阅读说明：本技术 一种基于质谱分析技术快速鉴定大西洋鲑鱼和虹鳟鱼的方法 (Method for rapidly identifying Atlantic salmon and rainbow trout based on mass spectrometry technology ) 是由 毕红燕 王成玉 于 2020-07-18 设计创作，主要内容包括：本发明公开了一种基于质谱分析技术快速鉴定大西洋鲑鱼和虹鳟鱼的方法,包括如下步骤：步骤(1)准备大西洋鲑鱼样本和虹鳟鱼样本；步骤(2)向两种鱼肉样本冷却后制得样本；步骤(3)得到滤液样本；步骤(4)取步骤(3)待干后进入质谱仪分析；步骤(5)对步骤(4)质谱分析后的两种质谱数据采集；步骤(6)在市场上购买大西洋鲑鱼,并按照上述步骤(1)-(4)的方法处理,然后进行质谱数据采集；步骤(8)比较步骤(6)中的购买大西洋鲑鱼和步骤(5)中的虹鳟鱼样本的质谱图的相似性得分。本发明用基质辅助激光解吸/电离质谱技术区分和鉴定大西洋鲑鱼和虹鳟鱼,达到高效率、省时、快速、灵敏和高通量的检测效果。(The invention discloses a method for rapidly identifying Atlantic salmon and rainbow trout based on a mass spectrometry technology, which comprises the following steps: preparing an Atlantic salmon sample and an oncorhynchus mykiss fish sample; cooling the two fish samples to obtain samples; obtaining a filtrate sample in the step (3); step (4), the dried product obtained in the step (3) is put into a mass spectrometer for analysis; step (5) collecting two kinds of mass spectrum data after the mass spectrum analysis in the step (4); step (6) purchasing Atlantic salmon in the market, processing according to the method of the steps (1) to (4), and then performing mass spectrum data acquisition; and (8) comparing the similarity scores of the mass spectrograms of the purchased atlantic salmon in the step (6) and the rainbow trout sample in the step (5). The invention uses the matrix-assisted laser desorption/ionization mass spectrometry technology to distinguish and identify the Atlantic salmon and the rainbow trout, and achieves the detection effects of high efficiency, time saving, rapidness, sensitivity and high flux.)

1. A method for rapidly identifying Atlantic salmon and rainbow trout based on a mass spectrometry technology is characterized by comprising the following steps:

preparing an Atlantic salmon sample and a rainbow trout sample, weighing 5-10g of each sample, and respectively placing the samples in two containers, wherein the Atlantic salmon and the rainbow trout have close genetic relationship, the fish samples are similar, the color and texture of fish are similar, but the prices are different;

respectively adding trifluoroacetic acid aqueous solution into the two fish samples, then heating on an electric hot plate, cooling to obtain samples, wherein the trifluoroacetic acid aqueous solution is used for dissolving the fish tissue protein or peptide fragment extract and has excellent compatibility with an ion source of a mass spectrometer, and the heating on the electric hot plate is used for dissolving the protein more quickly;

step (3) diluting the two cooled samples with deionized water respectively, centrifuging, taking supernate, diluting the supernate, and filtering to obtain filtrate samples;

step (4) taking two filtrate samples obtained in the step (3) and respectively dripping 1-2 microliters of the two filtrate samples on a mass spectrum target plate, and after drying, feeding the two filtrate samples into a mass spectrometer for analysis;

step (5) collecting two kinds of mass spectrum data after the mass spectrum analysis in the step (4);

step (6) purchasing Atlantic salmon in the market, processing according to the method of the steps (1) to (4), and then performing mass spectrum data acquisition;

step (7) carrying out baseline and normalization processing on the three mass spectrum data collected in the steps (5) and (6);

step (8) comparing similarity scores of mass spectrograms of the Atlantic salmon purchased in step (6) and the rainbow trout sample purchased in step (5), and when the similarity score is zero, judging that the Atlantic salmon purchased is not the rainbow trout;

and (9) comparing the similarity scores of the mass spectrograms of the Atlantic salmon purchased in the step (6) and the Atlantic salmon samples purchased in the step (5), and judging that the Atlantic salmon purchased is the Atlantic salmon when the similarity score is more than 95%.

