Characteristic marker and mineral element combination-based method for tracing producing area of Shuangshui Ma goats

Abstract

The invention discloses a characteristic marker and mineral element combination-based Shenshuaoma sheep producing area traceability method, which comprises the following steps of: detecting high-resolution mass spectrum, detecting ICP-MS (Inductively Coupled Plasma Mass Spectrometry), establishing a characteristic marker model, and finally setting a content threshold value of each mineral element according to the content of the mineral element, and carrying out secondary judgment to realize high-precision identification of Shenshuaoma sheep. The comprehensive traceability rate can reach more than 99.5%, the technical problems of single traceability dimension and poor anti-interference capability of a single technology are solved, and the detection accuracy is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of food detection and origin traceability, and particularly relates to a method for tracing the origin of Xishui Ma goats based on the combined use of characteristic markers and mineral elements. Background Art

[0002] Xishui Ma goats are a local fine breed across the country. Xishui County, Zunyi City, Guizhou Province is the origin, and it has now become a national geographical indication product in China. At present, there is little research on the origin traceability of Xishui Ma goats. There are many such research methods for other different samples, such as protein detection technology, spectral detection technology, gene detection technology, isotope detection technology, and mass spectrometry detection technology, etc. The above-mentioned related traceability methods require complex pretreatment of samples, which takes a long time, cannot achieve fast, efficient, and real-time traceability, and do not meet the detection requirements of a large number of samples. And there are limitations of single indicators: traditional traceability methods rely on single element analysis or stable isotope ratios, and are easily affected by the migration of the breeding environment or feed standardization, resulting in misjudgment. Insufficient data integration: metabolomics and elementomics data are analyzed independently, lacking a multi-dimensional correlation model, and it is difficult to distinguish the influence of complex geographical environments. Low efficiency of biomarker screening: relying on manual screening of metabolite characteristic peaks, ignoring the dynamic association of metabolic pathways, and having poor resistance to individual differences and seasonal fluctuations. The literature report in "Science and Technology of Food Industry" in 2022 on the research of tracing the origin of Xishui Ma goats, a geographical indication protected product, using a method of connecting rapid evaporative ionization mass spectrometry (REIMS) with high-resolution mass spectrometry to quickly identify Xishui Ma goats. Although the monitoring time of the method of the present invention is short, there are certain false positive errors, and organic markers are easily affected by factors such as storage conditions, further affecting the accuracy of traceability. Summary of the Invention

[0003] The technical problem to be solved by the present invention is a series of problems existing in the method for tracing the origin of Xishui Ma goats in the prior art. The present invention provides a method for tracing the origin of Xishui Ma goats based on the combined use of characteristic markers and mineral elements, which well solves the above-mentioned existing problems and improves the accuracy of traceability.

[0004] The method for tracing the origin of Xishui Ma goats based on the combined use of characteristic markers and mineral elements of the present invention includes the following steps:

[0005] (1) Detection by high-resolution mass spectrometry

[0006] Cut the mutton sample into slices, store it frozen, and when in use, thaw the mutton and place it on the tin foil for use. Cut the mutton sample with a high-frequency electric knife, and the generated aerosol enters the in-situ ionization source time-of-flight mass spectrometer through the ion source, and collect data in the negative ion mode;

[0007] (2) Detection by ICP-MS: Accurately weigh the homogenized mutton samples, place them in digestion vessels for digestion, remove the acid after digestion, make up the volume, and determine the content of mineral elements;

[0008] (3) Establishment of the characteristic marker model: Use LiveID software to establish different groups according to the origin of mutton, select the TIC threshold of the collected sample spectra, upload the data to each group, and establish a model combining principal component analysis and linear discriminant analysis for discrimination;

[0009] (4) Set the content thresholds of each mineral element according to the content of mineral elements for secondary discrimination.

[0010] The modeling parameters adopted in the present invention using LiveID software: The characteristic markers are selected as sphingosine, phosphatidylinositol, and succinic acid, and the mineral elements are selected as Se, Fe, and Zn.

[0011] During secondary discrimination, the mineral elements are selected as Se, Fe, and Zn, and the content thresholds of each mineral element are Fe > 25 mg / kg, Zn > 35 mg / kg, and Se > 300 μg / kg respectively.

