CN111257462A - Method for detecting selenium compound in tea - Google Patents

Method for detecting selenium compound in tea Download PDF

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CN111257462A
CN111257462A CN202010115541.4A CN202010115541A CN111257462A CN 111257462 A CN111257462 A CN 111257462A CN 202010115541 A CN202010115541 A CN 202010115541A CN 111257462 A CN111257462 A CN 111257462A
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solution
tea
polypropylene
centrifugal tube
detecting
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陈孟君
严国军
杨钦沾
陆冬萍
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Zijin Tianyuan Tea Farmers' Professional Cooperative
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/146—Preparation by elimination of some components using membranes

Abstract

A method for detecting selenium compounds in tea comprises: taking 1 unit of tea sample, and placing the tea sample in a polypropylene centrifugal tube; adding a mixed enzyme reagent into the polypropylene centrifugal tube; adding water into the polypropylene centrifugal tube to a constant volume; ultrasonically extracting the solution of the polypropylene centrifugal tube at the temperature of 30-40 ℃ for 25-35 min, and oscillating the polypropylene centrifugal tube 1-3 times in the process; centrifuging the solution in the polypropylene centrifugal tube at a rotating speed of 3000-4000 r/min for 3-5 min; taking the supernatant in the polypropylene centrifuge tube, and filtering the supernatant through a filter membrane with the aperture of 0.22 mu m or 0.45 mu m to obtain a solution to be detected; separating the liquid to be detected by a plasma mass spectrometer to obtain a mixture solution of selenious acid root, selenate, selenocysteine, methylselenocysteine and selenomethionine, and detecting the mixture solution. According to the method for detecting the selenium compounds in the tea, the mixture solution containing the selenium organic matters can be fully extracted, and the content of various organic selenium compounds in the tea can be qualitatively and quantitatively detected.

Description

Method for detecting selenium compound in tea
Technical Field
The application relates to the technical field of component detection, in particular to a method for detecting selenium compounds in tea and a preparation method thereof.
Background
Selenium is a nutrient element which is necessary for human bodies and cannot be synthesized by the human bodies, and the biological functions of the selenium mainly comprise: improving immunity, resisting oxidation, delaying aging, protecting and repairing cells, preventing and resisting cancer, removing toxic substances from heavy metals, and protecting eyes. Therefore, the reasonable intake of selenium element has important effects on human health and prevention and treatment of diseases.
Selenium-enriched tea is a common selenium-enriched product in the market at present. The total selenium compounds in tea can be divided into inorganic selenium and organic selenium, wherein the inorganic selenium comprises selenite and selenate compounds; there are hundreds of organic selenium compounds, and 3 kinds of organic selenium compounds are commonly used, including selenocysteine, methylselenocysteine, selenomethionine, and the like. The functional role of selenium in human health depends on its total content and more on its chemical form. However, at present, much research on the detection of selenium in tea leaves only remains on the detection of total selenium and inorganic chemicals such as selenite and selenate, and no detection method related to the specific chemical form of organic chemicals containing selenium exists, and in the prior art, the content of inorganic selenium in tea leaves is detected first, and then the total content of organic selenium is calculated by using a differential subtraction method, so that the respective contents of various organic selenium compounds such as selenocysteine, methylselenocysteine and selenomethionine cannot be directly measured.
Disclosure of Invention
Based on this, it is needed to provide a method for detecting selenium compounds in tea to solve the deficiencies of the prior art.