2. The method for rapidly identifying Atlantic salmon and rainbow trout based on mass spectrometry as claimed in claim 1, wherein the dilution with deionized water in step (4) is designed to prevent the stacking of mass spectrum peaks of protein or peptide fragments and to obtain better mass spectrum peaks in mass spectrometry.

3. The method for rapidly identifying Atlantic salmon and rainbow trout based on mass spectrometry as claimed in claim 1, wherein the centrifugation of step (4) is performed by using a centrifuge of type TGL-18C.

4. The method for rapidly identifying Atlantic salmon and rainbow trout based on mass spectrometry as claimed in claim 1, wherein the filtration of step (4) is a 0.22 μm Millipore cellulose membrane.

5. The method for rapidly identifying Atlantic salmon and rainbow trout based on mass spectrometry as claimed in claim 1, wherein the mass spectrometer of step (4) is of the type Micro Flex LRF.

Technical Field

The invention relates to the technical field of mass spectrometry, and provides a method for rapidly identifying Atlantic salmon and rainbow trout based on a mass spectrometry technology.

Background

The aquaculture industry is one of the important and rapidly developing industries in the world, and as a rapidly developing industry, aquatic products contain many nutrients and physiologically active substances beneficial to human health, so that the demand of people for aquatic products, particularly marine products, is increasing. According to the hundred-degree encyclopedia introduction, the commercial value of salmon (Oncorhynchus) is more than 30, and at present, the most common are 2 kinds of trout (salmon and golden trout) and 4 kinds of salmon (Pacific salmon, Atlantic salmon, arctic white salmon and silver salmon).

Pacific salmon and Atlantic salmon are imported frequently in China. Atlantic salmon (Salmo salar), a raw seafood, is eaten by humans all over the world because it tastes delicious and is rich in proteins, amino acids, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), vitamin D and trace elements such as iron, copper, zinc, manganese, etc. Atlantic salmon has high edible value and large market demand. The cultivation cost of the rainbow trout (Oncorhynchus mykiss) is lower than that of the Atlantic salmon. On the market, the labels are rather fuzzy, and rainbow trout is sometimes also sold as salmon. Rainbow trout is often labeled as salmon in the market to earn profit. In fact, in order to earn benefits, the phenomenon of mislabeling aquatic products to be sub-full exists in the world. Scientists have suggested to label the scientific name of fish in labels as a distinction. The identification of fish species meets the needs of consumers. The rainbow trout and the Atlantic salmon belong to the same salmonidae, and the two kinds of fishes have similar meat color and texture, so that the interests of consumers are damaged when the rainbow trout with lower value is sold as Atlantic salmon to earn a riot. When Atlantic salmon can be used as supplementary food for infants, if the Atlantic salmon is purchased and eaten due to the mislabeling and the like, the health of the infants can be threatened due to factors such as pathogenic microorganisms. The effective discrimination of the Atlantic salmon and rainbow trout can reflect the necessity of identifying the variety of the fish.

The traditional aquatic product identification method is mainly based on identification of morphological characteristics, and the method for distinguishing fish species by observing specimen anatomy and according to the morphological characteristics of fish is practical, rapid and low in cost, but generally needs professionals and products to keep complete. When processed into finished or semi-finished products, the morphological characteristics of the fish disappear or partially disappear, thus increasing the difficulty of identification. Today, assays for classifying fish species based on morphological features still exist. Different fish have otoliths of different shapes and sizes, and otolith morphology is also used to identify fish. But requires the identifier to have the skills and knowledge of fish anatomy. Molecular biology techniques have been widely used to identify fish. However, this technique involves time-consuming and cumbersome steps of deoxyribonucleic acid (DNA) extraction, Polymerase Chain Reaction (PCR) amplification, sequence analysis, and the like. In addition, the relationships of some species are very close, and there are few nucleotide differences in the sequence, increasing the difficulty of identification.

