CN104034837A - Method for determining fatty-acid components of tissues of fish body - Google Patents

Abstract

The invention discloses a method for determining the fatty acid components of fish body tissue. The method comprises the steps of taking the weight percentage of fish body tissue as abdominal fat 0.2-0.5, liver 0.8-1.0, muscle 1-2 and gill 1.5-1.8, and then adding Grinding with chloroform-methanol mixture; then extracting the fat solution by reflux filtration; then drying the solution with nitrogen to extract crystallized fat; then dissolving the crystallized fat in methanolic potassium hydroxide solution, filling with nitrogen to prevent oxidation and cooling the container wall to reduce volatilization ; Then the fat is esterified into fatty acid, and then the fatty acid is dissolved with n-heptane; then the supernatant is taken for centrifugation and then sent to a gas chromatograph to obtain a chromatogram, which can be normalized by the area of the chromatogram Calculate relative amounts of fatty acid components. Thereby, the understanding of the fat structure of fish can be realized, and the protection and feeding of fish can be further carried out.

Description

A method for determining fatty acid components of fish body tissue

technical field

The invention relates to the field of fish tissue fatty acids, in particular to a method for determining fatty acid components of fish tissue.

Background technique

At present, wild fish are facing more and more threats, many of which are facing extinction, or the farming of other common fish encounters difficulties in feeding and matching nutrition. Although the fish body is rich in fatty acids, some fatty acids can be biosynthesized by the fish itself, but some cannot be synthesized or the amount of synthesis is very small, far from meeting the needs of fish growth and development stages, and must be supplemented by external sources. Therefore, it is of great significance to study and measure the fatty acid composition, physiological function and requirement of fish to improve the yield and quality of farmed fish.

In order to protect the race and long-term survival of wild fish and fish farming, it is necessary not only to constantly pay attention to the living environment of fish, but also to continuously study the structure of fish and its corresponding living needs, so it is necessary Accurate extraction and determination of the composition of fatty acids in its tissues can further protect wild fish and artificial breeding, so as to prevent the species of wild fish from being extinct and solve the difficulties of artificial breeding. But the current method of extracting fish tissue fat adopts the method of nitrogen blowing to dry the chloroform-methanol mixture, and does not do other treatments to the chloroform-methanol mixture, which causes the nitrogen blowing time to be too long, and the chloroform-methanol mixture is difficult to dry due to the refrigeration effect of nitrogen. Volatility affects fatty acid determination results, and when extracting fish tissue fatty acids, there is no uniform standard for tissue sampling volume, which affects the determination of fatty acid components in fish tissues, wastes resources, and has low accuracy.

Contents of the invention

The technical problem to be solved in the present invention is to reasonably extract the proportion components of each tissue of the fish body, quickly and comprehensively grind and extract the fatty acid, and then use the gas chromatograph to obtain the chromatogram and the area normalization method to calculate the relative content of the fatty acid components. Finally, the understanding of the fat structure of fish can be realized, so that the protection and feeding of fish can be further carried out.

In order to solve the above-mentioned technical problems, the invention provides a kind of method for measuring fish body tissue fatty acid composition, and it comprises the steps:

(1) Take a mature fish in the first class of fish and cut it open, take out the weight percentage of the fish body tissue as abdominal fat 0.2-0.5, liver 0.8-1.0, muscle 1-2 and gill 1.5-1.8, then add 4-6ml chloroform methanol Mixed solution, mixed and ground for 5-10 minutes;

(2) Transfer the grinding solution to a heatable container, add 15ml of chloroform-methanol mixture to the heatable container to cover the bottom of the container, connect the container to the condenser and place it in a constant temperature water bath, and reflux for 2-2.5 h, keep the temperature of the water bath at 50°C to 55°C;

(3) Filter the liquid into a container to remove the residue;

(4) Take out 10ml of filtrate in container b, dry the filtrate with nitrogen, and extract the crystalline fat;

(5) Add 2ml of 0.5mol/L potassium hydroxide methanol solution to the crystallized fat, fill it with nitrogen, shake the container b for 2-4 minutes until the crystallized fat is completely dissolved, and cool the outer wall of the container with tap water at the same time;

