Crab field rice authenticity identification method based on element and stable isotope conjoint analysis

Abstract

The invention provides a crab field rice authenticity identification method based on element and stable isotope conjoint analysis, and belongs to the technical field of crab field rice identification. According to the method, the rice field rice and the crab field rice are subjected to multi-element and stable isotope conjoint analysis, characteristic symbolic elements and stable isotopes which can be used for distinguishing the crab field rice and the rice field rice are screened in combination with stoichiometric analysis, then an orthogonal partial least squares discriminant analysis (OPLS-DA) model is established, the model can achieve 100% distinguishing of the rice to be detected, and the method has the advantages that the method is simple and convenient to operate, and the accuracy is high. The cross validation result of the model shows that R2Y is equal to 0.852, Q2 is equal to 0.802, the data interpretation capability and prediction capability of the model are good, and the 100-time replacement test result shows that the corresponding P values of R2Y and Q2 are smaller than 0.01, and the model is optimal.

Description

Technical Field

[0001] This invention belongs to the field of crab-field rice identification technology, specifically involving a method for identifying the authenticity of crab-field rice based on joint analysis of elements and stable isotopes. Background Technology

[0002] Panjin rice is a well-known rice brand. In the Panjin area, rice cultivation mainly adopts two different models: conventional planting (paddy field rice) and rice-crab co-cultivation (crab field rice). The main difference between these two models lies in crab feed management and nitrogen fertilizer application. In the rice-crab co-cultivation model, farmers provide crabs with a formulated feed consisting of soybean meal, wheat bran, corn flour, and fishmeal. This feed is often placed on the ridges near the paddy fields, rather than applied directly to the fields. This allows aquaculture personnel to monitor the crabs' feeding and adjust the feed intake accordingly. During the topdressing stage, the use of ammonium-based fertilizers (NH4+) is strictly prohibited in rice-crab co-cultivation fields. + Urea is often used as the sole nitrogen source. In terms of pest and disease control, the use of chemical pesticides is lower than in traditionally grown fields, and the use of highly toxic pesticides is strictly prohibited. Apart from this, all other measures are the same for both. Therefore, crab-field rice has better safety and taste compared to paddy field rice. However, there is currently no method to identify the authenticity of crab-field rice. Summary of the Invention

[0003] The purpose of this invention is to provide a method for identifying the authenticity of crab-field rice based on joint analysis of elements and stable isotopes. The method provided by this invention can identify the authenticity of crab-field rice.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for identifying the authenticity of crab-field rice based on joint analysis of elements and stable isotopes, comprising the following steps: (1) Collect rice samples from paddy fields and crab-field rice respectively; (2) Detect the content and stable isotope ratio of each element in the paddy rice and crab rice samples collected in step (1); the elements include Cu, Zn, K, P, Mg, Ca, Mn, Fe, As, Cd, Pb, Al, Rb, Se, Sr, V, Co, Ni, Ga, Cs, Ag, Cr and Ba; the stable isotope ratio includes the ratio of hydrogen, oxygen, carbon and nitrogen isotopes; (3) Perform orthogonal partial least squares discriminant analysis on all element contents and stable isotope ratios obtained in step (2) to obtain the projection values ​​of each element and stable isotope, and screen elements and stable isotopes with projection values ​​> 1.0; (4) Perform multiple rounds of orthogonal partial least squares discriminant analysis on the element content screened in step (3) to obtain the positional relationship diagram of sample points and confidence intervals. Screen the characteristic elements that can separate the two sets of sample points and confidence intervals in the positional relationship diagram of sample points and confidence intervals. (5) Using the content of the characteristic marker elements screened in step (4) and the ratio of the stable isotopes screened in step (3) as variables, an orthogonal partial least squares discriminant analysis model is jointly established to obtain the identification model; (6) Detect the content of characteristic marker elements and stable isotope ratios in the rice sample to be tested, and then use the identification model obtained in step (5) to identify whether the rice to be tested is crab field rice.

[0005] Preferably, the method for detecting the content of each element in step (2) includes: 1) The sample to be tested was successively crushed and hydrolyzed with nitric acid to obtain a digest solution; 2) Mix the digestion solution obtained in step 1) with water and filter to obtain the test solution; 3) Perform inductively coupled plasma mass spectrometry and inductively coupled plasma emission spectroscopy on the test liquid obtained in step 2) to obtain the content of each element.

[0006] Preferably, the nitric acid used in step 1) of nitric acid hydrolysis has a mass concentration of 65%.

[0007] Preferably, in step 1), during nitric acid hydrolysis, the mass ratio of the sample to the volume of nitric acid is 0.5 g: (9~11) mL.

