CN110702483B - A pretreatment method for ultrafast identification of seafood or meat - Google Patents

Abstract

The invention discloses a pretreatment method for identifying seafood or meat at a super-fast speed, which comprises the following steps: step 1, putting 5g of seafood muscle tissue sample and 5mL of 0.1M TFA aqueous solution into a beaker, and fully mixing the seafood muscle tissue sample and the TFA aqueous solution by using a glass rod to obtain a mixture, step 2, putting the mixture obtained in the step 1 on an electric heating plate, stirring and heating the mixture for 5min to boil, then cooling the mixture to room temperature, step 3, diluting the cooled sample mixture to 10mL, and centrifuging the mixture for five minutes at 1006g to obtain a supernatant; and 4, taking 50 mu L of the supernatant obtained in the step 3, diluting the supernatant to 2.5mL by using deionized water, taking out 2mL, filtering the supernatant by using a 0.22 mu m Millipore membrane, and taking 1 mu L of the filtered sample to be dripped on a target plate in the step 5, and after the sample is dried, putting the sample into a mass spectrometer for analysis. The invention can achieve the detection effects of high efficiency, rapidness and high flux.

Description

A pretreatment method for ultrafast identification of seafood or meat

technical field

The invention relates to the fields of pretreatment technology, mass spectrometry technology and chemometrics, and provides an efficient and reliable pretreatment method capable of identifying fish, seafood muscle tissue or muscle tissue of other edible animals.

Background technique

The pretreatment of the sample is a key step in the analysis of the sample, and the pretreatment is one of the important factors restricting the analysis efficiency. In recent years, the study of sample pretreatment methods has attracted extensive attention.

The pretreatment technology is mainly to effectively separate the complex components to improve the separation degree of each component, and then quantitatively or qualitatively analyze the target sample. In addition, when the sample to be tested is analyzed, the unreasonable pretreatment of the sample will also affect the test results. Therefore, reasonable preprocessing methods need to be developed.

There are many methods for pretreatment (extraction) of seafood muscle tissue protein, such as organic solvent method, ultrasonic-assisted method, mechanical crushing method, water enzymatic method, heat treatment method, water-soluble method, and the combination of these methods, etc. ² . One of the purposes of pretreatment is to remove excess fat and other non-protein substances to achieve protein extraction.

Acid-base method is a commonly used method for protein extraction. Muscle tissue will be dissolved to the greatest extent under acidic or alkaline conditions. After dissolution, centrifugation. The upper layer is the fat layer, the middle layer is the protein solution, the lower layer is insoluble matter, and the middle layer is Usually it is the desired extracted target protein.

In recent years, proteomics has been widely used in species identification and traceability. Measurement of peptide mass fingerprints (PMFs) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) is the main method in identifying differential proteins. Studies have shown that matrix, salt ions, and sample pretreatment can affect mass spectrometry results. Sample pretreatment can affect ionic strength, number of peaks, etc.

Domestic patents (patent application number: CN201310080097.7, publication number: CN104046683A) have reported a method for species identification, which utilizes the DNA barcode gene PCR amplification technology in the nucleus and the high-resolution melting curve method to identify close relatives species or its hybrid progeny. There are essential differences between this research and the methods and techniques involved in this project.

The prior art involves pre-processing steps such as extracting target individual DNA, designing primers, PCR amplification, and sequence alignment.

SUMMARY OF THE INVENTION

The present invention provides a pretreatment method for ultra-fast identification of seafood or meat, the purpose of which is to adopt a time-saving and efficient pretreatment technology, which can be used to identify and identify seafood only based on a small number of samples, and the biological substance extraction step takes at most five minute.

This application is achieved through the following technical solutions:

A kind of pretreatment method of ultra-fast identification of seafood or meat, is characterized in that, comprises the steps:

Step 1, take 0.1g～50kg seafood muscle tissue sample and 0.1mL～50L 0.1M TFA aqueous solution into a beaker, and mix them thoroughly with a glass rod to obtain a sample mixture;

In step 2, the sample mixture in step 1 is heated on an electric hot plate, heated to boiling while stirring, and then cooled to room temperature;

Step 3, dilute the cooled sample mixture in Step 2 to 10 mL, and centrifuge for a certain period of time to obtain a supernatant;

Step 4, take 50 μL of the supernatant obtained in step 3, dilute it to 2.5 mL with deionized water, take out 2 mL, and filter it with a 0.22 μm Millipore membrane;

In step 5, 1 μL to 2 μL of the sample filtered in step 4 is dropped on the target plate, and after drying, the matrix-assisted laser desorption ionization mass spectrometer is used for analysis.

Further, the electric hot plate can be other heating devices that can cook the sample tissue out of lysate.

