Method for preparing squalene from liver for breeding takifugu obscurus
By extracting squalene from the breeding of dark-textured oriental livers, the problem of insufficient utilization of by-products is solved, the extraction of high-purity squalene and efficient utilization of resources are achieved, and environmental pollution is reduced.
Patent Information
- Application Number
- CN202411395380.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-08
- Publication Date
- 2025-01-10
AI Technical Summary
The prior art is difficult to effectively utilize the liver by-products of farmed dark-textured oriental squalene, resulting in waste of resources and environmental pollution, and lacks a method for extracting high-purity squalene from it.
Using liver oil preparation, saponification, extraction, purification and purification steps, sharks were extracted and purified from the liver of the breeding of dark-formed oriental saplings through n-hexane, sodium hydroxide-ethanol, silica gel column chromatography and high-performance liquid chromatography. ene.
The extraction of high-purity squalene is achieved, with simple process and convenient operation, which improves the processing value of by-products and reduces resource waste and environmental pollution.
Smart Images
Figure CN119285429A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of preparation of functional active substances from aquatic product by-products, and in particular to a method for preparing squalene from the liver of cultured pufferfish. Background Art
[0002] Squalene is an open-chain triterpene compound with six isoprene double bonds. Its chemical name is 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,20-tetracosahexene. It is a highly unsaturated aliphatic hydrocarbon compound. At room temperature, squalene is a colorless or slightly yellow transparent oily liquid with a molecular formula of C 30 H 50 . Squalene is a non-polar molecule, insoluble in water, poorly soluble in glacial acetic acid, methanol and ethanol, and easily soluble in organic solvents such as n-hexane, ether and acetone. Squalene is a biosynthetic precursor of steroids, a component of cell membrane structure, and is commonly found in various biological organisms. Squalene has antioxidant, anti-cancer and anti-tumor effects, immunity-enhancing effects, anti-bacterial and anti-inflammatory effects, anti-cardiovascular and cerebrovascular disease effects, and hypoxia tolerance, which makes it widely used in food, medicine, cosmetics and other fields. At present, shark liver oil is still the richest natural source of squalene, but due to the excessive capture of sharks in recent years, some deep-sea shark species are on the verge of extinction, which will be detrimental to the protection of species diversity in the long run. In addition, its reproduction cycle is long, its growth is slow, and pollutants, pathogens and toxic substances are easily accumulated in the liver. Therefore, it is necessary to explore new natural sources of squalene.
[0003] In recent years, the aquatic product processing industry has been developing continuously, and by-products have been increasing. In order to promote the high-value utilization of aquatic product processing by-products, the extraction of squalene from aquatic product processing by-products has also become a research hotspot. The dark-striped pufferfish (Takifugus pp.) belongs to the family Tetraodontidae. As one of the main pufferfish species currently cultivated, it has formed a linkage development model of the entire industrial chain from seedlings, breeding, processing, sales, catering, etc., and has developed diversified pufferfish processing frozen pre-products and snack foods. With the rapid growth of processing demand, by-products such as fish heads, fish skins, and fish livers are increasing. Due to the lack of deep processing technology, enterprises mainly use waste landfill, low-value processing into fish meal and harmless treatment, causing environmental pollution, waste of resources, and increased operating costs. Therefore, it is urgent to develop deep processing technology and high-value products based on the characteristics of aquaculture pufferfish processing by-products, so as to achieve the green transformation and development of aquaculture pufferfish industry with pollution reduction and efficiency increase. Among them, fish liver is one of the most important by-products of cultured dark-striped pufferfish. There is no report on the method of extracting squalene from the liver of cultured dark-striped pufferfish and further separating and purifying it to obtain pure squalene. Summary of the invention
[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for preparing squalene from the liver of cultured Takifugu obscurus.
