一种基于irms、lc-q-tof-ms和多元素分析相结合的大豆产地溯源识别方法

Soybean origin tracing identification method based on combination of IRMS, LC-Q-TOF-MS and multi-element analysis

文档序号：6249 发布日期：2021-09-17 浏览：41次中文

1. A soybean origin tracing and identifying method based on combination of IRMS, LC-Q-TOF-MS and multi-element analysis is characterized by comprising the following steps:

s1, preparing a standard sample: selecting soybean samples of different producing areas with definite regions, squeezing the soybean samples of each producing area to obtain soybean oil samples, grinding the soybean oil samples to obtain standard soybean powder samples, extracting water-soluble protein of the standard soybean powder samples to serve as a target object, diluting the soybean oil samples by 100-200 times step by using a diluting solvent to obtain the standard soybean oil samples, wherein the soybean samples are from at least two different producing areas;

s2, acquiring mass spectrum data, mineral element content data and stable isotope ratio data of the standard sample: respectively acquiring mass spectrum data information of the standard soybean oil samples in different regions by using a liquid chromatogram-quadrupole time-of-flight mass spectrometer to obtain IDA-MS high-resolution mass spectrum data of triglyceride compounds of the standard soybean oil samples; acquiring mass spectrum data information of the standard soybean powder samples of different producing areas by using a mineral element content analyzer to obtain mineral element content data of the standard soybean powder samples; acquiring mass spectrum data information of the water-soluble protein of the standard soybean powder samples in different producing areas by using an isotope mass spectrometer to obtain stable isotope ratio data of the water-soluble protein;

s3, determining a standard soybean oil sample targeting compound: matching triglyceride compound mass spectrum data in the IDA-MS high-resolution mass spectrum data obtained in the step S2 with standard triglyceride compound data in a lipid compound database to determine a triglyceride compound targeting compound of the standard soybean oil sample;

s4, establishing a soybean producing area traceability identification model: after triglyceride targeting compounds in standard soybean oil samples are determined, the IDA-MS high-resolution mass spectrum data is analyzed through analysis software to obtain triglyceride compound marker observed peak data of the standard soybean oil samples, triglyceride compound marker observed peak data in the standard soybean oil samples of the same producing area, mineral element content data of the standard soybean powder samples and stable isotope ratio data of water-soluble proteins of the standard soybean powder samples are simultaneously introduced into modeling software to be processed in one or more modes of a main component analysis method, a partial least square method-discriminant analysis method and an orthogonal partial least square method regression analysis method, and triglyceride compound marker observed peak data of the standard soybean oil samples of different producing areas, mineral element content data of the standard soybean powder samples, a sample identification method and a sample identification method, And introducing the stable isotope ratio data of the water-soluble protein of the standard soybean powder sample into modeling software to obtain characteristic distribution rules of soybean oil in the standard soybean oil samples of different producing areas, mineral elements of the standard soybean powder sample and the stable isotope distribution rules of the water-soluble protein of the standard soybean powder sample, and constructing a soybean producing area traceability identification model based on the combination of LC-Q-TOF-MS, mineral element content and stable isotope ratio analysis;

s5, result prediction: respectively squeezing a soybean sample to be detected to obtain a soybean oil sample, grinding the soybean oil sample to be detected to obtain a soybean oil sample to be detected, extracting the soybean oil sample to be detected to obtain soybean water-soluble protein to be detected, diluting the soybean oil sample by a diluent by 100-200 times step by step to obtain the soybean oil sample to be detected, collecting IDA-MS high-resolution mass spectrum data of the soybean oil sample to be detected by a base liquid chromatography-quadrupole flight time mass spectrometer, collecting mineral element content data of the soybean oil sample to be detected by a mineral element content analyzer, and collecting stable isotope ratio data of the soybean water-soluble protein to be detected by an isotope mass spectrometer; and simultaneously introducing IDA-MS high-resolution mass spectrum data, mineral element content data and stable isotope ratio data of the soybean sample to be detected into the soybean origin tracing and identifying model in the step S4, and performing origin tracing prediction on the soybean to be detected.

2. The method for tracing to soybean origin according to claim 1, wherein the diluting solvent is a methanol-ethyl acetate mixture, and the ratio of methanol to ethyl acetate in the diluting solvent is 1: 1, gradually diluting the soybean oil sample by 200 times by using a methanol-ethyl acetate mixed solution.

3. The method for identifying soybean origin tracing according to claim 1, wherein the step of acquiring mass spectrum data of standard soybean oil sample in step S2 is as follows:

4. The method for source tracing identification of soybean origin according to claim 1, characterized in that the method for collecting mineral element content data of standard soybean meal sample in step S2 is as follows: weighing 0.5g of standard soybean meal sample into an inner tank of a microwave digestion tank, adding 4ml of nitric acid, covering the inner tank cover, and soaking for 6-12 hours; after soaking, screwing an outer tank of a microwave digestion tank, placing the outer tank into a microwave digestion instrument for digestion, setting a temperature raising program for digestion of a standard soybean powder sample in the digestion instrument, taking out an inner tank after digestion and cooling, driving up acid on an electric heating plate, transferring digestive juice in the inner tank into a 50mL polypropylene centrifugal tube after acid driving, washing the inner tank of the microwave digestion tank for many times by using a small amount of deionized water, combining washing liquids, fixing the volume of a mixed liquid of the digestive juice in the polypropylene centrifugal tube and the deionized water to 25mL by using the deionized water, uniformly mixing for later use, simultaneously performing a blank contrast test, and then determining and analyzing the mineral element content in the standard soybean powder sample by using ICP-MS and/or ICP-AES through a mineral element content analyzer.

5. The method for source tracing identification of soybean origin according to claim 1, characterized in that the method for collecting mineral element content data of standard soybean meal sample in step S2 is as follows: the method for acquiring the mineral element content data of the standard soybean powder sample in the step S2 comprises the following steps: weighing 0.5g of standard soybean powder sample, placing the standard soybean powder sample in a polyethylene digestion tube, adding 4ml of nitric acid, covering a sealing cover, and soaking for 6-12 hours; after soaking, placing the sealed polyethylene digestion tube in a digestion instrument, adding inert gas in an inner tank body of the digestion instrument in advance, wherein the pressure of the inert gas is 4000KPa, setting a temperature raising program to digest a standard soybean powder sample in the digestion instrument, taking out the inner tank after cooling after digestion, driving up acid on a hot plate, transferring digestive juice in the inner tank to a 50mL polypropylene centrifuge tube after acid driving, washing the inner tank of the microwave digestion tank for multiple times by using a small amount of deionized water, combining washing liquids, fixing the volume of a mixed liquid of the digestive juice and the deionized water in the polypropylene centrifuge tube to 25mL by using the deionized water, uniformly mixing for later use, and simultaneously performing a blank comparison test, wherein a mineral element content analyzer adopts ICP-MS and/or ICP-AES to determine and analyze the mineral element content in the standard soybean powder sample.

6. The method for tracing to soybean origin according to claim 1, wherein the water-soluble protein extraction process of the standard soybean meal sample in step S1 is as follows: grinding and crushing a soybean sample to prepare a 30g standard soybean powder sample, weighing 1g standard soybean powder sample, adding 10mL of ultrapure water, performing ultrasonic treatment for 30min under the rotation condition of 10000 revolutions, centrifuging for 5min, extracting 1mL of supernatant protein extract, adding the supernatant protein extract into a 3K ultrafiltration centrifugal tube, centrifuging to obtain proteins with molecular weights of more than 3K Da respectively, transferring the centrifuged proteins into a sample injection bottle, and freeze-drying for more than 8 hours until all the proteins are dried to obtain solid protein powder of water-soluble proteins;

the method for acquiring stable isotope ratio data of the water-soluble protein of the standard soybean meal sample in the step S2 comprises the following steps:

a1. selecting an element analysis-isotope mass spectrometer EA-IRMS, placing solid protein powder of water-soluble protein in a tin cup and a silver cup, and respectively detecting the isotope ratio of stable carbon and nitrogen and the isotope ratio of stable hydrogen and oxygen in the water-soluble protein;

a2. tuning a correction element analysis-isotope mass spectrometer EA-IRMS;

a3. introducing water-soluble protein wrapped by tin cups into a 980 ℃ high-temperature combustion furnace to obtain CO₂And N₂Gas of CO₂And N₂Lead-in isotope mass spectrometer hostDelta in water-soluble proteins¹⁵N and delta¹³C, detecting; introducing water soluble protein wrapped by silver cup into 1380 deg.C pyrolysis furnace to obtain CO and H₂Gas, CO and H₂Introduction into isotope mass spectrometer host for water-soluble protein delta²H and delta¹⁸And (4) detecting.

7. The method for identifying soybean origin tracing according to claim 3, wherein the method for determining the target compound of step S3 comprises: according to the molecular weight range of the target object in the IDA-MS high-resolution mass spectrum data, dividing the IDA-MS high-resolution mass spectrum data in the standard soybean oil sample into 3 regions: a first region with the molecular weight of 800-;

8. The method for identifying soybean origin and source tracing of claim 1, wherein said step S4 further comprises a blind sample verification step, said blind sample verification step comprising: selecting a plurality of soybean verification samples in the same area, respectively squeezing the soybean verification samples in a definite area to obtain a soybean oil sample to be verified and water-soluble protein of the soybean oil sample to be verified after grinding and extraction, acquiring IDA-MS high-resolution mass spectrum data of the soybean oil sample to be verified by a liquid chromatogram-quadrupole time-of-flight mass spectrometer, acquiring mineral element content data of the soybean oil sample to be verified by a mineral element content analyzer, and acquiring water-soluble protein stable isotope ratio data of the soybean oil sample to be verified by an isotope mass spectrometer; and simultaneously introducing IDA-MS high-resolution mass spectrum data, mineral element content data and water-soluble protein stable isotope ratio data of the soybean verification sample into the soybean origin tracing and identifying model in the step S4, and verifying the origin tracing accuracy of the soybean verification sample.

