Method for determining intracellular beta-glucosidase in acid soil
By using phosphate buffer combined with chloroform fumigation and salicin colorimetric method in acidic soil, intracellular and extracellular β-glucosidase were successfully distinguished, solving the problems of accuracy and feasibility of the determination method in acidic soil and realizing high-precision enzyme activity determination.
Patent Information
- Application Number
- CN202610121327.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-29
- Publication Date
- 2026-04-28
AI Technical Summary
Existing technologies are insufficient to effectively distinguish between intracellular and extracellular β-glucosidases in acidic soils, resulting in inadequate accuracy and feasibility of the assay methods.
Phosphate buffer (pH=6.0, 0.263 mol/L sodium dihydrogen phosphate dihydrate and 0.025 mol/L disodium hydrogen phosphate dodecahydrate) was used as the buffer for enzyme activity assay. The enzyme activity was determined by combining chloroform fumigation method and salicin colorimetric method, and the supernatant was collected by pre-culture and centrifugation.
This study enables highly accurate and feasible determination of intracellular β-glucosidase in acidic soils, improving the stability and reproducibility of the results and reducing the deviation of the measured values.
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Figure CN121933506A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of soil chemical analysis technology, and relates to a method for determining intracellular β-glucosidase, particularly a method for determining intracellular β-glucosidase in acidic soil. Background Technology
[0002] β-glucosidases are important hydrolytic enzymes that break down β-1,4-glycosidic bonds, participating in the catalytic degradation of various cellulose components in plant debris and playing a crucial role in soil carbon cycling. β-glucosidases function in the final stages of cellulose degradation by hydrolyzing cellulosic disaccharide residues; the final product of these reactions is glucose, an important carbon and energy source for soil microbial growth and metabolism. Because soil β-glucosidases participate in soil carbon cycling and play a vital role in soil function, they can serve as indicators of soil quality. Compared to extracellular β-glucosidases, intracellular β-glucosidases constitute the largest pool of β-glucosidases in soil and are more closely linked to microbial activity, making them a potentially sensitive indicator of soil quality. However, current methods for distinguishing between intracellular and extracellular β-glucosidases in soil are not yet mature, especially in acidic soils, where successful differentiation is difficult, severely hindering the discovery and utilization of related indicators.
[0003] The chloroform fumigation method is a commonly used method for distinguishing between intracellular and extracellular enzymes in soil. This method involves placing a weighed soil sample in an aluminum box, which is then placed in a vacuum desiccator. A beaker containing ethanol-free chloroform, along with a small amount of anti-bumping glass beads, is placed in the desiccator. A small beaker containing NaOH solution is also placed in the desiccator to absorb CO2 released during fumigation, and a beaker containing a small amount of water is placed in the desiccator to maintain humidity. The desiccator is then covered and evacuated to a vacuum. After the chloroform boils for 3-5 minutes, the desiccator valve is closed, and the sample is incubated at 25°C in the dark for 24 hours. A non-fumigated control is also included. After fumigation, the small beaker containing chloroform is removed, the desiccator is cleaned, and the vacuum process is repeated 3-5 times until the soil no longer smells of chloroform. Chloroform fumigation causes soil microbial cells to lyse and die, releasing intracellular enzymes. The total enzyme activity is then obtained. Subtracting this total enzyme activity from the extracellular enzyme activity measured in the unfumigated soil sample yields the intracellular enzyme activity.
[0004] The salicin colorimetric method is a commonly used method for determining β-glucosidase activity. This method involves adding 10 mL of 4 mmol / L salicin solution and 20 mL of pH 5.8 acetate buffer to a weighed soil sample and incubating at 37 °C for 12 h. After filtration, 1 mL of the supernatant is taken, and 0.25 mL of pH 9.8 NH4OH-NH4Cl buffer, 0.5 mL of 2% 4-aminoantipyrine solution, and 0.5 mL of 8% potassium ferricyanide solution are added sequentially for color development. The soil β-glucosidase activity is then measured colorimetrically at 510 nm. However, in acidic soils, this method cannot distinguish between extracellular and intracellular β-glucosidase activity using acetate buffer. Summary of the Invention
[0005] To address the aforementioned technical problems in the background art, the present invention provides a method for determining intracellular β-glucosidase in acidic soil that is easy to operate, highly accurate, and feasible.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: Application of phosphate buffer in the determination of intracellular β-glucosidase.
