CN112255328A - Optimized method for measuring fatty acids in different tissues of fish body - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 28
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- 229930195729 fatty acid Natural products 0.000 title claims abstract description 19
- 150000004665 fatty acids Chemical class 0.000 title claims abstract description 19
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- 239000000243 solution Substances 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 5
- 239000012159 carrier gas Substances 0.000 claims description 4
- 238000004587 chromatography analysis Methods 0.000 claims description 4
- 230000000630 rising effect Effects 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 210000000577 adipose tissue Anatomy 0.000 claims description 3
- 210000005013 brain tissue Anatomy 0.000 claims description 3
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4055—Concentrating samples by solubility techniques
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2866—Grinding or homogeneising
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4055—Concentrating samples by solubility techniques
- G01N2001/4061—Solvent extraction
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Abstract
The invention discloses an optimized method for measuring fatty acid of different tissues of a fish body, which comprises the steps of optimizing a processing method of tissue samples of brain, intestinal tract, muscle and the like of the fish and meteorological chromatographic conditions, obtaining the optimal flow rate of 1.4mL/min under the condition of a split flow ratio of 10:1, and finally obtaining the measuring method which is accurate, time-saving and less in sample quantity demand. The method can provide a theoretical basis for determination of fatty acid components of fishes and small aquatic products, and also provides a technical support for research of lipid metabolism mechanisms of aquatic products.
Description
Technical Field
The invention belongs to the technical field of fish tissue fatty acid determination methods, and particularly relates to an optimized method for determining fatty acids of different tissues of a fish body.
Background
At present, the development of fishery and aquaculture industry plays a vital role in employment, food and nutrition of people all over the world. The fish is a favorite food for human, and the fish body contains fatty acid components such as EPA and DHA beneficial to human health, and the content of the essential fatty acid components determines the nutritional value of different fishes. How to determine the components and types of fatty acids in fish muscle and how to regulate the content of polyunsaturated fatty acid components in fish muscle is a hot spot of current research. Therefore, it is important to determine the fatty acid content and composition of different tissues of fish. However, the research on the determination of fish fatty acid is relatively few, most of the determination methods are performed according to the national standard GB/T9695.2-2008 (meat and meat product-fatty acid determination), the methods are relatively single, and certain limitations exist, such as the large amount of samples is needed when extracting trace tissue samples of fish brain, fat and liver, and the like, and the problems of resource waste and low determination accuracy exist. Therefore, this method is often applied to the measurement of a large amount of tissues such as fish muscle, and it is still necessary to improve how to measure fatty acid components in minute amounts of tissues and small fishes. In addition, the time currently required to use a conventional HP-88(AgILENTce112-88A7) gas chromatography column is relatively long.
Disclosure of Invention
The invention solves the technical problem of providing an optimized method for measuring fatty acid of different tissues of fish body, which can effectively save samples and save measuring time.
