CN113549575A - Bacillus subtilis FM-20 strain for high yield of protease and application thereof - Google Patents
Bacillus subtilis FM-20 strain for high yield of protease and application thereof Download PDFInfo
- Publication number
- CN113549575A CN113549575A CN202110819990.1A CN202110819990A CN113549575A CN 113549575 A CN113549575 A CN 113549575A CN 202110819990 A CN202110819990 A CN 202110819990A CN 113549575 A CN113549575 A CN 113549575A
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- bacillus subtilis
- protease
- strains
- microbial inoculum
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Links
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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
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)2S) 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 has low temperature, high humidity and poor ventilation effect, and the concentration of harmful gases such as ammonia gas and hydrogen sulfide is higher, 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 the regulation of the daily ration structure of livestock and poultry to control the generation of odor, such as the application of enzyme preparations, plant type extracts, microecological preparations, compounds such as ferrous sulfate and the like, functional oligosaccharides such as soybean oligosaccharides and the like, and reduce pollution gas and improve the internal environment of a chicken house in a spice masking type mode such as zeolite powder and the like adsorption and natural aromatic oil and the like so as to improve the growth performance and the 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 embodiment of the invention provides a Bacillus subtilis FM-20 strain with high protease yield, which is classified as Bacillus subtilis and preserved in China general microbiological culture Collection center (CGMCC) at 12 days 5 months 2021 with the preservation number of CGMCC No. 22509; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.
Harmful gases such as ammonia gas, hydrogen sulfide and the like are mainly generated by undigested nutrient substances and some 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, amylase and the like generated by the bacillus subtilis FM-20 strain can enhance the intestinal function of poultry animals, promote the digestion and absorption of the poultry animals on nutrient substances, and improve the protease activity of a digestion system of the poultry animals, so that the digestion and absorption rate of the nutrient substances such as protein and the like is improved, nitrogen and sulfur-containing substances are reduced to be discharged in the form of excrement, 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 a sterilized protease identification medium plate by using a puncher, respectively taking 75 mu L of obtained supernatant liquid into a filling hole, culturing at the constant temperature of 37 ℃ for 24h, 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;
and thirdly, screening through protease activity determination, an ammonia inhibition characteristic test, a sulfur inhibition characteristic test, an inhibition test on pathogenic bacteria of escherichia coli, preliminary identification of the property of an antibacterial substance, a cholate resistance characteristic test, an artificial gastric juice tolerance test, an artificial intestinal juice tolerance test and a high-temperature tolerance test 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 ℃ for 24-48h at 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 of fowls.
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: fermenting the bacillus subtilis FM-20 strain to obtain bacterial sludge, drying the bacterial sludge, and mixing the bacterial sludge with a filler or respectively mixing the bacterial sludge with the filler and 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 cultivation at 35-37 ℃ and 180-220r/min for 24-48h, centrifuging a fermentation liquid to obtain bacterial sludge, mixing the obtained bacterial sludge with a filler, and performing flash evaporation drying to obtain the microbial inoculum.
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, wherein the feed additive comprises the microbial inoculum and other acceptable nutrient components in feed.
The other nutrient components include at least one of probiotics, minerals, vitamins, and herbal extracts.
Drawings
FIG. 1 is a 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 further described in detail with reference to the following 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、10-60.1mL 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. Will be provided withThe bacterial colonies of 22 strains are transferred to the slant of the NA culture medium, the numbers of FM-1 to FM-22 (the ratio of the diameter of a hydrolysis ring of FM-20 to the diameter of the bacterial colonies is maximum and reaches 5.6), the mixture is cultured for 24 hours at the constant temperature of 37 ℃, and the mixture is stored for standby 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 a sterilized protease identification medium plate by using a puncher, respectively taking 75 mu L of supernate into injection holes, culturing at the constant temperature of 37 ℃ for 24h, and 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 production capacity, 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
And (3) transferring 15 strains with larger transparent rings in the re-screened strains to an NA culture medium inclined plane, and placing the inclined plane at the constant temperature of 37 ℃ for culturing for 24 hours 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 measuring the activity of neutral acidic protease in GB/T28715-2012 feed additives.
