CN102033011A - Soil phytic acid extracting solution and application thereof - Google Patents
Soil phytic acid extracting solution and application thereof Download PDFInfo
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Abstract
The invention relates to the field of chemical industry, particularly a soil phytic acid extracting solution and a method for measuring the phytic acid content in soil by using the same. The soil phytic acid extracting solution comprises the following components in percentage by mass: 20-30% of sodium sulfate and 10-15% of organic acid; and the solvent is ultrapure water. The method comprises the following steps: establishing a standard curve of phytic acid content and absorbance, coarsely extracting phytic acid in the soil, separating the phytic acid by using anion exchange resin, measuring the absorbance, and computing the phytic acid content. The method has the advantages of high separation efficiency and high accuracy, is simple for operation, and is suitable for separating and measuring the phytic acid content of various types of soil.
Description
Technical field
The present invention relates to chemical field, be specifically related to a kind of soil phytic acid extract and use the assay method that this extract carries out phytic acid content in the soil.
Background technology
Phosphorus can be divided into organophosphorus and Phos two big classes in the soil.Soil accounts for 20%～50% of full phosphorus based on organophosphorus, wherein about half again the form with phytate exist, so the number percent that phytic acid accounts for content of tatal phosphorus is 10～25%.The soil organophosphorus is a very complicated problems, and many The Nomenclature Composition and Structure of Complexes are not clear, and most of organophosphorus exists with the macromolecule form, and validity is not high.Improve the absorption of plant,, and then promote the plain Sustainable utilization of resources of phosphorus to have great importance for the plain nutrition of the phosphorus that improves plant to organic phosphorus such as phytates in the soil.
Richardson etc. discover the arabidopsis transgenic lines of overexpression aspergillus niger phytase PhyA, secrete the phytase (phyatase) to the rhizosphere medium under the guiding of signal peptide, and the phytate of effectively degrading discharges inorganic states phosphorus (Pi).To be phosphorus at phytate obviously improve for suction phosphorus quantity and plant strain growth characteristic under the condition of source transfer-gen plant, shows that phytase gene has important value improving in the utilization of plant to organic phosphorus.But up to now, relevant some that obtain are changeed phytase crop (arabidopsis, tobacco, potato, clover), and to the also rarely seen report of research of organic phosphate use aspects such as phytate in the soil, its major technique bottleneck is the assay method that yet there are no a kind of soil phytic acid content of standard.In addition, the plant species of high phytic acid utilization factor or the screening of kind also are based on the accurate mensuration of soil phytic acid content, therefore need badly and set up phytic acid content assay method in a kind of soil.
Summary of the invention
In order to solve the above problems, the present invention aims to provide a kind of soil phytic acid extract and uses the assay method that this extract carries out phytic acid content in the soil.
One object of the present invention is to provide a kind of soil phytic acid extract, comprises the sodium sulphate of 20%～30% mass percent and the organic acid of 10%～15% mass percent, and solvent is a water.
Wherein, described sodium sulfate quality number percent is preferably 20%, and the organic acid mass percent is preferably 10%.
Wherein, described organic acid is preferably trichloroacetic acid, chloroacetic acid, dichloroacetic acid, dichloro ethane diacid, most preferably is trichloroacetic acid.
Another object of the present invention is to provide uses described soil phytic acid extract, and phytic acid content in the soil is carried out method for measuring.
The phytic acid content assay method comprises the steps: in the soil provided by the invention
1) sets up the typical curve of plant acid solution
The phytic acid standard solution of configuration variable concentrations series, 0.3wt% ferric trichloride-3wt% sulfosalicylic acid the aqueous solution that adds equal volume in the above-mentioned series standard solution, measure the absorbance at 500nm place after the reaction respectively, set 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 above-mentioned phytic acid extract, vibration is filtered, and collects filtrate;
3) with step 2) gained filtrate separates phytic acid with anion exchange resins, add ferric trichloride-sulfosalicylic acid solution with step 1) equal volume same concentrations, 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.Wherein, computing formula is:
X＝(CV
1V
3)/(V
2V
4m)
In the formula: X, phytic acid content/mgmg in the sample
-1C, sample liquid contains phytic acid amount/mg; M, the quality/g of sample; V
1, the volume/mL behind the extract constant volume; V
2, get the volume/mL of extract after the separation; V
3, the volume/mL behind the parting liquid constant volume; V
4, when test solution is measured, get the volume/mL of parting liquid.