Further, the matrix-assisted laser desorption ionization mass spectrometer may be a mass spectrometer with other ionization modes.

Further, the sample mixture in the step 2 can dissolve the protein after being heated and boiled.

Further, the matrix-assisted laser desorption ionization mass spectrometer is combined with chemometric methods to identify seafood or meat.

Further, the selection of the seafood muscle tissue samples in the step 1 adopts a blind sample identification method, and one or more are arbitrarily selected from the seafood muscle tissue samples.

Further, the unknown samples are identified according to the similarity of the mass spectra of each species, or the principal component analysis chart is obtained according to the chemometrics method, and the blind sample is identified by judging the aggregation of the blind samples and each sample.

Further, in the step 1, 5g of seafood muscle tissue samples and 5mL of 0.1M TFA were mixed in the same proportion.

Further, in the step 1, 5g of seafood muscle tissue sample and 2.5mL of 0.2M TFA were mixed in the same proportion.

Further, the centrifugation in the step 3 is centrifugation at 1006g.

Beneficial effects:

There are many types of seafood, many of which are closely related or not similar in appearance or meat quality, or are difficult to distinguish after being made into minced meat. Therefore, some traders will mislabel or use low-priced seafood instead of high-priced seafood. In order to make profits, some people are allergic to specific seafood. If these seafoods are mislabeled or substituted, it will damage the economic interests and even health of consumers. The present invention utilizes non-enzymatic pretreatment techniques combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) analysis to obtain more than 190 mass spectrometry data, while combining chemometric techniques including principal component analysis (PCA) and partial least squares Multiplicative regression discriminant analysis (PLS-DA) is further used to analyze the similarities and differences between different seafood species, which can achieve high-efficiency, rapid and high-throughput detection.

Description of drawings

FIG. 1 is a schematic diagram of the identification of different seafood according to the first embodiment of the present invention (including FIGS. 1A, 1B, 1C, and 1D).

FIG. 2 is a schematic diagram of identifying seafood with similar species according to the first embodiment of the present invention (including FIGS. 2A and 2B ).

FIG. 3 is a schematic diagram of identification by the blind sample identification method according to the second embodiment of the present invention.

Detailed ways

Below in conjunction with the accompanying drawings, the embodiments of the present invention are described in detail: the present embodiment is implemented on the premise of the technical solution of the present invention, and provides detailed embodiments and specific operation processes, but the protection scope of the present invention is not limited to the following described embodiment.

Example 1 (identification of large yellow croaker and small yellow croaker):

The large yellow croaker and the small yellow croaker belong to the same genus (Genus) in scientific classification, and their appearance and morphology are so similar that it is difficult for laymen to distinguish them. Therefore, they are used as analytical models. The current technology mainly relies on DNA technology, which is costly and relatively time-consuming. The present invention can successfully distinguish the two in the whole 1-2 hours based on the preprocessing of five minutes.

1. Sample preprocessing:

1. Put 5g of yellow croaker tissue sample mixture and 5mL of 0.1M TFA aqueous solution into a beaker, and mix thoroughly with a glass rod to obtain a mixture;

2. Place the mixture in step 1 on a hot plate, stir and heat for 5 minutes to boil, then cool to room temperature;

3. Dilute the cooled sample mixture in step 2 to 10 mL, and centrifuge at 1006g for five minutes to obtain a supernatant;

4. Take 50 μL of the supernatant from step 3, dilute to 2.5 mL with deionized water, remove 2 mL, and filter with 0.22 μM Millipore membrane.

Second, mass spectrometry analysis:

Take 1 μL of the sample filtered in the above stage and drop it on the target plate. After drying, enter the mass spectrometer (model: Micro FlexLRF, or other mass spectrometry or spectroscopic techniques) for analysis to obtain mass spectral data.

3. Distinguish species based on stoichiometry:

Principal component analysis (PCA) and partial least squares regression discriminant analysis (PLS-DA) were performed on the mass spectrometry data in the above stages.

1) Identification of different seafood:

As shown in FIG. 1, FIG. 1A and FIG. 1C are the mass spectra of seafood; FIG. 1B is the principal component analysis (PCA) diagram of five kinds of seafood; FIG. 1D is the principal component analysis (PCA) diagram of two overlapping fishes. Figures 1A and 1B are two fish of the same genus (Genus) scientifically. Figure 1A shows the different mass spectra of large yellow croaker. We can see that these six mass spectra are very similar, but due to systematic errors such as operation and instrumentation, the intensities of some proteins/peptides are different or new proteins appear. Similarly, the six mass spectra of the small yellow croaker in Fig. 1C also resulted in different intensities of certain proteins/peptides or the appearance of new proteins due to these systematic errors. Figure 1B is a principal component analysis (PCA) diagram of five types of seafood. Different seafoods are represented by different shapes. It can be seen that fish 1 and 2 in Figure 1B overlap due to their similar classifications, so fish 1 and 2 are separately distinguished, as shown in the figure As shown in 1D, when fish 1 and 2 are distinguished individually, the two can be well distinguished.

