CN111413436A - Method for identifying lamb mutton and adult mutton - Google Patents
Method for identifying lamb mutton and adult mutton Download PDFInfo
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- CN111413436A CN111413436A CN202010326945.8A CN202010326945A CN111413436A CN 111413436 A CN111413436 A CN 111413436A CN 202010326945 A CN202010326945 A CN 202010326945A CN 111413436 A CN111413436 A CN 111413436A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
Abstract
The invention discloses a method for identifying lamb mutton and adult mutton, which comprises the steps of extracting metabonomics data of a sample to be detected, and carrying out chemometrics analysis on the detection data to extract characteristic information; the invention provides a reliable and rapid identification method for distinguishing lamb mutton and adult mutton, the method is convenient, sensitive and accurate, and the rapid discrimination of mutton can be realized by screening the difference markers through the analysis model.
Description
Technical Field
The invention relates to the technical field of food authenticity detection, in particular to a method for identifying lamb mutton and adult mutton.
Background
Along with the development of social economy, the living standard of people is greatly improved. The consumer's consumption concept of meat products has been shifted from pure "quantity demand" to "quality demand". Mutton is deeply welcomed by consumers by virtue of the characteristics of high protein, low fat and delicious meat, so the demand of mutton is continuously increased, but some illegal vendors often use some cheap meat to sell as mutton under the drive of interests, such as duck meat and the like, and the rights and interests of consumers are seriously influenced. At present, researchers have developed many technologies aiming at the problem of mutton adulteration, such as PCR technology for nucleic acid detection, enzyme-linked immunosorbent assay technology, electronic nose technology for odor detection and the like. The PCR technology often needs to design primers and amplify genes aiming at different species, and false negative results are easy to occur; the development of enzyme-linked immunosorbent assay requires the finding of antigens with high specificity and thermal stability; the electronic nose technology mainly aims at detecting volatile flavor substances, and the sensor has selectivity on compounds. It is worth noting that the above techniques have certain limitations for identifying adulteration of the same species or tissue.
The lamb mutton has delicate and delicious meat, so the lamb mutton has higher price than adult mutton. In recent years, the use of inexpensive adult mutton to serve as premium lamb has become an increasing challenge to disrupt market order. Because the mutton belongs to the same species and the traditional biological detection technologies such as DNA, nucleic acid and the like fail, the development of an identification method for accurately identifying the mutton adulteration is urgently needed so as to maintain the market fairness and protect the rights of consumers.
Disclosure of Invention
The invention aims to solve the existing problems and provides a method for identifying lamb mutton and adult mutton by screening feature markers for identifying lamb mutton and adult mutton.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention comprises the following steps:
A. extracting a sample to be detected to obtain polar and nonpolar metabolite extracts, and carrying out metabonomics data acquisition on the extracts;
B. carrying out chemometric analysis on the metabonomics, establishing an analysis model and screening difference markers, wherein the chemometric analysis comprises principal component analysis and orthogonal partial least square analysis, judging whether two groups of samples have difference or not through the principal component analysis, and determining potential characteristic markers of the samples to be detected through the orthogonal partial least square analysis;
C. identifying the sample to be detected by using the screened characteristic marker;
further, the detection data includes relative peak areas and retention times of all metabolites.
Specifically, preprocessing the detection data, wherein the preprocessing comprises baseline correction, peak extraction, peak correction, standardization and normalization, obtaining relative peak areas and retention time of all metabolites, and eliminating compounds with variation coefficients exceeding 30%; the feature values with missing values over 50% were rejected, and the remaining missing values were filled in with half the minimum of the feature values in all samples.
Further, metabolites with variable importance greater than 1 while having a t-test significance level value less than 0.05 were identified as differential biomarkers based on an analytical model of principal component analysis.
Specifically, an analysis model based on orthogonal partial least squares analysis obtains potential feature markers by using Variable Importance (VIP) >1, significance level (P) <0.05 and difference multiple (FC) >2 as threshold values.
