CN106093270A - A kind of sweet three ester α position fatty acid determination methods of animal and plant fat - Google Patents
A kind of sweet three ester α position fatty acid determination methods of animal and plant fat Download PDFInfo
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Abstract
The invention provides a kind of sweet three ester α position fatty acid determination methods of animal and plant fat, including, three esters sweet in vegetable and animals oils are carried out chromatography and collect triglyceride, by it with after lipase hydrolysis, hydrolyzate is separated through thin layer chromatography, determine that α free fatty collection carry out derivatization treatment, enter gas chromatographic detection, measure composition and the content of α position fatty acid in triglyceride.The method have easy and simple to handle, reagent safety, need that solvent-oil ratio is few, analysis result accurately, detection limit is low, analysis time is fast advantage, sample requirements low.
Description
Technical field
The invention belongs to oils and fats and measure field, be specifically related to a kind of sweet three ester α position fatty acid determination sides of animal and plant fat
Method.
Background technology
The composition that mainly comprises of edible animals and plants oil is triglyceride, and its content accounts for more than the 95% of total lipid, and 5% is it
His lipid, such as diglyceride, monoglyceride, fatty acid, phospholipid, glycolipid and steroid etc..Triglyceride is by a part glycerol
Being generated by esterification with three molecules of fatty acids, the fatty acid of esterification lays respectively at α and β position, its chemical structural formula such as accompanying drawing
2。
Naturally occurring oils and fats due to source, fatty acid species and content, carbochain length, double bond number, position of double bond
Difference and there is different physicochemical properties and rheological behavior.Moreover, fatty acid position distribution in sweet three esters decides
The digestion of sweet three esters, absorbs, metabolism and nutritive value.Such as: Palmic acid (C16:0) is the main fatty acid component in breast milk
(accounting for total fatty acids 25%), wherein >=65% is positioned on β position, is hydrolyzed to form β-sweet ester by pancreatic lipase, forms breast with cholate
Rotten microgranule and be absorbed by organisms.And the Palmic acid in Lac Bovis seu Bubali and infant formula Ruzhong is mainly distributed on α position, it is hydrolyzed to by pancreatic lipase
Free fatty, easy and Ca 2+ forms calcium soap, it is impossible to absorbed well by human body.And the oleic acid (C18:1) in sweet three esters
It is easier to when being positioned at α position be utilized by body, it is provided that energy.It addition, stearic acid (C18:0) does not have a liter hyperlipidemia when being in α position
With the effect of cholesterol, when being positioned at β position, then as myristic acid (C 14:0) and Palmic acid (C 16:0), there is rising blood
Liquid cholesterol effect.Meanwhile, blood plasma fatty acid distribution is also had a major impact by acylation location, and therefore in oils and fats, fatty acid composition is solid
The most important, but the position distribution of fatty acid also can not be ignored, and this is accomplished by being measured the position distribution of fatty acid in oils and fats.
At present, carried out both at home and abroad substantial amounts of about sweet three ester α or the research work of β position fatyy acids technology.Conventional
The method of fatty acid position distribution in sweet three esters that measures have pancreatic lipase Hydrolyze method and grignard edman degradation Edman.Pancreatic lipase Hydrolyze method is root
According to the alpha specific of pancreatic lipase, by hydrolyzing the fatty acid on sweet three ester α positions, β-sweet ester is measured, analyzes fatty acid
A kind of conventional enzyme solution of position distribution.But, pancreatic lipase hydrolysis has fatty acid and three-dimensional regioselective, especially
To polyunsaturated fatty acid and Short-Chain Fatty Acids, so, it is impossible to accurately analyze the position distribution of these fatty acids.Another kind side
Method is grignard edman degradation Edman, and the DAG generated, by the sweet three bit esterified Fatty acid degradation of ester α, is measured by grignard reaction, thus
Calculate a kind of chemical method of fatty acid position distribution.The method can avoid the pancreatic lipase selective hydrolysis to fatty acid,
But, allylic bromination magnesium meets water electrode easy in inactivation, and has toxicity.Therefore, other lipase specific for hydrolysis animals and plants are explored
The method of the sweet three ester α position fatty acid compositions of oils and fats detection, significant to the structure analysing in depth sweet three esters of animals and plants.
Summary of the invention
The purpose of this part is to summarize some aspects of embodiments of the invention and briefly introduce some preferably to implement
Example.Make a summary in this part and the description of the present application and denomination of invention may be done a little simplification or omit to avoid making our department
Point, the purpose of specification digest and denomination of invention obscure, and this simplification or omission cannot be used for limiting the scope of the present invention.
In view of problem present in the sweet three ester α position fatty acid determination methods of above-mentioned and/or existing animal and plant fat, propose
The present invention.
Therefore, present invention aim to address the deficiencies in the prior art, it is provided that the sweet three ester α position fat of a kind of animal and plant fat
The assay method of acid.
