CN108872444A - A method of triglycerides sn-3 fatty acid composition of measurement - Google Patents
A method of triglycerides sn-3 fatty acid composition of measurement Download PDFInfo
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Abstract
The invention discloses a kind of methods of measurement triglycerides sn-3 fatty acid composition, belong to chemical analysis technology field and zymotechnic field.Method of the invention uses sn-3 specific lipases (such as rabbit stomach lipase, dog gastric lipase, fusarium cutinase and recombinant human gastric lipase) direct hydrolysis triglycerides to detect sn-3 fatty acid；Sn-3 fatty acid are detected using method of the invention, there are the advantages such as easy to operate, lipid dosage is low, accuracy is higher.
Description
Technical field
The present invention relates to a kind of methods of measurement triglycerides sn-3 fatty acid composition, belong to chemical analysis technology field
And zymotechnic field.
Background technique
Triglycerides (TAG) is essential aliphatic acid for human body, is the important composition ingredient of lipid, by glycerol and 3 fatty acid
It constitutes, plays the role of the function for giving stored energy source in human body, may also function as fixed and protection internal organ.Currently, it is made
For the main nutrients of a kind of edible oil and breast milk, there is critically important nutritive value.
The structure of triglycerides as shown in Figure 1, due to its 1 and 3 upper fatty acid difference, cause its carbon atom 2
Asymmetry, so that triglycerides can generate two kinds of steric isomers：1-FAA, 2FAB, 3-FACGlycerol and 1-FAC, 2FAB,
3-FAAGlycerol, therefore, triglycerides have changeability in stereotaxis distribution.
However, triglycerides stereotaxis distribution on changeability (i.e. on triglycerides fatty acid structure and composition
It is different) it will affect the absorption and metabolism of triglycerides, and then influence the nutritive value of triglycerides.For example, when palmitinic acid is located at
At sn-1 or sn-3 of triglycerides, the palmitinic acid of free form can be hydrolyzed by pancreatic lipase, they can be intracorporal with people
Calcium binding forms calcium soap, therefore leads to human body calcium loss；The structure that pinolenic acid (PLA) is evenly distributed in triglycerides skeleton
The absorption efficiency of its PLA of rouge is higher than PLA mainly in sn-3 sweet three esters.
The nutritive value of the different three-dimensional specific locations analytical technology evaluation different structure triglycerides of fat is industrially commonly used,
To improve the quality of triglycerides product, wherein mass-spectrometric technique is the most widely used detection technique in lipid analysis field, still,
Mass-spectrometric technique lacks isomerism standard items, it is difficult to distinguish 1-FAA, 2FAB, 3-FACGlycerol and 1-FAC, 2FAB, 3-FAAGlycerol
Two kinds of isomers.Therefore, we are badly in need of finding the fat that can efficiently detect triglycerides sn-1, sn-2 and sn-3
The method of sour structure and composition.
Currently, the detection of sn-2 fatty acid is highly developed at present, this detection method is mainly using to triglycerides
Sn-1, sn-3 have lipase (such as pancreatic lipase) hydrolyzing triglyceride of specificity, discharge sn-1, sn-3 fatty acid,
Then it is analyzed using low-polarity components and the composition of sn-1, sn-3 fatty acid is analyzed, to calculate triglycerides
The composition of sn-2 fat.
But sn-1 and sn-3 fatty acid of triglycerides are formed, it is only capable of measuring using Grignard Reagent method at present,
I.e.：Triglycerides is first degraded to diglyceride and monoglyceride with Grignard Reagent (such as ethylmagnesium bromide), passes through thin-layer chromatography
Method separation obtains 1,2- and 2, the mixture of 3- diglyceride, then uses chiral high performance liquid chromatography by 1,2- and 2,3- glycerol two
Ester separation obtains 1,2- and 2 by gas chromatographic analysis respectively, and the fatty acid of 3- diglyceride forms, meanwhile, using pancreas fat
Enzyme hydrolysis triglycerides obtains sn-2 fatty acid compositions, eventually by the fatty acid group for calculating acquisition sn-3 and sn-1
At.The method is related to repeatedly hydrolysis, separating for several times and multiple chromatography, and cumbersome, error is larger.
Therefore, it still needs to further study for the detection technique that sn-1, sn-3 are fatty acid, commonly uses analytical reagent composition at present
The structure of sweet three ester, but there is effluent and isomer altogether in the TAGs chromatographic peak of many natural lipids, it is accurate to determine
The composition and content of TAGs is extremely difficult.
Summary of the invention
To solve the above problems, the present invention provides a kind of methods of measurement triglycerides sn-3 fatty acid composition.This
Method uses sn-3 specific lipases (such as rabbit stomach lipase, dog gastric lipase, fusarium cutinase and recombinant human gastric lipase
Deng) direct hydrolysis triglycerides to be to detect sn-3 fatty acid；The easy to operate, lipid using the method sn-3 fatty acid of detection
Dosage is low, accuracy is higher.