9. The method for identifying soybean origin tracing according to claim 1, wherein in step S4, the triglyceride compound marker observed peak data in the standard soybean oil sample of the same soybean origin, the mineral element content data of the standard soybean flour sample, and the stable isotope ratio data of the water-soluble protein of the standard soybean flour sample are simultaneously introduced into modeling software to perform an orthogonal partial least squares regression analysis, so as to construct an OPLS-DA soybean origin tracing identification model based on LC-Q-TOF-MS, the combination of the mineral element content and the stable isotope ratio analysis.

10. The method for identifying soybean origin tracing according to claim 1, wherein in the process of preparing the standard samples in step S1, the soybean samples are taken from n different regions, the soybean samples are divided into n x (n-1)/2 groups, each group of soybean samples consists of two soybean samples from different origins, and a two-country soybean origin tracing identification model is established for identifying the corresponding soybean country or region in the two-country soybean origin tracing identification model according to steps S2, S3 and S4.

Background

The field of food quality safety detection relates to two major quality safety problems, namely the risk problem of toxic and harmful substances and the authenticity problem of products. Regarding the detection problem of harmful substances in food, a large number of standards and detection methods reported in literatures at home and abroad detect toxic and harmful substances in food, and regarding the authenticity problem of the quality of food, the detection method has attracted attention and attention of consumers at home and abroad in recent ten years, and gradually becomes a hot spot and a difficult problem in the field of food quality detection. Currently, the technology for detecting the authenticity of food mainly comprises fingerprint technology such as ultraviolet spectrum and infrared spectrum, atomic spectrum technology such as atomic absorption, emission and fluorescence, isotope mass spectrum technology, high-resolution mass spectrum technology, nuclear magnetic resonance technology, raman spectrum technology, and omics technology which is started in the nineteenth 20 th century, and the food omics in the omics technology comprise omics technology based on omics and epigenomics, transcriptomics, proteomics, metabonomics and lipidomics, and the like, and proteomics, metabonomics and lipidomics are commonly used in the field of food inspection, so that the problems of true and false functional components of food, food nutrient content and food origin tracing can be judged through the omics technology. In the field of origin tracing authenticity identification of food, isotope mass spectrometry technology and omics technology are two relatively reliable identification technologies, but the methods and patents for origin tracing of soybeans by using omics technology are few.

The patent application with the publication number of CN104360004A discloses a method for identifying the authenticity of cubilose by using LC-Q-TOF combined with statistical analysis. Adding a formic acid solution into a sample to be detected, then boiling in a water bath, cooling, and filtering by a filter membrane to obtain a treated sample to be detected; collecting mass spectrum information of the treated sample to be detected by using a liquid chromatogram-quadrupole time-of-flight mass spectrometer, and extracting a characteristic compound; and (4) calling the obtained characteristic compound information of the sample to be detected into a cubilose authenticity identification model for prediction. And judging to be a genuine bird's nest when the accuracy is 80% or more, or else, judging to be a counterfeit bird's nest. The invention also establishes a cubilose authenticity identification model, and only one authenticity identification model is needed during identification, so that the detection is simpler and easier to operate. The method is used for identifying the cubilose product, but because the cubilose and the soybean have different components, the method cannot extract the target compound of the soybean and further completes the tracking of the soybean production area.

Patent application No. CN111474275A discloses a method for tracing potato origin based on mineral elements and stable isotopes, which comprises: collecting potato samples of potato production bases in inner Mongolia, Heilongjiang, Xinjiang and Sichuan provinces respectively, and preparing analysis samples; determining the contents of aluminum, manganese, copper and zinc in the obtained analysis sample, and determining the ratio of the carbon stable isotope to the carbon stable isotope delta 13C and the ratio of the nitrogen stable isotope to the nitrogen stable isotope delta 15N; and (5) bringing the measurement result into a discrimination model to judge the production area of the potato sample. The method for tracing the potato origin of the invention is based on the mineral elements and stable isotope content of different origins, can use objective detection technical means to indicate the sources of different potato products, has the accuracy rate of not less than 95.3 percent, fills the gap of the lack of potato origin tracing technology, can provide data support for protecting potato regional brands with the advantages of origins, avoids the phenomenon of insufficient production, and has wide application prospect. The method cannot establish a soybean origin tracing model based on lipidomics.

The patent application with the publication number of CN106560698A discloses a plant production place identification method based on multiple detection technologies, and the invention discloses a method for identifying the production place of Wuyi rock tea by combining near infrared spectrum, stable isotope and trace element data, belongs to the technical field of authenticity identification of geographic marking products, and aims to solve the problems that single detection data cannot represent all key information of production place traceability and different types of detection data are matched in data used in a metrological method in a combined mode. The method is based on a least square support vector machine (LS-SVM) model, near infrared, stable isotopes and trace elements are fused together for modeling analysis, the recognition rate is the highest and reaches 100.0 percent, the recognition rate is far higher than that of an LS-SVM judgment result established by single data, the recognition rate of blind samples reaches 100 percent, and the method has a good application prospect. The invention is also suitable for identifying the production places of Chinese torreya, lotus root starch and other plant samples, and the invention is not suitable for plants except tea, but for plants rich in oil, a near infrared method is not suitable.

Disclosure of Invention

The invention aims to provide a soybean origin tracing identification method based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology, which aims to solve the technical problems, the soybean origin tracing identification method based on the analysis and fusion of LC-Q-TOF-MS, mineral elements and stable isotope content is established by combining an M liquid chromatography-quadrupole time-of-flight mass spectrometer LC-Q-TOF-MS and a stable isotope ratio analysis technology, the three data are fused and then introduced into a soybean origin tracing identification model, and the origin tracing prediction of the soybean to be detected is carried out through the model by referring to triglyceride compound high-resolution mass spectrum data of the soybean oil sample to be detected, the mineral element content of the soybean powder sample and the stable isotope ratio of water-soluble protein in the soybean powder sample, the accuracy of tracing the soybean producing area is improved.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a soybean origin tracing identification method based on combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology comprises the following steps:

s4, establishing a soybean producing area traceability identification model: after triglyceride targeting compounds in standard soybean oil samples are determined, the IDA-MS high-resolution mass spectrum data is analyzed through analysis software to obtain triglyceride compound marker observed peak data of the standard soybean oil samples, triglyceride compound marker observed peak data in the standard soybean oil samples of the same producing area, mineral element content data of the standard soybean powder samples and stable isotope ratio data of water-soluble proteins of the standard soybean powder samples are simultaneously introduced into modeling software to be processed in one or more modes of a main component analysis method, a partial least square method-discriminant analysis method and an orthogonal partial least square method regression analysis method, and triglyceride compound marker observed peak data of the standard soybean oil samples of different producing areas, mineral element content data of the standard soybean powder samples, a sample identification method, a, And introducing the stable isotope ratio data of the water-soluble protein of the standard soybean powder sample into modeling software to obtain characteristic distribution rules of soybean oil in the standard soybean oil samples of different producing areas, mineral elements of the standard soybean powder sample and the stable isotope distribution rules of the water-soluble protein of the standard soybean powder sample, and constructing a soybean producing area traceability identification model based on the combination of LC-Q-TOF-MS, mineral element content and stable isotope ratio analysis; the marker observation peak data is MarkerView peaks data, the MarkerView peaks data of triglyceride of the standard soybean oil sample is obtained by the analysis of MasterView analysis software,

Further preferably, in step S3, the lipid compound database is the lipid compound database LIPID MAPS Lipidomics Gateway published in the united states, the standard triglyceride compound data is 6899 triglyceride compounds of the tiranylglycerols in the lipid compound database LIPID MAPS Lipidomics Gateway, and 116 triglyceride compounds common to soybean oil samples are determined as the triglyceride compound targeting compounds of the standard soybean oil samples by the PeakView software qualitative analysis.

Preferably, the dilution solvent is a methanol-ethyl acetate mixed solution, and the ratio of methanol to ethyl acetate in the dilution solvent is 1: 1, gradually diluting the soybean oil sample by 200 times by using a methanol-ethyl acetate mixed solution.

Preferably, the step of collecting mass spectrum data of the standard soybean oil sample in step S2 is as follows:

Preferably, the liquid chromatography conditions of the liquid chromatograph in the liquid chromatography-quadrupole time-of-flight mass spectrometer are as follows: the flow rate is 0.5. mu.L/min, the column temperature is 40 ℃, Xbridge BEH C18 chromatographic column gradient elution is carried out, and the sample volume is 2. mu.L; the A phase in the mobile phase is isopropanol, and the B phase in the mobile phase is acetonitrile, wherein the content of the B phase in the mobile phase in different time periods is as follows: 0min, 70% B; 0-5min, 70-65% B; 5-8min, 65% B; 10-10.5min, 65-70% B; 10.5-15min, 70% B.

The mass spectrum condition of the quadrupole flight time of the mass spectrometer in the liquid chromatogram-quadrupole flight time mass spectrometer is as follows: the mass spectrometer adopts a positive ion mode to collect data, and the ion source is as follows: ESI and APCI complex sources; the positive ion scanning mode is as follows: APCI source connection automatic correction system, first grade TOF-MS scans accurate mass range: 100-2000 Da, data acquisition time of 100ms, DP of 100V and CE of 10V, wherein DP is declustering voltage, and CE is collision energy; secondary IDA-MS scan accurate mass range: 50-2000 Da, DP:100V, CE:35 +/-15V; the mass spectrometer adopts a high-sensitivity mode, the data acquisition time is 50ms, the signal threshold is 100cps, data are acquired for 6 times in each circulation, and dynamic background subtraction is adopted.