For solving above-mentioned technical problem, the technical scheme is that a kind of sweet three ester α position fat of animal and plant fat
The assay method of fat acid, it is characterised in that include, three esters sweet in vegetable and animals oils are carried out chromatography and collect triglyceride,
By it with after lipase hydrolysis, hydrolyzate is separated through thin layer chromatography, determines that α free fatty collection perform the derivatization place
Reason, enters gas chromatographic detection, measures composition and the content of α position fatty acid in triglyceride.
As a kind of preferred version of the sweet three ester α position fatty acid determination methods of animal and plant fat of the present invention, wherein:
Described chromatography, it is to use silica gel column chromatography to separate, and wherein, column chromatography eluent is petroleum ether and ether, its volume ratio
Being 80～90:10～15, elution speed is 1～3ml/min.
As a kind of preferred version of the sweet three ester α position fatty acid determination methods of animal and plant fat of the present invention, wherein:
Described lipase hydrolysis, it is to add lipase, sodium cholate, CaCl2:, at 37～55 DEG C, keep 15～25min, then exist
Under conditions of 3000～5000r/min, centrifugal 5min.
As a kind of preferred version of the sweet three ester α position fatty acid determination methods of animal and plant fat of the present invention, wherein:
Described lipase is the alpha specific lipase of Rhizopus oryzae fermenting and producing, and its addition is the 5～15% of triglyceride quality.
As a kind of preferred version of the sweet three ester α position fatty acid determination methods of animal and plant fat of the present invention, wherein:
Described lipase hydrolysis, is additionally added MgCl2, and meet Mg after interpolation2+Concentration in mixed liquor is 3～4mmol/L.
As a kind of preferred version of the sweet three ester α position fatty acid determination methods of animal and plant fat of the present invention, wherein:
Described thin layer chromatography separates, and it is that silica gel G plate is placed in 105 DEG C of baking ovens activation 1h, then satisfies by 0.4mol/L boric acid solution
And process, gravity-flow ventilation launches after drying, and the methanol of 2,7 dichlorofluoresceins uniformly spraying 0.2wt% on thin plate is molten
Liquid and under uviol lamp color development treatment, oleic acid, as with reference to standard specimen, determines α-free fatty position on high performance thin layer chromatography
Put, α-free fatty band is scraped, uses ether extraction.
As a kind of preferred version of the sweet three ester α position fatty acid determination methods of animal and plant fat of the present invention, wherein:
Described launch, wherein, the extension liquid of use be hexane, ether, acetic acid be 70～80:20～30:0.5～1 by volume
Mixed liquor, the process time is 40～50min.
As a kind of preferred version of the sweet three ester α position fatty acid determination methods of animal and plant fat of the present invention, wherein:
Described derivatization treatment, its be use Nitrogen evaporator solvent is vapored away, and in centrifuge tube add 1～2mL normal hexane and 0.5～
Sodium hydroxide-the methanol solution of 1mL 1～1.5mol/L, mixes 1min by mixed liquor vortex, and 4000～5000r/min are centrifuged 3
～5min, collect supernatant.
As a kind of preferred version of the sweet three ester α position fatty acid determination methods of animal and plant fat of the present invention, wherein:
Described enter gas chromatographic detection, its be Aspirate supernatant in sample injection bottle, use the detection of gas chromatogram-hydrogen flame ion, carrier gas
For purity more than 99.99% nitrogen, flow rate of carrier gas is 1.0mL/min, and split ratio is 100～50: 1, vaporizer temperature 250～
300 DEG C, fid detector temperature is 250～300 DEG C, sampling volume 1.0 μ L, gas chromatographic column specification be SP-2560,100m ×
0.25mm, 0.20 μm capillary column.
As a kind of preferred version of the sweet three ester α position fatty acid determination methods of animal and plant fat of the present invention, wherein:
Described gas chromatogram-hydrogen flame ion detection analysis condition is temperature programming, and its initial temperature is 80～100 DEG C, keep 2～
5min, rises to 160～200 DEG C with 5～15 DEG C/min, keeps 2～5min, then is warming up to 210～230 DEG C with 5～15 DEG C/min.
The present invention is had the advantages that
The present invention is by chromatography, lipase hydrolysis, thin layer chromatography, fatty acid derivitization, five rings of gas chromatographic detection
Save preferably in combination with and Parameter Conditions preferably optimize (including enzymatic hydrolysis condition, developing solvent correlative factor etc.), formed exclusive to dynamic
The method that the sweet three ester α position fatty acids of Vegetable oil lipoprotein are measured, the method has easy and simple to handle, reagent safety, needs solvent to disappear
Consumption is few, analysis result is accurate, detection limit is low, analysis time is fast advantage, sample requirements are low.