Further, potential signature acquisition was performed in chemometric analysis by positive and negative ion mode lamb and adult lamb.
Specifically, the spray voltages of the positive and negative ion modes were 5500V and-4500V, respectively, and the declustering voltage was 80V and-80V, respectively. The ion source temperature was 500 ℃. Atomizer pressure (GS1)50psi, heating assist gas pressure (GS2)50psi, collision energy 35 + -15V. The data acquisition range is 50-1500Da.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a reliable and rapid identification method for distinguishing lamb mutton and adult mutton, the method is convenient, sensitive and accurate, and rapid identification of mutton can be realized by screening difference markers through an analysis model.
Drawings
FIG. 1 is a flow chart of the operation process of the method for discriminating lamb mutton from adult mutton in the invention;
FIG. 2 is a PCA score chart of a method for discriminating lamb and adult mutton according to the present invention;
FIG. 3 is a model diagram of OP L S-DA showing the method for discriminating lamb from adult mutton according to the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
The invention provides a method for identifying the same variety of lamb mutton and adult mutton. Carrying out chemometric analysis on the detection data, extracting characteristic information, establishing an analysis model, determining the differential metabolites of the lamb mutton and the adult mutton, and identifying the lamb mutton and the adult mutton by using the screened characteristic markers.
Materials and methods
Methanol, acetonitrile, butanol, and dichloromethane were purchased from Fisher corporation; formic acid and sodium acetate were purchased from Dikma corporation; ultrapure water (18.2 M.OMEGA.) was used for the experiments prepared by Milli-Q system (Millipore Billerica, MA).
Preparation and extraction of samples
Samples of alpine merino fine-wool sheep were collected from the southern section of the province of Gansu. 4 adult mutton samples (3 years old, 50.05 + -6.96 kg) and 4 lamb mutton samples (8 months old, 24.48 + -2.23 kg). All samples were collected from the same pasture and were freely fed during grazing. Taking the longissimus dorsi of the same part of each sheep, mincing, and placing in a refrigerator at-80 deg.C for use.
Weighing 100mg of sample, placing the sample in a 10m L centrifuge tube, adding 3m L dichloromethane/methanol (2/1, v/v) mixed solution, then adding 2.5m L ultrapure water, performing ultrasonic treatment for 20min, centrifuging at 10000 rpm for 15min, removing lower layer clear solution, transferring the lower layer clear solution into a glass centrifuge tube, blowing nitrogen to the near-dry state, redissolving with 1m L methanol, filtering with a 0.22 mu m filter membrane, bottling to be tested, preparing a quality control sample (QC), mixing all samples to be tested by taking the same volume of liquid, and monitoring the state of the instrument and the stability of data in the analysis process of the instrument.
The experimental apparatus used was an Exion L C ultra high performance liquid chromatography tandem high resolution quadrupole time-of-flight mass spectrometry (Sciex, Redwood City, CA, USA), the equipped chromatographic columns were C18 columns (2.1 × mM,2.7 μm, Agilent, USA). The liquid phase conditions were water/acetonitrile (15/85, V/V) and butanol, respectively, both phases contained 0.1% formic acid and 5mM ammonium acetate. the mobile phase B elution gradient was 0min, 2%, 3min, 90%, 5min, 50%, 6min, 55%, 9min 60%, 11min 70%, 13min, 2%, 13-15min, held at 2%. sample introduction 5 μ L, flow rate 0.3m 2/min, column temperature 40 ℃ mass spectrometry conditions were electrospray ion source (ESI), acquisition mode was data correlation acquisition mode (IDA), spray voltage in air pressure subtraction, negative ion mode, and spray voltage, respectively, 0.3m 2/min, 50-35V, 50-35V, 50V, 35-15V, 50-50V, 50-50V, 150V, 50V, 150V, 387, respectively, and auxiliary heating pressure, 50V, 2, psi, 2, psi.
