A selenium speciation standard control sample in a nut matrix, and a preparation method and application thereof

Abstract

The application provides a selenium form standard quality control sample in a nut matrix and a preparation method and application thereof, and belongs to the technical field of standard substances. The kernel of abalone fruit is first crushed to obtain kernel particles; the kernel particles are sequentially subjected to defatting treatment and second crushing to obtain defatted powder; a selenite solution is sprayed on the defatted powder to obtain composite powder; and the composite powder is sequentially subjected to freeze-drying, third crushing and mixing to obtain the selenium form standard quality control sample in the nut matrix. The selenium form standard quality control sample in the nut matrix prepared by the method has good uniformity and stability, and the use of the standard quality control sample helps to improve the accuracy and reliability of the detection results of the selenium form (selenite and / or selenomethionine) content in plant products such as nuts.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of standard substance, in particular to a selenium speciation standard control sample in nut matrix and a preparation method and application thereof. BACKGROUND

[0002] As one of the essential trace elements for human body, selenium has physiological functions such as anti-oxidation, anti-aging, inhibition of tumor occurrence and protection of cardiovascular system. With the improvement of public awareness of selenium nutrition, selenium-rich foods (such as selenium-rich rice, selenium-rich walnuts and selenium-rich vegetables) are increasingly favored by the consumer market. However, the existence form of selenium in food has significant differences, and different forms of selenium also have different effects on human health. Studies have shown that inorganic selenium (such as sodium selenite and sodium selenate) has high toxicity, while organic selenium (including selenomethionine, selenocystine and selenocysteine) not only has lower toxicity, but also has higher bioavailability. It is worth noting that both inorganic selenium and organic selenium can be effectively absorbed and utilized by the human body within the appropriate concentration range, but the safe intake threshold of inorganic selenium is significantly lower than that of organic selenium.

[0003] Currently, selenium speciation detection technology has developed to a relatively mature stage. The conventional detection process usually first separates the target substance by liquid separation technology, and then completes the quantitative determination by atomic fluorescence spectrometry (AFS) or inductively coupled plasma mass spectrometry (ICP-MS). However, due to the lack of effective standard substances, the reliability of the selenium speciation detection results can only be indirectly verified by the recovery rate. However, the recovery rate index can only reflect the accuracy of the detection process (such as the instrument measurement link), and the extraction efficiency of the sample in the pretreatment process (i.e. the degree of effective release and retention of the target selenium speciation from the sample matrix) cannot be effectively evaluated. The lack of such quality control system can easily lead to deviations in the detection results, which may pose potential risks to the quality control of the selenium-rich product market.

[0004] Therefore, it is urgent to develop a selenium speciation standard control sample in plant matrix such as nut matrix with simple preparation process and accurate value results, so as to effectively verify the accuracy and reliability of the selenium speciation detection method. SUMMARY

[0005] The purpose of the present application is to provide a selenium speciation standard control sample in nut matrix and a preparation method and application thereof. The selenium speciation standard control sample in nut matrix prepared by the method of the present application has good uniformity and stability, and the use of the standard control sample helps to improve the accuracy and reliability of the selenium speciation (selenite and / or selenomethionine) content detection results in plant products such as nuts.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] The application provides a preparation method of a selenium form standard quality control sample in a nut matrix, comprising the following steps:

[0008] The seed kernels of abalone fruit are first crushed to obtain seed kernel particles;

[0009] The seed kernel particles are sequentially subjected to defatting treatment and second crushing to obtain defatted powder;

[0010] The defatted powder is sprayed with a selenite solution to obtain composite powder;

[0011] The composite powder is sequentially subjected to freeze-drying, third crushing and mixing to obtain the selenium form standard quality control sample in the nut matrix.

[0012] Preferably, the defatting reagent used in the defatting treatment comprises petroleum ether, and the use amount ratio of the seed kernel particles to the defatting reagent is 1 g:5-8 mL; the defatting treatment is performed at a temperature of 15-30 ℃; the defatting treatment is performed under oscillation conditions, the oscillation rate is 150-180 rpm, and the oscillation time is 8-12 h; the defatting treatment and the second crushing further comprise sequentially subjecting the obtained liquid after the defatting treatment to standing and solid-liquid separation, and collecting the solid material; the standing time is 25-35 min.

[0013] Preferably, the second crushing further comprises screening the obtained material after the second crushing, and collecting undersize as the defatted powder; the screen mesh used in the screening has a pore size of 0.250-0.425 mm.

[0014] Preferably, the concentration of the selenite solution is 0.2-2 mg / L, and the use amount ratio of the defatted powder to the selenite solution is 1-3 g:1 mL;

[0015] The preparation method of the composite powder comprises the following steps: the defatted powder is laid to form a laying layer, the selenite solution is sprayed in a spray form on the laying layer, and the composite powder is obtained after standing absorption;

[0016] The thickness of the laying layer is 0.3-0.6 mm; the spraying pressure is 0.2-0.4 MPa, and the height is 40-60 cm; the standing absorption temperature is 15-30 ℃, and the time is 1-3 h.

