CN110643550A - Bacillus subtilis J530 strain for producing urease inhibitor and application thereof - Google Patents
Bacillus subtilis J530 strain for producing urease inhibitor and application thereof Download PDFInfo
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Abstract
The invention relates to a urease inhibitor producing bacillus subtilis J530 strain and application thereof, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 9 and 17 months, the preservation address is No. 3 of Xilu No.1 of Beijing Korean Chen, and the preservation number is CGMCC NO: 18603. the bacillus subtilis J530 strain has high activity of producing urease inhibitor, and can inhibit urea or uric acid in livestock and poultry manure from being decomposed into ammonia by urease.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a bacillus subtilis J530 strain for producing a urease inhibitor and application thereof.
Background
In recent years, with the rapid development of intensive and large-scale breeding industry, environmental pollution caused by volatilization and emission of livestock and poultry excrement ammonia is more and more concerned by people, and the volatilization and emission of ammonia not only causes pollution to surface water and soil, but also brings serious air pollution and causes great harm to the health of raised animals, feeders and residents around raising farms. In the current cultivation manure treatment system, a large amount of urea (such as pigs and other livestock) or uric acid (such as chickens and other poultry which can be converted into urea after decomposition) contained in the manure of livestock and poultry is easily decomposed into ammonia by urease generated by microorganisms and volatilized, so that 75% of nitrogen in the manure is lost, and the N/P ratio of the manure of livestock and poultry is also disordered, for example, the N/P ratio of fresh pig manure is about 5: 1, while the N/P ratio of the stored pig manure is 1:1, so that a large amount of nitrogen element is lost, and the fertilizer efficiency is reduced. Therefore, the reduction of the ammonia emission of the livestock and poultry manure has important significance for environmental protection and reutilization of the livestock and poultry manure.
Currently, urease inhibitors have been used to reduce ammonia emissions in manufacturing practice. The existing urease inhibitor mainly comprises inorganic compounds (such as heavy metal salts, borate, chloride, fluoride, phosphate, nickel nitrate and the like), organic compounds (such as ectopic acids, urea derivatives, hydroxylamine, oximinoacids, paraformaldehyde, quinone, polyphenol, heterocyclic mercaptan and the like) and plant extracts (such as yucca extract, lauraceae plant extract, garlic extract and the like). Inorganic and organic compounds have safety problems, are toxic and harmful to people, livestock and environment, and are difficult to meet the requirements of the breeding industry. Plant extracts are relatively safe, but the plant growth period is long, the raw material source is unstable, the extraction process is complex, the large-scale production cost is high, the efficiency is low, the inhibition effect on urease is low, and the capacity of reducing ammonia volatilization is limited.
Disclosure of Invention
Aiming at the technical problems that the existing urease inhibitor has toxic hazard and pollution to human, livestock and environment and low inhibition rate on ammonia, the invention provides a bacillus subtilis J530 strain for producing the urease inhibitor.
The invention also provides application of the bacillus subtilis J530 strain in reducing fecal ammonia emission.
The invention also provides a composite microbial inoculum.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a urease inhibitor producing Bacillus subtilis J530 strain is classified as Bacillus subtilis, and is preserved in China general microbiological culture Collection center (CGMCC) on 9 and 17 days in 2019 with the preservation number of CGMCC NO: 18603; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.
The bacillus subtilis J530 strain provided by the invention is obtained by separating and screening healthy chicken manure in broiler farms, belongs to bacillus, is non-toxic and pollution-free, has high activity of producing urease inhibitors, and has a 16S rDNA sequence shown as SEQ ID No. 1. The bacillus subtilis J530 strain can grow and reproduce in livestock and poultry intestinal tracts and generate a urease inhibitor, urea or uric acid in livestock and poultry excrement is inhibited from being decomposed into ammonia by urease, the inhibition rate of the urease activity in the excrement of pigs, chickens and the like can reach more than 99%, and the ammonia content in the intestinal tracts and blood of the livestock and poultry is effectively reduced. Experiments prove that the bacillus subtilis J530 strain can well grow by utilizing a 'similar feed culture medium' (a culture medium with components close to those of livestock and poultry feed) under a facultative anaerobic condition, and can generate 65.82U/ml acid protease, so that the bacillus subtilis J530 strain can adapt to the intestinal environment of livestock and poultry and can play a probiotic role in promoting protein digestion in the intestinal tract. In addition, when the bacillus subtilis J530 strain is discharged out of the body along with excrement or is directly added into the excrement in the body, the urease activity can be continuously inhibited in the composting process, and the inhibition rate of the bacillus subtilis J530 strain on ammonia in an in-vitro excrement ammonia emission test can reach 90.8%, so that the effect of reducing the emission of ammonia is achieved.
On the other hand, the bacillus subtilis J530 strain can also generate antibacterial peptide substances, can generate antibacterial activity equivalent to 52.49U/ml of terramycin under the condition of a shake flask test, has the effect of inhibiting diarrhea pathogenic bacteria such as escherichia coli, salmonella, clostridium welchii and the like, and can prevent and treat livestock and poultry diarrhea.
The screening method of the bacillus subtilis J530 strain specifically comprises the following steps:
placing fresh chicken manure of healthy chicken flocks in a broiler farm at normal temperature for 24 hours to prepare a bacterial suspension, carrying out water bath at 80 ℃ for 10 minutes, coating the bacterial suspension on an NA culture medium after gradient dilution for conventional culture, and inoculating a bacterial colony obtained by culture to a flat primary screen culture medium for 24 hours; the plate primary screening medium contained 20mg/ml urea, 1mg/ml phenol red, 10mg/ml sucrose, 10mg/ml peptone, 3mg/ml beef powder, 5mg/ml sodium chloride and 20mg/ml agar, the balance being distilled water, and the pH was 6.4.
Picking out colonies which are not discolored at the periphery from the colonies obtained by the culture in the step one, performing slant culture for 36h by using NA culture media respectively, then inoculating the colonies into a shaking flask primary screening culture medium for shake cultivation for 1 day, and taking a non-turbid sample as a sample fermentation liquid to be selected; the shaking flask primary screening culture medium contains 20mg/ml urea, 10mg/ml sucrose, 5mg/ml potassium chloride, 0.5mg/ml magnesium sulfate, 0.5mg/ml calcium chloride, 1mg/ml sodium dihydrogen phosphate and 1mg/ml disodium hydrogen phosphate, the balance is distilled water, and the pH is 6-8.
