CN110699299B - A kind of Bacillus licheniformis X173 bacterial strain producing urease inhibitor and application thereof - Google Patents

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

A kind of Bacillus licheniformis X173 bacterial strain producing urease inhibitor and application thereof

technical field

The invention relates to the technical field of microbes, in particular to a urease inhibitor-producing Bacillus licheniformis X173 strain and an application thereof.

Background technique

In recent years, with the rapid development of intensive and large-scale aquaculture, the environmental pollution caused by the volatilization and discharge of ammonia from livestock and poultry excrement has attracted more and more attention. The volatilization and discharge of ammonia not only cause surface water and soil Pollution, and serious air pollution, will cause great harm to the health of animals, breeders and residents around the farm. In the current breeding manure treatment system, a large amount of urea (pigs and other livestock) or uric acid (chickens and other poultry, which can be converted into urea after decomposition) contained in livestock and poultry manure is easily decomposed into ammonia by the urease produced by microorganisms and volatilized. 75% of the nitrogen in the excrement is lost, and the N/P ratio of the livestock and poultry manure is out of balance. For example, the N/P ratio of fresh pig manure is about 5:1, while the stored pig manure N/P The ratio is 1:1, and a large amount of nitrogen is lost, which reduces the fertilizer efficiency. Therefore, reducing ammonia emissions from livestock and poultry manure is of great significance for environmental protection and reuse of livestock and poultry manure.

Currently, urease inhibitors have been used to reduce ammonia emissions in production practice. Existing urease inhibitors mainly include inorganic compounds (such as heavy metal salts, borates, chlorides, fluorides, phosphates, nickel nitrate, etc.), organic compounds (such as isotopic acids, urea derivatives, hydroxylamine , hydroxamic acids, paraformaldehyde, quinones, polyphenols, heterocyclic thiols, etc.) and plant extracts (such as yucca extracts, lauraceae plant extracts, garlic extracts, etc.). Inorganic and organic compounds have safety problems, are toxic and polluting to humans, animals and the environment, and are difficult to meet the needs of the breeding industry. Plant extracts are relatively safe, but the plant growth cycle is long, the source of raw materials is unstable, the extraction process is more complicated, the cost of large-scale production is high, the efficiency is low, and the inhibitory effect on urease is low, and the ability to reduce ammonia volatilization is limited .

Contents of the invention

Aiming at the technical problems that the existing urease inhibitors are toxic and polluting to humans, animals and the environment, and have a low inhibitory rate to ammonia, the invention provides a urease inhibitor-producing Bacillus licheniformis X173 strain.

And, the present invention also provides the application of the above Bacillus licheniformis X173 strain in reducing fecal ammonia discharge.

And, the present invention also provides a composite microbial agent.

In order to achieve the above-mentioned purpose of the invention, the embodiment of the present invention adopts the following technical solutions:

A urease inhibitor-producing Bacillus licheniformis X173 strain, whose taxonomic name is Bacillus licheniformis (Bacillus licheniformis), was preserved in the General Microorganism Center of China Committee for Microorganism Culture Collection on September 17, 2019, and its preservation number is CGMCC NO: 18602; the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

The Bacillus licheniformis X173 strain provided by the present invention is isolated and screened from the fresh feces of healthy boars in pig farms, belongs to the genus Bacillus, is non-toxic, non-polluting, and has high activity for producing urease inhibitors. Its 16S rDNA The sequence is shown in SEQ ID NO.1. The Bacillus licheniformis X173 strain can grow and reproduce in the intestines of livestock and poultry and produce urease inhibitors, which can inhibit the decomposition of urea or uric acid in livestock and poultry feces into ammonia by urease, and the inhibition rate of urease activity in pig, chicken and other feces can reach 98.5%, effectively reducing the content of ammonia in the intestines and blood of livestock and poultry. Tests have proved that the Bacillus licheniformis X173 strain can grow well under facultative anaerobic conditions using a "feed-like medium" (a medium whose composition is close to that of livestock and poultry feed), and can produce 51.27U/ml of acid protease, It shows that the 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. Moreover, when the Bacillus licheniformis X173 strain is excreted with feces or directly added to in vitro feces, it can also continue to inhibit urease activity during the composting process, and the inhibition rate of ammonia in the in vitro fecal ammonia emission test can reach 93.13%, thus playing a role To the role of ammonia emission reduction.

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

① Take fresh pig feces from healthy boars in pig farms and place them at room temperature for 36 hours to make a bacterial suspension, bathe in water at 80°C for 10 minutes, apply gradient dilutions, and then apply NA medium for routine culture, and inoculate the cultured colonies on plates The primary screening medium was cultured for 36 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 ml agar, the balance is distilled water, pH 6.4.

②Pick out the surrounding undiscolored colonies from the colonies obtained in step ①, and culture them on the slant surface of NA medium for 48 hours, then inoculate them in the primary screening medium of the shaker flask and culture them on a shaking table for 2 days, and take the samples that are not turbid as the candidate for selection. Sample fermentation broth; shake flask primary screening medium contains 20mg/ml urea, 10mg/ml sucrose, 5mg/ml potassium chloride, 0.5mg/ml magnesium sulfate, 0.5mg/ml calcium chloride, 1mg/ml dihydrogen phosphate Sodium and 1 mg/ml disodium hydrogen phosphate, the balance is distilled water, pH is 6-8.