(6) Add 2ml of 10% boron trifluoride ether or boron trifluoride methanol solution to container b, mix well, fill with nitrogen, place container b in a water bath at 50°C and let it stand for 3 minutes before cooling to room temperature;

(7) Add 2ml of n-heptane and oscillate evenly, then let it stand still, and rinse the supernatant with 15ml-20ml saturated saline;

(8) Take the supernatant and place it in a centrifuge tube, add anhydrous sodium sulfate to account for 0.2-0.5% of the supernatant mass;

(9) Get the supernatant and send it into a gas chromatograph to obtain a chromatogram;

(10) Calculate the relative content of the fatty acid components by using the area normalization method of the chromatogram.

This method extracts the various tissues of the fish body according to a certain proportion, and adds an appropriate amount of chloroform-methanol mixture to grind the various tissues of the fish body, so that the grinding is more complete, and the fat of each tissue is exuded more fully; then add chloroform-methanol mixture again The liquid is refluxed and heat-treated so that the fat is fully dissolved in the chloroform-methanol mixture to further extract a relatively pure fat solution; then in order to prevent the fat from being oxidized, the fat solution is blown dry with nitrogen to obtain crystallized fat, and then the prevention can be achieved through step (5). The fat is oxidized, and the crystallized fat can be quickly dissolved in the potassium hydroxide methanol solution to reduce the damage of the fat, and make the determination of the fatty acid component more accurate; and then through the step (6), the fat can be quickly esterified into a fatty acid, and it can be Greatly reduce the oxidation and damage of fat and fatty acid, improve the purity, and ensure the accuracy of the determination of fatty acid components; then through step (7) the fatty acid is dissolved in n-heptane and purified by removing bubbles with salt water; then through step (8) can Prevent external moisture from entering the supernatant, and then enter steps (9) and (10) to send the purified fatty acid solution into a gas chromatograph to obtain a chromatogram, and then use the area normalization method to calculate the fatty acid component from the chromatogram. The fatty acids that can be measured include myristic acid, myristic acid, palmitic acid, palmitoleic acid, heptadecanoic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid , eicosadienoic acid, eicosatrienoic acid, arachidonic acid, erucic acid, eicosapentaenoic acid and docosahexaenoic acid, through this determination method, each type can be determined The ratio of different fatty acid components of fish to the total fatty acids can also be calculated correspondingly to the specific content value of different fatty acid components of each type of fish, so as to realize the understanding of the fat structure of fish, so that further research on the protection and protection of fish can be carried out. Raising and formulating the feed suitable for each fish.

Preferably, the chloroform-methanol mixture is a chloroform-methanol ratio of 2:1.

Preferably, the fish is red tilapia, yellow catfish, freshwater shark, and spiny mahi.

Preferably, in the step (1), the percentage by weight of each tissue of each mature fish body is abdominal fat 0.5, liver 1.0, muscle 1.5 and gill 1.8, and the chloroform-methanol mixture added is 5ml.

Preferably, the heatable container in the step (2) is a round bottom flask.

Preferably, the reflux of the step (2) is a process of extracting fat components with a volatile organic solvent of a chloroform-methanol mixture, which is to mix the grinding liquid with an organic solvent, and heat the mixed liquid while cooling the volatile organic solvent. The solvent makes it return to a liquid state and mixes it into the grinding liquid to continue dissolving the fat, and repeatedly heats the liquid organic solvent and cools the gaseous organic solvent until the fat component is dissolved in the organic solvent.

Preferably, the step (4) nitrogen blowing process also includes placing the container b in a water bath at 30°C with a water depth of 5 cm, which can speed up the volatilization rate of the chloroform-methanol mixture and will not cause fat in the sample Volatilizes with the extract to ensure the activity and purity of the fat.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments, so that those skilled in the art can implement it with reference to the description.

