CN113549575B

CN113549575B - Bacillus subtilis FM-20 strain for high yield of protease and application thereof

Info

Publication number: CN113549575B
Application number: CN202110819990.1A
Authority: CN
Inventors: 王伟; 郭晓军; 王世英; 李佳; 朱宝成; 王飒
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2022-09-23
Anticipated expiration: 2041-07-20
Also published as: CN113549575A

Abstract

The invention relates to a Bacillus subtilis FM-20 strain with high protease yield and application thereof, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) on 12.5.2021, and the preservation number is CGMCC No. 22509; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang. The bacillus subtilis FM-20 strain has high activity of producing protease, can reduce the emission of nitrogen and sulfur-containing substances in the form of excrement, can inhibit the activity of harmful bacteria such as escherichia coli and the like, can resist gastric acid, bile salt and high temperature, has good safety, is non-toxic and pollution-free, and can be used as a feed additive for poultry.

Description

Bacillus subtilis FM-20 strain for high yield of protease and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a bacillus subtilis FM-20 strain for high yield of protease and application thereof.

Background

The excrement generated in the poultry raising process generates a large amount of harmful gas, ammonia (NH) ₃ ) Hydrogen sulfide (H) ₂ S) has the greatest influence on the production performance of poultry. After the content of ammonia gas and hydrogen sulfide in the poultry house reaches a certain concentration, the disease resistance of the poultry flock can be reduced sharply, and particularly in winter and spring, the poultry house is low in temperature, high in humidity and poor in ventilation effect, and the concentration of harmful gases such as ammonia gas and hydrogen sulfide is high, so that the production performance and the health level of the poultry flock are influenced. Meanwhile, the harmful gas in the chicken farm is finally discharged into the atmosphere, and the degree of environmental pollution is also increased.

Researchers adopt adjustment of the structure of livestock and poultry daily ration to control the generation of odor, such as application of enzyme preparations, plant type extracts, microecologics, compounds such as ferrous sulfate, functional oligosaccharides such as soybean oligosaccharides, and reduce pollution gas and improve the environment in a chicken house in a spice masking type mode of zeolite powder and other adsorbents and natural aromatic oil, so as to improve the growth performance and health level of broiler chickens.

At present, the development potential is the greatest, and the most studied method for reducing the generation of harmful gases is mainly a microbiological method. In recent years, bacillus probiotics are receiving much attention and are a hot spot for researching microecologics.

Disclosure of Invention

Aiming at the technical problem that harmful gases such as ammonia gas, hydrogen sulfide and the like influence the production performance and the health level of chicken flocks, the invention provides a bacillus subtilis FM-20 strain for high protease yield and application thereof.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

in a first aspect, the present invention provides a highly protease-producing Bacillus subtilis FM-20 strain, which is classified under the name Bacillus subtilis (B.) (Bacillus subtilis) The strain is preserved in China general microbiological culture Collection center on 12 th 5 th 2021 with the preservation number of CGMCC 22509; health-care productThe Tibetan address is No. 3 of Xilu No.1 Beijing, Chaoyang, Beicheng.

Harmful gases such as ammonia gas and hydrogen sulfide are mainly produced by undigested nutrients and organic compounds in poultry excrement under the action of microorganisms. Therefore, the protein digestibility is improved, the residual quantity of nitrogen and sulfur-containing substances in the poultry manure is reduced, and the emission of harmful gases such as ammonia gas, hydrogen sulfide and the like can be effectively reduced. The bacillus subtilis FM-20 strain provided by the invention is obtained by primary screening on a casein plate and secondary screening by a liquid shake flask fermentation method from excrement of healthy broiler chickens, belongs to bacillus, and exogenous enzymes such as protease and amylase generated by the bacillus strain can enhance the intestinal functions of poultry animals, promote the digestion and absorption of the poultry animals on nutrient substances, and improve the protease activity of a digestive system of the poultry animals, so that the digestion and absorption rate of the nutrient substances such as protein and the like is improved, the emission of nitrogen-containing and sulfur-containing substances in the excrement form is reduced, and the contents of ammonia gas and hydrogen sulfide in air in a chicken house are effectively reduced. Meanwhile, the bacillus subtilis FM-20 strain can inhibit the activity of harmful bacteria such as escherichia coli and the like, reduce the conversion of protein into ammonia, amine and other harmful substances or gases, and reduce air pollution. The bacillus subtilis FM-20 strain can resist gastric acid, bile salt and high-temperature environment in the microbial inoculum processing process, has good safety and no toxicity or pollution, and can be used as a feed additive for poultry. The 16S rDNA sequence is shown as follows (SEQ ID No. 1):

TGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAA。

the screening method of the bacillus subtilis FM-20 strain specifically comprises the following steps:

firstly, feces of healthy broiler chickens are subjected to heat treatment and then are subjected to gradient dilution, the feces are coated on a protease identification culture medium and cultured for 24 hours, single colonies which grow well and have obvious transparent circles are selected, and strains with the ratio of the diameter of a hydrolysis circle to the diameter of the colonies larger than 2.0 are selected as primary screening strains;

secondly, transferring the primary screened strain obtained in the first step to an NA culture medium slant for culturing for 24h of activated strain, respectively inoculating the activated strain to NB culture solution, performing shake culture for 48h, centrifuging, and taking supernatant; uniformly punching holes on a sterilized protease identification culture medium flat plate by using a puncher, respectively taking 75 mu L of obtained supernatant liquid into injection holes, culturing at the constant temperature of 37 ℃ for 24 hours, and selecting strains with the ratio of the diameter of a hydrolysis ring to the diameter of a bacterial colony being more than 2.0;

thirdly, screening through protease activity determination, ammonia inhibition characteristic tests, sulfur inhibition characteristic tests, inhibition tests on pathogenic bacteria of escherichia coli, primary identification of the property of an antibacterial substance, cholate resistance characteristic tests, artificial gastric juice tolerance tests, artificial intestinal juice tolerance tests and high temperature tolerance tests to obtain the bacillus subtilis FM-20 strain.

In a second aspect, the embodiment of the invention also provides the application of the bacillus subtilis FM-20 strain in reducing the emission of ammonia gas and hydrogen sulfide in poultry excrement.

The bacillus subtilis FM-20 strain provided by the invention has higher protease-producing activity which can reach 202.3U/mL, and the generated antibacterial substances can also inhibit the activity of harmful bacteria such as escherichia coli and the like, reduce the conversion of protein into ammonia, amine and other harmful substances or gases, can tolerate gastric acid, bile salt and high temperature, and can be taken by poultry animals in a mode of mixing the bacillus subtilis FM-20 strain with feed or drinking water, so that the discharge amount of ammonia and hydrogen sulfide in excrement of the poultry animals can be reduced, the air pollution is reduced, and the environment in a house is improved.

In a third aspect, the embodiments of the present invention also provide a fermentation product of the above-mentioned Bacillus subtilis FM-20 strain, wherein the fermentation product is prepared by fermenting the Bacillus subtilis FM-20 strain. The product obtained by fermentation can be further processed into products which can be used in feed or drinking water.

Preferably, the method for producing the fermentation product comprises: culturing the bacillus subtilis FM-20 strain on an NA culture medium for 16-24h, inoculating the cultured strain into an NB culture solution, performing shake culture at 35-37 ℃ and 180-. The fermentation supernatant contains fermentation product of Bacillus subtilis FM-20 strain, and can be added into feed for feeding animals or daily drinking water for fowls and animals.

Preferably, the centrifugation is 10000r/min, and the centrifugation is carried out for 5min at 4 ℃.

In a fourth aspect, the embodiment of the invention also provides a microbial inoculum, wherein the microbial inoculum is prepared by fermenting the bacillus subtilis FM-20 strain. The microbial inoculum can be used as a feed additive to reduce the emission of ammonia gas and hydrogen sulfide in poultry excrement.

The preparation method of the microbial inoculum can adopt the following modes: and fermenting the bacillus subtilis FM-20 strain to obtain bacterial sludge, and drying the bacterial sludge and then mixing with the filler or respectively mixing with the filler and then drying to obtain the microbial inoculum.

The following operation processes can be specifically adopted:

culturing the bacillus subtilis FM-20 strain on an NA culture medium for 16-24h, inoculating the cultured strain into an NB culture solution, performing shake culture at 35-37 ℃ for 24-48h at 180-220r/min, centrifuging to obtain bacterial sludge, performing spray drying to obtain high-concentration bacterial powder, adding a filler, and mixing to obtain the microbial inoculum;

or culturing the bacillus subtilis FM-20 strain on an NA culture medium for 16-24h, inoculating the cultured strain into an NB culture solution, performing shake culture at 35-37 ℃ for 24-48h at 180-220r/min, centrifuging fermentation liquor to obtain bacterial sludge, mixing the obtained bacterial sludge with a filler, and performing flash evaporation drying to obtain the microbial agent.