Raw data were preprocessed using Peakview 2.2 software (AB Sciex, USA) including baseline correction, peak extraction, peak correction, normalization and normalization to obtain the relative peak area and retention time of all metabolites the processed data were imported into Excel, calculated coefficient of variation (CV%) for QC samples would exceed 30% compound rejection, missing values exceed 50% feature rejection, the remaining missing values were filled with half the minimum of the feature in all samples, calculated fold difference (FC) of metabolites between lamb and adult lamb groups and single factor analysis of variance t-test on preprocessed data with SPSS22.0 software while data were exported to SIMCA14.1 software for visual analysis, Principal Component Analysis (PCA) and PCA cross-bias analysis (OP L S-DA) were used to identify potentially different biomarkers in lamb and adult lamb.
The two groups of samples have better separation degree, and the samples all fall within a 95% confidence interval, the cumulative contribution rates of the four components in the positive ion mode and the negative ion mode to the difference are respectively 61.5% and 60.1%, which shows that the PCA model has good identification capability on the two groups of samples, namely lamb mutton and adult mutton can be distinguished by the PCA and further shows that the two groups of samples have obvious difference, then orthogonal partial least square analysis is carried out to establish an OP L S-DA model (shown in figure 2, the orthogonal partial least square analysis score chart, a is the positive ion mode, and b is the negative ion mode) and to screen metabolites with obvious difference in the lamb mutton and the adult mutton2X (cum) and R2Y (cum) represents the model's ability to interpret the X and Y matrices, Q, respectively2Y (cum) represents the predictive power of the model when R2The smaller X, the smaller R2Y and Q2The larger Y is and the closer to 1, the more stable and reliable the model is, in the established OP L S-DA model, R is in positive ion mode2X(cum)、 R2Y (cum) and Q2Y (cum) is 43.3%, 97.7% and 88.9%, respectively, R in negative ion mode2X(cum)、R2Y (cum) and Q2Y (cum) is 41.7%, 97.4% and 81.4 respectively, which shows that the model is relatively stable and has good prediction capability, based on OP L S-DA model, VIP>1,P<0.05, FC>2 as threshold, 2 potential signatures were obtained in positive ion mode: ranunculin (Flavoxanthin) and Phosphatidic Acid (PA); 1 potential signature marker was obtained in negative ion mode: phosphatidylinositol (PI), detailed information is shown in table 1. The characteristic difference object is screened and identified from a large amount of data, has objectivity and accuracy, and can be used for identifying lamb mutton and adult mutton of alpine merino fine hair sheep.
TABLE 1 potential signature markers in lamb and adult mutton
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. A method for distinguishing lamb mutton from adult mutton, which is characterized by comprising the following steps:
A. extracting a sample to be detected to obtain polar and nonpolar metabolite extracts, and carrying out metabonomics data acquisition on the extracts;
B. carrying out chemometric analysis through the metabonomics data, establishing an analysis model and screening differential markers, wherein the chemometric analysis comprises principal component analysis and orthogonal partial least square analysis, judging whether two groups of samples have differences through the principal component analysis, and determining potential characteristic markers of the samples to be detected through the orthogonal partial least square analysis;
C. and identifying the sample to be detected by using the screened characteristic marker.
2. The method of claim 1, wherein the detection data comprises relative peak area and retention time of all metabolites.
3. The method for discriminating between lamb and adult mutton according to claim 1, wherein the detection data is preprocessed, the preprocessing comprises baseline correction, peak extraction, peak correction, standardization and normalization, the relative peak areas and retention times of all metabolites are obtained, and the coefficient of variation is more than 30% of the compounds are eliminated; the feature values with missing values over 50% were rejected, and the remaining missing values were filled in with half the minimum of the feature values in all samples.
4. The method of claim 1, wherein metabolites with Variable Importance (VIP) greater than 1 and single factor analysis of variance test (t-test) significance level (P) less than 0.05 are identified as differential biomarkers based on an analytical model of principal component analysis.
5. The method of claim 1, wherein the analysis model based on orthogonal partial least squares analysis is used to obtain the potential signature marker by using VIP >1, P <0.05 and multiple of difference (FC) >2 as threshold values.