[0008] Preferably, the method for detecting stable isotope ratios in step (2) includes: After the sample to be tested is crushed, it is detected by an elemental analyzer-stable isotope ratio mass spectrometer to obtain the stable isotope ratio.

[0009] Preferably, when detecting the carbon and nitrogen isotope ratio, the detection parameters include: a combustion furnace temperature of 910~930℃, a reduction furnace temperature of 590~610℃, a carrier gas of helium, a helium purging flow rate of 240~260mL / min, a detection time of 540~560s, a carbon dioxide trapping current of 90~110μA, and a nitrogen trapping current of 390~410μA. When detecting the hydrogen and oxygen isotope ratio, the detection parameters include: pyrolysis furnace temperature of 1440~1460℃, carrier gas of helium, helium purging flow rate of 140~160mL / min, detection time of 940~960s, hydrogen trapping current of 390~410μA, and carbon monoxide trapping current of 190~210μA.

[0010] Preferably, the elements screened in step (3) are Se, Rb, Cu, Cd, Ag, V, Zn, Fe, Co and Cs.

[0011] Preferably, the stable isotopes screened in step (3) are d 15 N and d 13 C.

[0012] Preferably, the multi-round variable combination orthogonal partial least squares discriminant analysis in step (4) includes: sequentially removing one element from the elements screened in step (3), performing orthogonal partial least squares discriminant analysis with the remaining elements, then sequentially removing two elements, performing orthogonal partial least squares discriminant analysis with the remaining elements, repeating the above process, removing one more element each time, until the number of remaining elements is 5, and screening out the characteristic elements that can separate the two groups of sample points and confidence intervals.

[0013] Preferably, the characteristic elements in step (4) are Se, Rb, Cu, Cd, Ag, V and Zn.

[0014] This invention provides a method for identifying the authenticity of rice from crab-field fields based on joint analysis of elements and stable isotopes, comprising the following steps: (1) collecting rice samples from paddy fields and rice samples from crab-field fields respectively; (2) detecting the content of each element and the stable isotope ratio in the rice samples from paddy fields and rice samples collected in step (1) respectively; the elements include Cu, Zn, K, P, Mg, Ca, Mn, Fe, As, Cd, Pb, Al, Rb, Se, Sr, V, Co, Ni, Ga, Cs, Ag, Cr and Ba; the stable isotope ratio includes the ratio of hydrogen, oxygen, carbon and nitrogen isotopes; (3) performing orthogonal partial least squares discriminant analysis on all element contents and stable isotope ratios obtained in step (2) to obtain the projection values ​​of each element and stable isotope. (3) Screen elements and stable isotopes with projection values ​​> 1.0; (4) Perform multi-round variable combination orthogonal partial least squares discriminant analysis on the content of elements screened in step (3) to obtain the positional relationship diagram of sample points and confidence intervals, and screen characteristic elements that can separate two sets of sample points and confidence intervals in the positional relationship diagram of sample points and confidence intervals; (5) Use the content of characteristic elements screened in step (4) and the ratio of stable isotopes screened in step (3) as variables to jointly establish an orthogonal partial least squares discriminant analysis model to obtain an identification model; (6) Detect the content of characteristic elements and the ratio of stable isotopes in the rice sample to be tested, and then use the identification model obtained in step (5) to identify whether the rice to be tested is crab field rice. This invention conducted multi-element and stable isotope joint analysis on paddy rice and crab-field rice, and screened biomarkers that can distinguish between them using chemometric analysis, including characteristic marker elements and stable isotopes. Then, an orthogonal partial least squares discriminant analysis (OPLS-DA) model was established. This model can achieve 100% distinction between the tested rice varieties. The cross-validation results of this model show R... 2 Y=0.852, Q 2 =0.802, indicating that the model has good data interpretation and predictive ability. The results of 100 permutation tests show that R... 2 Y and Q 2 The corresponding P-values ​​are all less than 0.01, indicating that the model is optimal. Attached Figure Description

[0015] Figure 1 The VIP values ​​of each element in step (3) of Example 1; Figure 2 The VIP values ​​of each stable isotope in step (3) of Example 1; Figure 3 The OPLS-DA identification model established in Example 1 based on the content of characteristic marker elements and the ratio of stable isotopes, along with the model's cross-validation variance and the results of 100 permutation tests; Figure 4 The OPLS-DA identification model established based on the content of characteristic elements in Comparative Example 1, along with the model's cross-validation variance and the results of 100 permutation tests; Figure 5 The identification results are for the two samples of rice (DC1 and DC2) to be tested in Example 1. Detailed Implementation