Technical scheme is as follows：
The present invention provides a kind of method of measurement triglycerides sn-3 fatty acid composition, the method is that will use glycerol
Triglycerides after the three enzyme-specific hydrolysising purifications of ester sn-3 or the determinand containing triglycerides, obtain steatolysis product；By rouge
It is extracted after solution product is cooling, and with TLC separation, obtains free fatty acid；Free fatty acid is subjected to esterification, is obtained
Esterification free fatty acid；With gas chromatographic analysis esterification free fatty acid to get sn-3 fatty acid compositions of triglycerides；
The triglycerides sn-3 enzyme-specifics include rabbit stomach lipase, dog gastric lipase, fusarium cutinase and recombinant human gastric lipase.
In one embodiment of the invention, the purification process of the triglycerides or the determinand containing triglycerides is
By triglycerides or determinand capillary containing triglycerides on silica gel plate point sample, solvent is added and is unfolded, scrapes
Triglycerides band, then extract triglycerides band with organic solvent and be centrifuged, organic solvent is removed, glycerol after purification is obtained
Three esters or determinand containing triglycerides.
In one embodiment of the invention, the ingredient of the solvent includes n-hexane, ether and acetic acid.
In one embodiment of the invention, in the solvent, the volume ratio of n-hexane, ether and acetic acid is
(50~70):(30~50):(1~2).
In one embodiment of the invention, in the solvent, the volume ratio of n-hexane, ether and acetic acid is
70:30:2。
In one embodiment of the invention, it is petroleum ether or ether that the organic solvent, which is organic solvent,.
In one embodiment of the invention, the additional amount of the organic solvent is to flood the triglycerides band scraped
Powder.
In one embodiment of the invention, the glycerol after described sn-3 enzyme-specific hydrolysising purifications with triglycerides
Three esters or determinand containing triglycerides are that I first is added in triglycerides after purification or the determinand containing triglycerides
It is emulsified after primary gum-solution, the triglycerides after being emulsified or the determinand containing triglycerides；After emulsification sweet again
Water is carried out after being separately added into sn-3 enzyme-specifics of enzyme stabilizers and triglycerides in oily three esters or determinand containing triglycerides
Solution reaction obtains hydrolyzed fat acid；
When the triglycerides sn-3 enzyme-specifics are dog gastric lipase, the ingredient of enzyme stabilizers is NaCl solution；
Or when the triglycerides sn-3 enzyme-specifics are rabbit stomach lipase, human gastric lipase or fusarium cutinase, enzyme
The ingredient of stabilizer includes calcium chloride, bovine serum albumin(BSA) and sodium chloride.
In one embodiment of the invention, when the triglycerides sn-3 enzyme-specifics are dog gastric lipase, enzyme
The ingredient of stabilizer is the NaCl solution of mass percentage concentration 0.5~1.5%；
Or when the triglycerides sn-3 enzyme-specifics are rabbit stomach lipase, human gastric lipase or fusarium cutinase, enzyme
The ingredient of stabilizer includes 1~5mmol/L calcium chloride, 1~50mmol/L bovine serum albumin(BSA) and 1~300mmol/L chlorination
Sodium.
In one embodiment of the invention, when the triglycerides sn-3 enzyme-specifics are dog gastric lipase, enzyme
The ingredient of stabilizer is the NaCl solution of mass percentage concentration 0.9%；
Or when the triglycerides sn-3 enzyme-specifics are rabbit stomach lipase, human gastric lipase or fusarium cutinase, enzyme
The ingredient of stabilizer includes 3.5mmol/L calcium chloride, 30mmol/L bovine serum albumin(BSA) and 150mmol/L sodium chloride.
In one embodiment of the invention, the pH of the enzyme stabilizers is 2.0~7.0.
In one embodiment of the invention, the pH of the enzyme stabilizers is 6.0.
In one embodiment of the invention, the gum arabic solution is with triglycerides after purification or containing glycerol
The volume ratio of the determinand of three esters should be maintained at 1:1~100.
In one embodiment of the invention, the gum arabic solution is with triglycerides after purification or containing glycerol
The volume ratio of the determinand of three esters should be maintained at 1:10.
In one embodiment of the invention, the mass percentage concentration of the gum arabic solution be 0.1%~
20%.
In one embodiment of the invention, the mass percentage concentration of the gum arabic solution is 10%.
In one embodiment of the invention, the method for the emulsification is ultrasound or homogeneous.
In one embodiment of the invention, the time of the emulsification is 0.1~120min.
In one embodiment of the invention, the time of the emulsification is 30min.
In one embodiment of the invention, the triglycerides of the triglycerides sn-3 enzyme-specifics after emulsification
Or the additive amount in the determinand containing triglycerides should be maintained at 1~100U/ μ g.
In one embodiment of the invention, the triglycerides of the triglycerides sn-3 enzyme-specifics after emulsification
Or the additive amount in the determinand containing triglycerides should be maintained at 10U/ μ g.
In one embodiment of the invention, the enzyme stabilizers are with the triglycerides after emulsification or containing triglycerides
The volume ratio of determinand should be maintained at 1:100~1000.