[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:
[0006] Provided is a method for preparing squalene from the liver of cultured Takifugu obscurus, comprising the following steps:
[0007] Step 1. Preparation of liver oil: taking out the liver from cultured Takifugu obscurus, adding n-hexane, n-heptane, petroleum ether or ether-water mixture, homogenizing, ultrasonically extracting, centrifuging, filtering, and obtaining cultured Takifugu obscurus liver oil;
[0008] Step 2. Saponification: adding sodium hydroxide-ethanol to the cultured pufferfish liver oil and reflux in a water bath;
[0009] Step 3. Extraction: transfer the saponified liquid to a separatory funnel and add n-hexane, n-heptane or cyclohexane for extraction;
[0010] Step 4. Purification: dissolve the sample collected in step 3 in dichloromethane, purify it by chromatography on a silica gel column, and collect the fraction containing the squalene component;
[0011] Step 5. Purification: dissolving the sample collected in step 4 in methanol and purifying and separating by high performance liquid chromatography;
[0012] Step 6. Freeze drying: Freeze drying technology is used to remove the solvent and solidify the squalene.
[0013] Furthermore, the method of step 1 is specifically as follows:
[0014] The liver is taken out from cultured dark-striped pufferfish, and impurities and blood stains on the surface are cleaned with distilled water. After the water is drained, the liver is minced with a tissue crusher; the minced sample is weighed and placed in a container, and n-hexane, n-heptane, petroleum ether or ether-water mixture is added, and the mixture is homogenized with a homogenizer for 5-10 minutes; n-hexane, n-heptane, petroleum ether or ether-water mixture is added again, and the mixture is homogenized for 1-5 minutes; ultrasonic extraction is performed for 10 minutes; centrifugation is performed at a low temperature and high speed of 8000-12000 r / min for 10-15 minutes; the upper layer solution is transferred to another container, the water in the solvent is removed, the solution is filtered, and the solution is rotary evaporated to dryness to obtain cultured dark-striped pufferfish liver oil.
[0015] Furthermore, in step 2, the pH is controlled between 8 and 9, and refluxed in a water bath for 40 to 60 minutes.
[0016] Furthermore, in step 3, the extraction is performed by shaking for 5 to 10 minutes; the sample is extracted twice more, the three extracts are combined, and the extracts are rotary evaporated in a water bath at 30 to 60° C., and dried in a vacuum dryer at 50 to 65° C. for later use.
[0017] Furthermore, in step 4, chromatography purification is performed through a silica gel 100-400 mesh column at a flow rate of 1-2 mL / min.
[0018] Furthermore, in step 5, a C18 chromatographic column is used, the mobile phase is methanol, the flow rate is 2-15 mL / min, and the detection wavelength is 210 nm; the main peak effluent is collected and rotary evaporated in a water bath at 30-60° C.
[0019] The beneficial effects of the present invention are:
[0020] The method of the invention has the advantages of simple process, convenient operation, high extraction efficiency and high purity of the obtained product, and is a good method for fully utilizing the processing value of the liver byproduct of cultured dark-striped pufferfish to obtain high-purity squalene. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 is a gas chromatogram of squalene prepared in Example;
[0022] Figure 2 is a standard curve diagram of squalene prepared in Example;
[0023] Figure 3 It is the quantitative spectrum of squalene prepared in Example. DETAILED DESCRIPTION
[0024] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.
[0025] Example
[0026] A method for preparing squalene from the liver of cultured Takifugu obscurus, specifically comprising the following steps:
[0027] 1. Preparation of liver oil: Take out the liver from the cultured dark-striped pufferfish, clean the surface impurities, blood stains, etc. with distilled water, drain the water, and mince it with a tissue crusher. Weigh 100g of the minced sample and place it in a container, add 150-250mL of n-hexane (n-heptane, petroleum ether, ether)-water mixture (v:v, 2:1-5:1), and use a homogenizer to average for 5-10min. Add 50-100mL of n-hexane (n-heptane, petroleum ether, ether)-water mixture again and homogenize for 1-5min. Ultrasonic extraction for 10min. Centrifuge at low temperature and high speed at 8000-12000r / min for 10-15min. Transfer the upper layer solution to another container, add an appropriate amount of anhydrous sodium sulfate (anhydrous magnesium sulfate) to remove the water in the solvent, filter, and rotary evaporate to dryness to obtain cultured dark-striped pufferfish liver oil.