[0016] This invention provides a method for identifying the authenticity of crab-field rice based on joint analysis of elements and stable isotopes, comprising the following steps: (1) Collect rice samples from paddy fields and crab-field rice respectively; (2) Detect the content and stable isotope ratio of each element in the paddy rice and crab rice samples collected in step (1); the elements include Cu, Zn, K, P, Mg, Ca, Mn, Fe, As, Cd, Pb, Al, Rb, Se, Sr, V, Co, Ni, Ga, Cs, Ag, Cr and Ba; the stable isotope ratio includes the ratio of hydrogen, oxygen, carbon and nitrogen isotopes; (3) Perform orthogonal partial least squares discriminant analysis on all element contents and stable isotope ratios obtained in step (2) to obtain the projection values ​​of each element and stable isotope, and screen elements and stable isotopes with projection values ​​> 1.0; (4) Perform multiple rounds of orthogonal partial least squares discriminant analysis on the element content screened in step (3) to obtain the positional relationship diagram of sample points and confidence intervals. Screen the characteristic elements that can separate the two sets of sample points and confidence intervals in the positional relationship diagram of sample points and confidence intervals. (5) Using the content of the characteristic marker elements screened in step (4) and the ratio of the stable isotopes screened in step (3) as variables, an orthogonal partial least squares discriminant analysis model is jointly established to obtain the identification model; (6) Detect the content of characteristic marker elements and stable isotope ratios in the rice sample to be tested, and then use the identification model obtained in step (5) to identify whether the rice to be tested is crab field rice.

[0017] The identification method of this invention is applicable to Panjin rice.

[0018] This invention collects rice samples from paddy fields and crab fields, respectively.

[0019] In one implementation method, this invention uses a five-point quincunx sampling method to collect composite samples from each experimental area, collecting a total of 60 rice sub-samples. Of these, 30 were collected from conventionally planted fields and 30 from rice-crab co-culture ecological fields. Five sub-samples were systematically collected from the east, south, west, north, and center of each plot, and then merged to form a single representative composite sample for each plot. This sampling method ensures the capture of spatial heterogeneity within individual plots. There are a total of 12 experimental fields (including 6 rice-crab co-culture fields and 6 conventional rice fields), thus forming 12 composite samples after integrating the sub-samples from each plot. To eliminate interference from variety in identifying the authenticity of rice planting patterns, all rice samples in this invention selected the main cultivated variety Yanfeng 47 from the Panjin area.

[0020] After obtaining samples of paddy rice and crab rice, this invention detects the content and stable isotope ratio of each element in the collected samples of paddy rice and crab rice respectively.

[0021] In this invention, the elements include Cu, Zn, K, P, Mg, Ca, Mn, Fe, As, Cd, Pb, Al, Rb, Se, Sr, V, Co, Ni, Ga, Cs, Ag, Cr, and Ba; the stable isotope ratios include the ratios of hydrogen, oxygen, carbon, and nitrogen isotopes.

[0022] In this invention, the method for detecting the content of each element preferably includes: 1) The sample to be tested was successively crushed and hydrolyzed with nitric acid to obtain a digest solution; 2) Mix the digestion solution obtained in step 1) with water and filter to obtain the test solution; 3) Perform inductively coupled plasma mass spectrometry and inductively coupled plasma emission spectroscopy on the test liquid obtained in step 2) to obtain the elemental content.

[0023] The present invention preferably involves sequentially pulverizing and hydrolyzing the sample to be tested with nitric acid to obtain a digest solution.

[0024] In this invention, the particle size of the pulverized sample to be tested is preferably ≤0.149 mm.

[0025] The present invention does not impose any special limitations on the pulverization operation. Any pulverization technique known to those skilled in the art can be used to ensure that the particle size of the sample to be tested is within the required range after pulverization.

[0026] In this invention, the mass concentration of nitric acid used in the nitric acid hydrolysis is preferably 65%.

[0027] In this invention, during the acid hydrolysis of nitric acid, the preferred ratio of the mass of the sample to the volume of nitric acid is 0.5 g: (9~11) mL, more preferably 0.5 g: 10 mL.

[0028] In this invention, the temperature of the nitric acid hydrolysis is preferably 150~200℃, more preferably 180℃; the volume of the system after nitric acid hydrolysis is preferably 4~6% of the volume of the system before nitric acid hydrolysis, more preferably 5%.

[0029] This invention controls the parameters of nitric acid hydrolysis within the above-mentioned range, which enables the hydrolysis to proceed fully and further improves the accuracy of identification.

