CN102033011A - Soil phytic acid extracting solution and application thereof - Google Patents

Abstract

The invention relates to the field of chemical industry, in particular to a soil phytic acid extracting solution and a method for determining the content of phytic acid in soil by applying the extracting solution. The soil phytic acid extracting solution provided by the invention comprises 20-30% of sodium sulfate and 10-15% of organic acid by mass, and the solvent is ultrapure water. The method for measuring the phytic acid content of the soil comprises the steps of establishing a standard curve of the phytic acid content and the absorbance, roughly extracting the phytic acid in the soil, separating the phytic acid by using anion exchange resin, measuring the absorbance, and calculating the phytic acid content. The method for measuring the phytic acid content in the soil provided by the invention has the advantages of high separation efficiency, simple operation and high accuracy, and is suitable for separating and measuring the phytic acid content of various soil types.

Description

A kind of soil phytic acid extract and application thereof

technical field

The invention relates to the field of chemical industry, in particular to a soil phytic acid extraction solution and a method for measuring phytic acid content in soil using the extraction solution.

Background technique

Phosphorus in soil can be divided into organic phosphorus and inorganic phosphorus. The soil is dominated by organic phosphorus, accounting for about 20% to 50% of the total phosphorus, about half of which exists in the form of phytate, so the percentage of phytic acid in the total phosphorus content is 10 to 25%. Soil organic phosphorus is a very complicated issue, and many components and structures are still unclear. Most organic phosphorus exists in the form of polymers, and its effectiveness is not high. Improving the absorption of organic phosphorus such as phytate in the soil by plants is of great significance for improving the phosphorus nutrition of plants and promoting the sustainable utilization of phosphorus resources.

Richardson et al. studied the Arabidopsis transgenic line overexpressing Aspergillus niger phytase PhyA and found that the phytase (phyatase) secreted into the rhizosphere medium under the guidance of the signal peptide can effectively degrade phytate and release inorganic Phosphorus (Pi). The amount of phosphorus uptake and plant growth characteristics of the transgenic plants were significantly improved under the condition of phytate as the phosphorus supply source, indicating that the phytase gene has an important value in improving the utilization of organic phosphorus by plants. But so far, there are few reports on the utilization of organic phosphorus such as phytate in soil by some obtained transphytase crops (Arabidopsis thaliana, tobacco, potato, clover). There is no standard method for the determination of soil phytic acid content. In addition, the screening of plant species or varieties with high phytic acid utilization rate is also based on the accurate determination of soil phytic acid content, so it is urgent to establish a method for the determination of phytic acid content in soil.

Contents of the invention

In order to solve the above problems, the present invention aims to provide a soil phytic acid extract and a method for determining the content of phytic acid in soil using the extract.

One object of the present invention is to provide a soil phytic acid extraction solution, comprising 20%-30% by mass of sodium sulfate and 10%-15% by mass of organic acid, and the solvent is water.

Wherein, the mass percentage of the sodium sulfate is preferably 20%, and the mass percentage of the organic acid is preferably 10%.

Wherein, the organic acid is preferably trichloroacetic acid, chloroacetic acid, dichloroacetic acid, dichlorooxalic acid, most preferably trichloroacetic acid.

Another object of the present invention is to provide a method for determining the content of phytic acid in soil using the soil phytic acid extract.

The method for measuring phytic acid content in soil provided by the invention comprises the following steps:

1) Establish the standard curve of phytic acid solution

Prepare phytic acid standard solutions of different concentration series, add the same volume of 0.3wt% ferric chloride-3wt% sulfosalicylic acid aqueous solution to the above series of standard solutions, measure the absorbance at 500nm after the reaction, and establish the phytic acid content Standard curve with its absorbance;

2) Rough extraction of phytic acid from soil

Weigh the soil sample and place it in the above-mentioned phytic acid extract, vibrate, filter, and collect the filtrate;

3) Separate phytic acid from the filtrate obtained in step 2) with an anion exchange resin, add ferric chloride-sulfosalicylic acid solution with the same volume and concentration as in step 1), and measure the absorbance at 500 nm after the reaction, according to the phytic acid content Calculate the phytic acid content from the standard curve of its absorbance. Among them, the calculation formula is:

X＝(CV ₁ V ₃ )/(V ₂ V ₄ m)

In the formula: X, the phytic acid content in the sample/mg·mg ^-1 ; C, the phytic acid content in the sample solution/mg; m, the mass of the sample/g; V ₁ , the volume of the extract after constant volume/mL; V ₂ , the volume/mL of the extract after separation; V ₃ , the volume/mL of the separation liquid after constant volume; V ₄ , the volume/mL of the separation liquid when measuring the test solution.

