CN111982874A - Method for detecting selenium element in grains - Google Patents
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- CN111982874A CN111982874A CN202010820691.5A CN202010820691A CN111982874A CN 111982874 A CN111982874 A CN 111982874A CN 202010820691 A CN202010820691 A CN 202010820691A CN 111982874 A CN111982874 A CN 111982874A
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- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 136
- 239000011669 selenium Substances 0.000 title claims abstract description 132
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 132
- 238000000034 method Methods 0.000 title claims abstract description 25
- 150000004676 glycans Chemical class 0.000 claims abstract description 29
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 29
- 239000005017 polysaccharide Substances 0.000 claims abstract description 29
- 102000008114 Selenoproteins Human genes 0.000 claims abstract description 24
- 108010074686 Selenoproteins Proteins 0.000 claims abstract description 24
- 235000013339 cereals Nutrition 0.000 claims abstract description 15
- 239000002002 slurry Substances 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 19
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 239000007787 solid Substances 0.000 claims description 15
- 238000001816 cooling Methods 0.000 claims description 14
- 238000000926 separation method Methods 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 13
- 230000029087 digestion Effects 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 238000000502 dialysis Methods 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 238000005238 degreasing Methods 0.000 claims description 6
- 239000012074 organic phase Substances 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 238000007872 degassing Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 238000005185 salting out Methods 0.000 claims description 5
- 229920006395 saturated elastomer Polymers 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 238000005516 engineering process Methods 0.000 claims description 4
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 3
- 238000000184 acid digestion Methods 0.000 claims description 3
- 238000001391 atomic fluorescence spectroscopy Methods 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- TVBSSDNEJWXWFP-UHFFFAOYSA-N nitric acid perchloric acid Chemical compound O[N+]([O-])=O.OCl(=O)(=O)=O TVBSSDNEJWXWFP-UHFFFAOYSA-N 0.000 claims description 3
- 239000002994 raw material Substances 0.000 abstract description 7
- 238000001514 detection method Methods 0.000 abstract description 6
- 238000004140 cleaning Methods 0.000 abstract description 3
- 238000003756 stirring Methods 0.000 abstract description 2
- 239000011573 trace mineral Substances 0.000 abstract description 2
- 235000013619 trace mineral Nutrition 0.000 abstract description 2
- 229940091258 selenium supplement Drugs 0.000 description 97
- 239000000523 sample Substances 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229910017604 nitric acid Inorganic materials 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000001079 digestive effect Effects 0.000 description 2
- 238000005562 fading Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 2
- -1 potassium ferricyanide Chemical compound 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- PMYDPQQPEAYXKD-UHFFFAOYSA-N 3-hydroxy-n-naphthalen-2-ylnaphthalene-2-carboxamide Chemical compound C1=CC=CC2=CC(NC(=O)C3=CC4=CC=CC=C4C=C3O)=CC=C21 PMYDPQQPEAYXKD-UHFFFAOYSA-N 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 244000062793 Sorghum vulgare Species 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 125000003748 selenium group Chemical group *[Se]* 0.000 description 1
- 229960001881 sodium selenate Drugs 0.000 description 1
- 239000011655 sodium selenate Substances 0.000 description 1
- 235000018716 sodium selenate Nutrition 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
- G01N21/6404—Atomic fluorescence
Landscapes
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Optics & Photonics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention belongs to the technical field of trace element detection, and particularly relates to a method for detecting selenium element in cereals, which comprises the steps of cleaning cereals, airing, crushing, homogenizing at 8000r/min for 20min to prepare a plurality of sample slurries, and directly digesting to obtain the total selenium content; separating organic selenium and inorganic selenium, and respectively digesting and measuring the total amount of the inorganic selenium and the organic selenium; and thirdly, separating the organic selenium on the basis of the second step to obtain selenoprotein and polysaccharide selenium, and digesting and determining the content of the selenoprotein and the polysaccharide selenium respectively. The raw material treatment of the invention adopts a homogenizing method, and the raw materials are easier to be analyzed out in the grains than the traditional crushing, stirring and other modes; and the measured selenium is various and has high precision.
Description
Technical Field
The invention belongs to the technical field of trace element detection, and particularly relates to a method for detecting selenium element in cereals.