Thirdly, selecting colonies which turn red around from the colonies obtained by the culture in the step one, respectively culturing the colonies for 48 hours by using an NA culture medium, and then inoculating the colonies in an NB culture medium for shake cultivation for 48 hours to obtain fermentation liquor of each colony; adding urea aqueous solution and phenol red indicator into the obtained bacterial colony fermentation liquor respectively, shaking up, and taking fermentation liquor with shortest pink red appearance time, namely urease-producing strain fermentation liquor; adding 10ml of urea buffer solution into 200 mu l of urease-producing strain fermentation liquor, fully shaking and mixing, immediately placing in a constant-temperature water bath at 30 +/-0.5 ℃ for 30 +/-10 min, adding 10ml of hydrochloric acid solution, quickly cooling to below 20 ℃ after shaking, and titrating with a sodium hydroxide standard solution until the solution is blue-green; adding 10ml hydrochloric acid solution into 200 μ l of the urease producing strain fermentation broth inactivated by 100 deg.C water bath for 10min, shaking, adding 10ml urea buffer solution, shaking thoroughly, immediately placing in 30 + -0.5 deg.C constant temperature water bath for 30min + -10 s, rapidly cooling to below 20 deg.C, and titrating with sodium hydroxide standard solution until the solution is blue-green; judging the activity of the urease-producing strain fermentation liquor according to the volume of the sodium hydroxide standard solution consumed by the two urease-producing strain fermentation liquors; wherein the urea buffer solution is a phosphate buffer solution containing 0.5mol/L urea and having a pH of 7.0 +/-0.1, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1 mol/L.
Mixing the fermentation liquor of each sample to be selected obtained in the step (c) with the fermentation liquor of the urease-producing strain obtained in the step (c), adding urea and phenol red indicator, diluting and shaking up to be used as a test article; preparing a sample without the fermentation liquor of the sample to be selected by the same method as a positive control; comparing the time of the test products changing into pink with the positive control, and taking the strain corresponding to the test product with the color changing time longer than the positive control for 1.5min, namely the strain producing the urease inhibitor.
Fifthly, detecting the activity of the strain obtained in the fourth step by using a method for measuring the urease activity in the soybean product for the feed GB/T8622-2006, and re-screening to obtain the bacillus subtilis J530 strain.
The embodiment of the invention also provides application of the bacillus subtilis J530 strain in reducing fecal ammonia emission.
The bacillus subtilis J530 strain provided by the invention has high-efficiency and stable ammonia-reducing capability, can effectively control the activity of urease in excrement by adding daily ration or directly adding the daily ration into the excrement, reduces the decomposition of urea nitrogen or uric acid nitrogen in the excrement, can reduce the fertilizer efficiency loss of compost by reducing the emission of ammonia, and has great application potential in the aspects of recycling, harmlessly treating livestock waste, treating environmental pollution and promoting the healthy and sustainable development of livestock industry.
Preferably, the bacillus subtilis J530 strain is prepared into a microbial agent for feed additives or a fecal composting microbial agent, and the bacillus subtilis J530 strain is prepared into a microorganismThe method of the biological agent comprises the following steps: liquid submerged fermentation is carried out firstly, then the obtained fermentation liquor is continuously centrifuged, and finally the precipitate obtained by centrifugation is spray-dried. The daily ration or the daily ration is directly added into the excrement, so that the intestinal ammonia concentration of the poultry and the ammonia concentration of the environment of the poultry house can be reduced, the environment in the livestock and poultry colony house is improved, the ammonia stress is reduced, and the immunity and the production performance of the livestock and poultry are improved. When the microbial inoculum is used for the manure compost, the reduction of the N/P ratio of the compost can be reduced, and the compost maturity is promoted. The bacillus subtilis J530 strain is subjected to a process of combining liquid deep fermentation, continuous centrifugation and spray drying, and the viable bacteria (spore) content of the obtained bacterial powder can reach 5.0 multiplied by 1011CFU/g. The obtained bacterial powder can be mixed with soluble starch to make into feed additive, so that the final prepared microbial feed additive has viable bacteria (spore) content of 1.0 × 1010CFU/g, water content less than or equal to 8%, and the obtained feed additive can be added into feed and can also be used for drinking water, and has a shelf life of 24 months.
The embodiment of the invention also provides a composite microbial inoculum, which is a bacterial strain containing the bacillus subtilis J530 bacterial strain and other urease inhibitors. The microorganisms producing urease in the excrement are various, the structure and the property of the produced urease have certain difference, different urease inhibitors are combined to have a synergistic effect on the ammonia emission reduction function, and the effect of improving the ammonia emission reduction is achieved.
Preferably, the screening method of the bacterial agent containing other strains capable of producing urease inhibitors specifically comprises the following steps:
step a, preparing feces into bacterial suspension, conventionally culturing by using an NA culture medium, and inoculating a bacterial colony obtained by culture into a primary screening culture medium containing urea and an acid-base indicator for culture;
b, picking surrounding non-discolored colonies from the colonies obtained by culturing in the step a, and screening strains which do not produce urease to prepare a sample fermentation liquor to be selected;
c, selecting surrounding discolored colonies from the colonies obtained by culturing in the step a to prepare urease-producing strain fermentation liquor;
and d, mixing the fermentation liquor of the urease-producing strain obtained in the step c with the fermentation liquor of the urease-non-producing strain obtained in the step b, and screening the urease inhibitor-producing strain.
The screening method is simple and easy to implement, does not need expensive and complicated equipment, has low cost, can be realized in a common microorganism laboratory, and has high production efficiency and low cost of large-scale production. The bacterial strain in the fermentation liquor of the sample to be selected, which is obtained in the step b, is a bacterial strain which does not produce urease, the fermentation liquor of the bacterial strain which is produced in the step c is urease solution produced by microorganisms, and the method combining the two steps of screening in the step b and the step d can improve the screening efficiency, facilitate the screening of the bacterial strain which has the inhibiting effect on urease and is more suitable for reducing the ammonia emission of livestock and poultry manure, and save a large amount of manpower and material resources.
Preferably, the livestock and poultry manure in the step a is manure prepared by placing fresh livestock and poultry manure for 24-36 hours at normal temperature.