[0017] Preferably, the freeze-drying step comprises laying the composite powder to form a laying layer, and then sequentially performing pre-freezing, first freeze-drying and second freeze-drying;

[0018] The thickness of the laying layer is 2-3 cm; the pre-freezing temperature is -40 to -60 DEG C, the pressure is normal pressure, and the time is 2-6 h; the first freeze-drying temperature is -10 to -30 DEG C, the vacuum degree is 5-15 Pa, and the time is 8-16 h; and the second freeze-drying temperature is 20-40 DEG C, the vacuum degree is 3-8 Pa, and the time is 3-6 h.

[0019] Preferably, the third pulverization is further followed by screening the material obtained after the third pulverization to collect undersize material; and the screen used in the screening has a mesh size of 0.075-0.180 mm.

[0020] Preferably, the mixing is carried out by three-dimensional rotary mixing at a rotation speed of 18-30 rpm for 6-10 h.

[0021] The application provides a selenium form standard quality control sample in a nut matrix prepared by the preparation method.

[0022] The application provides an application of the selenium form standard quality control sample in the nut matrix in detecting selenite content and / or selenomethionine content in a plant sample.

[0023] Preferably, the plant sample includes a nut sample.

[0024] Advantages: the selenium form standard quality control sample in the nut matrix prepared by the method has good uniformity and stability, and the use of the standard quality control sample helps to improve the accuracy and reliability of the detection result of selenium form (selenite and / or selenomethionine) content in a plant product such as a nut. Specifically, the application uses abalone fruit as raw material, which has a relatively high natural selenium content and a relatively high organic selenium such as selenomethionine content, and the proportion of other organic selenium forms that are easily degraded is extremely low, so that the interference on the overall stability of the standard quality control sample can be effectively avoided; after defatting treatment, the sample matrix effect is significantly reduced, then selenite is added as an inorganic selenium source, and finally the prepared standard quality control sample contains stable inorganic selenium (selenite) and organic selenium (selenomethionine) two forms, and the two selenium forms are the main existing forms of selenium in plant products such as nuts, which can effectively meet the technical needs of accurate quality control in the detection of such products. At the same time, the method is simple to operate, and no heat treatment process is used in the preparation process, so that the chemical stability of selenite and selenomethionine is ensured, and the standard quality control sample can be stored stably at low temperature (4 DEG C) for more than 12 months. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The chromatogram of the standard solution;

[0026] Figure 2Chromatogram of the selenium speciation standard control sample in the nut matrix prepared for Example 1. DETAILED DESCRIPTION

[0027] The present application provides a preparation method of a selenium speciation standard control sample in a nut matrix, comprising the following steps:

[0028] The seed kernel of the baobab fruit is first crushed to obtain seed kernel particles.

[0029] The seed kernel particles are sequentially subjected to defatting treatment and second crushing to obtain defatted powder.

[0030] The defatted powder is sprayed with a selenite solution to obtain composite powder.

[0031] The composite powder is sequentially subjected to freeze-drying, third crushing and mixing to obtain the selenium speciation standard control sample in the nut matrix.

[0032] In the present application, the raw materials used are all commercially available or prepared by methods well known to those skilled in the art, unless otherwise specified.

[0033] In the present application, the seed kernel of the baobab fruit is first crushed to obtain seed kernel particles. As an embodiment of the present application, the baobab fruit can be from Brazil, specifically from Acre, Amazonas or Pará state; preferably, the seed kernel is obtained by shelling the baobab fruit, and then the seed kernel is first crushed. As an embodiment of the present application, the particle size of the seed kernel particles can be 1.5-2.5 mm; the present application does not have special limitations on the first crushing method, as long as the seed kernel particles with the above-mentioned particle size can be obtained; in the examples of the present application, the first crushing is specifically performed by using a crusher.

[0034] After obtaining the kernel granules, the kernel granules are subjected to defatting treatment and second pulverization in sequence to obtain defatted powder. As an embodiment of the present application, the defatting reagent used in the defatting treatment can include petroleum ether, and the use amount ratio of the kernel granules to the defatting reagent can be 1 g:5-8 mL, specifically 1 g:5 mL, 1 g:6 mL, 1 g:7 mL or 1 g:8 mL; the temperature of the defatting treatment can be 15-30 ℃, specifically 15 ℃, 20 ℃, 25 ℃ or 30 ℃; the defatting treatment can be carried out under oscillation, and the oscillation rate can be 150-180 rpm, specifically 150 rpm, 160 rpm, 170 rpm or 180 rpm; and the oscillation time can be 8-12 h, specifically 8 h, 9 h, 10 h, 11 h or 12 h. The present application removes oil and fat in the kernel granules through defatting treatment. In the embodiments of the present application, specifically, the kernel granules are placed in a conical flask, the defatting reagent is added, and then the conical flask is sealed by using a bottle cap provided with a gas-permeable small hole, and the conical flask containing the kernel granules and the defatting reagent is placed in a constant-temperature oscillator for defatting treatment. As an embodiment of the present application, the defatting treatment preferably further includes: sequentially subjecting the material obtained after the defatting treatment to standing and solid-liquid separation, and then collecting the solid material for the second pulverization; the standing time can be 25-35 min, specifically 30 min, and the standing in the embodiments of the present application facilitates the solid-liquid separation; and the solid-liquid separation can be performed by suction filtration. In the embodiments of the present application, the second pulverization is performed by using a pulverizer.