Background
The existence of selenium in nature is divided into two categories: inorganic selenium, which generally refers to sodium selenite and sodium selenate, including yeast selenium and malt selenium having a large amount of inorganic selenium residues, obtained from byproducts of metal mineral reserves, has great toxicity and is not easily absorbed and is not suitable for human and animals, and organic selenium, which is formed by combining amino acids, polysaccharides, nucleotides and the like through biotransformation, generally exists in the form of selenoprotein, polysaccharide selenium and the like, and is a selenium source which is allowed to be used by human and animals.
Selenium is added into a plurality of nutritional varieties, the detection method of selenium at present is various and has mature technology, the problem is how to separate selenium in different forms to be a detection problem, application number CN201210458379.1 discloses a detection method for directly measuring organic selenium, protein selenium or polysaccharide selenium in marine products by using a microwave digestion-inductively coupled plasma mass spectrometry (ICP-MS), and the invention has the advantages that: the organic selenium, the protein selenium and the polysaccharide selenium in the marine products can be efficiently, simply and quickly measured; however, the invention can easily remove the small molecular selenium in the separation process, and the chemical reaction separation is excessively used, so that the chemical bond breakage is easily caused, the substance solubility is influenced, and the test result is inaccurate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting selenium, which mainly aims at grains and the like.
The invention is realized by the following technical scheme:
a method for detecting selenium element in cereals comprises cleaning cereals, air drying, pulverizing, homogenizing at 8000r/min for 20min to obtain several sample slurries, and directly digesting to obtain total selenium content; separating organic selenium and inorganic selenium, and respectively digesting and measuring the total amount of the inorganic selenium and the organic selenium; and thirdly, separating the organic selenium on the basis of the second step to obtain selenoprotein and polysaccharide selenium, and digesting and determining the content of the selenoprotein and the polysaccharide selenium respectively.
The digestion method adopts a nitric acid-perchloric acid digestion method in the prior art: putting 1-2g of digestion raw material into a digestion tube, adding 2ml of nitric acid for multiple times, separating for 5 minutes between the two times until the liquid in the digestion tube is transparent and colorless, transferring the digestion tube into an acid remover to remove acid until a small amount of solid is separated out, then cooling, adding 2ml of perchloric acid, adding water to a constant volume of 10ml, heating the sample until the sample is colorless, and cooling the sample.
Further, the method for separating organic selenium and inorganic selenium comprises the following steps:
(1) putting the sample slurry into a dialysis bag, repeatedly dialyzing in distilled water until inorganic selenium cannot be detected in the dialyzed external liquid, collecting all the dialyzed external liquid, and then adding cyclohexane for extraction to obtain a water phase containing inorganic selenium and an organic phase containing water-soluble organic selenium;
(2) mixing organic phase containing water soluble organic selenium, dialyzing the inner solution, concentrating and drying to obtain organic selenium;
(3) separating the water phase containing inorganic selenium by membrane distillation technology to obtain inorganic selenium.
Further, the separation of selenoprotein:
s1, adding 15-25ml of normal hexane into 5g of organic selenium, soaking for 5-8 hours in ultrasonic waves at the frequency of 30-50KHz and the power of 60-90w, and centrifuging to remove supernatant; in the prior art, acetone is generally adopted, but compared with acetone, n-hexane functional groups are fewer, and chemical bond reactions are fewer except the mutual fusion between physical action and non-polarity;
s2, repeating the degreasing of the n-hexane for 1-3 times in the step S1, and degassing the remainder at 70-80 ℃ for 20-40min under the pressure of 0.5-0.7atm to obtain the degreased organic selenium;
s3, adding the degreased organic selenium into a mixed solution of 18ml of Tris-HCl buffer solution and 2ml of glycerol, centrifuging to obtain insoluble non-protein solids, adding ammonium sulfate into the supernatant until the mixture is saturated, mixing uniformly, standing for 4-6h at 4 ℃ for salting out, and cooling and drying the obtained precipitate to obtain the selenoprotein.