Preferably, the liquid chromatography-quadrupole time-of-flight mass spectrometer is an shimadzu LC20AD liquid chromatograph, the mass spectrometer is a Triple TOF5600+ mass spectrometer, the automatic correction system is a CDS system, and the conditions of the quadrupole time-of-flight mass spectrometer further include: the calibration is carried out for 1 time per 10 samples, the flow rate of APCI positive ion calibration solution is 0.3mL/min, and the pressure of an air curtain is as follows: 40psi, ion source atomization gas pressure: at 50psi, ion source assist gas pressure: 50psi, ion source temperature: the ion source voltage is 5500V at 500 ℃, all IDA-MS high-resolution mass spectrum data collected by a mass spectrometer are collected in analysis TF 1.6 software of ABSciex company, and after being qualitatively and quantitatively processed and analyzed on PeakView and MasterView software, the IDA-MS high-resolution mass spectrum data are introduced into SIMCA14.0 software (Umetrics company of Switzerland) to be processed in one mode of principal component analysis, partial least square method discriminant analysis and orthogonal partial least square method discriminant analysis.

Preferably, the mineral element content data of the standard soybean flour sample in step S2 is acquired by: weighing 0.5g of standard soybean meal sample into an inner tank of a microwave digestion tank, adding 4ml of nitric acid, covering the inner tank cover, and soaking for 6-12 hours; after soaking, screwing an outer tank of a microwave digestion tank, placing the outer tank into a microwave digestion instrument for digestion, setting a temperature raising program for digestion of a standard soybean powder sample in the digestion instrument, taking out an inner tank after digestion and cooling, driving up acid on an electric heating plate, transferring digestive juice in the inner tank into a 50mL polypropylene centrifugal tube after acid driving, washing the inner tank of the microwave digestion tank for many times by using a small amount of deionized water, combining washing liquids, fixing the volume of a mixed liquid of the digestive juice in the polypropylene centrifugal tube and the deionized water to 25mL by using the deionized water, uniformly mixing for later use, simultaneously performing a blank contrast test, and then determining and analyzing the mineral element content in the standard soybean powder sample by using ICP-MS and/or ICP-AES through a mineral element content analyzer.

Preferably, the blank comparison tests comprise two groups, wherein 25mL of deionized water is placed in a polypropylene centrifuge tube in one group of blank comparison tests; another set of blank controls contained 25mL of nitric acid in polypropylene centrifuge tubes.

Preferably, the temperature raising program of the microwave digestion tank in the microwave digestion instrument comprises the following steps:

a1. heating the microwave digestion instrument to 120 ℃ within 8min, and then keeping the temperature of 120 ℃ for 5 min;

a2. the temperature of the microwave digestion apparatus was then increased from 120 ℃ to 180 ℃ over 10min, and then maintained at 180 ℃ for 10 min.

Preferably, the mineral element content data of the standard soybean flour sample in step S2 is acquired by: the method for acquiring the mineral element content data of the standard soybean powder sample in the step S2 comprises the following steps: weighing 0.5g of standard soybean powder sample, placing the standard soybean powder sample in a polyethylene digestion tube, adding 4ml of nitric acid, covering a sealing cover, and soaking for 6-12 hours; after soaking, placing the sealed polyethylene digestion tube in a digestion instrument, adding inert gas in an inner tank body of the digestion instrument in advance, wherein the pressure of the inert gas is 4000KPa, setting a temperature raising program to digest a standard soybean powder sample in the digestion instrument, taking out the inner tank after cooling after digestion, driving up acid on a hot plate, transferring digestive juice in the inner tank to a 50mL polypropylene centrifuge tube after acid driving, washing the inner tank of the microwave digestion tank for multiple times by using a small amount of deionized water, combining washing liquids, fixing the volume of a mixed liquid of the digestive juice and the deionized water in the polypropylene centrifuge tube to 25mL by using the deionized water, uniformly mixing for later use, and simultaneously performing a blank comparison test, wherein a mineral element content analyzer adopts ICP-MS and/or ICP-AES to determine and analyze the mineral element content in the standard soybean powder sample.

Preferably, the inert gas is nitrogen, and the temperature of an external cavity of the digestion instrument is less than or equal to 40 ℃.

Preferably, the temperature raising program of the polyethylene digestion tube in the digestion instrument comprises the following steps:

a1. under the conditions of 15000KPa pressure and 1500W power, the temperature of the microwave digestion instrument is increased to 120 ℃ within 8 min;

a2. then under the conditions of 15000KPa pressure and 1500W power, the temperature of the microwave digestion instrument is increased from 120 ℃ to 240 ℃ within 8 min;

a3. further, the temperature of 240 ℃ was maintained for 20min under the conditions of 15000KPa pressure and 1500W power.

Preferably, 0.5g of a standard soybean powder sample is weighed to the accuracy of 0.001g, and the temperature set by the electric hot plate is 140-160 ℃.

More preferably, the electric heating plate is set to a temperature of 150 ℃.

Preferably, the water-soluble protein extraction process of the standard soybean meal sample in step S1 is as follows: grinding and crushing a soybean sample to prepare a 30g standard soybean powder sample, weighing 1g standard soybean powder sample, adding 10mL of ultrapure water, performing ultrasonic treatment for 30min under the rotation condition of 10000 revolutions, centrifuging for 5min, extracting 1mL of supernatant protein extract, adding the supernatant protein extract into a 3K ultrafiltration centrifugal tube, centrifuging to obtain proteins with molecular weights of more than 3K Da respectively, transferring the centrifuged proteins into a sample injection bottle, and freeze-drying for more than 8 hours until all the proteins are dried to obtain solid protein powder of water-soluble proteins; the isotope mass spectrometer is one of an on-line gas analysis-stable isotope mass spectrometer GasBench-IRMS, an element analysis-isotope mass spectrometer EA-IRMS, a gas chromatography-isotope mass spectrometer GC-IRMS and a liquid chromatography-isotope mass spectrometer LC-IRMS.

The method for acquiring stable isotope ratio data of the water-soluble protein of the standard soybean meal sample in the step S2 comprises the following steps:

a2. tuning a correction element analysis-isotope mass spectrometer EA-IRMS;

a3. introducing water-soluble protein wrapped by tin cups into a 980 ℃ high-temperature combustion furnace to obtain CO₂And N₂Gas of CO₂And N₂Introduction into isotope mass spectrometer host for delta in water-soluble protein¹⁵N and delta¹³C, detecting; introducing water soluble protein wrapped by silver cup into 1380 deg.C pyrolysis furnace to obtain CO and H₂Gas, CO and H₂Introduction into isotope mass spectrometer host for water-soluble protein delta²H and delta¹⁸And (4) detecting.

Preferably, in the step a2, the method for tuning the calibration elemental analysis-isotope mass spectrometer EA-IRMS is as follows: introducing high-purity CO, H2, N2 and CO2 into the elemental analysis-isotope mass spectrometer EA-IRMS as reference gases, and then using the known delta²H、δ¹⁸O、δ¹⁵N and delta¹³And C, calibrating the delta value of the stable isotope of the reference gas by using the standard substance with the C value, and using the delta value of the stable isotope calibrated by the reference gas as a detection standard to correct the delta value of the stable isotope in the water-soluble protein of the standard soybean powder sample detected in the step a3.

Preferably, the processing and analysis of the data during calibration is performed by Isodat3.0 software of Thermo Scientific, USA.

Preferably, the method for determining the target compound of step S3 includes: according to the molecular weight range of the target object in the IDA-MS high-resolution mass spectrum data, dividing the IDA-MS high-resolution mass spectrum data in the standard soybean oil sample into 3 regions: a first region with the molecular weight of 800-;

the step S4 further includes an optimization step of the soybean origin tracing and identifying model, where the optimization step includes: and determining partial triglyceride compounds with large contribution degree in the target compounds through the VIP value of the soybean origin tracing identification model, and deleting abnormal value samples exceeding 99% confidence intervals and all soybean oil samples in the area with small number in the identification model according to hotelling's and DModx indexes so as to optimize the soybean origin tracing identification model. And determining partial mineral elements with large contribution degree in the standard soybean powder sample through a VIP value of a soybean origin tracing identification model, wherein the VIP value is a variable projection importance analysis value, selecting 27 elements as optimized characteristic contribution degree elements, and establishing the optimized soybean origin tracing identification model based on LC-Q-TOF-MS, mineral element content and stable isotope ratio analysis combination by using the optimized triglyceride targeting compound and the characteristic contribution degree element data.

Preferably, the triglyceride compounds with large VIP value contribution degree are selected from the triglyceride compounds with the molecular weight range of 766-930Da as the preferred triglyceride targeting compounds, and comprise the triglyceride compounds with the following molecular weights: 873.6967, 875.7123, 851.7123, 877.7280, 853.7280, 865.7280, 913.7280, 829.7280, 915.7436, 767.6184, 835.6810, 879.7436, 917.7593, 859.7749, 855.7436, 895.779, 825.6967, 921.7906, 887.8062 and 869.7593; the optimized soybean characteristic contribution elements are Sr, As, Ni, Cd, Mo, Se, Cs, Cr, Mn, Tl, Zr, Co, Be, U, V, Al, Eu, Na, Ce, Cu, Nd, P, Gd, Sn, Sc, I and Sm elements.

