Screening method of urease inhibitor producing bacteria

Abstract

The invention particularly relates to a screening method of urease inhibitor producing bacteria. The screening method comprises the following steps: screening ammonia nitrogen efficient bacteria; screening strains which do not produce urease; screening of urease inhibitor producing strains, wherein the strains with high urease activity inhibition rate are selected as the urease inhibitor producing strains. According to the screening method of the urease inhibitor producing bacteria, ammonia nitrogen efficient bacteria are firstly screened, then urease inhibitor producing strains are screened, generation of ammonia gas is controlled from the source, NH3 is decomposed from the terminal, and therefore the problem of stink of the ammonia gas can be more efficiently solved; according to the secondary screening method for the strains not producing urease, the defect that alkali-producing strains not producing urease are excluded by a urea-phenol red plate can be avoided, the strains not producing urease can be relatively comprehensively screened, overflowing NH3 is detected, and the secondary screening method is relatively suitable for an odor generation scene; a urease material of a urease inhibitor production strain is a supernatant obtained after centrifugation of a bacterial liquid, mainly a bio-enzyme rather than a thallus, and interference of antagonism between bacteria can be reduced.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for screening urease inhibitor-producing bacteria. Background Art

[0002] Ammonia is one of the most common malodor pollutants that is easily soluble in water and has a very low olfactory threshold. Ammonia not only causes unpleasant effects on the senses of organisms, but also has a great impact on the health of organisms. Uric acid (which will be converted into urea) and urea in feces and urine are decomposed into a large amount of ammonia under the action of urease. Therefore, toilets, garbage dumps, livestock and poultry farms and other places with excrement have a relatively serious problem of ammonia odor pollutants. At present, the deodorization market mainly decomposes NH by absorption, adsorption and utilization. 3 To remove the ammonia smell, but if the activity of urease in microorganisms can be inhibited by urease inhibitors, the rate of urea conversion to ammonia can be greatly slowed down. Starting from the source and the terminal together, the ammonia pollution problem can be solved more quickly and efficiently.

[0003] At present, urease inhibitor products are mainly divided into inorganic compounds, organic compounds and plant extracts. The former two have the problem of large environmental pollution and side effects on organisms, while the latter have the problem of slow effect, low inhibition effect and high extraction cost. Therefore, it is possible to consider developing strains that can produce urease inhibitors. However, there are few studies on urease inhibitors, such as Aspergillus ochraceus I36 discovered by Lin Xinjian and others from the Institute of Soil and Fertilizer of Fujian Academy of Agricultural Sciences in 1997; Bacillus licheniformis X173, Bacillus coagulans P1-678, and Bacillus subtilis J530, a total of 3 strains discovered by Zhang Ying and others from Hebei Agricultural University in 2019; and Priestia filamentosa GW10 discovered by Wang Wenbo, Shi Shangbai and others in 2022.

[0004] In the prior art, the screening experiments for strains producing urease inhibitors all use plates added with urea and phenol red in the acid-base indicator to initially screen out non-urease-producing strains, and then compare the inhibition rate of the fermentation broth of the non-urease-producing strains on the urease activity of the mixture of urease extract and urea buffer solution to screen the urease inhibitor-producing strains.

[0005] In the initial screening, urease-producing strains used the urea in the plate to decompose and generate alkaline NH 3 Phenol red will turn red when the pH rises. If the plate turns red, it means the strain produces urease, and if it does not, it means the strain does not produce urease. However, the key to the plate turning red in this method is that the strain produces alkali, not necessarily NH 3 The urea-phenol red plate will also exclude the alkaline-producing strains that do not produce urease, and the initial screening method is not perfect. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies in the prior art, the present invention aims to provide a method for screening urease inhibitor producing bacteria, wherein the method first screens ammonia nitrogen efficient bacteria and then screens urease inhibitor producing strains, thereby controlling the production of ammonia from the source and decomposing NH 3 , thereby being able to more efficiently solve the stench problem of ammonia. The screening method for non-urease-producing strains can avoid the disadvantage that urea-phenol red plates exclude alkali-producing non-urease-producing strains, and can screen non-urease-producing strains more comprehensively.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solution: a method for screening urease inhibitor-producing bacteria, comprising the following steps: (1) Screening of efficient bacteria for ammonia nitrogen removal: (A1) Collecting samples containing livestock and poultry feces and / or urine from the soil of a breeding farm, and performing enrichment culture of ammonia-nitrogen-degrading bacteria to obtain an enriched bacterial solution of ammonia-nitrogen-degrading bacteria; (A2) diluting the enriched bacterial solution of ammonia nitrogen-degrading bacteria, applying it to a separation medium for cultivation, and obtaining ammonia nitrogen-degrading preliminary screening bacteria after separation and purification; (A3) After the initial screening of ammonia nitrogen degradation bacteria is activated, it is inoculated into the screening culture medium to test the degradation of ammonia nitrogen in each bacterial solution. The strain with the highest ammonia nitrogen degradation rate is selected as the efficient ammonia nitrogen degradation bacteria to enter the next round of experiments; (2) Screening of strains that do not produce urease: (B1) After the ammonia nitrogen efficient degradation bacteria are activated, they are inoculated into the urea nutrient solution for cultivation. The turbidity of each bacterial solution is observed and the OD of the bacterial solution is detected. 600 Value, pick OD 600 The strains with the highest values ​​were selected as the urease-producing strains for initial screening; the strains with clarified bacterial liquid were selected as the non-urease-producing strains for initial screening and entered the next round of experiments; (B2) Take a bacterial liquid container, connect a water-blocking microporous membrane filter to the air inlet of the bacterial liquid container, and connect the air outlet to an air filter; sterilize the bacterial liquid container at high temperature, and then fill it with culture medium under aseptic operation, and inoculate the urease-producing primary screening strain and the non-urease-producing primary screening strain obtained in step (B1); connect wet cotton soaked in phenol red solution behind the air filter at the air outlet of the bacterial liquid container, and connect an air pump to the air inlet of the bacterial liquid container; turn on the air pump to ventilate the bacterial liquid container, keep the cotton at the air outlet moist, observe whether the cotton turns red, select the several strains whose cotton turns red the fastest among the urease-producing primary screening strains as representative urease-producing strains, and select the strains whose cotton does not turn red as non-urease-producing strains to enter the next round of experiments; (3) Screening of strains producing urease inhibitors: (C1) After activating the representative urease-producing strains obtained in step (B1) with culture medium, taking equal amounts of bacterial liquid and mixing them, refrigerating and storing them, centrifuging them, and taking the supernatant as the crude bacterial urease extract solution; activating the non-urease-producing strains obtained in step (B2) with culture medium to obtain a urease inhibitor test solution; (C2) Mix the bacterial urease extract solution and the urease inhibitor test solution, add urea buffer solution and shake well, place in a constant temperature water bath, and add hydrochloric acid solution to terminate the reaction; add a mixed indicator to the solution, and titrate with sodium hydroxide solution until the solution turns blue-green; set the mixed solution without adding the urease inhibitor test solution as the positive control group, set the mixed solution without adding the urease inhibitor test solution, and add urea buffer solution and hydrochloric acid solution in reverse order as the blank control group; calculate the urease activity of the positive control group and the sample, and select the strain with the highest urease activity inhibition rate as the urease inhibitor producing strain, that is, the urease inhibitor producing strain; the calculation formula of urease activity inhibition rate is as follows: Urease activity inhibition rate = (urease activity of positive control group - urease activity of sample) * 100% / urease activity of positive control.

