CN110699299B

CN110699299B - Bacillus licheniformis X173 strain for producing urease inhibitor and application thereof

Info

Publication number: CN110699299B
Application number: CN201911115482.4A
Authority: CN
Inventors: 翟蔚; 王志刚; 李毅; 王世英; 翟荣生
Original assignee: Urad Qianqi Rongsheng Dadi Biotech Feed Co ltd; Hebei Agricultural University
Current assignee: Urad Qianqi Rongsheng Dadi Biotech Feed Co ltd; Hebei Agricultural University
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2023-01-17
Anticipated expiration: 2039-11-14
Also published as: CN110699299A

Abstract

The invention relates to a Bacillus licheniformis X173 strain for producing urease inhibitor and application thereof, wherein the strain is preserved in China general microbiological culture Collection center in 17.9.2019 with the preservation address of No. 3 Xilu No.1 Beijing of the Beijing Chaoyang district, and the preservation number is CGMCC NO:18602. the Bacillus licheniformis X173 strain has high activity of producing urease inhibitor, and can inhibit urea or uric acid in livestock and poultry feces from being decomposed into ammonia by urease.

Description

Bacillus licheniformis X173 strain for producing urease inhibitor and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and in particular relates to a bacillus licheniformis X173 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 system for treating breeding manure, a large amount of urea (such as pig livestock) or uric acid (such as chicken livestock which can be converted into urea after decomposition) contained in the manure is easily decomposed into ammonia by urease generated by microorganisms and volatilized, so that 75% of nitrogen in the manure is lost, and N/P ratio of the manure 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, nitrogen element is greatly lost, and 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 urease inhibition effect is low, and the ammonia volatilization reduction capability 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 licheniformis X173 strain for producing the urease inhibitor.

The invention also provides application of the bacillus licheniformis X173 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 Bacillus licheniformis X173 strain for producing urease inhibitor, which is classified as Bacillus licheniformis (Bacillus licheniformis), is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 9, 17 days, 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 bacillus licheniformis X173 strain provided by the invention is obtained by separating and screening fresh excrement of healthy male pigs in a pork pig farm, belongs to bacillus, is non-toxic and pollution-free, has high activity of producing urease inhibitors, and has a 16S rDNA sequence shown in SEQ ID No. 1. The bacillus licheniformis X173 strain can grow and reproduce in intestinal tracts of livestock and poultry and generate a urease inhibitor, urea or uric acid in excrement of livestock and poultry is inhibited from being decomposed into ammonia by urease, the inhibition rate of the urease activity in excrement of pigs, chickens and the like can reach 98.5%, and the ammonia content in intestinal tracts and blood of livestock and poultry is effectively reduced. Experiments prove that the bacillus licheniformis X173 strain can well grow by utilizing a 'similar diet culture medium' (a culture medium with components close to those of livestock and poultry feeds) under a facultative anaerobic condition, and can generate 51.27U/ml acidic protease, so that the bacillus licheniformis X173 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 licheniformis X173 strain is discharged out of the body along with excrement or directly added into the excrement in the body, the urease activity can be continuously inhibited in the composting process, and the inhibition rate on ammonia in an in-vitro excrement ammonia emission test can reach 93.13 percent, so that the effect of reducing the emission of ammonia is achieved.

The screening method of the bacillus licheniformis X173 strain specifically comprises the following steps:

(1) placing fresh pig feces of healthy male pigs in a pork pig farm at normal temperature for 36h to prepare a bacterial suspension, carrying out water bath at 80 ℃ for 10min, coating the bacterial suspension on an NA culture medium after gradient dilution for conventional culture, and inoculating bacterial colonies obtained by culture to a flat primary screen culture medium for culture for 36h; 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.

(2) Selecting colonies which are not discolored at the periphery from the colonies obtained by the culture in the step (1), performing slant culture for 48h by using NA culture media respectively, then inoculating the colonies into a shaking flask primary screening culture medium for shake cultivation for 2 days, 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.

(3) Selecting colonies which turn red around from the colonies obtained by the culture in the step (1), respectively culturing for 36h by using an NA culture medium, and then inoculating into an NB culture medium for shake cultivation for 36h to obtain fermentation liquor of each colony; adding urea aqueous solution and phenol red indicator into each bacterial colony fermentation liquid, shaking up, and taking fermentation liquid with shortest pink color appearance time, namely urease-producing strain fermentation liquid; adding 10ml of urea buffer solution into 200 mu l of urease producing strain fermentation liquor inactivated by 100 ℃ water bath for 10min, fully shaking and mixing, immediately placing in 30 ℃ +/-0.5 ℃ constant temperature water bath for 30min +/-10 s, adding 10ml of hydrochloric acid solution, shaking, rapidly cooling to below 20 ℃, and titrating with sodium hydroxide standard solution until the solution is blue-green; adding 10ml hydrochloric acid solution into 200 mul fermentation liquor of the urease producing strain, shaking, adding 10ml urea buffer solution, shaking fully, immediately placing in 30 +/-0.5 ℃ constant temperature water bath for 30min +/-10 s, cooling to below 20 ℃ rapidly, 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 0.1mol/L.