The filler in the microbial inoculum can be at least one of bran, rice bran or rice chaff, and other common microbial inoculum excipients can also be adopted.

In a fifth aspect, the embodiment of the invention also provides a feed additive, and the feed additive comprises the microbial inoculum and other acceptable nutrient components in feed.

The other nutritional components comprise at least one of probiotics, minerals, vitamins, and herbal extracts.

Drawings

FIG. 1 is the colony morphology of FM-20 strain of example 1;

FIG. 2 shows spore staining of FM-20 strain in example 1;

FIG. 3 shows the bacterial cell staining of FM-20 strain in example 1;

FIG. 4 is a phylogenetic tree of FM-20 strain of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

The embodiment of the invention provides a bacillus subtilis FM-20 strain with high protease yield, which is obtained by screening through the following steps:

1. isolation and screening of protease producing Bacillus

1.1 Medium formulation

Protease identification medium: 5g of beef extract, 30g of casein, 5g of NaCl, 20g of agar and 1000mL of distilled water are added, and the pH value is natural.

NA medium: 10mg/ml peptone, 3mg/ml beef extract, 5mg/ml sodium chloride, 20mg/ml agar and the balance of distilled water, wherein the pH value is 7.2-7.4.

NB culture solution: 10mg/ml peptone, 3mg/ml beef extract, 5mg/ml sodium chloride and the balance of distilled water, wherein the pH value is 7.2-7.4.

LA medium: 10mg/ml peptone, 5mg/ml yeast extract, 10mg/ml sodium chloride, 20mg/ml agar, pH 7.0-7.2 and the balance of distilled water.

All media were prepared in distilled water and autoclaved at 121 ℃ for 20 min.

1.2 isolation and Primary screening of protease producing Bacillus

Taking 1.0g of a healthy broiler feces sample, placing the sample in a test tube of 9.0mL sterile water, and heating at 80 ℃ for 15 min. After mixing uniformly, 1.0mL of the solution is taken for gradient dilution to obtain a series of diluents with different concentrations. Respectively take 10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 0.1mL of the diluted solution was applied to a protease identification medium plate and incubated in a 37 ℃ incubator for 24 hours. Selecting a plate with the colony number of 30-300, and picking out a single colony which grows well and has an obvious transparent ring, namely a protease-producing strain. And co-screening to obtain 56 strains, wherein the ratio of the hydrolysis circle diameter to the colony diameter of 22 strains is more than 2.0. The bacterial colony of the 22 strains is transferred to an NA culture medium inclined plane, the serial number is FM-1-FM-22 (the ratio of the diameter of a hydrolysis ring of FM-20 to the diameter of the bacterial colony is maximum and reaches 5.6), the bacterial colony is cultured for 24 hours at the constant temperature of 37 ℃, and the bacterial colony is stored for later use at the temperature of 4 ℃.

1.3 rescreening of protease-producing functional strains

Transferring the 22 strains obtained by primary screening to an NA culture medium slant, and culturing at the constant temperature of 37 ℃ for 24h to activate the strains. Respectively inoculating the activated strains into NB culture solution, shake culturing at 37 deg.C and 220r/min for 48 hr, centrifuging at 4 deg.C and 10000r/min for 5min, and collecting supernatant. Uniformly punching holes on a sterilized protease identification culture medium flat plate by using a puncher, respectively taking 75 mu L of supernate into injection holes, culturing for 24h at the constant temperature of 37 ℃, screening out strains with the ratio of the diameter of a hydrolysis ring to the diameter of a bacterial colony being more than 2.0, namely strains with stronger protease-producing capacity, and taking the strains as re-screening strains, wherein the ratio of the diameter of the hydrolysis ring of FM-20 to the diameter of the bacterial colony is the largest and reaches 3.8.

1.4 determination of enzyme-producing Activity of fermentation broth of protease-producing functional Strain

15 strains with larger transparent circles in the re-screened strains are transferred to the slant of the NA culture medium and are cultured for 24 hours at the constant temperature of 37 ℃ to activate the strains. Inoculating the activated strain into NB culture solution, shake culturing at 37 deg.C and 220r/min for 48 hr, centrifuging at 4 deg.C and 10000r/min for 5min, and collecting supernatant. And (3) carrying out protease activity determination on the obtained fermentation supernatant by adopting a forskolin-phenol method.

Protease activity determination: the method adopts a forskolin-phenol method in a spectrophotometric method for determining the activity of neutral acid protease in the GB/T28715-2012 feed additive.