[0035] As an embodiment of the present application, the second pulverization preferably further includes: subjecting the material obtained after the second pulverization to screening, and collecting undersize as the defatted powder; and the screen mesh used in the screening can have a pore size of 0.250-0.425 mm, specifically 0.250 mm. The screening in the embodiments of the present application is performed by using the screen mesh having the above pore size, which is beneficial to ensuring sample uniformity; if the screen mesh has too large a pore size, it can lead to insufficient sample uniformity in the subsequent process; and if the screen mesh has too small a pore size, it can cause too low undersize yield and result in excessive sample loss.

[0036] After obtaining the defatted powder, the application sprays a selenite solution on the defatted powder to obtain a composite powder. The application uses selenite as an inorganic selenium source, which is conducive to improving the stability of the sample; if selenate is used, it is easy to be reduced in the nut matrix, resulting in unstable detection results; in addition, the main component of inorganic selenium in the selenium-enriched product of plant matrix is selenite. As an embodiment of the application, the selenite solution is prepared by selenite and water; the selenite can be sodium selenite; the water can be ultrapure water. As an embodiment of the application, the concentration of the selenite solution can be 0.2-2 mg / L, specifically 0.2 mg / L, 0.5 mg / L, 0.8 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L or 2 mg / L; the use amount ratio of the defatted powder to the selenite solution can be 1-3 g: 1 mL, specifically 1 g: 1 mL, 1.5 g: 1 mL, 2 g: 1 mL, 2.5 g: 1 mL or 3 g: 1 mL.

[0037] As an embodiment of the application, the preparation method of the composite powder can include the following steps: laying the defatted powder to form a laying layer, spraying the selenite solution in the form of spray on the laying layer, and obtaining the composite powder after standing absorption. As an embodiment of the application, the thickness of the laying layer can be 0.3-0.6 mm, specifically 0.3 mm, 0.4 mm, 0.5 mm or 0.6 mm; if the laying layer is too thin, the laying operation is significantly increased in difficulty and time-consuming and labor-consuming; if the laying layer is too thick, such as ≥0.8 mm, it will cause insufficient spraying of the selenite solution, resulting in uneven sample processing. In the embodiment of the application, the defatted powder is laid on a stainless steel tray to form a laying layer.

[0038] As an embodiment of the present application, the spraying pressure can be 0.2-0.4 MPa, specifically 0.2 MPa, 0.25 MPa, 0.3 MPa, 0.35 MPa or 0.4 MPa; the height can be 40-60 cm, specifically 40 cm, 45 cm, 50 cm, 55 cm or 60 cm. In the embodiment of the present application, the selenite solution is placed in an electric sprayer, and the selenite solution is sprayed on the layer in the form of spray; the spraying time of the present application is based on ensuring that the selenite solution is sprayed completely. In the embodiment of the present application, the working area is divided into several independent units, and each unit is kept fully isolated to avoid cross interference; after each unit completes the work independently, the samples are combined, and this method can effectively ensure that the subsequent spraying operation of the working area will not affect the completed area; wherein the continuous spraying time of each unit during the spraying process can be 2-6 s, specifically 2 s, 3 s, 4 s, 5 s or 6 s. In the embodiment of the present application, the spraying is carried out under the above conditions, which is beneficial to ensure the uniform spraying of the selenite solution.

[0039] As an embodiment of the present application, the temperature of the standing absorption can be 15-30℃, specifically 15℃, 20℃, 25℃ or 30℃; the time can be 1-3 h, specifically 1 h, 2 h or 3 h. The standing absorption is carried out under the above conditions in the present application, which can make the target substance fully contact with the absorption medium and reach the ideal mass transfer equilibrium state.

[0040] After obtaining the composite powder, the present application sequentially carries out freeze-drying, third crushing and mixing on the composite powder to obtain the selenium form standard quality control sample in the nut matrix. As an embodiment of the present application, the step of freeze-drying can include: laying the composite powder to form a layer, and then sequentially carrying out pre-freezing, first freeze-drying and second freeze-drying.

