Method for extracting cholesterol from fish oil and high-purity cholesterol

Abstract

The application discloses a fish oil cholesterol extraction method and high-purity cholesterol, relates to the technical field of natural sterol extraction and oil refining by-product resource utilization, and comprises the following steps: S1. filtering, dewatering and degumming pretreatment are carried out on fish oil raw materials to obtain pretreated fish oil; S2. the pretreated fish oil is mixed with an alcohol solvent, and under the protection of inert gas, an alkali liquor is added to carry out a controlled saponification reaction, so that glyceride, cholesterol ester and fatty acid ester in the fish oil are converted into saponification products, and at the same time, cholesterol enters the saponification system in a free state; through the controlled saponification reaction, glyceride, cholesterol ester and fatty acid ester in the fish oil are fully converted, so that cholesterol is converted from a combined state into a free state, and an acid value or a saponification rate is used as an end point control index, and the process repeatability is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of natural sterol extraction and resource utilization of by-products from oil refining, and particularly to a method for extracting cholesterol from fish oil and the high-purity cholesterol prepared by this method. Background Technology

[0002] Cholesterol is an important sterol compound, widely found in animal tissues, animal fats, and their processing byproducts. Due to its typical steroidal ring structure and hydroxyl active site, cholesterol serves as a crucial raw material in drug synthesis, vitamin D derivative preparation, steroid compound research, cell culture, and liposome material preparation. Commonly used industrial sources of cholesterol include lanolin, animal brain tissue, egg yolks, and animal fat byproducts; however, some sources suffer from issues such as fluctuating raw material availability, strong off-odors, complex impurities, high separation costs, or insufficient sustainability.

[0003] Fish oil is an important lipid resource produced during aquatic product processing and oil refining. Its main component is triglycerides, while it also contains certain amounts of cholesterol, cholesterol esters, free fatty acids, phospholipids, pigments, oxidation products, and other sterol impurities. Cholesterol and other unsaponifiable matter are relatively concentrated, especially in fish oil deodorization distillates and fish oil refining byproducts, making further cholesterol recovery valuable. However, the fatty acid composition of fish oil is complex, with a high content of unsaturated fatty acids, and cholesterol often coexists with structurally similar sterol impurities, fat-soluble pigments, and oxidation byproducts, making it difficult to obtain high-purity cholesterol through direct solvent extraction or ordinary crystallization.

[0004] Existing cholesterol extraction methods typically include steps such as saponification, solvent extraction, decolorization, and recrystallization. While these methods can extract cholesterol from lipid systems, they still have the following shortcomings: First, if the saponification reaction is not properly controlled, cholesterol esters may not be released completely, and residual fatty acids and saponification byproducts may easily enter the subsequent extract, resulting in a decrease in product purity. Second, relying solely on organic solvent extraction and ordinary crystallization makes it difficult to effectively distinguish cholesterol from other structurally similar sterol impurities. Third, rapid cooling or one-time water crystallization can easily form inclusion crystals, causing fatty acids, pigments, other sterols, and solvent residues to be trapped.

[0005] Therefore, how to achieve efficient release, selective enrichment, purification and high-purity crystallization of cholesterol in fish oil while ensuring the authenticity, operability and feasibility of the process and industrial scale-up is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a method for extracting cholesterol from fish oil and a high-purity cholesterol product. Through a combination of processes including controlled saponification, salting out, organic solvent extraction, selective inclusion of cyclodextrin, uninclusion, and antisolvent gradient crystallization, the cholesterol in fish oil is fully released and effectively separated from fatty acids, saponification products, pigments, oxidative impurities, and other sterol impurities, thereby obtaining a high-purity cholesterol product with high cholesterol content, low levels of other sterol impurities, low fatty acid residue, low solvent residue, and stable appearance.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting cholesterol from fish oil, comprising the following steps: S1. The fish oil raw material is pretreated by filtration, dehydration and degumming to obtain pretreated fish oil.

[0008] The fish oil raw material can be crude fish oil, refined fish oil, deodorized fish oil distillate, fish oil refining byproducts, or mixtures thereof. The purpose of pretreatment is to reduce the interference of moisture, colloids, mechanical impurities, and some polar impurities on saponification, extraction, and inclusion separation. Preferably, the moisture content in the pretreated fish oil is controlled below 0.20 wt%, the acid value is controlled below 15 mg KOH / g, and the peroxide value is controlled below 10 mmol / kg.