The result shows that 3 strains have higher enzyme activity, namely FM-2, FM-8 and FM-20 strains, wherein the activity of protease produced by the FM-20 strain is the maximum and reaches 202.3U/mL.

1.5 characteristic study of Bacillus to reduce the release of ammonia and hydrogen sulfide

1.5.1 characteristic test for Ammonia-suppressing ability of Bacillus

(1) Weighing 50g of fresh chicken manure, adding the four parts into a 250mL triangular flask respectively, wherein the three parts are inoculated with 10% of inoculum size (namely 10mL of fermentation broth supernatant is inoculated to every 100g of chicken manure, the same applies below) into FM-2, FM-8 and FM-20 fermentation broth supernatants, and fresh chicken manure without inoculation is used as a reference;

(2) measuring 20mL of the boric acid absorption solution, placing in a 50mL triangular flask, and dropping indicator (methyl red: methylene blue = 2: 1) for absorbing NH ₃ ；

(3) The 250mL triangular bottle filled with fresh chicken manure is connected with the 50mL triangular bottle filled with absorption solution by a rubber tube, so that the whole system has good sealing property. Culturing at 25 deg.C, and measuring once for 24h and 48h respectively;

(4) absorbing NH by the absorption liquid ₃ Then, the solution is mixed with blueChanging purple into green, titrating with calibrated 0.05N hydrochloric acid to restore transparent bluish purple before absorption liquid, and obtaining ammonia amount according to the amount of hydrochloric acid. The results are shown in Table 1.

TABLE 1 Ammonia emission from broiler faeces inoculated with different strains

The results show that 3 strains all have strong ammonia inhibition effect.

1.5.2 Bacillus Sulfur inhibition Property test

(1) Weighing 50g of fresh chicken manure, respectively adding the four fresh chicken manure into a 250mL triangular flask, wherein the three fresh chicken manure is inoculated into FM-2, FM-8 and FM-20 fermentation broth supernatants in an inoculation amount of 10%, and fresh chicken manure without inoculation is used as a reference;

(2) 20mL of H is measured ₂ The S absorption solution, namely the alkaline zinc ammine salt solution is placed in a 50mL triangular flask and is used for absorbing H ₂ S；

(3) A250 mL triangular bottle filled with fresh chicken manure is connected with a 50mL triangular bottle filled with absorption solution through a rubber tube, and the whole system is good in sealing performance. Culturing at constant temperature of 25 deg.C, and measuring once in 24h and 48h respectively;

(4) 10mL of H was taken ₂ S absorption solution (stock solution or diluted by a certain multiple) is placed in a test tube, 1mL of p-aminodimethylaniline solution is added into the test tube and shaken evenly, 1 drop of FeCl is added after 1min ₃ Shaking the solution evenly, reacting for 30min, and adding 1 drop of ammonium dihydrogen phosphate solution to terminate the reaction. The absorbance of each solution was measured at a wavelength of 760nm, and the amount of hydrogen sulfide discharged was calculated by recording data. The results are shown in Table 2.

TABLE 2 hydrogen sulfide emission from broiler excrements inoculated with different bacterial strains

The results show that FM-20 and FM-8 both have strong effect of inhibiting the emission of hydrogen sulfide, but FM-2 is not obvious.

1.6 inhibition test of bacillus on pathogenic bacteria of escherichia coli and preliminary identification of property of bacteriostatic substance

1.6.1 inhibition test of Bacillus on pathogenic bacteria of Escherichia coli

Pouring 5.0mL of sterile water into activated escherichia coli pathogen inclined test tubes in sequence, lightly scraping the pathogens by using a sterilized bamboo stick, pouring the formed bacterial suspension into the original sterile water test tubes, oscillating for 1min by using a vortex oscillator, and pouring into triangular flasks filled with 100mL of NA culture medium at about 50 ℃. And pouring the mixture into double-disc plates (the diameter is 9 cm) immediately after mixing, pouring about 15mL of the mixture into each plate, and cooling the plates for later use to obtain the pathogenic bacteria plates.

Transferring the screened FM-2, FM-8 and FM-20 strains to an NA culture medium slant, and culturing at a constant temperature of 37 ℃ for 24-36 h to activate the strains. Inoculating the activated strain into NB culture solution, shaking for 48h at 35-38 ℃ and 220r/min, centrifuging for 5min at 4 ℃ and 10000r/min, and taking supernatant. Taking 100 μ L of supernatant, applying to pathogenic bacteria plate (perforating with sterile perforator at certain interval for use), standing for 20min, culturing at constant temperature of 35-37 deg.C for 24-48h, and observing the size of inhibition zone.