Preferably, the step S4 further includes a blind sample verification step, where the blind sample verification step includes: selecting a plurality of soybean verification samples in the same area, respectively squeezing the soybean verification samples in a definite area to obtain a soybean oil sample to be verified and water-soluble protein of the soybean oil sample to be verified after grinding and extraction, acquiring IDA-MS high-resolution mass spectrum data of the soybean oil sample to be verified by a liquid chromatogram-quadrupole time-of-flight mass spectrometer, acquiring mineral element content data of the soybean oil sample to be verified by a mineral element content analyzer, and acquiring water-soluble protein stable isotope ratio data of the soybean oil sample to be verified by an isotope mass spectrometer; and simultaneously introducing IDA-MS high-resolution mass spectrum data, mineral element content data and water-soluble protein stable isotope ratio data of the soybean verification sample into the soybean origin tracing and identifying model in the step S4, and verifying the origin tracing accuracy of the soybean verification sample.

Preferably, in step S4, the triglyceride compound marker observed peak data, the mineral element content data of the standard soybean flour sample, and the stable isotope ratio data of the water-soluble protein of the standard soybean flour sample of the same soybean are simultaneously introduced into the modeling software for the orthogonal partial least squares regression analysis, so as to construct the OPLS-DA soybean origin tracing identification model based on LC-Q-TOF-MS and the combination of the mineral element content and the stable isotope ratio analysis.

Preferably, in the process of preparing the standard sample in step S1, the soybean samples are from n different regions, the soybean samples are divided into n × (n-1)/2 groups, each group of soybean samples consists of two soybean samples from different origins, and each group of soybean samples is used for establishing a two-country soybean origin tracing identification model according to steps S2, S3 and S4, so as to identify the corresponding soybean country or region in the two-country soybean origin tracing identification model.

Preferably, in the preparation of the standard sample in step S1, the soybean samples originate from at least three different countries or regions, so that the step S4 establishes a multi-country soybean origin tracing model.

Preferably, in the preparation process of the standard sample in step S1, the soybean sample is from usa, brazil, argentina, canada, yerba mate, russia, and china, and a traceability model of soybean origin in usa-brazil, usa-argentina, usa-canada, usa-yerba mate, usa-russia, and usa-china is established according to steps S2, S3, and S4 in sequence, and is used for identifying the soybean origin of soybean origin in usa and other countries.

Has the advantages that:

according to the soybean origin tracing identification method based on the combination of the IRMS, the LC-Q-TOF-MS and the multi-element analysis technology, IDA-MS high-resolution mass spectrum data of a soybean oil sample to be detected is referred, MarkerView Peaks data of a triglyceride compound is obtained through analysis software, MarkerView Peaks data of the triglyceride compound in the soybean oil sample are subjected to preprocessing, mineral element content data in the soybean powder sample and stable isotope ratio data of water-soluble protein in the soybean powder sample are fused to establish a soybean origin tracing identification model, the three data collected by the sample to be detected are introduced into the soybean origin tracing identification model, origin tracing prediction of the soybean to be detected is carried out, and the accuracy of soybean origin tracing is improved; LC-Q-TOF-MS utilizes the high resolution mass spectrum data fusion of less triglyceride targeting compounds other two kinds can establish the multivariate statistical analysis discrimination prediction model, and further improves the accuracy of the soybean origin tracing identification by combining the multi-country and two-country soybean origin tracing identification models. Compared with a method for singly using stable isotope ratio tracing, the method for identifying the American soybean has the advantages that the accuracy is high, is improved by more than 10 percent, and is also improved compared with the accuracy of singly using an LC-Q-TOF-MS tracing method, so that the method can effectively identify the source between the American soybean and other main soybean producing countries, and avoids the situation that the American soybean is mixed with the soybeans of other countries and then enters China.

Drawings

FIG. 1 shows a LC-Q-TOF-MS-based multi-national OPLS-DA soybean origin tracing and identifying model before optimization;

FIG. 2 is a graph showing the VIP value distribution of triglyceride compounds in a LC-Q-TOF-MS-based multi-national OPLS-DA soybean provenance identification model before optimization;

FIG. 3 shows an optimized LC-Q-TOF-MS-based multi-national OPLS-DA soybean origin tracing and identification model;

FIG. 4 shows an OPLS-DA multi-national soybean origin tracing and identification model based on mineral content analysis before optimization;

FIG. 5 is a graph showing a VIP value distribution of mineral elements in an OPLS-DA multi-national soybean provenance identification model based on mineral element content analysis;

FIG. 6 shows an optimized OPLS-DA multi-national soybean origin tracing and identification model based on mineral content analysis;

FIG. 7 shows an isotope ratio-based multi-national OPLS-DA soybean origin tracing identification model established with water-soluble protein of soybean flour samples as a target object;

FIG. 8 shows a Brazil-American two-country OPLS-DA soybean origin tracing identification model based on combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology

FIG. 9 shows a traceability identification model of soybean origin of Argentina-American two countries OPLS-DA based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology;

FIG. 10 shows a Canada-United states two-country OPLS-DA soybean origin tracing identification model based on IRMS, LC-Q-TOF-MS and multi-element analysis technology;

FIG. 11 shows a Russian-American two-country OPLS-DA soybean origin tracing identification model based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technologies;

FIG. 12 shows a traceability identification model of OPLS-DA soybean origin of China-United states based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology;

FIG. 13 shows a stable isotope ratio-based multi-national OPLS-DA soybean origin tracing identification model established with soybean meal samples as target targets;

FIG. 14 shows a stable isotope ratio-based multi-national OPLS-DA soybean origin tracing identification model established with soybean oil samples as target targets;

FIG. 15 shows a PCA identification model and an OPLS-DA identification model of triglyceride compounds of soybean oil samples constructed based on three solvents.

Detailed Description

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following description will be made with reference to the accompanying drawings. It is obvious that the drawings in the following description are only some examples of the invention, and that for a person skilled in the art, other drawings and embodiments can be derived from them without inventive effort.

The technical solution of the present invention is described in detail with specific examples below.

The experimental device in the soybean oil sample data acquisition process includes: the experimental device comprises: selecting a Triple TOF5600+ high resolution mass spectrometer of ABsciex corporation of America; HPLC 20AD high performance liquid chromatograph of Shimadzu corporation, Japan; an Xbridge BEH C18 column (100 mm. times.2.1 mm,3 μm) from Waters, USA; VORTEX 4 VORTEX mixer from IKA, germany; and an oil press;

selecting a reagent: methanol, acetonitrile, acetone, ethyl acetate (chromatographically pure, semer feishel, usa);

the experimental device in the soybean powder sample data acquisition process comprises: ICP 5300 inductively coupled plasma emission spectrometer (PE corporation, usa); elane-drc-e inductively coupled plasma mass spectrometer (PE company, USA); an ETHosA type microwave digestion instrument (Milestone, Italy); ultra WAVE super microwave digestion platform (Milestone, italy); DS-360 graphite digestion instrument (cantonese analysis testing center); Milli-Q ultrapure water system (resistivity 18.2M Ω · cm); all glassware was treated with 20% HNO₃Soaking for 24h, repeatedly washing with water, and finally washing with deionized water for later use.

IV-ICP-MS-71A standard solution (10. mu.g/mL, inorganic vents); CCS-5 Standard solution: (100. mu.g/mL, inorganic vents); GNM-M292793-2013: ICP measurement with multi-element standard solution: national nonferrous metals and analysis testing center; nitric acid: superior grade pure, manufactured by Suzhou Crystal Ray, Inc. Mixed internal standard of Rh, Re and In: (100. mu.g/mL, inorganic vents); water: preparing by an ultrapure water machine (18.3M omega. Cm); other used reagents are superior pure; nitrogen gas: more than 99.99 percent.

Instruments used in data acquisition of soy water-soluble protein: flash EA2000 elemental analysis(iii) instrument, DeltaV Advantage isotope mass spectrometer and ConFlo IV interface (Thermo Scientific, USA); Milli-Q ultra pure water instruments (Millipore, USA); model SW 30H ultrasound (SWISS SONO SWISS); SIGMA3-30KS high speed refrigerated centrifuge (Sigma centrifuge, Germany); sartorius BT 223S thousandth balance (Sartorius, germany); an oil press; a vacuum freeze dryer (Alpha 1-2LD PLUS, Christ, Germany); a pulverizer (Shanghai Jiading grain and oil Co., Ltd., JL 064); silver cups (company iva alalysentechnik, germany); tin cups (Sammer. USA); he gas (purity more than 99%) as carrier gas, and CO₂Standard reference gas (purity greater than 99.9995%), CO standard reference gas (purity greater than 99.9995%), and H₂Standard reference gas (purity greater than 99.9995%); n is a radical of₂Standard reference gas (purity greater than 99.9995%); ultrafiltration centrifuge tubes (Millipore, USA, Centrifugal Filter Units, 3K/10K).

EA-IRMS uses carbon and nitrogen isotope standard substances: IAEA-600 caffeine: delta¹³C＝-27.771‰，δ¹⁵N ═ 1.00 ‰, based on V-PDB, purchased from international atomic energy agency; EA-IRMS with hydrogen-oxygen isotope standard substance: EMA-P1 polyamide: delta²H＝-25.3‰，δ¹⁸20.99% O based on V-SMOW, available from elementary Microanalysis, england; the experimental water was ultrapure water, and the impedance value was 18.2 M.OMEGA.cm.

Sources of soybean and soybean oil samples: the soybean standard sample is obtained from 807 soybean samples collected by the related directly-affiliated customs and foreign purchase of the whole country, wherein the quantity of the soybean samples of each production place is as follows: wherein 152 of the us samples, 423 of the brazil samples, 96 of the canada samples, 68 of the argentina samples, 14 of the yerba mate samples, 25 of the russian samples, and 29 of the chinese samples; soybean oil samples 334: of these, 36 us samples, 226 brazil samples, 7 ukraine samples, 46 argentina samples, 1 mexico sample, and 16 russian samples.