[0008] Further, in step (A1), the collected sample is added to the enrichment medium, the sample addition amount is 0.1-0.15g / mL, and after shaking and culturing in a shaker at 27-33°C for 2-4 days, the enriched bacterial liquid is inoculated into the enrichment medium at a volume fraction of 1-1.5%, and shaken and cultured in a shaker at a speed of 160-180r / min and 27-33°C for 2-4 days, and then transferred to a new enrichment medium. After 8-12 generations of transfer culture, an enriched bacterial liquid of ammonia-nitrogen-degrading bacteria is obtained.

[0009] Further, in step (A2), the enriched bacterial solution of ammonia-nitrogen-degrading bacteria is diluted by 10 -4 , 10 -5 and 10 -6 There are 3 gradients in total, and the diluted bacterial solution is spread on the separation medium. After culturing at 27-33°C for 2-3 days, the strain on the spread plate is streaked and separated and purified to obtain the initial screening bacteria for ammonia nitrogen degradation.

[0010] Furthermore, in step (A3), the ammonia nitrogen-degrading bacteria screened initially are activated with NA medium and then inoculated into the screening medium. The degradation of ammonia nitrogen in each bacterial solution is tested by Nessler's reagent spectrophotometry, and strains with high ammonia nitrogen degradation rates are selected as efficient ammonia nitrogen-degrading bacteria to enter the next round of experiments.

[0011] Further, in step (B1), the ammonia nitrogen efficient degrading bacteria are activated with NA medium and inoculated into urea nutrient solution, and cultured in a shake flask at a speed of 160-180 r / min and 27-33° C. for 2-4 days, and the turbidity of each bacterial solution is observed and the OD of the bacterial solution is detected.600 Value, pick OD 600 The strain with the highest value was selected as the urease-producing strain for initial screening; the strain with clarified bacterial liquid was selected as the non-urease-producing strain for initial screening and entered the next round of experiments.

[0012] Furthermore, in step (B2), a washing bottle is used as a bacterial liquid container, filled with urea-NA culture medium, and inoculated with urease-producing primary screening strains and non-urease-producing primary screening strains, with an inoculation amount of 0.8-1.2% by volume; wet cotton soaked in 0.08-0.12% phenol red solution is connected to the rear of the air filter, and an air pump is connected to the air inlet; the air pump is turned on to ventilate the washing bottle for 4-5 days, and the cotton at the air outlet is kept moist; the strains of the urease-producing primary screening bacteria whose cotton turns red the fastest are used as representative urease-producing strains; the strains whose cotton does not turn red are used as non-urease-producing strains for the next round of experiments.

[0013] Furthermore, in step (C1), the multiple representative urease-producing strains obtained in step (B1) are activated with NA culture medium respectively, and then equal amounts of bacterial liquid are mixed, stored at 3-5°C for 22-26 hours, and then centrifuged at 4000-5000 rpm for 12-15 minutes. The supernatant is the crude bacterial urease extract solution; the non-urease-producing strain obtained in step (B2) is activated with NA culture medium to obtain a urease inhibitor test solution.

[0014] Furthermore, in step (C2), the calculation formula of urease activity X is as follows: X=14*c(V0-V) / tm Where, c: concentration of Na(OH) standard titration solution, in mol / L; V0: blank consumption of sodium hydroxide calibration solution volume, unit is mL; V: volume of sodium hydroxide calibration solution consumed by the sample, in mL; t is the reaction time, in min; m is the sample volume, in mL; 14 is the molar mass of nitrogen.

[0015] Further, in step (C2), 0.2 mL of bacterial urease extract solution and 0.2 mL of urease inhibitor test solution are mixed in a test tube, 10 mL of urea buffer is added, the stopper is tightly closed and shaken, and after a water bath at 28-32° C. for 30 minutes, 10 mL of hydrochloric acid solution is added to terminate the reaction; the obtained solution is rinsed with pure water and transferred to another container, a mixed indicator is added, and NaOH solution is titrated until the solution turns blue-green; the urease activity of the positive control group and the sample is calculated, and the calculation formula of the urease activity X is as follows: X=14*c(V0-V) / 30m Where, c: concentration of Na(OH) standard titration solution, in mol / L; V0: blank consumption of sodium hydroxide calibration solution volume, unit is mL; V: volume of sodium hydroxide calibration solution consumed by the sample, in mL; 14 is the molar mass of nitrogen; 30 is the reaction time, in min; m is the sample volume, 0.2 mL.

[0016] The beneficial effects of the present invention are: (1) The present invention provides a method for screening urease inhibitor-producing bacteria, in which efficient ammonia nitrogen-producing bacteria are first screened and then urease inhibitor-producing strains are screened, thereby controlling the production of ammonia from the source and decomposing NH 3 , thus being able to more efficiently solve the odor problem of ammonia.

[0017] (2) The method for screening the non-urease-producing strains of the present invention can avoid the disadvantage that the urea-phenol red plate excludes the non-urease-producing strains that produce alkali, and can screen the non-urease-producing strains more comprehensively. In addition, the method detects the overflowed NH 3 , which can be more suitable for the odor generation scenario.