Accompanying drawing explanation
In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, required use in embodiment being described below
Accompanying drawing be briefly described, it should be apparent that, below describe in accompanying drawing be only some embodiments of the present invention, for
From the point of view of those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to obtain it according to these accompanying drawings
Its accompanying drawing.Wherein:
Fig. 1 be in embodiment 1～5 esterification derivant in SP-2560,100m × 0.25mm, 0.20 μm capillary tube gas phase
Separating spectrum in chromatographic column, determines the retention time of various fatty acid methyl ester according to peak sequence.
Fig. 2 is the chemical structural formula of triglyceride.
Detailed description of the invention
Understandable, below in conjunction with specific embodiment pair for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from
The detailed description of the invention of the present invention is described in detail.
Elaborate a lot of detail in the following description so that fully understanding the present invention, but the present invention is all right
Using other to be different from alternate manner described here to implement, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by following public specific embodiment.
Secondly, " embodiment " or " embodiment " referred to herein refers to may be included at least one realization side of the present invention
Special characteristic, structure or characteristic in formula.Different in this manual local " in one embodiment " occurred not refer both to
Same embodiment, is not single or the most mutually exclusive with other embodiments embodiment.
Thin layer chromatography is the separation method of a kind of physics, utilizes the difference of each component physicochemical properties in mixture, no
With degree be distributed in two the most miscible mutually in.One of them is fixing phase, and another is the flowing phase flowing through fixing phase,
Each component moves with friction speed, thus reaches to separate.The principle of adsorption chromatography relies primarily in sample each component to adsorbent
Different affinitys and the separation that the different dissolubility of solvent (developing solvent) is obtained.Solvent is due to capillary tube upon deployment
Effect move up, those to adsorbent affinity by force, dissolubility is little in a solvent component, move up slower.And certain
The component that a little in a solvent dissolubility weak to adsorbent affinity are big, moves up the most quickly.So, speed is moved due to material
The difference of degree, various components in sample, just can be separated.Sample size in the selection of fixing phase, chromatography, the pole of eluant
Final result all can be impacted by property, the flow velocity etc. of eluant.The selection of developing solvent is an important key of thin layer chromatography,
Mainly determine according to sample and the polarity of solvent or sample dissolubility in a solvent.Based on this, the present invention is through further
Preferably optimize, use extension liquid be hexane, ether, acetic acid be the mixed liquor of 70～80:20～30:0.5～1 by volume,
The process time is 40～50min, now can realize the highly purified separation of α free fatty.
Embodiment 1
The separation and Extraction of triglyceride in step 1 soybean oil
The method using silica gel column chromatography separates and obtains triglyceride.Column chromatography eluent is petroleum ether: ether (80～
90:10～15, V/V).Weigh oil sample about 1～5g, be transferred in 10～50ml volumetric flasks, use eluent constant volume.Take chromatography
Post (dimensions 1～3cm × 30～50cm), puts a little glass or sea sand, eluent and silica gel is stirred in bottom
Mixing, adds dress post.Draw oil sample 20～30ml, be slowly added into along chromatography post jamb, then carry out with 100～500ml eluents
Eluting, speed is about 1～3ml/min, and eluting is complete, and gleanings is triglyceride, and nitrogen blows down and removes organic solvent.
Step 2 lipase Lipase DF hydrolyzes sweet three esters
Weigh separating obtained triglyceride 0.2g and fill in test tube in 10mL tool, add Lipase DF lipase 10mg, then add
Enter 0.2mol/L Tris-HCl buffer 2mL, carefully shake, be subsequently adding 0.5mL sodium cholate solution, 0.2mL CaCl2Solution
With 2.5mg MgCl2Cover stopper carefully to shake, immediately test tube is put in 37 DEG C of thermostat water baths, keep reaction 15min, take
Go out, with agitator strenuous vibration 2min, add 1mL hydrochloric acid and 1mL ether immediately, cover stopper, and acutely shake with agitator,
5000r/min is centrifuged 5min and separates, and draws ether layer, with in 0.45 μm membrane filtration to sample bottle.
Step 3 High Performance Thin Layer Chromatography chromatography isolated and purified α free fatty
High performance thin layer chromatography (TLC) is utilized to separate the β-sweet ester in hydrolyzate and α-free fatty.Thin layer chromatography
Separation condition is: silica gel G plate is placed in 105 DEG C of baking ovens activation 1h, then with the saturated process of 0.4mol/L boric acid solution, naturally logical
Wind uses after drying.Extension liquid used is: normal hexane-ether-acetic acid (75:25:0.5, V/V/V), launches after about 45min
Complete, thin plate uniformly sprays the 2 of 0.2wt%, the methanol solution of 7-dichlorofluorescein color development treatment under uviol lamp.Oil
Acid, as with reference to standard specimen, determines α-free fatty position on TLC, is scraped by α-free fatty band, carry with ether
Take three times, merge stand-by.
