A method for detecting organic selenium content in high-selenium corn germ powder

Abstract

The invention relates to the technical field related to organic selenium detection, and discloses a method for detecting the organic selenium content of high-selenium corn germ powder. The method comprises the following steps: S1: sample crushing, S2: adding an enzymatic hydrolysis buffer into a centrifuge tube, then adding trypsin and proteinase K, mixing and performing ultrasonic enzymatic hydrolysis extraction, S3: centrifuging the sample after extraction and taking a supernatant, S4: using a chromatography-mass spectrometry system and an atomic fluorescence photometer to determine the total selenium content in the supernatant, and calculating the organic selenium content by an improved difference subtraction method. The invention can significantly improve the extraction rate of organic selenium in a sample by using an optimized buffer, enzyme dosage and ultrasonic conditions in the enzymatic hydrolysis process, and combining EDTA and sodium thiosulfate as a protective agent and a chelating agent. These measures effectively reduce selenium oxidation and interference of heavy metal ions in the sample during the treatment process, and ensure the integrity and stability of the sample.

Description

Technical Field

[0001] The invention belongs to the technical field related to organic selenium detection, and more specifically, particularly relates to a method for detecting the organic selenium content of high-selenium corn germ powder. Background Art

[0002] High-selenium corn germ powder is a functional food ingredient rich in selenium. It has gradually attracted widespread attention due to its good antioxidant, immune regulation and other health benefits. Selenium is an essential trace element, and moderate intake is of great significance to human health. High-selenium corn germ powder has been widely used in the field of food and health products due to its rich nutrients and high selenium content.

[0003] However, in the prior art, there are still some problems with the detection methods of organic selenium in high-selenium corn germ powder. Conventional detection methods, such as atomic fluorescence spectrometry and chromatographic analysis technology, although highly sensitive, are often insufficient in accuracy and repeatability when faced with complex matrices. In addition, during the sample processing process, selenium oxidation, interference from heavy metal ions, and low extraction efficiency of organic selenium will all have an adverse effect on the final test results. These problems make the detection of organic selenium in high-selenium corn germ powder face many challenges. It is urgent to develop an efficient and accurate detection method to ensure the reliability of the test results, so as to give full play to the health benefits of high-selenium corn germ powder.

[0004] The present invention aims to solve the above problems. By optimizing the enzymatic hydrolysis, centrifugation, precipitation and detection steps, an efficient method for detecting the organic selenium content in high-selenium corn germ powder is proposed to improve the detection accuracy and repeatability and ensure the accurate determination of organic selenium in complex matrices. Summary of the invention

[0005] The invention provides a method for detecting the organic selenium content of high-selenium corn germ powder, which is used to overcome the above-mentioned defects in the prior art.

[0006] The purpose and efficacy of the method for detecting the organic selenium content of high-selenium corn germ powder of the present invention are achieved by the following specific technical means:

[0007] A method for detecting the organic selenium content of high-selenium corn germ powder comprises the following steps:

[0008] S1: Grind the sample to be tested into powder, weigh 0.5-1g and put it into a centrifuge tube with a lid;

[0009] S2: Add enzymatic hydrolysis buffer to the centrifuge tube, then add trypsin and proteinase K, mix well and perform ultrasonic enzymatic hydrolysis extraction. The extraction process is as follows:

[0010] S21: Selection of enzymatic buffer: Use tris(hydroxymethyl)aminomethane-HCl buffer solution with a concentration of 0.03-0.06 mol / L and a pH value controlled between 7.2 and 7.5 to maintain optimal enzyme activity;

[0011] S22: Enzyme type and dosage: The dosage of trypsin is 50-100 mg, and the dosage of proteinase K is 8-15 mg to ensure efficient decomposition of protein;

[0012] S23: Ultrasonic conditions: the extraction temperature is controlled at 40-50°C, the extraction time is 1.5-2 hours, and the ultrasonic power is 200-250W to improve the enzymatic hydrolysis efficiency;