Wherein, the soil is preferably soil that has passed through a 100-mesh sieve.

Wherein, in step 2), after adding the extract solution, shake for 1-3 hours, preferably 2 hours.

Wherein, the anion exchange resin is selected from D201, D301 or D315, preferably D201, D301, most preferably D201.

The method for measuring phytic acid content in soil provided by the invention has high separation efficiency, simple operation and high accuracy, and is suitable for separation and determination of phytic acid content in various soil types.

Description of drawings

The standard curve of Fig. 1 phytic acid content and its absorbance

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1

Weigh 200g of sodium sulfate and 100g of trichloroacetic acid in a beaker, dissolve with ultrapure water, and use ultrapure water to make up to 1000ml.

Example 2

Weigh 150g of sodium sulfate and 150g of trichloroacetic acid in a beaker, dissolve them with ultrapure water, and dilute to 1000ml with ultrapure water.

Example 3

Weigh 500g of sodium sulfate and 200g of trichloroacetic acid in a beaker, dissolve them with ultrapure water, and dilute to 2000ml with ultrapure water.

Example 4

Weigh 200g of sodium sulfate and 100g of chloroacetic acid in a beaker, dissolve them with ultrapure water, and dilute to 1000ml with ultrapure water.

Example 5

Weigh 500g of sodium sulfate and 200g of dichloroacetic acid in a beaker, dissolve them with ultrapure water, and adjust the volume to 2000ml with ultrapure water.

Example 6

Weigh 200g of sodium sulfate and 100g of dichlorodiacetic acid in a beaker, dissolve them with ultrapure water, and dilute to 1000ml with ultrapure water.

Example 7

After air-drying the soil samples taken from Zaoqiang County, Hebei Province, pass through a 100-mesh sieve, weigh 1.0000±0.0001g of soil, and add phytic acid in the amount of 0, 0.025, 0.05, 0.07, 0.09, 0.12mg .

Accurately draw 0.1mg·mL ^-1 phytic acid standard solution 0.0, 1.0, 2.0, 3.0, 4.0, 5.0mL into six 10mL centrifuge tubes, make up to 5mL with distilled water, add ferric chloride-sulfosalicylic acid to react Solution 4mL, shake well, and centrifuge at 5000rpm for 10min. After standing for 15 minutes, pour the supernatant liquid into the cuvette, measure the absorbance at 500nm, take the absorbance as the ordinate, and the phytic acid content as the abscissa, draw the working curve and calculate the regression equation and correlation coefficient (as shown in Figure 1) .

Place the soil sample treated above in a 250mL Erlenmeyer flask with a stopper, add 100mL of the extract prepared in Example 1, shake for 2 hours, then filter, and collect the filtrate to make up to 200mL.

Weigh 10g of anion exchange resin D201, soak in distilled water for 3 hours, then soak in 2 times the amount of 4% sodium hydroxide solution for 2 hours, then soak in 2 times the amount of 4% hydrochloric acid solution for 2 hours, finally rinse with distilled water, soak in distilled water in spare.

The pretreated anion resin was wet-packed in an exchange column (1.8 cm×20 cm), and the exchange column was washed with deionized water until there was no chloride ion. Take 5mL of the sample extract, add 2mL of 30g·L ^-1 sodium hydroxide solution, add distilled water to a total volume of 30mL, mix well and pour into the ion exchange column. Then wash the exchange column with 30 mL of water and 30 mL of 0.05 mol·L ^-1 sodium chloride washing solution at a flow rate of 0.5 mL·min ^-1 , and discard the washing solution. Finally, the exchange column was eluted with 0.7mol·L ^-1 sodium chloride elution solution, collected in a 50mL graduated Erlenmeyer flask with a stopper, and the volume was adjusted to the mark.