Combined with mass spectrometry (MS) technology and chemometrics technology, the identification of different types of seafood can be achieved. Figure 1C shows that Species 1 and Species 2 overlap because they are fish of the same genus (Genus). FIG. 1A is the mass spectrum of the five kinds of seafood, and FIG. 1C is the principal component analysis diagram of the five kinds of seafood; 1, 2, 3, 4, and 5 in the figure represent seafood variety 1, seafood variety 2, seafood variety 3, and seafood variety 4, respectively. , seafood varieties 5.

2) Identification of seafood with similar species:

In order to realize the identification of seafood products with similar species, we use two species of fish belonging to the same genus as a model. Figure 2 is its mass spectrum and principal component analysis. It was found that although the mass spectrometry data patterns of these two fish were similar, their PCA classification maps were significantly different. We also analyzed other seafood samples of similar species, and a large amount of data showed that our preprocessing method can realize the identification of seafood products of similar species.

As shown in Figure 2, the identification of similar seafood products. FIG. 2A is a mass spectrum of two fish of the same genus, and FIG. 2B is a principal component analysis diagram of the two fish.

Example 2 (blind identification):

As shown in Figure 3, 5 g of fish meat tissue samples were taken, and a sample was randomly selected from the obtained fish meat samples for blind sample testing to further verify the reliability of the method. The results showed that the single-blind samples overlapped with the actual samples within the 95% confidence interval, which further demonstrated the feasibility of blind sample identification by this method. The mass spectrum of the blind sample has higher similarity than the mass spectrum of seafood 5. The applicant believes that the blind sample is from seafood 5, and the unknown samples can be identified by directly comparing the mass spectrometry data patterns. For unknown samples of similar species, in order to improve the identification accuracy, a large amount of data is synthesized, and based on chemometric methods, it can be obtained that the location sample is from a certain sample at a certain confidence level (eg 99% or 95%).

The invention is a pretreatment method for ultra-fast identification of seafood, which is also applicable to other non-seafood protein meat products.

The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and the descriptions in the above-mentioned embodiments and the description are only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have Various changes and modifications fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

1. A pretreatment method for identifying seafood or meat at ultra-fast speed is characterized by comprising the following steps:

step 1, putting 0.1 g-50 kg of seafood muscle tissue sample and 0.1 mL-50L of 0.1MTFA aqueous solution into a beaker, and fully mixing with a glass rod to obtain a sample mixture;

step 2, placing the sample mixture obtained in the step 1 on an electric hot plate for heating, heating to boiling while stirring, and then cooling to room temperature;

step 3, diluting the sample mixture cooled in the step 2 to 10mL, and centrifuging for a certain time to obtain a supernatant;

step 4, taking 50 mu L of the supernatant obtained in the step 3, diluting the supernatant to 2.5mL by using deionized water, taking out 2mL, and filtering the supernatant by using a 0.22 mu m Millipore membrane;

step 5, dripping 1-2 mu L of the sample filtered in the step 4 on a target plate, and analyzing by using a matrix-assisted laser desorption ionization mass spectrometer after drying;

the sample mixture in the step 2 can dissolve protein after being heated and boiled;

the matrix-assisted laser desorption ionization mass spectrometer is combined with a chemometrics method to identify seafood or meat;

selecting the seafood muscle tissue samples in the step 1 by adopting a blind sample identification method, and optionally selecting one or more from the seafood muscle tissue samples;

and identifying unknown samples by comparing the similarity of mass spectrograms of various species, or obtaining a principal component analysis chart by a chemometric method, and judging the aggregation condition of the blind samples and various samples to carry out blind sample identification.

2. The pretreatment method of claim 1, wherein the electric heating plate is a heating device capable of boiling the sample tissue to obtain a solution.

3. The pretreatment method of claim 1, wherein the matrix assisted laser desorption ionization mass spectrometer is a mass spectrometer with other ionization modes.

4. The pretreatment method of claim 1, wherein 5g of seafood muscle tissue sample is mixed with 5mL of 0.1M TFA at the same ratio in step 1.

5. The pretreatment method of claim 1, wherein 5g of seafood muscle tissue sample is mixed with 2.5mL of 0.2M TFA at the same ratio in step 1.

6. The pre-treatment method for ultra-fast identification of seafood or meat as claimed in claim 1, wherein the centrifugation in step 3 is at 1006 g.

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