Wherein, described soil was preferably the soil of 100 mesh sieves.
Wherein, step 2) vibrated 1～3 hour after adding extract in, be preferably 2 hours.
Wherein, described anion exchange resins is selected from D201, D301 or D315, is preferably D201, D301, most preferably is D201.
Phytic acid content assay method separation efficiency height in the soil provided by the invention, assay method is simple to operate, accuracy is high, is applicable to the separation and the mensuration of multiple soil types phytic acid content.
Description of drawings
The typical curve of Fig. 1 phytic acid content and its absorbance
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
Take by weighing the sodium sulphate of 200g and 100g trichloroacetic acid in beaker, with the ultrapure water dissolving, and with the ultrapure water constant volume to 1000ml.
Embodiment 2
Take by weighing the sodium sulphate of 150g and 150g trichloroacetic acid in beaker, with the ultrapure water dissolving, and with the ultrapure water constant volume to 1000ml.
Embodiment 3
Take by weighing the sodium sulphate of 500g and 200g trichloroacetic acid in beaker, with the ultrapure water dissolving, and with the ultrapure water constant volume to 2000ml.
Embodiment 4
Take by weighing the sodium sulphate of 200g and 100g chloroacetic acid in beaker, with the ultrapure water dissolving, and with the ultrapure water constant volume to 1000ml.
Embodiment 5
Take by weighing the sodium sulphate of 500g and 200g dichloroacetic acid in beaker, with the ultrapure water dissolving, and with the ultrapure water constant volume to 2000ml.
Embodiment 6
Take by weighing the sodium sulphate of 200g and 100g dichloro oxalic acid in beaker, with the ultrapure water dissolving, and with the ultrapure water constant volume to 1000ml.
Embodiment 7
To Zaoqiang County, Hebei province institute soil sampling air-dry after, cross 100 mesh sieves, take by weighing 1.0000 ± 0.0001g soil, add the processing of phytic acid respectively, addition is respectively 0,0.025,0.05,0.07,0.09,0.12mg.
Accurately draw 0.1mgmL
-1Phytic acid standard solution 0.0,1.0,2.0,3.0,4.0,5.0mL complements to 5mL with distilled water in 6 10mL centrifuge tubes, add ferric trichloride-sulfosalicylic acid reaction solution 4mL, shakes up, with the centrifugal 10min of 5000rpm rotating speed.After placing 15min, upper strata liquid being poured in the cuvette, measured absorbance in the 500nm place, is ordinate with the absorbance, and phytic acid content is a horizontal ordinate, and the drawing curve also calculates regression equation and related coefficient (as shown in Figure 1).
The pedotheque of above-mentioned processing is placed 250mL tool plug triangular flask, add the extract of 100mL embodiment 1 preparation respectively, filter after during vibration 2h, collect the filtrate constant volume to 200mL.
Take by weighing 10g anion exchange resins D201, be dipped in 3h in the distilled water, 4% sodium hydroxide solution with 2 times of amounts soaks 2h again, is soaked in 4% hydrochloric acid solution 2h of 2 times of amounts then, uses distilled water flushing at last, is soaked in the distilled water standby.
Will through pretreated resin anion (R.A.) wet-filling in exchange column (among the 1.8cm * 20cm), with the deionized water wash exchange column to there not being chlorion.Get the 5mL sample extracting solution, add 30gL
-1Sodium hydroxide solution 2mL adds distilled water to cumulative volume 30mL, pours in the ion exchange column behind the mixing.Use 30mL water and 30mL 0.05molL then respectively
-1The sodium chloride wash solution is with 0.5mLmin
-1Flow velocity washing exchange column, discard cleansing solution.Use 0.7molL at last
-1Sodium chloride elute soln wash-out exchange column is collected in the 50mL scale tool plug triangular flask, and is settled to scale.
Get eluent 1mL in the 10mL centrifuge tube, add 0.3wt% ferric trichloride-3wt% sulfosalicylic acid aqueous solution 4mL, shake up, all the other steps are with the making of working curve, survey its absorbance, and calculate phytic acid content in the sample liquid from regression equation, the result is as shown in table 1.