In one embodiment of the invention, the enzyme stabilizers are with the triglycerides after emulsification or containing triglycerides
The volume ratio of determinand should be maintained at 1:400.
In one embodiment of the invention, the temperature of the hydrolysis is 20 DEG C~45 DEG C, the time is 0~5h.
In one embodiment of the invention, the temperature of the hydrolysis be 37 DEG C, time 0.5h.
In one embodiment of the invention, it is extracted as after the cooling by hydrolyzed fat acid first that hydrolyzed fat acid is cold
But it extracts to the organic solvent that volume is 20~4000 times of hydrolyzed fat acid is added after 15 DEG C~25 DEG C immediately, then will extract
Hydrolyzed fat acid afterwards is centrifuged, and is removed organic solvent, is obtained free fatty acid.
In one embodiment of the invention, it is extracted as after the cooling by hydrolyzed fat acid first that hydrolyzed fat acid is cold
But it extracts to the organic solvent that volume is 100 times of hydrolyzed fat acid is added after 20 DEG C immediately, then by the hydrolysis rouge after extraction
Fat acid is centrifuged, and is removed organic solvent, is obtained free fatty acid.
In one embodiment of the invention, it is described to isolated free fatty acid carry out esterification refer to point
It is reacted from a certain amount of sulfuric acid-methanol solution is added in obtained free fatty acid, obtains reaction solution；Reaction solution is cooling
The organic solvent that volume is 50~500 times of reaction solution is added after to 15 DEG C~25 DEG C to extract, obtains extracting solution；In extracting solution
Middle addition water, takes supernatant to cross film, obtains esterification free fatty acid.
In one embodiment of the invention, it is described to isolated free fatty acid carry out esterification refer to point
It is reacted from a certain amount of sulfuric acid-methanol solution is added in obtained free fatty acid, obtains reaction solution；Reaction solution is cooling
The organic solvent that volume is 100 times of reaction solution is added after to 20 DEG C to extract, obtains extracting solution；Water is added in extracting solution,
It takes supernatant to cross film, obtains esterification free fatty acid.
In one embodiment of the invention, the concentration of the sulfuric acid-methanol solution be mass percentage concentration be 1~
10%.
In one embodiment of the invention, it is 5% that the concentration of the sulfuric acid-methanol solution, which is mass percentage concentration,.
In one embodiment of the invention, the body of the sulfuric acid-methanol solution and isolated free fatty acid
Product ratio should be maintained at 1:10~1000.
In one embodiment of the invention, the body of the sulfuric acid-methanol solution and isolated free fatty acid
Product ratio should be maintained at 1:100.
The present invention provides a kind of methods of above-mentioned measurement triglycerides sn-3 fatty acid composition in measurement triglycerides
The application of sn-3 fatty acid composition aspects.
Beneficial effect：
(1) method of the invention uses sn-3 specific lipases (such as rabbit stomach lipase, dog gastric lipase, fusarium cutin
Enzyme and recombinant human gastric lipase etc.) direct hydrolysis triglycerides to be to detect sn-3 fatty acid；
(2) sn-3 fatty acid are detected using method of the invention, with easy to operate, lipid dosage is low, accuracy compared with
High advantage；
(3) method of the invention can be widely applied to vegetable fat, animal fat (including from milk and each device
The grease of official's tissue) and microbial oil etc. sn-3 fatyy acids of triglycerides.
Detailed description of the invention
Fig. 1：The structural schematic diagram of triglycerides；
Fig. 2：Mix the thin-layer chromatographic analysis result of lipid.
Specific embodiment
Embodiment 1：The sn-3 fatty acid of soybean oil triglycerides forms
(1) three ester of purification of glycerol
Weigh 100mg soybean oil, with capillary on silica gel plate point sample, with n-hexane/ether/acetic acid (70:30:2, v/
V/v it is) solvent, scrapes triglycerides band, with extracted by ether and be transferred in centrifuge tube of having weighed, nitrogen is blown to constant weight；
(2) rabbit stomach lipase hydrolysis triglycerides
The gum arabic solution that 0.25mL mass concentration is 10%, ultrasonic emulsification are added in three ester of purification of glycerol
30min is separately added into 4mL enzyme stabilizers (3.5mmol/L calcium chloride, 30mmol/L bovine serum albumin(BSA) and 150mmol/L chlorination
Sodium, pH4.0) and 100U rabbit stomach lipase, 37 DEG C of water-baths shake 0~3h, are rinsed cool down with circulating water after reaction, immediately plus
Enter extracted by ether, nitrogen obtains steatolysis product after blowing；
(3) pass through TLC separation free fatty acid
With capillary by the grease point after hydrolysis on silica gel plate, with n-hexane/ether/acetic acid (70:30:2, v/v/v)
It for solvent, scrapes free fatty acid band (band is shown in Fig. 2), extracted by ether is added and is transferred to the screw socket glass tube weighed
In, nitrogen obtains the position the sn-3 fatty acid released after blowing, weigh and calculate the quality for obtaining free fatty acid；
(4) free fatty acid methyl esters
5% sulfuric acid-methanol solution of 1mL is added in screw socket glass tube, 60 DEG C of water-bath 60min are cooled to after taking-up
0.5mL n-hexane mechanical shaking extraction is added in room temperature, and distilled water is added to bottleneck, supernatant is taken to cross film storage；
(5) high temperature gas chromatography detection free-fat acid derivative is constituted
The actual conditions of high temperature gas chromatography are：Gas chromatograph is furnished with flame ionization detector and Trace TR-
FAME capillary column (60m × 0.25mm × 0.25 μm)；Split ratio 50:1；Nitrogen is carrier gas, flow velocity 1.2mL/min；Detector
It is 250 DEG C with injector temperature；2 μ L of sampling volume；Temperature program is：60 DEG C of initial column temperature, 3min is kept, with 5 DEG C/min
Speed rise to 175 DEG C, maintain 15min, rise to 220 DEG C with the speed of 2 DEG C/min, keep 10min.
