CN107356688A - A kind of method for distinguishing multiple geographical population jumbo flying squids using muscle essential fatty acid composition - Google Patents

A kind of method for distinguishing multiple geographical population jumbo flying squids using muscle essential fatty acid composition Download PDF

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CN107356688A
CN107356688A CN201710552376.7A CN201710552376A CN107356688A CN 107356688 A CN107356688 A CN 107356688A CN 201710552376 A CN201710552376 A CN 201710552376A CN 107356688 A CN107356688 A CN 107356688A
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fatty acid
muscle
squid
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CN107356688B (en
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贡艺
陈新军
李云凯
陈玲
高小迪
高春霞
李建华
王少琴
张海亭
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Shanghai Maritime University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The present invention relates to a kind of method for distinguishing multiple geographical population jumbo flying squids using muscle essential fatty acid composition：Specifically include (1) extraction jumbo flying squid muscle fatty acid；(2) GC MS measure aliphatic acid is utilized；(3) muscle fatty acid data are analyzed；(4) the serial unrighted acid discriminant coefficient of n 3 and n 6 and constant of multiple geographical population jumbo flying squids are calculated；(5) differentiate：Jumbo flying squid muscle n 3 to be discriminated and the serial unsaturated fatty acid content percentages of n 6 are obtained using above-mentioned steps (1) (3), multiple discriminant scores are calculated with the discriminant coefficient and constant of step (4), its affiliated geographical population is represented with geographic area corresponding to maximal function value.The method of this method has the advantages of quick, accuracy is high.

Description

It is a kind of to distinguish multiple geographical population jumbo flying squids using muscle essential fatty acid composition Method
Technical field
It is more using the composition differentiation of muscle essential fatty acid more specifically to one kind the present invention relates to Fisheries Science field The method of individual geographical population jumbo flying squid.
Background technology
Aliphatic acid is a kind of special compound, is mainly existed in the form of triglyceride and phosphatide, it is in biology Feeding activity during it is relatively stable be not easy to change, be that one of natural biological mark (is permitted available for food source is analyzed By force, application [J] ocean journal of the Yang Hongsheng fatty acid biomarkers in the research of marine ecosystems nutrition relationship, 2011,33 (1):1-6).As biomarker, aliphatic acid has several big superiority：First, aliphatic acid can store fat by being formed, storage In vivo, so as to reflect situation of ingesting biological in one period, relative to traditional analysis of stomach method, reduce Judge the contingency of biological feeding habits；Second, it is relatively stable in metabolic processes, it is basic with structure in assimilation process ingesting Keep constant；3rd, the aliphatic acid in organism in triglyceride mostlys come from taken in food (Henderson RJ,Sargent JR.Lipid biosynthesis in rainbow trout,Salmo gairdnerii,fed diets of differing lipid content[J].Comparative Biochemistry&Physiology Part C Comparative Pharmacology,1981,69(1):31-37)。
The aliphatic acid of marine organisms is divided into saturated fatty acid and the class of unrighted acid two.Wherein n-3 and n-6 series of height Unrighted acid can only obtain from bait, it is impossible to itself synthesis, therefore these aliphatic acid are referred to as essential fatty acid (Olsen Y.Lipids and Essential Fatty Acids in Aquatic Food Webs:What Can Freshwater Ecologists Learn from Mariculture[M]//Lipids in Freshwater Ecosystems.Springer New York,1999:161-202), these essential fatty acids are ground frequently as marine food relation Fatty acid biomarkers (Gordon BJ, Tocher DR, Macdonald FM, et al.Effects of diets in studying carefully rich in linoleic(18:2n-6)and α-linolenic(18:3n-3)acids on the growth,lipid class and fatty acid compositions and eicosanoid production in juvenile turbot(Scophthalmus maximus L.)[J].Fish Physiology and Biochemistry,1994,13 (2):105；Smith GI,Atherton P,Reeds DN,et al.Dietary omega-3fatty acid supplementation increases the rate of muscle protein synthesis in older adults:a randomized controlled trial[J].American Journal of Clinical Nutrition,2011,93(2):402-412).Therefore jumbo flying squid different geographic populations are understood using fatty acid biomarkers method Feeding habits and nutrition status.