[0009] S2. The pretreated fish oil is mixed with an alcohol solvent, and an alkaline solution is added under inert gas protection to carry out a controlled saponification reaction, so that the glycerides, cholesterol esters and fatty acid esters in the fish oil are converted into saponification products, and cholesterol enters the saponification system in a free state.

[0010] The alcohol solvent is preferably ethanol, isopropanol, or a mixture of ethanol and isopropanol. The mass-to-volume ratio of the pretreated fish oil to the alcohol solvent is preferably 1 g:(3 mL-8 mL). The alkali solution is preferably a potassium hydroxide ethanol solution, a sodium hydroxide ethanol solution, a potassium hydroxide isopropanol solution, or a sodium hydroxide isopropanol solution, with a concentration preferably between 0.8 mol / L and 2.5 mol / L, and the amount of alkali used is preferably 1.05 to 1.50 times the theoretical amount required for the saponification value of the fish oil.

[0011] The controlled saponification reaction is carried out at a temperature of 50℃-75℃ for 1.0h-3.5h. The endpoint of the controlled saponification reaction is marked by the acid value of the reaction system dropping below 1.0mgKOH / g or the saponification rate reaching 99% or higher. By controlling the reaction endpoint as described above, cholesterol ester residues due to insufficient saponification can be avoided, and the risk of cholesterol oxidation degradation and color changes caused by excessively high temperature and long-term treatment can be reduced.

[0012] Preferably, before the controlled saponification reaction, an antioxidant is added to the reaction system. The antioxidant is one or more of tocopherol, ascorbyl palmitate, and tert-butylhydroquinone, and the amount added is 0.01%-0.10% of the mass of the pretreated fish oil. The inert gas is nitrogen or argon.

[0013] S3. Add a saline phase to the saponification system to perform salting-out and separation, obtaining a saponified aqueous phase and a cholesterol-containing unsaponifiable phase.

[0014] The saline phase is preferably a sodium chloride aqueous solution with a mass concentration of 5%-20%, added in an amount 0.3-1.2 times the volume of the saponification system. During salting-out, the system temperature is controlled at 35℃-55℃, and the pH of the cholesterol-containing unsaponifiable phase after separation is controlled at 8.0-10.5. This step improves oil-water separation by increasing the ionic strength of the aqueous phase, reducing the entrainment of saponification products and polar impurities in the organic phase, and lowering the risk of emulsification during subsequent extraction.

[0015] S4. The cholesterol-containing unsaponifiable phase is extracted using a non-polar or moderately polar organic solvent. The organic phases are combined, washed with water, dried, and concentrated under reduced pressure to obtain a crude cholesterol extract.

[0016] The organic solvent is preferably one or more selected from n-hexane, petroleum ether, ethyl acetate, and methyl tert-butyl ether. The extraction is performed 2-4 times, with the volume ratio of organic solvent to cholesterol-containing unsaponifiable phase used in each extraction being (0.8-2.0):1. The combined organic phases are washed with water until the pH of the washing solution reaches 6.5-7.5, and then dried using anhydrous sodium sulfate or molecular sieves. This step transfers cholesterol from the saponification system to the organic phase while reducing the residue of water-soluble salts, soaps, and alkalis.

[0017] S5. Dissolve the crude cholesterol extract in an alcohol-water mixed solvent, add a cyclodextrin-based inclusion agent for selective inclusion, so that cholesterol is enriched in the inclusion precipitate, while some other unsaponifiable impurities are retained in the mother liquor, forming a cholesterol-cyclodextrin inclusion precipitate. Separate the inclusion precipitate and wash it with a low-temperature alcohol-water mixed solvent.

[0018] The cyclodextrin-based inclusion agent is preferably β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or methyl-β-cyclodextrin. The molar ratio of the cyclodextrin-based inclusion agent to cholesterol in the crude cholesterol extract is (1.0-2.5):1. The volume fraction of alcohol in the alcohol-water mixed solvent is 40%-75%. The inclusion reaction temperature is 35℃-60℃, and the reaction time is 0.5h-2.5h. After inclusion, the mixture is cooled to 0℃-15℃ and kept at this temperature to precipitate the inclusion precipitate.