Immunological techniques such as enzyme-linked immunosorbent assay identify Atlantic salmon and rainbow trout based on the recognition of proteins by antibodies, and although these detection methods are effective in identifying Atlantic salmon and rainbow trout, they do not meet the high throughput requirements when large-scale detection and identification is required. The simple, rapid, high-flux and time-saving analysis technology is found for identifying the Atlantic salmon and the rainbow trout, and the requirements of aquatic product markets, food safety and human health are met. Mass Spectrometry (MS) is used in different fields such as analytical chemistry, nucleoside biochemistry, oligonucleotide and nucleic acid analysis, organometallic chemistry, nutrition, food residues and organic adulteration analysis due to its advantages of rapidity, sensitivity and high throughput.

The traditional aquatic product identification method is mainly based on identification of morphological characteristics, and the method for distinguishing fish species by observing specimen anatomy and according to the morphological characteristics of fish is practical, rapid and low in cost, but generally needs professionals and products to keep complete. When processed into finished or semi-finished products, the morphological characteristics of the fish disappear or partially disappear, thus increasing the difficulty of identification. Today, assays for classifying fish species based on morphological features still exist. Different fish have otoliths of different shapes and sizes, and otolith morphology is also used to identify fish. But requires the identifier to have the skills and knowledge of fish anatomy. Molecular biology techniques have been widely used to identify fish. However, this technique involves time-consuming and cumbersome steps of deoxyribonucleic acid (DNA) extraction, Polymerase Chain Reaction (PCR) amplification, sequence analysis, and the like. In addition, the relationships of some species are very close, and there are few nucleotide differences in the sequence, increasing the difficulty of identification. Immunological techniques such as enzyme-linked immunosorbent assay identify Atlantic salmon and rainbow trout based on the recognition of proteins by antibodies, and although these detection methods are effective in identifying Atlantic salmon and rainbow trout, they do not meet the high throughput requirements when large-scale detection and identification is required. The simple, rapid, high-flux and time-saving analysis technology is found for identifying the Atlantic salmon and the rainbow trout, and the requirements of aquatic product markets, food safety and human health are met.

By means of multivariate analysis technology, through carrying out dimension reduction analysis on a large amount of data, for example, a principal component analysis score chart can be obtained to establish a standard chart library, and identification of unknown samples is realized through comparison. However, the multivariate analysis technology relied at present relates to processing methods such as first derivative, second derivative, mean centralization, multivariate scattering correction, standard normal variable transformation and normalization, and the steps are complex. The method for identifying the salmon can realize the identification of the salmon only by carrying out baseline and normalization on the original mass spectrum data and then directly comparing the data, and has the characteristics of rapidness, simplicity and convenience.

In China, a patent (the patent name: a rapid identification method for Atlantic salmon and rainbow trout, the patent application number: 201910126179.8, the publication number: CN109765196A) reports an identification method for Atlantic salmon and rainbow trout, and the method adopts near infrared spectroscopy (NIR) technology to collect and pretreat data, and then adopts a chemometric method to establish a rapid identification model for Atlantic salmon and rainbow trout. This study is essentially different from the present project involving methods and techniques. The invention is based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) technology, directly analyzes the obtained mass spectrum data, and can accurately and quickly distinguish Atlantic salmon and rainbow trout in a short time.

Disclosure of Invention

The invention provides a method for rapidly identifying Atlantic salmon and rainbow trout based on a mass spectrometry technology, which can realize high-throughput, rapid, sensitive and simple fish species identification by adopting the mass spectrometry technology and only needing to be based on a small amount of samples and simple data processing.