[0013] S24: Sodium thiosulfate was added as a protective agent to prevent oxidation and loss of selenium in the sample;

[0014] S25: During the enzymatic hydrolysis process, EDTA (disodium ethylenediaminetetraacetic acid) was added as a chelating agent at a dosage of 0.01-0.05 mol / L to remove possible interference from heavy metal ions in the sample;

[0015] S3: After extraction, the sample is centrifuged at a speed of 10,000 to 12,000 rpm for 20 to 30 minutes, with the temperature maintained below 4°C, and the supernatant is collected;

[0016] S4: Determine the total selenium content in the supernatant using a chromatography-mass spectrometry system and an atomic fluorescence spectrometer, and calculate the organic selenium content by an improved subtraction method. The specific steps are as follows:

[0017] S41: pretreating the supernatant with a specific selective precipitant (e.g. sodium phosphate or silver nitrate) to precipitate and filter out inorganic selenium;

[0018] S42: converting the organic selenium in the filtrate into a hydride form, and detecting it by an atomic fluorescence spectrophotometer, and the signal after eliminating interference is the organic selenium content;

[0019] S43: Calibration is performed by combining the external standard method with the internal standard method to ensure the accuracy and sensitivity of the test results.

[0020] According to a further technical solution, during the enzymatic hydrolysis process, the ultrasonic conditions include intermittent ultrasonic extraction, with each ultrasonic extraction lasting 30 minutes, an interval of 10 minutes, and three cycles to further improve the enzymatic hydrolysis efficiency and the extraction rate of organic selenium.

[0021] A further technical solution is to add silver nitrate as a selective precipitant during the centrifugation process at a concentration of 0.1 to 0.2 mol / L, filter through a 0.22 μm microporous filter membrane after pretreatment, and add 0.5 to 2 times the volume of the supernatant methanol to remove inorganic selenium and improve the purity of the filtrate.

[0022] According to a further technical solution, in step S4, the standard selenium forms of the external standard method are selenocystine, selenomethionine and selenocysteine, and the calibration substance of the internal standard method is methylselenocysteine, so as to improve the stability and repeatability of the test results.

[0023] As a further technical solution, the sample is pre-cooled with liquid nitrogen before enzymatic hydrolysis, with a freezing temperature of ~196°C and a freezing time of 15 minutes. During the pulverization process, a double-layer heat insulation device is used to keep warm to prevent the volatilization of organic selenium due to temperature fluctuations.

[0024] In a further technical solution, the enzymatic hydrolysis buffer further includes 1% to 2% glycerol as a stabilizer to protect enzyme activity.

[0025] According to a further technical solution, in the chromatography-mass spectrometry system, the mobile phase is methanol and diammonium citrate buffer, the volume fraction of methanol is 2%, and the concentration of the buffer is 5 to 10 mmol / L.

[0026] According to a further technical solution, the standard selenium forms used in the external standard method include selenocysteine, methylselenocysteine ​​and selenomethionine, and the concentrations of the standard solutions are 0, 20, 50 and 100 ug / L.

[0027] As a further technical solution, the chromatographic column used in the chromatography-mass spectrometry analysis is a C18 chromatographic column with a size of 2.1 mm x 50 mm and a particle size of 1.7 μm, so as to achieve efficient separation of selenium compounds.

[0028] According to a further technical solution, the sample is filtered using a 0.22 μm aqueous filter membrane before chromatography-mass spectrometry analysis to remove possible particulate impurities and prevent clogging of the chromatographic column.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] By using optimized buffer, enzyme dosage and ultrasonic conditions during the enzymatic hydrolysis process, and combining EDTA and sodium thiosulfate as protective agents and chelating agents, the extraction rate of organic selenium in the sample can be significantly improved. These measures effectively reduce selenium oxidation and interference from heavy metal ions during sample processing, ensuring the integrity and stability of the sample. Finally, combined with the use of subtraction and internal and external standard methods, the precision and accuracy of the detection are greatly improved, so that reliable organic selenium content data can be obtained even in complex matrices.

