Soil extracellular polymeric substance extraction and determination method based on cation exchange resin method

Abstract

The invention discloses a soil extracellular polymeric substance extraction and determination method based on a cation exchange resin method, and relates to the technical field of soil analysis, and the method specifically comprises the following steps: firstly, cleaning cation exchange resin with hot water, then sequentially adding HC l, NaC l and NaOH solutions, oscillating, and airing to obtain CER resin particles; the method comprises the following steps: treating a fresh soil sample by using a CaC12 solution and a buffer solution to obtain a pretreated soil sample; weighing CER resin particles, adding the CER resin particles into a centrifugal tube filled with the pretreated soil sample, oscillating, centrifuging and filtering to obtain an EPS extracting solution; and finally, detecting the absorbance of the EPS extracting solution under different wavelengths by using a microplate reader to respectively obtain the polysaccharide content and the protein content. According to the EPS extraction and determination method, the extraction efficiency of the EPS in the soil is effectively improved by optimizing the pretreatment steps of the cation exchange resin and accurately controlling parameter conditions such as the resin dosage, the extraction time and the rotating speed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of soil analysis, and particularly relates to a method for extracting and determining soil extracellular polymers based on a cation exchange resin method. BACKGROUND

[0002] Soil extracellular polymers (EPS) are high molecular weight natural polymers secreted by various microorganisms such as bacteria, fungi and algae in the soil, mainly including polysaccharides and proteins. The structural diversity of polysaccharides endows EPS with multiple functions such as adhering to the surface, obtaining nutrients and providing stable structure, and can regulate EPS synthesis, etc. Part of the extracellular enzymes participate in EPS degradation, acting on different polymers and organic particles, and part of them interact with polysaccharides and remain in the biofilm. Therefore, EPS plays an important role in the soil, can act as a microbial protective layer, regulate biological responses in the soil environment, alleviate abiotic stress in the environment and improve the stability of soil aggregates, etc. Accurate extraction of EPS in the soil and determination of the polysaccharide and protein contents are of great significance for in-depth understanding of the functions and mechanisms of the soil ecosystem.

[0003] Traditional EPS extraction methods such as centrifugation, ultrasonic method and formaldehyde-NaOH extraction method may not be able to fully release EPS, resulting in low extraction efficiency or easy cell lysis, causing serious pollution of the intracellular environment, mixing of intracellular substances in the extracted EPS and affecting the accuracy of subsequent analysis and interfering with the detection results. The cation exchange resin (CER) method gradually becomes the mainstream extraction technology due to its mild action and small damage to cells. However, the existing CER method still has problems such as the influence of resin residue impurities on the extraction purity, the low sensitivity of the polysaccharide and protein detection method and the inability to accurately quantify the EPS components. Therefore, it is urgent to provide a method for extracting and determining soil extracellular polymers based on a cation exchange resin method to expand the research and application of soil EPS. SUMMARY

[0004] The purpose of the present application is to provide a method for extracting and determining soil extracellular polymers based on a cation exchange resin method to solve the problem of low EPS extraction purity and the inability to accurately quantify EPS components in the prior art cation exchange resin method.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: a method for extracting and determining soil extracellular polymers based on a cation exchange resin method, specifically comprising the following steps:

[0006] S1, cation exchange resin pretreatment: first, use 70-80℃ hot water to clean Na type cation exchange resin until the supernatant is colorless and no foam, then add HCl solution and shake, wash with ultrapure water, then add NaCl solution and shake, then add NaOH solution and shake, finally wash with ultrapure water until the solution is neutral, dry to obtain CER resin particles;

[0007] S2, soil sample pretreatment: weigh the fresh soil and place it in a centrifuge tube, add CaCl2 solution, then place the centrifuge tube in a shaker and shake, then centrifuge and discard the supernatant, then add pre-cooled buffer solution to the centrifuge tube containing soil particles to obtain the pretreated soil sample;

[0008] S3, extraction of soil extracellular polymers: weigh the CER resin particles and add them to the centrifuge tube containing the pretreated soil sample, then place the centrifuge tube in a shaker and shake, then centrifuge to separate the supernatant and soil particles, and finally filter the supernatant to obtain the EPS extract;

[0009] S4, determination of polysaccharide content: mix the EPS extract with phenol solution in equal volumes, then add concentrated sulfuric acid and vortex to mix, then perform water bath heating in the dark, cool to room temperature, and then use an enzyme marker to detect the absorbance to obtain the polysaccharide content;

[0010] S5, determination of protein content: first prepare compound reagent A, then mix the EPS extract with compound reagent A and incubate in the dark, then add Folin phenol reagent and incubate in the dark again, and finally use an enzyme marker to detect the absorbance to obtain the protein content.