[0018] (3) The urease material of the urease inhibitor producing strain used in the present invention is the supernatant after centrifugation of bacterial culture, which is mainly biological enzyme rather than bacterial body, and can reduce the interference of antagonism between bacteria. DETAILED DESCRIPTION

[0019] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0020] In some embodiments of the present invention, a method for screening urease inhibitor-producing bacteria comprises the following steps: (1) Screening of efficient bacteria for ammonia nitrogen removal: (A1) Collecting samples containing livestock and poultry feces and / or urine from the soil of a breeding farm, and performing enrichment culture of ammonia-nitrogen-degrading bacteria to obtain an enriched bacterial solution of ammonia-nitrogen-degrading bacteria; (A2) diluting the enriched bacterial solution of ammonia nitrogen-degrading bacteria, applying it to a separation medium for cultivation, and obtaining ammonia nitrogen-degrading preliminary screening bacteria after separation and purification; (A3) After the initial screening of ammonia nitrogen degradation bacteria is activated, it is inoculated into the screening culture medium to test the degradation of ammonia nitrogen in each bacterial solution. The strain with the highest ammonia nitrogen degradation rate is selected as the efficient ammonia nitrogen degradation bacteria to enter the next round of experiments; (2) Screening of strains that do not produce urease: (B1) After the ammonia nitrogen efficient degradation bacteria are activated, they are inoculated into the urea nutrient solution for cultivation. The turbidity of each bacterial solution is observed and the OD of the bacterial solution is detected.600 Value, pick OD 600 The strains with the highest values ​​were selected as the urease-producing strains for initial screening; the strains that did not produce urease after clarification were selected for the next round of experiments; (B2) Take a bacterial liquid container, connect a water-blocking microporous membrane filter to the air inlet of the bacterial liquid container, and connect the air filter to the air outlet; sterilize the bacterial liquid container at high temperature, and then fill it with culture medium under aseptic operation, and inoculate the primary screening strain; connect the wet cotton soaked in phenol red solution behind the air filter at the air outlet of the bacterial liquid container, and connect the air pump to the air inlet of the bacterial liquid container; turn on the air pump to ventilate the bacterial liquid container, keep the cotton at the air outlet moist, and observe whether the cotton turns red. The three strains with the fastest cotton turning red in the primary screening of urease-producing bacteria are selected as representative strains of urease production; the strains whose cotton does not turn red are selected as non-urease-producing strains and enter the next round of experiments. ; (3) Screening of strains producing urease inhibitors: (C1) After activating the representative urease-producing strains obtained in step (B1) with culture medium, taking equal amounts of bacterial liquid and mixing them, storing them at 4°C for 24 hours, centrifuging them at 4000-5000 rpm, and taking the supernatant to obtain a crude bacterial urease extract solution; activating the non-urease-producing strain obtained in step (B2) with culture medium to obtain a urease inhibitor test solution; (C2) Mix the bacterial urease extract solution and the urease inhibitor test solution, add urea buffer solution and shake well, place in a constant temperature water bath, and add hydrochloric acid solution to terminate the reaction; add a mixed indicator to the solution, and titrate with sodium hydroxide solution until the solution turns blue-green; set the mixed solution without adding the urease inhibitor test solution as the positive control group, set the mixed solution without adding the urease inhibitor test solution, and add urea buffer solution and hydrochloric acid solution in reverse order as the blank control group; calculate the urease activity of the positive control group and the sample, and select the strain with the highest urease activity inhibition rate as the urease inhibitor producing strain; the calculation formula of urease activity inhibition rate is as follows: Urease activity inhibition rate = (urease activity of positive control group - urease activity of sample) * 100% / urease activity of positive control.

[0021] In some embodiments of the present invention, in step (A1), the collected sample is added to the enrichment medium, the sample addition amount is 0.1-0.15 g / mL, and after shaking and culturing in a shaker at 27-33°C for 2-4 days, the enriched bacterial liquid is inoculated into the enrichment medium at a volume fraction of 1-1.5%, and the culture is shaken in a shaker at a speed of 160-180 r / min and 27-33°C for 2-4 days, and then transferred to a new enrichment medium. After 8-12 generations of transfer culture, an enriched bacterial liquid of ammonia nitrogen degrading bacteria is obtained.

[0022] In some embodiments of the present invention, in step (A2), the enriched bacterial solution of ammonia-nitrogen-degrading bacteria is diluted by 10 -4 , 10 -5 and 10 -6 There are 3 gradients in total, and the diluted bacterial solution is spread on the separation medium. After culturing at 27-33°C for 2-3 days, the strain on the spread plate is streaked and separated and purified to obtain the initial screening bacteria for ammonia nitrogen degradation.

[0023] In some embodiments of the present invention, in step (A3), the ammonia nitrogen-degrading bacteria screened are activated with NA medium and then inoculated into the screening medium, and the degradation of ammonia nitrogen in each bacterial liquid is tested by Nessler's reagent spectrophotometry, and strains with high ammonia nitrogen degradation rates are selected as efficient ammonia nitrogen-degrading bacteria to enter the next round of experiments.

[0024] In some embodiments of the present invention, in step (B1), the ammonia nitrogen efficient degrading bacteria are activated with NA medium and then inoculated into urea nutrient solution, and the rotation speed is 160-180r / min, and the shaking flask is cultured at 27-33°C for 2-4 days, and the turbidity of each bacterial solution is observed and the OD of the bacterial solution is detected. 600 Value, pick OD 600 The strain with the highest value was selected as the urease-producing initial screening strain; the strain with clarified bacterial liquid was selected as the non-urease-producing initial screening strain for the next round of experiments.

[0025] In some embodiments of the present invention, in step (B2), a washing bottle is used as a bacterial liquid container, urea-NA culture medium is added, and the primary screening strain is inoculated with an inoculation amount of 0.8-1.2% by volume; wet cotton soaked in 0.08-0.12% phenol red solution is connected to the rear of the air filter, and an air pump is connected to the air inlet; the air pump is turned on to ventilate the washing bottle for 4-5 days, and the cotton at the air outlet is kept moist; the three strains of urease-producing primary screening bacteria whose cotton turns red the fastest are selected as representative urease-producing strains; the strains whose cotton does not turn red are regarded as non-urease-producing strains and enter the next round of experiments.

[0026] In some embodiments of the present invention, in step (C1), the multiple representative urease-producing strains obtained in step (B1) are activated with NA culture medium respectively, and then equal amounts of bacterial liquid are mixed, stored at 3-5°C for 22-26 hours, and then centrifuged at 4000-5000 rpm for 12-15 minutes, and the supernatant is the crude bacterial urease extract solution; the non-urease-producing strain obtained in step (B2) is activated with NA culture medium to obtain a urease inhibitor test solution.