[0012] The specific parameters adopted by the high-resolution mass spectrometer are as follows: the cutting voltage is set at 40 V, the cutting length is 2 cm, the incision depth is 0.5 cm, the cutting duration is 2 - 3 s, each sample is repeatedly collected 10 - 15 cuts, the in-situ ionization source time-of-flight mass spectrometer uses leucine enkephalin, m / z = 554.2615, as the mass number calibration internal standard, methanol is used as the auxiliary solution, the flow rate is 200 μL / min, the temperature of the electrothermal probe is 500 °C, the mass number scanning range is 50 - 1200 m / z, and the scanning time is 1.0 s.

[0013] The detection conditions of ICP-MS: RF power: 1550 W, carrier gas flow rate: 1.05 L / min, detection mode: KED mode, He gas flow rate: 4.3 ml / min.

[0014] Use Progenesis Bridge software to segment the REIMS raw data, import it into Progenesis QI software for peak extraction and differential analysis, examine the clustering of samples, find the differential compounds with VIP > 1 through EZinfo software, and further screen and identify them through the database.

[0015] The parameters adopted by using LiveID software: The main components are 25 compounds, preferably sphingosine, phosphatidylinositol, and succinic acid, the linear discriminant is 3, and the unrecognized threshold is 20%.

[0016] Beneficial effects achieved by the present invention compared with the prior art: The present invention adopts a method for tracing the origin of Xishui Ma goats by combining characteristic markers and mineral elements, realizing high-precision identification of Xishui Ma goats. The comprehensive tracing rate can reach more than 99.5%, solving the technical problems of single tracing dimension and poor anti-interference ability of single technology, and significantly improving the detection accuracy. Detailed implementation manners

[0017] Example 1

[0018] 1. Materials and instruments

[0019] Methanol of mass spectrometry purity, from Merck, Germany; Leucine Enkephalin: Leu-Enkaphalin, from Waters, USA. All mutton samples were collected locally by a special person to ensure the accuracy and authenticity of the origin. The origin of mutton: Xishui County, Zunyi City; other areas in Guizhou Province except Xishui County; Tongliao City and Ulanqab City in Inner Mongolia Autonomous Region; Baicheng City and Songyuan City in Jilin Province.

[0020] In-situ ionization source time-of-flight mass spectrometer: Xevo G2-XS REMIS Q-TOF: from Waters, USA; ICPMS: from Thermo Fisher Scientific, USA; High-frequency electrosurgical knife: product of Erbe, Germany; Milli-Q Reference ultrapure water generator: from Millipore, USA.

[0021] 2. Experimental methods

[0022] 2.1 Sample data collection

[0023] The cutting size of mutton samples is: flakes with length, width and height of 10 cm × 4 cm × 8 mm, placed in a frozen dumpling box for storage. When needed, it is thawed in the refrigerator and placed flat on tin foil. The erbe high-frequency electrosurgical knife is used to collect data by cutting the sample. The cutting voltage is selected from 10 - 50 V, the length is selected from 0.5 - 3 cm, the depth is selected from 0.2 - 1.0 cm, and the time is selected from 1 - 5 s. Each sample is repeatedly collected 10 - 15 cuts.

[0024] Every time about 10 - 15 samples are tested or when the baseline noise is found to be too large, the ion source, scalpel and connecting pipeline need to be cleaned to ensure the accuracy and reliability of the collected sample data.

[0025] 2.2 Detection conditions

[0026] The in-situ ionization source time-of-flight mass spectrometer uses Leucine Enkephalin, m / z = 554.2615, as the mass number calibration internal standard, uses methanol as the auxiliary solution, with a flow rate of 200 μL / min, the temperature of the electrothermal probe is 500 °C, the mass number scanning range is 50 - 1200 m / z, the scanning time is 1.0 s, and the samples are collected in negative ion mode.

[0027] Detection conditions of ICP-MS: RF power: 1550 W, carrier gas flow rate: 1.05 L / min, detection mode: KED mode, He gas flow rate: 4.3 ml / min, measuring common mineral elements, with emphasis on measuring the contents of Se, Fe, and Zn.

[0028] 3. Data processing

[0029] First, use the LiveID software to select the TIC threshold according to the actual situation, establish 4 different groups based on the origin of mutton, and upload the collected data of mutton from different origins to each group, namely Xishui Mayang, Guizhou sheep, Jilin sheep, and Inner Mongolia sheep. The parameters used are: principal component analysis, linear discriminant is 3, and the unrecognized threshold is 20%. Establish a model combining principal component analysis (PCA) and linear discriminant analysis (LDA), and observe the clustering analysis of mutton from different origins under different groups.