The soybean origin tracing and identifying method based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology comprises the following steps:

s4, establishing a soybean producing area traceability identification model: after triglyceride targeting compounds in standard soybean oil samples are determined, the IDA-MS high-resolution mass spectrum data is analyzed through analysis software to obtain triglyceride compound marker observed peak data of the standard soybean oil samples, triglyceride compound marker observed peak data in the standard soybean oil samples of the same producing area, mineral element content data of the standard soybean powder samples and stable isotope ratio data of water-soluble proteins of the standard soybean powder samples are simultaneously introduced into modeling software to be processed in one or more modes of a main component analysis method, a partial least square method-discriminant analysis method and an orthogonal partial least square method regression analysis method, and triglyceride compound marker observed peak data of the standard soybean oil samples of different producing areas, mineral element content data of the standard soybean powder samples, a sample identification method, a, And introducing the stable isotope ratio data of the water-soluble protein of the standard soybean powder sample into modeling software to obtain characteristic distribution rules of soybean oil in the standard soybean oil samples of different producing areas, mineral elements of the standard soybean powder sample and the stable isotope distribution rules of the water-soluble protein of the standard soybean powder sample, and constructing a soybean producing area traceability identification model based on the combination of LC-Q-TOF-MS, mineral element content and stable isotope ratio analysis; the marker observation peak data is MarkerView peaks data, the MarkerView peaks data of triglyceride of the standard soybean oil sample is obtained by the analysis of MasterView analysis software,

Multi-country OPLS-DA soybean origin tracing and identifying model example 1

Selecting soybean samples of different producing areas with definite regions, squeezing the soybean samples of each producing area to obtain soybean oil samples, grinding to obtain standard soybean powder samples, extracting water-soluble protein of the standard soybean powder samples to be used as a target object, diluting the soybean oil samples by 200 times by using a diluting solvent to obtain the standard soybean oil samples, selecting a methanol-ethyl acetate mixed solution as the diluting solvent, wherein the ratio of methanol to ethyl acetate in the diluting solvent is 1: 1.

Collecting standard soybean oil sample quality spectrum data:

placing the diluted standard soybean oil sample into a sample injector of the liquid chromatogram-quadrupole time-of-flight mass spectrometer, performing separation analysis on the standard soybean oil sample by a liquid chromatograph in the liquid chromatograph-quadrupole time-of-flight mass spectrometer, then performing mass spectrum data acquisition of the standard soybean oil sample by a mass spectrometer in the liquid chromatogram-quadrupole time-of-flight mass spectrometer, respectively obtaining primary mass spectrum information and secondary mass spectrum information of the standard soybean oil sample through primary TOF-MS scanning and secondary IDA-MS scanning of a mass spectrometer, wherein the secondary mass spectrum information is IDA-MS high-resolution mass spectrum data, determining a triglyceride compound targeting compound of the standard soybean oil sample by IDA-MS high resolution mass spectrometry data, the method for determining the targeting compound comprising: according to the molecular weight range of the target object in the IDA-MS high-resolution mass spectrum data, dividing the IDA-MS high-resolution mass spectrum data in the soybean oil sample into 3 regions: the method comprises a first region with the molecular weight of 800-1000, a second region with the molecular weight of 550-800 and a third region with the molecular weight of less than 550, wherein the first region and the second region are soybean oil lipid metabolism characteristic regions, animal and plant derived triglyceride compounds with the molecular weight range of 700-950 in a lipid compound database and soybean oil triglyceride compounds in the first region and the second region are matched and screened, and 114 soybean oil triglyceride compounds with the molecular weight range of 766-920Da are determined to be used as lipidomic analysis target compounds, and the method is applied to identification model research of the origin of the lipidosome origin in the soybean oil.

The lipid compound database is a lipid compound database LIPID MAPS Lipidomics Gateway published in the United states, the standard triglyceride compound data is 6899 triglyceride compounds of Triradylglycerides in the lipid compound database LIPID MAPS Lipidomics Gateway, and 116 common triglyceride compounds in a soybean oil sample are determined to be the triglyceride compound targeting compounds of the standard soybean oil sample through qualitative analysis of PeakView software.

The liquid chromatogram condition of the liquid chromatograph in the liquid chromatogram-quadrupole time-of-flight mass spectrometer is as follows: the flow rate is 0.5. mu.L/min, the column temperature is 40 ℃, Xbridge BEH C18 chromatographic column gradient elution is carried out, and the sample volume is 2. mu.L; the A phase in the mobile phase is isopropanol, and the B phase in the mobile phase is acetonitrile, wherein the content of the B phase in the mobile phase in different time periods is as follows: 0min, 70% B; 0-5min, 70-65% B; 5-8min, 65% B; 10-10.5min, 65-70% B; 10.5-15min, 70% B.

The mass spectrum condition of the quadrupole flight time of the mass spectrometer in the liquid chromatogram-quadrupole flight time mass spectrometer is as follows: the mass spectrometer adopts a positive ion mode to collect data, and the ion source is as follows: ESI and APCI complex sources; the positive ion scanning mode is as follows: APCI source connection automatic correction system, first grade TOF-MS scans accurate mass range: 100-2000 Da, 100ms of data acquisition time, 100V of DP and 10V of CE; secondary IDA-MS scan accurate mass range: 50-2000 Da, DP:100V, CE:35 +/-15V; the mass spectrometer adopts a high-sensitivity mode, the data acquisition time is 50ms, the signal threshold is 100cps, data are acquired for 6 times in each circulation, and dynamic background subtraction is adopted.

The liquid chromatogram-quadrupole time-of-flight mass spectrometer is an Shimadzu LC20AD liquid chromatograph, the mass spectrometer is a Triple TOF5600+ mass spectrometer, the automatic correction system is a CDS system, and the conditions of the quadrupole time-of-flight mass spectrometer further include: the calibration is carried out for 1 time per 10 samples, the flow rate of APCI positive ion calibration solution is 0.3mL/min, and the pressure of an air curtain is as follows: 40psi, ion source atomization gas pressure: at 50psi, ion source assist gas pressure: 50psi, ion source temperature: the method comprises the steps of collecting all IDA-MS high-resolution mass spectrum data collected by a mass spectrometer at 500 ℃, collecting the IDA-MS high-resolution mass spectrum data by analysis TF 1.6 software of ABSciex company, qualitatively and quantitatively processing and analyzing the IDA-MS high-resolution mass spectrum data by PeakView and MasterView software, introducing the IDA-MS high-resolution mass spectrum data into SIMCA14.0 software (Umetrics company, Switzerland), and carrying out orthogonal partial least squares regression analysis on triglyceride compound marker observation peak data in a standard soybean oil sample to obtain the distribution rules of triglyceride and metabolites of soybeans of different origins.

Collecting mineral element content of a standard soybean powder sample:

weighing 0.5g of standard soybean meal sample into an inner tank of a microwave digestion tank, adding 4ml of nitric acid, covering the inner tank cover, and soaking for 6-12 hours; after soaking, screwing an outer tank of a microwave digestion tank, placing the outer tank into a microwave digestion instrument for digestion, setting a temperature raising program for digestion of a standard soybean powder sample in the digestion instrument, taking out an inner tank after digestion and cooling, driving up acid on an electric heating plate, transferring digestive juice in the inner tank into a 50mL polypropylene centrifugal tube after acid driving, washing the inner tank of the microwave digestion tank for many times by using a small amount of deionized water, combining washing liquids, fixing the volume of a mixed liquid of the digestive juice in the polypropylene centrifugal tube and the deionized water to 25mL by using the deionized water, uniformly mixing for later use, simultaneously performing a blank contrast test, and then determining and analyzing the mineral element content in the standard soybean powder sample by using ICP-MS and/or ICP-AES through a mineral element content analyzer.

The blank comparison tests comprise two groups, wherein 25mL of deionized water is placed in a polypropylene centrifuge tube in one group of blank comparison tests; another set of blank controls contained 25mL of nitric acid in polypropylene centrifuge tubes.

The microwave digestion tank heating program in the microwave digestion instrument comprises the following steps as shown in table 1:

a1. heating the microwave digestion instrument to 120 ℃ within 8min, and then keeping the temperature of 120 ℃ for 5 min;

a2. the temperature of the microwave digestion apparatus was then increased from 120 ℃ to 180 ℃ over 10min, and then maintained at 180 ℃ for 10 min.

TABLE 1 microwave digestion tank temperature rise program in the microwave digestion instrument

Step (ii) of	Temperature/. degree.C	Temperature rise time/min	Retention time/min
				1	120	8	5
2	180	10	10

The inductively coupled plasma emission spectrometry ICP-AES has the working conditions that: the detection wavelength is 396.152 nm; the power is 1500W; plasma gas flow rate: 10.0L/min; the auxiliary gas flow is 0.55L/min; flow rate of atomizing gas: 0.55L/min; the rotating speed of the peristaltic pump is 50r/min, and the observation mode is as follows: axial direction; carrier gas: 99.996% high purity argon;

the operating conditions of inductively coupled plasma mass spectrometry ICP-MS are as follows: the mode adopted is Standard (STD); argon (more than or equal to 99.998 percent, high purity); 20, scanning/reading; reading/repetition number is 1; the repetition times are 3; the flow rate of atomizing gas is 0.84L/min; the auxiliary air flow is 1.2L/min; the plasma gas flow rate is 1500L/min; ICP radio frequency power is 1500W; peristaltic pump 20 rpm. Sampling cone and intercepting cone: a platinum cone.