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 adding dropwise indicator (methyl red blood/methylene blue 2/1) for absorbing NH3;
(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 liquid3Then, the solution changes from blue-purple to green, and the calibrated hydrochloric acid of 0.05N is used for titration, so that the transparent blue-purple is recovered from the absorption liquid, and the ammonia amount is obtained according to the using amount of the 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 characterization of Bacillus for Sulfur inhibition
(1) Weighing 50g of fresh chicken manure, adding the four parts into a 250mL triangular flask respectively, wherein the three parts are inoculated with 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 control;
(2) 20mL of H was measured2The S absorption solution, namely the alkaline zinc ammine salt solution is placed in a 50mL triangular flask and is used for absorbing H2S;
(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) 10mL of H was taken2Placing S absorption solution (stock solution or diluted by a certain multiple) in a test tube, adding 1mL of p-aminodimethylaniline solution into the test tube, shaking up, adding 1 drop of FeCl after 1min3Shaking 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 9cm) 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-butanol respectively, naturally volatilizing the extract liquor in a ventilation 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 an agar punching and diffusion method, and taking distilled water as a negative control and taking 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 antibiotic of the FM-20 Strain 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.
According to the preliminary analysis of the results, the bacteriostatic substances produced by the FM-2, FM-8 and FM-20 strains are antibacterial proteins or antibacterial peptides.
1.7 probiotic Property testing of Bacillus strains
1.7.1 test of the cholate resistance of Bacillus Strain
Inoculating FM-2, FM-8 and FM-20 strains into NA solid plate culture medium containing cholate 0.3% by cross-hatching inoculation method, culturing at 37 deg.C, and observing after 5 d. As a result, it was found that the FM-2 and FM-20 strains grew well on the plate containing 0.3% cholate NA, and that the FM-8 strain did not grow and 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 according to the inoculum size of 1.0% (v/v), respectively, performing shake culture at 37 deg.C 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 Bacillus Strain tolerance test to Artificial intestinal juice
Take KH2PO46.8g, 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 20min incubation 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 morphology characteristics of FM-20 Strain
The bacterial colony of the strain is irregular in shape, irregular in edge, unsmooth in surface, wrinkled and free of pigment generation;
gram-positive, rod-like, and oval spores (see FIGS. 1-3).
2.2 sequencing 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, 9 standard strains having the highest homology similarity with the 16S rDNA sequence of the strain FM-20 belong to the genus Bacillus, wherein the homology similarity between the strain FM-20 and the 16S rDNA sequence of Bacillus subtilis is 100%, and the homology between the other species Bacillus calbranelisii, Bacillus tequilensis, Bacillus mojavensis, Bacillus vallisportis, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus aereiformis, Bacillus amyloliquefaciens and strain FM-20 is 97.12-99.93%, so that the strain FM-20 can be preliminarily judged to belong to the genus 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 identification.
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 No. 22509; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.
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 ℃ for 48 hours at 200r/min, 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 multiplied by 10)10CFU/g)。
Example 4
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 example 1 on an NA culture medium for 24 hours, inoculating the cultured strain into an NB culture solution, performing shake cultivation for 48 hours at 35-37 ℃ at 200r/min, centrifuging fermentation liquor to obtain bacterial sludge, mixing the bacterial sludge with bran, and performing flash evaporation drying to obtain a microbial inoculum (1.19 multiplied by 10)10CFU/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 are fed for 7d, 14d and 21d, 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 the test, the content of ammonia gas and hydrogen sulfide measured in the afternoon is obviously increased relative to the value measured in the morning, and the content of ammonia gas and hydrogen sulfide is obviously reduced after manure cleaning. The results show that the activities of the chicken flocks and the manure cleaning have a significant influence on the content of harmful gases in the henhouse.
2.4 detection result of harmful gas release amount of fresh excrement
TABLE 4 amount of harmful gas released from excrements of broiler chickens
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 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 (10)
1. A Bacillus subtilis FM-20 strain for highly producing protease is characterized in that the Bacillus subtilis FM-20 strain is classified as Bacillus subtilis, is preserved in China general microbiological culture Collection center (CGMCC) on 5-month and 12-month 2021, and has the preservation number of CGMCC No. 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 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.
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CN114672437B (en) * | 2022-04-15 | 2023-08-11 | 河北谷旺金莱生物科技有限公司 | Bacillus subtilis G-1 for producing organic acid and application thereof |
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