[0019] This step is crucial for achieving highly selective purification in this invention. Cyclodextrin-based inclusion agents possess a hydrophobic cavity structure. Under specific alcohol-to-water ratios, temperatures, and concentrations, they can utilize the differences in spatial size, hydrophobic interactions, and molecular arrangement of different sterol molecules to increase the inclusion precipitation ratio of cholesterol, thereby achieving selective enrichment of cholesterol. Free fatty acids, pigments, oxidation byproducts, and some structurally mismatched sterol impurities are largely retained in the mother liquor. Washing the inclusion precipitate with a low-temperature alcohol-to-water mixed solvent further removes entrained impurities.

[0020] S6. The cholesterol-cyclodextrin inclusion precipitate is placed in an inclusion-uninclusion solvent for uninclusion treatment, so that cholesterol is released from the inclusion precipitate, and cholesterol enrichment solution is obtained after separating the cyclodextrin inclusion agent.

[0021] The inclusion-unpacking solvent is preferably an aqueous solution of ethanol, an aqueous solution of isopropanol, or an ethyl acetate-ethanol mixture with a volume fraction of 75%-95%. The inclusion-unpacking temperature is 45℃-70℃, and the inclusion-unpacking time is 0.5h-2.0h. The separated cyclodextrin-based inclusion agent is washed with water and dried before being returned to step S5 for reuse. This step can reduce the consumption of inclusion agent and improve the economic efficiency of the process.

[0022] S7. The cholesterol enrichment solution is subjected to antisolvent gradient crystallization, the crystals are separated and vacuum dried to obtain high-purity cholesterol.

[0023] The antisolvent gradient crystallization involves gradually adding water as an antisolvent to a cholesterol enrichment solution and cooling the solution at a rate of 0.1°C / min to 1.0°C / min for crystallization. By gradually changing the solvent polarity and slowly cooling the solution, cholesterol can precipitate in a more complete crystal form, reducing the embedding of other sterols, fatty acids, and pigments, thereby improving crystal purity and batch stability.

[0024] Preferably, the water is added as an antisolvent in 2-5 stages, and after each stage, the mixture is stirred and balanced for 10-30 minutes before the next stage of water addition is carried out; more preferably, the water is added in stages according to 20%-40% of the total water addition, 20%-40% of the total water addition, and the remaining amount.

[0025] The present invention also provides a high-purity cholesterol, which is prepared by the above-mentioned extraction method of cholesterol from fish oil; the high-purity cholesterol has a cholesterol content of not less than 98.5 wt%, a moisture content of not more than 0.5 wt%, a total fatty acid residue of not more than 0.5 wt%, a total organic solvent residue of not more than 5000 ppm, and a total content of other sterol impurities of not more than 0.3 wt%, and the high-purity cholesterol is a white to off-white crystalline powder.

[0026] Compared with the prior art, the present invention provides a method for extracting cholesterol from fish oil and high-purity cholesterol, which has the following beneficial effects: by controlling the saponification reaction, the glycerides, cholesterol esters and fatty acid esters in fish oil are fully converted, so that cholesterol is converted from the bound state to the free state, and the acid value or saponification rate is used as the endpoint control index, which significantly improves the repeatability of the process.

[0027] This invention reduces the entrainment of soap, free fatty acids, inorganic salts and water-soluble impurities into crude cholesterol extract through salting-out layering and multiple organic solvent extractions, thereby reducing the load on subsequent inclusion and crystallization.

[0028] This invention utilizes the cavity matching and hydrophobic interaction between cyclodextrin inclusion agents and cholesterol molecules to enrich cholesterol in inclusion precipitation and reduce the residue of some structurally similar sterol impurities and other non-saponifiable impurities in the final product.

[0029] This invention combines unpacking and antisolvent gradient crystallization to precipitate cholesterol in a high-purity crystalline form, reducing impurity encapsulation, increasing cholesterol content, and reducing other sterol impurities, fatty acid residues, and solvent residues.