Step 4 α-free fatty acid methyl esters
Use Nitrogen evaporator to be vapored away by solvent, and in centrifuge tube, add the NaOH-CH of 1mL 0.5mol/L3OH solution,
It is placed in 65 DEG C of vibration saponification 30min to without oil droplet, then heats to 70 DEG C, addition 2mL BF3-CH3OH solution (volume ratio 1:3,
Now with the current), heat 3min, be cooled to after room temperature add 2mL normal hexane, mixed liquor vortex is mixed 1min, 4000～
5000r/min is centrifuged 3～5min, collects supernatant.
Step 5GC method measures α free fatty composition
Aspirate supernatant, in gas chromatographic sample introduction bottle, uses gas chromatographic analysis α position fatty acid composition, analysis condition
For: Shimadzu GC-2014 gas chromatograph, chromatographic column SP-2560,100m × 0.25mm, 0.20 μm capillary column, use program liter
Temperature, initial temperature 80 DEG C, keep 4min, rise to 215 DEG C with 15 DEG C/min, keep 20min；Carrier gas be high pure nitrogen (>=
99.99%)；Flow rate of carrier gas is 1.0mL/min；Split ratio 100～50:1；Vaporizer temperature 260 DEG C；Fid detector temperature is
300℃；Sampling volume 1.0 μ L.Mix target retention time by fatty acid methyl ester and identify that fatty acid species, relative amount represent
For mol%.
Embodiment 2
The separation and Extraction of triglyceride in step 1 Semen Lini oil
The method using silica gel column chromatography separates and obtains triglyceride.Column chromatography eluent is petroleum ether: ether (80～
90:10～15, V/V).Weigh oil sample about 1～5g, be transferred in 10～50ml volumetric flasks, use eluent constant volume.Take chromatography
Post (dimensions 1～3cm × 30～50cm), puts a little glass or sea sand, eluent and silica gel is stirred in bottom
Mixing, adds dress post.Draw oil sample 20～30ml, be slowly added into along chromatography post jamb, then carry out with 100～500ml eluents
Eluting, speed is about 1～3ml/min, and eluting is complete, and gleanings is triglyceride, and nitrogen blows down and removes organic solvent.
Step 2 lipase Lipase DF hydrolyzes sweet three esters
Weigh separating obtained triglyceride 0.2g and fill in test tube in 10mL tool, add Lipase DF lipase 20mg, then add
Enter 0.2mol/L Tris-HCl buffer 2mL, carefully shake, be subsequently adding 0.5mL sodium cholate solution, 0.2mL CaCl2Solution
With 1.5mg MgCl2Cover stopper carefully to shake, immediately test tube is put in 40 DEG C of thermostat water baths, keep reaction 15min, take
Go out, with agitator strenuous vibration 2min, add 1mL hydrochloric acid and 1mL ether immediately, cover stopper, and acutely shake with agitator,
4000r/min is centrifuged 5min and separates, and draws ether layer, with in 0.45 μm membrane filtration to sample bottle；
Step 3 High Performance Thin Layer Chromatography chromatography isolated and purified α free fatty
High performance thin layer chromatography (TLC) is utilized to separate the β-sweet ester in hydrolyzate and α-free fatty.Thin layer chromatography
Separation condition is: silica gel G plate is placed in 105 DEG C of baking ovens activation 1h, then with the saturated process of 0.4mol/L boric acid solution, naturally logical
Wind uses after drying.Extension liquid used is: normal hexane-ether-acetic acid (80:20:1, V/V/V), has launched after about 50min
Finish, thin plate uniformly sprays the 2 of 0.2wt%, the methanol solution of 7-dichlorofluorescein color development treatment under uviol lamp.Oleic acid
As with reference to standard specimen, determine α-free fatty position on TLC, α-free fatty band is scraped, uses ether extraction
Three times, merge stand-by.
Step 4 α-free fatty acid methyl esters
Use Nitrogen evaporator to be vapored away by solvent, and in centrifuge tube, add the NaOH-CH of 1mL 0.5mol/L3OH solution,
It is placed in 65 DEG C of vibration saponification 30min to without oil droplet, then heats to 70 DEG C, addition 2mL BF3-CH3OH solution (volume ratio 1:3,
Now with the current), heat 3min, be cooled to after room temperature add 2mL normal hexane, mixed liquor vortex is mixed 1min, 4000～
5000r/min is centrifuged 3～5min, collects supernatant.