[0041] As an embodiment of the present application, the thickness of the layer can be 2-3 cm. As an embodiment of the present application, the pre-freezing temperature can be -40 to -60 DEG C, specifically -40 DEG C, -45 DEG C, -50 DEG C, -55 DEG C or -60 DEG C; the pressure can be normal pressure; and the time can be 2-6 h, specifically 2 h, 3 h, 4 h, 5 h or 6 h. As an embodiment of the present application, the first freeze-drying temperature can be -10 to -30 DEG C, specifically -10 DEG C, -15 DEG C, -20 DEG C, -25 DEG C or -30 DEG C; the vacuum degree can be 5-15 Pa, specifically 5 Pa, 10 Pa or 15 Pa; and the time can be 8-16 h, specifically 8 h, 10 h, 12 h, 14 h or 16 h. As an embodiment of the present application, the second freeze-drying temperature can be 20-40 DEG C, specifically 25 DEG C, 30 DEG C, 35 DEG C or 40 DEG C; the vacuum degree can be 3-8 Pa, specifically 3 Pa, 4 Pa, 5 Pa, 6 Pa, 7 Pa or 8 Pa; and the time can be 3-6 h, specifically 3 h, 4 h, 5 h or 6 h.

[0042] In the embodiment of the present application, the third crushing is specifically performed by a pulverizer. As an embodiment of the present application, the third crushing and the mixing preferably further comprise: screening the material obtained after the third crushing, and collecting undersize. The screen mesh used for the screening can have a pore size of 0.075-0.180 mm, specifically 0.150 mm.

[0043] As an embodiment of the present application, the mixing can be three-dimensional rotary mixing. The three-dimensional rotary mixing can have a rotation speed of 18-30 rpm, specifically 18 rpm, 22 rpm, 24 rpm, 26 rpm or 30 rpm, and a time of 6-10 h, specifically 6 h, 8 h or 10 h. In the embodiment of the present application, the three-dimensional rotary mixing can be performed by a high-efficiency mixer. The three-dimensional rotary mixing under the above conditions can realize uniform dispersion and sufficient contact of the material in the radial direction, axial direction and tangential direction, and effectively avoid the problems of local concentration unevenness or mixing dead angle.

[0044] After the mixing, the selenium speciation standard control sample in the nut matrix is preferably packaged in a glass bottle, and stored in a refrigerator (temperature: 0-4 DEG C).

[0045] The present application provides a selenium speciation standard control sample in a nut matrix prepared by the preparation method.

[0046] The present application provides an application of the selenium speciation standard control sample in a nut matrix in detecting the content of selenite and / or selenomethionine in a plant sample.

[0047] As an embodiment of the present application, the plant sample can include a nut sample, and the nut sample can include a baobab fruit.

[0048] The present application does not have special limitations on the method and conditions for detecting the content of selenite and / or selenomethionine in the plant sample using the selenium speciation standard quality control sample in the nut matrix. The conventional method and conditions in the art can be used. Specifically, for the detection of the selenium speciation content in the plant product, the nut matrix selenium speciation standard quality control sample and the sample to be tested are subjected to synchronous full-process pretreatment and instrument determination; when the determination result of the nut matrix selenium speciation standard quality control sample is within the specified allowable error range, it can be determined that the determination result of the sample to be tested has passed effective quality control, and the determination data is accurate and reliable.

[0049] The technical solutions in the present application will be described clearly and completely in combination with the embodiments in the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0050] The present application determines the total selenium content and selenium speciation types of different kinds of nuts. Table 1 is the determination result of the total selenium content and selenium speciation types of different kinds of nuts. As can be seen from Table 1, the total selenium content of the baobab fruit produced in Brazil is significantly higher than that of nuts produced in other places, and the selenium speciation is mainly selenomethionine with high stability; in contrast, although the baobab fruits produced in Peru and Bolivia only contain selenomethionine, the selenium content is relatively low. In addition, the selenium content of other common nuts such as pumpkin seeds, hazelnuts, pine nuts and sunflower seeds is generally insufficient, which cannot meet the requirements of the quality control sample raw material. Sunflower seeds and pumpkin seeds also contain easily degradable selenocystine components. Although some walnut samples produced in selenium-rich areas can reach a high selenium content level, which meets the basic requirements for the preparation of quality control samples, but due to the presence of selenocystine, it may affect the long-term stability of the quality control sample. Based on the above analysis, the present application finally selects the baobab fruit produced in Brazil as the preferred raw material for the preparation of the standard quality control sample.