[0011] Further, the concentration of HCl solution in S1 is 0.1-1 mol / L; the concentration of NaOH solution in S1 is 0.1-1 mol / L; the concentration of NaCl solution is 1%-5%; the volume ratio of Na type cation exchange resin to HCl solution, NaOH solution and NaCl solution in S1 is 1:3:3:3.

[0012] Further, the concentration of CaCl2 solution in S2 is 0.01-0.05 mol / L; the buffer solution in S2 is at least one of phosphate buffer solution, citrate buffer solution and acetate buffer solution; the relative centrifugal force during centrifugation in S2 is set to 3000-3500xg, and the temperature is set to 0-4℃.

[0013] Further, the relative centrifugal force during centrifugation in S3 is set to 3000-4000xg, and the temperature is set to 0-4℃.

[0014] Further, the volume ratio of the EPS extract solution in S4 to concentrated sulfuric acid is 1:1-5; the concentration of the phenol solution in S4 is 1%-5%; the temperature of the water bath heating in S4 is set to 90-100 DEG C; and the wavelength detection of the enzyme label instrument in S4 is set to 400-500 nm.

[0015] Further, the composite reagent A in S5 is a mixed solution of CuSO4.5H2O solution, potassium sodium tartrate solution and sodium carbonate-sodium hydroxide solution, and the volume ratio of CuSO4.5H2O solution, potassium sodium tartrate solution and sodium carbonate-sodium hydroxide solution is 1:1:100.

[0016] Further, the light-avoiding incubation time in S5 is 10-30 min; and the concentration of the Folin phenol reagent in S5 is 0.1-0.2 mol / L.

[0017] Further, the wavelength detection of the enzyme label instrument in S5 is set to 700-800 nm.

[0018] Compared with the prior art, the method for extracting and determining soil EPS based on the cation exchange resin method has the following beneficial effects:

[0019] (1) By optimizing the pretreatment step of the cation exchange resin and precisely controlling the extraction conditions, the extraction efficiency of EPS in the soil is effectively improved; compared with the traditional extraction method, more EPS can be extracted in a shorter time, which provides sufficient samples for subsequent analysis and research.

[0020] (2) By precisely controlling the resin dosage, extraction time and rotation speed and other parameters, the intracellular pollution caused by the mixing of intracellular substances into the extract solution is avoided, and the purity and accuracy of the extracted EPS are ensured.

[0021] (3) The phenol-sulfuric acid method and the improved Lowery microplate method are used to determine the polysaccharide and protein content by using the enzyme label instrument, which has the advantages of simple operation, high accuracy and good repeatability, and can provide reliable technical means for the quantitative analysis of EPS and accurate data support for the research and application of the soil ecosystem. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments or the prior art, the drawings needed in the embodiments will be briefly introduced below, and obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can also be obtained by those skilled in the art according to these drawings.

[0023] Figure 1The flowchart of the method for extracting and determining soil extracellular polymers based on the cation exchange resin method provided by the embodiment of the present application is shown. DETAILED DESCRIPTION

[0024] In order to make the skilled in the art better understand the technical solutions of the present application, the present application will be further described in detail below with reference to the drawings.

[0025] Embodiment:

[0026] Please refer to Figure 1 The method for extracting and determining soil extracellular polymers based on the cation exchange resin method specifically comprises the following steps:

[0027] S1, cation exchange resin pretreatment: first, wash the Na-type cation exchange resin with hot water at 70-80℃ until the supernatant is colorless and free of foam, then add HCl solution and shake, wash with ultrapure water, then add NaCl solution and shake, then add NaOH solution and shake, and finally wash with ultrapure water until the solution is neutral, and dry to obtain CER resin particles; the concentration of HCl solution in S1 is 0.1-1 mol / L; the concentration of NaOH solution in S1 is 0.1-1 mol / L; the concentration of NaCl solution is 1%-5%; the volume ratio of Na-type cation exchange resin to HCl solution, NaOH solution and NaCl solution in S1 is 1:3:3:3.