The insoluble non-protein solid obtained by centrifugation contains abundant polysaccharide selenium, can be separated in the next step, and has dispersed raw material components in the separation process because the insoluble non-protein solid is subjected to homogenization and ultrasonic waves are adopted in the degreasing process, and the insoluble non-protein solid does not need to be separated in a chemical mode.
Further, separation of polysaccharide selenium: and (4) adding 20ml of water into the non-protein solid in the step S3, carrying out ultrasonic treatment with 20KHz for 3 hours at the power of 6w and the temperature of 80-90 ℃, removing insoluble substances, transferring the mixed solution into a dialysis bag, repeatedly dialyzing in distilled water for 3-6 times for 3-5 hours each time, and finally cooling and drying the dialyzed inner liquid to obtain the polysaccharide selenium.
Polysaccharide selenium is extracted by ultrasound high temperature water, and other inorganic salts, in-process buffers, etc. are removed by dialysis.
The method for detecting the selenium element adopts the atomic fluorescence spectrometry to detect the selenium.
Compared with the prior art, the invention has the following technical advantages:
1. the raw material treatment adopts a homogenizing method, and the components in the grains are easier to be analyzed compared with the traditional crushing, stirring and other modes;
2. the invention has the advantages of various selenium types and high precision.
Detailed Description
The invention is further illustrated by the following examples, which illustrate the processes required to be used in the prior art.
Characterization of inorganic selenium: adding 15% cysteine solution into the solution to be detected, uniformly mixing, adding 1 drop of methylene blue solution, fading inorganic selenium for 30-60s, and fading organic selenium for 5min +.
Determination of selenium by atomic fluorescence spectrometry:
drawing a measurement standard curve: selenium standard stock solution (100 μ g/mL) 100.0mg of elemental selenium (spectrally pure) was accurately weighed, dissolved in a small amount of nitric acid, added with 2mL of perchloric acid, heated in a boiling water bath for 3.5h, cooled, added with 8.4mL of hydrochloric acid, and then boiled in a boiling water bath for 2 min. The diluted solution is accurately diluted to 1000mL, and the hydrochloric acid concentration of the diluted solution is 0.1 mol/L. The stock solution concentration was 100. mu.g/mL. Diluting a proper amount of selenium standard stock solution into solutions of 0 mug/L, 2 mug/L, 4 mug/L, 8 mug/L, 12 mug/L and 20 mug/L, wherein 3mL of the solutions are respectively added with 1.6mL of hydrochloric acid and 0.8mL of 80g/L potassium ferricyanide solution, and then the selenium standard use solution is obtained.
The selenium standard use solution is injected and tested under the following conditions: the negative high pressure is 300V; the lamp current is 90 mA; the atomization temperature is 800 ℃; the furnace height is 9 mm; the flow rate of the carrier gas is 450 mL/min; the shielding airflow rate is 850 mL/min; the injection volume was 1.5 mL. And drawing a standard curve according to the measurement result and establishing a regression equation. The regression equation is: y is 66.32x +6.375 and R2 is 0.999.
Taking 3mL of digestive juice to be detected, adding 1.6mL of hydrochloric acid and 0.8mL of potassium ferricyanide solution of 80g/L, uniformly mixing, measuring, and calculating the content of selenium by using a regression equation. And (4) averaging the two identification data, if the two identification data are greatly different, adding a test sample, and taking an average value of similar results.
X ═ C-C0 × V/m, where: x is the selenium content, mu g/kg; c is the concentration of selenium in digestive juice, mu g/L; c0 is blank sample concentration, μ g/L; v is the volume of the sample with constant volume, mL; m is the sample weight, g. The results are shown in table 1:
a nitric acid-perchloric acid digestion method: putting 1-2g of digestion raw material into a digestion tube, adding 2ml of nitric acid for multiple times, separating for 5 minutes between the two times until the liquid in the digestion tube is transparent and colorless, transferring the digestion tube into an acid remover to remove acid until a small amount of solid is separated out, then cooling, adding 2ml of perchloric acid, adding water to a constant volume of 10ml, heating the sample until the sample is colorless, and cooling the sample.
