阅读说明：本技术 富硒木耳中硒形态的检测方法 (Method for detecting form of selenium in selenium-rich agaric ) 是由 魏春雁 刘笑笑 宋志峰 张振都 金秋 张奇 马虹 王泽武 于 2020-05-26 设计创作，主要内容包括：本发明提供一种富硒木耳中硒形态的检测方法,包括:步骤1,样品处理：富硒干木耳反复磨碎,以得到木耳粉末；步骤2,样品酶解：称取木耳粉末作为待测样品,装于离心管中,向该离心管中加入浓度不低于0.0033g/ml的蛋白水解酶溶液,以得到样品溶液,其中,待测样品与蛋白水解酶的质量比为1：(40-80)；步骤3,样品提取：将上述样品溶液采取超声波提取,在超声波期间,每间隔预设时间内摇晃所述离心管；步骤4,样品离心：将经过超声波提取的样品溶液离心,以得到上清液；步骤5,过滤：将所述上清液通过滤膜过滤后,得到待测样液；步骤6,仪器测定：对步骤5中得到的待测样液采用高效液相色谱法与氢化物发生-原子荧光光谱法测定样品中的硒形态。(The invention provides a method for detecting the form of selenium in selenium-rich agaric, which comprises the following steps of 1, sample treatment: repeatedly grinding selenium-rich dry Auricularia to obtain Auricularia powder; step 2, sample enzymolysis: weighing agaric powder as a sample to be detected, filling the sample to a centrifuge tube, adding a proteolytic enzyme solution with the concentration not less than 0.0033g/ml into the centrifuge tube to obtain a sample solution, wherein the mass ratio of the sample to be detected to the proteolytic enzyme is 1: (40-80); step 3, sample extraction: extracting the sample solution by adopting ultrasonic waves, and shaking the centrifuge tube at preset time intervals during the ultrasonic wave; and 4, centrifuging the sample: centrifuging the sample solution subjected to ultrasonic extraction to obtain a supernatant; and 5, filtering: filtering the supernatant through a filter membrane to obtain a sample solution to be detected; step 6, measuring by an instrument: and (3) determining the selenium form in the sample by adopting a high performance liquid chromatography and a hydride generation-atomic fluorescence spectrometry for the sample liquid to be detected obtained in the step (5).)

1. A method for detecting the form of selenium in selenium-rich agaric is characterized by comprising the following steps:

step 1, sample treatment: repeatedly grinding selenium-rich dry Auricularia to obtain Auricularia powder;

step 2, sample enzymolysis: weighing agaric powder as a sample to be detected, filling the sample to a centrifuge tube, adding a proteolytic enzyme solution with the concentration not less than 0.0033g/ml into the centrifuge tube to obtain a sample solution, wherein the mass ratio of the sample to be detected to the proteolytic enzyme is 1: (40-80);

step 3, sample extraction: extracting the sample solution by adopting ultrasonic waves, and shaking the centrifuge tube at preset time intervals during the ultrasonic wave;

and 4, centrifuging the sample: centrifuging the sample solution subjected to ultrasonic extraction to obtain a supernatant;

and 5, filtering: filtering the supernatant through a filter membrane to obtain a sample solution to be detected;

step 6, measuring by an instrument: and (3) determining the selenium form in the sample by adopting a high performance liquid chromatography and a hydride generation-atomic fluorescence spectrometry for the sample liquid to be detected obtained in the step (5).

2. The detection method according to claim 1, wherein the detection conditions of the instrument used in the step 6 are as follows:

chromatographic conditions are as follows: hamilton PRP-X100 ion exchange chromatographic column (250mm 4.1mm, 10 μm), mobile phase of 40mmol/L diammonium hydrogen phosphate, pH value adjusted to 5.8-6.0 with 20% formic acid, flow rate of 1.0mL/min, sample injection volume of 100 μ L;

atomic fluorescence spectrum conditions: 300V of negative high voltage, 90mA of total lamp current, 400mL/min of carrier gas flow, 600mL/min of shielding gas flow and 8mm of atomizer height;

hydride generation conditions: potassium iodide 0.4% + potassium hydroxide 0.35% (oxidant), potassium borohydride 2% + potassium hydroxide 0.35% (reductant), hydrochloric acid 7% (current-carrying), and pump speed 80 r/min.

3. The method for detecting according to claim 1, wherein the proteolytic enzyme solution is a streptokinase enzyme solution.

4. The method according to claim 3, wherein the solution of pronase is prepared by dissolving 0.01g of pronase E in 3ml of water.