6. The method for discriminating between lamb and adult mutton according to claim 1, wherein the positive and negative ion modes are selected according to the polarity or non-polarity of the metabolite extract in the chemometric analysis to obtain the lamb and adult mutton for potential feature markers.
7. The method for discriminating between lamb and adult mutton according to claim 1, wherein the chromatogram of the sample to be tested is ultra high performance liquid chromatogram; the ultra-high performance liquid chromatography is subjected to gradient elution by a mobile phase A and a mobile phase B; the mobile phases A and B were water/acetonitrile (15/85, v/v) and butanol, respectively, both containing 0.1% formic acid and 5mM ammonium acetate.
8. The method of claim 7, wherein the gradient elution conditions comprise 0min, 2% (B), 3min, 90% (B), 5min, 50% (B), 6min, 55% (B), 9min, 60% (B), 11min, 70% (B), 13min, 2% (B), 13-15min, 2% (B), sample size 5 μ L, flow rate 0.3m L/min, and column temperature 40 ℃.
9. The method for discriminating lamb and adult mutton according to claim 1, wherein the mass spectrum of the sample to be tested is quadrupole time-of-flight high-resolution mass spectrum; the method is characterized in that the spraying voltages of the positive ion mode and the negative ion mode are 5500V and-4500V respectively, and the declustering voltage is 80V and-80V respectively. The ion source temperature was 500 ℃. Atomizer pressure (GS1)50psi, heating assist gas pressure (GS2)50psi, collision energy 35 + -15V. The data acquisition range is 50-1500Da.
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Cited By (6)
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| CN112215277A (en) * | 2020-10-09 | 2021-01-12 | 内蒙古农业大学 | Method and system for distinguishing beef and mutton species and feeding mode authenticity |
| CN112858558A (en) * | 2021-01-22 | 2021-05-28 | 陕西科技大学 | Triglycerides-based method for identifying adulteration of cow milk and sheep milk |
| CN112881563A (en) * | 2021-02-08 | 2021-06-01 | 中国检验检疫科学研究院 | Method for detecting an overheated meat product |
| CN114942286A (en) * | 2022-05-17 | 2022-08-26 | 复旦大学 | Detection method of hydrophilic polypeptide |
| CN114965796A (en) * | 2022-06-21 | 2022-08-30 | 陕西科技大学 | Method for analyzing irradiated mutton metabonomics by ultra-high performance liquid chromatography tandem mass spectrometry |
| CN116933160A (en) * | 2023-09-19 | 2023-10-24 | 中国农业科学院农产品加工研究所 | Meat variety and part identification method based on lipid characteristic-machine learning |
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Cited By (9)
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| CN112215277A (en) * | 2020-10-09 | 2021-01-12 | 内蒙古农业大学 | Method and system for distinguishing beef and mutton species and feeding mode authenticity |
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| CN112858558A (en) * | 2021-01-22 | 2021-05-28 | 陕西科技大学 | Triglycerides-based method for identifying adulteration of cow milk and sheep milk |
| CN112881563A (en) * | 2021-02-08 | 2021-06-01 | 中国检验检疫科学研究院 | Method for detecting an overheated meat product |
| CN112881563B (en) * | 2021-02-08 | 2023-02-17 | 中国检验检疫科学研究院 | Method for detecting an overheated meat product |
| CN114942286A (en) * | 2022-05-17 | 2022-08-26 | 复旦大学 | Detection method of hydrophilic polypeptide |
| CN114965796A (en) * | 2022-06-21 | 2022-08-30 | 陕西科技大学 | Method for analyzing irradiated mutton metabonomics by ultra-high performance liquid chromatography tandem mass spectrometry |
| CN116933160A (en) * | 2023-09-19 | 2023-10-24 | 中国农业科学院农产品加工研究所 | Meat variety and part identification method based on lipid characteristic-machine learning |
| CN116933160B (en) * | 2023-09-19 | 2024-01-09 | 中国农业科学院农产品加工研究所 | Meat variety and part identification method based on lipid characteristic-machine learning |
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