Preferably, the method for preparing the fermentation liquor of the sample to be selected by screening the strains which do not produce urease in the step b comprises the following steps: and respectively using the peripheral non-discolored colonies for 24-48 h by using an NA culture medium, then inoculating the colonies in a shake flask primary screening culture medium containing urea for shake cultivation for 1-2 days, wherein the non-turbid sample is the fermentation liquor of the sample to be selected. If the strain is not turbid in a shake flask culture medium containing urea, the strain does not grow, and the strain is proved to be incapable of producing urease and utilizing urea as a nitrogen source. Therefore, the strain corresponding to the non-turbid sample is a non-urease-producing strain, and the non-turbid sample is a fermentation broth of the non-urease-producing strain. The composition of the shake flask prescreening culture medium is preferably as follows: 20mg/ml urea, 10mg/ml sucrose, 5mg/ml potassium chloride, 0.5mg/ml magnesium sulfate, 0.5mg/ml calcium chloride, 1mg/ml sodium dihydrogen phosphate and 1mg/ml disodium hydrogen phosphate, and the balance of distilled water, wherein the pH value is 6-8. The culture method using NA medium is preferably slant culture.
Preferably, the method for preparing the urease producing strain fermentation broth in step c comprises the following steps: selecting colonies which turn red around from the colonies obtained by the culture in the step a, respectively culturing the colonies for 24-48 h by using an NA (Nurse number) culture medium, and then inoculating the colonies in an NB (NB) culture medium for shake cultivation for 24-48 h to obtain fermentation liquor of each strain; and (3) adding a urea aqueous solution and a phenol red indicator into the obtained strain fermentation liquor respectively, shaking up, and taking the fermentation liquor with the shortest pink red appearance time, namely the urease-producing strain fermentation liquor. The colony with the periphery changed into red is the colony of the urease-producing strain, the larger the red range is spread, the stronger the urease-producing activity is, and the colony with the red range as large as possible is preferred in the step, so that the activity of the obtained urease-producing strain is stronger. The culture method using NA medium is preferably slant culture. The shorter the occurrence time of the pink color of the fermentation liquor is, the higher the urease production activity of the corresponding urease production strains is, and the occurrence time of the pink color of the strains with high activity is less than 15 min. The urease activity of the strain corresponding to the fermentation liquor with the shortest pink color occurrence time can reach 0.165U/g, and the strain can be used as a screening tool for screening the bacillus subtilis J530 strain in the step d.
Preferably, step c further comprises rescreening the obtained urease-producing strain fermentation broth, wherein the rescreening method comprises the following steps: and mixing the urease-producing strain fermentation liquor with a urea buffer solution, and judging the activity of the urease-producing strain fermentation liquor strain according to the consumption condition of urea in the urea buffer solution. The specific method may preferably be: adding 10ml of urea buffer solution into 200 mu l of inactivated urease-producing strain fermentation liquor, fully shaking and mixing, immediately placing in a constant-temperature water bath at 30 +/-0.5 ℃ for 30 +/-10 s, adding 10ml of hydrochloric acid solution, quickly cooling to below 20 ℃ after shaking, and titrating with sodium hydroxide standard solution until the solution is blue-green; adding 10ml of hydrochloric acid solution into 200 mu l of urease-producing strain fermentation liquor, shaking, adding 10ml of urea buffer solution, shaking fully, immediately placing in a constant-temperature water bath at 30 +/-0.5 ℃ for 30min +/-10 s, rapidly cooling to below 20 ℃, and titrating with sodium hydroxide standard solution until the solution is blue-green; judging the activity of the urease-producing strain fermentation liquor according to the volume of the sodium hydroxide standard solution consumed by the two urease-producing strain fermentation liquors; wherein the urea buffer solution is a phosphate buffer solution containing 0.5mol/L urea and having a pH of 7.0 +/-0.1, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1 mol/L. The method for inactivating the fermentation liquor of the urease-producing strain preferably adopts 100 ℃ water bath for 10 min.
Wherein, the rapid cooling can adopt ice bath or cold water to wash the outer wall of the container and the like. The method for determining the activity can be calculated by the following formula:in the formula: x is the urease activity (U/g) of the sample; c is the concentration (mol/L) of the sodium hydroxide standard titration solution; v0Consuming a standard titration solution volume (ml) of sodium hydroxide for a second urease producing strain fermentation broth; v is the volume (ml) of the sodium hydroxide standard titration solution consumed by the fermentation liquor of the first urease-producing strain; 14 is the molar mass of nitrogen; 30 is reaction time (min); m is the sample volume (ml). The specific urease activity of the urease-producing strain can be calculated according to the method.
Preferably, the specific operation method of step d is: b, mixing the fermentation liquor of each sample to be selected obtained in the step a with the fermentation liquor of the urease-producing strain respectively, adding urea and a phenol red indicator, diluting, and shaking up to be used as a test article; preparing a sample without the fermentation liquor of the sample to be selected by the same method as a positive control; and comparing the time for changing the color of each test article and the positive control into pink, wherein the strain corresponding to the test article with the color change time at least 0.5min longer than that of the positive control is the strain for producing the urease inhibitor.
Preferably, the strain in the other microbial inoculum capable of producing the urease inhibitor is Bacillus licheniformis X173 strain, the classification name of the strain is Bacillus licheniformis (Bacillus licheniformis), the strain is preserved in China general microbiological culture Collection center on 9-17.2019, and the preservation number is CGMCC NO: 18602; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang. The strain and the bacillus subtilis J530 strain have obvious synergistic effect, and have better effect on reducing the emission of the ammonia in the livestock and poultry manure. The 16S rDNA sequence is shown in SEQ ID NO. 2.
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 J530 strain for producing urease inhibitor, which is obtained by screening through the following steps:
1. screening of non-urease-producing strains
1.1 Medium formulation
Plate prescreening culture medium: 20mg/ml urea, 1mg/ml phenol red, 10mg/ml sucrose, 10mg/ml peptone, 3mg/ml beef powder, 5mg/ml sodium chloride and 20mg/ml agar, the balance being distilled water, pH 6.4.
Shaking flask primary screening culture medium: 20mg/ml urea, 10mg/ml sucrose, 5mg/ml potassium chloride, 0.5mg/ml magnesium sulfate, 0.5mg/ml calcium chloride, 1mg/ml sodium dihydrogen phosphate and 1mg/ml disodium hydrogen phosphate, and the balance of distilled water, wherein the pH value is 6-8.
