CN108896646A - The Fast Classification and identification method of seafood similar in affiliation - Google Patents
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Abstract
The present invention provides the Fast Classification and identification method of seafood, and the classification and identification between difference seafood similar in kind may be implemented.The part meat or skin for taking seafood, as sample to be tested；Sample to be tested is placed in alkaline solution and is handled；Solution is adjusted to neutral or faintly acid；Solution desalination；Solution is detected with mass spectrum and records mass signal；Chemometrics application is carried out to the mass signal of acquisition, to distinguish different seafood.The method of the invention is simple and easy, is based on simple sample pretreatment, the scientific verification of the seafood of same category, good resolution, high sensitivity can be realized within a few hours.
Description
Technical field
The present invention relates to analytical chemistry, mass spectrum and quick analysis fields, provide a kind of quick point for can be used for seafood
The analysis method of class and identification.
Background technique
The fraud driven in aquatic products industry by economic interests is a global problem, wherein food labelling accidentally pastes
Phenomenon is very universal, replaces expensive seafood for aquatic products, including with inexpensive seafood, replaces object in imminent danger with normal legal seafood
Kind, replace needing to carry out the seafood etc. of toxin and allergy detection with normal seafood, economy and health, whole world sea to consumer
The ecological environments such as ocean, lake and river can produce negative effect.Random searching is carried out to 19 national market fishes samples
It has been shown that, accidentally patch fingerling is up to 25% to average label, wherein the label mistake patch rate of China and Turkey is more up to 86%.According to《When
For weekly》Report there is 58% to need to screen by toxin and allergy in the aquatic products of U.S.'s label mistake patch, label mistake patch is
In order to escape coherent detection.FAO (Food and Agriculture Organization of the United Nation) (FAO) estimates that the annual output total value of global aquatic food industry is 120,000,000,000 dollars,
Label mistake patch, which is equivalent to, cheats this value at 400,000,000,000 dollars1.It can be seen that economic problems of the label mistake discount as initiation
It is quite serious with potential health problem, it need to cause more to pay attention to.
On the other hand, statistics finds global about more than 32500 kinds of fishes, and quantity is also increasing1.According to incomplete system
Meter, the fish and shellfish for probably having 110 kinds or so are commonly used for seafood.It is ambiguous to develop high-throughput, time saving, suitable identification
The method of the seafood of kind is particularly important.
In addition to shape observation, the method for identifying that species are most praised highly is DNA test, and gene is present in any tissue,
It is not limited by age and state, and can seafood sample is analyzed to treated；But the extraction of DNA is related to very much
Step, time-consuming and laborious, the design of primer is most important during PCR amplification, and whether the species for being related to seafood can succeed quilt
Identification, and the design of appropriate primer is not easy to.
Proteomic techniques are developed to the identification of seafood kind, e.g., the two of water-solubility protein in fish myogen
Dimension chromatography (2-DE) is used for the identification of fish, and Bossier etc. is directed to water-soluble fish myogen with isoelectric focusing (IEF) method and is divided
Analysis is used to be formed a database with the IEF of family or same fish flesh sample water solubility Protein Separation figure and makees to identify fish
For reference, the identification to realize fingerling is compared with database by unknown sample.But they have found the IEF of the fish of some types
Scheme closely similar, or even close to the similarity between different samples, they suggest making more samples to build library.But it does more
Few sample is able to reflect the real conditions of a sample, they do not provide more specific description2.Ortea etc. is by reverse phase hair
Capillary liquid-phase chromatographic and tandem mass spectrometry reflect to different shrimps to being analyzed with the fish flesh peptide hydrolysis of ultrasonic wave added
Not3.Palmblad etc. proposed the identification that fish is realized using the comparison of a large amount of tandem mass spectrometry data in 2013.Based on protein
The data that the fish of omics technology or the identification of marine product are normally based on tandem mass spectrometry find the specific peptide fragment of species4, 2016
Year, Palmblad etc. omits the step of looking for specific peptide fragment, but has collected a large amount of mass spectrometric datas, with these mass spectrum figures
At a mass spectrum graphics and text library (mass spectral library), by unknown sample mass spectrogram and the progress of mass spectrum graphics and text library
It compares, realizes the identification of unknown sample5。
Under the distant situation of seafood race relation, identify relatively easily, still, the seafood products of the same category are because of it
The similitude of shape, for layman, however it is often difficult to distinguish.If for the same category of the purpose reaped staggering profits seafood that
This pretends to be, and is more difficult to be found.The scientific method classified and identified specifically for seafood similar in affiliation waits
Exploitation.