A method for detecting selenium compounds in tea leaves comprises the following steps:
taking 1 unit of tea sample, and placing the tea sample in a polypropylene centrifugal tube;
adding a mixed enzyme reagent into the polypropylene centrifugal tube, wherein the mixed enzyme reagent comprises any combination of one to three of a neutral protease solution, a pronase protease solution and a proteinase k solution;
adding water into the polypropylene centrifugal tube to a constant volume;
ultrasonically extracting the solution of the polypropylene centrifugal tube at the temperature of 30-40 ℃ for 25-35 min, and oscillating the polypropylene centrifugal tube 1-3 times in the process;
centrifuging the solution in the polypropylene centrifugal tube at a rotating speed of 3000-4000 r/min for 3-5 min;
taking the supernatant in the polypropylene centrifuge tube, and filtering the supernatant through a filter membrane with the aperture of 0.22 mu m or 0.45 mu m to obtain a solution to be detected;
separating the liquid to be detected by a plasma mass spectrometer to obtain a mixture solution of selenious acid root, selenate, selenocysteine, methylselenocysteine and selenomethionine, and detecting the mixture solution.
Preferably, the enzyme amount of the neutral protease solution is: 30-60 mg, the enzyme amount of the pronase solution is 3-10 mg, and the enzyme amount of the proteinase k solution is 4-10 mg.
Preferably, the ultrasonic extraction of the solution of the polypropylene centrifuge tube at the temperature of 30-40 ℃ for 25-35 min, during which the polypropylene centrifuge tube is vibrated for 1-3 times, comprises:
and ultrasonically extracting the solution of the polypropylene centrifugal tube by using an ultrasonic cleaner at the temperature of 30-40 ℃ for 25-35 min, and vibrating the polypropylene centrifugal tube for 1-3 times in the process.
Preferably, the taking of 1 unit tea leaf sample is carried out in a polypropylene centrifuge tube, which comprises:
grinding the tea sample into powder, mixing uniformly, preparing into a sample to be detected, and placing into a polypropylene centrifuge tube.
Preferably, the plasma mass spectrometer is a high performance liquid chromatography series inductively coupled plasma mass spectrometer.
Preferably, the plasma mass spectrometer uses a citric acid solution as the mobile phase of the detection solution, the concentration of the citric acid solution is 10mmol/L, the pH value of the citric acid solution is 4.5, and the flow rate of the citric acid solution is 1.0 mL/min.
Compared with the prior art, the method has the following beneficial effects:
the method for detecting the selenium compound in the tea takes the mixed enzyme reagent as an extracting agent, is assisted by operating means of ultrasonic extraction, heating, oscillation, centrifugation and filtration, can sufficiently extract a mixture solution of selenium-containing organic matters, and then separates and detects the mixture solution by using a plasma mass spectrometer, so that the content of various organic selenium compounds such as selenocysteine, methyl selenocysteine, selenomethionine and the like in the tea can be qualitatively and quantitatively detected.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
Fig. 1 is a schematic flow chart of a preparation method of a method for detecting selenium compounds in tea according to an embodiment of the present application.
Detailed Description
To facilitate an understanding of the present application, the present application will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present application are given in the accompanying drawings. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like as used herein are for illustrative purposes only and do not represent the only embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
A method for detecting selenium compounds in tea leaves comprises the following steps:
taking 1 unit of tea sample, and placing the tea sample in a polypropylene centrifugal tube;
adding a mixed enzyme reagent into the polypropylene centrifugal tube, wherein the mixed enzyme reagent comprises any combination of one to three of a neutral protease solution, a pronase protease solution and a proteinase k solution;
adding water into the polypropylene centrifugal tube to a constant volume;
ultrasonically extracting the solution of the polypropylene centrifugal tube at the temperature of 30-40 ℃ for 25-35 min, and oscillating the polypropylene centrifugal tube 1-3 times in the process;
centrifuging the solution in the polypropylene centrifugal tube at a rotating speed of 3000-4000 r/min for 3-5 min;
taking the supernatant in the polypropylene centrifuge tube, and filtering the supernatant through a filter membrane with the aperture of 0.22 mu m or 0.45 mu m to obtain a solution to be detected;
separating the liquid to be detected by a plasma mass spectrometer to obtain a mixture solution of selenious acid root, selenate, selenocysteine, methylselenocysteine and selenomethionine, and detecting the mixture solution.