The content of mineral elements in the standard soybean flour sample is determined by ICP-AES and ICP-MS, wherein the ICP-AES is used for detecting 13 major elements such as Si, Mg, Al, P, K, Na, Ca, Mn, Fe, Cu, Sr and Zn in the standard soybean flour sample, and the ICP-MS is used for detecting and analyzing other 39 trace elements. ICP-AES and ICP-MS are used for detecting and analyzing to obtain 52 kinds of mineral element content data, an excel list is summarized and sorted by describing a statistical analysis method, and a primary identification model is constructed by introducing the excel list into SIMCA 14.1 software (Umetrics, Switzerland) for main component analysis, partial least square method discriminant analysis and orthogonal partial least square method discriminant analysis. In this embodiment, 6 major elements, such as Ca, Mg, P, Si, Mn, and Zn, are further selected, and content correlation between two elements in the soybean sample is discussed, where a correlation coefficient between Mg and Ca in the soybean sample is 0.81, a correlation coefficient between P and Si in the soybean sample is 0.72, and a correlation coefficient between Zn and Mn in the soybean sample is 0.36, so that it can be known that the major elements with relatively high content in the soybean sample have intrinsic consistency, especially the major elements Mg and Ca in the soybean sample, and thus, the content of mineral elements in the soybeans in different countries and regions can be presumed to have biological intrinsic consistency, so that the soybeans in different producing areas can be identified and distinguished by mineral elements.

In the determination process of the mineral element content, for the accuracy of data, a soybean sample is optimized, some modeling sample data in the preliminarily constructed origin and origin tracing identification model based on the mineral element content have a remarkable outlier, meanwhile, the origin tracing model parameter index Hotelling's T2 value also shows that some modeling data are abnormal value data, and abnormal value data need to be removed according to the mathematical modeling technical route principle, so that the origin tracing identification model for the multi-element analysis of the soybean is constructed, the abnormal value data are deleted, and the origin tracing identification model is optimized.

The water soluble protein extraction procedure for the standard soy flour sample was as follows: grinding and crushing a soybean sample to prepare a 30g standard soybean powder sample, weighing 1g standard soybean powder sample, adding 10mL of ultrapure water, performing ultrasonic treatment for 30min under the rotation condition of 10000 revolutions, centrifuging for 5min, extracting 1mL of supernatant protein extract, adding the supernatant protein extract into a 3K ultrafiltration centrifugal tube, centrifuging to obtain proteins with molecular weights of more than 3K Da respectively, transferring the centrifuged proteins into a sample injection bottle, and freeze-drying for more than 8 hours until all the proteins are dried to obtain solid protein powder of the water-soluble proteins.

The isotope mass spectrometer is an on-line gas analysis-stable isotope mass spectrometer GasBench-IRMS, an element analysis-isotope mass spectrometer EA-IRMS, a gas chromatography-isotope mass spectrometer GC-IRMS, a liquid chromatography-homogeneous mass spectrometerOne of the site-specific mass spectrometers LC-IRMS. In the present example, an elemental analysis-isotope mass spectrometer EA-IRMS was selected to analyze the stable isotope ratio of the soybean water-soluble protein. The stable isotope ratio analysis of the soybean water-soluble protein is realized by an element analyzer and a tandem isotope mass spectrometer. EA-IRMS allows the high-precision determination of the average composition of stable isotopes of hydrogen, carbon, oxygen, nitrogen, sulfur, etc. in organic or inorganic samples. The EA-IRMS method comprises wrapping sample in tinfoil, weighing, directly placing into EA automatic sample injector, and converting into NO by high temperature combustion in combustion tube_x，CO₂，SO₂Or H₂O, and the like. According to the detection requirement, the mixed gas is separated by a gas chromatographic column and the like to remove interfering gas, and the target gas finally enters an isotope mass spectrometer for detection. In the carbon isotope mass spectrometry, the mixed gas generated by the combustion of the sample enters a reduction tube along with the helium flow, and NO is contained in the reduction tube_xConversion to N₂Excess of O₂Is removed, and then the mixed gas is passed through a drying tube to remove H₂O, then realizing CO through a gas chromatographic column₂And N₂To obtain single CO₂A gas.

The method for acquiring the stable isotope ratio data of the water-soluble protein of the standard soybean powder sample comprises the following steps:

a1. selecting an elemental analysis-isotope mass spectrometer EA-IRMS, placing weighed solid protein powder of the water-soluble protein in a tin cup and a silver cup, and respectively detecting the isotope ratio of stable carbon and nitrogen and the isotope ratio of stable hydrogen and oxygen in the water-soluble protein;

a2. tuning a correction element analysis-isotope mass spectrometer EA-IRMS;

a3. putting the water-soluble protein wrapped by the tin cup into an EA automatic sample injector, and introducing the water-soluble protein into a 980 ℃ high-temperature combustion furnace to obtain CO₂And N₂Gas of CO₂And N₂Introducing helium mixture into isotope mass spectrometer host to perform delta in water-soluble protein¹⁵N and delta¹³C, detecting; introducing water soluble protein wrapped by silver cup into 1380 deg.C pyrolysis furnace to obtain CO and H₂Gas, CO and H₂Introducing mixed helium into isotope mass spectrometer host to perform delta of water-soluble protein²H and delta¹⁸And (4) detecting.

In the step a2, the method for tuning the correction element analysis-isotope mass spectrometer EA-IRMS is as follows: introducing high-purity CO, H2, N2 and CO2 as reference gases and introducing high-purity He gas as a drainage gas into the elemental analysis-isotope mass spectrometer EA-IRMS, and then using the known delta²H、δ¹⁸O、δ¹⁵N and delta¹³And C, calibrating the delta value of the stable isotope of the reference gas by using the standard substance with the C value, and using the delta value of the stable isotope calibrated by the reference gas as a detection standard to correct the delta value of the stable isotope in the water-soluble protein of the standard soybean powder sample detected in the step a3. The processing and analysis of the data during calibration was performed by Isodat3.0 software from Thermo Scientific, USA. Known as delta²H、δ¹⁸O、δ¹⁵N and delta¹³The standard substance of the C value is: carbon and nitrogen isotope standard substance IAEA-600 caffeine: delta¹³C＝-27.771‰，δ¹⁵N ═ 1.00 ‰, based on V-PDB, purchased from international atomic energy agency; hydroxide isotope standard EMA-P1 polyamide: delta²H＝-25.3‰，δ¹⁸O20.99 per mill based on V-SMOW.

Element analysis-isotope mass spectrometer EA-IRMS isotope mass spectrum is different from organic mass spectrum for conventionally determining molecular weight of target object in sample by CO₂、H₂、CO、N₂And indirectly detecting the small molecular compound to determine the isotopic abundance ratio of carbon, hydrogen, oxygen and nitrogen. By CO₂Detection of water-soluble protein delta of standard soybean meal sample by target gas¹³C value, H₂Detection of water-soluble protein delta of standard soybean meal sample by target gas²Detection of water-soluble protein delta of standard soybean powder sample by H and CO target gas¹⁸O，N₂Determination of the Delta of Water-soluble proteins of Standard Soybean flour samples with target gas¹⁵N。

Collecting delta of water-soluble protein of standard soybean powder sample by element analysis-isotope mass spectrometer EA-IRMS²H、δ¹⁸O、δ¹⁵N and delta¹³And C value is obtained by describing a statistical analysis method, summarizing and sorting an excel list, introducing the excel list into SIMCA 14.1 software, and performing treatment in an orthogonal partial least square regression analysis method mode to obtain the distribution rules of the soybean isotopes in different production areas, thereby constructing an isotope identification model for tracing the soybean production areas. As shown in FIG. 7, the delta of water soluble protein for the standard soy flour sample was selected²H、δ¹⁸O、δ¹⁵N and delta¹³The result of the multi-country identification model of OPLS-DA constructed by C shows that the discrimination of soybean sample producing areas in the countries such as Brazil, America, Argentina, Uraguay and Canada is relatively good. By 4 variables delta to water soluble protein in soybean²H、δ¹⁸O、δ¹⁵N and delta¹³C analysis of the degree of contribution to the differentiation of the producing area, delta¹⁵N contributes most to the source tracing of the place of origin, followed by delta²H and delta¹⁸O，δ¹³C contributes the least.

In the embodiment, Brazil soybean samples, American soybean samples, Chinese soybean samples, Argentina soybean samples, Canadian soybean samples, Utray soybean samples and Russian soybean samples with specific regions are selected to prepare standard samples, IDA-MS high-resolution mass spectrum data of soybeans in different regions in the standard samples are respectively collected through a liquid chromatogram-quadrupole flight time mass spectrometer, and triglyceride compound MarkerView peaks data of the soybean samples in different production regions are obtained after qualitative and quantitative processing and analysis are carried out on PeakView and MasterView software; and collecting mineral element content of the optimized standard soybean powder sample by ICP-MS and ICP-AES, and collecting delta of water-soluble protein of the standard soybean powder sample by element analysis-isotope mass spectrometer EA-IRMS²H、δ¹⁸O、δ¹⁵N and delta¹³C value, the processed triglyceride compound marker observation peak data, mineral element content data and stable isotope ratio data of water-soluble protein are subjected to induction and arrangement of an excel list, SIMCA 14.1 software is introduced for orthogonal partial least squares regression analysis, and thus soybean origin tracing identification based on combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology is constructedAnd (4) modeling.