Summary of the invention
The present invention in order to overcome the prior art at least one is insufficient, a kind of quick differentiating method of seafood is provided, is realized
The Fast Classification of seafood.The method is also applied for the differentiation between seafood similar in affiliation.
Another object of the present invention is to establish seafood analytical database, obtains the Fast Classification of seafood and the side of identification
Method realizes that the method based on mass-spectrometric technique carries out Fast Classification and identification to seafood, can quickly screen the type of seafood, guard against
The purpose of personation and wrong mark seafood.
To achieve the goals above, the present invention uses following technical scheme：
The present invention provides a kind of quick differentiating method of seafood, includes the following steps：
Step a takes a small amount of meat or skin of seafood, as sample to be tested；
Step b, sample to be tested is placed in alkaline solution and is handled；
The solution of step b is adjusted to neutral or faintly acid after the completion of processing by step c；
The solution that step c is obtained is detected with mass spectrum and records mass signal by step d；
Step e carries out chemometrics application to the mass signal of acquisition, to distinguish different seafood.
Further, the alkaline solution in step b is sodium hydroxide solution, potassium hydroxide solution etc. with the molten of strong basicity
Liquid.
Further, in step b, after sample to be tested processing, the molecular weight after protein resolution is less than 10k Da.
Further, the mass spectrum be Matrix assisted laser desorption ionization mass spectrum, ion trap mass spectrometry, flight time mass spectrum,
One of level four bars mass spectrum.
Further, the seafood is one of fish, shellfish, shrimps, crab class and aquatic mammality or a variety of.
Further, the seafood is fish.
Further, fish-skin is as sample to be tested.The material of the analysis fish used is from fish-skin, including its back, abdomen etc.
Position, or the other parts from fish.Subcutaneous tissue is removed when fish-skin use；When analyzed fingerling has fish scale,
Remove fish scale.
Further, the concentration of the sodium hydroxide or potassium hydroxide solution is 0.5 mole every liter -1.3 moles every liter.Concentration
Can also be lower or higher, the lower words of concentration can be by extending the reaction time, and the concentration higher words reaction time can contract
It is short, to obtain biological sample.Other alkaline solutions can be salting liquid, such as sodium carbonate liquor or solution of potassium carbonate.Other are borrowed
Helping the means such as enzyme or/and ultrasound can also be used to obtain biological substance from seafood and is further classified and identification and analysis.
Further, before step d, it is also necessary to be purified to solution, the main purpose of purification is to remove in solution
Extra salinity；The method of purification can rotate de-sludging pillar, magnetic bead absorption, ultrafiltration auxiliary sample preparation method, acetone precipitation
Deng.
Further, solution is purified using de-sludging pillar.
Fast Classification described further and identification method between same category or the seafood and any seafood of same section into
Row classification.
The present invention also provides a kind of method for quickly identifying of seafood, include the following steps：
Step a takes the part meat or skin of seafood, as sample to be tested；
Sample to be tested is placed in alkaline solution and handled by step b；
Obtained solution is detected with mass spectrum and records mass signal by step c；
Step d repeats above step, obtains the number of principal components evidence of other seafood, establish a database；
Step e repeats the mass signal that step a-c obtains sample to be tested for seafood to be measured；
The mass signal of acquisition is compared with database by step f, obtains the information of seafood to be measured.
After the completion of step b processing, obtained solution is adjusted into pH value to neutral or faintly acid.