[0030] Use the Progenesis Bridge software to segment the REIMS raw data, import it into the Progenesis QI software for peak extraction and differential analysis, and examine the clustering of samples. Use the EZinfo software to find differential compounds with VIP>1, further screen and identify them through the database. Use the EZinfo software to perform OPLS-DA model analysis on Xishui Mayang and other sheep, where other sheep are the sum of Inner Mongolia sheep, Jilin sheep, and Guizhou sheep. Select marker compounds under the conditions of variable weight value VIP>1 and one-way analysis of variance non-parametric test P value ≤ 0.05. Generate the abundance map of the main differential compounds using the Progenesis QI software, match and identify the remaining compounds through the LIPID MAPS database, sort them in descending order according to the maximum fold change (Max fold change), and select the markers with large changes through LIPID Perform information matching through the Lipidomics Gateway website, and a total of 17 differentially large characteristic markers are screened out. The preferred principal component characteristic markers are sphingosine, phosphatidylinositol, and succinic acid.

[0031] The selected mineral elements are Se, Fe, and Zn, and the content thresholds of each mineral element are Fe>25 mg / kg, Zn>35 mg / kg, and Se>300 μg / kg. The setting of the thresholds for Se, Fe, and Zn is determined based on the long-term test results of Xishui Mayang.

[0032] Example 2

[0033] In the Live ID software, overall cross-model between-group verification analysis was carried out on samples such as Xishui Ma goats, Inner Mongolia sheep, Jilin sheep, and Guizhou sheep. They were randomly divided into 8 groups, and 7 groups were arbitrarily selected to establish a new model. This model was used to verify the remaining 1 group of data, repeated 3 times to measure the accuracy of the prediction model, and the recognition rate was 96.5%. Secondary discrimination was carried out on the unrecognized samples. The mineral elements selected were Se, Fe, and Zn, and the content thresholds of each mineral element were Fe > 25 mg / kg, Zn > 35 mg / kg, and Se > 300 μg / kg. The results showed that the comprehensive discrimination success rate was over 99.5%.

Claims

1. A method for tracing the origin of Xishui Ma sheep based on the combination of characteristic markers and mineral elements, characterized in that: The following steps are involved: (1) High-resolution mass spectrometry detection The mutton samples were cut into slices and frozen for storage. When used, the mutton was thawed and placed on tin foil for later use. The mutton samples were cut with a high-frequency electric knife, and the generated aerosol entered the in-situ ionization source time-of-flight mass spectrometer through an ion source, and data were collected in negative ion mode. (2) ICP-MS detection Accurately weigh the homogenized mutton sample, place it in a digestion tank for digestion, remove the acid after digestion, fix the volume, and determine the mineral element content; (3) Establishment of characteristic marker model: LiveID software was used to establish different groups according to the origin of mutton, the TIC threshold of the collected sample spectra was selected, the data was uploaded to each group, and a model combining principal component analysis and linear discriminant analysis was established for discrimination; (4) According to the content of mineral elements, the threshold value of each mineral element content is set to perform secondary discrimination.

2. The method for detecting origin tracing using IKnife-REIMS technology according to claim 1, characterized in that: The modeling parameters of LiveID software were applied: sphingosine, phosphatidylinositol and succinic acid were selected as characteristic markers, and Se, Fe and Zn were selected as mineral elements.

3. The method for detecting origin tracing using IKnife-REIMS technology according to claim 2, characterized in that: The mineral elements selected are Se, Fe and Zn, and the content thresholds of each mineral element are Fe>25mg / kg, Zn>35mg / kg, and Se>300ug / kg respectively.

4. The method for detecting origin tracing using IKnife-REIMS technology according to claim 1, characterized in that: The specific parameters used in the high-resolution mass spectrometry were as follows: the cutting voltage was set to 40 V, the cutting length was 2 cm, the incision depth was 0.5 cm, the cutting duration was 2 to 3 s, 10 to 15 cuts were repeated for each sample, and the in situ ionization source time-of-flight mass spectrometer used leucine enkephalin, m / z = 554.2615, as the internal standard for mass number correction, methanol was used as the auxiliary solution, the flow rate was 200 μL / min, the electric heating probe temperature was 500°C, the mass number scanning range was 50 to 1200 m / z, and the scanning time was 1.0 s.

5. The method for detecting origin tracing using IKnife-REIMS technology according to claim 1, characterized in that: The detection conditions of ICP-MS were as follows: RF power: 1550 W, carrier gas flow rate: 1.05 L / min, detection mode: KED mode, He gas flow rate: 4.3 ml / min.