5. The detection method as claimed in claim 1, wherein in the step 3, the centrifuge tube containing the sample solution is placed in an ultrasonic cleaner, the ultrasonic extraction time is 45min, and the centrifuge tube is shaken every 10 min.

6. The detection method according to claim 5, wherein a water temperature in the washer is set to 37 ℃ at the time of the ultrasonic extraction.

7. The detection method according to claim 1, wherein in the step 4, the sample solution is centrifuged at 8000r/min for 20 min.

8. The detection method according to claim 1, wherein in the step 5, the supernatant is filtered through a 0.45um microporous water system filter.

9. The detection method as claimed in claim 1, wherein in step 1, selenium-enriched dried Auricularia is repeatedly ground and sieved with 80-120 mesh sieve to obtain Auricularia powder.

10. The assay of claim 1, wherein the black fungus powder is black fungus powder.

Technical Field

The invention relates to the technical field of detection, and particularly relates to a method for detecting the form of selenium in selenium-enriched black fungus.

Background

As an essential trace element for human and animal bodies, selenium (Se) is the active center of more than 30 enzymes such as glutathione peroxidase, and has no substitution in the biochemical process of eliminating various oxygen free radicals!

Based on the antioxidant function, selenium may play an important role in the prevention and treatment of various human diseases, such as keshan disease, Kaschin-Beck disease, arthritis; preventing and treating cardiovascular and cerebrovascular diseases and diabetes; enhancing immunity and resisting aging; preventing and resisting cancer, improving the effect of radiotherapy and chemotherapy and reducing the side effect of radiotherapy and chemotherapy; heavy metal toxicity relieving, etc.

As a trace element necessary for human body, the deficiency of selenium (Se) intake brings harm to human health, and the selenium intake deficiency of different degrees exists in 72% of people in China. The American food and nutrition Committee recommends that the average intake of people is 50-200 mug/d; the international society for selenium science recommends that the per-person intake is 60 mug/d; the Chinese Nutrition Association recommends an adult intake of 60 μ g/d (2013). The results of nutritional diet survey conducted by the Chinese Nutrition society on part of cities show that the selenium intake of adults in China is less than 27 mug/d.

As China is a selenium-deficient country, 1094 counties and cities in China have soil selenium background values which are investigated, only 1/3 is reached to reach an internationally recognized critical value (0.1mg/kg), and the content of the selenium is less than or equal to 0.02mg/kg and accounts for 29 percent. From the three provinces of northeast, the selenium-rich region obliquely penetrates to the cloud plateau and is a low-selenium zone, and the selenium-rich region occupies 70% of the territorial area. Jilin province is a particularly severe selenium-deficient area! On the selenium-deficient belt, residents are susceptible to selenium deficiency.

The knowledge of the lack of selenium promotes the development of selenium-enriched food in China. Selenium-rich tea, selenium-rich cereal products, selenium-rich bean products, selenium-rich dairy products, selenium-rich edible fungi, selenium-rich beverages, mineral water and the like. Some are added into food in the processing link in the form of additives, some are food raw materials planted in limited selenium-rich areas, and other areas without selenium-rich are added with inorganic selenium in culture mediums (soil) or are sprayed with selenide on leaf surfaces, so that the effect of selenium-rich agricultural products is achieved. The livestock and poultry product selenium enrichment is to add selenide into the feed or inject selenium-containing medicines.

At present, the selenium-rich agricultural products available on the market are: selenium-rich garlic, selenium-rich rice, selenium-rich wheat, selenium-rich mung bean, selenium-rich potato, selenium-rich peanut, selenium-rich pumpkin, selenium-rich sesame, selenium-rich rapeseed, selenium-rich radish, selenium-rich edible fungi and the like. Selenium-rich agricultural products in our province include selenium-rich rice, selenium-rich agaric, selenium-rich apple and the like.

Because of the general lack of selenium, China abolishes the limit of selenium in all foods such as GB13105-1991 'hygienic Standard for Limited selenium in foods'.

However, the uncontrolled enriched selenium in all foods can cause the selenium intake of Chinese people to be excessive, the safe content of selenium in the body exists in a small range, and the beneficial and toxic effects of selenium mainly depend on the chemical form and content of the selenium, so that the establishment of a selenium form detection method which has high sensitivity, good stability, accuracy and reliability is particularly important.

Disclosure of Invention

In view of the above, the invention provides a method for detecting the form of selenium in selenium-rich black fungus, which has the advantages of high sensitivity, good stability, accuracy and reliability and is an effective method for determining the form content of selenium in the selenium-rich black fungus.