Compared with prior art, the present invention has the following advantages that：
The present invention utilizes the meat or skin of seafood, is handled by strong base solution, the biological sample solution after being cleared up,
Using the polypeptide or protein of the small-molecular-weight in Mass Spectrometer Method solution, not only realized pair in conjunction with chemometrics application method
Variety classes seafood is classified, and realizes the identification to unknown seafood kind.The method of the invention is simple and easy, can
To carry out Fast Classification and identification to seafood, and without carrying out complicated pretreatment to sample.
Molecule of the present invention using MALDI MS to molecular weight less than 10k Da detects, and using testing result to not
Same fingerling class distinguishes.In the molecular weight ranges, MALDI MS can provide preferable resolution ratio and sensitivity.This hair
It is suitable for other animals in addition to seafood, including birds, mammal etc. on bright such taxonomic identification theoretical method.
Further, the present invention is handled fish-skin using sodium hydroxide solution, can be in 1 hour or even shorter time
Inside obtain specific sample；Interminable digestion process in the prior art is omitted, however enzyme, as a kind of albumen, cost is much high
In sodium hydroxide.
Further, the present invention extracts protein sample from fish-skin and analyzes and identifies to fish, and the cost of fish-skin is far below
The flesh of fish.
The present invention, which develops one kind, quickly discriminate method for distinguishing, it can be achieved that Larimichthys crocea and little yellow croaker by the fish to same category
Classification and Identification (95% level of confidence).Meanwhile four kinds of fishes of Cynoglossidae are also used to screen, data show that we develop
Method the Classification and Identifications (95% level of confidence) of four kinds of tongue soles may be implemented.A kind of work is chosen from the fish analyzed
For blind sample, the identification of blind sample is found, the classification that blind sample fish may be implemented in the method that we develop identifies.
For above and other objects of the present invention, feature and advantage can be clearer and more comprehensible, preferred embodiment is cited below particularly,
And cooperate attached drawing, and it elaborates, it is as follows：
Detailed description of the invention
Fig. 1 is the flow diagram of Fast Classification of the present invention and identification method.
Fig. 2 is the mass spectrum full figure of two kinds of fishes in embodiment 1.Wherein A) be Larimichthys crocea, B) it is little yellow croaker.
Fig. 3 is examination figure of the embodiment 1 to two kinds of fishes.Wherein the ellipse on the left side is LC Larimichthys crocea, the ellipse on the right
For LP little yellow croaker.
Fig. 4 is examination figure of the embodiment 2 to blind sample fish.Blind sample fish another four kinds of fish belonged to examination as a result, and
Examination result of the blind sample fish in yellow croaker belongs to.The ellipse of lower-left is CA Cynolossus abbreviatus (Cynoglossus in left hand view in Fig. 4
Abbreviates), the ellipse of upper left is the narrow body tongue sole of CG (Cynoglossus gracilis), and the ellipse of upper right is CJ
Red tongue sole (Cynoglossus joyneri) and the ellipse of bottom right are CS Cynoglossus semilaevis (Cynoglossus
Semilaevis), two free points of the upper right corner are blind sample fish US；In right part of flg, the ellipse on the left side is LC Larimichthys crocea, the right
Ellipse be LP little yellow croaker, two points that X is labeled as in figure are blind sample fish US.
Specific embodiment
Fig. 1 is the flow diagram of Fast Classification of the present invention and identification method.Specifically, the present invention about fish point
Class and identity process include：
Step a takes the part meat or skin of seafood, as sample to be tested；
Sample to be tested is placed in the alkaline solutions such as sodium hydroxide or potassium hydroxide and handled by step b；
Obtained solution is detected with mass spectrum and records mass signal by step c；
Step d repeats above step, obtains the number of principal components evidence of other seafood, establish a database；
Step e repeats the mass signal that step a-c obtains sample to be tested for seafood to be measured；
The mass signal of acquisition is compared with database by step f, obtains the information of seafood to be measured.
The present invention is described in detail below by specific embodiment.