The result shows that the screened FM-2, FM-8 and FM-20 strains all show the inhibition effect on the pathogenic bacteria of the escherichia coli, wherein the FM-20 inhibition zone is the largest, and the diameter reaches 32.5 mm.

1.6.2 preliminary characterization of the Properties of the bacteriostatic substance

(1) Solvent extraction

Activating FM-2, FM-8 and FM-20 strains, and standing in NB medium for liquid fermentation. Taking four fermentation liquor portions of 48h, each portion being 100mL, centrifuging, extracting with 1/3 volumes of chloroform, diethyl ether, ethyl acetate and n-butyl alcohol respectively, naturally volatilizing the extract liquor in a ventilated place until the extract liquor is dried, dissolving with 5.0mL of distilled water respectively, measuring the antibacterial activity of the extract liquor, raffinate and ammonium sulfate precipitate on a pathogenic bacteria plate by using an agar punching diffusion method, and taking distilled water as a negative control and the fermentation liquor as a positive control.

The results show that the extracts of the 4 organic solvents have no obvious bacteriostatic activity, the raffinate still has obvious bacteriostatic activity, and the ammonium sulfate salting-out precipitates have higher bacteriostatic activity, which is shown in Table 3.

TABLE 3 inhibitory Effect of the antibacterial extracts on Escherichia coli

Note: the "+" shows that the inhibition zone is larger, the "+" shows that the inhibition zone is present, the "+" shows that the inhibition zone is not obvious, and the "-" shows that the inhibition zone is not present

(2) Salting out of ammonium sulfate

200mL of fermentation liquor of FM-2, FM-8 and FM-20 strains in 48 hours are respectively taken, ammonium sulfate is slowly added to 80 percent of saturation degree under the condition of continuous stirring after centrifugation, the mixture is precipitated overnight, supernatant liquid is poured out, and the precipitate is dissolved by 50mL of distilled water and then is put into a 300-Da dialysis bag to be dialyzed overnight by the distilled water. The antibacterial activity of the dialysate is measured on a pathogen plate by an agar perforation diffusion method, and the negative control of distilled water and the positive control of fermentation liquor are simultaneously carried out on the same plate.

The results show that the ammonium sulfate salting-out precipitates have higher bacteriostatic activity, and are shown in Table 4.

TABLE 4 bacteriostatic activity of ammonium sulfate salting-out precipitate

Note: + indicates a larger zone of inhibition, + indicates a general zone of inhibition, -indicates no zone of inhibition

(3) Susceptibility testing of bacteriostats to trypsin

And (3) subpackaging fermentation supernatants of FM-2, FM-8 and FM-20 strains into an EP tube, adding 2.0% trypsin solution, taking the fermentation supernatant added with normal saline as a control, incubating at constant temperature of 37 ℃ for 0h, 0.5h, 1.0h, 1.5h and 2.0h, and observing the antibacterial effect of the fermentation liquor treated by trypsin for different times by using an agar punching and diffusion method. The results are shown in Table 5.

TABLE 5 sensitivity of the FM-20 Strain antibacterial to Trypsin

After trypsin is added into the supernatant obtained by fermenting the FM-20 strain, the bacteriostatic activity of the fermentation liquor is gradually reduced along with the increase of time until the bacteriostatic activity disappears. And the physiological saline added into the fermentation liquor is used as a control, and the bacteriostatic activity is not lost along with the increase of time.

(4) Autoclaving test

After high-pressure steam sterilization, the bacteriostatic activity of the FM-2, FM-8 and FM-20 strains is completely lost, and white precipitates are generated, which indicates that the bacteriostatic substances generated by the FM-2, FM-8 and FM-20 strains are sensitive to high temperature and high pressure.

From the above results, the bacteriostatic substances produced by the FM-2, FM-8 and FM-20 strains were antimicrobial proteins or antimicrobial peptides.

1.7 probiotic Property testing of Bacillus strains

1.7.1 test of the cholate resistance of Bacillus Strain

The FM-2, FM-8 and FM-20 strains are inoculated into an NA solid plate culture medium containing cholate of 0.3 percent by adopting a cross-shaped line-drawing inoculation method, cultured at 37 ℃ and observed after 5 days. As a result, it was found that FM-2 and FM-20 strains grew well on NA plates containing 0.3% cholate, that FM-8 strain did not grow, and that it was not tolerant to cholate.