The document of Feeding Ecology research is carried out also not to jumbo flying squid different geographic populations currently with fatty acid biomarkers Appear in the newspapers.Mainly to the Analysis of Nutritive Composition of its muscle and peduncle, (Yang Xianshi, Wang Lili, Li Xueying etc., is secret for studies in China Shandong squid and sea of Japan nutrient components of squid A+E [J] modern foods science and technology, 2013,29 (9):2247-2251) and (beautiful squid foots instant leisure typical local food primary research [D] Dalian Ocean University of Wang Li, 2014) is studied in food processing, external (Saito H, Sakai M, the Wakabayashi T.Characteristics of the lipid and such as only Saito fatty acid compositions of the Humboldt squid,Dosidicus gigas:The trophic relationship between the squid and its prey[J].European Journal of Lipid Science and Technology,2014,116(3):360-366) to waters off Peru jumbo flying squid different tissues and stomach inclusion Aliphatic acid composition is studied.Numerous studies are found, can accurately be disclosed using the Detectable effects of fatty acid biomarkers Specific monoid monoid biology of the same race in food source difference and a certain specific complicated habitat in single or different niches Food source situation.
The content of the invention
It is an object of the invention to provide one kind multiple geographical population jumbo flying squids are distinguished using muscle essential fatty acid composition Method, this method has the advantages of quick, accuracy is high.
To solve the above problems, the present invention adopts the following technical scheme that：
A kind of method for distinguishing multiple geographical population jumbo flying squids using muscle essential fatty acid composition, it includes following step Suddenly：
(1) jumbo flying squid muscle fatty acid is extracted, obtains the solution containing jumbo flying squid muscle fatty acid；
(2) GC-MS measure muscle fatty acids are utilized；
(3) muscle fatty acid data are analyzed：
A. quantitative analysis muscle fat acid content, the ultimate density (a of n kind aliphatic acid in sample is obtained1……an), according to Formula：an× extension rate × original solution volume, the content of n kind aliphatic acid is calculated, wherein original solution volume is step (1) The liquor capacity containing jumbo flying squid muscle fatty acid；
B. the content divided by total fatty acid content of each aliphatic acid, calculates percentage composition shared by each aliphatic acid；
C. p kind n-3 and n-6 series unsaturated fatty acid content percentages (W is filtered out1……Wp) as the sample must Need fatty acid analysis result；
(4) n-3 the and n-6 series unrighted acid discriminant function coefficient and often of multiple geographical population jumbo flying squids is calculated Amount：
A. using geographical population as packet variable, n-3 and n-6 series unsaturated fatty acid contents percentage is used as from change Amount, to known p kind n-3 and n-6 the series unsaturated fatty acid content percentages from multiple (1 ... m) geographical population jumbo flying squids Than carrying out linear discriminant analysis；
B. analysis draws the corresponding differentiation of p kind n-3 and n-6 the series unrighted acids of multiple (1 ... m) geographical populations Coefficient (K1-1……K1-p, K2-1……K2-p, Km-1……Km-p) and m geographical population constant (C1……Cm)；
Wherein n, p, m are integer,
(5) differentiate：P kind n-3 and n-6 series is obtained in jumbo flying squid muscle to be discriminated using above-mentioned steps (1)-(3) not Saturated fatty acid content percentage (W1……Wp), m discriminant score is calculated according to the discriminant coefficient of step (4) and constant (F1……Fm),
Wherein functional value F1、F2……FmComputational methods be：
F1=K1-1×W1-1+K1-2×W1-2+……+K1-n×W1-p+C1,
F2=K2-1×W2-1+K2-2×W2-2+…+K2-p×W2-p+C2,
……
Fm=Km-1×Wm-1+Km-2×Wm-2+……+Km-p×Wm-p+Cm,
M functional value F is calculated1……Fm, its affiliated geographical population is represented with geographical population corresponding to maximal function value (that is if F1Value is maximum, then it is the 1st geographical population to judge the jumbo flying squid；If F2Value is maximum, then judges that the stem is soft Fish is the 1st geographical population ... ..., if FmValue is maximum, then it is m-th of geographical population to judge the jumbo flying squid.).
In a preferred embodiment of the invention, step (1) extraction jumbo flying squid muscle fatty acid comprises the following steps：
I. sample：Clip muscle at funnel lock cartilage, with ultra-pure water cleaning sample surface, then removes skin, is put into centrifugation Freezen protective in pipe；
Ii. it is freeze-dried：Sample after freezing is put into freeze drier, -50 DEG C to -60 DEG C freeze-drying 24-48 Hour；
Iii. grind：Dried sample grind into powder；
Iv. aliphatic acid is extracted：Utilize chloroform-methanol (volume ratio 1.5-2.5:1) it is small to soak above-mentioned powder 20-40 When, centrifugation, supernatant is washed with 0.6-1.5% sodium-chloride water solution, liquid separation after standing 1.5-2.5 hours, removes a layer solution And solvent therein is removed, obtained aliphatic acid flows back 8-12min in 0.3-0.6mol/L sodium hydroxides-methanol solution, then Boron chloride-methanol solution (methanol solution of 10-20% boron chlorides) backflow 25-35min is added, n-hexane is eventually adding and returns 1-3min is flowed, saturated sodium-chloride water solution is added after cooling, 20-30min is stood after mixing, finally obtains containing aliphatic acid just Upper hexane.