[0031] This method has a simple operation process and is easy to implement while maintaining high sensitivity and high precision. The optimization of ultrasonic enzymatic hydrolysis, reasonable centrifugation treatment and mature atomic fluorescence photometer detection technology make this method widely used in laboratory conditions for the detection of organic selenium in different types of selenium-rich foods and their products. In addition, the use of selective precipitation and hydride form conversion steps effectively avoids the matrix interference problem in traditional methods, making the detection process more suitable for the analysis of various complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0034] Figure 1 It is a schematic diagram of the overall steps of the present invention. DETAILED DESCRIPTION

[0035] The following is a further detailed description of the embodiments of the present invention in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0036] In the description of the present invention, unless otherwise specified, "plurality" means two or more than two; the orientations or positional relationships indicated by the terms "upper", "lower", "left", "right", "inner", "outer", "front end", "rear end", "head", "tail", etc. are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", "third", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0037] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0038] A method for detecting the organic selenium content of high-selenium corn germ powder comprises the following steps:

[0039] S1: Grind the sample to be tested into powder, weigh 0.5-1g and put it into a centrifuge tube with a lid;

[0040] S2: Add enzymatic hydrolysis buffer to the centrifuge tube, then add trypsin and proteinase K, mix well and perform ultrasonic enzymatic hydrolysis extraction. The extraction process is as follows:

[0041] S21: Selection of enzymatic buffer: Use tris(hydroxymethyl)aminomethane-HCl buffer solution with a concentration of 0.03-0.06 mol / L and a pH value controlled between 7.2 and 7.5 to maintain optimal enzyme activity;

[0042] S22: Enzyme type and dosage: The dosage of trypsin is 50-100 mg, and the dosage of proteinase K is 8-15 mg to ensure efficient decomposition of protein;

[0043] S23: Ultrasonic conditions: the extraction temperature is controlled at 40-50°C, the extraction time is 1.5-2 hours, and the ultrasonic power is 200-250W to improve the enzymatic hydrolysis efficiency;

[0044] S24: Sodium thiosulfate was added as a protective agent to prevent oxidation and loss of selenium in the sample;

[0045] S25: During the enzymatic hydrolysis process, EDTA (disodium ethylenediaminetetraacetic acid) was added as a chelating agent at a dosage of 0.01-0.05 mol / L to remove possible interference from heavy metal ions in the sample;

[0046] S3: After extraction, the sample is centrifuged at a speed of 10,000 to 12,000 rpm for 20 to 30 minutes, with the temperature maintained below 4°C, and the supernatant is collected;

[0047] S4: Determine the total selenium content in the supernatant using a chromatography-mass spectrometry system and an atomic fluorescence spectrometer, and calculate the organic selenium content by an improved subtraction method. The specific steps are as follows:

[0048] S41: pretreating the supernatant with a specific selective precipitant (e.g. sodium phosphate or silver nitrate) to precipitate and filter out inorganic selenium;

[0049] S42: converting the organic selenium in the filtrate into a hydride form, and detecting it by an atomic fluorescence spectrophotometer, and the signal after eliminating interference is the organic selenium content;

[0050] S43: Calibration is performed by combining the external standard method with the internal standard method to ensure the accuracy and sensitivity of the test results.

[0051] Preferably, during the enzymatic hydrolysis process, the ultrasonic conditions include intermittent ultrasonic extraction, each ultrasonic extraction lasts 30 minutes, with an interval of 10 minutes, and is cycled 3 times to further improve the enzymatic hydrolysis efficiency and the extraction rate of organic selenium.

[0052] Preferably, during the centrifugation process, silver nitrate is added as a selective precipitant at a concentration of 0.1 to 0.2 mol / L, filtered through a 0.22 μm microporous filter membrane after pretreatment, and methanol 0.5 to 2 times the volume of the supernatant is added to remove inorganic selenium and improve the purity of the filtrate.