③Pick the colonies that turn red around from the colonies obtained in step ①, culture them with NA medium for 36 hours, and then inoculate them in NB medium for 36 hours on a shaking table to obtain the fermentation liquid of each colony; Add urea aqueous solution and phenol red indicator respectively, shake well, take the fermentation broth with the shortest time for the pink color to appear, which is the fermentation broth of the urease-producing strain; take 200 μl of the fermentation broth of the urease-producing strain that has been inactivated in a 100°C water bath for 10 minutes, and add 10ml of urea Buffer solution, after shaking and mixing, immediately place it in a constant temperature water bath at 30°C±0.5°C for 30min±10s, add 10ml of hydrochloric acid solution, shake and quickly cool to below 20°C, titrate with sodium hydroxide standard solution until the solution turns blue Green; take another 200μl of the fermentation broth of the urease-producing strain, add 10ml of hydrochloric acid solution and shake, then add 10ml of urea buffer solution and shake, after shaking fully, immediately place it in a constant temperature water bath at 30℃±0.5℃ for 30min±10s, and cool it down rapidly To below 20°C, titrate with sodium hydroxide standard solution until the solution is blue-green; judge the activity of the fermentation broth of the urease-producing strain according to the volume of the sodium hydroxide standard solution consumed by two parts of the fermentation broth of the urease-producing strain; wherein the urea buffer The solution is a phosphate buffer solution containing 0.5mol/L urea and a pH of 7.0±0.1, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1mol/L.

④Mix the fermented liquid of each sample to be selected obtained in step ② with the fermented liquid of the urease-producing strain obtained in step ③ respectively, add urea and phenol red indicator, dilute and shake well, and use it as a test product; prepare the fermented liquid without the sample to be selected in the same way The sample was used as a positive control; compare the time for each test product to turn pink with that of the positive control, and the strain corresponding to the test product whose discoloration time was longer than the positive control 3min was the strain that produced the urease inhibitor.

⑤Using GB/T 8622-2006 Determination of urease activity in soybean products for feed to detect the activity of the strain obtained in step ④, and re-screened to obtain the Bacillus licheniformis X173 strain.

And, the embodiment of the present invention also provides the application of the above-mentioned Bacillus licheniformis X173 strain in reducing fecal ammonia discharge.

The Bacillus licheniformis X173 strain provided by the present invention has efficient and stable ammonia reduction ability, can effectively control the activity of urease in the feces by adding it to the diet or directly adding it to the feces, and reduce the decomposition of urea nitrogen or uric acid nitrogen in the feces. At the same time, ammonia The reduction of emissions can also reduce the loss of compost fertilizer efficiency, and has great application potential in resourceful and harmless treatment of animal husbandry waste, control of environmental pollution, and promotion of healthy and sustainable development of animal husbandry.

Preferably, the Bacillus licheniformis X173 strain is made into a microbial inoculum for feed additives or feces composting inoculum, and the method for making the Bacillus licheniformis X173 bacterial strain into a microbial inoculum is: first liquid submerged fermentation, and then The obtained fermentation broth is subjected to continuous centrifugation, and finally the centrifuged precipitate is subjected to spray drying. Adding it to the diet or directly adding it to the manure can reduce the intestinal ammonia concentration of poultry and the ammonia concentration of the poultry house environment, thereby improving the environment in the livestock and poultry pen, reducing ammonia stress, and improving the immunity and production performance of livestock and poultry. It can reduce the reduction of N/P ratio of compost and promote the maturity of compost when used as manure composting agent. It can reduce the N/P ratio imbalance of compost and promote the maturity of compost when it is used as manure composting agent. The Bacillus licheniformis X173 strain is combined with liquid submerged fermentation, continuous centrifugation and spray drying, and the content of live bacteria (spores) in the obtained bacteria powder can reach 4.5×10 ¹¹ CFU/g. The obtained bacterium powder can be mixed with soluble starch to make a feed additive, so that the content of viable bacteria (spores) of the finally prepared microbial feed additive is 1.0×10 ¹⁰ CFU/g, and the water content is ≤8%. The obtained feed additive can be used for adding in feed, It can also be used for drinking water, and the shelf life is 18 months.

And, the embodiment of the present invention also provides a composite microbial agent, which contains the above-mentioned Bacillus licheniformis X173 strain and other strains producing urease inhibitors. There are various urease-producing microorganisms in feces, and there are also certain differences in the structure and properties of urease. The combination of different urease inhibitors has a synergistic effect on the ammonia emission reduction function, which can enhance the effect of ammonia emission reduction.

Preferably, the screening method for other bacterial strains capable of producing urease inhibitors specifically includes the following steps:

Step a, making feces into bacterial suspension, using NA medium for routine culture, and inoculating the cultured colony into the primary screening medium containing urea and acid-base indicator to cultivate;

Step b, picking the surrounding undiscolored colonies from the colonies cultivated in step a, and screening the strains that do not produce urease to make the fermentation broth of the sample to be selected;

Step c, picking the surrounding discolored colonies from the colonies cultivated in step a to make a fermentation broth of urease-producing strains;

Step d, mixing the fermentation liquid of the urease-producing strain obtained in step c with the fermentation liquid of the non-urease-producing strain obtained in step b, and screening out the urease inhibitor-producing strain.

The screening method is simple and easy to implement, does not require expensive and complicated equipment, and has low cost, which can be realized in general microbiological laboratories, and has high production efficiency and low cost for large-scale production. The bacterial strain in the fermented liquid of the sample to be selected obtained in step b is a strain that does not produce urease, and the fermented liquid of the urease-producing strain obtained in step c is the urease solution produced by microorganisms, and the two-step screening method of step b and d is combined, The screening efficiency can be improved, and the bacterial strain which has an inhibitory effect on urease and is more suitable for reducing the ammonia discharge of livestock and poultry manure can be easily screened out, thereby saving a lot of manpower and material resources.

Preferably, the primary screening medium described 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 ml agar, the balance is distilled water, pH 6.4.