[0007] Application of phosphate buffer in the determination of intracellular β-glucosidase in acidic soil.
[0008] As described above, the phosphate buffer solution comprises sodium dihydrogen phosphate dihydrate with a final concentration of 0.263 mol / L and disodium hydrogen phosphate dodecahydrate with a final concentration of 0.025 mol / L; the pH of the phosphate buffer solution is 6.0.
[0009] A method for determining intracellular β-glucosidase in acidic soil, characterized in that the method comprises the following steps: 1) Prepare reagents for the chloroform fumigation method, reagents for the salicin colorimetric method, and phosphate buffer as described above; 2) Select soil samples for testing and pre-culture the soil samples; 3) The pre-cultured soil samples were divided into a fumigation group and a non-fumigation group. The fumigation group was treated with chloroform fumigation using the reagent prepared in step 1). 4) Phosphate buffer and salicin colorimetric reagent were added to the unfumigated soil samples obtained in step 3) and the soil samples after chloroform fumigation treatment, respectively, for incubation. 5) After the culture is completed, the absorbance of the unfumigated soil sample and the fumigated soil sample at 510 nm is determined by the salicin colorimetric method prepared in step 1) using reagents. The salicin concentration of the unfumigated soil sample and the salicin concentration of the fumigated soil sample are obtained. 6) Calculate intracellular β-glucosidase based on the salicylol concentration obtained in step 5).
[0010] The reagents used in the chloroform fumigation method described in step 1) above include an ethanol-free chloroform solution and a 0.5 mol / L NaOH solution; the reagents used in the salicin colorimetric method include a salicin solution with a molar concentration of 4 mmol / L, an ammonium chloride-ammonium hydroxide buffer solution, a potassium ferricyanide solution with a mass-to-volume ratio of 8%, and a 4-aminoantipyrine solution with a mass-to-volume ratio of 2%; the phosphate buffer solution includes sodium dihydrogen phosphate dihydrate with a final concentration of 0.263 mol / L and disodium hydrogen phosphate dodecahydrate with a final concentration of 0.025 mol / L; the pH of the phosphate buffer solution is 6.0.
[0011] The soil sample tested in step 2) above is acidic soil; the pre-cultivation in step 2) involves adjusting the soil moisture content of the soil sample to 60%±2% of field capacity and pre-cultivating it in a 25 ℃ pre-cultivation box for 7±0.5 days.
[0012] The specific implementation method of chloroform fumigation treatment in step 3) above is as follows: Place the soil sample of the fumigation group into an aluminum box, place the aluminum box into a vacuum desiccator, and place a beaker containing an ethanol-free chloroform solution into it. Place anti-boiling glass beads into the beaker, and at the same time place a small beaker containing 0.5 mol / L NaOH solution into it, and then place a beaker containing a small amount of water into it. Cover the vacuum desiccator and use a vacuum pump to evacuate the vacuum, so that the chloroform boils for 3-5 minutes. Close the vacuum desiccator valve and incubate it in the dark at 25°C for 24 hours. After the fumigation is completed, open the vacuum desiccator valve, take out the small beaker containing chloroform, clean the desiccator, and repeat the vacuuming 3-5 times until the soil sample has no chloroform smell.
[0013] The specific implementation method of step 4) above is as follows: the unfumigated test soil sample obtained in step 3) and the test soil sample after chloroform fumigation are respectively added to the phosphate buffer prepared in step 1) and the salicin solution with a molar concentration of 4 mmol / L for incubation; the mass-volume ratio of the test soil sample to the salicin solution and the phosphate buffer is 3±0.03g:10±0.1mL:20±0.2mL; the incubation conditions are to shake and mix evenly and then incubate in a 37 ℃ incubator for 12±0.1h.