Embodiment 1
Testing and appraisal example：
Step a collects sample；
For the present embodiment by taking the identification of Larimichthys crocea and little yellow croaker as an example, the shape of both fishes is very close, it is easy to obscure,
But the economic value difference of two kinds of fishes is huge.
(1) it takes the fish-skin of Larimichthys crocea and little yellow croaker as sample to be tested, removes spare after subcutaneous tissue and fish scale.
(2) two kinds of fish-skin 0.2g are taken respectively, and the sodium hydroxide solution that 2mL 1.3mol/L is added in every kind of fish-skin puts 15ml's
Centrifuge tube rocks overnight at room temperature, and the vertical mixed instrument of HS-3 is adjusted to maximum and shakes speed, takes 150 μ L solution 6mol/L's later
PH value is adjusted to neutral or faintly acid by HCl solution.
(3) using Thermo Scientific'sC18 Spin Column is done except salt treatment.(4) sample is examined
It surveys, is detected using Matrix assisted laser desorption ionization mass spectrum.2 μ L sample of the present embodiment midpoint and 1 μ L matrix.Detection makes
With linear positive ion mode, matrix is done with the sinapic acid (SA) of 15mg/mL.Use cromoci and Myoglobin as correction
Sample.
(5) same fish takes 6 as skin sample is taken, and each sample carries out 3 Mass Spectrometer Methods.Obtained mass spectrometric data is used
Statistical software carries out chemometrics application, extracts Differential variable.
In the present embodiment, test sample n times (n >=9) are repeated with the same fish, obtain the full spectrogram of mass spectrum (Fig. 2).
It carries out data processing by RStudio software and obtains data matrix to export to Excel, it is then soft by SIMCA
Part carries out PLS-DA analysis (Fig. 3).
It can be seen that by the PLS-DA analysis chart of each sample and belong to the fish of same category and may be implemented effectively to distinguish.It is logical
It crosses and more fishes is handled and detected, the PLS-DA of obtained different fishes can be schemed to be used as a database.
It, can also be using principal component analysis, just using offset minimum binary discriminating projection analysis (PLS-DA) in the present embodiment
Friendship-offset minimum binary discriminating projection method etc..
95% confidence interval is shown in oval part in Fig. 3, it can be seen that it is repeated as many times under experimental conditions,
With the differentiation and identification Larimichthys crocea that are apparent and little yellow croaker.
Embodiment 2
Blind sample identification
Take unknown fish and Larimichthys crocea in Cynolossus abbreviatus, narrow body tongue sole, red tongue sole and Cynoglossus semilaevis and embodiment and small
Yellow croaker Identification.
Taking unknown fish first and kissing the fish-skin of tongue sole, narrow body tongue sole, red tongue sole and Cynoglossus semilaevis is sample to be tested, respectively
And immersion treatment is carried out with sodium hydroxide solution.
The sample that takes that treated is a little (100 to 200 microlitres).With hydrochloric acid solution tune pH to neutral or faintly acid；It uses later
De-sludging pillar handles (process is as shown in Figure 1).
Step b, sample detection are detected using Matrix assisted laser desorption ionization mass spectrum.
The obtained sample drop of step a is added on MALDI target plate, matrix is done using SA, carries out Mass Spectrometer Method.
Step c, same fish take 6 as skin sample is taken, and each sample carries out 3 Mass Spectrometer Methods.Obtained spectra count
Chemometrics application is carried out according to statistical software, extracts Differential variable；
It carries out data processing by RStudio software and obtains data matrix to export to Excel, it is then soft by SIMCA
Part carries out PLS-DA analysis
The spectrogram one of the spectrogram of blind sample and the fish in the database to be compared is reinstated RStudio software and counted by step d
Export to Excel according to handling and obtaining data matrix, then by MetaboAnalyst 3.0 (McGill University,
Montreal, Canada) online software progress PLS-DA analysis, Fig. 4 is obtained, is belonged to by the blind sample of analysis result ownership any
Fish.