[0051] Table 1 Determination results of total selenium content and selenium speciation types of different kinds of nuts

[0052]

[0053] Example 1

[0054] The preparation method of the selenium speciation standard quality control sample in the nut matrix is as follows:

[0055] S1, taking baobab fruit (the producing area is specific to Acre in Brazil) as raw material, after shelling treatment, the seed kernels were obtained, and the seed kernels were preliminarily crushed by a pulverizer to obtain seed kernel particles (particle size of 1.5-2.5 mm);

[0056] S2, the seed kernel particles were placed in a conical flask, petroleum ether was added to the conical flask according to the ratio of 1g:6mL, and the conical flask was sealed with a bottle cap equipped with a breathable hole, then the conical flask containing the seed kernel particles and petroleum ether was placed in a constant temperature oscillator for defatting treatment, the defatting treatment conditions included: temperature of 20℃, oscillation speed of 150rpm, oscillation time of 8h, after oscillation, standing for 30min, the obtained liquid was filtered to remove oil, and the collected solid material was defatted material;

[0057] S3, the defatted material was crushed by a pulverizer, and sieved by a screen with a pore size of 0.250mm, and the undersize material was collected to obtain defatted powder;

[0058] S4, the selenite (specifically sodium selenite) was mixed with ultrapure water to prepare a selenite solution with a concentration of 0.5mg / L; the defatted powder was spread on a stainless steel tray to form a layer with a thickness of 0.5mm, the selenite solution was uniformly sprayed on the defatted powder according to the ratio of 3g:1mL (specifically, the working area was divided into several independent units, and each unit was kept isolated to avoid cross interference; after the work in each unit was completed, the samples were combined), the spraying conditions included: spraying pressure of 0.3MPa, spraying height of 50cm, and continuous spraying time of each unit of 4s; after spraying, standing absorption was carried out at a temperature of 20℃ for 1h to obtain a composite powder;

[0059] S5, the composite powder was placed in a freeze-drying tray and spread to form a layer with a thickness of 2cm, pre-frozen for 3h at a temperature of-50℃ and a pressure of atmospheric pressure, then first freeze-dried for 10h at a temperature of-10℃ and a vacuum degree of 10Pa, and then second freeze-dried for 4h at a temperature of 25℃ and a vacuum degree of 5Pa to obtain a freeze-dried powder;

[0060] S6, the freeze-dried powder was crushed by a pulverizer, and sieved by a screen with a pore size of 0.15mm, the undersize material was collected, and then three-dimensional rotational mixing was carried out by a high-efficiency mixer at a speed of 24rpm for 8h to obtain a nut matrix selenium form standard control sample, the nut matrix selenium form standard control sample was packaged in a glass bottle and stored in a refrigerator (0-4℃).

[0061] Test Example 1

[0062] The test example determines the content of selenium forms (selenomethionine and selenite) in the sample, as follows:

[0063] (1) Test sample: selenium form standard control sample in nut matrix prepared in Example 1.

[0064] (2) Reagents and selenium form standard substance:

[0065] Reagents: experimental water meets the first grade water requirements in GB / T6682-2008 “Water Specifications and Test Methods for Analytical Laboratories”; citric acid (analytical pure), ammonia water (analytical pure), hexane sulfonic acid sodium (analytical pure), methanol (chromatographic pure), and streptomyces proteinase E;

[0066] Selenium form standard substance: selenomethionine (purity > 99wt%, Acros Organics Company) and selenite standard solution (GBW10032, China Institute of Metrology).

[0067] (3) Instrument equipment: ultrasonic cleaner (KQ2200E, Kunshan Ultrasonic Instrument Co., Ltd.), liquid chromatography coupled with inductively coupled plasma tandem mass spectrometer (HPLC-ICP-MS / MS, model 8900, Agilent Technologies).

[0068] (4) Instrument conditions:

[0069] Chromatographic conditions: Agilent ZORBAX SB-Aq reversed-phase ion pair chromatographic column (250mm×4.6mm, 5μm); mobile phase includes mobile phase A and mobile phase B; mobile phase A: citric acid and hexane sulfonic acid sodium in methanol aqueous solution, not adjusted pH value, wherein the concentration of citric acid is 10.00mmol / L, the concentration of hexane sulfonic acid sodium is 5.00mmol / L, and the volume fraction of methanol in methanol aqueous solution is 2%; mobile phase B: citric acid and hexane sulfonic acid sodium in methanol aqueous solution, and adjusted pH value to 7.00 with ammonia water, wherein the concentration of citric acid is 10.00mmol / L, the concentration of hexane sulfonic acid sodium is 5.00mmol / L, and the volume fraction of methanol in methanol aqueous solution is 2%; flow rate is 1.50mL / min; column temperature is 25℃; injection volume is 10μL;

[0070] ICP-MS / MS conditions: RF power is 1500W; plasma gas (Ar) flow rate is 15L / min; reaction gas (O2) flow rate is 0.8mL / min; Rpq value is 0.6; nebulization gas flow rate is 1.1L / min; SeO 96+ Isotope data.