Preferably, the livestock and poultry manure in step a is fresh livestock and poultry manure left at room temperature for 24-36 hours.

Preferably, the method for screening the strains that do not produce urease in step b to prepare the fermentation broth of the candidate sample is as follows: respectively use NA medium for 24-48 hours on the surrounding undiscolored colonies, and then inoculate them in shake flasks containing urea Incubate on a shaker in the primary screening medium for 1-2 days, and the sample that is not turbid is the fermentation broth of the sample to be selected. The lack of turbidity in the shake flask medium containing urea indicates that the strain does not grow, which proves that the strain cannot produce urease and cannot use 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 the fermentation broth of a non-urease-producing strain. The ingredients of the shaking flask primary screening medium are preferably: 20mg/ml urea, 10mg/ml sucrose, 5mg/ml potassium chloride, 0.5mg/ml magnesium sulfate, 0.5mg/ml calcium chloride, 1mg/ml dihydrogen phosphate Sodium and 1 mg/ml disodium hydrogen phosphate, the balance is distilled water, pH is 6-8. Among them, the method of culturing with NA medium is preferably cultured on a slant.

Preferably, the method for preparing the fermentation broth of the urease-producing strain in step c is: pick the colonies that turn red around the colonies cultured in step a, culture them with NA medium for 24-48 hours, and then inoculate them in NB medium for shaking Bed culture for 24 to 48 hours to obtain the fermentation liquid of each strain; add urea aqueous solution and phenol red indicator to the obtained fermentation liquid of each strain, shake well, and take the fermentation liquid with the shortest time for the pink color to appear, which is the fermentation liquid of the urease-producing strain. The bacterium colony that becomes red around is the strain bacterium colony that produces urease, and its red range spreads bigger, and it shows that its urease activity is stronger, this step preferably the bacterium colony that red range is as large as possible, to ensure that the activity of the bacterial strain that produces urease is more powerful. The method of culturing with NA medium is preferably slant culture. The shorter the pink appearance time of the fermentation broth, the higher the urease activity of the corresponding urease-producing strains, and the pink appearance time of the strains with high activity is less than 15 minutes. The urease activity of the strain corresponding to the fermentation broth with the shortest pink appearance time can reach 0.165U/g, which can be used as a screening tool for the screening of the Bacillus licheniformis X173 strain in step d.

Preferably, step c also includes re-screening the fermented liquid of the obtained urease-producing strain, the re-screened method is: mixing the fermented liquid of the urease-producing strain with a urea buffer, and consuming the urea in the urea buffer The situation judges the activity of the fermented liquid bacterial strain of described urease-producing strain. The specific method can be preferably as follows: take 200 μl of the inactivated fermentation broth of the urease-producing strain, add 10ml of urea buffer solution, shake and mix thoroughly, immediately place it in a constant temperature water bath at 30°C±0.5°C for 30min±10s, add 10ml of hydrochloric acid Solution, after shaking, cool down rapidly to below 20°C, titrate with sodium hydroxide standard solution until the solution is blue-green; take another 200 μl of the fermentation broth of the urease-producing strain, add 10ml of hydrochloric acid solution for shaking, and then add 10ml of urea buffer solution for shaking Shake, after fully shaking, immediately place it in a constant temperature water bath at 30°C±0.5°C for 30min±10s, cool it down to below 20°C rapidly, titrate with sodium hydroxide standard solution until the solution turns blue-green; according to the two copies of the urease-producing strain The volume of sodium hydroxide standard solution consumed by the fermentation broth judges the activity of the fermentation broth of the urease-producing strain; wherein the urea buffer is a phosphate buffer containing 0.5mol/L urea and a pH of 7.0±0.1, and the hydrochloric acid solution The concentration of hydrogen chloride is 0.1mol/L. The method of inactivating the fermentation broth of the urease-producing strain is preferably in a water bath at 100° C. for 10 minutes.

式中：X为试样的尿素酶活性(U/g)；c为氢氧化钠标准滴定溶液浓度(mol/L)；V₀为第二份产脲酶菌株发酵液消耗氢氧化钠标准滴定溶液体积(ml)；V为第一份产脲酶菌株发酵液消耗氢氧化钠标准滴定溶液体积(ml)；14为氮的摩尔质量；30为反应时间(min)；m为试样体积(ml)。根据此方法可以计算出产脲酶菌株的具体脲酶活力。Among them, the rapid cooling can adopt methods such as ice bath or cold water showering on the outer wall of the container. The method for judging activity can be calculated by the following formula:

In the formula: X is the urease activity (U/g) of sample; C is the sodium hydroxide standard titration solution concentration (mol/L) _; Volume (ml); V is the volume (ml) of sodium hydroxide standard titration solution consumed by the fermentation broth of the first urease strain; 14 is the molar mass of nitrogen; 30 is the reaction time (min); m is the sample volume (ml) . According to this method, the specific urease activity of the urease-producing strain can be calculated.

Preferably, the specific operation method of step d is as follows: mix the fermentation liquid of each of the candidate samples obtained in step a with the fermentation liquid of the urease-producing strain, add urea and phenol red indicator, shake well after dilution, and use it as a test product Prepare in the same way the sample that does not contain the fermentation liquid of the sample to be selected, as a positive control; compare the time when the color of each described test product and the positive control turns pink, and the color change time is longer than the described test of the positive control at least 0.5min The strain corresponding to the product is the strain that produces the urease inhibitor.