[0030] The solvents used in this invention, such as ethanol, isopropanol, n-hexane, ethyl acetate, and methyl tert-butyl ether, are all common industrial solvents. The cyclodextrin inclusion agent can be recycled and reused, and the process has good scalability. Attached Figure Description

[0031] Figure 1 This is an HPLC purity chromatogram of the high-purity cholesterol obtained in Example 3 of the present invention; Figure 2 This is an HPLC purity chromatogram of the cholesterol product obtained in Comparative Example 1 of this invention. Figure 3 This is a detection chart of other sterol impurities in the high-purity cholesterol obtained in Example 3 of the present invention; Figure 4 This is a SEM image of the high-purity cholesterol crystals obtained in Example 3 of the present invention. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Please see Figures 1-4 This invention provides a method for extracting cholesterol from fish oil and a technical solution for obtaining high-purity cholesterol. Test Standard Description In the testing, cholesterol content can be detected according to GB 5009.128-2016 "National Food Safety Standard - Determination of Cholesterol in Food", and quantitative analysis can be performed using high-performance liquid chromatography. Moisture content can be detected according to GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food" or the Pharmacopoeia Moisture Determination Method. Residual solvents can be detected according to GB 5009.262-2016 "National Food Safety Standard - Determination of Residual Solvents in Food" or the Pharmacopoeia Residual Solvent Determination Method. Acid value can be detected according to GB 5009.229 "National Food Safety Standard - Determination of Acid Value in Food". Peroxide value can be detected according to GB 5009.227 "National Food Safety Standard - Determination of Peroxide Value in Food". Total fatty acid residue can be detected according to GB 5009.168 "National Food Safety Standard - Determination of Fatty Acids in Food".

[0034] The total content of other sterol impurities can be calculated by establishing a calibration curve or calibration factor based on the cholesterol determination method in GB 5009.128-2016, using standards such as cholesterol, campesterol, stigmasterol, β-sitosterol, 7-dehydrocholesterol, and cholesterol, and then normalizing the calculation using high performance liquid chromatography or gas chromatography.

[0035] The structural integrity of cyclodextrin before and after recovery can be characterized using Fourier transform infrared spectroscopy and X-ray powder diffraction. Fourier transform infrared spectroscopy can be used to observe whether the characteristic absorption peaks of the cyclodextrin skeleton undergo significant shifts or disappearances; X-ray powder diffraction can be used to observe whether the crystal form characteristic peaks of the recovered cyclodextrin remain stable.

[0036] Raw materials and general testing methods In the examples and comparative examples, the cholesterol content and total content of other sterol impurities were determined by high-performance liquid chromatography (HPLC). A C18 column was used, the mobile phase was acetonitrile-isopropanol, and the detector was an evaporative light scattering detector or a differential refractive index detector. An external standard curve was established using cholesterol standards, and normalization calculations were performed using campesterol, stigmasterol, β-sitosterol, 7-dehydrocholesterol, and cholesterol as representative impurities.

[0037] Moisture content was determined using the Karl Fischer volumetric method. Residual solvents were detected using headspace gas chromatography. Acid value was determined using acid-base titration. Peroxide value was determined using iodometric titration. Total fatty acid residues were determined by gas chromatography after methylation.

[0038] The structural integrity of cyclodextrin was characterized using Fourier transform infrared spectroscopy and X-ray powder diffraction. The Fourier transform infrared spectroscopy measurement range was 4000 cm⁻¹. - ¹-400cm- ¹, the OH stretching vibration peak, CH stretching vibration peak, COC glycosidic bond stretching vibration peak, and CO stretching vibration peak were recorded. X-ray powder diffraction was performed using Cu-Kα radiation, with a scanning range of 5°–40° and a scanning rate of 5° / min. The positions and relative intensities of the main diffraction peaks of fresh cyclodextrin and recycled cyclodextrin were compared.

[0039] Cholesterol yield is calculated using the following formula: Cholesterol yield (%) = Cholesterol mass in final product / Total cholesterol mass in pretreated fish oil feedstock × 100%.