1.7.2 tolerance test of Bacillus strains to Artificial gastric juice

Adding 16.4mL of 9.5-10.5% hydrochloric acid, and diluting with water to make pH values reach 2.0, 3.0 and 4.0 respectively; adding 1.0g pepsin into 100mL of liquid, mixing, and sterile filtering with 0.22 μm microporous membrane.

Inoculating activated FM-2, FM-8 and FM-20 strains into the artificial gastric juice with different pH values according to the inoculation amount of 1.0% (v/v), respectively, performing shake culture at 37 ℃ and 220r/min, and performing viable bacteria plate colony counting at 0h, 2h and 4 h. The results show that the FM-2 and FM-20 strains have stronger tolerance capability to gastric juice environment.

1.7.3 tolerance test of Bacillus strains to Artificial intestinal fluids

Take KH ₂ PO ₄ 6.8g, adding 500mL of water for dissolution, adjusting the pH value to 6.8 by using 0.4% sodium hydroxide solution, adding water for dilution to 1000mL, adding 1.0g of trypsin into each 100mL of the liquid, uniformly mixing, and performing sterile filtration by using a 0.22 mu m microporous membrane for later use.

Inoculating activated FM-2, FM-8 and FM-20 strains to the artificial intestinal juice according to the inoculation amount of 1.0% (v/v), performing shake culture at 37 ℃ at 220r/min, and performing viable bacteria plate colony counting at 0h, 2h, 4h and 6 h. The test results show that the artificial intestinal juice has little influence on the growth of the FM-2 and FM-20 strains, which indicates that the FM-2 and FM-20 strains have strong tolerance to the artificial intestinal juice. The FM-8 strain has low tolerance to artificial intestinal juice.

1.8 high temperature tolerance test of Bacillus strains

The following tests were carried out by inoculating the strains FM-2, FM-8 and FM-20 to LB medium and culturing at 37 ℃ for 48 hours to form a large number of spores: placing in 80 deg.C water bath for incubation, and sampling before incubation and during incubation for 40min to determine bacterial spore survival rate; placing in 90 ℃ water bath for incubation, and sampling before incubation and after incubation for 20min to determine the survival rate of bacterial spores; the culture was incubated in a water bath at 100 ℃ and samples were taken before and after 10min of incubation to determine the survival rate of bacterial spores.

Test results show that the strains FM-2, FM-8 and FM-20 almost completely survive in water bath at 80 ℃ for 40min, 74.65% -75.75% survive in water bath at 90 ℃ for 20min, and 42.00% -50.62% survive in water bath at 100 ℃ for 10 min. The screened FM-2, FM-8 and FM-20 strains have stronger tolerance to high temperature.

2. Species identification of Bacillus subtilis FM-20 strain

And (3) identifying the species of the strain FM-20 with better performance in all aspects.

2.1 colony and thallus morphological characteristics of FM-20 Strain

The bacterial colony of the strain is in an irregular shape, the edge is irregular, the surface is not smooth and has wrinkles, and no pigment is generated;

gram-positive, rod-shaped, and oval-shaped spores (see FIGS. 1-3).

2.2 sequencing results of 16S rDNA of FM-20 Strain

TABLE 6 16S rDNA sequence similarity of FM-20 and reference strains

A phylogenetic tree of the FM-20 strain is shown in FIG. 4.

From the above results, it was found that the 9 standard strains having the highest homology with the 16S rDNA sequence of the strain FM-20 belong to the genus Bacillus, wherein the strains FM-20 and FM-20 are derived from the sameBacillus subtilisThe corresponding homology similarity of the 16S rDNA sequence is 100%, and other speciesBacillus cabrialesii、Bacillus tequilensis、Bacillus mojavensis、 Bacillus vallismortis、Bacillus amyloliquefaciens、Bacillus atrophaeus、Bacillus licheniformis、Bacillus aerius、Bacillus stratosphericusThe homologous similarity of the sequence of the strain FM-20 is 97.12-99.93%, so that the strain FM-20 can be preliminarily judged to belong to the bacillus.

2.3 physiological and Biochemical Properties of FM-20 Strain

The strain is positive for catalase; glucose and mannitol can be utilized, and glucose is not utilized for generating gas; the reaction of V-P, gelatin liquefaction, casein hydrolysis, nitrate reduction and starch hydrolysis is positive, and the reaction of urease is negative; can tolerate 10 percent of NaCl and has positive nitrate reduction reaction.

In conclusion, the FM-20 is identified as a bacillus subtilis strain by combining the results of 16S rDNA sequence analysis and physiological and biochemical identificationBacillus subtilis) And (3) strain.