(4) Mixing the fermentation liquor of each sample to be selected obtained in the step (2) with the fermentation liquor of the urease-producing strain obtained in the step (3), adding urea and phenol red indicators, 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, namely the strain producing the urease inhibitor.

(5) And (5) detecting the activity of the strain obtained in the step (4) by using a method for determining the urease activity in the soybean product for feed GB/T8622-2006, and re-screening to obtain the bacillus licheniformis X173 strain.

The embodiment of the invention also provides application of the bacillus licheniformis X173 strain in reducing fecal ammonia emission.

The bacillus licheniformis X173 strain provided by the invention has high-efficiency and stable ammonia reduction 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 loss of compost fertilizer efficiency 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 licheniformis X173 strain is prepared into a microbial agent for a feed additive or a fecal composting microbial agent, and the method for preparing the bacillus licheniformis X173 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. 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 manure compost, the reduction of the N/P ratio of compost can be reduced, and the compost maturity is promoted. The microbial inoculum for the manure compost can reduce the imbalance condition of the N/P ratio of the compost and promote the compost to become thoroughly decomposed. The Bacillus licheniformis X173 strain is subjected to liquid deep fermentation, continuous centrifugation and spray drying to obtain bacterial powder with viable bacteria (spore) content of 4.5 × 10 ¹¹ CFU/g. The obtained bacterial powder can be mixed with soluble starch to make into feed additive, and the final microbial feed additive has viable bacteria (spore) content of 1.0 × 10 ¹⁰ CFU/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 the shelf life is 18 months.

The embodiment of the invention also provides a composite microbial inoculum which contains the bacillus licheniformis X173 strain and other strains for producing 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 can be achieved.

Preferably, the screening method of the other strains capable of producing the urease inhibitor 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;

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 primary screening medium in 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 being distilled water, and the pH is 6.4.

Preferably, the livestock and poultry manure in the step a is manure prepared by placing fresh livestock and poultry manure for 24-36 h 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 NA culture media for 24-48 h for the surrounding non-discolored colonies, then inoculating the colonies into a shake flask primary screening culture medium containing urea, and carrying out shake culture 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 method of culturing with NA medium preferably employs slant culture.

Preferably, the method for preparing the urease producing strain fermentation broth in step c comprises the following steps: selecting colonies with red peripheries from the colonies obtained by culturing in the step a, respectively culturing 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 each strain fermentation liquor; and (3) respectively adding urea aqueous solution and phenol red indicator into the obtained strain fermentation liquor, shaking up, and taking fermentation liquor with shortest pink color 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 method of culturing with NA medium preferably employs 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 15min. And d, 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 licheniformis X173 strain in the step d.

Preferably, the step c further comprises the step of rescreening the fermentation liquor of the urease-producing strain, 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 value of 7.0 +/-0.1, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1mol/L. The method for inactivating the urease-producing strain fermentation liquor is preferably performed in a water bath at 100 ℃ for 10min.

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; v ₀ Consuming 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 obtain a test sample; 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 inoculums capable of producing the urease inhibitor is a Bacillus subtilis J530 strain with a classification name of Bacillus subtilis, which is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 9, 17 and the preservation number of CGMCC NO:18603; the preservation address is No. 3 of Xilu No.1 of Beijing, chaoyang, beijing. The strain has obvious synergistic effect with the Bacillus licheniformis X173 strain, and has 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 described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

The embodiment of the invention provides a Bacillus licheniformis X173 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, the balance of distilled water, pH 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 pig manure sample of a healthy male pig in a certain pork pig farm in Baoding city, and placing the fresh pig manure sample at room temperature for 36 hours to ensure that bacillus in the fresh pig manure sample fully forms 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 ^-1 The 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 ^-6 And 10 ^-7 The bacterial suspension of (4). Taking the concentration as 10 ^-4 、10 ^-5 、10 ^-6 And 10 ^-7 The bacterial suspension was applied to 5 NA plate media, each 0.1ml, and inverted at 37 ℃ for 36 hours.