[0028] 2. Saponification: Weigh 0.5 g of oily sample (farmed dark-striped pufferfish liver oil), add 15-50 mL of sodium hydroxide (potassium hydroxide)-ethanol (methanol) (v:v, 1:50-1:25), control the pH between 8 and 9, reflux in a water bath for 40-60 min, add 10-20 mL of water after cooling, and shake well.
[0029] 3. Extraction: Transfer the saponified liquid from step 2 to a separatory funnel, add 10-15 mL of n-hexane (n-heptane, cyclohexane) to wash the flask and transfer it to a separatory funnel, shake and extract for 5-10 minutes. Repeat the sample extraction twice, combine the three extracts, evaporate to 0.5-1 mL in a 30-60°C water bath, and dry in a vacuum dryer at 50-65°C for later use.
[0030] 4. Purification: Dissolve the sample collected in step 3 in dichloromethane and purify it by chromatography through a silica gel (100-400 mesh) column. The eluent is dichloromethane (it can also be a mixture of chloroform-methanol = 9:1 or ethyl acetate-methanol = 8:2 in volume ratio), with a flow rate of 1-2 mL / min. The fractions are collected by an automatic fraction collector, and the collection time for each tube is set to 15 min. At the same time, a portion of the collected liquid from each tube is filtered through a 0.45 μm filter membrane into a sample injection vial and analyzed by a liquid chromatograph. The fractions containing the squalene component are collected, then combined, concentrated to near dryness by reduced pressure distillation at 30-60°C.
[0031] 5. Purification: The sample collected in step 4 was dissolved in 0.5-1 mL of methanol and purified and separated by high performance liquid chromatography using a C18 column, methanol as the mobile phase, a flow rate of 2-15 mL / min, and a detection wavelength of 210 nm. The main peak effluent was collected and rotary evaporated to 0.5-1 mL in a water bath at 30-60°C.
[0032] 6. Freeze drying: Freeze drying technology is used to remove the solvent and solidify squalene to maintain its stability and biological activity.
[0033] In this embodiment, the obtained squalene product is qualitatively and purely analyzed by gas chromatography, and the obtained squalene product is quantitatively analyzed by gas chromatography-tandem mass spectrometry. The gas chromatography-tandem mass spectrometry analysis method of squalene refers to the detection method of Zhou Min (Zhou Min, Sun Wenshan, Dong Yeqing, Zhao Yuejun, Zhong Hanhui, Wang Chuanpi. Determination of squalene in tea by solid phase extraction column purification-gas chromatography-triple quadrupole mass spectrometry [J]. Physical and Chemical Testing-Chemical Section, 2022, 58(11): 1309-1314.), using an HP-5MS chromatographic column (30m×0.25mm, 0.25μm); the injection port temperature is 250℃; the carrier gas is helium with a purity of not less than 99.999%; the collision gas is argon with a purity of not less than 99.999%; the flow rate is 1.00mL·min -1 Column heating program: initial temperature 80°C, maintained for 2.00 min; 40°C·min -1 The temperature was raised to 250℃ and maintained for 1.00min; the temperature was raised to 20℃·min -1 The temperature was raised to 290°C at a rate of 3.75 min and maintained for 13.00 min. The injection volume was 1 μL and the injection was splitless. The purity of the squalene standard product was ≥ 99.0%, purchased from Dr. Ehrenstorfer, Germany.