[0014] The specific implementation method of step 5) above is as follows: After the culture is completed, after centrifugation at 5000 rpm for 5 min, the supernatant is taken into a 25 mL graduated test tube. 0.25 mL of ammonium chloride-ammonium hydroxide buffer solution with pH 9.80, 0.5 mL of 2% 4-aminoantipyrine, and 0.5 mL of 8% potassium ferricyanide solution are added to the test tube in sequence. The mixture is shaken thoroughly, and finally, the volume is adjusted to 25 mL with distilled water. The absorbance values of the unfumigated soil sample and the fumigated soil sample are measured at 510 nm using a spectrophotometer within 15 min to obtain the salicylol concentrations of the unfumigated soil sample and the fumigated soil sample.
[0015] The specific implementation method of step 6) above is as follows: 6.1) Calculate the β-glucosidase activity of the fumigated soil based on the salicylol concentration of the tested soil samples obtained in step 5), wherein the β-glucosidase activity of the fumigated soil is the total enzyme activity; 6.2) Calculate the β-glucosidase activity of the unfumigated soil based on the salicylol concentration of the unfumigated soil samples obtained in step 5), wherein the β-glucosidase activity of the unfumigated soil is the extracellular enzyme activity; 6.3) Calculate intracellular enzyme activity based on the total enzyme activity obtained in step 6.1) and the extracellular enzyme activity obtained in step 6.2); the intracellular enzyme activity = total enzyme activity - extracellular enzyme activity; the intracellular enzyme activity is the intracellular β-glucosidase activity in acidic soil.
[0016] The advantages of this invention over the prior art are as follows: This invention provides a method for determining intracellular β-glucosidase in acidic soil. The method includes: 1) preparing reagents for chloroform fumigation, reagents for salicin colorimetry, and phosphate buffer as described above; 2) selecting test soil samples and pre-culturing them; 3) dividing the pre-cultured soil samples into fumigation and non-fumigation groups, and fumigating the fumigation group with the chloroform fumigation reagent prepared in step 1); 4) adding phosphate buffer and salicin colorimetric reagent to the non-fumigation group and the chloroform-fumigated soil samples obtained in step 3), respectively, for culturing; 5) after culturing, using the salicin colorimetric reagent prepared in step 1), measuring the salicin colorimetric value of the non-fumigation group and the fumigation group on a spectrophotometer at 510 nm. The absorbance value at nm was used to obtain the salicylol concentration in the unfumigated soil sample and the salicylol concentration in the fumigated soil sample; 6) Based on the salicylol concentration obtained in step 5), the intracellular β-glucosidase was calculated. This invention first pre-cultures the soil samples under suitable conditions for 7 days to restore microbial activity, resulting in more stable enzyme activity; simultaneously, using phosphate buffer as the buffer for enzyme activity determination allows for successful differentiation between extracellular and intracellular β-glucosidase, thus enabling the measurement of intracellular β-glucosidase activity. Furthermore, this invention uses 50 mL centrifuge tubes for culture and enzyme activity determination, and the supernatant is ablated for colorimetric analysis, which is more convenient than filtration. In summary, when measuring the intracellular β-glucosidase activity in acidic soil using the method provided by this invention and the classical method, the values obtained by this invention are 10.64, 8.63, and 8.02, respectively, with a standard value of 9.40±0.97. The values obtained by the conventional method are 1.54, 1.60, and 2.80, respectively, with a standard value of 1.98±1.01. Obviously, the intracellular β-glucosidase activity measured by this invention is high, has good parallelism, high accuracy, and high feasibility. Attached Figure Description