In a more preferred embodiment of the present invention, in step ii, the sample after freezing is put into freeze drier In, -50 DEG C of freeze-dryings.
In a more preferred embodiment of the present invention, in step iii, dried sample uses freezing mixing and ball milling Instrument MM400 (Retsch, Germany)
In a more preferred embodiment of the present invention, in step iv, in chloroform-methanol used chloroform and The volume ratio of methanol is 2:1.In a more preferred embodiment of the present invention, the mass concentration of sodium-chloride water solution is 0.9%.In a more preferred embodiment of the present invention, in step iv, sodium hydroxide-methanol solution is 0.5mol/L hydrogen-oxygens Change the methanol solution of sodium.In a more preferred embodiment of the present invention, in step iv, boron chloride-methanol solution is The methanol solution of 14% boron chloride.
In a more preferred embodiment of the present invention, 0.9% aqueous sodium chloride is added in step iv, in supernatant Liquid separation after liquid stands 2 hours.In a more preferred embodiment of the present invention, in step iv, obtained aliphatic acid is in hydrogen-oxygen Change backflow 10min in sodium-methanol solution (methanol solution of 0.5mol/L sodium hydroxides).One in the present invention is more preferably implemented In scheme, in step iv, boron chloride-methanol solution (methanol solution of 14% boron chloride) backflow 30min is added.In this hair In a bright more preferred, in step iv, n-hexane backflow 2min is added.
In a more preferred embodiment of the present invention, sodium hydroxide-methanol solution, boron chloride-first in step iv The dosage of alcoholic solution and n-hexane, is counted by volume, is 1:1:1.
In a more preferred embodiment of the present invention, step (2) GC-MS measure aliphatic acid, which utilizes, contains aliphatic acid Hexane solution, wherein adding Nonadecanoic acid methylester internal standard.
In a more preferred embodiment of the present invention, the volume ratio of Nonadecanoic acid methylester and hexane solution is 1:1.
In a preferred embodiment of the invention, step (2) GC-MS determines aliphatic acid and utilizes 7890B/5977A gas Matter combined instrument (Agilent, the U.S.) determines, and chromatographic column is HP-88 (60m × 0.25mm × 0.20 μm, Agilent, the U.S.), surveys Sample heating schedule is initial 125 DEG C, is raised to 145 DEG C with 8 DEG C/min, keeps 26min, is raised to 220 DEG C with 2 DEG C/min, keeps 1min, then 227 DEG C are raised to 1 DEG C/min, keep 1min, 250 DEG C of injector temperature, 280 DEG C of auxiliary heater temperature, split ratio 10：1, carrier gas is helium.
In a preferred embodiment of the invention, step (3) a using Masshunter Work Station according to The muscle fatty acid quantitative analysis content of fatty acid of step (2) GC-MS measure.
In a preferred embodiment of the invention, step (4) uses SPSS softwares to p kind n-3 and n-6 series insatiable hungers Linear discriminant analysis is carried out with content of fatty acid percentage.It is " soft using SPSS in a more preferred embodiment of the present invention Part carries out linear discriminant analysis to p kind n-3 and n-6 series unsaturated fatty acid contents percentage " it is by by multiple proles N-3 the and n-6 series unsaturated fatty acid contents data of body import SPSS softwares, select " differentiation " in " analysis " instrument, with Geographical population is as packet variable, p kind n-3 and n-6 series unsaturated fatty acid contents (W1……Wp) independent variable is used as, " statistics " function coefficients " selection " Fisher " of amount " in option, " output " module in " classification " option select " abstract ", " figure " mould Block selects " merging group ", and other are default setting, obtain p kind n-3 and n-6 the series unrighted acids of multiple geographical populations Corresponding discriminant coefficient and m geographical population constant.
In a preferred embodiment of the invention, n-3 and n-6 series unrighted acid is C18:2n6c、C18: 2n6t、C18:3n3、C18:3n6、C20:3n3、C20:3n6、C20:4n6、C20:5n3、C22:2n6 and C22:6n3.