[0071] (5) Sample pretreatment method: about 0.1 g of sample was accurately weighed in a 15 mL centrifuge tube, 10 mL of Tris / HCl buffer with a concentration of 50 M and 10 mg of streptomyces protease E were added, and after uniform mixing by vortex oscillator, the centrifuge tube was placed in an ultrasonic extractor, and extracted at a temperature of 40-50 DEG C for 5 h. After extraction, centrifugation was performed at 8000 rpm for 3 min, and the sample was filtered through a 0.45 μm filter membrane and determined by the instrument.

[0072] Figure 1 The chromatogram of the standard solution is shown in Figure 1, Figure 2 The chromatogram of the selenium form standard quality control sample prepared in the nut matrix of Example 1 is shown in Figure 2. The results show that the chromatographic peak symmetry of selenite and selenomethionine in the selenium form standard quality control sample prepared in the nut matrix of Example 1 is good. Compared with the standard chromatographic peak, each target analyte chromatographic peak and other impurity chromatographic peaks (non-target chromatographic peaks) have good separation degree, which fully meets the quality requirements of the quality control sample.

[0073] Test Example 2

[0074] The sample uniformity was tested in this test example, as follows:

[0075] Ten samples were randomly selected from the selenium form standard quality control sample prepared in the nut matrix of Example 1 and packaged in a glass bottle, and the selenite and selenomethionine contents were determined in duplicate under repeated conditions.

[0076] Table 2 shows the sample uniformity test and calculation results. The single factor variance analysis method (F test) was used for uniformity test and evaluation, F The value was calculated using Formulas 1-3: F

[0077] Formula 1;

[0078] Formula 2

[0079] Formula 3;

[0080] wherein, r is the number of measurements per group, is the first group average value in the group, is the total average value, is the average value in the group, is a single measurement value; K 1 is the inter-group degree of freedom, K 2 is the intra-group degree of freedom; Q 1 is the inter-group sum of squares, Q 2 is the intra-group sum of squares, with a degree of freedom of 9 and a significance level of 0.05; α ​= 0.05, degrees of freedom critical value table, it is known that F 0.05 (9,10) = 3.02.

[0081] It is calculated and known that: selenite F = 2.50, selenomethionine F = 2.78, selenite and selenomethionine statistical calculation F The value is less than the critical value (3.02), the sample meets the uniformity requirement, indicating that the selenium form content uniformity in the sample is good.

[0082] Table 2 Sample uniformity test and calculation results (mg / kg)

[0083]

[0084] Test Example 3

[0085] The sample meeting the uniformity requirement is subjected to stability detection in this test example. The stability detection includes two dimensions: long-term stability (storage stability, under the condition of 4°C) and short-term stability (transport stability, under the condition of 30°C). The sampling process follows the principle of "dense first and sparse later". Six sampling time points are set for short-term stability, and six sampling time points are also set for long-term stability detection. Three packaged samples are randomly taken at each sampling time point, and each sample is determined twice in parallel. The average value of the determination results of 6 times of 3 samples is taken as the final value, which is used to analyze the content of selenite and selenomethionine. According to the provisions of "JJF 1343-2022 Standard Material Valuation and Uniformity, Stability Evaluation", t-test analysis method is used as the statistical method for stability test in this test example. The curve of characteristic value changing with time is drawn to evaluate whether there is a one-way change trend of the characteristic value of the sample. The stability of the standard sample is quantitatively evaluated based on the linear fitting model. According to the model calculation result, the stability trend is analyzed: when the |β1| value of a certain index is less than the critical value t· S ( β 1), it is determined that the index remains stable within the observation period, indicating that the sample has good consistency. The specific calculation formulas are shown in formulas 4-6:

[0086] Formula 4;

[0087] Formula 5;

[0088] Formula 6;

[0089] Among them: is the time interval, is the average value of the time interval, is the actual measured value, is the average of the actual measured values, β 1 is the slope estimate of the straight line fitted by linear regression; S r is the residual standard deviation; S β 1) is the standard error of the slope, is the characteristic value representing the predicted value at time point according to the linear fitting model.

[0090] Table 3 is the stability result of selenite analog samples in samples during transportation at 30°C, and Table 4 is the stability result of selenomethionine analog samples in samples during transportation at 30°C; it can be seen that after 7 days of investigation at 30°C, the values of selenite and selenomethionine are 0.0031 (less than the critical value t· β 1=0.0039) and 0.0028 (less than the critical value t· S 1=0.0044), respectively, which confirms that both indicators exhibit good stability during transportation. β S β

[0091] Table 3 Stability result of selenite analog samples in samples during transportation at 30°C

[0092]

[0093] Table 4 Stability result of selenomethionine analog samples in samples during transportation at 30°C

[0094]

[0095] Table 5 Stability result of selenite analog samples in samples during long-term storage at 4°C, and Table 6 is the stability result of selenomethionine analog samples in samples during long-term storage at 4°C. It can be seen that after 12 months of storage at 4°C, the values of selenite and selenomethionine are 0.00040 (less than the critical value t· β 1=0.0025) and 0.0019 (less than the critical value t· S 1=0.0061), respectively, which confirms that both indicators exhibit good stability during the storage period. β S β