Take eluent 1mL in a 10mL centrifuge tube, add 0.3wt% ferric chloride-3wt% sulfosalicylic acid aqueous solution 4mL, shake well, the remaining steps are the same as the making of the working curve, measure its absorbance, and from the regression equation The phytic acid content in the sample liquid was calculated, and the results are shown in Table 1.

Table 1 Soil phytic acid content in standard samples

As can be seen from the results in Table 1, adding quantitative phytic acid to the soil, and then measuring with the method provided by the invention, the result accuracy is higher.

Example 8

Soil samples were collected from Shenzhou County, Xinji County, Wei County and Jing County, Hebei Province, respectively, including loam soil, clay soil, sandy loam soil and sandy soil. Air-dry the collected soil samples, pass through a 100-mesh sieve, and weigh 1.0000±0.0001g of soil for later use.

Put the soil sample treated above into a 250mL Erlenmeyer flask with a stopper, add 100mL of the extract prepared in Example 1, shake at 5000rpm for 3h, then filter, and collect the filtrate to make up to 200mL.

Weigh 10g of anion exchange resin D301, soak in distilled water for 3 hours, then soak in 2 times the amount of 4% sodium hydroxide solution for 2 hours, then soak in 2 times the amount of 4% hydrochloric acid solution for 2 hours, finally rinse with distilled water, soak in distilled water in spare.

The pretreated anion resin was wet-packed in an exchange column (1.8 cm×20 cm), and the exchange column was washed with deionized water until there was no chloride ion. Take 5mL of the sample extract, add 2mL of 30g·L ^-1 sodium hydroxide solution, add distilled water to a total volume of 30mL, mix well and pour into the ion exchange column. Then wash the exchange column with 30 mL of water and 30 mL of 0.05 mol·L ^-1 sodium chloride washing solution at a flow rate of 0.5 mL·min ^-1 , and discard the washing solution. Finally, the exchange column was eluted with 0.7mol·L ^-1 sodium chloride elution solution, collected in a 50mL graduated Erlenmeyer flask with a stopper, and the volume was adjusted to the mark.

Take 1mL of the eluent in a 10mL centrifuge tube, add 4mL of ferric chloride-sulfosalicylic acid reaction solution, shake well, and the rest of the steps are the same as that of the working curve, measure its absorbance, and calculate the sample solution from the regression equation Medium phytic acid content.

The results showed that the phytic acid contents of the soil samples in Shenzhou County, Xinji County, Wei County and Jing County were 0.225±0.001, 0.247±0.002, 0.253±0.003, 0.267±0.001mg/g, respectively.

The total phosphorus content in these four counties was measured by NaOH alkali-melted molybdenum-antimony colorimetric method to be 1.168±0.002, 0.921±0.003, 1.052±0.012 and 1.006±0.015 mg/g, respectively. According to the percentage of phytic acid in the total phosphorus content (10-25%), the phytic acid content measured in the soil samples of the four counties is basically within this range (19%-26%), so the accuracy of this method is relatively high. high.

Example 9

Air-dry the soil samples taken from Quzhou County, Qing County, Hejian County, Gucheng County and Gaoyang County in Hebei Province, pass through a 100-mesh sieve, and weigh 1.0000±0.0001g of soil for use.

Put the soil sample treated above into a 250mL Erlenmeyer flask with a stopper, add 100mL of the extract prepared in Example 2 respectively, shake at 4000rpm for 5h, then filter, and collect the filtrate to make up to 200mL.

Weigh 10g of anion exchange resin D315, soak in distilled water for 3 hours, then soak in 2 times the amount of 4% sodium hydroxide solution for 2 hours, then soak in 2 times the amount of 4% hydrochloric acid solution for 2 hours, finally rinse with distilled water, soak in distilled water in spare.

