CN110702483B

CN110702483B - Pretreatment method for identifying seafood or meat at ultra-fast speed

Info

Publication number: CN110702483B
Application number: CN201911083556.0A
Authority: CN
Inventors: 毕红燕; 王成玉
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2019-11-07
Filing date: 2019-11-07
Publication date: 2022-05-10
Anticipated expiration: 2039-11-07
Also published as: CN110702483A

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

Pretreatment method for identifying seafood or meat at ultra-fast speed

Technical Field

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

Background

The pretreatment of the sample is a key step for analyzing the sample, and the pretreatment is one of important factors for limiting the analysis efficiency. In recent years, research on methods for pretreating samples has been attracting much attention.

The pretreatment technology is mainly used for effectively separating complex components so as to improve the separation degree of each component and further quantitatively or qualitatively analyze a target sample. In addition, when a sample to be detected is analyzed, the detection result is also influenced by the irrational pretreatment of the sample. Therefore, reasonable pretreatment methods need to be developed.

The pretreatment (extraction) method of seafood muscle tissue protein is many, such as organic solvent method, ultrasonic-assisted method, mechanical disruption method, aqueous enzymatic method, heating treatment method, aqueous dissolution method, and combination thereof². One of the purposes of the pretreatment is to remove non-protein substances such as excess fat and to extract protein.

The acid-base method is a common method for extracting protein, the protein can be dissolved to the maximum extent in muscle tissue under the acidic or alkaline condition, the protein is dissolved and centrifuged, the upper layer is a fat layer, the middle layer is a protein solution, the lower layer is insoluble substances, and the middle layer is the target protein which needs to be extracted.

In recent years, proteomics is widely applied to species identification, root tracing and the like. The method for measuring Peptide Mass Fingerprint (PMF) by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) is a main method for identifying differential protein. Research shows that pretreatment of matrix, salt ions and samples can affect the mass spectrometry result. Sample pretreatment can affect ion intensity, peak count, etc.

There are patents in China (patent application No.: CN201310080097.7, publication No.: CN104046683A) which report methods for species identification, which utilize the PCR amplification technology of DNA barcode genes in the nucleus in combination with a high resolution melting curve method to identify closely related species or their hybrid progeny. This study is essentially different from the present project involving methods and techniques.

The prior art relates to pretreatment steps of extracting target individual DNA, designing primers, PCR amplification, sequence comparison and the like, and the technology adopts a water boiling method to extract the DNA of each individual, wherein the extraction step takes 30 minutes.

Disclosure of Invention

The invention provides a pretreatment method for identifying seafood or meat at a super-fast speed, which aims to adopt a time-saving and efficient pretreatment technology, can be used for identifying and identifying seafood only on the basis of a small amount of samples, and takes five minutes at most in a biological substance extraction step.

The application is realized by the following technical scheme:

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

step 1, taking 0.1g to 50kg of seafood muscle tissue sample and 0.1mL to 50L of 0.1M TFA aqueous solution, putting the seafood muscle tissue sample and the 0.1mL to 50L TFA aqueous solution into a beaker, and fully mixing the seafood muscle tissue sample and the 0.1L TFA aqueous solution by using 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;

and 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.

Further, the electric heating plate may be other heating devices capable of boiling the sample tissue out of the lysate.

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

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

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

Further, the seafood muscle tissue samples in the step 1 are selected by adopting a blind sample identification method, and one or more of the seafood muscle tissue samples are selected optionally.

Further, an unknown sample is identified by comparing the similarity of mass spectrograms of various species, or a principal component analysis chart is obtained by a chemometric method, and the aggregation condition of the blind sample and various samples is judged to carry out blind sample identification.

Further, 5g of seafood muscle tissue sample was mixed with 5mL of 0.1M TFA at the same ratio in step 1.

Further, 5g of seafood muscle tissue sample was mixed with 2.5mL of 0.2M TFA at the same ratio in step 1.

Further, the centrifugation in step 3 was carried out at 1006 g.

Has the advantages that:

many seafood types, wherein many seafood with similar or dissimilar relatives are difficult to distinguish due to similar appearance or meat quality and appearance, or meat paste, so some vendors can mark or use low-price seafood to replace high-price seafood to make profits, and some people are allergic to specific seafood, and if the seafood is marked or replaced by mistake, the economic benefit and even health of consumers can be damaged. The invention combines non-enzymatic pretreatment technology with matrix-assisted laser desorption/ionization (MALDI) Mass Spectrometry (MS) analysis to obtain more than 190 mass spectrometry data, and combines chemometrics technology including Principal Component Analysis (PCA) and partial least squares regression discriminant analysis (PLS-DA) to further analyze the similarity and difference between different marine products, thereby achieving the detection effects of high efficiency, rapidness and high flux.