Example 1, the enzyme amount of the neutral protease solution was: 30-60 mg, the enzyme amount of the pronase solution is 3-10 mg, and the enzyme amount of the proteinase k solution is 4-10 mg.
In particular, proteinase K is a potent proteolytic enzyme isolated from Candida albicans, has high specific activity, and is a key reagent for DNA extraction. Pronase is a mixture of serine enzyme and acid protease isolated from Streptococcus (Streptomyces griseus). The neutral protease is obtained by fermenting and extracting bacillus subtilis, belongs to an endonuclease, and can be used for various protein hydrolysis treatments. At a certain temperature and pH value, macromolecular protein can be hydrolyzed into amino acid and other products.
The mixed enzyme reagent can extract 2-4 forms of selenium. The combined use of different proteases can lead to different forms of selenium extraction and different extraction rates.
Preferably, the concentration of the neutral protease solution is: 20mg/mL, the concentration of the pronase solution is 10mg/mL, and the concentration of the proteinase k solution is 10 mg/mL.
Further, 1.0g of tea leaf sample is placed in a 50ml polypropylene centrifuge tube, mixed enzyme reagent with the weight of 50mg is added, water is added to fix the volume to 20ml, ultrasonic extraction is carried out for 30min in the environment of 37 ℃, oscillation is carried out for 2 times during the period, each time is carried out for 1min, the solution in the polypropylene centrifuge tube is centrifuged for 4min at the rotating speed of 4000r/min, aqueous solution with obvious layering effect is obtained, supernatant liquid is taken, the liquid to be detected is obtained by filtering through a filter membrane with the aperture of 0.45 mu m, then a plasma mass spectrometer is used for separating and detecting the liquid to be detected, and the content of total selenium and each selenium-containing compound is obtained, wherein each selenium-containing compound comprises selenocysteine (SeCys2), methylselenocysteine (SeMeCys), selenite (SeIV), selenomethionine (SeMet) and selenate (Se VI). The plasma mass spectrometer uses a citric acid solution as a mobile phase of the detection solution, the concentration of the citric acid solution is 10mmol/L, the pH value of the citric acid solution is 4.5, and the flow rate of the citric acid solution is 1.0 mL/min.
Example 1a, the mixed enzyme reagent was completely neutral protease solution, extracted selenocysteine content 0.304mg/Kg, selenite content 0.083mg/Kg, selenomethionine content 0.102mg/Kg, selenate content 0.110mg/Kg, total selenium content 0.748mg/Kg, and extraction rate 80.08%.
In example 1b, the mixed enzyme reagent was pronase solution, and the extracted selenomethionine content was 0.227mg/Kg, selenate content was 0.308mg/Kg, total selenium content was 0.748mg/Kg, and the extraction rate was 71.52%.
In example 1c, the mixed enzyme reagent was completely proteinase k solution, extracted selenomethionine content 0.146mg/Kg, selenate content 0.291mg/Kg, total selenium content 0.748mg/Kg, and extraction rate 58.42%.
Example 1d, the enzyme reagent was a mixture of 40mg neutral protease solution and 10mg proteinase K solution, extracted selenocysteine content 0.197mg/Kg, selenite content 0.067mg/Kg, selenate content 0.287mg/Kg, total selenium content 0.748mg/Kg, and extraction rate 73.66%.
Example 1e, the enzyme reagent was a mixture of 40mg of neutral protease solution and 10mg of pronase solution, and extracted to have a selenocysteine content of 0.197mg/Kg, a selenomethionine content of 0.165mg/Kg, a selenate content of 0.311mg/Kg, a total selenium content of 0.748mg/Kg, and an extraction rate of 89.57%.