In the present invention, the discriminant coefficient of different n-3 and n-6 series unrighted acids is different, and different geographical The same n-3 and n-6 series unrighted acid of colony is also different." discriminant coefficient (K in the present invention1-1……K1-p, K2-1……K2-p, Km-1……Km-p) " represent Different population different n-3 and n-6 series unrighted acids differentiation system Number.For example, " K1-1" represent m geographical population in the 1st geographical population jumbo flying squid p kind n-3 and n-6 series unsaturated lipids The discriminant coefficient of the 1st kind of unrighted acid in fat acid, " Km-p" represent that m-th of geographical population stem in m geographical population is soft The discriminant coefficient of pth kind unrighted acid in p kind n-3 and n-6 the series unrighted acids of fish.
In the present invention, " constant " is constant for a geographical population, and m kind geographical populations have m constant.This hair " constant (C in bright1……Cm) " represent m geographical population m constant.For example, " C1" represent m geographical population in the 1st The constant of geographical population, " Cm" represent m geographical population in m-th of geographical population constant.
In the present invention, " discriminant score (F1……Fm) " it is by a variety of n-3 and n-6 series unsaturated fatty acid contents hundred Divide what is obtained than being multiplied by constant that the discriminant coefficient of corresponding geographical population obtains and along with the geographical population, such as " F1" be The discriminant coefficient of corresponding 1st geographical population is multiplied by by a variety of (p kinds) n-3 and n-6 series unsaturated fatty acid content percentages What constant obtain and along with the 1st geographical population obtained.“Fm" it is unsaturated by a variety of (p kinds) n-3 and n-6 series It is that the discriminant coefficient that content of fatty acid percentage is multiplied by corresponding m-th of geographical population obtains and along with m-th geographical population Constant obtain.
The present invention creates a kind of letter by the jumbo flying squid muscle essential fatty acid ratio of components pair of a large amount of Different populations Single practical method, is only formed by measuring jumbo flying squid muscle essential fatty acid, just quickly can exactly be distinguished and differentiate jumbo flying squid Geographical population, accuracy rate may be up to 96%.
Embodiment
In order that the technical means, the inventive features, the objects and the advantages of the present invention are easy to understand, tie below Concrete example is closed, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention rather than the limitation present invention Scope.The experimental method of unreceipted actual conditions in example below, generally according to normal condition, or according to institute of manufacturer It is recommended that condition.
Embodiment 1
It is a kind of that the method for distinguishing jumbo flying squid geographical population is formed using muscle essential fatty acid, comprise the following steps：
66 tail jumbo flying squids of the random acquisition from 3 known geographical areas, respectively (0 ° of the tail of Eastern Pacific's Equatorial sea region 19 28'N~1 ° 00'S, 115 ° of 01'W~119 ° 00'W), the tail of waters off Peru 27 (9 ° of 50'S~15 ° 42'S, 79 ° of 45'W~85 ° 03' ) and the tail of seas off Chile 20 (37 ° of 06'S~38 ° 00'S, 79 ° of 01'W~83 ° 00'W) W.
(1) jumbo flying squid muscle fatty acid is extracted：
I. sample：Cut off by the trunk outside of belly, locked in funnel and surgical scissors clip 2 × 2cm muscle is used at cartilage, use is ultrapure Water cleaning sample surface, reuse tweezers and remove skin, be put into freezen protective in 10ml centrifuge tubes；
Ii. it is freeze-dried：Sample after freezing is put into freeze drier, -55 DEG C are freeze-dried 30 hours；
Iii. grind：Dried sample uses freezing mixing and ball milling instrument MM400 (Retsch, Germany) grind into powder If (not using immediately, the powder can be fitted into centrifuge tube, be preserved using vacuum compression bag normal temperature)；
Iv. aliphatic acid is extracted：
0.2g powder is taken in 15ml centrifuge tubes,
Add 12ml chloroforms-methanol solution (volume ratio 2:1), soak 30 hours,
Supernatant 10ml is taken after centrifugation, adds 4ml 0.9% sodium-chloride water solution, stands 2 hours,
Remove layer solution heating water bath in round-bottomed flask and obtain aliphatic acid,
4ml 0.5mol/L sodium hydroxide-methanol solution is added in round-bottomed flask, water-bath backflow dress is connected after mixing Put, heating water bath 10min,
4ml boron chlorides methanol solution (methanol solution of 14% boron chloride) water-bath backflow 30min is added,
4ml n-hexane water-bath reflux extraction 2min are added,