As can be seen from fig. 1, the LC-Q-TOF-MS-based multi-national OPLS-DA soybean origin tracing and identification model is established independently, soybean samples in brazil, russia, argentina and usa origins can be distinguished significantly, and the soybean and soybean oil origin tracing and identification model needs to be further optimized to avoid that the samples in part of countries or regions are distributed together in the identification model to affect the prediction and identification accuracy of the model, and therefore the soybean and soybean oil origin tracing and identification model needs to be further optimized, wherein the optimization steps include: and determining partial triglyceride compounds with large contribution degree in the target compounds through VIP values of the soybean origin tracing identification model, and deleting abnormal value samples exceeding 99% confidence intervals and all soybean oil samples in the areas with small number in the identification model according to hotelling's and DModx indexes so as to optimize the soybean and soybean oil origin tracing identification model. As shown in FIG. 2, the VIP values of the identification models are used for determining which variables have large contribution degrees, and finally triglyceride compounds with molecular weights of 873.6967, 875.7123, 851.7123, 877.7280, 853.7280, 865.7280, 913.7280, 829.7280, 915.7436, 767.6184, 835.6810, 879.7436, 917.7593, 859.7749, 855.7436, 895.779, 825.6967, 921.7906, 887.8062 and 869.7593 are determined to have large contribution degrees on the traceability identification models of the production places of the multi-country OPLS-DA soybean and soybean oil, and the traceability identification models of the production places of the OPLS-DA soybean and the soybean oil are optimized according to hotelling's and DModx indexes and deleting a small number of Chinese and Uray samples. As shown in fig. 3, the optimized lipidomics-based multi-country OPLS-DA soybean origin tracing and identifying model can significantly distinguish samples of different countries, especially samples of brazil, russia, usa and other origins;

as shown in fig. 5, Sr, As, Ni, Cd, Mo, Se, Cs, Cr, Mn, Tl, Zr, Co, Be, U, V, Al, Eu, Na, Ce, Cu, Nd, P, Gd, Sn, Sc, I, and Sm elements in the soybean sample are elements with a large contribution degree, these 27 elements are selected As optimized characteristic contribution degree elements, As shown in fig. 4 and 6, the optimized mineral element content-based multinational OPLS-DA soybean origin-location traceability model can significantly distinguish samples of different countries, especially, brazilian and non-brazilian soybean samples; and samples from brazil, russia, usa, argentina, and canadian origins.

Therefore, the final soybean origin tracing identification model based on LC-Q-TOF-MS, mineral element content and stable isotope ratio analysis is composed of the optimized triglyceride targeting compound, characteristic contribution degree mineral element data and stable isotope delta¹⁵N、δ²H、δ¹⁸O and delta¹³C is constructed. It is capable of distinguishing significantly between samples of soybean from brazil, russia, usa, argentina and canadian origins.

After the multi-country OPLS-DA soybean origin tracing and identifying model is established, blind sample verification needs to be carried out on the model, and the blind sample verification steps comprise: selecting a plurality of soybean verification samples in the same area, respectively squeezing the soybean verification samples in a definite area to obtain a soybean oil sample to be verified and water-soluble protein of the soybean oil sample to be verified after grinding and extraction, acquiring IDA-MS high-resolution mass spectrum data of the soybean oil sample to be verified by a liquid chromatogram-quadrupole time-of-flight mass spectrometer, acquiring mineral element content data of the soybean oil sample to be verified by a mineral element content analyzer, and acquiring water-soluble protein stable isotope ratio data of the soybean oil sample to be verified by an isotope mass spectrometer; and simultaneously introducing IDA-MS high-resolution mass spectrum data, mineral element content data and water-soluble protein stable isotope ratio data of the soybean verification sample into the optimized soybean origin tracing and identifying model, and verifying the origin tracing accuracy of the soybean verification sample.

Respectively squeezing a soybean sample to be detected to obtain a soybean oil sample, grinding the soybean oil sample to be detected to obtain a soybean oil sample to be detected, extracting the soybean oil sample to be detected to obtain soybean water-soluble protein to be detected, diluting the soybean oil sample by a diluent by 100-200 times step by step to obtain the soybean oil sample to be detected, collecting IDA-MS high-resolution mass spectrum data of the soybean oil sample to be detected by a base liquid chromatography-quadrupole flight time mass spectrometer, collecting mineral element content data of the soybean oil sample to be detected by a mineral element content analyzer, and collecting stable isotope ratio data of the soybean water-soluble protein to be detected by an isotope mass spectrometer; and simultaneously introducing IDA-MS high-resolution mass spectrum data, mineral element content data and stable isotope ratio data of the soybean sample to be detected into the optimized soybean origin tracing and identifying model, and performing origin tracing prediction on the soybean to be detected.

Multi-country soybean producing area tracing and identifying model example 2

This example describes only the differences from the above example, in this example, the mineral content data of the standard soybean flour sample was collected by: the method for acquiring the mineral element content data of the standard soybean powder sample in the step S2 comprises the following steps: weighing 0.5g of standard soybean powder sample, placing the standard soybean powder sample in a polyethylene digestion tube, weighing 0.5g of standard soybean powder sample, accurately weighing 0.001g, adding 4ml of nitric acid, covering a sealing cover, and soaking for 6-12 hours; after soaking, placing the sealed polyethylene digestion pipe in a digestion instrument, pre-adding inert gas into an inner tank body of the digestion instrument, wherein the pressure of the inert gas is 4000KPa, setting a temperature-raising program to digest a standard soybean powder sample in the digestion instrument, taking out the inner tank after cooling after digestion, driving acid on an electric hot plate, and setting the temperature of the electric hot plate to be 150 ℃. And after acid dispelling is finished, transferring the digestive juice in the inner tank to a 50mL polypropylene centrifuge tube, washing the inner tank of the microwave digestion tank for multiple times by using a small amount of deionized water, combining washing solutions, fixing the volume of a mixed solution of the digestive juice and the deionized water in the polypropylene centrifuge tube to 25mL by using the deionized water, uniformly mixing for later use, simultaneously performing a blank comparison test, and determining and analyzing the mineral element content in the standard soybean powder sample by using an ICP-MS and/or ICP-AES (inductively coupled plasma-atomic emission Spectrometry) analyzer.

The inert gas is nitrogen, and the temperature of an outer cavity of the digestion instrument is less than or equal to 40 ℃.

The temperature program of the polyethylene digestion tube in the digestion instrument comprises the following steps as shown in table 2:

a1. under the conditions of 15000KPa pressure and 1500W power, the temperature of the microwave digestion instrument is increased to 120 ℃ within 8 min;

a2. then under the conditions of 15000KPa pressure and 1500W power, the temperature of the microwave digestion instrument is increased from 120 ℃ to 240 ℃ within 8 min;

a3. further, the temperature of 240 ℃ was maintained for 20min under the conditions of 15000KPa pressure and 1500W power.

TABLE 2 super microwave temperature program

Two-country soybean producing area tracing and identifying model example 1

In this embodiment, assuming that the soybean samples are from n different regions during the preparation of the standard soybean oil sample in step S1, the soybean samples are divided into n × (n-1)/2 groups, each group of soybean samples consists of two soybean samples with different origins, and a two-country soybean origin traceability system model is established for identifying the corresponding soybean country or region in the two-country soybean origin traceability system model according to steps S2, S3 and S4.

In the embodiment, Brazilian soybean samples, American soybean samples, with specific regions are selected to prepare standard soybean oil samples, standard soybean powder samples and water-soluble proteins of the standard soybean powder samples, the soybean oil samples in the two different regions are respectively subjected to liquid chromatography-quadrupole flight time mass spectrometer to acquire IDA-MS high-resolution mass spectrum data, the IDA-MS high-resolution mass spectrum data are qualitatively and quantitatively processed and analyzed on PeakView and MasterView software to obtain triglyceride compound marker observation peak data in the standard soybean oil samples, the soybean powder samples in the two different regions are respectively subjected to ICP-MS and/or ICP-AES to acquire mineral element content data, and the water-soluble proteins of the soybean powder samples in the two different regions are respectively subjected to EA-IRMS to acquire stable isotope ratio data, and (3) introducing the triglyceride compound marker observed peak data, mineral element content data and stable isotope ratio data into SIMCA 14.1 software to perform orthogonal partial least squares regression analysis processing, thereby establishing a soybean production area traceability model based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology. As can be seen from fig. 8, in the brazil-U.S. two-country OPLS-DA soybean origin tracing and identifying model, the U.S. and brazil soybean samples can be significantly distinguished, in order to further verify the determination accuracy of the two-country pressed soybean oil origin tracing and identifying model, the U.S. soybean sample and the brazil soybean sample are selected to be respectively pressed and ground to extract water-soluble protein, and then model blind sample verification is performed, and the verification result shows that the soybean oil sample determination accuracy of the bas source is 95%, and the sample determination accuracy of the U.S. source is 95%.

Two-country soybean producing area tracing and identifying model example 2

In the embodiment, American soybean samples of Argentina soybean samples with clear areas are selected to prepare standard soybean oil samples, standard soybean powder samples and water-soluble proteins of the standard soybean powder samples, IDA-MS high-resolution mass spectrum data are respectively collected by the soybean oil samples of the two different areas through a liquid chromatogram-quadrupole flight time mass spectrometer, after the IDA-MS high-resolution mass spectrum data are qualitatively and quantitatively processed and analyzed on PeakView and MasterView software, triglyceride compound marker observation peak data in the standard soybean oil samples are obtained, mineral element content data are respectively collected by the soybean powder samples of the two different areas through ICP-MS and/or ICP-AES, and stable isotope ratio data are respectively collected by the water-soluble proteins of the soybean powder samples of the two different areas through EA-IRMS, and (3) introducing the triglyceride compound marker observed peak data, mineral element content data and stable isotope ratio data into SIMCA 14.1 software to perform orthogonal partial least squares regression analysis processing, thereby establishing a soybean production area traceability model based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology. As can be seen from fig. 9, in the argantin-american two-country OPLS-DA soybean origin tracing and identifying model, american and argantin soybean samples can be significantly distinguished, and in order to further verify the determination accuracy of the two-country pressed soybean oil origin tracing and identifying model, the american soybean sample and the argantin soybean sample are selected to be pressed and ground respectively to extract water-soluble protein, and then the model blind sample verification is performed, and the verification result shows that the determination accuracy of the argantin-derived soybean oil sample is 95%, and the determination accuracy of the american-derived sample is 95%.