Preferably, the strains in the other bacterial agents capable of producing urease inhibitors are the Bacillus subtilis J530 strain, and its classification name is Bacillus subtilis (Bacillus subtilis), which was deposited in the China Microbiological Culture Collection on September 17, 2019. General Microbiology Center of the Management Committee, its deposit number is CGMCC NO: 18603; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The strain has a significant synergistic effect with the above-mentioned Bacillus licheniformis X173 strain, and has a better effect for reducing ammonia emission from livestock and poultry manure. Its 16S rDNA sequence is shown in SEQ ID NO.2.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Example 1

The embodiment of the present invention provides a urease inhibitor-producing Bacillus licheniformis X173 strain, which is screened through the following steps:

1. Screening of non-urease-producing strains

1.1 Medium formula

Plate initial screening 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 is distilled water, The pH is 6.4.

Shake flask primary screening 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, the balance is distilled water, pH is 6-8.

NA medium: 10mg/ml peptone, 3mg/ml beef extract, 5mg/ml sodium chloride, 20mg/ml agar, the balance is distilled water, pH7.2～7.4

All media were prepared with distilled water and sterilized by high-pressure steam at 121°C for 20 minutes.

1.2 Plate initial screening

Fresh pig feces samples from healthy male pigs in a pig farm in Baoding City were taken and placed at room temperature for 36 hours to allow the bacillus in them to fully form spores. Weigh 5 g of feces samples after standing still, add them to a 250 ml Erlenmeyer flask filled with 45 ml of sterile water and 15 glass beads, shake for 5 min on a shaker at 150 r/min, and bathe in 80 ° C for 10 min to obtain a concentration of 10 ⁻¹ bacterial suspension. Then 10-fold serial dilution was performed with sterile water to obtain bacterial suspensions with concentrations of 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 and 10 ^-7 . Take bacterial suspensions with concentrations of 10 ^-4 , 10 ^-5 , 10 ^-6 , and 10 ^-7 and spread 5 NA plate medium respectively, 0.1ml per plate, and incubate upside down at 37°C for 36h.

Use sterilized bamboo sticks to pick up the colonies growing on the NA plate medium, inoculate with a "cross" on the plate primary screening medium plate, and observe the color change around the "cross" bacterial lawn after inverting at 37°C for 36 hours. Screen and pick the colonies that are still yellow around (do not turn red) into the NA slant medium, culture at 37°C for 48 hours, and store them for later use.

1.3 Shake flask primary screening

Use a sterilized bamboo stick to pick up the slant bacterial lawn obtained in the 1.2-plate primary screening, inoculate it into a 50ml shake flask primary screening medium (250ml Erlenmeyer flask), culture on a shaker at 37°C and 150r/min for 2 days, and observe the turbidity of the medium Condition. The fermented liquid that is not turbid is the fermented liquid of the strain that does not produce urease, and it is preserved for future use. A total of 126 non-urease-producing strains were screened out from more than 5000 strains.

2. Screening of urease-producing strains

2.1 Reagent and medium formula

Urea: analytically pure.

Phenol red indicator: Weigh 0.1g of phenol red, add 1.43ml of 0.1mol/L sodium hydroxide solution, grind in a mortar to promote dissolution, then transfer to a 250ml volumetric flask, add distilled water to the mark, and shake well for later use.

Plate initial screening 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 is distilled water, pH is 6.4;

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

NB medium: 10mg/ml peptone, 3mg/ml beef extract, 5mg/ml sodium chloride, the balance is distilled water, pH is 7.2-7.4;

All media were prepared with distilled water and sterilized by high-pressure steam at 121°C for 20 minutes.

Urea buffer solution (pH7.0±0.1): Weigh 8.95g disodium hydrogen phosphate, 3.40g potassium dihydrogen phosphate dissolved in water and dilute to 1000ml, then dissolve 30g urea in this buffer solution, valid for one month.

Hydrochloric acid solution [c(HCl)=0.1mol/L]: Pipette 8.3ml of hydrochloric acid and dilute to 1000ml with water.

Sodium hydroxide solution [c(NaOH)=0.1mol/L]: Weigh 4g of sodium hydroxide, dissolve it in water and dilute to 1000ml.

Mixed indicator: methyl red, bromocresol green mixed ethanol solution: weigh 0.1g methyl red, dissolve in 95% ethanol and dilute to 100ml, then weigh 0.5g bromocresol green, dissolve in 95% ethanol And diluted to 100ml, the two solutions were mixed in equal volumes and stored in brown bottles.

2.2 Primary screening of urease-producing strains

Take the "cross" on the primary screening medium plate in 1.2 and inoculate the colonies that turn red around the bacterial lawn and spread in a large range to the NA slant medium, culture at 37°C for 36 hours, and store it for later use. These strains were inoculated into a 250ml Erlenmeyer flask containing 50ml of NB medium, and cultured on a shaker at 150r/min for 24h to prepare a fermentation broth.

Take 1.0ml of the above-mentioned fermentation broth, add it to a 25ml colorimetric tube, add 0.02g urea (1.0ml of a 2% (m/v) urea aqueous solution that is now prepared), add 2 drops of phenol red indicator, and add 20ml of water to fully Shake well for 15 seconds. Record the time when the pink color appears, and judge the urease activity according to the time. A sample blank (without urea) and a reagent blank (without sample) were also set up. The color of the sample blank and reagent blank did not change after adding the phenol red indicator, indicating that the test results were reliable. Record the fermentation broth with the shortest pink appearance time (less than 15 minutes), which is the fermentation broth of the urease-producing strain of the present invention, and the corresponding strain has the highest urease activity, and it is stored for future use.

2.3 Re-screening of urease-producing strains

Measure 200μl±1μl of urease-producing strain fermentation broth into a colorimetric tube, add 10ml of urea buffer, immediately cover the colorimetric tube and shake vigorously, then immediately place the colorimetric tube in a constant temperature water bath at 30°C±0.5°C, and time Keep it for 30min±10s. When the reaction is stopped, add 10ml of hydrochloric acid solution, shake and cool to 20°C rapidly. Transfer all the contents of the colorimetric tube into a 250ml Erlenmeyer flask, add 8-10 drops of mixed indicator, titrate with sodium hydroxide standard solution until the solution turns blue-green, and record the volume of sodium hydroxide consumed.