10ml saturated aqueous sodium chlorides are added after cooling, are rocked, are further continued for adding saturated aqueous sodium chloride to flask Neck, stratification 30min, isolated n-hexane layer (upper strata) 4ml containing aliphatic acid；
(2) GC-MS measure muscle fatty acids are utilized：
Take the filtering of 1ml upper solutions syringe filter to be placed in sample injection bottle, add 1ml Nonadecanoic acid methylester solution, GC-MS measure is carried out after mixing, using 7890B/5977A gas chromatograph-mass spectrometers (Agilent, the U.S.), chromatographic column is HP-88 (60m × 0.25mm × 0.20 μm, Agilent, the U.S.), test sample heating schedule is initial 125 DEG C, is raised to 145 DEG C with 8 DEG C/min, protects 26min is held, 220 DEG C are raised to 2 DEG C/min, keeps 1min, then 227 DEG C are raised to 1 DEG C/min, keeps 1min, injector temperature 250 DEG C, 280 DEG C of auxiliary heater temperature, split ratio 10:1, carrier gas is helium；
(3) muscle fatty acid data are analyzed
A. Masshunter Work Station quantitative analysis content of fatty acid is used, obtains n kind aliphatic acid in sample Ultimate density (a1……an), according to formula an(mg/L) × extension rate × 0.004L calculates the content of n kind aliphatic acid；
B. the content divided by total fatty acid content of each aliphatic acid, percentage composition (table 1) shared by each aliphatic acid is calculated；
C. 10 kinds of n-3 and n-6 series unrighted acids (C18 are filtered out:2n6c、C18:2n6t、C18:3n3、C18: 3n6、C20:3n3、C20:3n6、C20:4n6、C20:5n3、C22:2n6 and C22:Percentage composition 6n3) is as the sample Essential fatty acid analysis result；
The jumbo flying squid fatty acid data of table 1, wherein n.d. represent not detecting discovery.
(4) the discriminant function coefficient and constant of n-3 the and n-6 series unrighted acids of multiple geographical populations are calculated：
A. packet variable, 10 kinds of n-3 and n-6 series unsaturated fatty acid contents (W are used as using geographical population1……W 10) As independent variable, using SPSS softwares by 10 kinds of n-3 and n-6 series of the known 66 tail jumbo flying squids from 3 geographical populations not Saturated fatty acid content percentage (W1-1……W1-10, W2-1……W2-10, W3-1……W3-10) carry out linear discriminant analysis；
Carry out linear discriminant analysis specific operation process be：By 10 kinds of n-3 and n-6 series insatiable hungers of 3 geographical populations SPSS softwares are imported with content of fatty acid data, select " differentiation " in " analysis " instrument, using geographical population as packet variable, 10 kinds of n-3 and n-6 series unsaturated fatty acid contents (W1……W10) it is used as independent variable, " the function system in " statistic " option Number " selection " Fisher ", " output " module in " classification " option select " abstract ", and " figure " module selects " merging group ", its He is default setting；
B. analysis draws the discriminant coefficient of 10 kinds of n-3 and n-6 series unrighted acids of 3 geographical population jumbo flying squids (K1-1……K1-10, K2-1……K2-10, K3-1……K3-10) and constant (C1, C2, C3),
The wherein K of Eastern Pacific's Equatorial sea region1-1To K1-10Respectively 24.52,40.85, -23.51,15.89,12.29, - 1.13rd, 30.32,13.59,8.96,10.23), constant C1For -416.35；The K of waters off Peru2-1To K2-10Respectively 28.20, 43.56th, -6.51,14.06,6.16,9.51,19.12,11.17,4.88,9.67, constant C2For -342.23；Seas off Chile K3-1To K3-10Respectively 14.59,68.39, -17.22,24.31, -11.64,6.49,25.86,11.52,4.96,10.96, often Measure C3For -439.81；
Discriminant coefficient (the K of 10 kinds of n-3 and n-6 series unrighted acids of 3 kinds of geographical population jumbo flying squids1-1To K1-10, K2-1To K2-10, K3-1To K3-10) and constant (C1, C2, C3) referring to table 2；
(5) it is to prove discriminating accuracy rate of the invention simultaneously, and 24 tail jumbo flying squids of crawl (tail of Eastern Pacific's Equatorial sea region 8, it is secret The tail of Shandong off-lying sea 8, the tail of seas off Chile 8), after marking its actual geographical population respectively, then with the present invention method carry out discriminating area Point.24 tails jumbo flying squid muscle n-3 and n-6 series unsaturated fatty acid content to be discriminated is obtained using above-mentioned steps (1)-(3) Percentage (is shown in Table 3), utilizes every tail jumbo flying squid n-3 and n-6 series unsaturated fatty acid content percentage and 3 geographical population stems 3 functional value F are calculated in the discriminant coefficient and constant of squid1、F2And F3, represented with geographical population corresponding to maximal function value Its affiliated geographical population.If that is, F1Value is maximum, then it is Equatorial sea region colony of Eastern Pacific to judge the jumbo flying squid；Such as Fruit F2Value is maximum, then it is waters off Peru colony to judge the jumbo flying squid；If F3Value is maximum, then it is seas off Chile to judge the jumbo flying squid Colony.