The bacillus subtilis FM-20 strain is preserved in China general microbiological culture Collection center (CGMCC) on 2021, 5 months and 12 days, and the preservation number is CGMCC 22509; the preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang, Beijing.

Example 2

This example provides a fermentation product of Bacillus subtilis FM-20 strain, which is prepared by the following steps: culturing the bacillus subtilis FM-20 strain obtained in example 1 on an NA culture medium for 24h, inoculating the cultured strain into an NB culture solution, performing shake cultivation for 48h at 37 ℃ and 220r/min, centrifuging, and taking supernatant to obtain the bacillus subtilis FM-20 strain.

Example 3

This example provides a microbial inoculum prepared by fermenting the Bacillus subtilis FM-20 strain obtained in example 1. The preparation method comprises the following steps:

culturing the bacillus subtilis FM-20 strain obtained in the example 1 on an NA culture medium for 24 hours, inoculating the cultured strain into an NB culture solution, performing shake culture at 35-37 ℃ and 200r/min for 48 hours, centrifuging by using a disc centrifuge to obtain bacterial sludge, performing spray drying to obtain high-concentration bacterial powder, and adding bran to mix to obtain the microbial inoculum (1.12 x 10) ¹⁰ CFU/g）。

Example 4

culturing the bacillus subtilis FM-20 strain obtained in the example 1 on an NA culture medium for 24 hours, inoculating the cultured strain into an NB culture solution, performing shake culture at 35-37 ℃ and 200r/min for 48 hours, centrifuging fermentation liquor to obtain bacterial sludge, mixing the bacterial sludge with bran, and performing flash evaporation drying to obtain the microbial inoculum (1.19 multiplied by 10) ¹⁰ CFU/g）。

Example 5

The present example provides the use of a strain of Bacillus subtilis FM-20 as a feed additive for reducing ammonia and hydrogen sulfide emissions from poultry excreta.

1. Procedure of the test

The 14-day-old healthy Ross 308 broiler chickens are randomly divided into two groups, the microbial inoculum obtained in example 3 is added into chicken feed according to the addition amount of 0.1% (w/w) to serve as a test group, chicken groups which only eat the same feed and are not added with the microbial inoculum are used as a control group, and the conventional feeding management is carried out in a pre-feeding period of 7d and a testing period of 21 d. And respectively measuring the growth performance and the health level of the broiler chickens in the control group and the test group, and simultaneously detecting the protease activity, the total nitrogen and ammonia nitrogen content, the ammonia and hydrogen sulfide release amount for 48 hours, the ammonia and hydrogen sulfide content in the air of the chicken coop and the microorganism content in the air in the two groups of broiler chickens.

2. Test results and analysis

2.1 feeding test results

TABLE 1 feeding test Effect

As can be seen from Table 1, in the feeding test, the diarrhea rate of the test group is reduced by 76.07% (P < 0.01) and the mortality rate is reduced by 35.31% (P < 0.01) compared with the control group by 11.37% (P < 0.05).

2.2 determination results of digestive enzyme activity, total nitrogen and ammoniacal nitrogen in broiler manure

TABLE 2 detection results of protease activity in broiler manure

As can be seen from Table 2, after FM-20 spore probiotics 7d, 14d and 21d are fed, the protease activity in the chicken manure of the test group is respectively improved by 56.06%, 60.42% and 61.44%, and is respectively improved by 52.37%, 44.89% and 36.96% compared with the control group; the total nitrogen in the test group broiler manure is respectively reduced by 41.79%, 47.76% and 49.25%, and compared with the control group broiler manure is respectively reduced by 45.07%, 52.05% and 54.05%; the ammonia nitrogen in the test group broiler manure is respectively reduced by 71.82%, 76.82% and 79.55%, and compared with the control group broiler manure is respectively reduced by 76.15%, 84.06% and 87.14%.

2.3 detection results of Ammonia and Hydrogen sulfide gas in the House

The measuring time is selected when the person moves less, and the door and the window are closed in advance, so that the henhouse is in a closed state.

TABLE 3 gas concentration values in broiler chicken coop at different measurement times

As is clear from table 3, the content of harmful gases such as ammonia and hydrogen sulfide in the chicken house slightly increased with the age of the day. After FM-20 spore microecologics 7d, 14d and 21d are fed, the ammonia gas content in the test house is respectively reduced by 61.84%, 62.50% and 63.16%, and compared with the control group, the ammonia gas content is respectively reduced by 64.20%, 67.98% and 70.05%; the hydrogen sulfide content in the test group housing is respectively reduced by 60%, 60.77% and 62.31%, and compared with the control group, the hydrogen sulfide content is respectively reduced by 67.50%, 70% and 71.18%. Meanwhile, in tests, the content of ammonia and hydrogen sulfide measured in the afternoon is obviously increased relative to the value measured in the morning, and the content of ammonia and hydrogen sulfide is obviously reduced after excrement is cleaned. The results show that the activities of the chicken flocks and the manure cleaning have obvious influence on the content of harmful gases in the henhouse.