The application is realized by the following technical scheme:

a method for rapidly identifying Atlantic salmon and rainbow trout based on a mass spectrometry technology comprises the following steps:

preparing an Atlantic salmon sample and a rainbow trout sample, weighing 5-10g of each sample, and respectively placing the samples in two containers, wherein the Atlantic salmon and the rainbow trout have close genetic relationship, the fish samples are similar, the color and texture of fish are similar, but the prices are different;

respectively adding trifluoroacetic acid aqueous solution into the two fish samples, then heating on an electric hot plate, cooling to obtain samples, wherein the trifluoroacetic acid aqueous solution is used for dissolving the fish tissue protein or peptide fragment extract and has excellent compatibility with an ion source of a mass spectrometer, and the heating on the electric hot plate is used for dissolving the protein more quickly;

step (3) diluting the two cooled samples with deionized water respectively, centrifuging, taking supernate, diluting the supernate, and filtering to obtain filtrate samples;

step (4) taking two filtrate samples obtained in the step (3) and respectively dripping 1-2 microliters of the two filtrate samples on a mass spectrum target plate, and after drying, feeding the two filtrate samples into a mass spectrometer for analysis;

step (5) collecting two kinds of mass spectrum data after the mass spectrum analysis in the step (4);

step (6) purchasing Atlantic salmon in the market, processing according to the method of the steps (1) to (4), and then performing mass spectrum data acquisition;

step (7) carrying out baseline and normalization processing on the three mass spectrum data collected in the steps (5) and (6);

step (8) comparing similarity scores of mass spectrograms of the Atlantic salmon purchased in step (6) and the rainbow trout sample purchased in step (5), and when the similarity score is zero, judging that the Atlantic salmon purchased is not the rainbow trout;

and (9) comparing the similarity scores of the mass spectrograms of the Atlantic salmon purchased in the step (6) and the Atlantic salmon samples purchased in the step (5), and judging that the Atlantic salmon purchased is the Atlantic salmon when the similarity score is more than 95%.

Further, the purpose of the dilution with deionized water in the step (4) is to prevent the stacking of mass spectrum peaks of the protein or peptide fragment during mass spectrometry, so that good mass spectrum peaks can be obtained more easily.

Further, the centrifugation in the step (4) is performed by adopting a centrifuge with the model number of TGL-18C.

Further, the filtration of the step (4) is a filtration with a 0.22 μm Millipore cellulose membrane.

Further, the model of the mass spectrometer in the step (4) is Micro Flex LRF.

Has the advantages that: the meat color and texture of a plurality of fishes with similar species such as Atlantic salmon and rainbow trout are similar, and particularly the fish fillets, fish steaks or surimi are processed to increase the distinguishing difficulty, so that a rapid, sensitive and reliable mass spectrum technology is established to distinguish and identify the fishes and the rainbow trout. The invention can achieve the purpose of distinguishing the Atlantic salmon and the rainbow trout only by a small amount of samples. The method can distinguish and identify the Atlantic salmon and the rainbow trout by directly comparing mass spectrograms only by carrying out basic processing on the obtained original mass spectrometric data. The invention uses matrix-assisted laser desorption/ionization (MALDI) Mass Spectrometry (MS) technology to distinguish and identify Atlantic salmon and rainbow trout, and achieves the detection effects of high efficiency, time saving, rapidness, sensitivity and high flux.

Drawings

FIG. 1 is a graph showing the mass spectral similarity scores of Atlantic salmon and rainbow trout in this application.

FIG. 2 is a graph showing the mass spectrum similarity scores of rainbow trout and blind samples in the present application.

FIG. 3 is a schematic representation of mass spectral similarity scores for Atlantic salmon and blind samples in this application.

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 protection scope of the present invention is not limited to the following embodiments.