[0027] In some embodiments of the present invention, in step (C2), the calculation formula of urease activity X is as follows: X=14*c(V0-V) / tm Where, c: concentration of Na(OH) standard titration solution, in mol / L; V0: blank consumption of sodium hydroxide calibration solution volume, unit is mL; V: volume of sodium hydroxide calibration solution consumed by the sample, in mL; t is the reaction time, in min; m is the sample volume, in mL; 14 is the molar mass of nitrogen.

[0028] In some embodiments of the present invention, the enrichment medium comprises the following raw materials at the following concentrations: glucose 4.5-5.5 g, (NH 4 ) 2 SO 4 0.35-0.45 g, K 2 HPO 4 0.8-1.2 g, MgSO 4 7H 2 O 0.35-0.45 g, trace element solution 0.7-1.3 mL, water 1000 mL; the pH of the enrichment medium is 7.2-7.4, and it is sterilized at 120-124°C for 15-25 min.

[0029] In some embodiments of the present invention, the separation medium comprises raw materials at the following concentrations: glucose 4.0-6.0 g, (NH 4 ) 2 SO 4 0.4 g, K 2 HPO 4 1.0 g, MgSO 4 7H 2 O 0.5 g, trace element solution 1 mL, agar 2.0 g, water 1000 mL; the pH of the separation medium is 7.2-7.4, and it is sterilized at 120-124°C for 15-25 min.

[0030] In some embodiments of the present invention, the screening medium comprises raw materials at the following concentrations: glucose 10.0 g, (NH 4 ) 2 SO 4 0.5 g, NaCl 1.0 g, K 2 HPO 4 0.5 g MgSO 4 7H 2O 0.25 g, trace element solution 1 mL, water 1000 mL; the pH of the screening medium is 7.2-7.4, sterilized at 120-124°C for 15-25 min; the trace element solution includes the following raw materials: EDTA 9.0-11.0 g, ZnSO 4 1.0-1.4g, CaCl 2 1.4-1.6g, MnCl 2 ·4H 2 O 0.8-1.2g, FeSO 4 7H 2 O 1.8-2.2g, (NH 4 ) 2 Mo 7 O 24 ·4H 2 O 0.8-1.2g, CuSO 4 ·5H 2 O 0.8-1.2g, CoCl 2 6H 2 O 0.8-1.2g, water 1000 mL.

[0031] In some embodiments of the present invention, the urea buffer comprises raw materials with the following concentrations: Na 2 HPO 4 12H 2 O8-9.5g, KH 2 PO 4 3-3.8g, urea 27-33g, water 1000 mL.

[0032] In some embodiments of the present invention, the preparation method of the mixed indicator comprises the following steps: dissolving 0.8-0.12 g of methyl red in 90-96% ethanol and diluting to 100 mL, then dissolving 0.4-0.6 g of bromocresol green solution in 90-96% ethanol and diluting to 100 mL, mixing the two solutions in a volume ratio of 0.8-1.2:0.8-1.2 and storing them in a brown reagent bottle.

[0033] Example 1 This embodiment provides a method for screening urease inhibitor-producing bacteria, comprising the following steps: (1) Screening of efficient bacteria for ammonia nitrogen removal: (A1) Enrichment and purification Collect samples containing feces and urine of livestock and poultry from the soil of the farm, take 10 g of the collected samples and add them to 90 mL of enrichment medium, shake and culture them in a shaker at a speed of 180 / min and a temperature of 30°C for 3 days, take 1 mL of the enriched bacterial liquid and inoculate it into 90 mL of enrichment medium, shake and culture them in a shaker at a speed of 180 / min and a temperature of 30°C for 3 days, and then transfer them to a new enrichment medium, and after 10 generations of transfer culture, obtain an enriched bacterial liquid of ammonia nitrogen-degrading bacteria; The enrichment medium includes raw materials at the following concentrations: glucose 5.0 g, (NH 4 ) 2 SO 4 0.4 g, K 2 HPO 4 1.0 g, MgSO 4 7H 2 O 0.5 g, trace element solution 1 mL, water 1000 mL; the pH of the enrichment medium is 7.2-7.4, and it is sterilized at 121°C for 20 min.

[0034] (A2) Initial screening The enriched bacterial solution of ammonia-nitrogen-degrading bacteria was diluted to 10 -4 , 10 -5 and 10 -6 There are 3 gradients in total, and 0.1 mL of the diluted bacterial solution is spread on the separation medium. After culturing at 30°C for 3 days, the strains on the spread plate are streaked and separated and purified to obtain the initial screening bacteria for ammonia nitrogen degradation; The separation medium includes raw materials of the following concentrations: glucose 5.0 g, (NH 4 ) 2 SO 4 0.4 g, K 2 HPO 4 1.0 g, MgSO 4 7H 2 O 0.5 g, trace element solution 1 mL, agar 2.0 g, water 1000 mL; the pH of the separation medium is 7.2-7.4, and it is sterilized at 121°C for 20 min.

[0035] (A3) Rescreening The ammonia nitrogen degradation bacteria were activated with NA medium and then inoculated into the screening medium. The degradation of ammonia nitrogen in each bacterial solution was tested by Nessler's reagent spectrophotometry. The strains with high ammonia nitrogen degradation rate were selected as ammonia nitrogen efficient degradation bacteria for the next round of experiments. The screening medium includes raw materials of the following concentrations: glucose 10.0 g, (NH 4 ) 2 SO4 0.5 g, NaCl 1.0 g, K 2 HPO 4 0.5 g MgSO 4 7H 2 O 0.25 g, trace element solution 1 mL, water 1000 mL; the pH of the screening medium is 7.2-7.4, and it is sterilized at 121°C for 20 min; The trace element solution includes raw materials with the following concentrations: EDTA 10.0 g, ZnSO 4 1.2g, CaCl 2 1.5g, MnCl 2 ·4H 2 O 1.0g, FeSO 4 7H 2 O 2.0g, (NH 4 ) 2 Mo 7 O 24 ·4H 2 O 1.0g, CuSO 4 ·5H 2 O 1.0g, CoCl 2 6H 2 O 1.0g, water 1000 mL; pH 7~7.4.