Drawings

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

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

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

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.

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), have similar appearance forms and are difficult to distinguish in the case of layman, and thus are used as analytical models. The current technology relies mainly on DNA technology, which is costly and relatively time consuming. The invention can successfully distinguish the two in the whole 1-2 hours based on the pretreatment of five minutes.

Firstly, sample pretreatment:

1. placing 5g of the tissue sample mixture of the large and small yellow croakers and 5mL of 0.1M TFA aqueous solution into a beaker, and sufficiently mixing with a glass rod to obtain a mixture;

2. placing the mixture obtained in the step 1 on an electric hot plate, stirring and heating for 5min to boil, and then cooling to room temperature;

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

4. 50 μ L of the supernatant from step 3 was taken, diluted to 2.5mL with deionized water, 2mL removed, and filtered through a 0.22 μm Millipore membrane.

Secondly, mass spectrometry analysis:

and dripping 1 mu L of the sample filtered in the stage on a target plate, and after drying, analyzing the sample in a mass spectrometer (model: Micro Flex LRF, or other mass spectra or spectral techniques) to obtain mass spectrum data.

And thirdly, distinguishing species based on chemometrics:

the mass spectral data from the stages above was subjected to Principal Component Analysis (PCA) and to partial least squares regression discriminant analysis (PLS-DA).

1) Identification of different seafood:

as shown in fig. 1, fig. 1A and 1C are mass spectrograms of seafood; FIG. 1B is a Principal Component Analysis (PCA) diagram of five seafood products; FIG. 1D is a graph of Principal Component Analysis (PCA) of two fish species overlaid on each other. FIGS. 1A and 1B are two species of fish of the same Genus (Genus) in scientific classification. FIG. 1A is a diagram showing different mass spectra of large yellow croaker, and we see that the six mass spectra are very similar, but the intensity of some proteins/peptide fragments is different or new proteins appear due to systematic errors of operation, instruments and the like. Similarly, the six mass spectra of the small yellow croaker in FIG. 1C resulted in different intensities of some proteins/peptide fragments or new proteins due to these systematic errors. FIG. 1B is a Principal Component Analysis (PCA) chart of five seafood products, different seafood products are shown in different shapes, and it can be seen that

fish

1 and 2 in FIG. 1B overlap due to similar classifications, and thus

fish

1 and 2 are distinguished individually, as shown in FIG. 1D, and when

fish

1 and 2 are distinguished individually, they can be distinguished well.

The identification of different seafood species can be realized by combining the Mass Spectrum (MS) technology and the chemometrics technology. FIG. 1C shows that fingerling 1 and fingerling 2 overlap due to fish of the same Genus (Genus). FIG. 1A is a mass spectrum diagram of five seafood, and FIG. 1C is a main component analysis diagram of five seafood; in the figure, 1,2,3,4 and 5 respectively represent seafood variety 1, seafood variety 2, seafood variety 3, seafood variety 4 and seafood variety 5.

2) Identifying seafood with similar species:

in order to realize the identification of seafood products with similar species, two fish species belonging to the same genus are used as models. FIG. 2 is a mass spectrum and a principal component analysis chart thereof. The two fish were found to have distinct differences in their PCA classification profiles, although the mass spectra data patterns were very close. Other seafood samples with similar species are analyzed, and a large amount of data show that the pretreatment method can realize the identification of seafood products with similar species.

As shown in FIG. 2, it is the identification of similar seafood products. FIG. 2A is a mass spectrum of two species of fish belonging to the same genus, and FIG. 2B is a principal component analysis chart of the two species of fish.

Example 2 (blind identification):

as shown in FIG. 3, 5g of a fish tissue sample was taken, and any one of the obtained fish samples was subjected to a blind test for further verifying the reliability of the method. The results show that single blind samples overlap with actual samples within 95% confidence intervals, further illustrating the feasibility of blind identification using this method. The mass spectrogram of the blind sample has higher similarity than that of seafood 5, and the applicant believes that the blind sample is from seafood 5 and can directly compare a mass spectrum data pattern to identify an unknown sample. For closely related unknown samples, to improve identification accuracy, a large number of data are combined, and based on chemometric methods, it can be obtained that the location sample is from a certain sample at a certain confidence level (e.g., 99% or 95%).

The invention relates to a pretreatment method for identifying seafood with ultra-fast speed, which is also suitable for other non-seafood protein meat products.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

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 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.