The invention provides a method for detecting the form of selenium in selenium-enriched black fungus, which comprises the following steps:

step 1, sample treatment: repeatedly grinding selenium-rich dry Auricularia to obtain Auricularia powder;

step 2, sample enzymolysis: weighing Auricularia powder as a sample to be detected, placing the sample into a centrifuge tube, adding a proteolytic enzyme solution with the concentration of 0.002-0.004g/ml into the centrifuge tube to obtain a sample solution, wherein the mass ratio of the sample to be detected to the proteolytic enzyme is 1: (40-80), preferably, 1: 60;

step 3, sample extraction: extracting the sample solution by adopting ultrasonic waves, and shaking the centrifuge tube at preset time intervals during the ultrasonic wave;

and 4, centrifuging the sample: centrifuging the sample solution subjected to ultrasonic extraction to obtain a supernatant;

and 5, filtering: filtering the supernatant through a filter membrane to obtain a sample solution to be detected;

step 6, measuring by an instrument: and (3) determining the selenium form in the sample by adopting a high performance liquid chromatography and a hydride generation-atomic fluorescence spectrometry for the sample liquid to be detected obtained in the step (5).

Preferably, the detection conditions of the instrument used in the step 6 are as follows:

chromatographic conditions are as follows: hamilton PRP-X100 ion exchange chromatographic column (250mm 4.1mm, 10 μm), mobile phase of 40mmol/L diammonium hydrogen phosphate, pH value adjusted to 5.8-6.0 with 20% formic acid, flow rate of 1.0mL/min, sample injection volume of 100 μ L;

atomic fluorescence spectrum conditions: 300V of negative high voltage, 90mA of total lamp current, 400mL/min of carrier gas flow, 600mL/min of shielding gas flow and 8mm of atomizer height;

hydride generation conditions: potassium iodide 0.4% + potassium hydroxide 0.35% (oxidant), potassium borohydride 2% + potassium hydroxide 0.35% (reductant), hydrochloric acid 7% (current-carrying), and pump speed 80 r/min. The potassium iodide is used for reducing selenic acid into selenious acid, the potassium borohydride is used for reacting with hydrochloric acid to generate active hydrogen, and selenium is reduced into gaseous hydride, but the potassium borohydride only can react with selenite but not with selenate, so that the potassium iodide is required to be used for detecting the selenic acid, and the concentration of the potassium iodide directly influences the fluorescence intensity of the selenic acid.

Preferably, the proteolytic enzyme solution is a streptokinase protease solution.

Preferably, the solution of the pronase protease is prepared by dissolving 0.01g of pronase protease E in 3ml of water.

Preferably, in the step 3, the centrifuge tube with the sample solution is placed into an ultrasonic cleaner, the ultrasonic extraction time is 45min, and the centrifuge tube is shaken every 10 min.

Preferably, the temperature of water in the washer is set at 37 ℃ at the time of the ultrasonic extraction.

Preferably, in the step 4, the centrifugation conditions of the sample solution are 8000r/min of rotation speed and 20min of centrifugation time.

Preferably, in the step 5, the supernatant is filtered through a 0.45um microporous water system filter membrane.

Preferably, in the step 1, the selenium-rich dried agaric is repeatedly ground and sieved by a sieve of 80-120 meshes to obtain the agaric powder.

Preferably, the black fungus powder is black fungus powder.

The technical scheme of the invention has the following beneficial effects:

according to the method for detecting the selenium form in the selenium-enriched black fungus, a new method for measuring 5 selenium forms in the selenium-enriched black fungus by ultrasonic-assisted enzymatic extraction and atomic fluorescence spectrometry is established. The method completely achieves baseline separation of selenocysteine, methyl-selenocysteine, selenious acid, selenomethionine and selenic acid within 16min, and the detection limit of 5 selenium forms is 0.68 mu g/L of selenocysteine, 0.85 mu g/L of methyl-selenocysteine, 0.39 mu g/L of selenious acid, 1.22 mu g/L of selenomethionine and 23.21 mu g/L of selenic acid; the linear range is 0.9990-0.9999; the method is applied to analysis of the selenium form in the selenium-enriched black fungus, and the standard addition recovery rate is 79.78-105.26%; the detection method has high sensitivity, good stability, accuracy and reliability, and is an effective method for determining the selenium form content in the selenium-enriched black fungus.