2.4 detection result of harmful gas release amount of fresh feces

TABLE 4 harmful gas release amount of meat chicken manure

As is clear from table 4, the content of harmful gases such as ammonia and hydrogen sulfide in the chicken house slightly increased with the age of the day. After the FM-20 spore microecologics 7d, 14d and 21d are fed, the ammonia gas release amount of the fresh excrement in the test group for 48 hours is respectively reduced by 23.33%, 30.90% and 32.06% compared with the control group; compared with a control group, the release amount of hydrogen sulfide of the fresh feces of the test group for 48 hours is respectively reduced by 16.92 percent, 14.69 percent and 15.99 percent.

2.5 microbial content in air in henhouse

TABLE 5 Total bacteria count and Escherichia coli genus in broiler chicken coop

Table 5 shows that the total number of bacteria in the test colony house is respectively reduced by 48.96%, 48.17% and 48.37% after FM-20 spore probiotics 7d, 14d and 21d are fed, and the total number of bacteria in the test colony house is respectively reduced by 56.94%, 56.49% and 57.33% compared with the control colony; the numbers of Escherichia coli in the test group house were respectively reduced by 75.74%, 74.56% and 72.19%, and were respectively reduced by 78.31%, 77.95% and 77.83% compared with the control group.

Example 6

The embodiment of the invention provides a feed additive, which comprises the microbial inoculum obtained in the embodiment 3 or 4.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

SEQUENCE LISTING

<110> university of agriculture in Hebei

<120> bacillus subtilis FM-20 strain with high protease yield and application thereof

<130> 2021.7.13

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1422

<212> DNA

<213> FM-20

<400> 1

tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60

acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120

atggttgttt gaaccgcatg gttcaaacat aaaaggtggc ttcggctacc acttacagat 180

ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag 240

ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300

ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360

gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtac cgttcgaata 420

gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480

ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540

tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600

aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660

tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720

gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780

agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840

gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900

catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960

tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020

gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080

ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140

tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200

caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260

gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320

cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380

cccgaagtcg gtgaggtaac cttttaggag ccagccgccg aa 1422

Claims

1. A protease-producing Bacillus subtilis FM-20 strain characterized by its classification name of Bacillus subtilis (Bacillus subtilis: (B))Bacillus subtilis) The strain is preserved in China general microbiological culture Collection center on 12 th 5 th 2021 with the preservation number of CGMCC 22509; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.

2. Use of the bacillus subtilis FM-20 strain of claim 1 for reducing ammonia and hydrogen sulfide emissions from poultry excreta.

3. The fermentation product of the bacillus subtilis FM-20 strain of claim 1, wherein the fermentation product is produced by fermentation of the bacillus subtilis FM-20 strain.

4. The fermentation product of bacillus subtilis FM-20 strain according to claim 3, wherein the fermentation product is prepared by a process comprising: culturing the bacillus subtilis FM-20 strain on an NA culture medium for 16-24h, inoculating the cultured strain into an NB culture solution, performing shake cultivation at 35-37 ℃ for 24-48h at 180-220r/min, centrifuging, and taking a supernatant.

5. The fermentation product of Bacillus subtilis FM-20 strain according to claim 4, wherein the centrifugation is 10000r/min, 5min at 4 ℃.

6. A microbial inoculum, which is prepared by fermenting the bacillus subtilis FM-20 strain of claim 1.

7. The microbial inoculum according to claim 6, which is prepared by the following method: fermenting the bacillus subtilis FM-20 strain to obtain bacterial mud, drying the bacterial mud and then mixing the bacterial mud with a filler or mixing the bacterial mud and the filler and then drying to obtain the microbial inoculum.

8. The microbial inoculant according to claim 7, wherein said bulking agent comprises at least one of bran, rice bran or rice chaff.

9. A feed additive, which is characterized by comprising the microbial inoculum of claim 6 and other acceptable nutrient components in feed.

10. The feed additive of claim 9 wherein the other nutritional components comprise at least one of probiotics, minerals, vitamins, herbal extracts.