Two-country soybean producing area tracing and identifying model example 3

In the embodiment, Canadian soybean samples and American soybean samples in specific regions are selected to prepare standard soybean oil samples, standard soybean powder samples and water-soluble proteins of the standard soybean powder samples, the soybean oil samples in the two different regions are respectively subjected to liquid chromatography-quadrupole flight time mass spectrometer to acquire IDA-MS high-resolution mass spectrum data, the IDA-MS high-resolution mass spectrum data are qualitatively and quantitatively processed and analyzed on PeakView and MasterView software to obtain triglyceride compound marker observation peak data in the standard soybean oil samples, the soybean powder samples in the two different regions are respectively subjected to acquisition of mineral element content data through the PeakMS and/or ICP-AES, and the water-soluble proteins of the soybean powder samples in the two different regions are respectively subjected to acquisition of stable isotope ratio data through the EA-IRMS, and (3) introducing the triglyceride compound marker observed peak data, mineral element content data and stable isotope ratio data into SIMCA 14.1 software to perform orthogonal partial least squares regression analysis processing, thereby establishing a soybean production area traceability model based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology. As can be seen from fig. 10, in the canadian-U.S. two-country OPLS-DA soybean origin tracing and identifying model, the U.S. soybean samples and the canadian soybean samples can be significantly distinguished, and in order to further verify the determination accuracy of the two-country pressed soybean oil origin tracing and identifying model, the U.S. soybean samples and the canadian soybean samples are selected to be pressed and ground respectively to extract water-soluble proteins, and then the model blind sample verification is performed, and the verification result shows that the determination accuracy of the canadian soybean oil samples is 90%, and the determination accuracy of the U.S. soybean samples is 90%.

Two-country soybean producing area tracing and identifying model example 4

In the embodiment, Russian and Chinese soybean samples with specific regions are selected to prepare standard soybean oil samples, standard soybean powder samples and water-soluble proteins of the standard soybean powder samples, the soybean oil samples in two different regions are respectively subjected to liquid chromatography-quadrupole flight time mass spectrometer to acquire IDA-MS high-resolution mass spectrum data, the IDA-MS high-resolution mass spectrum data are qualitatively and quantitatively processed and analyzed on PeakView and MasterView software to obtain triglyceride compound marker observation peak data in the standard soybean oil samples, the soybean powder samples in the two different regions are respectively subjected to ICP-MS and/or ICP-AES to acquire mineral element content data, and the water-soluble proteins of the soybean powder samples in the two different regions are respectively subjected to EA-IRMS to acquire stable isotope ratio data, and (3) introducing the triglyceride compound marker observed peak data, mineral element content data and stable isotope ratio data into SIMCA 14.1 software to perform orthogonal partial least squares regression analysis processing, thereby establishing two soybean origin tracing models of Russia-America and China-America based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology. As can be seen from fig. 11 and 12, in the russian-usa and china-usa two-country OPLS-DA soybean origin tracing identification model, american and russian, american and chinese soybean samples can be significantly distinguished, in order to further verify the determination accuracy of the two-country pressed soybean oil origin tracing identification model, the american soybean sample, the russian soybean sample and the chinese soybean sample are selected to be respectively pressed, ground and extracted with water-soluble protein, and then model blind sample verification is performed, and the verification result shows that the russian-china-derived soybean oil sample determination accuracy is 100%, and the american-derived sample determination accuracy is more than 95%.

As can be seen from the four embodiments of the two-country soybean origin traceability identification model and the multi-country identification model, the two-country soybean origin traceability identification model has higher traceability accuracy.

Comparative example 1

In this comparative example, stable isotope-based target pair establishment was performed to verify different types of targeted targets for soybeanIn the comparative example, soybean samples with definite production places are selected and respectively ground to obtain soybean powder samples as target objects, and EA-IRMSS is used for collecting delta in the soybean powder samples²H、δ¹⁸O、δ¹⁵N and delta¹³And C, establishing a multi-country OPLS-DA soybean origin tracing identification model by using an orthogonal partial least square regression analysis method, wherein the established model is shown in FIG. 13.

Comparative example 2

In the comparative example, in order to verify the influence of different types of target objects of soybeans on the establishment of an OPLS-DA identification model based on stable isotope ratio analysis, soybean oil samples with definite production places are selected to be used as the target objects after being squeezed respectively, and EA-IRMSS is used for collecting delta in the soybean oil samples²H、δ¹⁸O、δ¹⁵N and delta¹³And C, establishing a multi-country OPLS-DA soybean origin tracing identification model by using an orthogonal partial least squares regression analysis method, wherein the established model is shown in FIG. 14.

As shown in FIGS. 7, 13 and 14, the comparative analysis of the delta of water-soluble protein of the multi-national OPLS-DA soybean origin tracing model based on the standard soybean flour sample in example 1²H、δ¹⁸O、δ¹⁵N and delta¹³C, comparing the OPLS-DA multinational identification model constructed by C with that of comparative example 1 and comparative example 2 to distinguish the influence of different target objects on the stable isotope traceability identification model. The comparison result shows that the isotope identification models of the same sample, the soybean powder sample, the soybean oil sample and the water-soluble protein can basically distinguish the soybean and the soybean oil sample in the United states and Brazil, but the selection of the soybean water-soluble protein as the target object is obviously superior to the soybean powder sample and the soybean oil sample in the distinction between the samples in other countries and the American sample. For this purpose, the delta of the water-soluble protein of the soybean flour sample was selected separately²H、δ¹⁸O、δ¹⁵N and delta¹³C establishing origin tracing of soybean based on combination of MALDI-TOF/TOF and stable isotope technology, and can be applied to Brazil, America, Argentina, Uyery, Canada, etcNational soybean samples are clearly distinguished.

Comparative example 3

In the comparative example, in order to verify the influence of different dilution solvents on the triglyceride lipidomics origin tracing-based identification model clustering effect of a soybean oil sample, the soybean oil sample is obtained by squeezing the soybean sample with a definite origin respectively, acetone is used as a dilution solvent to dilute the soybean oil sample, then IDA-MS high-resolution mass spectrum data of triglyceride compounds in the soybean oil sample are collected by a liquid chromatogram-quadrupole time-of-flight mass spectrometer LC-Q-TOF-MS, and the triglyceride high-resolution mass spectrum data are processed by two multivariate statistical analysis models of principal component analysis PCA and orthogonal partial least squares regression analysis.

Comparative example 4

In the comparative example, soybean samples with definite production places are selected and respectively squeezed to obtain soybean oil samples, isopropyl triglyceride is used as a diluting solvent to dilute the soybean oil samples, then IDA-MS high-resolution mass spectrum data of triglyceride compounds in the soybean oil samples are collected by a liquid chromatogram-quadrupole time-of-flight mass spectrometer LC-Q-TOF-MS, and the triglyceride high-resolution mass spectrum data are processed by two multivariate statistical analysis models of principal component analysis PCA and orthogonal partial least squares regression analysis OPLS-DA.

Comparative analysis of multi-national OPLS-DA soybean origin tracing model examples 1, comparative example 3 and comparative example 4 the influence of different dilution solvents on origin tracing of soybean oil triglycerides was analyzed. As shown in fig. 15, it can be seen from the PCA model and the OPLS-DA model constructed after diluting the soybean oil sample with solvents such as acetone (acetone), methanol-ethyl acetate mixed solvent (EA-MEOH), and Isopropanol (Isopropanol), that the sample aggregation effect is synchronized when the soybean oil sample is diluted with the solvents such as acetone, methanol, and ethyl acetate, and the aggregation effect of the Isopropanol-diluted soybean oil sample is significantly different from that of the other two solvent dilution methods. Particularly, the analysis result of an OPLS-DA model shows that the aggregation area of the isopropanol diluted soybean oil sample of the same sample is obviously distinguished from the methanol and ethyl acetate mixed solution and the isopropanol diluted soybean oil sample. In order to further examine the influence of different solvents on the lipidomics analysis of triglyceride in soybean oil, a PCA-class model is constructed as shown in FIG. 11, and the problem of the source tracing and identification result of soybean oil samples diluted by three solvents is judged.

The isopropanol diluted soybean oil traceability identification model can only distinguish the difference between the pressed soybean oil and the finished soybean oil and can not obviously identify soybean oil samples from different national sources, the methanol and ethyl acetate mixed solution diluted soybean oil sample can identify the sample sources of the pressed soybean oil and the finished soybean oil without difference, the acetone diluted soybean oil sample can also identify the origin of the soybean oil sample, but the sample has higher aggregation dispersity, and the methanol and ethyl acetate mixed solution is not suitable to be used as a diluting solvent of the soybean oil sample because the acetone has larger influence on a liquid chromatographic column and is obviously superior to the other two diluting solvents.

The embodiments of the soybean origin tracing identification method based on the combination of IRMS, LC-Q-TOF-MS and multi-element analysis technology provided by the invention are explained in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the core concepts of the present invention. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

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Soybean origin tracing identification method based on combination of IRMS, LC-Q-TOF-MS and multi-element analysis

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