Sample preparation: cleaning grains, air drying, pulverizing, and homogenizing at 8000r/min for 20min to obtain sample slurry;
first, organic selenium and inorganic selenium are separated
(1) Putting 30g of sample slurry into a dialysis bag, repeatedly dialyzing in distilled water, detecting inorganic selenium in the dialyzed external liquid after 5 times of 120 hours, collecting all the dialyzed external liquid, and then adding cyclohexane for extraction to obtain an inorganic selenium-containing water phase and a water-soluble organic selenium-containing organic phase;
(2) mixing organic phase containing water soluble organic selenium, dialyzing the inner solution, concentrating and drying to obtain organic selenium;
(3) separating the water phase containing inorganic selenium by membrane distillation technology to obtain inorganic selenium.
Various organic forms of selenium were extracted as described in examples 1-3;
example 1 separation of selenoprotein, polysaccharide selenium
And (3) separating selenoprotein:
s1, adding 20ml of n-hexane into 5g of organic selenium, soaking for 6 hours by ultrasonic waves with the frequency of 40KHz and the power of 80w, and centrifuging to remove supernatant;
s2, repeating the n-hexane degreasing step in the S1 step for 2 times, and degassing the remainder at 75 ℃ for 30min under the pressure of 0.6atm to obtain degreased organic selenium;
s3, adding the degreased organic selenium into a mixed solution of 18ml of Tris-HCl buffer solution and 2ml of glycerol, centrifuging to obtain insoluble non-protein solids, adding ammonium sulfate into the supernatant until the mixture is saturated, mixing uniformly, standing for 5 hours at 4 ℃ for salting out, and cooling and drying the obtained precipitate to obtain the selenoprotein.
Separation of polysaccharide selenium
And (4) adding 20ml of water into the non-protein solid in the step S3, carrying out ultrasonic treatment with 20KHz for 3 hours at the power of 6w and the temperature of 85 ℃, removing insoluble substances, transferring the mixed solution into a dialysis bag, repeatedly dialyzing for 5 times in distilled water for 4 hours each time, and finally cooling and drying the dialyzed inner liquid to obtain the polysaccharide selenium.
Example 2 separation of selenoprotein, polysaccharide selenium
And (3) separating selenoprotein:
s1, adding 25ml of n-hexane into 5g of organic selenium, soaking for 5 hours by ultrasonic waves, wherein the frequency is 50KHz, the power is 90w, and centrifuging to remove supernate;
s2, repeating the n-hexane degreasing step in the S1 step for 1 time, and degassing the remainder at 80 ℃ for 20min under the pressure of 0.7atm to obtain degreased organic selenium;
s3, adding the degreased organic selenium into a mixed solution of 18ml of Tris-HCl buffer solution and 2ml of glycerol, centrifuging to obtain insoluble non-protein solids, adding ammonium sulfate into the supernatant until the mixture is saturated, mixing uniformly, standing for 4 hours at 4 ℃ for salting out, and cooling and drying the obtained precipitate to obtain the selenoprotein.
Separation of polysaccharide selenium
And (4) adding 20ml of water into the non-protein solid in the step S3, carrying out ultrasonic treatment with 20KHz for 3 hours at the power of 6w and the temperature of 90 ℃, removing insoluble substances, transferring the mixed solution into a dialysis bag, repeatedly dialyzing in distilled water for 3 times, carrying out 5 hours each time, and finally cooling and drying the dialyzed inner liquid to obtain the polysaccharide selenium.
Example 3 separation of selenoprotein, polysaccharide selenium
And (3) separating selenoprotein:
s1, adding 15ml of n-hexane into 5g of organic selenium, soaking for 8 hours by ultrasonic waves with the frequency of 30KHz and the power of 60w, and centrifuging to remove supernatant;
s2, repeating the n-hexane degreasing step in the S1 step for 3 times, and degassing the remainder at 70 ℃ for 40min under the pressure of 0.5atm to obtain degreased organic selenium;
s3, adding the degreased organic selenium into a mixed solution of 18ml of Tris-HCl buffer solution and 2ml of glycerol, centrifuging to obtain insoluble non-protein solids, adding ammonium sulfate into the supernatant until the mixture is saturated, mixing uniformly, standing for 6 hours at 4 ℃ for salting out, and cooling and drying the obtained precipitate to obtain the selenoprotein.