[0034] from Figure 1 It can be seen that the retention time of the squalene chromatographic peak in the sample is consistent with the retention time of the squalene standard, indicating that the squalene product was extracted from the sample in this embodiment. And by integrating the peak areas of the target chromatographic peak and other impurity peaks on the spectrum, it can be concluded that the purity of squalene in the sample is 92%. Figure 2 The standard curve for quantitative determination of squalene has a concentration range of 0.5 μg / mL to 10 μg / mL and a correlation coefficient of 0.9999. Under this standard curve, the concentration of the squalene sample on the machine is 0.97 μg / mL. After calculation, the extraction efficiency of squalene in this example is 70.6%.
[0035] In summary, the method provided by the present invention can prepare squalene from the liver of cultured Takifugu obscurus, and high-purity functional active substance squalene can be obtained through a simple processing technique. In particular, efficient extraction can be achieved through the selection of a pre-treatment solvent.
[0036] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.
[0037] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A method for preparing squalene from the liver of cultured Takifugu obscurus, characterized in that: The following steps are involved: Step 1. Preparation of liver oil: taking out the liver from cultured Takifugu obscurus, adding n-hexane, n-heptane, petroleum ether or ether-water mixture, homogenizing, ultrasonically extracting, centrifuging, filtering, and obtaining cultured Takifugu obscurus liver oil; Step 2. Saponification: adding sodium hydroxide-ethanol to the cultured pufferfish liver oil and reflux in a water bath; Step 3. Extraction: transfer the saponified liquid to a separatory funnel and add n-hexane, n-heptane or cyclohexane for extraction; Step 4. Purification: dissolve the sample collected in step 3 in dichloromethane, purify it by chromatography on a silica gel column, and collect the fraction containing the squalene component; Step 5. Purification: dissolving the sample collected in step 4 in methanol and purifying and separating by high performance liquid chromatography; Step 6. Freeze drying: Freeze drying technology is used to remove the solvent and solidify the squalene.
2. The method for preparing squalene from the liver of cultured Takifugu obscurus according to claim 1, characterized in that: The specific method of step 1 is: The liver is taken out from cultured dark-striped pufferfish, and impurities and blood stains on the surface are cleaned with distilled water. After the water is drained, the liver is minced with a tissue crusher; the minced sample is weighed and placed in a container, and n-hexane, n-heptane, petroleum ether or ether-water mixture is added, and the mixture is homogenized with a homogenizer for 5-10 minutes; n-hexane, n-heptane, petroleum ether or ether-water mixture is added again, and the mixture is homogenized for 1-5 minutes; ultrasonic extraction is performed for 10 minutes; centrifugation is performed at a low temperature and high speed of 8000-12000 r / min for 10-15 minutes; the upper layer solution is transferred to another container, the water in the solvent is removed, the solution is filtered, and the solution is rotary evaporated to dryness to obtain cultured dark-striped pufferfish liver oil.
3. The method for preparing squalene from the liver of cultured Takifugu obscurus according to claim 1, characterized in that: In step 2, the pH is controlled between 8 and 9, and refluxed in a water bath for 40 to 60 minutes.
4. The method for preparing squalene from the liver of cultured Takifugu obscurus according to claim 1, characterized in that: In step 3, the extraction is performed by shaking for 5 to 10 minutes; the sample is extracted twice more, and the three extracts are combined, rotary evaporated in a water bath at 30 to 60° C., and dried in a vacuum dryer at 50 to 65° C. for later use.
5. The method for preparing squalene from the liver of cultured Takifugu obscurus according to claim 1, characterized in that: In step 4, the product is purified by chromatography through a silica gel column with a mesh size of 100 to 400 at a flow rate of 1 to 2 mL / min.
6. The method for preparing squalene from the liver of cultured Takifugu obscurus according to claim 1, characterized in that: In step 5, a C18 chromatographic column is used, the mobile phase is methanol, the flow rate is 2-15 mL / min, and the detection wavelength is 210 nm; the main peak effluent is collected and rotary evaporated in a water bath at 30-60° C.