Wet-pack the pretreated anion resin into an exchange column (1.8cmx20cm), and wash the exchange column with deionized water until there is no chloride ion. Take 5mL of the sample extract, add 2mL of 30g·L ^-1 sodium hydroxide solution, add distilled water to a total volume of 30mL, mix well and pour into the ion exchange column. Then wash the exchange column with 30 mL of water and 30 mL of 0.05 mol·L ^-1 sodium chloride washing solution at a flow rate of 0.5 mL·min ^-1 , and discard the washing solution. Finally, the exchange column was eluted with 0.7mol·L ^-1 sodium chloride elution solution, collected in a 50mL graduated Erlenmeyer flask with a stopper, and the volume was adjusted to the mark.

Take 1mL of the eluent in a 10mL centrifuge tube, add 4mL of ferric chloride-sulfosalicylic acid reaction solution, shake well, and the rest of the steps are the same as that of the working curve, measure its absorbance, and calculate the sample solution from the regression equation Medium phytic acid content.

The results showed that the phytic acid contents of soil samples in Quzhou County, Qing County, Hejian County, Gucheng County and Gaoyang County were 0.220±0.002, 0.258±0.004, 0.218±0.001, 0.228±0.002, 0.267±0.003mg/ g.

The total phosphorus content of the five counties measured by NaOH alkali-melted molybdenum-antimony anti-colorimetry method was 1.253±0.014, 0.914±0.015, 0.938±0.021, 1.059±0.031 and 1.036±0.011mg/g. According to the percentage of phytic acid in the total phosphorus content (10-25%), the phytic acid content measured in the soil samples of the five counties is basically within this range (17%-28%), so the reliability of the method higher.

Example 10

The soil samples taken from Nangong County, Jizhou County and Xian County of Hebei Province were air-dried, passed through a 100-mesh sieve, and 1.0000±0.0001g of soil was weighed for use.

Put the soil sample treated above into a 250mL Erlenmeyer flask with a stopper, add 100mL of the extract prepared in Example 5, shake at 5000rpm for 1 hour, then filter, and collect the filtrate to make up to 200mL.

Weigh 10g of anion exchange resin D201, soak in distilled water for 3 hours, then soak in 2 times the amount of 4% sodium hydroxide solution for 2 hours, then soak in 2 times the amount of 4% hydrochloric acid solution for 2 hours, finally rinse with distilled water, soak in distilled water in spare.

Wet-pack the pretreated anion resin into an exchange column (1.8cmx20cm), and wash the exchange column with deionized water until there is no chloride ion. Take 5mL of the sample extract, add 2mL of 30g·L ^-1 sodium hydroxide solution, add distilled water to a total volume of 30mL, mix well and pour into the ion exchange column. Then wash the exchange column with 30 mL of water and 30 mL of 0.05 mol·L ^-1 sodium chloride washing solution at a flow rate of 0.5 mL·min ^-1 , and discard the washing solution. Finally, the exchange column was eluted with 0.7mol·L ^-1 sodium chloride elution solution, collected in a 50mL graduated Erlenmeyer flask with a stopper, and the volume was adjusted to the mark.

Take 1mL of the eluent in a 10mL centrifuge tube, add 4mL of ferric chloride-sulfosalicylic acid reaction solution, shake well, and the rest of the steps are the same as that of the working curve, measure its absorbance, and calculate the sample solution from the regression equation Medium phytic acid content.

The results showed that the phytic acid contents of soil samples from Nangong County, Jizhou County and Xian County were 0.261±0.004, 0.272±0.001 and 0.274±0.003 mg/g, respectively.

The total phosphorus content in these three counties was measured by NaOH alkali-melted molybdenum-antimony anticolorimetric method to be 1.116±0.034, 1.309±0.035 and 1.434±0.031 mg/g, respectively. According to the percentage of phytic acid in the total phosphorus content (10-25%), the phytic acid content measured in the soil samples of the three counties is basically within this range (19%-28%), so the reliability of the method higher.

Example 11

The soil samples taken in Julu County, Linxi County and Qinghe County of Hebei Province were air-dried, passed through a 100-mesh sieve, and 1.0000±0.0001g of soil was weighed for use.