[0040] Example 1 1000g of deodorized fish oil distillate was used as the raw material. Mechanical impurities were removed by plate and frame filtration. The oil was then dehydrated for 40 minutes at 60℃ and a vacuum of -0.090MPa, followed by hydration and degumming to obtain pretreated fish oil. The pretreated fish oil was found to have a moisture content of 0.18wt%, an acid value of 12.6mgKOH / g, and a peroxide value of 8.7mmol / kg, meeting the pretreatment control requirements.

[0041] Pretreated fish oil was added to a reaction vessel, followed by 3000 mL of ethanol at a ratio of 1 g: 3 mL. Nitrogen gas was bubbled through the vessel for 15 min, and then 0.10 g of tocopherol was added. Subsequently, a 0.8 mol / L potassium hydroxide ethanol solution was added, with the amount of alkali used being 1.05 times the theoretical amount required for the fish oil saponification value. The reaction was stirred at 50 °C for 1.0 h. After the reaction, the acid value of the reaction system was measured to be 0.92 mg KOH / g, and the saponification rate was 99.1%.

[0042] A 5% sodium chloride aqueous solution was added to the saponification system, with the amount added being 0.3 times the volume of the saponification system. The mixture was allowed to stand at 35°C to separate into two phases: a soap-containing aqueous phase and a cholesterol-containing unsaponifiable phase. The pH of the unsaponifiable phase after separation was 10.3.

[0043] The unsaponifiable phase was extracted twice with n-hexane, with a volume ratio of n-hexane to unsaponifiable phase of 0.8:1 each time. The organic phases were combined, washed with deionized water until the pH of the washings reached 7.3, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude cholesterol extract.

[0044] Crude cholesterol extract was dissolved in a 40% (v / v) aqueous ethanol solution, and β-cyclodextrin was added at a molar ratio of β-cyclodextrin to cholesterol in the crude extract of 1.0:1. The mixture was stirred at 35°C for 0.5 h to induce inclusion, and then cooled to 0°C in an ice-water bath and kept at this temperature for 1 h to precipitate cholesterol-β-cyclodextrin inclusion. The inclusion precipitate was separated by filtration and washed with a 40% aqueous ethanol solution at 0°C.

[0045] The inclusion precipitate was placed in a 75% (v / v) aqueous ethanol solution and the inclusion was detached at 45°C for 0.5 h. β-cyclodextrin was recovered by hot filtration, and the filtrate was used as a cholesterol enrichment solution. Water was added to the cholesterol enrichment solution in three separate portions as an antisolvent, and the solution was cooled to 5°C at a rate of 0.1°C / min. After holding at this temperature for 2 h, the crystals were collected by filtration and vacuum dried at 45°C for 8 h to obtain a white to off-white crystalline powder, i.e., high-purity cholesterol.

[0046] Example 2 1000g of fish oil refining byproducts were filtered, vacuum dehydrated, and degummed to obtain pretreated fish oil. The moisture content was found to be 0.06wt%, the acid value was 4.8mgKOH / g, and the peroxide value was 3.4mmol / kg.

[0047] Pretreated fish oil was mixed with isopropanol at a ratio of 1 g: 8 mL under argon protection, and 1.00 g of ascorbate palmitate was added. A 2.5 mol / L sodium hydroxide isopropanol solution was added, with the alkali dosage being 1.50 times the theoretical saponification amount. The reaction was stirred at 75 °C for 3.5 h. The final acid value was measured to be 0.35 mg KOH / g, and the saponification rate was 99.8%.

[0048] A 20% sodium chloride aqueous solution was added, at a volume 1.2 times that of the saponification system. The mixture was allowed to stand at 55°C to separate into layers, with the unsaponified phase having a pH of 8.2. The mixture was extracted four times with ethyl acetate, each time at a volume ratio of 2.0:1 for the unsaponified phase. The organic phases were combined, washed with water until the pH reached 6.8, dried through a molecular sieve, and concentrated under reduced pressure to obtain a crude cholesterol extract.

[0049] Crude cholesterol extract was dissolved in a 75% (v / v) isopropanol aqueous solution, and hydroxypropyl-β-cyclodextrin was added. The molar ratio of cyclodextrin inclusion agent to cholesterol was 2.5:1. Inclusion was carried out at 60°C for 2.5 h, and then cooled to 15°C to precipitate the inclusion precipitate. The precipitate was washed with a 75% isopropanol aqueous solution at 15°C.