Example 1

This technical scheme provides a kind of method of measuring fatty acid composition of fish body tissue, and it comprises the steps:

(1) Get a mature red tilapia by laparotomy, take out the weight percentage of the fish body tissue as abdominal fat 0.5, liver 1.0, muscle 1.5 and gill 1.8, then add 5ml of chloroform-methanol mixture, mix and grind for 7 minutes;

(2) Grinding liquid is transferred in the round-bottomed flask, in this round-bottomed flask, add 15ml chloroform-methanol mixed liquor again to cover the bottom of round-bottomed flask, round-bottomed flask is connected with condensing tube and placed in constant temperature water bath, Reflux for 2 hours and keep the temperature of the water bath at 55°C. The reflux is a process of extracting fat components with volatile organic solvents such as chloroform-methanol mixtures. The organic solvent is mixed in the grinding liquid to continue dissolving the fat, and the liquid organic solvent is heated and the gaseous organic solvent is cooled repeatedly until the fat component is dissolved in the organic solvent. The organic solvent used can be a mixture of chloroform and methanol.

(3) Filter the liquid into a container to remove the residue;

(4) Take out 10ml of filtrate in container b, and place container b in a water bath at 30°C with a water depth of 5 cm. Dry the filtrate with nitrogen to extract crystalline fat, which can speed up the volatilization rate of the chloroform-methanol mixture without It will cause the fat in the sample to volatilize with the extract, ensuring the activity and purity of the fat;

(5) Add 2ml of 0.5mol/L potassium hydroxide methanol solution to the crystallized fat, fill it with nitrogen, shake the container b up and down, left and right for 2 minutes until the crystallized fat is completely dissolved, and at the same time cool the outer wall of the container with tap water;

(6) Add 2ml of 10% boron trifluoride ether or boron trifluoride methanol solution to container b, mix well, fill with nitrogen, place container b in a water bath at 50°C and let it stand for 3 minutes before cooling to room temperature;

(7) Add 2ml of n-heptane and oscillate evenly, then let it stand still, and rinse the supernatant with 15ml of saturated saline;

(8) Take the supernatant and place it in a centrifuge tube, add anhydrous sodium sulfate to account for 0.3% of the supernatant mass;

(9) Get the supernatant and send it into a gas chromatograph to obtain a chromatogram;

(10) Calculate the relative content of the fatty acid components by using the area normalization method of the chromatogram.

Wherein the chloroform-methanol mixture used is chloroform and methanol in a ratio of 2:1.

Example 2

This technical scheme provides a kind of method of measuring fatty acid composition of fish body tissue, and it comprises the steps:

(1) Take a mature yellow catfish and laparotomy, take out the weight percentage of the fish body tissue as abdominal fat 0.3, liver 0.9, muscle 1.5 and gill 1.5, then add 4ml of chloroform-methanol mixture, mix and grind for 6 minutes;

(2) Transfer the grinding liquid to a heatable container, add 15ml of chloroform-methanol mixture to the heatable container to cover the bottom of the container, connect the container to the condenser and place it in a constant temperature water bath, and reflux for 2.5 hours. Keep the temperature of the water bath at 53°C;

(3) Filter the liquid into a container to remove the residue;

(4) Take out 10ml of filtrate in container b, and place container b in a water bath at 30°C with a water depth of 5 cm. Dry the filtrate with nitrogen to extract crystalline fat, which can speed up the volatilization rate of the chloroform-methanol mixture without It will cause the fat in the sample to volatilize with the extract, ensuring the activity and purity of the fat;

(5) Add 2ml of 0.5mol/L potassium hydroxide methanol solution to the crystallized fat, fill it with nitrogen, shake the container b for 3 minutes until the crystallized fat is completely dissolved, and cool the outer wall of the container with tap water at the same time;

(6) Add 2ml of 10% boron trifluoride ether or boron trifluoride methanol solution to container b, mix well, fill with nitrogen, place container b in a water bath at 50°C and let it stand for 3 minutes before cooling to room temperature;

(7) Add 2ml of n-heptane and oscillate evenly, then let it stand still, and rinse the supernatant with 17ml of saturated saline;

(8) Take the supernatant and place it in a centrifuge tube, add anhydrous sodium sulfate to account for 0.2% of the supernatant mass;

(9) Get the supernatant and send it into a gas chromatograph to obtain a chromatogram;

(10) Calculate the relative content of the fatty acid components by using the area normalization method of the chromatogram.