Such as the computational methods for three functional values that numbering is 1 jumbo flying squid are：
Functional value F1=1.37 (W1-1)×24.52(K1-1)+1.76(W1-2)×40.85(K1-2)+0.39(W1-3)×(- 23.51)(K1-3)+0.71(W1-4)×15.89(K1-4)+1.73(W1-5)×12.29(K1-5)+0.00(W1-6)×(-1.13) (K1-6)+4.88(W1-7)×30.32(K1-7)+8.11(W1-8)×13.59(K1-8)+2.04(W1-9)×8.96(K1-9)+39.60 (W1-10)×10.23(K1-10)+(-416.35)(C1)=394.08
Functional value F2=1.37 (W2-1)×28.20(K2-1)+1.76(W2-2)×43.56(K2-2)+0.39(W2-3)×(- 6.51)(K2-3)+0.71(W2-4)×14.06(K2-4)+1.73(W2-5)×6.16(K2-5)+0.00(W2-6)×9.51(K2-6)+ 4.88(W2-7)×19.12(K2-7)+8.11(W2-8)×11.17(K2-8)+2.04(W2-9)×4.88(K2-9)+39.60(W2-10) ×9.67(K2-10)+(-342.23)(C2)=367.95
Functional value F3=1.37 (W3-1)×14.59(K3-1)+1.76(W3-2)×68.39(K3-2)+0.39(W3-3)×(- 17.22)(K3-3)+0.71(W3-4)×24.31(K3-4)+1.73(W3-5)×(-11.64)(K3-5)+0.00(W3-6)×6.49 (K3-6)+4.88(W3-7)×25.86(K3-7)+8.11(W3-8)×11.52(K3-8)+2.04(W3-9)×4.96(K3-9)+39.60 (W3-10)×10.96(K3-10)+(-439.81)(C3)=354.71
Contrast numbering is that 1 jumbo flying squid, 3 discriminant scores are understood, functional value F1(394.08)>Functional value F2(367.95)> Functional value F3(354.71), i.e., No. 1 jumbo flying squid belongs to Eastern Pacific's Equatorial sea region.
Table 2n-3 and n-6 series unrighted acid, the discriminant coefficient and constant of corresponding geographical population
See, 24 jumbo flying squids there is the affiliated geographic area of 23 jumbo flying squids to differentiate correct, differentiate rate of accuracy reached from the above 96%.It can be seen that method of the invention can provide quick, reliable discrimination method for fish production and management organization.So fishing The industry producer is in sale or regulatory authorities detection jumbo flying squid, it is only necessary to which it is measured from large batch of jumbo flying squid must fat Fat acid content, you can differentiate the geographical population belonging to it.
Carry out not having conditional description to the present invention by example according to the preferred form of production of the present invention, still It should be appreciated that in the range of appended claims definition, expert, which can do, to change and/or modification, without departing from correlation Protection domain.