Step 5GC method measures α free fatty composition
Aspirate supernatant, in gas chromatographic sample introduction bottle, uses gas chromatographic analysis α position fatty acid composition, analysis condition
For: Shimadzu GC-2014 gas chromatograph, chromatographic column SP-2560,100m × 0.25mm, 0.20 μm capillary column, use program liter
Temperature, initial temperature 100 DEG C, keep 3min, rise to 220 DEG C with 10 DEG C/min, keep 30min；Carrier gas be high pure nitrogen (>=
99.99%)；Flow rate of carrier gas is 1.0mL/min；Split ratio 100～50:1；Vaporizer temperature 260 DEG C；Fid detector temperature is
300℃；Sampling volume 1.0 μ L.Mix target retention time by fatty acid methyl ester and identify that fatty acid species, relative amount represent
For mol%.
Embodiment 3
The separation and Extraction of triglyceride in step 1 butter
The method using silica gel column chromatography separates and obtains triglyceride.Column chromatography eluent is petroleum ether: ether (80～
90:10～15, V/V).Weigh oil sample about 1～5g, be transferred in 10～50ml volumetric flasks, use eluent constant volume.Take chromatography
Post (dimensions 1～3cm × 30～50cm), puts a little glass or sea sand, eluent and silica gel is stirred in bottom
Mixing, adds dress post.Draw oil sample 20～30ml, be slowly added into along chromatography post jamb, then carry out with 100～500ml eluents
Eluting, speed is about 1～3ml/min, and eluting is complete, and gleanings is triglyceride, and nitrogen blows down and removes organic solvent.
Step 2 lipase Lipase DF hydrolyzes sweet three esters
Weigh separating obtained triglyceride 0.1g and fill in test tube in 10mL tool, add Lipase DF lipase 10mg, then add
Enter 0.2mol/L Tris-HCl buffer 2mL, carefully shake, be subsequently adding 0.5mL sodium cholate solution, 0.2mL CaCl2Solution
With 2.0mg MgCl2Cover stopper carefully to shake, immediately test tube is put in 40 DEG C of thermostat water baths, keep reaction 15min, take
Go out, with agitator strenuous vibration 2min, add 1mL hydrochloric acid and 1mL ether immediately, cover stopper, and acutely shake with agitator,
3000r/min is centrifuged 5min and separates, and draws ether layer, with in 0.45 μm membrane filtration to sample bottle；
Step 3 High Performance Thin Layer Chromatography chromatography isolated and purified α free fatty
High performance thin layer chromatography (TLC) is utilized to separate the β-sweet ester in hydrolyzate and α-free fatty.Thin layer chromatography
Separation condition is: silica gel G plate is placed in 105 DEG C of baking ovens activation 1h, then with the saturated process of 0.4mol/L boric acid solution, naturally logical
Wind uses after drying.Extension liquid used is: normal hexane-ether-acetic acid (70:30:1, V/V/V), has launched after about 40min
Finish, thin plate uniformly sprays the 2 of 0.2wt%, the methanol solution of 7-dichlorofluorescein color development treatment under uviol lamp.Oleic acid
As with reference to standard specimen, determine α-free fatty position on TLC, α-free fatty band is scraped, uses ether extraction
Three times, merge stand-by.
Step 4 α-free fatty acid methyl esters
Use Nitrogen evaporator to be vapored away by solvent, and in centrifuge tube, add the NaOH-CH of 1mL 0.5mol/L3OH solution,
It is placed in 65 DEG C of vibration saponification 30min to without oil droplet, then heats to 70 DEG C, addition 2mL BF3-CH3OH solution (volume ratio 1:3,
Now with the current), heat 3min, be cooled to after room temperature add 2mL normal hexane, mixed liquor vortex is mixed 1min, 4000～
5000r/min is centrifuged 3～5min, collects supernatant.
Step 5GC method measures α free fatty composition
Aspirate supernatant, in gas chromatographic sample introduction bottle, uses gas chromatographic analysis α position fatty acid composition, analysis condition
For: Shimadzu GC-2014 gas chromatograph, chromatographic column SP-2560,100m × 0.25mm, 0.20 μm capillary column, use program liter
Temperature, initial temperature 80 DEG C, keep 4min, rise to 220 DEG C with 15 DEG C/min, keep 20min；Carrier gas be high pure nitrogen (>=
99.99%)；Flow rate of carrier gas is 1.0mL/min；Split ratio 100～50:1；Vaporizer temperature 260 DEG C；Fid detector temperature is
300℃；Sampling volume 1.0 μ L.Mix target retention time by fatty acid methyl ester and identify that fatty acid species, relative amount represent
For mol%.
Embodiment 4
The separation and Extraction of triglyceride in step 1 Petiolus Trachycarpi oil
The method using silica gel column chromatography separates and obtains triglyceride.Column chromatography eluent is petroleum ether: ether (80～
90:10～15, V/V).Weigh oil sample about 1～5g, be transferred in 10～50ml volumetric flasks, use eluent constant volume.Take chromatography
Post (dimensions 1～3cm × 30～50cm), puts a little glass or sea sand, eluent and silica gel is stirred in bottom
Mixing, adds dress post.Draw oil sample 20～30ml, be slowly added into along chromatography post jamb, then carry out with 100～500ml eluents
Eluting, speed is about 1～3ml/min, and eluting is complete, and gleanings is triglyceride, and nitrogen blows down and removes organic solvent.