Wherein the chloroform-methanol mixture used is chloroform and methanol in a ratio of 2:1.

Example 3

This technical scheme provides a kind of method of measuring fatty acid composition of fish body tissue, and it comprises the steps:

(1) Get a mature freshwater shark laparotomy, take out the weight percentage of the fish body tissue as abdominal fat 0.5, liver 1.0, muscle 2 and gill 1.8, then add 6ml of chloroform-methanol mixture, mix and grind for 10 minutes;

(2) Transfer the grinding liquid to a heatable container, add 15ml of chloroform-methanol mixture to the heatable container to cover the bottom of the container, connect the container to the condenser and place it in a constant temperature water bath, and reflux for 2.5 hours. Keep the temperature of the water bath at 55°C;

(3) Filter the liquid into a container to remove the residue;

(4) Take out 10ml of filtrate in container b, dry the filtrate with nitrogen, and extract the crystalline fat;

(5) Add 2ml of 0.5mol/L potassium hydroxide methanol solution to the crystallized fat, fill it with nitrogen, shake the container b for 4 minutes until the crystallized fat is completely dissolved, and cool the outer wall of the container with tap water at the same time;

(6) Add 2ml of 10% boron trifluoride ether or boron trifluoride methanol solution to container b, mix well, fill with nitrogen, place container b in a water bath at 50°C and let it stand for 3 minutes before cooling to room temperature;

(7) Add 2ml of n-heptane and oscillate evenly, then let it stand still, and rinse the supernatant with 20ml of saturated saline;

(8) Get the supernatant and place it in a centrifuge tube, add anhydrous sodium sulfate to account for 0.5% of the supernatant mass;

(9) Get the supernatant and send it into a gas chromatograph to obtain a chromatogram;

(10) Calculate the relative content of the fatty acid components by using the area normalization method of the chromatogram.

Example 4

This technical scheme provides a kind of method of measuring fatty acid composition of fish body tissue, and it comprises the steps:

(1) Get a mature big spiny loach for laparotomy, take out the weight percentage of the fish body tissue as abdominal fat 0.2, liver 0.8, muscle 1 and gill 1.5, then add 4ml of chloroform-methanol mixture, mix and grind for 5 minutes;

(2) Transfer the grinding liquid to a heatable container, add 15ml of chloroform-methanol mixture to the heatable container to cover the bottom of the container, connect the container to the condenser and place it in a constant temperature water bath, reflux for 2h, and keep The temperature of the water bath is 50°C;

(3) Filter the liquid into a container to remove the residue;

(4) Take out 10ml of filtrate in container b, dry the filtrate with nitrogen, and extract the crystalline fat;

(5) Add 2ml of 0.5mol/L potassium hydroxide methanol solution to the crystallized fat, fill it with nitrogen, shake the container b for 2-4 minutes until the crystallized fat is completely dissolved, and cool the outer wall of the container with tap water at the same time;

(6) Add 2ml of 10% boron trifluoride ether or boron trifluoride methanol solution to container b, mix well, fill with nitrogen, place container b in a water bath at 50°C and let it stand for 3 minutes before cooling to room temperature;

(7) Add 2ml of n-heptane and oscillate evenly, then let it stand still, and rinse the supernatant with 15ml of saturated saline;

(8) Take the supernatant and place it in a centrifuge tube, add anhydrous sodium sulfate to account for 0.2% of the supernatant mass;

(9) Get the supernatant and send it into a gas chromatograph to obtain a chromatogram;

(10) Calculate the relative content of the fatty acid components by using the area normalization method of the chromatogram.