[0096] Table 5 Stability result of selenite analog samples in samples during long-term storage at 4°C

[0097] ​​​​​​​

[0098] Table 6 Stability results of selenomethionine analog samples in samples stored at 4°C for a long time

[0099]

[0100] Test Example 4

[0101] This test example is based on the technical specification requirements of JJF 1343-2022 Standard Material Value and Uniformity, Stability Evaluation, and the standard value of the selenium form standard control sample in the nut matrix prepared in Example 1 is determined. Specifically, a multi-laboratory joint value method is used to organize 8 detection institutions with certified qualifications (including national scientific research institutes, universities and third-party commercial detection laboratories certified by different types of institutions) to jointly carry out the value determination of the standard control sample. In the data processing stage, first, the original test data is subjected to normality test; then Grubbs test method is used to identify and eliminate abnormal values of each laboratory internal data; at the same time, Cochran test method is used to evaluate the equal precision characteristics of the measurement data between laboratories. Only when all statistical tests meet the specified requirements, the relevant data can be considered to have statistical representativeness, and be included in the final value result statistical analysis.

[0102] Table 7 is the inter-laboratory test data of the standard control sample (selenite), and Table 8 is the inter-laboratory test data of the standard control sample (selenomethionine). The normality test uses the Shapiro-Wilk test method, and the table shows that W(n, p) = 0.767 (where n = 3, p = 0.95), and the results of selenite and selenomethionine of each laboratory in Table 7 and Table 8 are greater than 0.767, so it can be considered that the detection data of each laboratory is normally distributed. Grubbs test method is used to test whether the detection results of each laboratory group have suspicious values, and the Grubbs critical value table shows that λ(0.05, 3) = 1.155, and the results of selenite and selenomethionine of each laboratory in Table 7 and Table 8 show that the maximum residual value of the detection results is less than 1.155, so it can be considered that there is no suspicious value in the detection results of each laboratory group. UmaxThe absolute values of the standard values of each laboratory are less than λ (0.05, 3)XS, indicating that there are no abnormal values between the value results of each laboratory, and there is no suspicious value, so all the data are retained and can participate in the statistical analysis of the value results. The Cochran test is used to determine whether the value results of each laboratory are equal in precision. The Cochran test requires that the C value (Cochran test statistic) be less than or equal to C (α, m, n), indicating that the average values between groups of data are equal in precision, otherwise it is determined that the data is an outlier, and the data should be removed when calculating the value results; according to the critical table, C (0.05, 8, 3) = 0.516, and the results of selenite and selenomethionine of each laboratory in Table 7 and Table 8 show that the C value of the Cochran test is 0.2510 and 0.3462, which is less than the critical value, so the data between the groups of laboratory test results is equal in precision, and all the data should be retained and can participate in the statistical analysis of the value results.

[0103] Table 9 is the average value of selenite and selenomethionine of 8 laboratories, which is 0.802 mg / kg and 0.500 mg / kg, respectively, and this result is used for the value of the results.

[0104] Table 7 Laboratory inter-laboratory test data (selenite)

[0105]

[0106] Table 8 Laboratory inter-laboratory test data (selenomethionine)

[0107]

[0108] Table 9 Average value of selenite and selenomethionine of 8 laboratories

[0109]

[0110] After the suspicious value is removed and the equal precision test of the value results of each laboratory is performed, the average value of the value results of each laboratory needs to be further tested for significant difference and normality. According to the statistical analysis, there is no significant difference between the average values of the test results of 8 laboratories (p>0.05), and the normal distribution characteristics are met (p>0.05), indicating that the data meet the requirements of statistical analysis. Therefore, the standard value of the selenium form standard quality control sample in the nut matrix prepared in Example 1 of the present application is the arithmetic mean of the average values of each laboratory, and the standard values of selenite (calculated as Se) and selenomethionine (calculated as Se) are 0.802 mg / kg and 5.00 mg / kg, respectively.

[0111] According to JJF 1343-2022 Evaluation of the value and uniformity and stability of standard materials, the uncertainty of the value of the standard material is composed of three parts: the uncertainty caused by the non-uniformity of the standard material Ubb Uncertainty due to instability of the reference material U sts Uncertainty due to the process of assigning value to the reference material U char The combined uncertainty is determined by calculating the uncertainty of each part U （y） The expanded uncertainty is twice the combined uncertainty U at the 95% confidence level U （y） Where U （y） And U According to formula 7~8:

[0112] Formula 7

[0113] × Formula 8

[0114] Uncertainty due to the inhomogeneity of the reference material U bb The calculation method is shown in formula 9:

[0115] Formula 9

[0116] Where, s bb is the inter-bottle standard deviation, MS between is the inter-bottle mean square (between-group variance), MS within is the intra-bottle mean square (within-group variance), n is the number of repeated measurements for each bottle.