The sn-3 position fatty acid that table 1-1 soybean oil is measured at different hydrolysis degrees (wt%) forms (wt%)
| Fatty acid | 1% | 2% | 3% | 4% | 5% |
| C8:0 | 1.04±0.45a | 0.51±0.06a | 1.69±0.20b | 0.91±0.21a | 1.04±0.16a |
| C14:0 | 0.67±0.07a | 0.54±0.06b | 0.47±0.05b | 0.35±0.04c | 0.29±0.01c |
| C16:0 | 23.34±0.68a | 21.65±0.56b | 18.67±0.84c | 17.09±0.46d | 16.14±0.41d |
| C18:0 | 11.10±0.69a | 10.38±0.61a | 10.29±0.95a | 6.89±0.29b | 6.16±0.17b |
| C20:0 | 0.76±0.12a | 0.42±0.02b | 0.48±0.01b | 0.29±0.06c | 0.23±0.02c |
| C22:0 | 0.89±0.06a | 0.69±0.09b | 0.86±0.07a | 0.45±0.07c | 0.40±0.05c |
| C24:0 | 0.75±0.03a | 0.39±0.13b | 0.38±0.06b | 0.33±0.01b | 0.28±0.01b |
| C16:1n-9 | 0.42±0.03a | 0.57±0.43ab | 0.34±0.06ab | 0.21±0.03b | 0.21±0.01b |
| C18:1n-9 | 22.60±0.73a | 24.05±1.16b | 22.16±0.46a | 24.60±0.60b | 24.83±0.21b |
| C18:2n-6 | 34.96±0.64a | 37.08±2.00b | 39.44±1.75c | 42.80±0.28d | 44.45±0.43d |
| αC18:3n-3 | 3.47±0.64a | 3.33±0.30a | 5.22±0.27b | 5.75±0.16bc | 5.98±0.04c |
Note：Data representation is average value ± standard variance；Every row letter is different to indicate significant difference (p<0.05)
Table 1-1 is the composition that soybean oil measures sn-3 fatty acid under different hydrolysis degrees, when hydrolysis free-fat
When the quality of acid reaches 4% or more of original triglycerides, measures the horizontal of various fatty acid and conspicuousness variation no longer occurs, because
This selects 4% scale as soybean oil sn-3 fatty acid of detection.
As shown in table 1-1, there are 3 kinds of content of fatty acid to be greater than 10% in the position the sn-3 fatty acid of soybean triglycerides, successively
For C18:2n-6,C18:1n-9 and C16:0.
Embodiment 2：The hydrolysis degree of soybean oil triglycerides sn-3 fatty acid under different enzyme concentrations
(1) three ester of purification of glycerol
Weigh 100mg soybean oil, with capillary on silica gel plate point sample, with n-hexane/ether/acetic acid (70:30:2, v/
V/v it is) solvent, scrapes triglycerides band, with extracted by ether and be transferred in centrifuge tube of having weighed, nitrogen is blown to constant weight；
(2) rabbit stomach lipase hydrolysis triglycerides
The gum arabic solution that 0.25mL mass concentration is 10%, ultrasonic emulsification are added in three ester of purification of glycerol
30min is separately added into 4mL enzyme stabilizers (3.5mmol/L calcium chloride, 30mmol/L bovine serum albumin(BSA) and 150mmol/L chlorination
Sodium, pH4.0) and 10U, 50U, 100U, 150U or 200U rabbit stomach lipase, 37 DEG C of water-baths concussion 0.5h, after reaction with stream
Dynamic water rinses cooling, and extracted by ether is added immediately, and nitrogen obtains steatolysis product after blowing；
(3) pass through TLC separation free fatty acid
With capillary by the grease point after hydrolysis on silica gel plate, with n-hexane/ether/acetic acid (70:30:2, v/v/v)
For solvent, free fatty acid band is scraped, extracted by ether is added and is transferred in the screw socket glass tube weighed, nitrogen obtains after blowing
The position the sn-3 fatty acid that must be released weighs and calculates the quality for obtaining free fatty acid；
(4) free fatty acid methyl esters
5% sulfuric acid-methanol solution of 1mL is added in screw socket glass tube, 60 DEG C of water-bath 60min are cooled to after taking-up
0.5mL n-hexane mechanical shaking extraction is added in room temperature, and distilled water is added to bottleneck, supernatant is taken to cross film storage；
(5) high temperature gas chromatography detection free-fat acid derivative is constituted
The actual conditions of high temperature gas chromatography are：Gas chromatograph is furnished with flame ionization detector and Trace TR-
FAME capillary column (60m × 0.25mm × 0.25 μm)；Split ratio 50:1；Nitrogen is carrier gas, flow velocity 1.2mL/min；Detector
It is 250 DEG C with injector temperature；2 μ L of sampling volume；Temperature program is：60 DEG C of initial column temperature, 3min is kept, with 5 DEG C/min
Speed rise to 175 DEG C, maintain 15min, rise to 220 DEG C with the speed of 2 DEG C/min, keep 10min.