And (3) picking bacterial colonies growing on the NA plate culture medium by using a sterilized bamboo stick, performing cross-shaped inoculation on a plate of the plate primary screening culture medium, performing inverted culture at 37 ℃ for 36 hours, and observing color change around cross-shaped bacterial lawn. Screening and picking out colonies with the surrounding yellow color (not changing into red color) in an NA slant culture medium, culturing at 37 ℃ for 48h, and preserving for later use.

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 (250 ml triangular flask), shake-cultured at 37 deg.C and 150r/min for 2 days, 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, 126 strains which do not produce urease were screened from 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, ground in a mortar to promote dissolution, transferred to a 250ml volumetric flask, added with distilled water to the mark and shaken well for later use.

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 (ph 7.0 ± 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 1000ml.

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 of the medium plate primary screening culture medium of 1.2 to 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 shake cultivation is carried out at 150r/min for 24h, thus obtaining the 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 in addition, a sample blank (without urea) and a reagent blank (without a sample) are arranged, and 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 15 min), 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 ℃. Transferring the content of the colorimetric cylinder into a 250ml conical flask, adding 8-10 drops of mixed indicator, titrating with 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 colorimetric tube in 30 ℃ +/-0.5 ℃ constant-temperature water bath, and timing 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; v ₀ Consumption of sodium hydroxide standard titration solution volume (ml) for blank; v is the volume (ml) of the standard titration solution of the sodium hydroxide 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 the urease-non-producing strain fermentation liquor obtained from the 1.3, uniformly mixing, adding 0.02g of urea (1.0 ml of a 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 (4) taking the strain corresponding to the test product with the discoloration time longer than that of the positive control for 3 min.

4. Rescreening of urease inhibitor producing strains

The urease inhibitor producing activity of the primarily screened strain is quantitatively detected by using a method for detecting the urease activity in the soybean product for feed GB/T8622-2006, 200 mu l of urease inhibitor producing strain fermentation liquor is added into a sample test tube and a blank test tube, the urease activity of the urease inhibitor producing strain added with the urease inhibitor is detected, and the urease activity is compared with the urease activity of the known urease inhibitor-free urease producing strain to calculate a urease activity reduction value, so that the bacillus licheniformis X173 strain with the urease inhibitor can be screened.

5. Identification of Bacillus licheniformis X173 strain

The bacillus licheniformis X173 strain is identified, and the results of the physiological and biochemical identification characteristics are shown in table 1.

TABLE 1

16S rDNA sequence detection is carried out on the Bacillus licheniformis X173 strain, 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 X173 can be identified as the bacillus licheniformis.

The bacillus licheniformis X173 strain is preserved in 2019, 9 and 17 days and preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC NO:18602; the preservation address is No. 3 of Xilu No.1 of Beijing, chaoyang, beijing.

Example 2

This example provides the use of Bacillus licheniformis strain X173 as a feed additive for reducing fecal ammonia emissions.

The Bacillus licheniformis X173 strain obtained in example 1 was added to the 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	51.42％
Excrement and urine	48.28％
		Average	49.85％

As can be seen from the results in Table 2, the addition of Bacillus licheniformis X173 strain to the feed significantly increased the protease activity. 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 licheniformis X173 strain as a fecal composting microbial inoculum in reducing fecal ammonia emission.

1. Reagent and preparation method

Sulfuric acid,. Rho (H) ₂ SO ₄ )＝1.84g/ml；

Sulfuric acid absorbent, c (1/2H) ₂ SO ₄ ) =0.01mol/L: measuring 2.7ml of sulfuric acid, adding the sulfuric acid into water, diluting the sulfuric acid to 1L to obtain 0.1mol/L stock solution, and diluting the stock solution by 10 times when the stock solution is used temporarily;

a nano-grade reagent: weighing 12g of sodium hydroxide (NaOH) dissolved in 60ml of water, cooling, weighing 1.7g of mercuric dichloride (HgCl) ₂ ) Dissolving the mercuric chloride solution in 30ml of water, weighing 3.5g of potassium iodide (KI) in 10ml of water, slowly adding the mercuric chloride solution into the potassium iodide solution under stirring until a formed red precipitate is not dissolved, slowly adding a sodium hydroxide solution cooled to room temperature into a mixed solution of the mercuric chloride and the potassium iodide under stirring, adding the rest mercuric chloride solution, uniformly mixing, standing in a dark place for 24 hours, 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, ρ =500g/L: 50g of potassium sodium tartrate (KNaC) are weighed ₄ H ₆ O ₆ ·4H ₂ O), adding 60ml of distilled water, heating in a water bath to dissolve, cooling and fixing the volume to 100ml.