The invention adopts the following technical scheme for solving the technical problems, and the optimized method for measuring fatty acids in different tissues of the fish body is characterized by comprising the following steps of:
step S1: firstly, dissecting a fish body to be detected, respectively taking muscle tissue, brain tissue, liver tissue, intestinal tissue, gill tissue and adipose tissue of the fish body to be detected, putting the fish body into different centrifuge tubes, placing the centrifuge tubes in liquid nitrogen for 2-3min for quick freezing, and then placing the centrifuge tubes in a refrigerator with the temperature of-80 ℃ for low-temperature storage, or directly carrying out step S2 freeze drying after dissecting;
step S2: placing the sample on a culture dish, drying the sample in a freeze dryer (LaboGene ScanVac CoolSafe) for 16-24 hours, and then putting the freeze-dried sample in a mortar and grinding the sample into powder;
step S3: weighing 0.1g to 0.3g of the ground sample, transferring the weighed sample into a 15mL centrifuge tube, and adding 8mL of a mixed solution of chloroform and methanol with the volume ratio of 2: 1;
step S4: centrifuging with a refrigerated ultracentrifuge (Eppeddorf-5810R) at 5000rpm/min at 4 deg.C for 10min, repeatedly extracting the precipitate twice, combining the supernatants and adding 2mL of 1.6 wt% CaCl2Shaking the solution, uniformly mixing, centrifuging at 4 ℃ at 5000rpm/min for 10min, and finally adding the lower-layer chloroform solution into a new glass centrifuge tube;
step S5: concentrating the sample with nitrogen blower (ALLSHENG KD200), and concentrating the obtained lower layer chloroform solution with pure nitrogen to less than or equal to 0.5mL for 25-30 min;
step S6: adding 1mL of lipidization solution into the concentrated solution of the sample, wherein the lipidization solution is a mixed solution of methanol and 2.5 wt% of concentrated sulfuric acid, then placing the mixture in a water bath kettle at 70 ℃ for 4h in a water bath, naturally cooling the mixture for 2min, and then adding 0.3-0.5mL of n-hexane and 1.5mL of ddH2O, shaking and uniformly mixing, centrifuging at 3000rpm for 2min, extracting the methyl esterification solution on the upper layer, taking 80 mu L of supernatant into a gas phase special sample loading bottle, and carrying out chromatographic analysis;
step S7: the chromatographic column adopts AgILENTce112-88A 7: HP-88(100m 250 μm 0.2 μm) chromatographic conditions the carrier gas was high purity nitrogen, and the flow rate was set at 1.4mL/min at a split ratio of 10: 1; temperature rising procedure: the initial column temperature is 120 ℃, the temperature is maintained for 1min, the temperature is increased to 190 ℃ at the speed of 10 ℃/min, the temperature is maintained for 19min, the temperature is increased to 200 ℃ at the speed of 2 ℃/min, the temperature is maintained for 28min, and the total time is 60 min; the temperature of a heater at a sample inlet is 250 ℃, a front detector FID is constant in flow;
step S8: chromatograms were obtained for each chromatographic condition.
Compared with the prior art, the invention has the following beneficial effects: the invention improves the classical method for determining fatty acid by Folch, mainly optimizes the pretreatment method of fish tissue samples, and simultaneously selects different gas chromatographic columns to optimize the corresponding sample determination method. Through improvement and optimization, compared with the conventional method, the method has the advantages of saving samples, saving time and the like. The method has certain guiding significance for measuring fatty acid of different tissues of the fish and small aquatic products, and provides technical support for researching the lipid metabolism mechanism of the aquatic products.
Drawings
FIGS. 1-7 are chromatograms with flow rates set at 0.8/1.0/1.2/1.4/1.6/1.8/2.0mL/min, respectively, at a split ratio of 10:1, respectively;
FIGS. 8-10 are chromatograms with split ratios set to no split/50: 1/100:1, respectively, at a flow rate of 1.4mL/min, respectively.
Detailed Description
The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.