[0017] Figure 1 This is the standard curve of salicylol content used in this invention. Detailed Implementation
[0018] 1. Reagent preparation Salicin solution: Weigh 1.1532 g of salicin and dissolve in distilled water, then bring the volume to 1 L. Acetate buffer: Weigh 27.22 g of sodium acetate trihydrate (or 16.41 g of sodium acetate) and dissolve in distilled water, add 0.4 mL of glacial acetic acid, and bring the volume to 1 L. Phosphate buffer (pH=6.0): Weigh 41.05 g of sodium dihydrogen phosphate dihydrate and 8.81 g of disodium hydrogen phosphate dodecahydrate and dissolve in distilled water, then bring the volume to 1 L. 2% 4-Aminoantipyrine solution: Weigh 2.00 g of 4-aminoantipyrine and dissolve in distilled water, then bring the volume to 100 mL. It can be stored for one week at low temperature. 8% Potassium ferricyanide solution: Weigh 8.00 g of potassium ferricyanide and dissolve in distilled water, then bring the volume to 100 mL. It can be stored for one week at low temperature. Ammonium chloride-ammonium hydroxide buffer solution: Dissolve 20.00 g of ammonium chloride in 100 mL of ammonia water. Prepare this reagent fresh for each use according to the specified ratio. Ethanol-free chloroform solution: Extract chloroform from the chloroform by mixing it with water at a volume ratio of 1:2. Collect the lower chloroform layer and mix three times.
[0019] 2. Operating steps: 2.1) Adjust the soil moisture content of the test soil samples to 60% of the field capacity and pre-culture them in a 25℃ incubator for 7 days to restore microbial activity.
[0020] 2.2) After the pre-culture was completed, chloroform fumigation and non-fumigation treatments were performed to distinguish between intracellular and extracellular enzymes.
[0021] 2.3) Weigh 3.00 g of soil samples from the fumigated group / non-fumigated group into a 50 mL centrifuge tube, add 10 mL of salicin solution and 20 mL of phosphate buffer, shake to mix well, and incubate at 37 ℃. After incubation, take a certain volume of supernatant into a 25 mL graduated test tube, add 0.25 mL of ammonium chloride-ammonium hydroxide buffer (pH 9.80), 0.5 mL of 2% 4-aminoantipyrine, and 0.5 mL of 8% potassium ferricyanide solution, shake well, and finally dilute to 25 mL with distilled water. Measure the absorbance at 510 nm using a spectrophotometer within 15 min.
[0022] 2.4) A standard curve was prepared using salicylol standard solution. The enzyme activity unit was μg / g. -1 h -1 Each treatment was repeated three times, with substrate-free and soil-free treatments set up as controls.
[0023] 2.5) Calculate intracellular β-glucosidase.
[0024] The technical solution provided by the present invention will be described in detail below with reference to specific embodiments: The soil samples used in this embodiment were collected from Beibei District of Chongqing, Qiyang of Hunan, Hailun of Heilongjiang, Nanjing of Jiangsu, and Yueyang of Hunan, with three replicates for each soil sample. In Beibei District of Chongqing, the crop was sweet potato, and the soil pH was 5.74; in Qiyang of Hunan, the crop was rapeseed, and the soil pH was 4.89; in Hailun of Heilongjiang, the crop was maize, and the soil pH was 6.27; in Nanjing of Jiangsu, the crop was rice, and the soil pH was 6.62; and in Yueyang of Hunan, the crop was soybean, and the soil pH was 5.16.
[0025] To fully illustrate the feasibility and superiority of the measurement method provided by this invention, a comparison is made between Method A (existing measurement method) and Method B (the measurement method provided by this invention): Method A: Intracellular β-glucosidase activity in five soil samples was detected using the acetate buffer method.