2. Test procedure

The Bacillus licheniformis X173 strain obtained in example 1 was inoculated into NB medium and subjected to shake cultivation at 37 deg.C at 150r/min for 24h to obtain a fermentation broth. 200g of fresh pig manure is weighed and evenly placed in a 1000ml big beaker, the fermentation liquor of the bacillus licheniformis X173 strain is poured into the big beaker according to the inoculation amount of 20 percent (namely 40 ml) 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 cup wall of the big beaker to ensure the bonding property and the air tightness of the preservative film and the cup wall, 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 sulfuric acid absorption liquid. The above is the test group.

Meanwhile, a control group is set, the fermentation liquor of the strains is not inoculated in the control group, but the same amount of sterile water is added in the control group, 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. And after the culture is finished, determining the relative ammonia gas content of the test group and the control group by using a nano reagent spectrophotometry method for determining ammonia in ambient air and waste gas according to the national environmental protection standard HJ 533-2009 of the people's republic of China. And judging whether the bacillus licheniformis X173 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. Determination of Ammonia Release amount

Respectively putting 10ml of sulfuric acid absorption solution of each group after 7 days of culture 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 absorbance at a 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 ammonia gas in a test group compared with that in 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 generating the urease inhibitor on the livestock and poultry manure is shown. The absorbance value of the control group sample diluted 6 times was 0.502, and the absorbance of the test group sample added with the Bacillus licheniformis X173 strain was 0.207. According to calculation, the inhibition rate of the bacillus licheniformis X173 strain on ammonia gas in an in-vitro fecal test is 93.13%. The bacillus licheniformis X173 strain provided by the invention can greatly reduce the ammonia gas emission of livestock and poultry manure.

Example 4

The embodiment provides a composite microbial inoculum.

The preparation method of other strains for producing urease inhibitors comprises the following steps:

(1) taking fresh cattle manure of male cattle in a certain beef cattle farm in Baoding city, placing the fresh cattle manure at normal temperature for 36h to prepare a bacterial suspension, performing conventional culture by using an NA culture medium, and inoculating a bacterial colony obtained by culture to a primary screening culture medium for culture for 36h; the prescreening 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 distilled water, and the pH was 6.4.

(2) Selecting colonies which are not discolored at the periphery from the colonies obtained by the culture in the step (1), respectively using NA culture media for 36h, then inoculating the colonies into shaking flask primary screening culture media 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.

(3) Selecting colonies which turn red around from the colonies obtained by the culture in the step (1), respectively culturing for 24h by using an NA culture medium, and then inoculating the colonies in an NB culture medium for shake cultivation for 24h 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 0.1mol/L.

(4) Mixing the fermentation liquor of each sample to be selected obtained in the step (2) with the fermentation liquor of the urease-producing strain obtained in the step (3), adding urea and phenol red indicators, 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, and taking the strain corresponding to the test article with the color change time longer than the positive control for 2min to obtain the strain for 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 0.490 (3-fold dilution) 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 51.20%. The bacterial strain and a bacillus licheniformis X173 bacterial strain are compounded according to the mass ratio of 1. The result proves that under the condition of the same inoculation amount as that in example 3, the absorbance of the composite microbial inoculum test group provided by the embodiment is 0.533, namely, the inhibition rate of the composite microbial inoculum test group on ammonia gas in an in vitro fecal test is 82.30%, the inhibition rate is increased by 31.10% compared with the inhibition rate of the strain alone, and the inhibition rate is increased by 11.14% compared with the average inhibition rate of the strain and the bacillus licheniformis X173 strain, which indicates that the bacillus licheniformis X173 strain can improve the ammonia emission reduction effect of other strains for producing urease inhibitors.

Example 5

This example provides another complex inoculant.

(1) placing fresh chicken manure of healthy chicken flocks in a certain broiler farm in Baoding city at normal temperature for 24h to prepare a bacterial suspension, carrying out water bath at 80 ℃ for 10min, coating the bacterial suspension on an NA culture medium after gradient dilution for conventional culture, and inoculating the bacterial colony obtained by culture in a primary screening culture medium for 24h; the prescreening 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 distilled water, and the pH was 6.4.

(2) Selecting colonies which are not discolored at the periphery from the colonies obtained by culturing in the step (1), 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 culture 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.