Step 2 lipase Lipase DF hydrolyzes sweet three esters
Weigh separating obtained triglyceride 0.2g and fill in test tube in 10mL tool, add Lipase DF lipase 10mg, then add
Enter 0.2mol/L Tris-HCl buffer 2mL, carefully shake, be subsequently adding 0.5mL sodium cholate solution, 0.2mL CaCl2Solution
With 2.5mg MgCl2Cover stopper carefully to shake, immediately test tube is put in 45 DEG C of thermostat water baths, keep reaction 15min, take
Go out, with agitator strenuous vibration 2min, add 1mL hydrochloric acid and 1mL ether immediately, cover stopper, and acutely shake with agitator,
4000r/min is centrifuged 5min and separates, and draws ether layer, with in 0.45 μm membrane filtration to sample bottle.
Step 3 High Performance Thin Layer Chromatography chromatography isolated and purified α free fatty
High performance thin layer chromatography (TLC) is utilized to separate the β-sweet ester in hydrolyzate and α-free fatty.Thin layer chromatography
Separation condition is: silica gel G plate is placed in 105 DEG C of baking ovens activation 1h, then with the saturated process of 0.4mol/L boric acid solution, naturally logical
Wind uses after drying.Extension liquid used is: normal hexane-ether-acetic acid (80:20:0.5, V/V/V), launches after about 40min
Complete, thin plate uniformly sprays the 2 of 0.2wt%, the methanol solution of 7-dichlorofluorescein color development treatment under uviol lamp.Oil
Acid, as with reference to standard specimen, determines α-free fatty position on TLC, is scraped by α-free fatty band, carry with ether
Take three times, merge stand-by.
Step 4 α-free fatty acid methyl esters
Use Nitrogen evaporator to be vapored away by solvent, and in centrifuge tube, add the NaOH-CH of 1mL 0.5mol/L3OH solution,
It is placed in 65 DEG C of vibration saponification 30min to without oil droplet, then heats to 70 DEG C, addition 2mL BF3-CH3OH solution (volume ratio 1:3,
Now with the current), heat 3min, be cooled to after room temperature add 2mL normal hexane, mixed liquor vortex is mixed 1min, 4000～
5000r/min is centrifuged 3～5min, collects supernatant.
Step 5GC method measures α free fatty composition
Aspirate supernatant, in gas chromatographic sample introduction bottle, uses gas chromatographic analysis α position fatty acid composition, analysis condition
For: Shimadzu GC-2014 gas chromatograph, chromatographic column SP-2560,100m × 0.25mm, 0.20 μm capillary column, use program liter
Temperature, initial temperature 100 DEG C, keep 3min, rise to 220 DEG C with 15 DEG C/min, keep 30min；Carrier gas be high pure nitrogen (>=
99.99%)；Flow rate of carrier gas is 1.0mL/min；Split ratio 100～50:1；Vaporizer temperature 260 DEG C；Fid detector temperature is
300℃；Sampling volume 1.0 μ L.Mix target retention time by fatty acid methyl ester and identify that fatty acid species, relative amount represent
For mol%.
Embodiment 5
The extraction of step 1 chicken fat
Slough moisture in chicken fat by vacuum drying, add the mixture of chloroform/methanol (2:1, vol/vol), and fully
Homogenizing shakes.Then add the NaCl aqueous solution (0.86%, w/w) of 1/4 volume, fully shake balance.Collect organic facies, mistake
Filter, and use rotary evaporation to remove organic solvent, gained lipid storage is at-18 DEG C.
The separation and Extraction of triglyceride in step 2 chicken fat
The method using silica gel column chromatography separates and obtains triglyceride.Column chromatography eluent is petroleum ether: ether (80～
90:10～15, V/V).Weigh oil sample about 1～5g, be transferred in 10～50ml volumetric flasks, use eluent constant volume.Take chromatography
Post (dimensions 1～3cm × 30～50cm), puts a little glass or sea sand, eluent and silica gel is stirred in bottom
Mixing, adds dress post.Draw oil sample 20～30ml, be slowly added into along chromatography post jamb, then carry out with 100～500ml eluents
Eluting, speed is about 1～3ml/min, and eluting is complete, and gleanings is triglyceride, and nitrogen blows down and removes organic solvent.