Examples
An optimized method for measuring fatty acid in different tissues of a fish body comprises the following steps:
step S1: firstly, dissecting a fish body to be detected, respectively putting muscle tissue, brain tissue, liver tissue, intestinal tissue, gill tissue and adipose tissue of the fish body to be detected into different centrifuge tubes, placing the centrifuge tubes in liquid nitrogen for 2-3min for quick freezing, and then placing the centrifuge tubes in a refrigerator at minus 80 ℃ for low-temperature storage;
step S2: tissue disruption, placing the sample on a culture dish, drying the sample in a freeze dryer (LaboGene ScanVac CoolSafe) for 16-24 hours, and then putting the freeze-dried sample into a mortar and grinding the sample into powder;
step S3: weighing 0.1g of the ground sample, transferring the weighed sample into a 15mL centrifuge tube, and adding 8mL of a mixed solution of chloroform and methanol with the volume ratio of 2: 1;
step S4: centrifuging with a refrigerated ultracentrifuge (Eppeddorf-5810R) at 5000rpm/min at 4 deg.C for 10min, repeatedly extracting the precipitate twice, combining the supernatants and adding 2mL of 1.6 wt% CaCl2Shaking the solution, uniformly mixing, centrifuging at 4 ℃ at 5000rpm/min for 10min, and finally adding the lower-layer chloroform solution into a new glass centrifuge tube;
step S5: concentrating the sample with nitrogen blower (ALLSHENG KD200), and concentrating the obtained lower layer chloroform solution with pure nitrogen to less than or equal to 0.5mL for 25-30 min;
step S6: adding 1mL of lipidization solution into the concentrated solution of the sample, wherein the lipidization solution is a mixed solution of methanol and 2.5 wt% of concentrated sulfuric acid, then placing the mixture in a water bath kettle at 70 ℃ for 4h in a water bath, naturally cooling the mixture for 2min, and then adding 0.3-0.5mL of n-hexane and 1.5mL of ddH2O, shaking and uniformly mixing, centrifuging at 3000rpm for 2min, extracting the methyl esterification solution on the upper layer, taking 80 mu L of supernatant into a gas phase special sample loading bottle, and carrying out chromatographic analysis;
step S7: the chromatographic column adopts AgILENTce112-88A 7: HP-88(100m 250 μm 0.2 μm) chromatographic condition carrier gas is high-purity nitrogen, and flow rates are respectively set to be 0.8/1.0/1.2/1.4/1.6/1.8/2.0mL/min under the condition of a split ratio of 10: 1; under the condition that the flow rate is 1.4mL/min, the split ratio is respectively set as no split/10: 1/50:1/100: 1; temperature rising procedure: the initial column temperature is 120 ℃, the temperature is maintained for 1min, the temperature is increased to 190 ℃ at the speed of 10 ℃/min, the temperature is maintained for 19min, the temperature is increased to 200 ℃ at the speed of 2 ℃/min, the temperature is maintained for 28min, and the total time is 60 min; the temperature of a heater at a sample inlet is 250 ℃, a front detector FID is constant in flow;
step S8: chromatograms were obtained for each chromatographic condition.
The method for tissue disruption is a freeze-drying method, the sample loss amount of the method is small, and the method is not only suitable for tissue determination with large sample amount such as muscle, but also can be used for determining tissues of trace samples such as brain, liver and fat and fatty acid components of muscle of small fishes; the nitrogen blowing can quickly evaporate the chloroform in the sample to be treated, and the purity of the sample can be maintained while the effect of quick separation and purification is achieved. The nitrogen blowing method has the advantages of simple operation, short time consumption and the like. The esterification solution is a mixed solution of methanol and 2.5 wt% concentrated sulfuric acid, the acid catalysis esterification is generally applicable to all methyl esterification reactions due to good catalysis effect, the water bath time can be adjusted according to the content of the sample in the methyl esterification process, and if the sample is less, the content of fatty acid methyl ester can be increased by prolonging the water bath time.
Under the condition that other conditions are unchanged and appropriate, the invention respectively adopts the flow rates of 1.0mL/min, 1.2mL/min, 1.4mL/min and 1.6mL/min to grope the separation speed of the sample in the chromatographic column, and the result shows that the speed of 1.4mL/min can not only achieve the best separation effect, but also ensure shorter total determination time. We also blinded the optimal split ratios for our samples with four split ratios, 100:1, 50:1, 10:1 and 3:1, respectively. The highest peak signal value of the sample was found to be best around 250pA at a split ratio of 3:1 by volume. If the concentration of the fatty acid in the sample is too high, the split ratio is increased so as to avoid that the sample is not completely sampled and stays in the gas chromatographic column to influence the separation effect of the sample, and further shorten the service life of the gas chromatographic column. And after the flow rate and the sample split ratio are determined, further adjusting the temperature rise gradient to obtain the temperature rise program when the optimal separation degree is obtained.