[0026] The specific analytical steps for each type of soil are as follows: A.1) Accurately weigh 3.00 g of soil sample passing through a 2 mm sieve for chloroform fumigation treatment, and set up a non-fumigated control to distinguish intracellular / extracellular enzymes. Place the weighed soil sample into an aluminum box (3.00 g of soil sample for each treatment, set up parallel treatments). Place the aluminum box into a vacuum desiccator, and place a beaker containing ethanol-free chloroform prepared in step seven into the beaker. Add a small amount of anti-bumping glass beads to the beaker, and place a small beaker containing 0.5 mol / L NaOH solution prepared in step one into the beaker to absorb the CO2 released during fumigation. Then place a beaker containing a small amount of water into the beaker to maintain the humidity of the container. Cover the vacuum desiccator and use a vacuum pump to evacuate the system, allowing the chloroform to boil for 3-5 minutes. Close the vacuum desiccator valve and incubate at 25 ℃ in the dark for 24 hours, while setting up a non-fumigated control. After fumigation, open the vacuum desiccator valve, remove the small beaker containing chloroform, clean the desiccator, and repeat the vacuuming process 3-5 times until the soil has no chloroform odor.
[0027] A.2) The fumigated and unfumigated soil samples were undamagedly placed into 50 mL Erlenmeyer flasks, 10 mL of 4 mmol / L salicin solution and 20 mL of pH 5.8 acetate buffer were added, the mixture was shaken and mixed evenly, and then placed in an incubator at 37 ℃ for 12 h. A.3) After the culture is completed, filter the solution with qualitative filter paper, take a certain volume of filtrate into a 25 mL graduated test tube, add 0.25 mL of ammonium chloride-ammonium hydroxide buffer solution (pH=9.80), 0.5 mL of 2% 4-aminoantipyrine, and 0.5 mL of 8% potassium ferricyanide solution in sequence, shake well, and finally make up to 25 mL with distilled water. Measure the absorbance at 510 nm on a spectrophotometer within 15 min. A.4) A standard curve was prepared using salicylol standard solution, with enzyme activity units in μg / g. -1 h-1 Each treatment was repeated three times, with substrate-free and soil-free treatments as controls. The fumigation treatment and non-fumigation treatment corresponded to total β-glucosidase and extracellular β-glucosidase activities, respectively, with the difference between the two representing intracellular β-glucosidase activity.
[0028] Method B: Using the method provided by this invention: The specific analytical steps for each type of soil are as follows: B.1) Adjust the moisture content of the soil sample that has passed through a 2mm sieve to 60% of the field capacity, and pre-culture it in a 25℃ incubator for 7 days to restore soil microbial activity; B.2) After the pre-culture is completed, fumigation treatment is carried out (the fumigation treatment method is the same as A.1). B.3) Accurately weigh 3.00 g of fumigated soil sample and 3.00 g of unfumigated soil sample and put them into a 50 mL centrifuge tube. Add 10 mL of 4 mmol / L salicin solution and 20 mL of pH 6.0 phosphate buffer. Shake to mix well and then incubate at 37 ℃ for 12 h. B.4) After the culture is completed, centrifuge the centrifuge tube at 5000 rpm for 5 min, and then take a certain volume of supernatant into a 25 mL graduated test tube. Add 0.25 mL of ammonium chloride-ammonium hydroxide buffer solution with pH 9.80, 0.5 mL of 2% 4-aminoantipyrine, and 0.5 mL of 8% potassium ferricyanide solution in sequence. Shake well and finally make up to 25 mL with distilled water. Measure the absorbance at 510 nm on a spectrophotometer within 15 min. B.5) A standard curve was prepared using salicylol standard solution, with enzyme activity units in μg / g. -1 h -1 Each treatment was repeated three times, with substrate-free and soil-free treatments as controls. The fumigation and non-fumigation treatments corresponded to total β-glucosidase and extracellular β-glucosidase activities, respectively, with the difference representing intracellular β-glucosidase activity. It should be noted that the salicylol concentration was derived from absorbance values during the calculation, and the β-glucosidase activity was ultimately calculated based on the salicylol concentration. β-glucosidase activity (μg g) is... -1 h -1 = (a × V × n) / (m × t); where: a is the concentration of salicylol (μg / mL) obtained from the standard curve; V is the volume of the colorimetric solution (mL); n is the fractionation factor; m is the dry soil weight (g); and t is the incubation time (h).