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
All media were prepared in distilled water and autoclaved at 121 ℃ for 20 min.
1.2 Flat-bed prescreening
Taking a fresh chicken manure sample of a healthy chicken flock of a certain broiler farm in Baoding city, and standing at room temperature for 24 hours to enable bacillus in the fresh chicken manure sample to fully form spores. Weighing 5g of stood feces sample, adding into 250ml triangular flask containing 45ml of sterile water and 15 glass beads, shaking on a shaking table at 150r/min for 5min, and carrying out water bath at 80 ℃ for 10min to obtain a concentration of 10-1The bacterial suspension of (4). Then carrying out gradient dilution by 10 times by using sterile water to obtain the concentration of 10-2、10-3、10-4、10-5、10-6And 10-7The bacterial suspension of (4). Taking the concentration as 10-4、10-5、10-6And 10-7The bacterial suspension is respectively coated with 5 NA plate culture mediums, each plate is 0.1ml, and inverted and cultured for 24h at 37 ℃.
And picking colonies growing on the NA plate culture medium by using a sterilized bamboo stick, performing cross-scribing inoculation on a plate of the plate primary screening culture medium, performing inverted culture at 37 ℃ for 24 hours, and observing color change around cross lawn. Colonies which are still yellow (do not turn red) are selected and picked out on NA slant culture medium, and are preserved for later use after being cultured for 36h at 37 ℃.
1.3 Shake flask prescreening
The slant lawn obtained in 1.2 plate prescreening was picked with sterilized bamboo stick, inoculated into 50ml shake flask prescreening medium (250ml triangular flask), shake-cultured at 37 deg.C and 150r/min for 1 day, and the turbid condition of the medium was observed. The fermentation liquor which is not turbid is the fermentation liquor of the strain which does not produce urease, and is preserved for later use. In total, 125 strains which do not produce urease are screened from more than 5000 strains.
2. Screening of urease-producing strains
2.1 reagent and Medium formulations
Urea: and (5) analyzing and purifying.
Phenol red indicator: 0.1g of phenol red is weighed, 1.43ml of 0.1mol/L sodium hydroxide solution is added, the mixture is ground in a mortar to promote dissolution, then the mixture is transferred to a 250ml volumetric flask, distilled water is added to the scale mark, and the mixture is shaken up for standby.
Plate prescreening culture medium: 20mg/ml urea, 1mg/ml phenol red, 10mg/ml sucrose, 10mg/ml peptone, 3mg/ml beef powder, 5mg/ml sodium chloride and 20mg/ml agar, the balance being distilled water, the pH being 6.4;
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 medium: 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;
all the culture media are prepared by distilled water, and are sterilized by high-pressure steam at 121 ℃ for 20 min.
Urea buffer solution (ph7.0 ± 0.1): 8.95g of disodium hydrogen phosphate and 3.40g of potassium dihydrogen phosphate are dissolved in water and diluted to 1000ml, and 30g of urea is dissolved in this buffer for an expiration date of one month.
Hydrochloric acid solution [ c (hcl) 0.1mol/L ]: 8.3ml of hydrochloric acid are removed and diluted to 1000ml with water.
Sodium hydroxide solution [ c (naoh) ═ 0.1mol/L ]: 4g of sodium hydroxide are weighed out, dissolved in water and diluted to 1000 ml.
Mixing an indicator: namely, a mixed ethanol solution of methyl red and bromocresol green: 0.1g of methyl red is weighed, dissolved in 95% ethanol and diluted to 100ml, 0.5g of bromocresol green is weighed, dissolved in 95% ethanol and diluted to 100ml, the two solutions are mixed in equal volume and stored in a brown bottle.
2.2 preliminary screening of urease-producing strains
And (3) taking colonies which turn red around the lawn and have a large diffusion range after cross inoculation on the medium plate primary screening culture medium plate of the 1.2 medium plate, placing the colonies on an NA slant culture medium, culturing for 36h at 37 ℃, and preserving for later use. The strains are inoculated into a 250ml triangular flask containing 50ml NB medium and subjected to shaking culture at 150r/min for 24h to prepare fermentation liquor.
1.0ml of the fermentation liquid is taken and added into a 25ml colorimetric tube, 0.02g of urea (1.0 ml of the prepared 2% (m/v) urea aqueous solution) is added, 2 drops of phenol red indicator are added, 20ml of water is added, and the mixture is fully shaken for 15 seconds. The time of appearance of pink was recorded and urease activity was judged by time. And additionally arranging a sample blank (without urea) and a reagent blank (without a sample), wherein the color of the sample blank and the reagent blank is not changed after the phenol red indicator is added, so that the test result is reliable. Recording the fermentation liquor with the shortest pink color appearance time (less than 15min), namely the urease-producing strain fermentation liquor, wherein the corresponding strain has the highest urease activity and is preserved for later use.
2.3 rescreening of urease-producing strains
Measuring 200 mul +/-1 mul of urease-producing strain fermentation liquor in a colorimetric tube, adding 10ml of urea buffer solution, immediately covering the colorimetric tube, violently shaking, immediately placing the colorimetric tube in a thermostatic water bath at 30 +/-0.5 ℃, and timing for 30min +/-10 s. When the reaction was stopped, 10ml of hydrochloric acid solution was added, shaken and rapidly cooled to 20 ℃. And transferring the content of the colorimetric tube into a 250ml conical flask, adding 8-10 drops of mixed indicator, titrating with a sodium hydroxide standard solution until the solution is blue-green, and recording the volume of the consumed sodium hydroxide.
Taking another colorimetric tube for blank test, measuring 200 mu l +/-1 mu l of urease-producing strain fermentation liquor inactivated by 100 ℃ water bath for 10min into the colorimetric tube, adding 10ml of hydrochloric acid solution, shaking, adding 10ml of urea buffer solution, immediately covering the colorimetric tube, violently shaking, immediately placing the test tube in 30 ℃ +/-0.5 ℃ constant-temperature water bath, and keeping the time for 30min +/-10 s. And (3) rapidly cooling the colorimetric tube to 20 ℃ when the reaction is stopped, then transferring the content of the colorimetric tube into a 250ml conical flask, adding 8-10 drops of mixed indicator, titrating with a sodium hydroxide standard solution until the solution is blue-green, and recording the volume of the consumed sodium hydroxide.