Soil phytic acid content in table 1 standard model
From the result of table 1 as can be seen, add quantitative phytic acid in soil, measure with method provided by the invention, result precision is higher.
Embodiment 8
Respectively from Shenzhou City, Hebei province county, Xinji county, Wei County and Jing County soil sampling, wherein be respectively loam, clay, sandy loam and sandy soil.Institute's soil sampling is air-dry, cross 100 mesh sieves, it is stand-by to take by weighing 1.0000 ± 0.0001g soil.
The pedotheque of above-mentioned processing is placed 250mL tool plug triangular flask, add the extract of 100mL embodiment 1 preparation respectively, filter after during 5000rpm vibration 3h, collect the filtrate constant volume to 200mL.
Take by weighing 10g anion exchange resins D301, be dipped in 3h in the distilled water, 4% sodium hydroxide solution with 2 times of amounts soaks 2h again, is soaked in 4% hydrochloric acid solution 2h of 2 times of amounts then, uses distilled water flushing at last, is soaked in the distilled water standby.
Will through pretreated resin anion (R.A.) wet-filling in exchange column (among the 1.8cm * 20cm), with the deionized water wash exchange column to there not being chlorion.Get the 5mL sample extracting solution, add 30gL
-1Sodium hydroxide solution 2mL adds distilled water to cumulative volume 30mL, pours in the ion exchange column behind the mixing.Use 30mL water and 30mL 0.05molL then respectively
-1The sodium chloride wash solution is with 0.5mLmin
-1Flow velocity washing exchange column, discard cleansing solution.Use 0.7molL at last
-1Sodium chloride elute soln wash-out exchange column is collected in the 50mL scale tool plug triangular flask, and is settled to scale.
Get eluent 1mL in the 10mL centrifuge tube, add ferric trichloride-sulfosalicylic acid reaction solution 4mL, shake up, all the other steps are surveyed its absorbance with the making of working curve, and calculate phytic acid content in the sample liquid from regression equation.
The result shows, the phytic acid content of the pedotheque of Shenzhou City county, Xinji county, Wei County and Jing County is respectively 0.225 ± 0.001,0.247 ± 0.002,0.253 ± 0.003,0.267 ± 0.001mg/g.
The content of tatal phosphorus that adopts NaOH alkali fusion molybdenum antimony resistance colorimetric method to record these 4 counties is respectively 1.168 ± 0.002,0.921 ± 0.003,1.052 ± 0.012 and 1.006 ± 0.015mg/g.The number percent (10～25%) that accounts for content of tatal phosphorus according to phytic acid draws, and the phytic acid content that the pedotheque in this 4 county records is in this scope (19%～26%) substantially all, so this method accuracy is higher.
Embodiment 9
Air-dry to Quzhou County, Hebei province, Qing County, interchannel county, Gucheng County and Gaoyang County institute soil sampling, cross 100 mesh sieves, it is stand-by to take by weighing 1.0000 ± 0.0001g soil.
The pedotheque of above-mentioned processing is placed 250mL tool plug triangular flask, add the extract of 100mL embodiment 2 preparation respectively, filter after during 4000rpm vibration 5h, collect the filtrate constant volume to 200mL.
Take by weighing 10g anion exchange resins D315, be dipped in 3h in the distilled water, 4% sodium hydroxide solution with 2 times of amounts soaks 2h again, is soaked in 4% hydrochloric acid solution 2h of 2 times of amounts then, uses distilled water flushing at last, is soaked in the distilled water standby.
Will be in exchange column (1.8cmx20cm) through pretreated resin anion (R.A.) wet-filling, with the deionized water wash exchange column to there not being chlorion.Get the 5mL sample extracting solution, add 30gL
-1Sodium hydroxide solution 2mL adds distilled water to cumulative volume 30mL, pours in the ion exchange column behind the mixing.Use 30mL water and 30mL 0.05molL then respectively
-1The sodium chloride wash solution is with 0.5mLmin
-1Flow velocity washing exchange column, discard cleansing solution.Use 0.7molL at last
-1Sodium chloride elute soln wash-out exchange column is collected in the 50mL scale tool plug triangular flask, and is settled to scale.