[0036] (2) Screening of strains that do not produce urease: (B1) Initial screening The ammonia nitrogen efficient degrading bacteria were activated with NA medium and inoculated into urea nutrient solution. The culture was carried out in a shaking flask at a speed of 180 / min and a temperature of 30°C for 3 days. The turbidity of each bacterial solution was observed and the OD of the bacterial solution was detected. 600 Value, pick OD 600 After the urease activity of the ten strains with the highest values ​​was measured, the three strains with the highest urease activity were selected as representative urease-producing strains; the strains with clarified bacterial liquid were selected as non-urease-producing initial screening strains for the next round of experiments; The NA medium includes raw materials of the following concentrations: 10.0 g of peptone, 3.0 g of beef powder, 5.0 g of sodium chloride, and 1000 mL of water; the pH of the NA medium is 7-7.4, and it is sterilized at 121° C. for 20 min; The urea culture medium includes raw materials with the following concentrations: 20.0 g urea, 10.0 g sucrose, 5.0 g KCl, and MgSO 4 7H 2 O 0.5 g, CaCl 2 0.5g, Na 2 HPO 41.0 g, NaH 2 PO 4 1.0 g, 1000 mL of water; the pH of the urea medium is 6.0-8.0. Sterilize at 121°C for 20 min; after urea is filtered and sterilized, the medium composed of raw materials other than urea is sterilized and cooled before adding to the medium.

[0037] (B2) Rescreening Take a 100mL washing bottle as a container for bacterial solution and urea-NA culture solution, connect a water-blocking microporous filter with a pore size of 0.22μm to the air inlet of the washing bottle, and connect an air filter with a pore size of 0.22μm to the air outlet of the washing bottle; after high-temperature sterilization of the washing bottle connection device, fill it with 100mL of urea-NA culture solution under aseptic operation, and inoculate the primary screening strain with an inoculation volume of 1%; connect wet cotton soaked in 0.1% phenol red solution behind the air filter; after the washing bottle connection device is connected, connect an air pump to the air inlet of the washing bottle; turn on the air pump to ventilate the washing bottle for 5 days, and keep the cotton at the air outlet moist during the process, and observe whether the cotton turns red; select the three strains with the fastest red cotton among the urease-producing primary screening strains as representative urease-producing strains, and select the strains without red cotton as non-urease-producing strains to enter the next round of experiments; The urea-NA culture solution includes raw materials of the following concentrations: 20.0 g urea, 10.0 g peptone, 3.0 g beef powder, 5.0 g sodium chloride, and 1000 mL water; the pH of the urea-NA culture solution is 7-7.4, and it is sterilized at 121° C. for 20 min; after the urea is filtered and sterilized, the culture medium composed of raw materials other than urea is sterilized and cooled before being added to the culture medium; The preparation method of the phenol red indicator comprises the following steps: weighing 0.1 g of phenol red, dissolving it in 126.57 mL of anhydrous ethanol, and storing it in a brown reagent bottle.

[0038] (3) Screening of strains producing urease inhibitors (C1) Activating the three representative urease-producing strains obtained in step (B1) with NA medium respectively, taking equal amounts of bacterial liquid and mixing them, storing them at 4°C for 24 hours, centrifuging them at 4000 rpm for 15 minutes, and taking the supernatant after centrifugation to obtain a bacterial urease extract solution; activating each non-urease-producing strain obtained in step (B2) with NA medium to obtain a urease inhibitor test solution; (C2) Take 0.2mL of bacterial urease extract solution and 0.2mL of urease inhibitor test solution in a 25mL test tube and mix them evenly. Add 10mL of urea buffer solution, cover the stopper and shake well. After 30min in a 30℃ water bath, add 10mL of 0.1mol / L hydrochloric acid solution to terminate the reaction. Rinse the solution with 25mL of pure water and transfer it to a conical flask. Add 10 drops of mixed indicator and titrate with 0.1mol / L NaOH solution until the solution turns blue-green. Set the mixed solution without urease inhibitor test solution as the positive control group, set the mixed solution without urease inhibitor test solution and add urea buffer solution and hydrochloric acid solution in reverse order as the blank control group. Calculate the urease activity of the positive control group and the sample, and select the strain with high urease activity inhibition rate as the urease inhibitor producing strain, i.e., the urease inhibitor producing strain. The calculation formula of urease activity inhibition rate is as follows: Urease activity inhibition rate = (urease activity of positive control group - urease activity of sample) * 100% / urease activity of positive control.

[0039] Furthermore, in step (C2), the calculation formula of urease activity X is as follows: X=14*c(V0-V) / tm Where, c: concentration of Na(OH) standard titration solution, in mol / L; V0: blank consumption of sodium hydroxide calibration solution volume, unit is mL; V: volume of sodium hydroxide calibration solution consumed by the sample, in mL; t is the reaction time, in min; m is the sample volume, in mL; 14 is the molar mass of nitrogen.

[0040] The urea buffer comprises the following raw materials at the following concentrations: Na 2 HPO 4 12H 2 O 8.95g, KH 2 PO 4 3.4 g, urea 30.0 g, water 1000 mL.

[0041] The preparation of 0.1 mol / L hydrochloric acid solution is as follows: 8.3 mL of hydrochloric acid is diluted to 1000 mL with water; the preparation of 0.1 mol / L NaOH solution is as follows: 4 g of sodium hydroxide is dissolved in water and diluted to 1000 mL.

[0042] The preparation method of the mixed indicator comprises the following steps: dissolving 0.1 g of methyl red in 95% ethanol and diluting to 100 mL, then dissolving 0.5 g of bromocresol green solution in 95% ethanol and diluting to 100 mL, mixing the two solutions in equal volumes and storing them in a brown reagent bottle.

[0043] Example 2 This embodiment provides a method for screening urease inhibitor-producing bacteria, comprising the following steps: (1) Screening of efficient bacteria for ammonia nitrogen removal: (A1) Enrichment and purification Samples containing feces and urine of livestock and poultry were collected from the soil of the farm, and 10 g of the collected samples were added to 90 mL of enrichment culture medium. After shaking and culturing in a shaker at a speed of 180 / min and a temperature of 30°C for 3 days, 1 mL of the enriched bacterial liquid was inoculated into 90 mL of enrichment culture medium, and shaken and cultured in a shaker at a speed of 180 / min and a temperature of 30°C for 3 days, and then transferred to a new enrichment culture medium. After 10 generations of transfer culture, an enriched bacterial liquid of ammonia-nitrogen-degrading bacteria was obtained.