[0029] The preparation and plotting of the standard curve for salicylol standard solution: Prepare the salicylol standard solution and plot the standard curve with salicylol concentration as the x-axis and the measured average absorbance value as the y-axis. The preparation of the salicylol standard solution is as follows: Accurately weigh 0.1000 g of salicylol into a 100 mL beaker, add approximately 20 mL of anhydrous ethanol, and use an ultrasonic cleaner to assist dissolution, ensuring no visible precipitate. Transfer the dissolved solution to a 100 mL volumetric flask, wash the beaker several times with anhydrous ethanol, and dilute to the mark. Shake well and transfer to a brown reagent bottle, label it (indicating "Salicylol stock solution 1000 mg / L" and the preparation date), and store at 4°C protected from light. Pipette 1 mL of the stock solution into a 100 mL volumetric flask, add deionized water to the mark, shake well, and store in a brown bottle at 4°C. This is the 10 mg / L intermediate solution. Take eight 10 mL volumetric flasks, numbered 1-8, and add 0, 0.4, 1.2, 2.0, 2.8, 3.6, 4.4, and 5.2 mL of intermediate solution respectively. Dilute to the mark with deionized water and mix well to obtain standard solutions with concentrations of 0, 0.4, 1.2, 2.0, 2.8, 3.6, 4.4, and 5.2 mg / L. Measure the absorbance at 510 nm using a spectrophotometer. Plot a standard curve with salicylol concentration on the x-axis and the measured absorbance values on the y-axis, as shown below. Figure 1 As shown.
[0030] The experimental results are shown in Table 1: Table 1. Results of determination of intracellular β-glucosidase activity by different methods Note: "-" indicates that enzyme activity cannot be detected. As shown in Table 1, when using acetate buffer (Method A), the measured data exhibits significant deviations, even negative values, and the variation between repeated measurements is substantial, resulting in low accuracy. The method provided by this invention (Method B) eliminates the issue of undetectable activity, and the smaller standard deviation indicates less deviation between analytical results after using phosphate buffer, demonstrating higher accuracy and better reproducibility.
Claims
1. Application of phosphate buffer in the determination of intracellular β-glucosidase.
2. Application of phosphate buffer in the determination of intracellular β-glucosidase in acidic soil.
3. The application according to claim 1 or 2, characterized in that: The phosphate buffer solution comprises sodium dihydrogen phosphate dihydrate with a final concentration of 0.263 mol / L and disodium hydrogen phosphate dodecahydrate with a final concentration of 0.025 mol / L; the pH of the phosphate buffer solution is 6.
0.
4. A method for determining intracellular β-glucosidase in acidic soil, characterized in that: The method for determining intracellular β-glucosidase in acidic soil includes the following steps: 1) Prepare reagents for chloroform fumigation, reagents for salicin colorimetric method, and phosphate buffer solution as described in claim 1, 2, or 3; 2) Select soil samples for testing and pre-culture the soil samples; 3) The pre-cultured soil samples were divided into a fumigation group and a non-fumigation group. The fumigation group was treated with chloroform fumigation using the reagent prepared in step 1). 4) Phosphate buffer and salicin colorimetric reagent were added to the unfumigated soil samples obtained in step 3) and the soil samples after chloroform fumigation treatment, respectively, for incubation. 5) After the culture is completed, the absorbance of the unfumigated soil sample and the fumigated soil sample at 510 nm is determined by the salicin colorimetric method prepared in step 1) using reagents. The salicin concentration of the unfumigated soil sample and the salicin concentration of the fumigated soil sample are obtained. 6) Calculate intracellular β-glucosidase based on the salicylol concentration obtained in step 5).