[0050] The inclusion precipitate was placed in a 95% (v / v) isopropanol aqueous solution and the inclusion was detached at 70°C for 2.0 h. The cyclodextrin-based inclusion agent was recovered by filtration, and the filtrate was subjected to antisolvent gradient crystallization. Water was added to the filtrate in five portions, and the mixture was cooled to 10°C at a cooling rate of 1.0°C / min. After holding at this temperature for 2 h, the mixture was filtered, and the crystals were vacuum dried at 50°C for 6 h to obtain high-purity cholesterol.

[0051] Example 3 1000g of deodorized fish oil distillate was pretreated to obtain pretreated fish oil. The moisture content was found to be 0.10wt%, the acid value was 8.1mgKOH / g, and the peroxide value was 5.2mmol / kg.

[0052] Pretreated fish oil and ethanol were mixed at a ratio of 1 g: 5 mL, and the mixture was protected under nitrogen gas. 0.30 g of tocopherol was added. A 1.5 mol / L potassium hydroxide ethanol solution was then added, with the alkali dosage being 1.25 times the theoretical saponification amount. The reaction was carried out at 62 °C for 2.2 h. The final acid value was 0.48 mg KOH / g, and the saponification rate was 99.6%.

[0053] A 12% sodium chloride aqueous solution was added, at a volume 0.7 times that of the saponification system. Salting-out was performed at 45°C, and the unsaponified phase had a pH of 9.2. Extraction was performed three times with a mixed solvent of hexane and ethyl acetate (4:1 volume ratio), with the organic solvent to unsaponified phase volume ratio at 1.2:1 each time. The organic phases were combined, washed with water until the pH reached 7.0, dried, and concentrated under reduced pressure to obtain crude cholesterol extract.

[0054] Crude cholesterol extract was dissolved in a 60% (v / v) aqueous ethanol solution, and β-cyclodextrin was added at a molar ratio of 1.8:1 to cholesterol. Inclusion was carried out at 48°C for 1.5 h, followed by cooling to 8°C to precipitate the inclusion. The precipitate was washed with a low-temperature 60% (v / v) aqueous ethanol solution and then placed in an 85% (v / v) aqueous ethanol solution. The inclusion was detached at 58°C for 1.2 h, and the β-cyclodextrin was recovered by filtration.

[0055] The obtained cholesterol enrichment solution was added to water as an antisolvent in four portions and cooled to 8°C at a cooling rate of 0.5°C / min to crystallize. The crystals were filtered and dried under vacuum at 48°C for 7 hours to obtain high-purity cholesterol.

[0056] Example 4 1000g of crude fish oil was filtered through diatomaceous earth, vacuum dehydrated, and degummed by hydration to obtain pretreated fish oil. The moisture content was found to be 0.20wt%, the acid value was 14.7mgKOH / g, and the peroxide value was 9.6mmol / kg.

[0057] Pretreated fish oil was mixed with a 1:1 volume ratio of ethanol to isopropanol at a ratio of 1 g:6 mL. Under nitrogen protection, 0.50 g of tert-butylhydroquinone was added. A 1.2 mol / L potassium hydroxide ethanol solution was then added, with the alkali dosage being 1.30 times the theoretical saponification amount. The reaction was carried out at 58 °C for 2.8 h. The final acid value was 0.71 mg KOH / g, and the saponification rate was 99.3%.

[0058] A 15% sodium chloride aqueous solution was added, at a volume 0.9 times that of the saponification system. Salting-out was performed at 42°C, and the unsaponified phase had a pH of 9.8. Extraction was performed three times with a mixed solvent of methyl tert-butyl ether and n-hexane at a volume ratio of 1:3, with the organic solvent to unsaponified phase volume ratio being 1.5:1 each time. After washing with water, drying, and concentration, crude cholesterol extract was obtained.