Example 5

This technical scheme provides a kind of method of measuring fatty acid composition of fish body tissue, and it comprises the steps:

(1) Take a mature large spiny loach for laparotomy, take out the weight percentage of fish body tissue as abdominal fat 0.5, liver 1.0, muscle 1.5 and gill 1.8, then add 6ml of chloroform-methanol mixture, mix and grind for 5-10 minutes;

(2) Transfer the grinding liquid to a heatable container, add 15ml of chloroform-methanol mixture to the heatable container to cover the bottom of the container, connect the container to the condenser and place it in a constant temperature water bath, and reflux for 2.3h. Keep the temperature of the water bath at 55°C;

(3) Filter the liquid into a container to remove the residue;

(4) Take out 10ml of filtrate in container b, and place container b in a water bath at 30°C with a water depth of 5 cm. Dry the filtrate with nitrogen to extract crystalline fat, which can speed up the volatilization rate of the chloroform-methanol mixture without It will cause the fat in the sample to volatilize with the extract, ensuring the activity and purity of the fat;

(5) Add 2ml of 0.5mol/L potassium hydroxide methanol solution to the crystallized fat, fill it with nitrogen, shake the container b for 2-4 minutes until the crystallized fat is completely dissolved, and cool the outer wall of the container with tap water at the same time;

(6) Add 2ml of 10% boron trifluoride ether or boron trifluoride methanol solution to container b, mix well, fill with nitrogen, place container b in a water bath at 50°C and let it stand for 3 minutes before cooling to room temperature;

(7) Add 2ml of n-heptane and oscillate evenly, then let it stand still, and rinse the supernatant with 15ml of saturated saline;

(8) Take the supernatant and place it in a centrifuge tube, add anhydrous sodium sulfate to account for 0.4% of the supernatant mass;

(9) Get the supernatant and send it into a gas chromatograph to obtain a chromatogram;

(10) Calculate the relative content of the fatty acid components by using the area normalization method of the chromatogram.

This method extracts the various tissues of the fish body according to a certain proportion, and adds an appropriate amount of chloroform-methanol mixture to grind the various tissues of the fish body, so that the grinding is more complete, and the fat of each tissue is exuded more fully; then add chloroform-methanol mixture again The liquid is refluxed and heat-treated so that the fat is fully dissolved in the chloroform-methanol mixture to further extract a relatively pure fat solution; then in order to prevent the fat from being oxidized, the fat solution is blown dry with nitrogen to obtain crystallized fat, and then the prevention can be achieved through step (5). The fat is oxidized, and the crystallized fat can be quickly dissolved in the potassium hydroxide methanol solution to reduce the damage of the fat, and make the determination of the fatty acid component more accurate; and then through the step (6), the fat can be quickly esterified into a fatty acid, and it can be Greatly reduce the oxidation and damage of fat and fatty acid, improve the purity, and ensure the accuracy of the determination of fatty acid components; then through step (7) the fatty acid is dissolved in n-heptane and purified by removing bubbles with salt water; then through step (8) can Prevent extraneous moisture from entering the supernatant, and then enter steps (9) and (10) to send the purified fatty acid solution into a gas chromatograph to obtain a chromatogram, and then use the area normalization method to calculate the fatty acid component from the chromatogram. Among them, the fatty acids that can be determined in fish include myristic acid, myristic acid, palmitic acid, palmitoleic acid, heptadecanoic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, Arachidic acid, eicosadienoic acid, eicosatrienoic acid, arachidonic acid, erucic acid, eicosapentaenoic acid and docosahexaenoic acid, through the method of this determination, each The ratio of the different fatty acid components of a type of fish to the total fatty acids can also be calculated correspondingly to the specific content value of the different fatty acid components of each type of fish, so as to realize the understanding of the fat structure of fish, so that further research on the fat structure of fish can be carried out. Protection and feeding, preparation of feed suitable for each fish.