[0030] After the nitric acid hydrolysis is completed, the present invention preferably cools the product of the nitric acid hydrolysis to obtain a digestion solution.

[0031] The present invention does not impose any special limitations on the cooling operation; any technical solution known to those skilled in the art can be used to cool to room temperature.

[0032] After obtaining the digestive fluid, the present invention preferably mixes the digestive fluid with water and then filters it to obtain the test solution.

[0033] In this invention, the water is preferably deionized water.

[0034] In this invention, the volume ratio of the digestive fluid to water is preferably 1:(48~52), more preferably 1:49.

[0035] The present invention does not have any particular limitation on the way the digestive fluid and water are mixed; any technical solution known to those skilled in the art can be used to mix the two evenly.

[0036] The present invention preferably uses a 0.45μm aqueous membrane for filtration. The present invention does not impose any special limitations on the specific material and source of the aqueous membrane; commercially available products well known to those skilled in the art can be used.

[0037] After obtaining the test solution, the present invention preferably performs inductively coupled plasma mass spectrometry and inductively coupled plasma emission spectroscopy on the test solution to obtain the content of each element.

[0038] This invention uses inductively coupled plasma mass spectrometry (ICP-MS) to detect the contents of Cu, Zn, As, Cd, Pb, Rb, Se, Sr, V, Co, Ni, Ga, Cs, Ag, and Cr, and uses inductively coupled plasma optical emission spectrometry (ICP-OES) to detect the contents of K, P, Mg, Ca, Mn, Fe, Al, and Ba.

[0039] As one implementation method, the detection conditions of the ICP-MS (Agilent 7900) can be as follows: RF power 1200~1600W, plasma flow rate 15L / min, carrier gas flow rate 0.80L / min, auxiliary gas flow rate 0.40L / min, helium flow rate 4~5mL / min, nebulizer temperature 2℃, sample rise rate 0.1~0.3r / s, nebulizer is a high-salt / concentric nebulizer, sampling cone / cutoff cone is a nickel / platinum cone, sampling depth is 8~10mm, acquisition mode is spectrum, detection mode is automatic, number of measurement points per peak is 1~3, and number of repetitions is 2~3.

[0040] As one implementation method, the detection conditions of the ICP-OES (Agilent 5800) can be as follows: 3 repeat counts, pump speed 12 rpm, sample aspiration time 25 s, rinsing time 15 s, reading time 5 s, RF power 1.2 kW, stabilization time 15 s, observation mode axial, nebulizer gas flow rate 0.7 L / min, plasma gas flow rate 12 L / min, and auxiliary gas flow rate 1 L / min.

[0041] In this invention, the method for detecting the stable isotope ratio preferably includes: After the sample to be tested is crushed, it is detected by an elemental analyzer-stable isotope ratio mass spectrometer to obtain the stable isotope ratio.

[0042] In this invention, the particle size of the pulverized sample to be tested is preferably ≤0.149 mm.

[0043] The present invention does not impose any special limitations on the pulverization operation. Any pulverization technique known to those skilled in the art can be used to ensure that the particle size of the sample to be tested is within the required range after pulverization.

[0044] In one embodiment, the present invention places 5.0 mg of pulverized sample into a tin capsule (4 mm × 4 mm × 11 mm) and detects the C and N isotope ratios using an elemental analyzer-stable isotope ratio mass spectrometer.

[0045] In this invention, when detecting the C and N isotope ratio, the preferred detection parameters include: a combustion furnace temperature of 910-930°C, a reduction furnace temperature of 590-610°C, helium as the carrier gas (99.999% purity), a helium purge flow rate of 240-260 mL / min, an isotope ratio mass spectrometer detection time of 540-560 s, a carbon dioxide trapping current of 90-110 μA, and a nitrogen trapping current of 390-410 μA; more preferably: a combustion furnace temperature of 920°C, a reduction furnace temperature of 600°C, a helium purge flow rate of 250 mL / min, a detection time of 550 s, a carbon dioxide trapping current of 100 μA, and a nitrogen trapping current of 400 μA.

[0046] In one embodiment, the present invention places 1.0 mg of pulverized sample into a silver capsule (4 mm × 4 mm × 11 mm) and detects the H and O isotope ratio using an elemental analyzer-stable isotope ratio mass spectrometer.

[0047] In this invention, when detecting the H and O isotope ratio, the detection parameters preferably include: a pyrolysis furnace temperature of 1440~1460℃ for the elemental analyzer, helium as the carrier gas, a helium purge flow rate of 140~160mL / min, an isotope ratio mass spectrometer detection time of 940~960s, a hydrogen trapping current of 390~410μA, and a carbon monoxide trapping current of 190~210μA; more preferably: a pyrolysis furnace temperature of 1450℃, a helium purge flow rate of 150mL / min, a detection time of 950s, a hydrogen trapping current of 400μA, and a carbon monoxide trapping current of 200μA.