Claims (9)

1.一种利用肌肉必需脂肪酸组成区分多个地理群体茎柔鱼的方法，其包括如下步骤：1. A method utilizing muscle essential fatty acid composition to distinguish a plurality of geographic groups of squid, comprising the steps of: (1)提取茎柔鱼肌肉脂肪酸，得到含有茎柔鱼肌肉脂肪酸的溶液；(1) extracting the fatty acid of the squid muscle to obtain a solution containing the muscle fatty acid of the squid; (2)利用GC-MS测定肌肉脂肪酸；(2) Utilize GC-MS to measure muscle fatty acid; (3)分析肌肉脂肪酸数据：(3) Analyze muscle fatty acid data: a.定量分析肌肉脂肪酸含量，获得样品中n种脂肪酸的最终浓度(a1……an)，根据公式：an×稀释倍数×原始溶液体积，计算n种脂肪酸的含量，其中原始溶液体积为步骤(1)的含有茎柔鱼肌肉脂肪酸的溶液体积；a. Quantitatively analyze the content of fatty acids in muscle to obtain the final concentration of n fatty acids in the sample (a 1 ... a n ), and calculate the content of n fatty acids according to the formula: a n × dilution factor × original solution volume, where the original solution volume Be the volume of solution that contains stem squid muscle fatty acid of step (1); b.各脂肪酸的含量除以总脂肪酸含量，计算出各脂肪酸所占含量百分比；b. Divide the content of each fatty acid by the total fatty acid content to calculate the content percentage of each fatty acid; c.筛选出p种n-3和n-6系列不饱和脂肪酸含量百分比(W1……Wp)作为该样品的必需脂肪酸分析结果；c. Screen out the content percentages of p kinds of n-3 and n-6 series of unsaturated fatty acids (W 1 ... W p ) as the essential fatty acid analysis results of the sample; (4)计算多个地理群体茎柔鱼的n-3和n-6系列不饱和脂肪酸判别函数系数和常量：(4) Calculating the n-3 and n-6 series unsaturated fatty acid discriminant function coefficients and constants of multiple geographical groups of squid: a.以地理群体作为分组变量，n-3和n-6系列不饱和脂肪酸含量百分比作为自变量，对已知来自多个(1……m)地理群体茎柔鱼的p种n-3和n-6系列不饱和脂肪酸含量百分比进行线性判别分析；a. With geographic groups as grouping variables and n-3 and n-6 series unsaturated fatty acid content percentages as independent variables, p species n-3 and Linear discriminant analysis was performed on the content percentage of n-6 series unsaturated fatty acids; b.分析得出多个(1……m)地理群体的p种n-3和n-6系列不饱和脂肪酸的相应判别系数(K1-1……K1-p，K2-1……K2-p，Km-1……Km-p)和m个地理群体的常量(C1……Cm)；b. Analyze the corresponding discriminant coefficients (K 1-1 ... K 1-p , K 2-1 ... ... K 2-p , K m-1 ... K mp ) and constants for m geographic groups (C 1 ... C m ); 其中n，p，m都为整数，Where n, p, m are all integers, (5)判别：利用上述步骤(1)-(3)得到待判别的茎柔鱼肌肉中p种n-3和n-6系列不饱和脂肪酸含量百分比(W1……Wp)，根据步骤(4)的判别系数和常量计算m个判别函数值(F1、F2……Fm)，(5) Discrimination: Utilize the above steps (1)-(3) to obtain the content percentages of p kinds of n-3 and n-6 series unsaturated fatty acids (W 1 ... W p ) in the muscle of squid squid to be discriminated, according to the steps (4) discriminant coefficients and constants to calculate m discriminant function values (F 1 , F 2 ... F m ), 其中函数值F1、F2……Fm的计算方法为：The calculation method of the function value F 1 , F 2 ... F m is: F1＝K1-1×W1-1+K1-2×W1-2+……+K1-n×W1-p+C1，F 1 =K 1-1 ×W 1-1 +K 1-2 ×W 1-2 +...+K 1-n ×W 1-p +C 1 , F2＝K2-1×W2-1+K2-2×W2-2+…+K2-p×W2-p+C2，F 2 =K 2-1 ×W 2-1 +K 2-2 ×W 2-2 +...+K 2-p ×W 2-p +C 2 , ……... Fm＝Km-1×Wm-1+Km-2×Wm-2+……+Km-p×Wm-p+Cm，F m =K m-1 ×W m-1 +K m-2 ×W m-2 +...+K mp ×W mp +C m , 计算得到m个函数值F1……Fm，以最大函数值对应的地理群体表示其所属地理群体。Calculate m function values F 1 ... F m , and use the geographic group corresponding to the largest function value to represent the geographic group it belongs to. 2.根据权利要求1所述的方法，其中步骤(1)提取茎柔鱼肌肉脂肪酸包括以下步骤：2. The method according to claim 1, wherein step (1) extracting stem squid muscle fatty acid comprises the following steps: i.取样：漏斗锁软骨处剪取肌肉，清洗样品表面，去除皮肤，放入离心管中冷冻保存；i. Sampling: Cut the muscle from the clavicular cartilage of the funnel, clean the surface of the sample, remove the skin, and put it into a centrifuge tube for cryopreservation; ii.