Step 3 lipase Lipase DF hydrolyzes sweet three esters
Weigh separating obtained triglyceride 0.2g and fill in test tube in 10mL tool, add Lipase DF lipase 30mg, then add
Enter 0.2mol/L Tris-HCl buffer 2mL, carefully shake, be subsequently adding 0.5mL sodium cholate solution, 0.2mL CaCl2Solution
With 1.5mg MgCl2Cover stopper carefully to shake, immediately test tube is put in 55 DEG C of thermostat water baths, keep reaction 25min, take
Go out, with agitator strenuous vibration 2min, add 1mL hydrochloric acid and 1mL ether immediately, cover stopper, and acutely shake with agitator,
5000r/min is centrifuged 5min and separates, and draws ether layer, with in 0.45 μm membrane filtration to sample bottle.
Step 4 High Performance Thin Layer Chromatography chromatography isolated and purified α free fatty
High performance thin layer chromatography (TLC) is utilized to separate the β-sweet ester in hydrolyzate and α-free fatty.Thin layer chromatography
Separation condition is: silica gel G plate is placed in 105 DEG C of baking ovens activation 1h, then with the saturated process of 0.4mol/L boric acid solution, naturally logical
Wind uses after drying.Extension liquid used is: normal hexane-ether-acetic acid (80:20:1, V/V/V), has launched after about 50min
Finish, thin plate uniformly sprays the 2 of 0.2wt%, the methanol solution of 7-dichlorofluorescein color development treatment under uviol lamp.Oleic acid
As with reference to standard specimen, determine α-free fatty position on TLC, α-free fatty band is scraped, uses ether extraction
Three times, merge stand-by.
Step 5 α-free fatty acid methyl esters
Use Nitrogen evaporator to be vapored away by solvent, and in centrifuge tube, add the NaOH-CH of 1mL 0.5mol/L3OH solution,
It is placed in 65 DEG C of vibration saponification 30min to without oil droplet, then heats to 90 DEG C, addition 2mL BF3-CH3OH solution (volume ratio 1:3,
Now with the current), heat 3min, be cooled to after room temperature add 2mL normal hexane, mixed liquor vortex is mixed 1min, 4000～
5000r/min is centrifuged 3～5min, collects supernatant.
Step 6GC method measures α free fatty composition
Aspirate supernatant, in gas chromatographic sample introduction bottle, uses gas chromatographic analysis α position fatty acid composition, analysis condition
For: Shimadzu GC-2014 gas chromatograph, chromatographic column SP-2560,100m × 0.25mm, 0.20 μm capillary column, use program liter
Temperature, initial temperature 100 DEG C, keep 4min, rise to 225 DEG C with 10 DEG C/min, keep 30min；Carrier gas be high pure nitrogen (>=
99.99%)；Flow rate of carrier gas is 1.0mL/min；Split ratio 100～50:1；Vaporizer temperature 260 DEG C；Fid detector temperature is
300℃；Sampling volume 1.0 μ L.Mix target retention time by fatty acid methyl ester and identify that fatty acid species, relative amount represent
For mol%.
The control of hydrolysis temperature is extremely important, owing to enzymic catalytic reaction is a thermodynamic process, affects enzyme
Catalysis activity, the dissolved state of substrate and the mass transfer velocity etc. of viscosity, substrate and product.Temperature is the lowest, is unfavorable for reaching reaction
Required activation energy, lipase effectively can not be combined with substrate, and reaction rate is relatively low, and the response time extends, sweet three esters in product
Hydrolysis degree is low；Temperature is the highest, then can cause lipase degeneration, reduces enzyme and lives.
Size and the enzymolysis speed of enzyme concentration are directly related, if the amount of substrate can keep sufficiently large, and response speed
Also complying with kinetics relation with enzyme amount, when enzyme amount increases, enzymolysis speed increases the most therewith.Lipase addition very little, then drops
Low reaction speed.Lipase addition is too much, when namely substrate content is relatively low, although at this moment enzymolysis speed is also with increasing
Greatly, but the impact of the resistance to mass tranfer of substrate and product has started to the impact more than enzyme enzymolysis Catalysis Rate, the increasing of its enzymolysis speed
Amount reduces, and the catalytic efficiency of enzyme reduces.
Inventor studies discovery, and along with the carrying out of reaction, the formation of enzymatic clarification reaction is had by the stirring of given pace
Facilitation, but cross too high the carrying out being unfavorable for enzymolysis on the contrary of stir speed (S.S.).Under different stir speed (S.S.)s, the hardness of sample etc. is deposited
May be relevant with its microcosmic degree of cross linking in difference.The intensity of cross-linked structure and quantity also directly influence the viscoelasticity of system simultaneously
Energy.
The alpha specific lipase of Rhizopus oryzae fermenting and producing belongs to α/β hydrolytic enzyme, and α/β hydrolytic enzyme refers to that class of enzymes is lived and relies on
Enzyme in the catalytic triads formed by Ser, His and Asp residue.The alpha specific lipase preference of Rhizopus oryzae fermenting and producing
In water insoluble substrates, it is adsorbed in water/oil interface before hydrolysis, changes mutually with substantial amounts of structure during catalytic reaction.Inventor
Research finds, Mg in enzymolysis solution2+Content when being 3～4mmol/L, it is possible to accelerate on water/oil interface in the tetrahedron of moment
Between the formation of state, to improve the enzymolysis speed of sweet three esters.