[0048] In one implementation method, the present invention employs a two-point calibration method to process and calibrate test data, using USGS40, USGS90, and USGS91 standard material values ​​for calibration. d 13 C and d 15 The N-value is corrected using standard material values ​​from USGS55, USGS90, and USGS91. d 2 H and d 18 O value.

[0049] Information for each standard material is as follows: USGS90 ( d 2 H = -13.9‰ d 18 O = +35.90‰ d 13 C = -13.75‰ d 15N=+8.84‰); USGS91 ( d 2 H=-45.7‰, d 18 O = +21.13‰ d 13 C = -28.28‰ d 15 N=+1.78‰); USGS55 ( d 2 H = -28.2‰ d 18 O=+19.12‰); USGS40( d 13 C = -26.389‰ d 15 N = -4.5‰).

[0050] After obtaining the content and stable isotope ratio of each element in the collected rice and crab rice samples, this invention performs orthogonal partial least squares discriminant analysis on the content and stable isotope ratio of all elements to obtain the projection (VIP) value of each element and stable isotope, and filters out elements and stable isotopes with projection values ​​> 1.0.

[0051] As one implementation method, the present invention uses MetaboAnalyst 4.0 online software for orthogonal partial least squares discriminant analysis (OPLS-DA), plotting and analysis.

[0052] In this invention, the elements screened are Se, Rb, Cu, Cd, Ag, V, Zn, Fe, Co, and Cs; the stable isotopes screened are... d 15 N and d 13 C.

[0053] After screening elements and stable isotopes with projection values ​​> 1.0, this invention performs multi-round variable combination orthogonal partial least squares discriminant analysis on the content of the screened elements to obtain a positional relationship diagram of sample points and confidence intervals. In the positional relationship diagram of sample points and confidence intervals, characteristic elements that can separate two sets of sample points and confidence intervals are screened.

[0054] In this invention, the multi-round variable combination orthogonal partial least squares discriminant analysis preferably includes: sequentially removing one element from the screened elements, performing orthogonal partial least squares discriminant analysis with the remaining elements, then sequentially removing two elements, performing orthogonal partial least squares discriminant analysis with the remaining elements, repeating the above process, removing one more element each time, until the number of remaining elements is 5, and screening out the characteristic marker elements that can separate the two sets of sample points and confidence intervals.

[0055] In this invention, the characteristic elements are Se, Rb, Cu, Cd, Ag, V, and Zn.

[0056] After screening for characteristic marker elements that can separate two sets of sample points and confidence intervals, this invention uses the content of the screened characteristic marker elements and the ratio of the screened stable isotopes as variables to jointly establish an orthogonal partial least squares discriminant analysis model to obtain the identification model.

[0057] This invention uses a combination of the content of characteristic marker elements and the ratio of stable isotopes for modeling, which has a better recognition effect than modeling using only the content of characteristic marker elements.

[0058] After obtaining the identification model, the present invention detects the content of characteristic marker elements and stable isotope ratios in the rice sample to be tested, and then uses the identification model to identify whether the rice to be tested is crab-field rice.

[0059] In this invention, the detection method for the content of characteristic marker elements and the stable isotope ratio in the rice sample to be tested is the same as the detection method for the content of elements and the stable isotope ratio in the sample to be tested described in the above technical solution, and will not be repeated here.

[0060] This invention conducted a combined analysis of the content of 23 elements and stable isotope ratios (4 types) of crab-field rice and paddy rice from Panjin area. Combined with chemometric analysis, 9 markers were screened to distinguish between crab-field rice and paddy rice, including 7 characteristic marker elements (Se, Rb, Cu, Cd, Ag, V, and Zn) and 2 stable isotopes (…). d 15 N and d 13 C), and then jointly establish an orthogonal partial least squares discriminant analysis (OPLS-DA) model, which can achieve 100% distinction between rice from crab fields and paddy fields; cross-validation results show R 2 Y=0.852, Q 2 =0.802, indicating that the model has good data interpretation and predictive ability; the results of 100 permutation tests show that R 2 Y and Q 2 The corresponding P-values ​​were all less than 0.01, indicating that the current model is optimal. Among the biomarkers present in higher concentrations in crab-field rice were Ag, V, and... d 15 N.