(3) Selecting colonies which turn red around from the colonies obtained by the culture in the step (1), respectively culturing for 48h by using an NA culture medium, and then inoculating the colonies in an NB culture medium for shake cultivation for 48h to obtain fermentation liquor of each colony; adding urea aqueous solution and phenol red indicator into each bacterial colony fermentation liquid, shaking up, and taking fermentation liquid with shortest pink color appearance time, namely urease-producing strain fermentation liquid; adding 10ml of urea buffer solution into 200 mu l of urease producing strain fermentation liquor inactivated by 100 ℃ water bath for 10min, fully shaking and mixing, immediately placing in 30 ℃ +/-0.5 ℃ constant temperature water bath for 30min +/-10 s, adding 10ml of hydrochloric acid solution, shaking, rapidly cooling to below 20 ℃, and titrating with sodium hydroxide standard solution until the solution is blue-green; adding 10ml hydrochloric acid solution into 200 mul fermentation liquor of the urease producing strain, shaking, adding 10ml urea buffer solution, shaking fully, immediately placing in 30 +/-0.5 ℃ constant temperature water bath for 30min +/-10 s, cooling to below 20 ℃ rapidly, 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 0.1mol/L.

(4) Mixing the fermentation liquor of each sample to be selected obtained in the step (2) with the fermentation liquor of the urease-producing strain obtained in the step (3), adding urea and phenol red indicators, 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 this 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 this strain was 0.278 at the same inoculum size as in example 3, i.e., the inhibition of ammonia gas by this strain in the in vitro fecal test was 90.77%. The bacillus licheniformis X173 strain and the bacillus licheniformis X173 strain are compounded according to the mass ratio of 1, and the effect of the compound microbial inoculum in reducing the ammonia emission of excrement is examined in a mode of being used as an excrement composting microbial inoculum 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 0.097, namely, the inhibition rate of the bacterial strain in an in vitro fecal test is 96.78%, the inhibition rate is improved by 6.01% compared with the single bacterial strain, the inhibition rate is improved by 4.83% compared with the average inhibition rate of the bacterial strain and the Bacillus licheniformis X173 strain, and the inhibition rate is improved by 3.65% compared with the single Bacillus licheniformis X173 strain, which indicates that the Bacillus licheniformis X173 strain can improve the ammonia emission reduction effect of other strains producing urease inhibitor, and the bacterial strain can also improve the ammonia emission reduction effect of the Bacillus licheniformis X173 strain. The strain is identified as Bacillus subtilis and is preserved, the strain name is Bacillus subtilis J530, the classification name is Bacillus subtilis, 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:18603; the preservation address is No. 3 of Xilu No.1 of Beijing, chaoyang, beijing.

The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

SEQUENCE LISTING

<110> Wulat Qiang Yangsheng Chengsheng Dai Biotech fodder GmbH, hebei university of agriculture

<120> Bacillus licheniformis X173 strain for producing urease inhibitor and application thereof

<130> 2019.10.08

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1394

<212> DNA

<213> Bacillus licheniformis X173S rDNA

<400> 1

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

<210> 2

<211> 1371

<212> DNA

<213> Bacillus subtilis J530S rDNA

<400> 2

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

Claims

1. A Bacillus licheniformis X173 strain for producing urease inhibitor, which is classified as Bacillus licheniformis (Bacillus licheniformis), is preserved in China general microbiological culture Collection center in 2019, 9 and 17 days, 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.

2. Use of the bacillus licheniformis X173 strain according to claim 1 for reducing fecal ammonia emission.

3. The use of claim 2, wherein the bacillus licheniformis X173 strain is formulated as a microbial inoculant for use in a feed additive or a fecal composting inoculant by: 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 complex microbial preparation comprising the bacillus licheniformis X173 strain of claim 1 and other strains producing urease inhibitors; the other bacterial strains capable of producing the urease inhibitor are Bacillus subtilis J530 with classification names of Bacillus subtilis, which is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 9 and 17 days, and the preservation number of the Bacillus subtilis is CGMCC NO:18603; the preservation address is No. 3 of Xilu No.1 of Beijing, chaoyang, beijing.

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 out 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;

6. The complex microbial inoculant according to claim 5, wherein: the plate prescreening 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

In the step a, the excrement is prepared by placing fresh livestock excrement at normal temperature for 24-36 h.

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 NA culture medium for 24-48 h for the colonies without color change at the periphery, then inoculating the colonies in a shake flask primary screening culture medium containing urea for shake culture for 1-2 days, wherein the sample without turbidity 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 culture medium, and then inoculating the colonies in an 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 obtain a test sample; 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 with that of the positive control into pink, wherein the strain corresponding to the test article with the color change time longer than that of the positive control by at least 0.5min is the strain for producing the urease inhibitor.