It should be noted that above example is only in order to illustrate technical scheme and unrestricted, although with reference to preferably
The present invention has been described in detail by embodiment, it will be understood by those within the art that, can be to the technology of the present invention
Scheme is modified or equivalent, and without deviating from the spirit and scope of technical solution of the present invention, it all should be contained at this
In the middle of bright right.
Claims (10)
1. the sweet three ester α position fatty acid determination methods of animal and plant fat, it is characterised in that: include,
Three esters sweet in vegetable and animals oils are carried out chromatography and collect triglyceride；
By it with after lipase hydrolysis, hydrolyzate is separated through thin layer chromatography, determines that α free fatty collection derive
Change processes；
Enter gas chromatographic detection, measure composition and the content of α position fatty acid in triglyceride.
The sweet three ester α position fatty acid determination methods of animal and plant fat the most according to claim 1, it is characterised in that: described layer
Analysis separate, its be use silica gel column chromatography separate, wherein, column chromatography eluent is petroleum ether and ether, its volume ratio be 80～
90:10～15, elution speed is 1～3ml/min.
The sweet three ester α position fatty acid determination methods of animal and plant fat the most according to claim 1 or claim 2, it is characterised in that: described
Lipase hydrolysis, it is to add lipase, sodium cholate, CaCl2, at 37～55 DEG C, keep 15～25min, then 3000
～under conditions of 5000r/min, centrifugal 5min.
The sweet three ester α position fatty acid determination methods of animal and plant fat the most according to claim 1 or claim 2, it is characterised in that: described
Lipase is the alpha specific lipase of Rhizopus oryzae fermenting and producing, and its addition is the 5～15% of triglyceride quality.
The sweet three ester α position fatty acid determination methods of animal and plant fat the most according to claim 3, it is characterised in that: described fat
Fat enzyme hydrolysis, is additionally added MgCl2, and meet Mg after interpolation2+Concentration in mixed liquor is 3～4mmol/L.
6., according to the sweet three ester α position fatty acid determination methods of the arbitrary described animal and plant fat of claim 1,2 or 5, its feature exists
In: described thin layer chromatography separates, and it is that silica gel G plate is placed in 105 DEG C of baking ovens activation 1h, then uses 0.4mol/L boric acid solution
Saturated process, gravity-flow ventilation launches after drying, and uniformly sprays the 2 of 0.2wt%, the methanol of 7-dichlorofluorescein on thin plate
Solution and under uviol lamp color development treatment, oleic acid, as with reference to standard specimen, determines that α-free fatty is on high performance thin layer chromatography
Position, scrapes α-free fatty band, uses ether extraction.
The sweet three ester α position fatty acid determination methods of animal and plant fat the most according to claim 6, it is characterised in that enter described in:
Row launch, wherein, use extension liquid be hexane, ether, acetic acid be the mixing of 70～80:20～30:0.5～1 by volume
Liquid, the process time is 40～50min.
8. according to the sweet three ester α position fatty acid determination methods of the arbitrary described animal and plant fat of claim 1,2,5 or 7, its feature
Be: described derivatization treatment, its be use Nitrogen evaporator solvent is vapored away, and in centrifuge tube add 1～2mL normal hexane and
0.5～1mL 1～sodium hydroxide-the methanol solution of 1.5mol/L, mixed liquor vortex is mixed 1min, 4000～5000r/min
Centrifugal 3～5min, collect supernatant.
9. according to the sweet three ester α position fatty acid determination methods of the arbitrary described animal and plant fat of claim 1,2,5 or 7, its feature
Be: described in enter gas chromatographic detection, its be Aspirate supernatant in sample injection bottle, use the detection of gas chromatogram-hydrogen flame ion,
Carrier gas is the nitrogen that purity is more than 99.99%, and flow rate of carrier gas is 1.0mL/min, and split ratio is 100～50: 1, vaporizer temperature
250～300 DEG C, fid detector temperature is 250～300 DEG C, sampling volume 1.0 μ L, gas chromatographic column specification be SP-2560,
100m × 0.25mm, 0.20 μm capillary column.
The sweet three ester α position fatty acid determination methods of animal and plant fat the most according to claim 9, it is characterised in that: described gas
Phase chromatograph-hydrogen flame ion detection analysis condition is temperature programming, and its initial temperature is 80～100 DEG C, keeps 2～5min, with 5
～15 DEG C/min rises to 160～200 DEG C, keep 2～5min, then be warming up to 210～230 DEG C with 5～15 DEG C/min.
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