[0061] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0062] Example 1 A method for identifying the authenticity of crab-field rice based on joint analysis of elements and stable isotopes is as follows: (1) A five-point plum blossom sampling method was used to collect composite samples from each experimental area, resulting in a total of 60 rice sub-samples. 30 were collected from conventionally planted fields, and 30 from rice-crab co-culture ecological fields. Five sub-samples were systematically collected from the east, south, west, north, and center of each plot, and then merged to form a single representative composite sample for each plot. There were a total of 12 experimental fields (including 6 rice-crab co-culture fields and 6 conventional rice fields). Therefore, after integrating the sub-samples from each plot, 12 composite samples were formed. All rice samples were of the main cultivated variety Yanfeng 47 in Panjin area. (2) Weigh 0.5g of crushed rice sample powder (particle size ≤0.149mm), put it into a 60mL digestion tube that has been acid-washed, add 10.0mL of nitric acid (mass concentration of 65%, analytical grade, Merck), and then heat in the temperature-controlled mode of the electrothermal digestion system (DigitBlockEHD36, LabTech) at 180℃ until the volume of the system is 0.5mL. Cool to room temperature, add deionized water to the volume of 25.0mL, mix well, filter through a 0.45μm aqueous membrane to obtain the test solution, and then detect the content of Cu, Zn, As, Cd, Pb, Rb, Se, Sr, V, Co, Ni, Ga, Cs, Ag and Cr by ICP-MS, and detect the content of K, P, Mg, Ca, Mn, Fe, Al and Ba by ICP-OES. The detection conditions of ICP-MS and ICP-OES are shown in Table 1 and Table 2, respectively. 5.0 mg of crushed rice sample powder (particle size ≤ 0.149 mm) was placed in a tin capsule (4 mm × 4 mm × 11 mm), and the C and N isotope ratios were detected by an elemental analyzer (Vario PYRO cube, Elementar, Germany) - stable isotope ratio mass spectrometer (Isoprime100, Elementar). The detection parameters were as follows: the combustion furnace temperature of the elemental analyzer was 920℃, the reduction furnace temperature was 600℃, the carrier gas was helium (purity 99.999%), the helium purge flow rate was 250 mL / min, the detection time of the isotope ratio mass spectrometer was 550 s, the trapping current of carbon dioxide was 100 μA, and the trapping current of nitrogen was 400 μA. 1.0 mg of pulverized rice sample (particle size ≤ 0.149 mm) was placed in a silver capsule (4 mm × 4 mm × 11 mm), and the H and O isotope ratios were detected using an elemental analyzer (Vario PYRO cube, Elementar, Germany) - stable isotope ratio mass spectrometer (Isoprime100, Elementar). The detection parameters were as follows: the elemental analyzer pyrolysis furnace temperature was 1450 °C, the carrier gas was helium, the helium purge flow rate was 150 mL / min, the isotope ratio mass spectrometer detection time was 950 s, the hydrogen trapping current was 400 μA, and the carbon monoxide trapping current was 200 μA. Correction using USGS40, USGS90, and USGS91 standard material values d 13 C and d 15 The N-value is corrected using standard material values ​​from USGS55, USGS90, and USGS91. d 2 H and d 18 O value; Information for each standard material is as follows: USGS90 ( d 2 H = -13.9‰ d 18 O = +35.90‰ d 13 C = -13.75‰ d 15 N=+8.84‰); USGS91 ( d 2 H=-45.7‰, d 18 O = +21.13‰ d 13 C = -28.28‰ d 15 N=+1.78‰); USGS55 ( d 2 H = -28.2‰ d 18 O=+19.12‰); USGS40( d 13 C = -26.389‰ d 15 N=-4.5‰); (3) The content of all elements and stable isotope ratios obtained in step (2) were subjected to orthogonal partial least squares discriminant analysis (OPLS-DA) using MetaboAnalyst 4.0 online software, plotted and analyzed to obtain the projection (VIP) values ​​of each element and stable isotope. Elements Se, Rb, Cu, Cd, Ag, V, Zn, Fe, Co and Cs with projection values ​​>1.0 and stable isotopes were selected. d 15 N and d 13 C; The VIP values ​​of each element and stable isotope are as follows: Figure 1 and Figure 2 As shown, CG represents paddy rice and DX represents crab rice. (4) Perform multiple rounds of orthogonal partial least squares discriminant analysis on the element content screened in step (3). Remove one element from the screened elements in turn, and perform orthogonal partial least squares discriminant analysis on the remaining elements. Then remove two elements in turn, and perform orthogonal partial least squares discriminant analysis on the remaining elements. Repeat the above process, removing one more element each time, until the number of remaining elements is 5. Obtain the positional relationship diagram of sample points and confidence intervals. Screen the characteristic elements Se, Rb, Cu, Cd, Ag, V and Zn that can separate the two sets of sample points and confidence intervals in the obtained positional relationship diagram of sample points and confidence intervals. (5) The ratio of the content of the characteristic marker elements (Se, Rb, Cu, Cd, Ag, V and Zn) screened in step (4) to the stable isotopes screened in step (3) ( d 15 N and d 13 C) Using these variables as variables, an orthogonal partial least squares discriminant analysis model is jointly established to obtain the identification model; (6) Detect the contents of Se, Rb, Cu, Cd, Ag, V and Zn in the two samples of rice (crab-field rice) according to the detection method in step (2). d 15 N and d 13 The ratio of C stable isotopes is used to identify whether the rice to be tested is crab field rice, based on the identification model in step (5).