Take another colorimetric tube for blank test, measure 200μl±1μl of the fermentation broth of urease-producing strains inactivated in a 100°C water bath for 10 minutes, add 10ml of hydrochloric acid solution, shake, then add 10ml of urea buffer, immediately Cover the colorimetric tube cover and shake vigorously, then immediately place the colorimetric tube in a constant temperature water bath at 30°C±0.5°C, and keep the timer for 30min±10s. When the reaction is stopped, cool the colorimetric tube to 20°C quickly, then transfer all the contents of the colorimetric tube into a 250ml conical flask, add 8-10 drops of mixed indicator, and titrate with sodium hydroxide standard solution until the solution turns blue Green, record the volume of sodium hydroxide consumed.

In formula: X is the urease activity (U/g) of sample; C is sodium hydroxide standard titration solution concentration (mol/L); V ₀ is blank consumption sodium hydroxide standard titration solution volume (ml); V is The volume of sodium hydroxide standard titration solution consumed by the sample (ml); 14 is the molar mass of nitrogen; 30 is the reaction time (min); m is the sample volume (ml).

3. Screening of strains producing urease inhibitors

Get 1.0ml of urease-producing strain fermentation broth and add it to a 25ml colorimetric tube, add 1.0ml of the fermentation broth of the non-urease-producing strain obtained in 1.3 above, mix well, add 0.02g of urea (2% (m/v) Urea aqueous solution (1.0ml) and phenol red indicator 2 drops, add water 20ml, shake well for 15 seconds, as the test group. At the same time, the preparation method was the same as that of the test group except that the sample of the fermentation broth of the non-urease-producing strain was not added, which was used as a positive control group. Record test group colorimetric tube and positive control group colorimetric tube pink appearance time, and judge whether there is urease inhibitor in the fermented liquid according to time, if test group colorimetric tube pink appearance time is longer than positive control group colorimetric tube, It means that there is a urease inhibitor; if the pink color tube of the test group is the same as or shorter than that of the positive control group, it means that there is no urease inhibitor. According to this principle, the fermentation broth that can contain urease inhibitor can be selected , and its corresponding strain is the strain producing urease inhibitor. Take the strain corresponding to the test product whose discoloration time is longer than 3 minutes of the positive control.

4. Re-screening of strains producing urease inhibitors

With the urease inhibitor-producing bacterial strains initially screened out above, the urease inhibitor-producing bacterial strains of the obtained bacterial strains were quantitatively detected by using GB/T 8622-2006 Determination of Urease Activity in Soybean Products for Feed, in sample test tubes and Add 200 μl of urease inhibitor-producing bacterial strain fermentation broth to blank test tubes, measure the urease activity of the urease-producing bacterial strain after adding the urease inhibitor, and then compare this urease activity with the urease activity of the previously known urease-producing bacterial strain without urease inhibitor. Compared with the activity, the urease activity reduction value is calculated, so that the Bacillus licheniformis X173 strain with urease inhibitor can be screened out.

5. Identification of Bacillus licheniformis X173 strain

The Bacillus licheniformis X173 strain was identified, and the results of physiological and biochemical identification characteristics are shown in Table 1.

Table 1

The 16S rDNA sequence was detected on the Bacillus licheniformis X173 strain, and the obtained sequence is shown in SEQ ID NO.1. According to the identification characteristics in Table 1 and 16S rDNA sequence detection, the X173 strain can be identified as Bacillus licheniformis.

The Bacillus licheniformis X173 strain was preserved on September 17, 2019 in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, and its preservation number is CGMCC NO: 18602; the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing number 3.

Example 2

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

The Bacillus licheniformis X173 strain obtained in Example 1 was added to pig feed at an addition amount of 0.1% (m/m), and the changes in protease activity in pig feces and intestinal tract were detected. The results are shown in Table 2.

Table 2

Detection site Increase rate of protease activity intestine 51.42% stool 48.28% average 49.85%

It can be seen from the results in Table 2 that the protease activity can be significantly improved after adding the Bacillus licheniformis X173 strain to the feed. The improvement of protease activity can improve the utilization rate of feed protein, reduce the excretion of nitrogenous substances such as protein, and reduce the emission of fecal ammonia gas from the source of ammonia gas generation.

Example 3

This example provides the application of the Bacillus licheniformis X173 strain as a fecal composting bacterial agent in reducing fecal ammonia emissions.

1. Reagents and preparation methods

Sulfuric acid, ρ(H ₂ SO ₄ )=1.84g/ml;

Sulfuric acid absorption solution, c(1/2H ₂ SO ₄ )=0.01mol/L: Measure 2.7ml of sulfuric acid into water and dilute to 1L to prepare a 0.1mol/L stock solution, which should be diluted 10 times before use;

Nessler's reagent: Weigh 12g of sodium hydroxide (NaOH) and dissolve it in 60ml of water, cool it, weigh 1.7g of mercuric dichloride (HgCl ₂ ) and dissolve it in 30ml of water, weigh 3.5g of potassium iodide (KI) in 10ml of water, Slowly add the above mercuric dichloride solution into the potassium iodide solution under stirring until the formed red precipitate is no longer dissolved, then slowly add the sodium hydroxide solution cooled to room temperature into the above mercuric dichloride and potassium iodide solution under stirring Add the remaining mercuric dichloride solution to the mixed solution, mix well and let it stand in the dark for 24 hours, pour out the supernatant, store it in a brown bottle, plug it tightly with a rubber stopper, store it at 2°C to 5°C Use within 1 month;

Potassium sodium tartrate solution, ρ=500g/L: Weigh 50g of potassium sodium tartrate (KNaC ₄ H ₆ O ₆ ·4H ₂ O), add 60ml of distilled water, heat in a water bath to dissolve, and then dilute to 100ml after cooling.