Put the soil sample treated above into a 250mL Erlenmeyer flask with a stopper, add 100mL of the extract prepared in Example 6 respectively, shake at 5000rpm for 2h, then filter, and collect the filtrate to make up to 200mL.

Weigh 10g of anion exchange resin D301, soak in distilled water for 3 hours, then soak in 2 times the amount of 4% sodium hydroxide solution for 2 hours, then soak in 2 times the amount of 4% hydrochloric acid solution for 2 hours, finally rinse with distilled water, soak in distilled water in spare.

Wet-pack the pretreated anion resin into an exchange column (1.8cmx20cm), and wash the exchange column with deionized water until there is no chloride ion. Take 5mL of the sample extract, add 2mL of 30g·L ^-1 sodium hydroxide solution, add distilled water to a total volume of 30mL, mix well and pour into the ion exchange column. Then wash the exchange column with 30 mL of water and 30 mL of 0.05 mol·L ^-1 sodium chloride washing solution at a flow rate of 0.5 mL·min ^-1 , and discard the washing solution. Finally, the exchange column was eluted with 0.7mol·L ^-1 sodium chloride elution solution, collected in a 50mL graduated Erlenmeyer flask with a stopper, and the volume was adjusted to the mark.

Take 1mL of the eluent in a 10mL centrifuge tube, add 4mL of ferric chloride-sulfosalicylic acid reaction solution, shake well, and the rest of the steps are the same as that of the working curve, measure its absorbance, and calculate the sample solution from the regression equation Medium phytic acid content.

The results showed that the phytic acid contents of the soil samples in Julu County, Linxi County and Qinghe County were 0.356±0.004, 0.270±0.001, 0.241±0.001mg/g, respectively.

The total phosphorus content in these three counties was measured by NaOH alkali-melted molybdenum-antimony anti-colorimetric method to be 1.28±0.024, 1.153±0.025 and 1.018±0.011 mg/g, respectively. According to the percentage of phytic acid in the total phosphorus content (10-25%), the phytic acid content measured in the soil samples of the three counties is basically within this range (23%-27%), so the reliability of the method higher.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (10)

1. a soil phytic acid extract is characterized in that, comprises the sodium sulphate of 20%～30% mass percent and the organic acid of 10%～15% mass percent, and solvent is a water.

2. extract according to claim 1 is characterized in that, the mass percent of described sodium sulphate is 20%.

3. phytic acid extract according to claim 1 is characterized in that, described organic acid mass percent is 10%.

4. according to claim 1 or 3 described phytic acid extracts, it is characterized in that described organic acid is selected from trichloroacetic acid, chloroacetic acid, dichloroacetic acid, dichloro ethane diacid.

5. phytic acid extract according to claim 4 is characterized in that, described organic acid is a trichloroacetic acid.

6. an application rights requires 1～5 each described phytic acid extract to carry out phytic acid content assay method in the soil, comprises the steps:

1) set up the typical curve of phytic acid content and absorbance:

The phytic acid standard solution of configuration variable concentrations series adds ferric trichloride 0.3wt%-sulfosalicylic acid 3wt% aqueous solution in above-mentioned series standard solution, measure the absorbance at 500nm place after the reaction respectively, sets up the typical curve of phytic acid content and its absorbance;

2) phytic acid is slightly carried in the soil:

Take by weighing pedotheque and place each described phytic acid extract of claim 1～5, vibration is filtered, and collects filtrate;

3) with step 2) gained filtrate separates phytic acid with anion exchange resins, add the ferric trichloride-sulfosalicylic acid aqueous solution identical with step 1), the absorbance at 500nm place is measured in the reaction back, according to the typical curve of phytic acid content and its absorbance, calculates phytic acid content.

7. assay method according to claim 6 is characterized in that described soil is crossed 100 mesh sieves.

8. assay method according to claim 6 is characterized in that, described duration of oscillation is 1～3 hour.

9. assay method according to claim 6 is characterized in that described anion exchange resins is selected from D201, D301 or D315.

10. assay method according to claim 9 is characterized in that, described anion exchange resins is D201.

Images