冷冻干燥：将冷冻后的样品放入冷冻干燥机中，-50℃至-60℃冷冻干燥24-48小时；ii. Freeze-drying: put the frozen samples into a freeze dryer, and freeze-dry them at -50°C to -60°C for 24-48 hours; iii.研磨：干燥后的样品研磨成粉末；iii. Grinding: the dried sample is ground into powder; iv.提取脂肪酸：利用三氯甲烷-甲醇(体积比1.5-2.5:1)浸泡上述粉末20-40小时，离心，上清液用0.6-1.5％的氯化钠水溶液洗涤，静置1.5-2.5小时后分液，取下层溶液水浴加热以除去其中的溶剂，得到的脂肪酸在0.3-0.6mol/L氢氧化钠-甲醇溶液中回流8-12min，再加入三氯化硼-甲醇溶液(10-20％三氯化硼的甲醇溶液)回流25-35min，最后加入正己烷回流1-3min，冷却后加入饱和氯化钠水溶液，混匀后静置20-30min，最后得到含有脂肪酸的正己烷上层。iv. Fatty acid extraction: soak the above powder for 20-40 hours with chloroform-methanol (volume ratio 1.5-2.5:1), centrifuge, wash the supernatant with 0.6-1.5% sodium chloride aqueous solution, and let stand for 1.5-2.5 Separate the liquid after 1 hour, take the lower layer solution and heat it in a water bath to remove the solvent therein, the fatty acid obtained is refluxed in 0.3-0.6mol/L sodium hydroxide-methanol solution for 8-12min, then add boron trichloride-methanol solution (10- 20% methanol solution of boron trichloride) to reflux for 25-35min, finally add n-hexane to reflux for 1-3min, add saturated aqueous sodium chloride solution after cooling, mix well and let stand for 20-30min, finally get the n-hexane upper layer containing fatty acid . 3.根据权利要求2所述的方法，其中步骤iv中氢氧化钠-甲醇溶液、三氯化硼-甲醇溶液和正己烷的体积比为1:1:1。3. The method according to claim 2, wherein the volume ratio of sodium hydroxide-methanol solution, boron trichloride-methanol solution and normal hexane is 1:1:1 among the step iv. 4.根据权利要求1所述的方法，其中步骤(2)GC-MS测定脂肪酸利用含有脂肪酸的正己烷溶液，其中加入十九烷酸甲酯内标。4. The method according to claim 1, wherein step (2) GC-MS measures fatty acid and utilizes the normal hexane solution containing fatty acid, wherein adds nonadecanoic acid methyl ester internal standard. 5.根据权利要求4所述的方法，其中十九烷酸甲酯与正己烷溶液的体积比是1:1。5. The method according to claim 4, wherein the volume ratio of methyl nonadecanoate and n-hexane solution is 1:1. 6.根据权利要求1或4所述的方法，其中步骤(2)GC-MS测定脂肪酸利用7890B/5977A气质联用仪(Agilent，美国)测定，色谱柱为HP-88(60m×0.25mm×0.20μm，Agilent，美国)，测样升温程序为初始125℃，以8℃/min升到145℃，保持26min，以2℃/min升到220℃，保持1min，再以1℃/min升到227℃，保持1min，进样口温度250℃，辅助加热器温度280℃，分流比10:1，载气为氦气。6. according to the described method of claim 1 or 4, wherein step (2) GC-MS measures fatty acid and utilizes 7890B/5977A mass spectrometer (Agilent, the U.S.) to measure, and chromatographic column is HP-88 (60m * 0.25mm * 0.20μm, Agilent, the United States), the temperature program of the test sample is initially 125°C, rise to 145°C at 8°C/min, keep for 26min, rise to 220°C at 2°C/min, keep for 1min, and then rise at 1°C/min To 227°C, hold for 1min, the temperature of the injection port is 250°C, the temperature of the auxiliary heater is 280°C, the split ratio is 10:1, and the carrier gas is helium. 7.根据权利要求1所述的方法，其中步骤(3)a采用Masshunter Work Station定量分析脂肪酸含量。7. The method according to claim 1, wherein step (3) a adopts Masshunter Work Station to quantitatively analyze fatty acid content. 8.根据权利要求1所述的方法，其中步骤(4)使用SPSS软件对p种n-3和n-6系列不饱和脂肪酸含量百分比进行线性判别分析。8. The method according to claim 1, wherein step (4) uses SPSS software to carry out linear discriminant analysis to p kinds of n-3 and n-6 series unsaturated fatty acid content percentages. 9.根据权利要求1所述的方法，其中n-3和n-6系列不饱和脂肪酸为C18:2n6c、C18:2n6t、C18:3n3、C18:3n6、C20:3n3、C20:3n6、C20:4n6、C20:5n3、C22:2n6、C22:6n3。9. The method according to claim 1, wherein n-3 and n-6 series unsaturated fatty acids are C18:2n6c, C18:2n6t, C18:3n3, C18:3n6, C20:3n3, C20:3n6, C20: 4n6, C20:5n3, C22:2n6, C22:6n3.
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