[0117] The calculated uncertainties of selenite and selenomethionine are U bb 0.03 mg / kg and 0.079 mg / kg, respectively.

[0118] U sts The calculation can be performed according to the formula shown in formula 10:

[0119] Formula 10

[0120] Where R is the inter-laboratory range (maximum - minimum).

[0121] The calculated uncertainties of selenite and selenomethionine are U sts 0.0273 mg / kg and 0.0808 mg / kg, respectively.

[0122] U char The calculation can be performed according to the formula shown in formula 11:

[0123] Formula 11;

[0124] Wherein, the S value is the standard deviation of the mean value of the results of the 8 laboratories, P The value is the total number of laboratories participating in the calibration experiment, specifically 8.

[0125] The calculated selenite and selenomethionine U char The results are 0.011 mg / kg and 0.028 mg / kg, respectively.

[0126] According to the above results, the total synthesis uncertainty of selenite is calculated as U = 0.068 mg / kg (relative uncertainty 4.2%), and the total synthesis uncertainty of selenomethionine is U = 0.18 mg / kg (relative uncertainty 1.8%), the characteristic value of selenite in the selenium form standard control sample prepared in the nut matrix of the embodiment 1 of the present application is 0.802 ± 0.068 mg / kg; and the characteristic value of selenomethionine is 5.00 ± 0.18 mg / kg.

[0127] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a standard control sample of selenium speciation in a nut matrix, characterized by, The method comprises the following steps: The kernel of the baobab fruit is first crushed to obtain kernel particles; The kernel particles are sequentially subjected to defatting treatment and second crushing to obtain defatted powder; The defatted powder is sprayed with a selenite solution to obtain a composite powder; The composite powder is sequentially subjected to freeze-drying, third crushing and mixing to obtain the nut matrix selenium speciation standard control sample.

2. The production method according to claim 1, characterized by, The defatting reagent used in the defatting treatment comprises petroleum ether, and the use amount ratio of the kernel particles to the defatting reagent is 1 g: 5-8 mL; the defatting treatment is performed at a temperature of 15-30°C; the defatting treatment is performed under oscillation conditions, the oscillation rate is 150-180 rpm, and the oscillation time is 8-12 h; the defatting treatment and the second crushing further comprise sequentially subjecting the obtained liquid after the defatting treatment to standing and solid-liquid separation, and collecting the solid material; the standing time is 25-35 min.

3. The production method according to claim 1 or 2, characterized by, The second crushing further comprises screening the obtained material after the second crushing, and collecting undersize material as the defatted powder; the screen used in the screening has a mesh size of 0.250-0.425 mm.

4. The method of claim 1, wherein, The concentration of the selenite solution is 0.2-2 mg / L, and the use amount ratio of the defatted powder to the selenite solution is 1-3 g: 1 mL; The preparation method of the composite powder comprises the following steps: the defatted powder is laid to form a laying layer, the selenite solution is sprayed in the form of a spray on the laying layer, and the composite powder is obtained after standing absorption; The thickness of the laying layer is 0.3-0.6 mm; the spraying pressure is 0.2-0.4 MPa, and the spraying height is 40-60 cm; the standing absorption temperature is 15-30°C, and the standing absorption time is 1-3 h.

5. The method of claim 1, wherein, The freeze-drying step comprises laying the composite powder to form a laying layer, and then sequentially performing pre-freezing, first freeze-drying and second freeze-drying; The thickness of the laying layer is 2-3 cm; the pre-freezing temperature is -40--60°C, the pre-freezing pressure is normal pressure, and the pre-freezing time is 2-6 h; the first freeze-drying temperature is -10--30°C, the first freeze-drying vacuum degree is 5-15 Pa, and the first freeze-drying time is 8-16 h; the second freeze-drying temperature is 20-40°C, the second freeze-drying vacuum degree is 3-8 Pa, and the second freeze-drying time is 3-6 h.

6. The method of claim 1, wherein, The third crushing and the mixing further comprise screening the obtained material after the third crushing, and collecting undersize material; the screen used in the screening has a mesh size of 0.075-0.180 mm.

7. The preparation method according to claim 6, characterized in that, The mixing is performed in the form of three-dimensional rotary mixing, the three-dimensional rotary mixing speed is 18-30 rpm, and the three-dimensional rotary mixing time is 6-10 h.

8. The nut matrix selenium speciation standard control sample prepared by the preparation method in any one of claims 1-7.

9. The application of the nut matrix selenium speciation standard control sample in claim 8 in detecting the content of selenite and / or the content of selenomethionine in a plant sample.

10. Use according to claim 9, characterized in that, The plant sample comprises a nut sample.

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