2. Test steps

The Bacillus licheniformis X173 strain obtained in Example 1 was inoculated into NB medium, and cultured on a shaker at 150 r/min at 37° C. for 24 hours to prepare a fermentation broth of the strain. Weigh 200g of fresh pig manure, put it evenly into a 1000ml large beaker, pour the fermented liquid of Bacillus licheniformis X173 bacterial strain into the large beaker according to 20% inoculum size (i.e. 40ml) and mix evenly, then absorb 40ml of sulfuric acid into the large beaker. The 50ml small beaker of liquid is also put into the large beaker. First apply a layer of Vaseline on the outer wall of the beaker to ensure the adhesion and airtightness of the plastic wrap and the wall of the cup, and then seal the mouth of the large beaker with double layers of plastic wrap to ensure that the ammonia gas produced will not Leaked into the atmosphere, can be absorbed by sulfuric acid absorbent. The above is the test group.

A control group is set at the same time, the control group does not inoculate the fermented liquid of the strain but adds an equal amount of sterile water, and the rest are the same as the test group.

Then put the sealed large beaker into a biochemical incubator with a constant temperature of 30° C., and cultivate it for 7 days. After the cultivation, the relative ammonia content of the test group and the control group was determined by Nessler's reagent spectrophotometry according to the National Environmental Protection Standard of the People's Republic of China HJ 533-2009 Determination of Ammonia in Ambient Air and Waste Gas. By comparing the difference of ammonia gas produced between the test group and the control group, it can be judged whether the Bacillus licheniformis X173 strain has the effect of reducing ammonia emission on livestock and poultry manure.

3. Determination of ammonia release

Take 10ml of the sulfuric acid absorption solution of each group after 7 days of culture above, respectively, in 25ml colorimetric tubes, add 0.50ml potassium sodium tartrate solution, shake well, then add 0.50ml Nessler's reagent, shake well, after standing for 10min, wash with water For reference, use a 10mm cuvette to measure the absorbance at a wavelength of 420nm. Because dilute sulfuric acid solution absorbs ammonia in the air, the ammonium ions generated react with Nessler's reagent to form a yellow-brown complex. The absorbance at the wavelength of 420nm can be used to know how much the ammonia gas in the test group is reduced compared with the control group, so as to see the ammonia emission reduction effect of the strains screened by the strain screening method for producing urease inhibitors provided by the present invention for livestock and poultry manure. According to this method, the absorbance value of the sample of the control group diluted 6 times was 0.502, and the absorbance of the sample of the test group added with the above-mentioned Bacillus licheniformis X173 strain was 0.207. According to the calculation, it can be known that the inhibition rate of the Bacillus licheniformis X173 strain to ammonia gas in the in vitro feces test is 93.13%. It shows that the Bacillus licheniformis X173 strain provided by the invention can greatly reduce the ammonia gas emission of livestock and poultry manure.

Example 4

This embodiment provides a composite bacterial agent.

Prepare other bacterial strains producing urease inhibitors, the specific preparation method is:

① Take fresh cow manure from a beef cattle farm in Baoding City and place it at room temperature for 36 hours to make a bacterial suspension, use NA medium for routine culture, and inoculate the cultured colonies on the primary screening medium for 36 hours; 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, pH 6.4.

②Pick out the surrounding undiscolored colonies from the colonies cultivated in step ①, respectively use NA medium for 36 hours, and then inoculate them in the primary screening medium of shaker flasks and culture them on a shaking table for 1 day, take samples that are not turbid, and ferment them as samples to be selected liquid; shake flask primary screening 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, pH is 6-8.

③Pick out the colonies that turn red around from the colonies obtained in step ①, culture them with NA medium for 24 hours, and then inoculate them in NB medium for 24 hours on a shaking table to obtain the fermentation liquid of each colony; Add urea aqueous solution and phenol red indicator respectively, shake well, take the fermentation broth with the shortest time for the pink color to appear, which is the fermentation broth of the urease-producing strain; take 200 μl of the fermentation broth of the urease-producing strain, add 10ml of urea buffer, shake and mix thoroughly Immediately put it in a constant temperature water bath at 30°C±0.5°C for 30min±10s, add 10ml of hydrochloric acid solution, shake it and cool it down to below 20°C quickly, titrate with standard sodium hydroxide solution until the solution turns blue-green; another 200μl is heated at 100°C Add 10ml of hydrochloric acid solution to shake the fermented liquid of the urease-producing strain inactivated in water bath for 10 minutes, then add 10ml of urea buffer solution and shake, after shaking fully, immediately place it in a constant temperature water bath at 30℃±0.5℃ for 30min±10s, and cool it down rapidly To below 20°C, titrate with sodium hydroxide standard solution until the solution is blue-green; judge the activity of the fermentation broth of the urease-producing strain according to the volume of the sodium hydroxide standard solution consumed by two parts of the fermentation broth of the urease-producing strain; wherein the urea buffer The solution is a phosphate buffer solution containing 0.5mol/L urea and a pH of 7.0±0.1, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1mol/L.

④Mix the fermented liquid of each sample to be selected obtained in step ② with the fermented liquid of the urease-producing strain obtained in step ③ respectively, add urea and phenol red indicator, dilute and shake well, and use it as a test product; prepare the fermented liquid without the sample to be selected in the same way The sample was used as a positive control; compare the time when the color of each test product and the positive control turns pink, and take the strain corresponding to the test product whose discoloration time is longer than the positive control 2min, to obtain the bacterial strain producing urease inhibitor.