Testing result is：The quality that each enzyme concentration reaction system measures hydrolysis free fatty acid is respectively original glycerol three
0.5 ± 0.1%, 2.6 ± 0.3%, 5.2 ± 0.3%, 6.1 ± 0.2% and the 6.5 ± 0.5% of ester, method for comprehensive detection has
The cost of effect property and enzyme, the optimal enzyme concentration of rabbit stomach lipase are 100U.
Embodiment 3：The hydrolysis degree of soybean oil triglycerides sn-3 fatty acid under different emulsification systems
(1) three ester of purification of glycerol
Weigh 100mg soybean oil, with capillary on silica gel plate point sample, with n-hexane/ether/acetic acid (70:30:2, v/
V/v it is) solvent, scrapes triglycerides band, with extracted by ether and be transferred in centrifuge tube of having weighed, nitrogen is blown to constant weight；
(2) rabbit stomach lipase hydrolysis triglycerides
The gum arabic solution that 0.25mL mass concentration is 10%, ultrasound are added in wherein three ester of a purification of glycerol
Enzyme stabilizers (3.5mmol/L calcium chloride, 30mmol/L bovine serum albumin(BSA) and 150mmol/L chlorination is added after emulsifying 30min
Sodium, pH4.0), it is denoted as A system.2mL acetate buffer (pH 4.0) is sequentially added in another three ester of purification of glycerol,
0.5mL sodium cholate solution (1g/L) and 0.2mL calcium chloride solution (220g/L), are denoted as B system.Respectively add in two parts of reaction systems
Enter 100U rabbit stomach lipase, 37 DEG C of water-baths shake 0.5h, rinsed cool down with circulating water after reaction, and ether is added immediately and mentions
It takes, nitrogen obtains steatolysis product after blowing；
(3) pass through TLC separation free fatty acid
With capillary by the grease point after hydrolysis on silica gel plate, with n-hexane/ether/acetic acid (70:30:2, v/v/v)
For solvent, free fatty acid band is scraped, extracted by ether is added and is transferred in the screw socket glass tube weighed, nitrogen obtains after blowing
The position the sn-3 fatty acid that must be released weighs and calculates the quality for obtaining free fatty acid；
(4) free fatty acid methyl esters
5% sulfuric acid-methanol solution of 1mL is added in screw socket glass tube, 60 DEG C of water-bath 60min are cooled to after taking-up
0.5mL n-hexane mechanical shaking extraction is added in room temperature, and distilled water is added to bottleneck, supernatant is taken to cross film storage；
(5) high temperature gas chromatography detection free-fat acid derivative is constituted
The actual conditions of high temperature gas chromatography are：Gas chromatograph is furnished with flame ionization detector and Trace TR-
FAME capillary column (60m × 0.25mm × 0.25 μm)；Split ratio 50:1；Nitrogen is carrier gas, flow velocity 1.2mL/min；Detector
It is 250 DEG C with injector temperature；2 μ L of sampling volume；Temperature program is：60 DEG C of initial column temperature, 3min is kept, with 5 DEG C/min
Speed rise to 175 DEG C, maintain 15min, rise to 220 DEG C with the speed of 2 DEG C/min, keep 10min.
Testing result is：A, B system measure the quality of hydrolysis free fatty acid be respectively original triglycerides 5.2 ±
0.3% and 0.4% ± 0.1%, illustrate that the effect of gum arabic emulsification system is much better than sn-2 fatyy acids methods and adopts
The sodium taurocholate system received.
Embodiment 4：The sn-3 fatty acid of human milk triglycerides forms
1. human milk fat is extracted
The triglycerides in human milk is extracted referring to IS014156 method.