[0053] Preferably, in step S4, the standard selenium forms of the external standard method are selenocysteine, selenomethionine and selenocysteine, and the calibration substance of the internal standard method is methylselenocysteine, so as to improve the stability and repeatability of the detection results.

[0054] Preferably, the sample is pre-cooled with liquid nitrogen before enzymatic hydrolysis, the freezing temperature is ~196°C, the freezing time is 15 minutes, and a double-layer insulation device is used for heat preservation during the pulverization process to prevent the organic selenium from volatilizing due to temperature fluctuations.

[0055] Preferably, the enzymatic hydrolysis buffer further comprises 1% to 2% glycerol as a stabilizer to protect the enzyme activity.

[0056] Preferably, in the chromatography-mass spectrometry system, the mobile phase is methanol and diammonium citrate buffer, the volume fraction of methanol is 2%, and the concentration of the buffer is 5 to 10 mmol / L.

[0057] Preferably, the standard selenium forms used in the external standard method include selenocysteine, methylselenocysteine ​​and selenomethionine, and the concentrations of the standard solutions are 0, 20, 50, and 100 ug / L.

[0058] Preferably, the chromatographic column used in the chromatography-mass spectrometry analysis is a C18 chromatographic column with a size of 2.1 mm x 50 mm and a particle size of 1.7 μm, so as to achieve efficient separation of selenium compounds.

[0059] Preferably, the sample is filtered using a 0.22 μm aqueous filter membrane before chromatography-mass spectrometry analysis to remove possible particulate impurities and prevent clogging of the chromatographic column. Embodiment 1:

[0060] Sample treatment: Weigh 0.8g of high-selenium corn germ powder, add 25mL of pH=7.3 buffer, and add 80mg of trypsin and 12mg of proteinase K. Perform ultrasonic enzymolysis at 45℃ for 1.5 hours (power 220W), add 0.03% sodium thiosulfate and 0.03mol / L EDTA. After centrifugation, add an equal volume of methanol to the supernatant, centrifuge again and test.

[0061] After testing 1: the organic selenium content is 12.5μg / g.

[0062] Embodiment 2:

[0063] Sample treatment: Weigh 1.0g sample, precool in liquid nitrogen for 15 minutes and then crush. Add 30mL pH=7.4 buffer, 70mg trypsin, and 10mg proteinase K. Ultrasonicate for 1.8 hours (power 250W), add 0.05% sodium thiosulfate and 0.04mol / LEDTA. After centrifugation, add 1.5 times the volume of methanol to the supernatant, centrifuge again and detect.

[0064] After test 2: the organic selenium content is 13.2μg / g.

[0065] Embodiment 3:

[0066] Sample treatment: Weigh 1.2g of sample, freeze in liquid nitrogen for 10 minutes and then crush. Add 20mL pH=7.2 buffer and 100mg trypsin and 15mg proteinase K. Perform ultrasonic enzymolysis at 40℃ for 2 hours (power 240W), add 0.04% sodium thiosulfate and 0.02mol / L EDTA. Add 0.8 times volume of methanol to the supernatant after centrifugation, centrifuge again and detect.

[0067] After test 3: the organic selenium content is 11.8μg / g.

[0068] Comparative Example 1:

[0069] Sample treatment: Weigh 1.0g sample, crush directly without liquid nitrogen freezing. Add 25mL pH=7.3 buffer, use 50mg trypsin and 8mg proteinase K. Ultrasonic enzymatic hydrolysis at 40℃ for 1.5 hours (power 200W), no protective agent added. Detect directly after centrifugation.

[0070] Result 1: The organic selenium content is 8.7μg / g.

[0071] Comparative Example 2:

[0072] Sample treatment: 0.8 g of sample was weighed, and no methanol was added. The enzymatic hydrolysis process was the same as in Example 1, and the sample was directly tested after centrifugation.