According to the invention, by optimizing the gas chromatography conditions, the carrier gas is high-purity nitrogen under the HP-88(100m 250 μm 0.2 μm) chromatography conditions, and the flow rate is 1.4 mL/min; temperature rising procedure: the initial column temperature is 120 ℃, the temperature is maintained for 1min, the temperature is increased to 190 ℃ at the speed of 10 ℃/min, the temperature is maintained for 19min, the temperature is increased to 200 ℃ at the speed of 2 ℃/min, the temperature is maintained for 15min, and the total time is 47 min; the temperature of the heater at the injection port is 250 ℃, the FID of the front detector is constant in flow, and the split ratio is 10: 1. Under such gas chromatography conditions, the chromatogram shows the best peak appearance, and the measurement effect is preferable.
The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.
Claims (1)
1. An optimized method for measuring fatty acids in different tissues of a fish body is characterized by comprising the following steps:
step S1: firstly, dissecting a fish body to be detected, respectively taking muscle tissue, brain tissue, liver tissue, intestinal tissue, gill tissue and adipose tissue of the fish body to be detected, putting the fish body into different centrifuge tubes, placing the centrifuge tubes in liquid nitrogen for 2-3min for quick freezing, and then placing the centrifuge tubes in a refrigerator with the temperature of-80 ℃ for low-temperature storage, or directly carrying out step S2 freeze drying after dissecting;
step S2: placing the sample on a culture dish, drying the sample in a freeze dryer (LaboGene ScanVac CoolSafe) for 16-24 hours, and then putting the freeze-dried sample in a mortar and grinding the sample into powder;
step S3: weighing 0.1g to 0.3g of the ground sample, transferring the weighed sample into a 15mL centrifuge tube, and adding 8mL of a mixed solution of chloroform and methanol with the volume ratio of 2: 1;
step S4: centrifuging with a refrigerated ultracentrifuge (Eppeddorf-5810R) at 5000rpm/min at 4 deg.C for 10min, repeatedly extracting the precipitate twice, combining the supernatants and adding 2mL of 1.6 wt% CaCl2Shaking the solution, uniformly mixing, centrifuging at 4 ℃ at 5000rpm/min for 10min, and finally adding the lower-layer chloroform solution into a new glass centrifuge tube;
step S5: concentrating the sample with nitrogen blower (ALLSHENG KD200), and concentrating the obtained lower layer chloroform solution with pure nitrogen to less than or equal to 0.5mL for 25-30 min;
step S6: adding 1mL of lipidization solution into the concentrated solution of the sample, wherein the lipidization solution is a mixed solution of methanol and 2.5 wt% of concentrated sulfuric acid, then placing the mixture in a water bath kettle at 70 ℃ for 4h in a water bath, naturally cooling the mixture for 2min, and then adding 0.3-0.5mL of n-hexane and 1.5mL of ddH2O, shaking and uniformly mixing, centrifuging at 3000rpm for 2min, extracting the methyl esterification solution on the upper layer, taking 80 mu L of supernatant into a gas phase special sample loading bottle, and carrying out chromatographic analysis;
step S7: the chromatographic column adopts AgILENTce112-88A 7: HP-88(100m 250 μm 0.2 μm) chromatographic conditions the carrier gas was high purity nitrogen, and the flow rate was set at 1.4mL/min at a split ratio of 10: 1; temperature rising procedure: the initial column temperature is 120 ℃, the temperature is maintained for 1min, the temperature is increased to 190 ℃ at the speed of 10 ℃/min, the temperature is maintained for 19min, the temperature is increased to 200 ℃ at the speed of 2 ℃/min, the temperature is maintained for 28min, and the total time is 60 min; the temperature of a heater at a sample inlet is 250 ℃, a front detector FID is constant in flow;
step S8: chromatograms were obtained for each chromatographic condition.
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| CN115308078A (en) * | 2022-08-09 | 2022-11-08 | 上海海洋大学 | Method for measuring fatty acid mass fractions of different tissues of soft fishes |
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