2.sn-3 fatyy acids
(1) three ester of purification of glycerol
Weigh the lipid of 50mg extraction, point sample on silica gel plate, with n-hexane/ether/acetic acid (70:30:It 2, v/v/v) is exhibition
Agent is opened, triglycerides band is scraped, with extracted by ether and is transferred in centrifuge tube of having weighed, nitrogen is blown to constant weight；
(2) rabbit stomach lipase hydrolysis triglycerides
The gum arabic solution that 0.25mL mass concentration is 10%, ultrasonic emulsification are added in three ester of purification of glycerol
30min is separately added into 6mL enzyme stabilizers (3.5mmol/L calcium chloride, 30mmol/L bovine serum albumin(BSA) and 150mmol/L chlorination
Sodium, pH4.0) and 100U rabbit stomach lipase, 37 DEG C of 0~3h of water-baths concussion.After reaction with circulating water rinse cool down, immediately plus
Enter extracted by ether, nitrogen obtains steatolysis product after blowing.
(3) pass through TLC separation free fatty acid
With capillary by the grease point after hydrolysis on silica gel plate, with n-hexane/ether/acetic acid (70:30:2, v/v/v)
For solvent, free fatty acid band is scraped, extracted by ether is added and is transferred in the screw socket glass tube weighed, nitrogen obtains after blowing
The position the sn-3 fatty acid that must be released weighs and calculates the quality for obtaining free fatty acid.
(4) free fatty acid methyl esters
5% sulfuric acid-methanol solution of 1mL is added in screw socket glass tube, 60 DEG C of water-bath 60min are cooled to after taking-up
0.2mL n-hexane mechanical shaking extraction is added in room temperature, and distilled water is added to bottleneck, supernatant is taken to cross film storage.
(5) high temperature gas chromatography detection free-fat acid derivative is constituted
The actual conditions of high temperature gas chromatography are：Gas chromatograph is furnished with flame ionization detector and Trace TR-
FAME capillary column (60m × 0.25mm × 0.25 μm)；Split ratio 50:1；Nitrogen is carrier gas, flow velocity 1.2mL/min；Detector
It is 250 DEG C with injector temperature；2 μ L of sampling volume；Temperature program is：60 DEG C of initial column temperature, 3min is kept, with 5 DEG C/min
Speed rise to 175 DEG C, maintain 15min, rise to 220 DEG C with the speed of 2 DEG C/min, keep 10min.
The sn-3 position fatty acid that table 2-1 human milk triglycerides is measured at different hydrolysis degrees (wt%) forms (wt%)
| Fatty acid | 1% | 2% | 3% | 4% | 5% | 6% |
| C12:0 | 3.38±0.05b | 3.58±0.28b | 3.79±0.17b | 4.34±0.77a | 5.03±0.21a | 5.06±0.39a |
| C14:0 | 2.61±0.12b | 2.69±0.10b | 2.80±0.06ab | 2.80±0.29ab | 3.10±0.07a | 3.02±0.09a |
| C15:0 | 0.35±0.03a | 0.30±0.02b | 0.28±0.02b | 0.23±0.03c | 0.23±0.01c | 0.23±0.01c |
| C16:0 | 29.14±0.70a | 25.50±1.03b | 21.97±1.59c | 21.38±0.53c | 21.81±0.22c | 21.37±0.62c |
| C17:0 | 0.49±0.01a | 0.43±0.01b | 0.43±0.02b | 0.37±0.02c | 0.37±0.01c | 0.37±0.01c |
| C18:0 | 17.43±0.39a | 14.60±1.49b | 11.62±1.11c | 12.17±0.40c | 12.44±0.22c | 12.25±0.35c |
| C20:0 | 0.38±0.02a | 0.32±0.00b | 0.31±0.02b | 0.28±0.00c | 0.26±0.00c | 0.27±0.01c |
| C22:0 | 0.56±0.10a | 0.38±0.14b | 0.36±0.07b | 0.15±0.02c | 0.14±0.00c | 0.15±0.01c |
| C16:1n-9 | 1.77±0.01a | 1.56±0.41a | 1.45±0.03ab | 1.19±0.09b | 1.24±0.00b | 1.25±0.01b |
| C18:1n-9 | 24.10±0.57c | 28.47±0.92b | 30.83±0.47a | 30.85±0.43a | 30.18±0.57a | 31.04±0.67a |
| C20:1n-9 | 0.58±0.08a | 0.53±0.03ab | 0.54±0.06ab | 0.41±0.01c | 0.42±0.00c | 0.46±0.03bc |
| C18:3n-3 | 0.79±0.00a | 0.84±0.14a | 0.84±0.03a | 0.86±0.01a | 0.87±0.03a | 0.89±0.04a |
| C18:2n-6 | 17.68±0.32c | 20.19±0.71b | 22.10±0.75a | 23.02±0.26a | 22.98±0.17a | 23.23±0.47a |
| C20:4n-6 | 0.35±0.04a | 0.36±0.02a | 0.39±0.01a | 0.34±0.03a | 0.35±0.01a | 0.36±0.01a |
| C22:2n-6 | 0.37±0.06a | 0.26±0.09b | 0.20±0.07bc | 0.10±0.01c | 0.09±0.00c | 0.12±0.03c |
Note：Data representation is average value ± standard variance；Every row letter is different to indicate significant difference (p<0.05)
Table 2-1 is the composition that human milk triglycerides measures sn-3 fatty acid under different hydrolysis degrees, when hydrolysis is gone on a tour
When reaching 4% or more of original triglycerides from the quality of fatty acid, measures the horizontal of various fatty acid and conspicuousness no longer occurs
Variation, therefore select 4% scale as detection sn-3 fatty acid of human milk rouge.