[0044] (A2) Initial screening The enriched bacterial solution of ammonia-nitrogen-degrading bacteria was diluted to 10 -4 , 10 -5 and 10 -6 There were 3 gradients in total, and 0.1 mL of the diluted bacterial solution was spread on the separation medium. After culturing at 30°C for 2 days, the strains on the spread plate were streaked and separated and purified to obtain the initial screening bacteria for ammonia nitrogen degradation.

[0045] (A3) Rescreening The ammonia nitrogen-degrading bacteria screened initially were activated with NA medium and then inoculated into the screening medium. The degradation of ammonia nitrogen in each bacterial solution was tested by Nessler's reagent spectrophotometry. The strains with high ammonia nitrogen degradation rates were selected as efficient ammonia nitrogen-degrading bacteria for the next round of experiments.

[0046] (2) Screening of strains that do not produce urease: (B1) Initial screening The ammonia nitrogen efficient degrading bacteria were activated with NA medium and inoculated into urea nutrient solution. The culture was carried out in a shaking flask at a speed of 180 / min and a temperature of 30°C for 3 days. The turbidity of each bacterial solution was observed and the OD of the bacterial solution was detected. 600 Value, pick OD 600 The three strains with the highest values ​​were selected as representative strains producing urease; the strains with clarified bacterial liquid were selected to enter the next round of experiments.

[0047] (B2) Rescreening Take a 100mL washing bottle as a container for bacterial solution and urea-NA culture solution, connect a water-blocking microporous membrane filter with a pore size of 0.22μm to the air inlet of the washing bottle, and connect an air filter with a pore size of 0.22μm to the air outlet of the washing bottle; after high-temperature sterilization of the washing bottle connecting device, fill it with 100mL of urea-NA culture solution under aseptic operation, and inoculate the primary screening strain with an inoculation volume of 1%; connect wet cotton soaked in 0.1% phenol red solution behind the air filter; after the washing bottle connecting device is connected, connect an air pump to the air inlet of the washing bottle; turn on the air pump to ventilate the washing bottle for 4 days, and keep the cotton at the air outlet moist during the process to observe whether the cotton turns red; select the strain whose cotton does not turn red as the non-urease producing strain to enter the next round of experiments.

[0048] (3) Screening of strains producing urease inhibitors (C1) Activating the three representative urease-producing strains obtained in step (B1) with NA medium respectively, taking equal amounts of bacterial liquid and mixing them, storing them at 4°C for 24 hours, centrifuging them at 4000 rpm for 15 minutes, and taking the supernatant after centrifugation to obtain a crude bacterial urease extract solution; activating each non-urease-producing strain obtained in step (B2) with NA medium to obtain a urease inhibitor test solution; (C2) Take 0.2mL of bacterial urease extract solution and 0.2mL of urease inhibitor test solution in a 25mL test tube and mix them evenly. Add 10mL of urea buffer solution, cover the stopper and shake well. After 30min in a 30℃ water bath, add 10mL of 0.1mol / L hydrochloric acid solution to terminate the reaction. Rinse the solution with 25mL of pure water and transfer it to a conical flask. Add 10 drops of mixed indicator and titrate with 0.1mol / L NaOH solution until the solution turns blue-green. Set the mixed solution without urease inhibitor test solution as the positive control group, set the mixed solution without urease inhibitor test solution and add urea buffer solution and hydrochloric acid solution in reverse order as the blank control group. Calculate the urease activity of the positive control group and the sample, and select the strain with the highest urease activity inhibition rate as the urease inhibitor producing strain. The calculation formula of urease activity inhibition rate is as follows: Urease activity inhibition rate = (urease activity of positive control group - urease activity of sample) * 100% / urease activity of positive control.

[0049] Furthermore, in step (C2), the calculation formula of urease activity X is as follows: X=14*c(V0-V) / tm Where, c: concentration of Na(OH) standard titration solution, in mol / L; V0: blank consumption of sodium hydroxide calibration solution volume, unit is mL; V: volume of sodium hydroxide calibration solution consumed by the sample, in mL; t is the reaction time, in min; m is the sample volume, in mL; 14 is the molar mass of nitrogen.

[0050] The rest of the content of this embodiment is the same as that of Embodiment 1 and will not be repeated here.

[0051] Comparative Example 1 This comparative example provides a method for screening urease inhibitor-producing bacteria, comprising the following steps: (1) Screening of efficient bacteria for ammonia nitrogen removal: (A1) Enrichment and purification Samples containing feces and urine of livestock and poultry were collected from the soil of the farm, and 10 g of the collected samples were added to 90 mL of enrichment culture medium. After shaking and culturing in a shaker at a speed of 180 / min and a temperature of 30°C for 3 days, 1 mL of the enriched bacterial liquid was inoculated into 90 mL of enrichment culture medium, and shaken and cultured in a shaker at a speed of 180 / min and a temperature of 30°C for 3 days, and then transferred to a new enrichment culture medium. After 10 generations of transfer culture, an enriched bacterial liquid of ammonia-nitrogen-degrading bacteria was obtained.

[0052] (A2) Initial screening The enriched bacterial solution of ammonia-nitrogen-degrading bacteria was diluted to 10 -4 , 10 -5 and 10 -6 There were 3 gradients in total, and 0.1 mL of the diluted bacterial solution was spread on the separation medium. After culturing at 30°C for 2 days, the strains on the spread plate were streaked and separated and purified to obtain the initial screening bacteria for ammonia nitrogen degradation.

[0053] (A3) Rescreening The ammonia nitrogen-degrading bacteria screened initially were activated with NA medium and then inoculated into the screening medium. The degradation of ammonia nitrogen in each bacterial solution was tested by Nessler's reagent spectrophotometry. The strains with high ammonia nitrogen degradation rates were selected as efficient ammonia nitrogen-degrading bacteria for the next round of experiments.

[0054] (2) Screening of strains that do not produce urease: (B1) Initial screening After the ammonia nitrogen efficient degrading bacteria were activated with NA medium, they were inoculated on urea-phenol red plates using the cross method. After culturing in a 30°C incubator for 3 days, the color change of the colonies on the urea-phenol red plates was observed. The strains that did not turn red were selected and inoculated into NA medium for activation for 2 days and then stored for the next round of experiments.