Get eluent 1mL in the 10mL centrifuge tube, add ferric trichloride-sulfosalicylic acid reaction solution 4mL, shake up, all the other steps are surveyed its absorbance with the making of working curve, and calculate phytic acid content in the sample liquid from regression equation.
The result shows, the phytic acid content of the pedotheque of Quzhou County, Qing County, interchannel county, Gucheng County and Gaoyang County is respectively 0.220 ± 0.002,0.258 ± 0.004,0.218 ± 0.001,0.228 ± 0.002,0.267 ± 0.003mg/g.
The content of tatal phosphorus that adopts NaOH alkali fusion molybdenum antimony resistance colorimetric method to record these 5 counties is respectively 1.253 ± 0.014,0.914 ± 0.015,0.938 ± 0.021,1.059 ± 0.031 and 1.036 ± 0.011mg/g.The number percent (10～25%) that accounts for content of tatal phosphorus according to phytic acid draws, and the phytic acid content that the pedotheque in this 5 county records is in this scope (17%～28%) substantially all, so this method confidence level is higher.
Embodiment 10
Air-dry to Hebei province's Nangong county, Jizhou City county and Xian County institute soil sampling, cross 100 mesh sieves, it is stand-by to take by weighing 1.0000 ± 0.0001g soil.
The pedotheque of above-mentioned processing is placed 250mL tool plug triangular flask, add the extract of 100mL embodiment 5 preparation respectively, filter after during 5000rpm vibration 1h, collect the filtrate constant volume to 200mL.
Take by weighing 10g anion exchange resins D201, be dipped in 3h in the distilled water, 4% sodium hydroxide solution with 2 times of amounts soaks 2h again, is soaked in 4% hydrochloric acid solution 2h of 2 times of amounts then, uses distilled water flushing at last, is soaked in the distilled water standby.
Will be in exchange column (1.8cmx20cm) through pretreated resin anion (R.A.) wet-filling, with the deionized water wash exchange column to there not being chlorion.Get the 5mL sample extracting solution, add 30gL
-1Sodium hydroxide solution 2mL adds distilled water to cumulative volume 30mL, pours in the ion exchange column behind the mixing.Use 30mL water and 30mL 0.05molL then respectively
-1The sodium chloride wash solution is with 0.5mLmin
-1Flow velocity washing exchange column, discard cleansing solution.Use 0.7molL at last
-1Sodium chloride elute soln wash-out exchange column is collected in the 50mL scale tool plug triangular flask, and is settled to scale.
Get eluent 1mL in the 10mL centrifuge tube, add ferric trichloride-sulfosalicylic acid reaction solution 4mL, shake up, all the other steps are surveyed its absorbance with the making of working curve, and calculate phytic acid content in the sample liquid from regression equation.
The result shows, the phytic acid content of the pedotheque of Nangong county, Jizhou City county and Xian County is respectively 0.261 ± 0.004,0.272 ± 0.001 and 0.274 ± 0.003mg/g.
The content of tatal phosphorus that adopts NaOH alkali fusion molybdenum antimony resistance colorimetric method to record these 3 counties is respectively 1.116 ± 0.034,1.309 ± 0.035 and 1.434 ± 0.031mg/g.The number percent (10～25%) that accounts for content of tatal phosphorus according to phytic acid draws, and the phytic acid content that the pedotheque in this 3 county records is in this scope (19%～28%) substantially all, so this method confidence level is higher.
Embodiment 11
Air-dry to Julu County, Hebei province, Linxi County and Qinghe County institute soil sampling, cross 100 mesh sieves, it is stand-by to take by weighing 1.0000 ± 0.0001g soil.
The pedotheque of above-mentioned processing is placed 250mL tool plug triangular flask, add the extract of 100mL embodiment 6 preparation respectively, filter after during 5000rpm vibration 2h, collect the filtrate constant volume to 200mL.
Take by weighing 10g anion exchange resins D301, be dipped in 3h in the distilled water, 4% sodium hydroxide solution with 2 times of amounts soaks 2h again, is soaked in 4% hydrochloric acid solution 2h of 2 times of amounts then, uses distilled water flushing at last, is soaked in the distilled water standby.