As shown in table 2-1, there are 4 kinds of content of fatty acid to be greater than 10% in the position the sn-3 fatty acid of human milk triglycerides, successively
For C18:1n-9,C18:2n-6,C16:0 and C18:0.
Embodiment 5：The sn-3 fatty acid of lard triglycerides forms
(1) three ester of purification of glycerol
Weigh 80mg lard, with capillary on silica gel plate point sample, with n-hexane/ether/acetic acid (70:30:2, v/v/v)
For solvent, triglycerides band is scraped, with extracted by ether and is transferred in centrifuge tube of having weighed, nitrogen is blown to constant weight；
(2) rabbit stomach lipase hydrolysis triglycerides
The gum arabic solution that 0.25mL mass concentration is 10%, ultrasonic emulsification are added in three ester of purification of glycerol
30min is separately added into 4mL enzyme stabilizers (3.5mmol/L calcium chloride, 30mmol/L bovine serum albumin(BSA) and 150mmol/L chlorination
Sodium, pH4.0) and 100U rabbit stomach lipase, 37 DEG C of 0~3h of water-baths concussion.After reaction with circulating water rinse cool down, immediately plus
Enter extracted by ether, nitrogen obtains steatolysis product after blowing.
(3) pass through TLC separation free fatty acid
With capillary by the grease point after hydrolysis on silica gel plate, with n-hexane/ether/acetic acid (70:30:2, v/v/v)
For solvent, free fatty acid band is scraped, extracted by ether is added and is transferred in the screw socket glass tube weighed, nitrogen obtains after blowing
The position the sn-3 fatty acid that must be released weighs and calculates the quality for obtaining free fatty acid.
(4) free fatty acid methyl esters
5% sulfuric acid-methanol solution of 1mL is added in screw socket glass tube, 60 DEG C of water-bath 60min are cooled to after taking-up
0.5mL n-hexane mechanical shaking extraction is added in room temperature, and distilled water is added to bottleneck, supernatant is taken to cross film storage.
(5) high temperature gas chromatography detection free-fat acid derivative is constituted
The actual conditions of high temperature gas chromatography are：Gas chromatograph is furnished with flame ionization detector and Trace TR-
FAME capillary column (60m × 0.25mm × 0.25 μm)；Split ratio 20:1；Nitrogen is carrier gas, flow velocity 1.2mL/min；Detector
It is 250 DEG C with injector temperature；2 μ L of sampling volume；Temperature program is：60 DEG C of initial column temperature, 3min is kept, with 5 DEG C/min
Speed rise to 175 DEG C, maintain 15min, rise to 220 DEG C with the speed of 2 DEG C/min, keep 10min.
The sn-3 position fatty acid that table 3-1 lard is measured at different hydrolysis degrees (wt%) forms (wt%)
| Fatty acid | 2% | 3% | 4% | 5% |
| C14:0 | 1.74±0.11ab | 1.83±0.03a | 1.98±0.04b | 1.95±0.04ab |
| C16:0 | 27.15±0.45a | 26.34±0.48ab | 26.61±0.32ab | 25.39±0.67b |
| C17:0 | 0.45±0.02a | 0.48±0.16a | 0.37±0.01a | 0.38±0.02a |
| C18:0 | 20.77±2.41a | 18.71±0.97a | 18.18±2.22a | 18.06±1.17a |
| C20:0 | 0.33±0.06a | 0.34±0.11a | 0.23±0.03a | 0.23±0.01a |
| C16:1n-9 | 2.00±0.11a | 2.14±0.13ab | 2.36±0.07b | 2.32±0.01b |
| C18:1n-9 | 32.81±2.00a | 35.23±0.91a | 35.27±1.67a | 36.54±1.06a |
| C20:1n-9 | 0.76±0.03a | 0.70±0.03ab | 0.64±0.05b | 0.65±0.01b |
| C18:2n-6 | 12.77±0.75a | 13.10±0.22a | 13.26±0.81a | 13.38±0.50a |
| C18:3n-3 | 0.43±0.03a | 0.40±0.05a | 0.42±0.09a | 0.42±0.07a |
| C20:2n-6 | 0.40±0.09a | 0.38±0.05a | 0.35±0.04a | 0.38±0.06a |
| C20:3n-6 | 0.14±0.02a | 0.13±0.02a | 0.12±0.01a | 0.11±0.01a |
| C20:4n-6 | 0.26±0.04a | 0.22±0.02a | 0.22±0.01a | 0.20±0.01a |
Note：Data representation is average value ± standard variance；Every row letter is different to indicate significant difference (p<0.05)
Table 3-1 is the composition that lard measures sn-3 fatty acid under different hydrolysis degrees, when hydrolysis free fatty acid
Quality when reaching 4% or more of original triglycerides, measure the horizontal of various fatty acid and conspicuousness variation no longer occur, therefore
Select 4% scale as lard sn-3 fatty acid of detection.