A method for rapidly identifying Atlantic salmon and rainbow trout based on a mass spectrometry technology comprises the following steps:

preparing an Atlantic salmon sample and a rainbow trout sample, weighing 5-10g of each sample, and respectively placing the samples in two containers, wherein the Atlantic salmon and the rainbow trout have close genetic relationship, the fish samples are similar, the color and texture of fish are similar, but the prices are different;

respectively adding trifluoroacetic acid aqueous solution into the two fish samples, then heating on an electric hot plate, cooling to obtain samples, wherein the trifluoroacetic acid aqueous solution is used for dissolving the fish tissue protein or peptide fragment extract and has excellent compatibility with an ion source of a mass spectrometer, and the heating on the electric hot plate is used for dissolving the protein more quickly;

step (3) diluting the two cooled samples with deionized water respectively, centrifuging, taking supernate, diluting the supernate, and filtering to obtain filtrate samples;

step (4) dripping 1-2 microliters of two filtrate samples obtained in the step (3) on a mass spectrum target plate respectively, and allowing the two filtrate samples to enter a mass spectrometer for analysis after drying, wherein the purpose of diluting with deionized water in the step (4) is to prevent mass spectrum peaks of protein or peptide fragments from stacking and easily obtain good mass spectrum peaks in mass spectrum analysis, the centrifugation in the step (4) adopts a centrifuge with the model of TGL-18C for centrifugation, the filtration in the step (4) is filtration by a Millipore cellulose membrane with the size of 0.22 mu m, and the model of the mass spectrometer in the step (4) is Micro Flex LRF;

step (5) collecting two kinds of mass spectrum data after the mass spectrum analysis in the step (4);

step (6) purchasing Atlantic salmon in the market, processing according to the method of the steps (1) to (4), and then performing mass spectrum data acquisition;

step (7) carrying out baseline and normalization processing on the three mass spectrum data collected in the steps (5) and (6);

step (8) comparing similarity scores of mass spectrograms of the Atlantic salmon purchased in step (6) and the rainbow trout sample purchased in step (5), and when the similarity score is zero, judging that the Atlantic salmon purchased is not the rainbow trout;

and (9) comparing the similarity scores of the mass spectrograms of the Atlantic salmon purchased in the step (6) and the Atlantic salmon samples purchased in the step (5), and judging that the Atlantic salmon purchased is the Atlantic salmon when the similarity score is more than 95%.

In order to verify the correctness of the method of the present application, the applicant performed genetic identification on the atlantic salmon after the test, and found that the identified result is the atlantic salmon, which indicates that the method of the present application for identifying fish species is feasible. Similarly, in the method for determining the type of fish by using the similarity score of mass spectrograms according to the present application, the method may also be used for analyzing mass spectrogram data by selecting fish having close relationship with other fish species.

1) Differentiation of atlantic salmon and rainbow trout based on mass spectral similarity scores:

in order to distinguish the Atlantic salmon from the rainbow trout, the mass spectrograms of the Atlantic salmon and the rainbow trout are directly compared by software, as shown in figure 1, wherein 1 and 2 respectively represent the rainbow trout and the Atlantic salmon, and the mass spectrograms of the Atlantic salmon and the Atlantic salmon are relatively similar to each other, but the mass spectrograms of the Atlantic salmon and the Atlantic salmon are compared by the software, so that the similarity score is 0, and the data structure shows that the Atlantic salmon and the Atlantic salmon actually have relatively large difference. A number of results indicate that the method of the invention is capable of achieving the identification of Atlantic salmon and rainbow trout.

2) Identification of commercially available fish (blind sample):

as shown in FIGS. 2 to 3, 1,2 and 3 represent rainbow trout, Atlantic salmon and blindness, respectively. Fig. 2 shows a plot of mass spectral similarity scores for the blind sample and rainbow trout, and it can be seen that the mass spectral similarity score for the blind sample (score 3) and rainbow trout (score 1) is 0, and in addition, fig. 3 shows a plot of mass spectral similarity scores for the blind sample and atlantic salmon (score 2), and the results show that the mass spectrum of the blind sample (score 3) and atlantic salmon (score 2) has 98.0% similarity, which we believe is from atlantic salmon. In summary, we can identify unknown samples by directly comparing mass spectral data patterns.

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.