[0073] Result 2: The organic selenium content is 9.5μg / g.

[0074] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A method for detecting the organic selenium content of high-selenium corn germ powder, characterized in that: S1: Grind the sample to be tested into powder, weigh 0.5-1g and put it into a centrifuge tube with a lid; S2: Add enzymatic hydrolysis buffer to the centrifuge tube, then add trypsin and proteinase K, mix well and perform ultrasonic enzymatic hydrolysis extraction. The extraction process is as follows: S21: Selection of enzymatic buffer: use tris(hydroxymethyl)aminomethane-HCl buffer solution with a concentration of 0.03-0.06 mol / L and a pH value controlled between 7.2 and 7.5 to maintain optimal enzyme activity; S22: Enzyme type and dosage: The dosage of trypsin is 50-100 mg, and the dosage of proteinase K is 8-15 mg to ensure efficient decomposition of protein; S23: Ultrasonic conditions: the extraction temperature is controlled at 40-50°C, the extraction time is 1.5-2 hours, and the ultrasonic power is 200-250W to improve the enzymatic hydrolysis efficiency; S24: Sodium thiosulfate was added as a protective agent to prevent oxidation and loss of selenium in the sample; S25: During the enzymatic hydrolysis process, EDTA was added as a chelating agent at a dosage of 0.01-0.05 mol / L to remove possible interference from heavy metal ions in the sample; S3: After extraction, the sample is centrifuged at a speed of 10,000 to 12,000 rpm for 20 to 30 minutes, with the temperature maintained below 4°C, and the supernatant is collected; S4: Determine the total selenium content in the supernatant using a chromatography-mass spectrometry system and an atomic fluorescence spectrometer, and calculate the organic selenium content by an improved subtraction method. The specific steps are as follows: S41: adding silver nitrate as a selective precipitant at a concentration of 0.1 to 0.2 mol / L, filtering through a 0.22 μm microporous filter membrane after pretreatment, and adding 0.5 to 2 times the volume of the supernatant with methanol to remove inorganic selenium and improve the purity of the filtrate; S42: converting the organic selenium in the filtrate into a hydride form, and detecting it by an atomic fluorescence spectrophotometer, and the signal after eliminating interference is the organic selenium content; S43: Calibration is performed by combining external standard method with internal standard method to ensure the accuracy and sensitivity of the test results; During the enzymatic hydrolysis process, the ultrasonic conditions included intermittent ultrasonic extraction, with each ultrasonic extraction lasting 30 minutes, with an interval of 10 minutes, and three cycles to further improve the enzymatic hydrolysis efficiency and the extraction rate of organic selenium; In step S43, the standard selenium forms of the external standard method are selenocystine, selenomethionine and selenocysteine, and the calibration substance of the internal standard method is methylselenocysteine, so as to improve the stability and repeatability of the test results; The enzymatic hydrolysis buffer also includes 1% to 2% glycerol as a stabilizer to protect enzyme activity; The samples were precooled with liquid nitrogen before enzymatic hydrolysis, with a freezing temperature of ~196°C and a freezing time of 15 minutes. During the pulverization process, a double-layer heat insulation device was used to prevent the volatilization of organic selenium due to temperature fluctuations. In the chromatography-mass spectrometry system, the mobile phase is methanol and diammonium citrate buffer, the volume fraction of methanol is 2%, and the concentration of the buffer is 5-10 mmol / L; The standard selenium forms used in the external standard method include selenocysteine, methylselenocysteine ​​and selenomethionine, and the concentrations of the standard solutions are 0, 20, 50 and 100ug / L; The chromatographic column used in the chromatography-mass spectrometry analysis was a C18 column with dimensions of 2.1 mm x 50 mm and a particle size of 1.7 μm to achieve efficient separation of selenium compounds; Before chromatography-mass spectrometry analysis, the samples were filtered using a 0.22 μm aqueous filter membrane to remove possible particulate impurities and prevent clogging of the chromatographic column.

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