings for the 1 st time, fertilizing once every 7d in the later period, measuring basic data (plant height, leaf length, leaf width and stem thickness), and fertilizing 3 times totally until the test is finished. At the end of the test, the plant height, stem thickness, leaf length, leaf width, and plant dry weight of the cucumber plants were determined. And (4) measuring the natural plant height of the plant, namely the distance from the ground to the highest point in the natural state. Stem thickness: the thick stem one centimeter below the first true leaf. Leaf length: the length of the 3 rd true leaf of the plant was determined. Leaf width: the length of the 3 rd true leaf of the plant was determined. And (3) measuring the dry weight of the plants: deactivating enzyme at 105 deg.C for 30min, drying at 80 deg.C to constant weight, and weighing.

TABLE 1 growth index of potted cucumber

Note: leaf area calculation formula: 14.61-5L +0.94L²+0.47W+0.63W²0.62LW (L leaf length; W leaf width)

As can be seen from Table 1, the method disclosed by the invention has the effect of promoting the growth of the cucumber on the premise that the cucumber is diluted by 300 times with the fermentation broth containing 6-8% of polypeptide to irrigate roots of the cucumber, and the normal growth requirement of the cucumber can be met. The polypeptide in the fermentation liquor provides a nitrogen source for the growth of the polypeptide, so that the application of the fertilizer can be reduced.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.