In the formula: x is the urease activity (U/g) of the sample; c is the concentration (mol/L) of the sodium hydroxide standard titration solution; v0Consumption of sodium hydroxide standard titration solution volume (ml) for blank; v is the volume (ml) of the sodium hydroxide standard titration solution consumed by the sample; 14 is the molar mass of nitrogen; 30 is reaction time (min); m is the sample volume (ml).
3. Screening of urease inhibitor producing strains
Adding 1.0ml of urease-producing strain fermentation liquor into a 25ml colorimetric tube, adding 1.0ml of urease-producing strain fermentation liquor obtained from the step 1.3, uniformly mixing, adding 0.02g of urea (1.0 ml of prepared 2% (m/v) urea aqueous solution) and 2 drops of phenol red indicator, adding 20ml of water, and fully shaking for 15 seconds to obtain a test group. Meanwhile, except that the sample of the fermentation liquor of the strain which does not produce urease is not added, the other preparation methods are the same as the test group and are used as a positive control group. Recording the occurrence time of pink of the colorimetric tubes of the test group and the colorimetric tubes of the positive control group, judging whether the urease inhibitor exists in the fermentation liquor according to the occurrence time, and if the occurrence time of the pink of the colorimetric tubes of the test group is longer than that of the colorimetric tubes of the positive control group, indicating that the urease inhibitor exists; if the occurrence time of the pink color of the cuvette of the test group is the same as or shorter than that of the cuvette of the positive control group, the urease inhibitor does not exist, the fermentation liquid containing the urease inhibitor can be selected according to the principle, and the corresponding strain is the strain producing the urease inhibitor. And taking the strain corresponding to the test product with the color change time longer than that of the positive control by 1.5 min.
4. Rescreening of urease inhibitor producing strains
And quantitatively detecting the urease inhibitor producing activity of the preliminarily screened urease inhibitor producing strain by using a method for measuring the urease activity in the soybean product for feed GB/T8622-.
5. Identification of Bacillus subtilis J530 Strain
The bacillus subtilis J530 strain is identified, and the results of physiological and biochemical identification characteristics are shown in table 1.
TABLE 1
The bacillus subtilis J530 strain is subjected to 16S rDNA sequence detection, and the obtained sequence is shown in SEQ ID No. 1. According to the identification characteristics and 16S rDNA sequence detection of the strain shown in the table 1, the strain J530 can be identified as the bacillus subtilis.
The bacillus subtilis J530 strain is preserved in 2019, 9 and 17 days and preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO: 18603; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.
Example 2
This example provides the use of the strain Bacillus subtilis J530 as a feed additive for reducing fecal ammonia emissions.
The Bacillus subtilis J530 strain obtained in example 1 was added to pig feed in an amount of 0.1% (m/m) to detect changes in protease activity in pig manure and intestinal tract. The results are shown in Table 2.
TABLE 2
| Detection site | Rate of increase in protease activity |
| Intestinal tract | 70.36％ |
| Excrement and urine | 68.70％ |
| Average | 69.53％ |
As can be seen from the results in Table 2, the protease activity was significantly improved by adding Bacillus subtilis J530 strain to the feed. The improvement of the protease activity can improve the utilization rate of the protein of the feed, reduce the excretion of nitrogen-containing substances such as protein and the like, and reduce the emission of ammonia gas in excrement from an ammonia gas generation source.
Example 3
The embodiment provides application of a bacillus subtilis J530 strain as a fecal composting microbial inoculum in reducing fecal ammonia emission.
1. Reagent and preparation method
Sulfuric acid,. rho (H)2SO4)＝1.84g/ml；
Sulfuric acid absorption solution, c (1/2H)2SO4) 0.01 mol/L: weighing 2.7ml of sulfuric acid, adding the sulfuric acid into water, diluting to 1L to obtain 0.1mol/L stock solution, and diluting by 10 times when the stock solution is used;
a nano-grade reagent: 12g of sodium hydroxide (NaOH) are weighed out and dissolved in 60ml of water, cooled and 1.7g of mercury dichloride (HgCl) are weighed out2) Dissolving in 30ml water, weighing 3.5g potassium iodide (KI) in 10ml water, slowly adding the above mercuric dichloride solution into potassium iodide solution under stirring until the red precipitate formed is no longer dissolved, slowly adding sodium hydroxide solution cooled to room temperature into the above solution under stirringAdding the rest mercuric chloride solution into the mixed solution of mercuric chloride and potassium iodide, uniformly mixing, standing for 24h in a dark place, pouring out supernatant, storing in a brown bottle, plugging by a rubber stopper, storing at 2-5 ℃ and using within 1 month;
sodium potassium tartrate solution,. rho.500 g/L: 50g of potassium sodium tartrate (KNaC) are weighed4H6O6·4H2O), adding 60ml of distilled water, heating in a water bath to dissolve, and cooling to reach the volume of 100 ml.
2. Test procedure
The Bacillus subtilis J530 strain obtained in example 1 was inoculated into NB medium, and shake-cultured at 37 ℃ at 150r/min for 24 hours to prepare a fermentation broth of the strain. 200g of fresh pig manure is weighed and evenly placed in a 1000ml big beaker, the fermentation liquor of the bacillus subtilis J530 strain is poured into the big beaker according to the inoculation amount of 20 percent (namely 40ml) and evenly mixed, and then a 50ml small beaker containing 40ml of sulfuric acid absorption liquid is also placed in the big beaker. Firstly, coating a layer of vaseline on the outer wall of the big beaker to ensure the bonding property and the air tightness of the preservative film and the wall of the big beaker, and then sealing the opening of the big beaker by using a double-layer preservative film to ensure that the generated ammonia gas can not leak into the atmosphere and can be absorbed by a sulfuric acid absorption liquid. The above is the test group.
Meanwhile, a control group is set, the control group is not inoculated with the fermentation liquor of the strain but added with the same amount of sterile water, and the rest is the same as the test group.