[0063] Table 1 ICP-MS detection conditions

[0064] Table 2 Detection conditions for ICP-OES

[0065] Comparative Example 1 A method for identifying the authenticity of crab-field rice based on elemental analysis is as follows: (1) Same as in Example 1; (2) The content of Cu, Zn, As, Cd, Pb, Rb, Se, Sr, V, Co, Ni, Ga, Cs, Ag, Cr, K, P, Mg, Ca, Mn, Fe, Al and Ba elements in rice samples were only detected according to the method in step (2) of Example 1; (3) The contents of all elements obtained in step (2) are subjected to orthogonal partial least squares discriminant analysis (OPLS-DA), plotting and analysis using MetaboAnalyst 4.0 online software to obtain the projection (VIP) values ​​of each element, and the elements with projection values ​​> 1.0 are selected as Se, Rb, Cu, Cd, Ag, V, Zn, Fe, Co and Cs; (4) Perform multiple rounds of orthogonal partial least squares discriminant analysis on the element content screened in step (3). Remove one element from the screened elements in turn, and perform orthogonal partial least squares discriminant analysis on the remaining elements. Then remove two elements in turn, and perform orthogonal partial least squares discriminant analysis on the remaining elements. Repeat the above process, removing one more element each time, until the number of remaining elements is 5. Obtain the positional relationship diagram of sample points and confidence intervals. Screen the characteristic elements Se, Rb, Cu, Cd, Ag, V and Zn that can separate the two sets of sample points and confidence intervals in the obtained positional relationship diagram of sample points and confidence intervals. (5) Using the content of the characteristic marker elements (Se, Rb, Cu, Cd, Ag, V and Zn) screened in step (4) as variables, an orthogonal partial least squares discriminant analysis model is established to obtain the identification model; (6) Detect the content of Se, Rb, Cu, Cd, Ag, V and Zn in the rice to be tested according to the detection method in step (2), and use the identification model in step (5) to identify whether the rice to be tested is crab field rice.

[0066] The OPLS-DA identification model established in Example 1 based on the content of characteristic marker elements and the ratio of stable isotopes, along with the model's cross-validation variance and the results of 100 permutation tests, are shown below. Figure 3 As shown. The OPLS-DA recognition model established solely based on the content of characteristic marker elements in Comparative Example 1, along with the model's cross-validation variance and the results of 100 permutation tests, are shown below. Figure 4 As shown in the figure. P1 represents the variance contribution of the prediction set, reflecting the model's predictive ability on the prediction set data, i.e., the model's performance on unknown data; O1 represents the variance contribution of the orthogonal prediction set; O2 represents the variance contribution of the orthogonal residual set; R 2 X represents the cumulative variance explained by the model for the independent variable X; R 2 Y represents the cumulative variance explained by the model for the dependent variable Y (grouped); Q2 This reflects the model's predictive ability, specifically its ability to predict the variance of the data. From... Figure 3 and Figure 4 As can be seen, the OPLS-DA model, jointly established based on the content of characteristic marker elements and the ratio of stable isotopes, can effectively distinguish between crab-field rice and paddy field rice. The cross-validation variance plot shows that RV, reflecting the model's ability to interpret data, is... 2 The Y-value is 0.852, which is higher than the R-value of the OPLS-DA model based solely on 7-element fingerprints. 2 Y; Q reflects the model's predictive ability. 2 The value is 0.802, which is also higher than the Q value, which is based on a model with only 7 elements. 2 The value. The results of 100 permutation tests show that the R-value of the model in Example 1 is... 2 Y and Q 2 The corresponding p-values ​​are all less than 0.01, indicating that the current model is optimal. In summary, Se, Rb, Cu, Cd, Ag, V, Zn, d 15 N and d 13 C can be used in combination for the authenticity identification of crab-field rice, with an accuracy rate of up to 100%. Overall, it can be seen that the discriminant analysis effect of the model established by combining multiple elements and stable isotopes is better than the model based on elements alone.