By the method for embodiment 3, investigate the effect of this bacterial strain in reducing feces ammonia discharge as the form of feces composting bacterial agent, the result proves, this bacterial strain is under the situation of identical inoculum amount in embodiment 3 and its absorbance is 0.490 (diluted 3 times), that is, the inhibitory rate of the bacterial strain to ammonia in the in vitro feces test is 51.20%. The strain was compounded with the Bacillus licheniformis X173 strain at a mass ratio of 1:1. According to the method of Example 3, the effect of the composite bacterial agent in reducing feces ammonia emissions was investigated in the form of a fecal composting bacterial agent. Result proves, under the situation of the same inoculum amount as in embodiment 3, the absorbance of the compound microbial agent test group provided by the present embodiment is 0.533, that is, the inhibition rate to ammonia is 82.30% in in vitro feces test, compares The inhibitory rate of using this bacterial strain alone has improved 31.10%, and has improved 11.14% than the average value of the inhibitory rate of this bacterial strain and Bacillus licheniformis X173 bacterial strain, illustrates that Bacillus licheniformis X173 bacterial strain can improve the bacterial strain ammonia emission reduction of other urease inhibitors Effect.

Example 5

This embodiment provides another kind of composite bacterial agent.

Prepare other bacterial strains producing urease inhibitors, the specific preparation method is:

① Take fresh chicken manure from a healthy chicken group in a broiler farm in Baoding City and place it at room temperature for 24 hours to make a bacterial suspension, bathe in 80°C water for 10 minutes, apply gradient dilution and then use NA medium for routine culture, and inoculate the cultured colonies in The primary screening medium was cultured 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 is distilled water, pH 6.4.

②Pick the undiscolored colonies from the colonies obtained in step ①, and culture them on the slant surface of NA medium for 36 hours, then inoculate them in the primary screening medium of the shaker flask and culture them on a shaker for 1 day, and take the samples that are not turbid as the candidate for selection. Sample fermentation broth; shake flask primary screening medium contains 20mg/ml urea, 10mg/ml sucrose, 5mg/ml potassium chloride, 0.5mg/ml magnesium sulfate, 0.5mg/ml calcium chloride, 1mg/ml dihydrogen phosphate Sodium and 1 mg/ml disodium hydrogen phosphate, the balance is distilled water, pH is 6-8.

③Pick the colonies that turn red around from the colonies obtained in step ①, culture them with NA medium for 48 hours, and then inoculate them in NB medium for 48 hours on a shaking table to obtain the fermentation broth of each colony; Add urea aqueous solution and phenol red indicator respectively, shake well, take the fermentation broth with the shortest time for the pink color to appear, which is the fermentation broth of the urease-producing strain; take 200 μl of the fermentation broth of the urease-producing strain that has been inactivated in a 100°C water bath for 10 minutes, and add 10ml of urea Buffer solution, after shaking and mixing, immediately place it in a constant temperature water bath at 30°C±0.5°C for 30min±10s, add 10ml of hydrochloric acid solution, shake and quickly cool to below 20°C, titrate with sodium hydroxide standard solution until the solution turns blue Green; take another 200μl of the fermentation broth of the urease-producing strain, add 10ml of hydrochloric acid solution and shake, then add 10ml of urea buffer solution and shake, after shaking fully, immediately place it in a constant temperature water bath at 30℃±0.5℃ for 30min±10s, and cool it down rapidly To below 20°C, titrate with sodium hydroxide standard solution until the solution is blue-green; judge the activity of the fermentation broth of the urease-producing strain according to the volume of the sodium hydroxide standard solution consumed by two parts of the fermentation broth of the urease-producing strain; wherein the urea buffer The solution is a phosphate buffer solution containing 0.5mol/L urea and a pH of 7.0±0.1, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1mol/L.

④Mix the fermented liquid of each sample to be selected obtained in step ② with the fermented liquid of the urease-producing strain obtained in step ③ respectively, add urea and phenol red indicator, dilute and shake well, and use it as a test product; prepare the fermented liquid without the sample to be selected in the same way The sample was used as a positive control; compare the time when the color of each test product and the positive control turns pink, and take the strain corresponding to the test product whose discoloration time is longer than the positive control 1.5min, to obtain the bacterial strain producing urease inhibitor. According to the method of Example 3, investigate the effect of the bacterial strain in reducing feces ammonia discharge as the form of feces composting bacterial agent, the results prove that the absorbance of the bacterial strain is 0.278 under the same inoculation amount as in Example 3, namely , the strain's inhibitory rate to ammonia was 90.77% in the in vitro stool test. The bacterium was compounded with the Bacillus licheniformis X173 strain at a mass ratio of 1:1, and the effect of the composite bacterium in reducing feces ammonia emissions was investigated in the form of a fecal composting bacterium according to the method of Example 3. Result proves, under the situation of the same inoculum amount as in embodiment 3, the absorbance of the compound microbial agent test group provided by the present embodiment is 0.097, that is, the inhibition rate to ammonia is 96.78% in in vitro feces test, compares The inhibitory rate of using this bacterial strain alone has improved 6.01%, and has improved 4.83% than the inhibitory rate average value of this bacterial strain and Bacillus licheniformis X173 bacterial strain, and has improved 3.65% than single-use Bacillus licheniformis X173 bacterial strain, illustrates that Bacillus licheniformis The X173 strain can improve the ammonia emission reduction effect of other strains producing urease inhibitors, and the bacterial strain can also improve the ammonia emission reduction effect of the Bacillus licheniformis X173 strain. The strain was identified as Bacillus subtilis, and it was preserved. The strain name is Bacillus subtilis J530, and its classification name is Bacillus subtilis. General Microbiology Center, its deposit number is CGMCC NO: 18603; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Any modification, equivalent replacement or improvement made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