[0055] (B2) Rescreening The strain obtained in step (B1) was activated with NA medium and inoculated into urea nutrient solution. The culture was shaken at a speed of 180 / min and a temperature of 30°C for 3 days. The turbidity of each bacterial solution was observed and the OD of the bacterial solution was detected. 600 The clarified strains were selected and inoculated into NA medium for activation for 2 days before being preserved for the next round of experiments.

[0056] (3) Screening of strains producing urease inhibitors (C1) Soaking the soybeans produced in the current year and clean water at a ratio of 1:10 overnight, using a pulper to make a homogenate, and then centrifuging at 5000r / min for 10 minutes, the supernatant is the soybean urease solution; activating each non-urease-producing strain obtained in step (B2) with NA culture medium to obtain a urease inhibitor test solution; (C2) Take 0.2mL of soybean urease solution and 0.2mL of urease inhibitor test solution in a 25mL test tube and mix them evenly. Add 10mL of urea buffer solution, cover the stopper and shake well. After 30min in a 30℃ water bath, add 10mL of 0.1mol / L hydrochloric acid solution to terminate the reaction. Rinse the solution with 25mL of pure water and transfer it to a conical flask. Add 10 drops of mixed indicator and titrate with 0.1mol / L NaOH solution until the solution turns blue-green. Set the mixed solution without urease inhibitor test solution as the positive control group, set the mixed solution without urease inhibitor test solution and add urea buffer solution and hydrochloric acid solution in reverse order as the blank control group. Calculate the urease activity of the positive control group and the sample, and select the strain with high urease activity inhibition rate as the urease inhibitor producing strain. The calculation formula of urease activity inhibition rate is as follows: Urease activity inhibition rate = (urease activity of positive control group - urease activity of sample) * 100% / urease activity of positive control.

[0057] Furthermore, in step (C2), the calculation formula of urease activity X is as follows: X=14*c(V0-V) / tm Where, c: concentration of Na(OH) standard titration solution, in mol / L; V0: blank consumption of sodium hydroxide calibration solution volume, unit is mL; V: volume of sodium hydroxide calibration solution consumed by the sample, in mL; t is the reaction time, in min; m is the sample volume, in mL; 14 is the molar mass of nitrogen.

[0058] The rest of the contents of this comparative example are the same as those of Example 1 and will not be described again here.

[0059] In Example 1, three urease inhibitor-producing strains (referred to as strains 1-3) were finally screened out; in Comparative Example 1, two urease inhibitor-producing strains (referred to as strains 4-5) were finally screened out.

[0060] Ammonia removal experiment: The No. 1-5 strains obtained by screening were inoculated into NA medium for activation. Six large beakers were taken, and 100g of fresh chicken manure was placed in each beaker. Then a small beaker containing 30mL of 1mol / L dilute sulfuric acid solution was placed. Five groups were inoculated with 10% of the bacterial solution of No. 1-5 strains, and the remaining CK group was inoculated with 10mL of clean water. The six large beakers were sealed and placed in a 30℃ incubator for 9 days. After being taken out, the concentration of ammonia nitrogen in the absorption liquid in each group of small beakers was tested by Nessler's reagent spectrophotometry to compare the release of ammonia nitrogen in each group of chicken manure, thereby verifying the comprehensive ability of the strains obtained under different screening methods to inhibit urease and degrade ammonia nitrogen.

[0061] Table 1 Ammonia nitrogen concentration of each group of absorption liquid on the 9th day in the ammonia removal experiment

[0062] It can be seen from the above table that the urease inhibitor producing strain in Example 1 of the present invention has a higher inhibition rate of ammonia nitrogen release. At the same time, through the analysis of the screening method of the urease inhibitor producing bacteria of the present invention, it can be seen that the screening method of the present invention can effectively solve the disadvantage that the screening method of Comparative Example 1 uses urea-phenol red plates to exclude alkali-producing non-urease-producing strains.

[0063] In summary, the screening method of urease inhibitor producing bacteria of the present invention first screens ammonia nitrogen efficient bacteria and then screens urease inhibitor producing strains, thereby controlling the production of ammonia from the source and decomposing NH 3 , thereby being able to more efficiently solve the stench problem of ammonia; the rescreening method of the non-urease-producing strain can avoid the disadvantage that the urea-phenol red plate excludes the alkali-producing non-urease-producing strains, and can screen the non-urease-producing strains more comprehensively, and the method detects the overflowed NH 3 , which can be more in line with the odor generation scene; the urease material of the urease inhibitor producing strain used in the present invention is the supernatant after centrifugation of the bacterial liquid, which is mainly biological enzyme rather than bacteria, and can reduce the interference of antagonism between bacteria.

[0064] The above specific embodiments are further explanations of the technical solutions and beneficial effects of the present invention, and are not limitations of the implementation methods. For those skilled in the art, any obvious replacements that do not depart from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A method for screening urease inhibitor-producing bacteria, characterized in that: The steps include: (1) Screening of efficient bacteria for ammonia nitrogen removal: (A1) Collecting samples containing livestock and poultry feces and / or urine from the soil of a breeding farm, and performing enrichment culture of ammonia-nitrogen-degrading bacteria to obtain an enriched bacterial solution of ammonia-nitrogen-degrading bacteria; (A2) diluting the enriched bacterial solution of ammonia nitrogen-degrading bacteria, applying it to a separation medium for cultivation, and obtaining ammonia nitrogen-degrading preliminary screening bacteria after separation and purification; (A3) After the initial screening of ammonia nitrogen degradation bacteria is activated, it is inoculated into the screening culture medium to test the degradation of ammonia nitrogen in each bacterial solution. The strain with the highest ammonia nitrogen degradation rate is selected as the efficient ammonia nitrogen degradation bacteria to enter the next round of experiments; (2) Screening of strains that do not produce urease: (B1) After the ammonia nitrogen efficient degradation bacteria are activated, they are inoculated into the urea nutrient solution for cultivation. The turbidity of each bacterial solution is observed and the OD of the bacterial solution is detected. 600 Value, pick OD 600 The strains with the highest values ​​were selected as the urease-producing strains for initial screening; the strains with clarified bacterial liquid were selected as the non-urease-producing strains for initial screening and entered the next round of experiments; (B2) Take a bacterial liquid container, connect a water-blocking microporous membrane filter to the air inlet of the bacterial liquid container, and connect the air outlet to an air filter; sterilize the bacterial liquid container at high temperature, and then fill it with culture medium under aseptic operation, and inoculate the urease-producing primary screening strain and the non-urease-producing primary screening strain obtained in step (B1); connect wet cotton soaked in phenol red solution behind the air filter at the air outlet of the bacterial liquid container, and connect an air pump to the air inlet of the bacterial liquid container; turn on the air pump to ventilate the bacterial liquid container, keep the cotton at the air outlet moist, and observe whether the cotton turns red; select the strains whose cotton turns red the fastest among the urease-producing primary screening strains as representative urease-producing strains, and select the strains whose cotton does not turn red as non-urease-producing strains to enter the next round of experiments; (3) Screening of strains producing urease inhibitors: (C1) activating the representative urease-producing strains obtained in step (B1) with culture medium, taking equal amounts of bacterial liquid and mixing them, centrifuging and taking the supernatant to obtain a bacterial urease extract solution; activating the non-urease-producing strain obtained in step (B2) with culture medium to obtain a urease inhibitor test solution; (C2) Mix the bacterial urease extract solution and the urease inhibitor test solution, add urea buffer solution and shake well, place in a constant temperature water bath, and add hydrochloric acid solution to terminate the reaction; add a mixed indicator to the solution, and titrate with sodium hydroxide solution until the solution turns blue-green; set the mixed solution without adding the urease inhibitor test solution as the positive control group, set the mixed solution without adding the urease inhibitor test solution, and add urea buffer solution and hydrochloric acid solution in reverse order as the blank control group; calculate the urease activity of the positive control group and the sample, and select the strain with the highest urease activity inhibition rate as the urease inhibitor producing strain; the calculation formula of urease activity inhibition rate is as follows: Urease activity inhibition rate = (urease activity of positive control group - urease activity of sample) * 100% / urease activity of positive control.