Then the sealed big beaker is put into a biochemical incubator with constant temperature of 30 ℃ for 7 days. After the culture is finished, the relative ammonia gas content of the test group and the control group is determined by utilizing a nano reagent spectrophotometry method for determining ammonia in ambient air and waste gas according to national environmental protection standard HJ 533-2009. And judging whether the bacillus subtilis J530 strain has the effect of reducing the emission of ammonia on the livestock and poultry manure by comparing the difference of ammonia generated by the test group and the control group.
3. Measurement of Ammonia Release amount
Respectively putting 10ml of the sulfuric acid absorption solution of each group after being cultured for 7 days into a 25ml colorimetric tube, respectively adding 0.50ml of potassium sodium tartrate solution, shaking up, respectively adding 0.50ml of Nashi reagent, shaking up, standing for 10min, using water as a reference, and measuring the absorbance at the wavelength of 420nm by using a 10mm cuvette. Because the dilute sulfuric acid solution absorbs ammonia in the air, the generated ammonium ions react with the Nalsberg reagent to generate a yellow-brown complex, and the absorbance of the complex at 420nm is in direct proportion to the content of ammonia, the reduction of the ammonia gas of a test group compared with that of a control group can be known by comparing the absorbance of the test group and the control group at the wavelength of 420nm, so that the ammonia emission reduction effect of the strains screened by the method for screening the strains producing the urease inhibitor on the livestock and poultry manure is shown. The absorbance value of the control group sample diluted 6 times was 0.497, and the absorbance of the test group sample added with the Bacillus subtilis J530 strain was 0.275. According to calculation, the inhibition rate of the bacillus subtilis J530 strain on ammonia gas in an in vitro fecal test is 90.78%. The bacillus subtilis J530 strain provided by the invention can greatly reduce the emission of ammonia gas in livestock and poultry manure.
Example 4
The embodiment provides a complex microbial inoculum.
The preparation method of other strains for producing urease inhibitors comprises the following steps:
placing fresh cow dung in a certain cow farm in a guaranteed market at normal temperature for 24 hours to prepare a bacterial suspension, performing conventional culture by using an NA (deoxyribonucleic acid) culture medium, and inoculating a bacterial colony obtained by culture to a primary screening culture medium for 24 hours; the primary screening medium contained 20mg/ml urea, 1mg/ml phenol red, 10mg/ml sucrose, 10mg/ml peptone, 3mg/ml beef powder, 5mg/ml sodium chloride and 20mg/ml agar, the balance being distilled water, and the pH was 6.4.
Secondly, selecting colonies which are not discolored at the periphery from the colonies obtained by the culture in the first step, respectively using NA culture media for 24 hours, then inoculating the colonies into a shaking flask primary screening culture medium for shaking table culture for 1 day, and taking a non-turbid sample as a sample fermentation liquor to be selected; the shaking flask primary screening culture medium contains 20mg/ml urea, 10mg/ml sucrose, 5mg/ml potassium chloride, 0.5mg/ml magnesium sulfate, 0.5mg/ml calcium chloride, 1mg/ml sodium dihydrogen phosphate and 1mg/ml disodium hydrogen phosphate, the balance is distilled water, and the pH is 6-8.
Thirdly, selecting colonies which turn red around from the colonies obtained by the culture in the step one, respectively culturing the colonies for 48 hours by using an NA culture medium, and then inoculating the colonies in an NB culture medium for shake cultivation for 48 hours to obtain fermentation liquor of each colony; adding urea aqueous solution and phenol red indicator into the obtained bacterial colony fermentation liquor respectively, shaking up, and taking fermentation liquor with shortest pink red appearance time, namely urease-producing strain fermentation liquor; adding 10ml of urea buffer solution into 200 mu l of urease-producing strain fermentation liquor, fully shaking and mixing, immediately placing in a constant-temperature water bath at 30 +/-0.5 ℃ for 30 +/-10 min, adding 10ml of hydrochloric acid solution, quickly cooling to below 20 ℃ after shaking, and titrating with a sodium hydroxide standard solution until the solution is blue-green; adding 10ml hydrochloric acid solution into 200 μ l of the urease producing strain fermentation broth inactivated by 100 deg.C water bath for 10min, shaking, adding 10ml urea buffer solution, shaking thoroughly, immediately placing in 30 + -0.5 deg.C constant temperature water bath for 30min + -10 s, rapidly cooling to below 20 deg.C, and titrating with sodium hydroxide standard solution until the solution is blue-green; judging the activity of the urease-producing strain fermentation liquor according to the volume of the sodium hydroxide standard solution consumed by the two urease-producing strain fermentation liquors; wherein the urea buffer solution is a phosphate buffer solution containing 0.5mol/L urea and having a pH of 7.0 +/-0.1, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1 mol/L.
Mixing the fermentation liquor of each sample to be selected obtained in the step (c) with the fermentation liquor of the urease-producing strain obtained in the step (c), adding urea and phenol red indicator, diluting and shaking up to be used as a test article; preparing a sample without the fermentation liquor of the sample to be selected by the same method as a positive control; comparing the time of the test products changing into pink with the positive control, and taking the strain corresponding to the test product with the color changing time longer than the positive control for 1.5min to obtain the strain producing the urease inhibitor.
The effect of the strain in reducing fecal ammonia emission was examined as a fecal composting agent as in example 3, and the results demonstrated that the absorbance of the strain was 0.525 (diluted 3-fold) at the same inoculum size as in example 3, i.e., the inhibition of ammonia by the strain in the in vitro fecal test was 47.18%. The bacterial strain and the bacillus subtilis J530 bacterial strain are compounded according to the mass ratio of 1:1, and the effect of the compound bacterial agent in reducing the emission of the ammonia in the excrement is examined in the form of being used as an excrement composting bacterial agent according to the method of the embodiment 3. The result proves that under the condition of the same inoculation amount as that in the embodiment 3, the absorbance of the composite microbial inoculum test group provided by the embodiment is 0.652, namely, the inhibition rate of the composite microbial inoculum test group on ammonia gas in an in vitro fecal test is 78.14%, the inhibition rate is improved by 30.96% compared with the inhibition rate of the strain alone, and the inhibition rate is improved by 9.16% compared with the average value of the inhibition rates of the strain and the bacillus subtilis J530 strain, which indicates that the bacillus subtilis J530 strain can improve the ammonia emission reduction effect of other strains for producing urease inhibitors.
Example 5
This example provides another complex inoculant.