5. The method for determining intracellular β-glucosidase in acidic soil according to claim 4, characterized in that: The reagents used in the chloroform fumigation method in step 1) include an ethanol-free chloroform solution and a 0.5 mol / L NaOH solution; the reagents used in the salicin colorimetric method include a salicin solution with a molar concentration of 4 mmol / L, an ammonium chloride-ammonium hydroxide buffer solution, a potassium ferricyanide solution with a mass-to-volume ratio of 8%, and a 4-aminoantipyrine solution with a mass-to-volume ratio of 2%; the phosphate buffer solution includes sodium dihydrogen phosphate dihydrate with a final concentration of 0.263 mol / L and disodium hydrogen phosphate dodecahydrate with a final concentration of 0.025 mol / L; the pH of the phosphate buffer solution is 6.
0.
6. The method for determining intracellular β-glucosidase in acidic soil according to claim 5, characterized in that: The soil sample used in step 2) is acidic soil; the pre-cultivation in step 2) involves adjusting the soil moisture content of the soil sample to 60%±2% of field capacity and pre-cultivating it in a 25 ℃ pre-cultivation box for 7±0.5 days.
7. The method for determining intracellular β-glucosidase in acidic soil according to claim 6, characterized in that: The specific implementation method of chloroform fumigation treatment in step 3) is as follows: Place the soil sample of the fumigation group into an aluminum box, place the aluminum box into a vacuum desiccator, and place a beaker containing an ethanol-free chloroform solution into it. Place anti-boiling glass beads into the beaker, and at the same time place a small beaker containing 0.5 mol / L NaOH solution into it, and then place a beaker containing a small amount of water into it. Cover the vacuum desiccator and use a vacuum pump to evacuate the vacuum, so that the chloroform boils for 3-5 minutes. Close the vacuum desiccator valve and incubate it in the dark at 25°C for 24 hours. After the fumigation is completed, open the vacuum desiccator valve, take out the small beaker containing chloroform, clean the desiccator, and repeat the vacuuming 3-5 times until the soil sample has no chloroform smell.
8. The method for determining intracellular β-glucosidase in acidic soil according to claim 7, characterized in that: The specific implementation method of step 4) is as follows: the unfumigated test soil sample obtained in step 3) and the test soil sample after chloroform fumigation are respectively added to the phosphate buffer prepared in step 1) and the salicin solution with a molar concentration of 4 mmol / L for incubation; the mass-volume ratio of the test soil sample to the salicin solution and the phosphate buffer is 3±0.03g:10±0.1mL:20±0.2mL; the incubation conditions are to shake and mix evenly and then incubate in a 37 ℃ incubator for 12±0.1h.
9. The method for determining intracellular β-glucosidase in acidic soil according to claim 8, characterized in that: The specific implementation method of step 5) is as follows: After the culture is completed, after centrifugation at 5000 rpm for 5 min, the supernatant is taken into a 25 mL graduated test tube. 0.25 mL of ammonium chloride-ammonium hydroxide buffer solution with pH 9.80, 0.5 mL of 2% 4-aminoantipyrine, and 0.5 mL of 8% potassium ferricyanide solution are added to the test tube in sequence. The mixture is shaken thoroughly, and finally, the volume is adjusted to 25 mL with distilled water. The absorbance values of the unfumigated soil sample and the fumigated soil sample are measured at 510 nm using a spectrophotometer within 15 min to obtain the salicylol concentrations of the unfumigated soil sample and the fumigated soil sample.
10. The method for determining intracellular β-glucosidase in acidic soil according to claim 9, characterized in that: The specific implementation method of step 6) is as follows: 6.1) Calculate the β-glucosidase activity of the fumigated soil based on the salicylol concentration of the tested soil samples obtained in step 5), wherein the β-glucosidase activity of the fumigated soil is the total enzyme activity; 6.2) Calculate the β-glucosidase activity of the unfumigated soil based on the salicylol concentration of the unfumigated soil samples obtained in step 5), wherein the β-glucosidase activity of the unfumigated soil is the extracellular enzyme activity; 6.3) Calculate intracellular enzyme activity based on the total enzyme activity obtained in step 6.1) and the extracellular enzyme activity obtained in step 6.2); the intracellular enzyme activity = total enzyme activity - extracellular enzyme activity; the intracellular enzyme activity is the intracellular β-glucosidase activity in acidic soil.