[0028] The specific implementation is that first, wash the Na-type cation exchange resin with hot water at 70-80℃ until the supernatant is colorless and free of foam, then add 3 times the volume of the resin of 1 mol / L HCl solution, place in a 180 r / min shaking table and shake for 2 h, wash with ultrapure water until the pH is 5.0, then add 3 times the volume of the resin of 5% NaCl solution, place in a 180 r / min shaking table and shake for 2 h, then add 3 times the volume of the resin of 1 mol / L NaOH solution, place in a 180 r / min shaking table and shake for 2 h, finally wash with ultrapure water until the solution is neutral (pH=7.0), and dry to obtain CER resin particles.

[0029] S2, soil sample pretreatment: weigh the fresh soil and place it in a centrifuge tube, add CaCl2 solution, then place the centrifuge tube in a shaking table and shake, centrifuge after shaking, discard the supernatant, then add a pre-cooled buffer solution to the centrifuge tube containing soil particles to obtain a pretreated soil sample; the concentration of CaCl2 solution in S2 is 0.01-0.05 mol / L; the buffer solution in S2 is at least one of a phosphate buffer solution, a citrate buffer solution and an acetate buffer solution; the relative centrifugal force during centrifugation in S2 is set to 3000-3500 x g, and the temperature is set to 0-4℃.

[0030] Specifically, 1 g of fresh soil is weighed and placed in a 10 mL centrifuge tube, 5 mL of 0.01 mol / L CaCl2 solution is added, and then the centrifuge tube is placed in a 4°C, 120 r / min shaker for 30 min. After shaking, centrifugation is performed at 4°C and 3200 x g for 30 min, the supernatant is discarded, and 5 mL of PBS pre-cooled in a 4°C refrigerator is added to the centrifuge tube containing the soil particles to obtain a pretreated soil sample, which is stored in the refrigerator for standby use.

[0031] S3, extraction of soil extracellular polymers: CER resin particles are weighed and added to a centrifuge tube containing the pretreated soil sample, then the centrifuge tube is placed in a shaker for shaking, and then centrifugation is performed to separate the supernatant and soil particles, and finally the supernatant is filtered to obtain an EPS extract; the relative centrifugal force during centrifugation in S3 is set to 3000-4000 x g, and the temperature is set to 0-4°C.

[0032] Specifically, 3.5 g of CER resin particles is weighed and added to a centrifuge tube containing the pretreated soil sample, then the centrifuge tube is placed in a 4°C, 180 r / min shaker for 2 h, then centrifugation is performed at 4°C and 4000 x g for 30 min to separate the supernatant and soil particles, and finally the supernatant is filtered through a 0.45 μm filter (a needle hole type filter is used) to obtain an EPS extract, which is stored in a 4°C refrigerator and analyzed within a week.

[0033] S4, determination of polysaccharide content: the EPS extract is mixed with an equal volume of phenol solution, then concentrated sulfuric acid is added and vortexed to mix, then water bath heating is performed in the dark, after cooling to room temperature, the absorbance is detected by an enzyme marker to obtain the polysaccharide content; in S4, the volume ratio of the EPS extract to concentrated sulfuric acid is 1:1-5; in S4, the concentration of the phenol solution is 1%-5%; in S4, the water bath heating temperature is set to 90-100°C; in S4, the wavelength for detection by the enzyme marker is set to 400-500 nm.

[0034] Specifically, first, an electronic balance weighing method is used to prepare a glucose standard stock solution with a concentration of 100 mg / L. Subsequently, using the stock solution, a glucose standard working solution with concentrations of 0, 10, 20, 30, 40, 60, 80, and 100 mg / L is prepared by gradient dilution. 150 μL of each concentration standard working solution is added to multiple 2 mL centrifuge tubes, and then 150 μL of 5% phenol solution is added. After shaking the centrifuge tubes, 750 μL of concentrated sulfuric acid is slowly added to each centrifuge tube below the liquid surface, vortexed for 10 s, and then placed in a 96°C constant temperature water bath for 20 min in the dark. Subsequently, it is placed in cold water for 10 min, and then cooled to room temperature in the air. The standard working sample solution for polysaccharide content determination is obtained.