Separation of polysaccharide selenium
And (4) adding 20ml of water into the non-protein solid in the step S3, carrying out ultrasonic treatment with 20KHz for 3 hours at the power of 6w and the temperature of 80 ℃, removing insoluble substances, transferring the mixed solution into a dialysis bag, repeatedly dialyzing for 6 times in distilled water for 3 hours each time, and finally cooling and drying the dialyzed inner liquid to obtain the polysaccharide selenium.
Comparative example 1 separation of selenoprotein, polysaccharide selenium
The difference from example 1 is that glycerol was replaced with Tris-HCl buffer, and the other steps are completely identical.
Experimental example 1 the results of digestion measurement of inorganic selenium and selenoprotein and polysaccharide selenium obtained in examples 1 to 3 and comparative example 1 are shown in table 1 (μ g/100 g): the test materials adopted in the examples 1-3 and the comparative example 1 are the same raw material (selenium-rich millet), the manufacturer gives data that the total selenium content is about 22-24 mug/100 g, the selenoprotein is about 15-17 mug/100 g, the polysaccharide selenium does not give data, and the inorganic selenium content is less than or equal to 1 mug/100 g.
TABLE 1 selenium content
As can be seen from Table 1, the detection of selenoprotein and polysaccharide selenium is more accurate, and most selenium elements can be detected by the method.
Claims (6)
1. A method for detecting selenium element in grains is characterized in that the grains are cleaned, aired and crushed, then homogenized for 20min at 8000r/min to prepare a plurality of sample slurries, and the total selenium content is directly measured by digestion; separating organic selenium and inorganic selenium, and respectively digesting and measuring the total amount of the inorganic selenium and the organic selenium; and thirdly, separating the organic selenium on the basis of the second step to obtain selenoprotein and polysaccharide selenium, and digesting and determining the content of the selenoprotein and the polysaccharide selenium respectively.
2. The method for detecting elemental selenium in cereals according to claim 1, wherein the digestion method is a nitric acid-perchloric acid digestion method.
3. The method for detecting elemental selenium in a grain according to claim 1, wherein the separating organic selenium and inorganic selenium comprises the steps of:
(1) putting the sample slurry into a dialysis bag, repeatedly dialyzing in distilled water until inorganic selenium cannot be detected in the dialyzed external liquid, collecting all the dialyzed external liquid, and then adding cyclohexane for extraction to obtain a water phase containing inorganic selenium and an organic phase containing water-soluble organic selenium;
(2) mixing organic phase containing water soluble organic selenium, dialyzing the inner solution, concentrating and drying to obtain organic selenium;
(3) separating the water phase containing inorganic selenium by membrane distillation technology to obtain inorganic selenium.
4. The method for detecting elemental selenium in cereals according to claim 1 or 3, wherein the separation of selenoprotein:
s1, adding 15-25ml of normal hexane into 5g of organic selenium, soaking for 5-8 hours in ultrasonic waves at the frequency of 30-50KHz and the power of 60-90w, and centrifuging to remove supernatant;
s2, repeating the degreasing of the n-hexane for 1-3 times in the step S1, and degassing the remainder at 70-80 ℃ for 20-40min under the pressure of 0.5-0.7atm to obtain the degreased organic selenium;
s3, adding the degreased organic selenium into a mixed solution of 18ml of Tris-HCl buffer solution and 2ml of glycerol, centrifuging to obtain insoluble non-protein solids, adding ammonium sulfate into the supernatant until the mixture is saturated, mixing uniformly, standing for 4-6h at 4 ℃ for salting out, and cooling and drying the obtained precipitate to obtain the selenoprotein.
5. The method of claim 4, wherein the separation of polysaccharide selenium comprises: and (4) adding 20ml of water into the non-protein solid in the step S3, carrying out ultrasonic treatment with 20KHz for 3 hours at the power of 6w and the temperature of 80-90 ℃, removing insoluble substances, transferring the mixed solution into a dialysis bag, repeatedly dialyzing in distilled water for 3-6 times for 3-5 hours each time, and finally cooling and drying the dialyzed inner liquid to obtain the polysaccharide selenium.
6. The method of claim 1, wherein the selenium is determined by atomic fluorescence spectrometry.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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