[0067] The identification results of the two samples of rice (DC1 and DC2) in Example 1 are as follows: Figure 5 As shown. From Figure 5 As can be seen, the sample points of both rice samples fell within the confidence circle of crab-field rice, indicating that both rice samples were crab-field rice.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for identifying the authenticity of crab-field rice based on joint elemental and stable isotope analysis, comprising the following steps: (1) Collect rice samples from paddy fields and crab-field rice respectively; (2) Detect the content and stable isotope ratio of each element in the paddy rice and crab rice samples collected in step (1); the elements include Cu, Zn, K, P, Mg, Ca, Mn, Fe, As, Cd, Pb, Al, Rb, Se, Sr, V, Co, Ni, Ga, Cs, Ag, Cr and Ba; the stable isotope ratio includes the ratio of hydrogen, oxygen, carbon and nitrogen isotopes; (3) Perform orthogonal partial least squares discriminant analysis on all element contents and stable isotope ratios obtained in step (2) to obtain the projection values ​​of each element and stable isotope, and screen elements and stable isotopes with projection values ​​> 1.0; (4) Perform multiple rounds of orthogonal partial least squares discriminant analysis on the element content screened in step (3) to obtain the positional relationship diagram of sample points and confidence intervals. Screen the characteristic elements that can separate the two sets of sample points and confidence intervals in the positional relationship diagram of sample points and confidence intervals. (5) Using the content of the characteristic marker elements screened in step (4) and the ratio of the stable isotopes screened in step (3) as variables, an orthogonal partial least squares discriminant analysis model is jointly established to obtain the identification model; (6) Detect the content of characteristic marker elements and stable isotope ratios in the rice sample to be tested, and then use the identification model obtained in step (5) to identify whether the rice to be tested is crab field rice.

2. The identification method according to claim 1, characterized in that, The methods for detecting the content of each element in step (2) include: 1) The sample to be tested was successively crushed and hydrolyzed with nitric acid to obtain a digest solution; 2) Mix the digestion solution obtained in step 1) with water and filter to obtain the test solution; 3) Perform inductively coupled plasma mass spectrometry and inductively coupled plasma emission spectroscopy on the test liquid obtained in step 2) to obtain the content of each element.

3. The identification method according to claim 2, characterized in that, In step 1), the nitric acid used for acid hydrolysis has a nitric acid concentration of 65%.

4. The identification method according to claim 3, characterized in that, In step 1), during nitric acid hydrolysis, the mass ratio of the sample to the volume of nitric acid is 0.5 g: (9~11) mL.

5. The identification method according to claim 1, characterized in that, The method for detecting stable isotope ratios in step (2) includes: After the sample to be tested is crushed, it is detected by an elemental analyzer-stable isotope ratio mass spectrometer to obtain the stable isotope ratio.

6. The identification method according to claim 5, characterized in that, When detecting the carbon and nitrogen isotope ratio, the detection parameters include: combustion furnace temperature of 910~930℃, reduction furnace temperature of 590~610℃, carrier gas of helium, helium purging flow rate of 240~260mL / min, detection time of 540~560s, carbon dioxide trapping current of 90~110μA, and nitrogen trapping current of 390~410μA. When detecting the hydrogen and oxygen isotope ratio, the detection parameters include: pyrolysis furnace temperature of 1440~1460℃, carrier gas of helium, helium purging flow rate of 140~160mL / min, detection time of 940~960s, hydrogen trapping current of 390~410μA, and carbon monoxide trapping current of 190~210μA.

7. The identification method according to claim 1, characterized in that, The elements screened in step (3) are Se, Rb, Cu, Cd, Ag, V, Zn, Fe, Co and Cs.

8. The identification method according to claim 1, characterized in that, The stable isotopes screened in step (3) are δ 15 N and δ 13 C.

9. The identification method according to claim 1, characterized in that, The multi-round variable combination orthogonal partial least squares discriminant analysis in step (4) includes: sequentially removing one element from the elements screened in step (3), performing orthogonal partial least squares discriminant analysis with the remaining elements, then sequentially removing two elements, performing orthogonal partial least squares discriminant analysis with the remaining elements, repeating the above process, removing one more element each time, until the number of remaining elements is 5, and screening out the characteristic elements that can separate the two groups of sample points and confidence intervals.

10. The identification method according to claim 1, characterized in that, The characteristic elements in step (4) are Se, Rb, Cu, Cd, Ag, V and Zn.

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