2. The method for screening urease inhibitor-producing bacteria according to claim 1, characterized in that: In step (A1), the collected sample is added to the enrichment medium, the sample addition amount is 0.1-0.15g / mL, and after shaking and culturing in a shaker at 27-33°C for 2-4 days, the enriched bacterial liquid is inoculated into the enrichment medium at a volume fraction of 1-1.5%, and the culture is shaken in a shaker at a speed of 160-180r / min and 27-33°C for 2-4 days, and then transferred to a new enrichment medium. After 8-12 generations of transfer culture, an enriched bacterial liquid of ammonia nitrogen degrading bacteria is obtained.

3. The method for screening urease inhibitor-producing bacteria according to claim 1, characterized in that: In step (A2), the enriched bacterial solution of ammonia-nitrogen-degrading bacteria is diluted by 10 -4 , 10 -5 and 10 -6 There are 3 gradients in total, and the diluted bacterial solution is spread on the separation medium. After culturing at 27-33°C for 2-3 days, the strain on the spread plate is streaked and separated and purified to obtain the initial screening bacteria for ammonia nitrogen degradation.

4. The method for screening urease inhibitor-producing bacteria according to claim 1, characterized in that: In step (A3), the ammonia nitrogen-degrading bacteria screened initially are activated with NA medium and then inoculated into the screening medium. The degradation of ammonia nitrogen in each bacterial solution is tested by Nessler's reagent spectrophotometry, and the strains with high ammonia nitrogen degradation rates are selected as efficient ammonia nitrogen-degrading bacteria to enter the next round of experiments.

5. The method for screening urease inhibitor-producing bacteria according to claim 1, characterized in that: In step (B1), the ammonia nitrogen efficient degrading bacteria are activated with NA medium and then inoculated into urea nutrient solution. The rotation speed is 160-180r / min and the shaking flask is cultured at 27-33°C for 2-4 days. The turbidity of each bacterial solution is observed and the OD of the bacterial solution is detected. 600 Value, pick OD 600 The strain with the highest value was selected as the urease-producing strain for initial screening; the strain with clarified bacterial liquid was selected as the non-urease-producing strain for initial screening and entered the next round of experiments.

6. The method for screening urease inhibitor-producing bacteria according to claim 1, characterized in that: In step (B2), take the washing bottle as the bacterial liquid container, fill it with urea-NA culture medium, and inoculate the urease-producing primary screening strain and the non-urease-producing primary screening strain at an inoculation volume of 0.8-1.2%; connect the wet cotton soaked in 0.08-0.12% phenol red solution behind the air filter, and connect the air inlet to the air pump; turn on the air pump to ventilate the washing bottle for 4-5 days, and keep the cotton at the air outlet moist.

7. The method for screening urease inhibitor-producing bacteria according to claim 1, characterized in that: In step (C1), the multiple representative urease-producing strains obtained in step (B1) are activated with NA culture medium respectively, and then equal amounts of bacterial liquid are mixed, stored at 3-5°C for 22-26 hours, and then centrifuged at 4000-5000 rpm for 12-15 minutes. The supernatant is the crude bacterial urease extract solution; the non-urease-producing strain obtained in step (B2) is activated with NA culture medium to obtain a urease inhibitor test solution.

8. The method for screening urease inhibitor-producing bacteria according to claim 1, characterized in that: In step (C2), the urease activity X is calculated as follows: X=14*c(V0-V) / tm Where, c: concentration of Na(OH) standard titration solution, in mol / L; V0: blank consumption of sodium hydroxide calibration solution volume, unit is mL; V: volume of sodium hydroxide calibration solution consumed by the sample, in mL; t is the reaction time, in min; m is the sample volume, in mL; 14 is the molar mass of nitrogen.

9. The method for screening urease inhibitor-producing bacteria according to claim 1, characterized in that: In step (C2), 0.2 mL of crude bacterial urease extract solution and 0.2 mL of urease inhibitor solution to be tested are mixed in a test tube, 10 mL of urea buffer is added, the stopper is tightly closed and shaken, and after a water bath at 28-32°C for 30 minutes, 10 mL of hydrochloric acid solution is added to terminate the reaction; the obtained solution is rinsed with pure water and transferred to another container, a mixed indicator is added, and NaOH solution is titrated until the solution turns blue-green; the urease activity of the positive control group and the sample is calculated, and the calculation formula of the urease activity X is as follows: X=14*c(V0-V) / 30m Where, c: concentration of Na(OH) standard titration solution, in mol / L; V0: blank consumption of sodium hydroxide calibration solution volume, unit is mL; V: volume of sodium hydroxide calibration solution consumed by the sample, in mL; 14 is the molar mass of nitrogen; 30 is the reaction time, in min; m is the sample volume, 0.2 mL.

Images