[0035] Into a 2 mL centrifuge tube, 150 μL of EPS extract and 150 μL of 5% phenol solution were added, mixed well, and then concentrated sulfuric acid was added. After vortexing for 10 s, the mixture was placed in a constant temperature water bath at 96°C for 20 min in the dark, then placed in cold water for 10 min, and then cooled to room temperature in air to obtain the sample to be tested. Each sample was repeated 3 times.

[0036] The absorbance of the standard working sample solution and the sample to be tested at 490 nm was measured using a microplate reader. The polysaccharide concentration of the standard working sample solution was used as the abscissa, and the absorbance was used as the ordinate to draw a standard curve and calculate the linear equation. The polysaccharide content of the sample to be tested was obtained.

[0037] S5, protein content determination: first, a composite reagent A was prepared, then the EPS extract was mixed with the composite reagent A and incubated in the dark, Folin phenol reagent was added and incubated in the dark again, and finally the absorbance was detected by a microplate reader to obtain the protein content; the composite reagent A in S5 was a mixture of CuSO4·5H2O solution, potassium sodium tartrate solution and sodium carbonate-sodium hydroxide solution, and the volume ratio of CuSO4·5H2O solution, potassium sodium tartrate solution and sodium carbonate-sodium hydroxide solution was 1:1:100; the incubation time in the dark in S5 was 10-30 min; the concentration of Folin phenol reagent in S5 was 0.1-0.2 mol / L; the wavelength setting of the microplate reader in S5 was 700-800 nm.

[0038] The specific implementation is that the protein content of EPS is determined by the improved Lowery microplate method. First, the working concentration of 100 mg / L bovine serum albumin standard mother liquor is prepared by electronic balance weighing method. Then, the mother liquor is used to prepare bovine serum protein standard working solutions with concentrations of 0, 10, 20, 30, 40, 60, 80 and 100 mg / L by gradient dilution. Then, composite reagent A and composite reagent B are prepared. Reagent A is a mixture of CuSO4·5H2O solution, potassium sodium tartrate solution and sodium carbonate-sodium hydroxide solution, and the volume ratio of CuSO4·5H2O solution, potassium sodium tartrate solution and sodium carbonate-sodium hydroxide solution is 1:1:100, which is prepared immediately before use; composite reagent B is a mixture of ultrapure water, potassium sodium tartrate solution and sodium carbonate-sodium hydroxide solution, and the volume ratio of ultrapure water, potassium sodium tartrate solution and sodium carbonate-sodium hydroxide solution is 1:1:100.

[0039] CuSO4·5H2O solution: 3.5 g of copper sulfate pentahydrate (CuSO4·5H2O) was dissolved in 100 mL of ultrapure water to obtain the solution;

[0040] Potassium sodium tartrate solution: 7.0 g of potassium sodium tartrate (NaKC4H4O6) was dissolved in 100 mL of ultrapure water to obtain the solution;

[0041] Sodium carbonate-sodium hydroxide solution: 70 g of sodium carbonate (Na2CO3) was dissolved in 1000 mL of 0.35 mol / L sodium hydroxide (NaOH) solution.

[0042] Sample preparation: 100 μL of the series of standard working solutions and 100 μL of EPS extract were added to two 96-well plates, respectively, and then 100 μL of composite reagent B and 100 μL of composite reagent A were added, respectively, and mixed well; then the two 96-well plates were incubated at room temperature for 10 min in the dark; then 100 μL of 0.2 mol / L Folin phenol reagent was added, respectively, and mixed well; then the two 96-well plates were incubated at room temperature for 20 min in the dark, to obtain the standard working sample solution and the sample to be tested.

[0043] Sample determination: the absorbance of the standard working sample solution and the sample to be tested at 750 nm was determined using a full-wavelength enzyme marker (USA).

[0044] The actual absorbance of the sample to be tested was calculated according to the following formula: A protein = 1.25 x (A total -A blind ),

[0045] Wherein, Atotal refers to the total absorbance measured using the Lowry reagent containing CuSO4 (i.e. composite reagent A), and Ablind refers to the control absorbance measured using the Lowry reagent without CuSO4 (i.e. composite reagent B).

[0046] The protein concentration of the standard working sample solution was taken as the abscissa, and the control absorbance was taken as the ordinate to draw the standard curve and calculate the linear equation, to obtain the protein content of the sample to be tested.