The principle of the gas chromatograph used in the present invention is to utilize the boiling point, polarity or adsorption performance of each component in the sample to be different, causing the distribution coefficients of each component to be different between the gas phase and the stationary liquid phase in the chromatographic column. When the sample is carried into the chromatographic column by the carrier gas, the components are repeatedly distributed between the two items. Due to the different adsorption or dissolution capabilities of the fixed relative components, each component in the chromatographic column The running speed is different. After a certain column length, they are separated from each other, and the signal generated by smoothly leaving the chromatographic column and entering the detector is amplified, and the chromatogram of each component is drawn on the recorder. The area normalization method is based on the peaks of each fatty acid component in the sample on the chromatogram, and there are about 12-17 fatty acids that can be detected in fish, and each of these fatty acids will form a chromatogram. Peak, the area of each peak adds up and is set as 100, how much the peak that each fatty acid forms accounts for in 100 is exactly the ratio that this fatty acid occupies in all fatty acids, both is the fish body fatty acid component that the present invention will measure relative content.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and implementation, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Therefore, the invention is not limited to the specific details without departing from the general concept defined by the claims and their equivalents.

Claims (7)

1. a method of measuring Fish tissue fatty acid component, is characterized in that, comprises the steps:

(1) a ripe fish of getting in a class fish is cut open the belly, and the percentage by weight that takes out Fish tissue is abdomen fat 0.2-0.5, liver 0.8-1.0, muscle 1-2 and gill 1.5-1.8, then adds 4-6ml chloroform methanol mixed liquor, mixed grinding 5-10 minute;

(2) lapping liquid is gone in heatable container, in this heatable container, add again 15ml chloroform methanol mixed liquor with covering container bottom, container and condenser pipe are connected and are placed in thermostat water bath, backflow 2-2.5h, keeping water-bath temperature is 50 DEG C～55 DEG C;

(3) by liquid filtering in a container, remove residue;

(4) take out 10ml filtrate in b container, dry up filtrate with nitrogen, extract crystallised fat;

(5) add 2ml0.5mol/L potassium hydroxide methanol solution in crystallised fat, be filled with nitrogen, jolting b container 2-4 minute to crystallised fat dissolves completely, uses tap water cooled containers outer wall simultaneously;

(6) add 2ml10% boron trifluoride diethyl etherate or boron trifluoride methanol solution in b container, mix, be filled with after b container being placed in after nitrogen to 50 DEG C of water-baths and leaving standstill 3 minutes and be cooled to normal temperature;

(7) leave standstill after adding the normal heptane of 2ml vibration evenly, with 15ml-20ml saturated common salt water wash supernatant liquor;

(8) get supernatant and be placed in centrifuge tube, add anhydrous sodium sulfate to account for the 0.2-0.5% of described supernatant quality;

(9) get supernatant liquor and send into gas chromatograph acquisition chromatogram;

(10) relative content with area normalization method calculating fatty acid component by chromatogram.

2. the method for measuring according to claim 1 Fish tissue fatty acid component, is characterized in that, the ratio that described chloroform methanol mixed liquor is chloroform and methyl alcohol is 2: 1.

3. the method for measuring according to claim 1 Fish tissue fatty acid component, is characterized in that, described fish is red tilapia, Pelteobagrus fulvidraco, Pangasius sutchi, large mastacembelus aculeatus.

4. measure according to claim 1 the method for Fish tissue fatty acid component, it is characterized in that, the percentage by weight that described step (1) is got the each tissue of every ripe fish body is abdomen fat 0.5, liver 1.0, muscle 1.5 and the gill 1.8, and the chloroform methanol mixed liquor adding is 15ml.

5. the method for measuring according to claim 1 Fish tissue fatty acid component, is characterized in that, the heatable container of described step (2) is round-bottomed flask.

6. measure according to claim 1 the method for Fish tissue fatty acid component, it is characterized in that, the backflow of described step (2) is the process of extracting fat constituent with the volatile organic solvent of chloroform methanol mixed liquor class, it is that lapping liquid is mixed with organic solvent, and heat mixed liquor, the organic solvent of simultaneously cooling volatilization makes it recover liquid state to sneak in lapping liquid and continue dissolved fat, repeatedly heat liquid organic solvent and cooling gaseous state organic solvent until fat constituent be dissolved in organic solvent.

7. the method for measuring according to claim 1 Fish tissue fatty acid component, is characterized in that, described step (4) nitrogen is blown in journey and also comprised b container is put to 30 DEG C of water-baths, and the placement depth of water is 5cm.