The preparation method of other strains for producing urease inhibitors comprises the following steps:
fresh pig manure of healthy male pigs in a certain pork pig farm in a guaranteed market is placed at normal temperature for 36 hours to prepare a bacterial suspension, the bacterial suspension is cultured by using an NA culture medium in a conventional way, and bacterial colonies obtained by culture are inoculated in a primary screening culture medium for 36 hours; the primary screening medium contained 20mg/ml urea, 1mg/ml phenol red, 10mg/ml sucrose, 10mg/ml peptone, 3mg/ml beef powder, 5mg/ml sodium chloride and 20mg/ml agar, the balance being distilled water, and the pH was 6.4.
Secondly, selecting colonies which are not discolored at the periphery from the colonies obtained by the culture in the first step, respectively using NA culture media for 48 hours, then inoculating the colonies into a shaking flask primary screening culture medium for shaking table culture for 2 days, and taking a non-turbid sample as a sample fermentation liquor to be selected; the shaking flask primary screening culture medium contains 20mg/ml urea, 10mg/ml sucrose, 5mg/ml potassium chloride, 0.5mg/ml magnesium sulfate, 0.5mg/ml calcium chloride, 1mg/ml sodium dihydrogen phosphate and 1mg/ml disodium hydrogen phosphate, the balance is distilled water, and the pH is 6-8.
Thirdly, selecting colonies which turn red around from the colonies obtained by the culture in the step one, respectively culturing the colonies for 36 hours by using an NA culture medium, and then inoculating the colonies in an NB culture medium for shake cultivation for 36 hours to obtain fermentation liquor of each colony; adding urea aqueous solution and phenol red indicator into the obtained bacterial colony fermentation liquor respectively, shaking up, and taking fermentation liquor with shortest pink red appearance time, namely urease-producing strain fermentation liquor; adding 10ml of urea buffer solution into 200 mu l of urease-producing strain fermentation liquor, fully shaking and mixing, immediately placing in a constant-temperature water bath at 30 +/-0.5 ℃ for 30 +/-10 min, adding 10ml of hydrochloric acid solution, quickly cooling to below 20 ℃ after shaking, and titrating with a sodium hydroxide standard solution until the solution is blue-green; adding 10ml hydrochloric acid solution into 200 μ l of the urease producing strain fermentation broth inactivated by 100 deg.C water bath for 10min, shaking, adding 10ml urea buffer solution, shaking thoroughly, immediately placing in 30 + -0.5 deg.C constant temperature water bath for 30min + -10 s, rapidly cooling to below 20 deg.C, and titrating with sodium hydroxide standard solution until the solution is blue-green; judging the activity of the urease-producing strain fermentation liquor according to the volume of the sodium hydroxide standard solution consumed by the two urease-producing strain fermentation liquors; wherein the urea buffer solution is a phosphate buffer solution containing 0.5mol/L urea and having a pH of 7.0 +/-0.1, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1 mol/L.
Mixing the fermentation liquor of each sample to be selected obtained in the step (c) with the fermentation liquor of the urease-producing strain obtained in the step (c), adding urea and phenol red indicator, diluting and shaking up to be used as a test article; preparing a sample without the fermentation liquor of the sample to be selected by the same method as a positive control; comparing the time of the test products changing into pink with the positive control, and taking the strain corresponding to the test product with the color changing time longer than the positive control for 3min to obtain the strain producing the urease inhibitor.
The effect of the strain in reducing fecal ammonia emission was examined as a fecal composting bacterial agent as in example 3, and the results demonstrated that the absorbance of the strain was 0.205 at the same inoculum size as in example 3, i.e., the inhibition of ammonia gas by the strain in the in vitro fecal test was 93.13%. The bacterial strain and the bacillus subtilis J530 bacterial strain are compounded according to the mass ratio of 1:1, and the effect of the compound bacterial agent in reducing the emission of the ammonia in the excrement is examined in the form of being used as an excrement composting bacterial agent according to the method of the embodiment 3. The result proves that under the condition of the same inoculation amount as that in the embodiment 3, the absorbance of the composite microbial inoculum test group provided by the embodiment is 0.096, namely, the inhibition rate of the composite microbial inoculum test on ammonia gas in an in vitro fecal test is 96.78%, the inhibition rate is improved by 3.65% compared with the inhibition rate of the strain alone, the inhibition rate is improved by 4.82% compared with the average inhibition rate of the strain and the bacillus subtilis J530 strain, and the inhibition rate is improved by 6.00% compared with the inhibition rate of the strain alone, which indicates that the bacillus subtilis J530 strain can improve the ammonia emission reduction effect of other strains producing urease inhibitor, and the strain can also improve the ammonia emission reduction effect of the bacillus subtilis J530 strain. The strain is identified as Bacillus licheniformis and preserved, the strain name is Bacillus licheniformis X173, the classification name is Bacillus licheniformis (Bacillus licheniformis), the strain is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 9 and 17 months, and the preservation number is CGMCC NO: 18602; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.