[0047] The above only describes certain exemplary embodiments of the present application by way of illustration, and it is needless to say that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present application. Therefore, the above figures and descriptions are illustrative in nature and should not be understood as limiting the scope of protection of the claims of the present application.

Claims

1. A method for extracting and determining extracellular polymers in soil based on cation exchange resin, characterized in that, Specifically, the following steps are included: S1. Pretreatment of cation exchange resin: First, wash the Na-type cation exchange resin with hot water at 70-80℃ until the supernatant is colorless and foam-free. Then, add HCl solution and shake. Wash with ultrapure water and then add NaCl solution and shake. Then add NaOH solution and shake. Finally, wash with ultrapure water until the solution is neutral. After drying, CER resin particles are obtained. S2. Soil sample pretreatment: Weigh fresh soil into a centrifuge tube, add CaCl2 solution, then place the centrifuge tube in a shaker and shake. After shaking, centrifuge, discard the supernatant, and then add a pre-chilled buffer solution to the centrifuge tube containing soil particles to obtain a pretreated soil sample. S3. Extraction of extracellular polymeric substances (EPS) from soil: Weigh CER resin particles and add them to a centrifuge tube containing pretreated soil samples. Then place the centrifuge tube in a shaker and oscillate. Centrifuge again to separate the supernatant and soil particles. Finally, filter the supernatant to obtain EPS extract. S4. Polysaccharide content determination: Mix EPS extract with phenol solution in equal volume, then add concentrated sulfuric acid and vortex mix, then heat in a light-protected water bath, cool to room temperature and detect absorbance with an enzyme-linked immunosorbent assay (ELISA) reader to obtain polysaccharide content. S5. Protein content determination: First, prepare compound reagent A, then mix EPS extract with compound reagent A and incubate in the dark. After adding Folin phenol reagent, incubate again in the dark. Finally, use an ELISA reader to detect the absorbance to obtain the protein content.

2. The method for extraction and determination of soil extracellular polymers based on cation exchange resin according to claim 1, characterized in that, The concentration of the HCl solution in S1 is 0.1–1 mol / L; the concentration of the NaOH solution in S1 is 0.1–1 mol / L; the concentration of the NaCl solution is 1%–5%; and the volume ratio of the Na-type cation exchange resin to the HCl solution, NaOH solution, and NaCl solution in S1 is 1:3:3:

3.

3. The method for extraction and determination of soil extracellular polymers based on cation exchange resin according to claim 1, characterized in that, The concentration of the CaCl2 solution in S2 is 0.01–0.05 mol / L; the buffer solution in S2 is at least one of phosphate buffer solution, citrate buffer solution, and acetate buffer solution; the relative centrifugal force during centrifugation in S2 is set to 3000–3500 × g, and the temperature is set to 0–4 °C.

4. The method for extraction and determination of soil extracellular polymers based on cation exchange resin according to claim 1, characterized in that, In S3, the relative centrifugal force during centrifugation is set to 3000-4000×g, and the temperature is set to 0-4℃.

5. The method for extraction and determination of soil extracellular polymers based on cation exchange resin according to claim 1, characterized in that, The volume ratio of EPS extract to concentrated sulfuric acid in S4 is 1:1 to 5; the concentration of phenol solution in S4 is 1% to 5%; the water bath heating temperature in S4 is set to 90 to 100°C; and the detection wavelength of the enzyme-linked immunosorbent assay (ELISA) in S4 is set to 400 to 500 nm.

6. The method for extraction and determination of extracellular polymers in soil based on cation exchange resin according to claim 1, characterized in that, In S5, the composite reagent A is a mixture of CuSO4·5H2O solution, potassium sodium tartrate solution, and sodium carbonate-sodium hydroxide solution, and the volume ratio of CuSO4·5H2O solution, potassium sodium tartrate solution, and sodium carbonate-sodium hydroxide solution is 1:1:

100.

7. The method for extraction and determination of soil extracellular polymers based on cation exchange resin according to claim 1, characterized in that, The incubation time in S5 is 10-30 min in the dark; the concentration of Folin phenol reagent in S5 is 0.1-0.2 mol / L.

8. The method for extraction and determination of soil extracellular polymers based on cation exchange resin according to claim 1, characterized in that, The detection wavelength of the ELISA reader in S5 is set to 700–800 nm.

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