Result is analyzed as shown in figure 4, blind sample is just fallen in 95% confidence interval of little yellow croaker.I.e. through this embodiment
Method has 95% confidence level to think that the sample of this unknown point comes from little yellow croaker, finally proves that this is tested with the present embodiment
The sample of Shi Suoyong is consistent.It can be seen that the analysis method developed herein can not only be used to distinguish different fingerlings, also
It can be used to identify unknown sample.
The above is a preferred embodiment of the present invention, it is noted that for the those of ordinary skill of this technology neighborhood
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Claims (10)
1. the differentiating method of seafood, which is characterized in that the described method comprises the following steps：
Step a takes the part meat or skin of seafood, as sample to be tested；
Step b, sample to be tested is placed in alkaline solution and is handled；
The solution of step b is adjusted to neutral or faintly acid after the completion of processing by step c；
The solution that step c is obtained is detected with mass spectrum and records mass signal by step d；
Step e carries out chemometrics application to the mass signal of acquisition, to distinguish different seafood.
2. the differentiating method of seafood according to claim 1, which is characterized in that the alkaline solution in step b is hydroxide
Sodium solution or potassium hydroxide solution.
3. the differentiating method of seafood according to claim 1, which is characterized in that in step b, after sample to be tested processing, egg
Molecular weight after white matter resolution is less than 10k Da.
4. the differentiating method of seafood according to claim 1, which is characterized in that the mass spectrum is Matrix Assisted Laser Desorption
One of ionization massspectrum, ion trap mass spectrometry, flight time mass spectrum, level four bars mass spectrum.
5. the differentiating method of seafood according to claim 1, which is characterized in that the seafood be fish, shellfish, shrimps,
One of crab class and aquatic mammality are a variety of.
6. the Fast Classification and identification method of seafood according to claim 5, which is characterized in that the seafood is fish.
7. the differentiating method of seafood according to claim 6, which is characterized in that take fish-skin or the flesh of fish as sample to be tested.
8. the differentiating method of seafood according to claim 1, which is characterized in that before step d, it is also necessary to be carried out to solution
Purification removes excess salt.
9. the differentiating method of seafood according to claim 8, which is characterized in that carried out using de-sludging pillar to solution net
Change.
10. the Fast Classification and identification method of a kind of seafood, which is characterized in that
Step a takes the part meat or skin of seafood, as sample to be tested；
Sample to be tested is placed in alkaline solution and handled by step b；
Obtained solution is detected with mass spectrum and records mass signal by step c；
Step d repeats above step, obtains the number of principal components evidence of variety classes seafood, establish a database；
Step e repeats the mass signal that step a-c obtains sample to be tested for seafood to be measured；
The mass signal of acquisition is compared with database by step f, obtains the ownership information of seafood to be measured.
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| CN110702483A (en) * | 2019-11-07 | 2020-01-17 | 上海海洋大学 | Pretreatment method for identifying seafood or meat at ultra-fast speed |
| CN111505094A (en) * | 2020-04-28 | 2020-08-07 | 上海交通大学 | Food feed species tracing identification method based on isoelectric focusing electrophoresis |
| CN111678973A (en) * | 2020-07-18 | 2020-09-18 | 上海海洋大学 | Method for rapidly identifying Atlantic salmon and rainbow trout based on mass spectrometry technology |
| CN112730410A (en) * | 2020-12-25 | 2021-04-30 | 上海海洋大学 | Method for quickly distinguishing seafood by using spectrometry |
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| CN111505094A (en) * | 2020-04-28 | 2020-08-07 | 上海交通大学 | Food feed species tracing identification method based on isoelectric focusing electrophoresis |
| CN111678973A (en) * | 2020-07-18 | 2020-09-18 | 上海海洋大学 | Method for rapidly identifying Atlantic salmon and rainbow trout based on mass spectrometry technology |
| CN112730410A (en) * | 2020-12-25 | 2021-04-30 | 上海海洋大学 | Method for quickly distinguishing seafood by using spectrometry |
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