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> agriculture university in Hebei province and animal husbandry general station in Hebei province
<120> urease inhibitor producing bacillus subtilis J530 strain and application thereof
<130> 2019.10.08
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1371
<212> DNA
<213> Bacillus subtilis J53016S rDNA
<400> 1
ccctgatgtt agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga 60
taactccggg aaaccggggc taataccgga tggttgtttg aaccgcatgg ttcaaacata 120
aaaggtggct tctgctacga cttacagatg ggcccgcggc gcattatcta gttggtgagg 180
taacggctca ccaaggcgac gatgcgtatc cgacctgaga gggtgatcgg ccacactggg 240
actgagacac ggcccacact cctacgggag gcagcagtag ggaatcttcc gcaatggaca 300
aaagtctgac ggagcaacgc cgcgtgagtg atgaaagttt tcggatcgta aagctctgtt 360
gttagggaag aaaaagtacc gttcgaatag ggcgggacct tgacggtacc taaccagaaa 420
gccacggcta actacgtgcc agcagccgcg gtaatacgta tgtgggaagc gttgtccgga 480
attattgggc gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct 540
ctcccgggga gggtcattgt aaactgggga acttgagtgc agaagaggag agtggaattc 600
cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc 660
tggtctgtaa ctgacgctga ggagcgaaag cgtggggagc gaaacaggat tagataccct 720
ggtagtccac gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct 780
gcagctaacg cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg 840
aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga 900
accttaccag gtcttgacat cctctgacaa tcctagagat aggacgtccc cttcgggggc 960
agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1020
cgcaacgagc gcaacccttg atcttagttg ccagcattca gttgggcact ctaaggtgac 1080
tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc 1140
tgggctacac acgtgctaca atggacagaa caaagggcag cgaaaccgcg aggttaagcc 1200
aatcccacaa atctgttctc agttcggatc gcagtctgca actcgcctgc gtgaagctgg 1260
aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca 1320
ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac c 1371
<210> 2
<211> 1394
<212> DNA
<213> Bacillus licheniformis X17316S rDNA
<400> 2
tgctaataca tgcaagtcga gcggaccgac gggagcttgc tcccttaggt cagcggcgga 60
cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg 120
ctaataccgg atgcttgatt gaaccgcatg gttcaattat aaaagggggc ttttagctac 180
cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg 240
acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 360
gccgcgtgag tgatgaaggt tttcggatcg taaaactctg ttgttaggga agaacaagta 420
ccgttcgaat agggcggtac cttgacggta cctaaccaga aagccacggc taactacgtg 480
ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaagcg 540
cgcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 600
ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 660
gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 720
gaggcgcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca cgccgtaaac 780
gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgcagcaaac gcattaagca 840
ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg gggcccgcac 900
aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca 960
tcctctgaca accctagaga tagggcttcc ccttcggggg cagagtgaca ggtggtgcat 1020
ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080
gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga caaaccggag 1140
gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac 1200
aatgggcaga acaaagggca gcgaagccgc gaggctaagc caatcccaca aatctgttct 1260
cagttcggat cgcagtctgc aactcgactg cgtgaagctg gaatcgctag taatcgcgga 1320
tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag 1380
agtttgtaac accc 1394
Claims (10)
1. A urease inhibitor producing Bacillus subtilis J530 strain is classified as Bacillus subtilis, and is preserved in China general microbiological culture Collection center (CGMCC) on 9 and 17 days in 2019 with the preservation number of CGMCC NO: 18603; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.
2. Use of the bacillus subtilis J530 strain of claim 1 for reducing fecal ammonia emissions.
3. The use of claim 2, wherein the bacillus subtilis J530 strain is prepared into a microbial agent for feed additives or manure composting microbial agents, and the method for preparing the bacillus subtilis J530 strain into the microbial agent comprises the following steps: liquid submerged fermentation is carried out firstly, then the obtained fermentation liquor is continuously centrifuged, and finally the precipitate obtained by centrifugation is spray-dried.
4. A compound bacterial agent, which is a bacterial strain containing the bacillus subtilis J530 strain of claim 1 and other urease inhibitors.
5. The complex microbial inoculant according to claim 4, wherein: the screening method of other strains capable of producing urease inhibitors specifically comprises the following steps:
step a, preparing feces into bacterial suspension, conventionally culturing by using an NA culture medium, and inoculating a bacterial colony obtained by culture into a flat plate primary screening culture medium containing urea and an acid-base indicator for culture;
b, picking surrounding non-discolored colonies from the colonies obtained by culturing in the step a, and screening strains which do not produce urease to prepare a sample fermentation liquor to be selected;
c, selecting surrounding discolored colonies from the colonies obtained by culturing in the step a to prepare urease-producing strain fermentation liquor;
and d, mixing the fermentation liquor of the urease-producing strain obtained in the step c with the fermentation liquor of the urease-non-producing strain obtained in the step b, and screening the urease inhibitor-producing strain.
6. The complex microbial inoculant according to claim 5, wherein: the plate primary screening culture medium in the step a contains 20mg/ml urea, 1mg/ml phenol red, 10mg/ml sucrose, 10mg/ml peptone, 3mg/ml beef powder, 5mg/ml sodium chloride and 20mg/ml agar, the balance is distilled water, and the pH is 6.4; and/or
And in the step a, the livestock and poultry manure is prepared by placing fresh livestock and poultry manure for 24-36 hours at normal temperature.
7. The complex microbial inoculum of claim 5, wherein the method for preparing the fermentation liquor of the sample to be selected by screening the bacterial strain which does not produce urease in the step b comprises the following steps: and respectively using the peripheral non-discolored colonies for 24-48 h by using an NA culture medium, then inoculating the colonies in a shake flask primary screening culture medium containing urea for shake cultivation for 1-2 days, wherein the non-turbid sample is the fermentation liquor of the sample to be selected.
8. The complex microbial inoculant according to claim 5, wherein the preparation method of the urease-producing strain fermentation broth in step c comprises the following steps: b, selecting surrounding discolored colonies from the colonies obtained by the culture in the step a, respectively culturing the colonies for 24-48 h by using an NA (Nurse number) culture medium, and then inoculating the colonies in an NB (NB) culture medium for shake cultivation for 24-48 h to obtain fermentation liquor of each strain; adding urea aqueous solution and phenol red indicator into the obtained strain fermentation liquor respectively, shaking up, and taking fermentation liquor with shortest pink red appearance time, namely the urease-producing strain fermentation liquor; and/or
Step c also comprises the step of re-screening the obtained urease-producing strain fermentation liquor, wherein the re-screening method comprises the following steps: and mixing the urease-producing strain fermentation liquor with a urea buffer solution, and judging the activity of the urease-producing strain fermentation liquor strain according to the consumption condition of urea in the urea buffer solution.
9. The complex microbial inoculant according to claim 5, wherein the specific operation method of the step d is as follows: b, mixing the fermentation liquor of each sample to be selected obtained in the step b with the fermentation liquor of the urease-producing strain respectively, adding urea and a phenol red indicator, diluting and shaking up to be used as a test article; preparing a sample without the fermentation liquor of the sample to be selected by the same method as a positive control; and comparing the time for changing the color of each test article and the positive control into pink, wherein the strain corresponding to the test article with the color change time at least 0.5min longer than that of the positive control is the strain for producing the urease inhibitor.
10. The composite microbial inoculum according to any one of claims 4 to 9, wherein the other bacterial strain capable of producing the urease inhibitor is Bacillus licheniformis X173, which is classified as Bacillus licheniformis (Bacillus licheniformis), and is deposited in the common microorganism center of the china committee for culture collection management of microorganisms No. CGMCC NO: 18602; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.
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