As shown in table 3-1, there are 4 kinds of content of fatty acid to be greater than 10% in sn-3 fatty acid of lard, be followed successively by C18:1n-9,
C16:0,C18:0 and C18:2n-6.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method of measurement triglycerides sn-3 fatty acid composition, which is characterized in that the method is that will use glycerol three
Triglycerides after sn-3 enzyme-specific hydrolysising purifications of ester or the determinand containing triglycerides, obtain steatolysis product；By steatolysis
It is extracted after product is cooling, and with TLC separation, obtains free fatty acid；Free fatty acid is subjected to esterification, obtains first
It is esterified free fatty acid；With gas chromatographic analysis esterification free fatty acid to get sn-3 fatty acid compositions of triglycerides；Institute
Stating sn-3 enzyme-specifics of triglycerides includes rabbit stomach lipase, dog gastric lipase, fusarium cutinase and recombinant human gastric lipase.
2. a kind of method of measurement as described in claim 1 triglycerides sn-3 fatty acid composition, which is characterized in that described
The purification process of triglycerides or the determinand containing triglycerides is with capillary by triglycerides or containing the to be measured of triglycerides
Solvent expansion is added in object point sample on silica gel plate, scrapes triglycerides band, then extract triglycerides band with organic solvent
And be centrifuged, organic solvent is removed, triglycerides after purification or the determinand containing triglycerides are obtained.
3. a kind of method of measurement as claimed in claim 2 triglycerides sn-3 fatty acid composition, which is characterized in that described
The ingredient of solvent includes n-hexane, ether and acetic acid.
4. the method for triglycerides sn-3 fatty acid of a kind of measurement composition as claimed in claim 2 or claim 3, which is characterized in that
In the solvent, the volume ratio of n-hexane, ether and acetic acid is (50~70):(30~50):(1~2).
5. the method that a kind of sn-3 fatty acid of measurement triglycerides as described in claim 1-4 is any forms, feature exist
In the triglycerides after described sn-3 enzyme-specific hydrolysising purifications with triglycerides or the determinand containing triglycerides are first to exist
It is emulsified after gum arabic solution is added in triglycerides after purification or the determinand containing triglycerides, after obtaining emulsification
Triglycerides or determinand containing triglycerides；Distinguish in triglycerides after emulsification or the determinand containing triglycerides again
Hydrolysis is carried out after sn-3 enzyme-specifics of enzyme stabilizers and triglycerides are added, obtains hydrolyzed fat acid；
When the triglycerides sn-3 enzyme-specifics are dog gastric lipase, the ingredient of enzyme stabilizers is NaCl solution；
Or when the triglycerides sn-3 enzyme-specifics are rabbit stomach lipase, human gastric lipase or fusarium cutinase, enzyme is stablized
The ingredient of agent includes calcium chloride, bovine serum albumin(BSA) and sodium chloride.
6. a kind of method of measurement a method as claimed in any one of claims 1 to 5 triglycerides sn-3 fatty acid composition, feature exist
In, when the triglycerides sn-3 enzyme-specifics are dog gastric lipase, the ingredients of enzyme stabilizers be mass percentage concentration 0.5~
1.5% NaCl solution；
Or when the triglycerides sn-3 enzyme-specifics are rabbit stomach lipase, human gastric lipase or fusarium cutinase, enzyme is stablized
The ingredient of agent includes 1~5mmol/L calcium chloride, 1~50mmol/L bovine serum albumin(BSA) and 1~300mmol/L sodium chloride.
7. the method that a kind of sn-3 fatty acid of measurement triglycerides as described in claim 1-6 is any forms, feature exist
In the volume ratio of the gum arabic solution and triglycerides after purification or the determinand containing triglycerides should be maintained at 1:
1~100.
8. a kind of method of measurement as claimed in claim 1 triglycerides sn-3 fatty acid composition, feature exist
In the volume ratio of the triglycerides after the enzyme stabilizers and emulsification or the determinand containing triglycerides should be maintained at 1:100~
1000。
9. a kind of method of measurement a method as claimed in any one of claims 1-8 triglycerides sn-3 fatty acid composition, feature exist
In additive amount in the triglycerides sn-3 triglycerides of enzyme-specific after emulsification or the determinand containing triglycerides
1~100U/ μ g should be maintained at.
10. a kind of method of any sn-3 fatty acid of the measurement triglycerides composition of claim 1-9 is in measurement glycerol
The application of three fatty acid of ester sn-3 composition aspect.
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| CN106153736A (en) * | 2015-03-12 | 2016-11-23 | 上海谱尼测试技术有限公司 | A kind of measure the detection method of OPO structure ester Sn-2 position fatty acid in baby formula milk powder |
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