Example 1f, the enzyme reagent was a mixture of 25mg pronase and 25mg proteinase K, extracted methylselenocysteine 0.054mg/Kg, selenite 0.067mg/Kg, selenate 0.307mg/Kg, total selenium 0.428mg/Kg, with an extraction rate of 57.22%.
Example 1g, the mixed enzyme reagent was a mixture of 40mg neutral protease solution, 5mg proteinase K solution and 5mg neutral protease solution, extracted to have selenocysteine content of 0.227mg/Kg, selenomethionine content of 0.147mg/Kg, selenate content of 0.296mg/Kg, total selenium content of 0.748mg/Kg, and extraction rate of 89.44%.
The above examples 1a-1g have different extraction rates of selenium compounds from tea leaves using different components of mixed enzyme reagents, and the extraction results of each example are shown in table 1:
Figure BDA0002391379410000051
Figure BDA0002391379410000061
TABLE 1
From the data comparison in table 1, it can be seen that: 4 forms of selenium can be extracted by neutral protease, but when the neutral protease is used alone to extract the selenium in the tea, the baseline is unstable, the matrix influence is huge, and accurate qualitative and quantitative determination cannot be realized. The protease k and pronase can only extract selenomethionine and selenate, and the extraction rates are 58.42% and 71.52% respectively. When protease k and pronase are mixed for use, three forms of selenium, namely methyl selenocysteine, selenate and selenite, can be extracted. The extraction rate only reaches 57.22 percent. When the neutral protease and the protease k are mixed for use, selenocysteine, selenite and selenate can be extracted, selenomethionine is not extracted, and the extraction rate is 73.66%. However, according to a large amount of data, the selenium in the food mostly exists in the form of selenomethionine. When the neutral protease and the pronase are mixed for use, selenocysteine, selenomethionine and selenate can be extracted, and the extraction rate is 89.57%. When the three enzymes are mixed for use, selenocysteine, selenomethionine and selenate can be extracted, the extraction efficiency is 89.44%, and compared with neutral protease combined pronase, the extraction type and the extraction rate are not greatly different.
Example 2, the ultrasonic extraction of the solution of the polypropylene centrifuge tube at a temperature of 30-40 ℃ for 25-35 min, during which the polypropylene centrifuge tube is oscillated for 1-3 times, comprises:
and ultrasonically extracting the solution of the polypropylene centrifugal tube by using an ultrasonic cleaner at the temperature of 30-40 ℃ for 25-35 min, and vibrating the polypropylene centrifugal tube for 1-3 times in the process.
Specifically, the ultrasonic extraction is carried out on an ultrasonic cleaner, and the main purpose is to increase the movement speed of medium molecules and increase the penetrating power of a medium by utilizing the special effects of cavitation, mechanical effect, cavitation effect, thermal effect and the like generated by ultrasonic waves, promote the cell tissue in the tea planting to break the wall or deform, increase the contact of protease and cells, fully extract effective components in a sample and shorten the extraction time. After the ultrasonic extraction was completed, the centrifuge tube was transferred to a centrifuge for centrifugation. The purpose of centrifugation is to separate the extract from the sample residue, while allowing the extract to better pass through the filter.
Example 3, a 1 unit sample of tea leaves was taken and placed in a polypropylene centrifuge tube comprising:
grinding the tea sample into powder, mixing uniformly, preparing into a sample to be detected, and placing into a polypropylene centrifuge tube.
Specifically, through grinding the tealeaves sample into the powder state for the tealeaves sample can fully contact the enzymolysis with protease in the polypropylene centrifuging tube, reduces the enzymolysis time, promotes enzymolysis efficiency.
Example 4, the plasma mass spectrometer is a high performance liquid chromatography tandem inductively coupled plasma mass spectrometer.
