CN111413436B

CN111413436B - Method for identifying lamb mutton and adult mutton

Info

Publication number: CN111413436B
Application number: CN202010326945.8A
Authority: CN
Inventors: 徐贞贞; 王济世; 徐雷; 杨曙明; 陈爱亮
Original assignee: Institute of Quality Standards and Testing Technology for Agro Products of Henan Academy of Agricultural Science
Current assignee: Institute of Quality Standards and Testing Technology for Agro Products of Henan Academy of Agricultural Science
Priority date: 2020-04-23
Filing date: 2020-04-23
Publication date: 2021-03-30
Anticipated expiration: 2040-04-23
Also published as: CN111413436A

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

Method for identifying lamb mutton and adult mutton

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 diagram of an OPLS-DA model of the method for discriminating lamb mutton 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.

The sample preparation process comprises the following steps: weighing 100mg of sample, placing the sample in a 10mL centrifuge tube, adding 3mL of dichloromethane/methanol (2/1, v/v) mixed solution, then adding 2.5mL of ultrapure water, carrying out ultrasonic treatment for 20min, centrifuging at 10000 rpm for 15min, removing the lower layer clear solution, transferring the lower layer clear solution into a glass centrifuge tube, blowing nitrogen to be nearly dry, redissolving with 1mL of methanol, filtering with a 0.22 mu m filter membrane, and bottling to be tested. Quality control samples (QC) were prepared by mixing all samples to be tested in the same volume of liquid. The role of the QC samples was to monitor the instrument status and stability of the data during the instrument analysis.

The instrument used for the experiment was an ExionLC ultra high performance liquid chromatography tandem high resolution quadrupole time-of-flight mass spectrometry (Sciex, Redwood City, Calif., USA) equipped with a C18 column (2.1X 150mm,2.7 μm, Agilent, USA). Liquid phase conditions: 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. Mobile phase B elution gradient: 0min, 2%; 3min, 90%; 5min, 50%; 6min, 55%; 9min 60%; 11min 70%; 13min, 2%; 13-15min, 2%, sample amount of 5 μ L, flow rate of 0.3mL/min, and column temperature of 40 deg.C. Mass spectrum conditions: the ion source is an electrospray ion source (ESI), the acquisition mode is a data-dependent acquisition mode (IDA), and dynamic background subtraction is performed. The spray voltage of the positive and negative ion modes is 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.

Raw data were pre-processed using Peakview 2.2 software (AB Sciex, USA) including baseline correction, peak extraction, peak correction, normalization and normalization to obtain relative peak areas and retention times for all metabolites. Introducing the processed data into Excel, calculating the coefficient of variation (CV%) of the QC sample, and removing more than 30% of compounds; 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. Fold difference (FC) of metabolites between lamb and adult lamb mutton groups was calculated and the one-way anova t-test was performed on the pre-processed data with the help of SPSS22.0 software. At the same time, the data was exported to SIMCA14.1 software for visual analysis. Principal Component Analysis (PCA) and orthogonal partial least squares analysis (OPLS-DA) were used for the identification of potentially differential biomarkers in lamb and adult lamb. Those metabolites whose variable importance (VIP variable importance) is greater than 1 while the one-way anova test (t-test) significance level P-value is less than 0.05 are considered differential biomarkers.

And (4) carrying out principal component analysis on the metabolite data, and judging whether the two groups of samples have difference. From the PCA score chart, as shown in FIG. 1, the principal component score chart, a in a positive ion mode and b in a negative ion mode, can find that the QC sample has high aggregation, indicating that the pretreatment and the instrument state are good. The two sets of samples had better separation, with all samples falling within the 95% confidence interval. The cumulative contribution rates of the first four components of the positive and negative ion modes to the difference are 61.5% and 60.1%, respectively. This demonstrates that the PCA model has good discriminatory power on the two groups of samples, i.e., lamb and adult mutton can be distinguished by PCA, and further demonstrates that there is a clear difference between the two groups of samples. Orthogonal partial least squares analysis was then performed to establish an OPLS-DA model (shown in fig. 2, orthogonal partial least squares analysis score plot, a is positive ion mode and b is negative ion mode) and to screen for metabolites with significant differences in lamb and adult lamb. In model R²X (cum) and R²Y(cum) Respectively representing the interpretability of the model on the X and Y matrices, Q²Y (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 OPLS-DA model, R is in a 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 the OPLS-DA model with 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

1. a method for identifying lamb and adult mutton, is characterized in that, comprises the following steps:

A. Extract the sample to be tested to obtain polar and non-polar metabolite extracts, and perform metabolomics data collection on the extracts;

B. Chemometric analysis is performed through the metabolomic data, and an analysis model is established to screen differential markers. The chemometric analysis includes principal component analysis and orthogonal partial least squares analysis, and judged by the principal component analysis Whether there is a difference between the two groups of samples, the potential characteristic markers of the samples to be tested are determined by orthogonal partial least squares analysis based on the analysis model of principal component analysis, and the variable importance VIP is greater than 1 and the one-way ANOVA test (t Test) metabolites with a significance level P less than 0.05 are determined as differential biomarkers;

C. Utilize the selected characteristic markers to identify the sample to be tested, and the chromatography is an ultra-high performance liquid chromatography; the ultra-high performance liquid chromatography carries out gradient elution with flow A and mobile phase B; Phase A is water/acetonitrile mixed in a volume ratio of 15/85, mobile phase B is butanol, both phases contain 0.1% formic acid and 5mM ammonium acetate, and the gradient elution conditions include: 0min, 2%B; 3min , 90%B; 5min, 50%B; 6min, 55%B; 9min 60%B; 11min 70%B; 13min, 2%B; 13-15min, keep 2%B; injection volume 5μL, flow rate 0.3mL /min, the column temperature is 40°C.

2 . The method for identifying lamb and adult mutton according to claim 1 , wherein the detection data includes relative peak areas and retention times of all metabolites. 3 .

3. A method for identifying lamb and adult mutton according to claim 1, wherein the detection data is preprocessed, and the preprocessing comprises baseline correction, peak extraction, peak correction, standardization and normalization , obtain the relative peak area and retention time of all metabolites, and the coefficient of variation will exclude more than 30% of the compounds; the eigenvalues with missing values exceeding 50% will be rejected, and the remaining missing values will be determined by using half of the minimum value of the eigenvalue in all samples. filling.

4. a kind of method for identifying lamb and adult mutton according to claim 1, is characterized in that, based on the analytical model of orthogonal partial least squares analysis, with VIP>1, P<0.05, difference multiple (FC) >2 was used as a threshold to obtain potential feature markers.

5. a kind of method for identifying lamb and adult mutton according to claim 1, is characterized in that, selects positive and negative ion mode according to the polarity or non-polarity of metabolite extract to obtain lamb and adult mutton to carry out potential. characteristic markers.

6. a kind of method for identifying lamb and adult mutton according to claim 1, is characterized in that, mass spectrometry is quadrupole time-of-flight high-resolution mass spectrometry; The declustering voltages were 80V and -80V, respectively, the ion source temperature was 500°C, the atomizer pressure was 50psi, the heating auxiliary gas pressure was 50psi, the collision energy was 35±15V, and the data acquisition range was 50-1500Da.