SEQUENCE LISTING

<110> Wulate Qianqi Rongsheng Dadi Biotechnology Feed Co., Ltd., Hebei Agricultural University

<120> A Bacillus licheniformis X173 strain producing urease inhibitor and its application

<130> 2019.10.08

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1394

<212> DNA

<213> Bacillus licheniformis X173 16S rDNA

<400> 1

tgctaataca tgcaagtcga gcggaccgac gggagcttgc tcccttaggt cagcggcgga 60

cgggtgagta aacacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg 120

ctaataccgg atgcttgatt gaaccgcatg gttcaattat aaaagggggc ttttagctac 180

cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg 240

acgatgcgta gccgacctga gagggtgatc ggccaacactg 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 J530 16S rDNA

<400> 2

ccctgatgtt agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga 60

taactccggg aaaccggggc taataccgga tggttgtttg aaccgcatgg ttcaaacata 120

aaaggtggct tctgctacga cttacagatg ggcccgcggc gcatttatcta gttggtgagg 180

taacggctca ccaaggcgac gatgcgtatc cgacctgaga gggtgatcgg ccaacactggg 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 (9)

1. A urease inhibitor-producing Bacillus licheniformis X173 strain, whose classification name is Bacillus licheniformis (Bacillus licheniformis), was preserved on September 17, 2019 in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and its preservation number It is CGMCC NO: 18602; the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. 2. the application of bacillus licheniformis X173 bacterial strain described in claim 1 in reducing feces ammonia discharge. 3. application according to claim 2, is characterized in that, said Bacillus licheniformis X173 bacterial strain is made into microbial inoculum for feed additive or feces composting inoculum, and described Bacillus licheniformis X173 bacterial strain is made into microbial inoculum The preparation method is as follows: submerged fermentation in liquid state first, then continuous centrifugation of the fermented liquid obtained, and finally spray drying of the precipitate obtained by the centrifugation. 4. a composite microbial agent, is characterized in that, described composite bacterial agent contains the bacterial strain of bacillus licheniformis X173 bacterial strain described in claim 1 and other urease inhibitors; described other bacterial strains capable of producing urease inhibitors are Bacillus subtilis Bacillus J530, whose taxonomic name is Bacillus subtilis, was deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on September 17, 2019, and its preservation number is CGMCC NO: 18603; the preservation address is Chaoyang, Beijing No. 3, Courtyard No. 1, Beichen West Road, District. 5. composite bacterial agent according to claim 4, is characterized in that: the screening method of the bacterial strain that described other can produce urease inhibitor specifically comprises the following steps: Step a, make feces into bacterial suspension, use NA medium for conventional culture, inoculate the cultured bacterial colony on the plate primary screening medium containing urea and acid-base indicator and cultivate; Step b, picking the surrounding undiscolored colonies from the colonies cultivated in step a, and screening the strains that do not produce urease to make the fermentation broth of the sample to be selected; Step c, picking the surrounding discolored colonies from the colonies cultivated in step a to make a fermentation broth of urease-producing strains; Step d, mixing the fermentation liquid of the urease-producing strain obtained in step c with the fermentation liquid of the non-urease-producing strain obtained in step b, and screening out the urease inhibitor-producing strain. 6. The composite microbial agent according to claim 5, characterized in that: the plate primary screening medium described in step a contains 20mg/ml urea, 1mg/ml phenol red, 10mg/ml sucrose, 10mg/ml peptone, 3mg/ml ground beef, 5mg/ml sodium chloride and 20mg/ml agar, the balance being distilled water, pH 6.4; and/or The feces in step a are fresh livestock and poultry feces left at room temperature for 24-36 hours. 7. The compound bacterial agent according to claim 5, wherein the method for making the sample fermentation liquid by screening the bacterial strain that does not produce urease as described in step b is as follows: the non-discolored bacterium colonies around the described area are respectively treated with NA Culture medium for 24-48 hours, and then inoculated in the primary screening medium of shake flasks containing urea and cultivated on a shaking table for 1-2 days. The sample that is not turbid is the fermentation broth of the sample to be selected. 8. composite bacterial agent according to claim 5, it is characterized in that, the method for preparing urease strain fermented liquid in the step c is: pick the bacterium colony that changes color around from step a culture gained bacterium colony, cultivate with NA culture medium respectively 24-48 hours, then inoculated in NB medium and cultured on a shaking table for 24-48 hours to obtain the fermentation liquid of each strain; add urea aqueous solution and phenol red indicator to the obtained fermentation liquid of each strain, shake well, and take the pink color with the shortest time to appear a fermentation broth, i.e. a fermentation broth of a urease-producing strain; and/or Step c also includes re-screening the fermented liquid of the obtained urease-producing strain. The re-screened method is: mixing the fermented liquid of the urease-producing strain with a urea buffer, and judging the urea consumption in the urea buffer. The activity of the fermented broth strain of the urease-producing strain was described. 9. The compound bacterial agent according to claim 5, characterized in that, the specific operation method of step d is: each described candidate sample fermented liquid of step b gained is mixed with described urease-producing strain fermented liquid respectively, adds urea and phenol red indicator, shake up after dilution, as the test product; prepare the sample that does not contain the fermentation liquid of the sample to be selected in the same way, as the positive control; compare the time when the color of each of the test products and the positive control turns pink, The strain corresponding to the test product whose discoloration time is at least 0.5 min longer than the positive control is the strain that produces the urease inhibitor.