Will be in exchange column (1.8cmx20cm) through pretreated resin anion (R.A.) wet-filling, with the deionized water wash exchange column to there not being chlorion.Get the 5mL sample extracting solution, add 30gL
-1Sodium hydroxide solution 2mL adds distilled water to cumulative volume 30mL, pours in the ion exchange column behind the mixing.Use 30mL water and 30mL 0.05molL then respectively
-1The sodium chloride wash solution is with 0.5mLmin
-1Flow velocity washing exchange column, discard cleansing solution.Use 0.7molL at last
-1Sodium chloride elute soln wash-out exchange column is collected in the 50mL scale tool plug triangular flask, and is settled to scale.
Get eluent 1mL in the 10mL centrifuge tube, add ferric trichloride-sulfosalicylic acid reaction solution 4mL, shake up, all the other steps are surveyed its absorbance with the making of working curve, and calculate phytic acid content in the sample liquid from regression equation.
The result shows, the phytic acid content of the pedotheque of Julu County, Linxi County, Qinghe County is respectively 0.356 ± 0.004,0.270 ± 0.001,0.241 ± 0.001mg/g.
The content of tatal phosphorus that adopts NaOH alkali fusion molybdenum antimony resistance colorimetric method to record these 3 counties is respectively 1.28 ± 0.024,1.153 ± 0.025 and 1.018 ± 0.011mg/g.The number percent (10～25%) that accounts for content of tatal phosphorus according to phytic acid draws, and the phytic acid content that the pedotheque in this 3 county records is in this scope (23%～27%) substantially all, so this method confidence level is higher.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as 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.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2516702A (en) * | 2013-07-19 | 2015-02-04 | Verenium Corp | Colorimetric Phytic Acid Assay |
| CN105177543A (en) * | 2015-08-19 | 2015-12-23 | 合肥市田源精铸有限公司 | Environment-friendly aqueous metal antirust agent and production method thereof |
| CN105203374A (en) * | 2015-09-16 | 2015-12-30 | 中国环境科学研究院 | Method for extracting small-molecule humic acid from soil through XAD resin |
| CN107422049A (en) * | 2017-04-19 | 2017-12-01 | 江西省农业科学院蔬菜花卉研究所 | A kind of extracting method of asparagus rhizosphere Organic Acids In Soil |
| CN109187383A (en) * | 2018-04-08 | 2019-01-11 | 渤海大学 | Phytic acid content and the method for eliminating tea polyphenols interference in a kind of measurement mixed solution |
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| EP1237639A1 (en) * | 1999-11-12 | 2002-09-11 | Aventis Cropscience S.A. | Multiple sorbent cartridges for solid phase extraction |
| CN101251451A (en) * | 2008-03-31 | 2008-08-27 | 河南农大迅捷测试技术有限公司 | Method for digesting available nutrient from acid soil as well as combined digestion agent thereof |
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| EP1237639A1 (en) * | 1999-11-12 | 2002-09-11 | Aventis Cropscience S.A. | Multiple sorbent cartridges for solid phase extraction |
| CN101251451A (en) * | 2008-03-31 | 2008-08-27 | 河南农大迅捷测试技术有限公司 | Method for digesting available nutrient from acid soil as well as combined digestion agent thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2516702A (en) * | 2013-07-19 | 2015-02-04 | Verenium Corp | Colorimetric Phytic Acid Assay |
| GB2516702B (en) * | 2013-07-19 | 2015-09-02 | Basf Enzymes Llc | Colorimetric Phytic Acid Assay |
| CN105177543A (en) * | 2015-08-19 | 2015-12-23 | 合肥市田源精铸有限公司 | Environment-friendly aqueous metal antirust agent and production method thereof |
| CN105203374A (en) * | 2015-09-16 | 2015-12-30 | 中国环境科学研究院 | Method for extracting small-molecule humic acid from soil through XAD resin |
| CN107422049A (en) * | 2017-04-19 | 2017-12-01 | 江西省农业科学院蔬菜花卉研究所 | A kind of extracting method of asparagus rhizosphere Organic Acids In Soil |
| CN109187383A (en) * | 2018-04-08 | 2019-01-11 | 渤海大学 | Phytic acid content and the method for eliminating tea polyphenols interference in a kind of measurement mixed solution |
| CN109187383B (en) * | 2018-04-08 | 2021-02-12 | 渤海大学 | Method for measuring phytic acid content in mixed solution and eliminating interference of tea polyphenol |
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Application publication date: 20110427 |