Specifically, the high performance liquid chromatography series inductively coupled plasma mass spectrometer (HPLC-ICP-MS) takes liquid as a mobile phase, a high-pressure infusion system is adopted, the mobile phase such as single solvents with different polarities or mixed solvents and buffer solutions with different proportions is pumped into a chromatographic column filled with a stationary phase, after components in the column are separated, the mobile phase enters ICP, high-power high-frequency radio-frequency signals on an inductance coil form high-temperature plasma in the coil, the balance and continuous ionization of the plasma are guaranteed through the promotion of gas, analyzed components form aerosol in an atomizer, the aerosol is brought into the central area of a plasma torch by carrier gas, and evaporation, decomposition, excitation and ionization are carried out. Ions in the plasma are effectively transmitted to a mass spectrometer through an interface of the ICP-MS; mass spectrometry analytically calculates the intensity of a component by selecting ions of different mass-to-nuclear ratios (m/z) to detect the intensity of an ion.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above embodiments only express a few embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (6)

1. A method for detecting selenium compounds in tea leaves, which is characterized by comprising the following steps:
taking 1 unit of tea sample, and placing the tea sample in a polypropylene centrifugal tube;
adding a mixed enzyme reagent into the polypropylene centrifugal tube, wherein the mixed enzyme reagent comprises any combination of one to three of a neutral protease solution, a pronase protease solution and a proteinase k solution;
adding water into the polypropylene centrifugal tube to a constant volume;
ultrasonically extracting the solution of the polypropylene centrifugal tube at the temperature of 30-40 ℃ for 25-35 min, and oscillating the polypropylene centrifugal tube 1-3 times in the process;
centrifuging the solution in the polypropylene centrifugal tube at a rotating speed of 3000-4000 r/min for 3-5 min;
taking the supernatant in the polypropylene centrifuge tube, and filtering the supernatant through a filter membrane with the aperture of 0.22 mu m or 0.45 mu m to obtain a solution to be detected;
separating the liquid to be detected by a plasma mass spectrometer to obtain a mixture solution of selenious acid root, selenate, selenocysteine, methylselenocysteine and selenomethionine, and detecting the mixture solution.
2. The method for detecting selenium compounds in tea as claimed in claim 1, wherein the enzyme amount of the neutral protease solution is: 30-60 mg, the enzyme amount of the pronase solution is 3-10 mg, and the enzyme amount of the proteinase k solution is 4-10 mg.
3. The method for detecting selenium compounds in tea leaves as claimed in claim 1, wherein the ultrasonic extraction of the solution in the polypropylene centrifuge tube at a temperature of 30-40 ℃ for 25-35 min while shaking the polypropylene centrifuge tube 1-3 times comprises:
and ultrasonically extracting the solution of the polypropylene centrifugal tube by using an ultrasonic cleaner at the temperature of 30-40 ℃ for 25-35 min, and vibrating the polypropylene centrifugal tube for 1-3 times in the process.
4. The method for detecting selenium compounds in tea as claimed in claim 1, wherein the step of taking 1 unit of tea sample and placing the tea sample in a polypropylene centrifuge tube comprises:
grinding the tea sample into powder, mixing uniformly, preparing into a sample to be detected, and placing into a polypropylene centrifuge tube.
5. The method for detecting selenium compounds in tea as claimed in claim 1, wherein the plasma mass spectrometer is a high performance liquid chromatography series inductively coupled plasma mass spectrometer.
6. The method for detecting selenium compounds in tea as claimed in claim 5, wherein the plasma mass spectrometer uses citric acid solution as the mobile phase of the detection solution, the concentration of the citric acid solution is 10mmol/L, the pH value of the citric acid solution is 4.5, and the flow rate of the citric acid solution is 1.0 mL/min.
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Citations (4)

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US20150132285A1 (en) * 2005-10-03 2015-05-14 Yong Qian Proteinase-engineered Cancer Vaccine Induces Immune Responses to Prevent Cancer and to Systemically Kill Cancer Cells
CN102721779A (en) * 2012-06-19 2012-10-10 浙江农林大学 Detection method of selenium forms in plants
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Application publication date: 20200609