Drawings

FIG. 1 is a diagram showing the state of the Auricularia powder with different material-to-liquid ratios before adding the aqueous solution;

FIG. 2 is a diagram showing the swelling state of Auricularia powders after adding aqueous solutions to Auricularia powders with different feed-liquid ratios;

FIG. 3 is a diagram showing the swelling state of Auricularia powders after adding aqueous solutions to Auricularia powders with different feed-liquid ratios;

FIG. 4 is a diagram showing the state of soaking and foaming of Auricularia powders at an interval of 15min after adding the aqueous solution into Auricularia powders with different feed-liquid ratios;

FIG. 5 is a diagram showing the state of soaking and foaming of Auricularia powders at an interval of 15min after adding the aqueous solution into Auricularia powders with different feed-liquid ratios;

FIG. 6 is a diagram showing the state of soaking and foaming of Auricularia powders at 30min intervals after adding the aqueous solution into Auricularia powders with different feed-liquid ratios;

FIG. 7 is a diagram showing the state of soaking and foaming of Auricularia powders at 30min intervals after adding the aqueous solution into Auricularia powders with different feed-liquid ratios;

FIG. 8 is a diagram showing the material-to-liquid ratio of 1/10 edible fungus powder in a foaming state;

FIG. 9 is a diagram showing the material-to-liquid ratio of 1/20 edible fungus powder in a foaming state;

FIG. 10 is a diagram showing the state of soaking and foaming of Auricularia powders at 45min intervals after adding the aqueous solution to Auricularia powders of different feed-liquid ratios;

FIG. 11 is a graph showing the ratio of feed to liquid after centrifugation of the sample solution of the present invention;

FIG. 12 is an external standard calibration Doppler plot of the flow curve for 5 concentration gradients of the inventive mixed standard solution;

FIG. 13 is a standard graph of selenocysteine according to the present invention;

FIG. 14 is a standard curve of methyl-selenocysteine according to the present invention;

FIG. 15 is a graph of selenious acid standard curve according to the present invention;

FIG. 16 is a standard curve of selenomethionine according to the invention;

fig. 17 is a selenic acid standard curve diagram of the present invention.

Detailed Description

The technical solution of the present application is specifically described below by way of examples. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The black fungus is rich in selenium, and the conventional detection of selenium form is not suitable for the detection of the selenium in the black fungus due to the high expansion rate and high viscosity of the black fungus in the soaking process.

The invention relates to a novel method for measuring 5 selenium forms in black fungus rich in selenium by utilizing ultrasonic-assisted enzymatic extraction-atomic fluorescence spectrometry. The method specifically comprises the following steps: processing a sample, performing enzymolysis, extracting, centrifuging, filtering, measuring and the like.

The method for detecting the form of selenium in the selenium-enriched black fungus provided by the embodiment of the invention is described below by combining specific embodiments.

Chemical agents of the invention;

93.5. mu.g/g selenocysteine solution (Selenocystine, Se-Cys), GBW 10087: the national institute of metrology science.

96.6 μ g/g methyl-selenocysteine solution (Se-methylselenocystein, MSeC), GBW 10088: the national institute of metrology science.

68.9 μ g/g selenious acid root solution (SeO32-), GBW 10032: the national institute of metrology science.

97.9. mu.g/g Selenomethionine solution (Selenomethionine, Se-Met), GBW 10034: (ii) a The national institute of metrology science.

75.1. mu.g/g selenate solution (SeO42-), GBW 10033: the national institute of metrology science.

Streptoase protease E (cat. nop8360) biochemical reagent, Solarbio life science.

Papain (CAS:9001-73-4), Solambio Life sciences Inc.

Potassium hydroxide (KOH): super pure, shinning-repairing fine chemical research institute of Tianjin.

Potassium borohydride (KBH 4): analytically pure, the institute of optometric and fine chemical engineering in Tianjin.

Diamine hydrogen phosphate ((NH4)2HPO 4): super pure, shinning-repairing fine chemical research institute of Tianjin.

Tetrabutylammonium bromide (C16H36 BrN): analytically pure, mcolin.

Methanol (CH3 OH): pure chromatography, Saimer Feishell science Co.

Hydrochloric acid (HCL): guaranteed reagent, Shi Gao Chun chemical Co., Ltd.

Potassium iodide (KI): pure analysis, Tianjin chemical reagent factory.

Formic acid (HCOOH): analytically pure, the institute of optometric and fine chemical engineering in Tianjin.

The experimental water is laboratory ultrapure water.

The apparatus and equipment of the invention:

SA-10 atomic fluorescence morphology analyzer: beijing Gitian Instrument Co., Ltd;

selenium atom fluorescent hollow cathode lamp: beijing institute for nonferrous metals;

KQ-500DE numerical control ultrasonic cleaner: kunshan ultrasonic instruments, Inc.;

BT124S electronic balance;

multifuge x 3R high speed refrigerated centrifuge: sammer Feishel technologies, Inc.;

the reagents and starting materials used in the present invention are commercially available.

The processes not mentioned in the preparation process of the present invention are all conventional processes.