[0059] Crude cholesterol extract was dissolved in a 55% (v / v) aqueous ethanol solution, and hydroxypropyl-β-cyclodextrin was added. The molar ratio of cyclodextrin inclusion agent to cholesterol was 1.6:1. Inclusion was carried out at 45°C for 1.8 h, followed by cooling to 5°C to precipitate the inclusion precipitate. The inclusion precipitate was washed with a low-temperature alcohol-water mixed solvent and then detached in an 80% aqueous ethanol solution at 55°C for 1.0 h. The resulting enriched solution was subjected to antisolvent gradient crystallization at a cooling rate of 0.3°C / min, filtered, and dried to obtain high-purity cholesterol.

[0060] Example 5 1000g of refined fish oil was filtered and vacuum dehydrated to obtain pretreated fish oil. The moisture content was found to be 0.04wt%, the acid value was 2.1mgKOH / g, and the peroxide value was 1.9mmol / kg.

[0061] Pretreated fish oil and isopropanol were mixed at a ratio of 1 g: 4 mL, and 0.20 g of tocopherol was added under nitrogen protection. A 1.8 mol / L potassium hydroxide isopropanol solution was added, with the alkali dosage being 1.20 times the theoretical saponification amount. The reaction was carried out at 68 °C for 1.8 h. The final acid value was 0.56 mg KOH / g, and the saponification rate was 99.4%.

[0062] A 10% sodium chloride aqueous solution was added, at a volume 0.6 times that of the saponification system. The mixture was allowed to separate into layers at 40°C, with the unsaponified phase having a pH of 8.9. The mixture was then extracted three times with petroleum ether at a volume ratio of 1.0:1 to the unsaponified phase each time. After washing with water until the pH reached 7.2, the crude cholesterol extract was obtained after drying and concentration.

[0063] Crude cholesterol extract was dissolved in a 65% (v / v) isopropanol aqueous solution, and β-cyclodextrin was added at a molar ratio of 2.0:1 to cholesterol. Inclusion was carried out at 52°C for 1.2 h, followed by cooling to 10°C to precipitate the inclusion. The precipitate was washed and then detached in a 90% ethanol aqueous solution at 60°C for 1.5 h. The resulting enriched solution was subjected to antisolvent gradient crystallization at a cooling rate of 0.7°C / min, filtered, and dried to obtain high-purity cholesterol.

[0064] Example 6 Fish oil deodorized distillate and fish oil refining byproducts were mixed at a mass ratio of 7:3, totaling 1000g. The mixture was then filtered, dehydrated, and degummed to obtain pretreated fish oil. The moisture content was found to be 0.09wt%, the acid value was 7.4mgKOH / g, and the peroxide value was 4.6mmol / kg.

[0065] Pretreated fish oil was mixed with ethanol at a ratio of 1 g: 5.5 mL. Under argon protection, 0.60 g of a mixed antioxidant consisting of tocopherol and ascorbate palmitate was added. A 1.6 mol / L sodium hydroxide ethanol solution was then added, with the alkali dosage being 1.35 times the theoretical saponification amount. The reaction was carried out at 64 °C for 2.4 h. The final acid value was 0.42 mg KOH / g, and the saponification rate was 99.7%.

[0066] An 18% sodium chloride aqueous solution was added, at a volume 0.8 times that of the saponification system. Salting-out was performed at 48°C, and the unsaponified phase had a pH of 9.5. Extraction was performed three times with a mixed solvent of ethyl acetate and n-hexane (volume ratio 1:2), with the organic solvent to unsaponified phase volume ratio being 1.6:1 each time. After washing with water, drying, and concentration, crude cholesterol extract was obtained.

[0067] Crude cholesterol extract was dissolved in a 62% (v / v) aqueous ethanol solution, and methyl-β-cyclodextrin was added at a molar ratio of 1.7:1 to cholesterol. Inclusion was carried out at 50°C for 1.6 h, followed by cooling to 6°C to precipitate the inclusion. The inclusion precipitate was then detached in an 85% aqueous ethanol solution at 62°C for 1.3 h. The resulting cholesterol-rich solution was subjected to antisolvent gradient crystallization at a cooling rate of 0.4°C / min, and after filtration and drying, high-purity cholesterol was obtained.

[0068] Comparative Example 1 is essentially the same as Example 3, except that the β-cyclodextrin selective inclusion step in step S5 is omitted, and the crude cholesterol extract is directly subjected to antisolvent crystallization after concentration. The resulting product is denoted as D1.

[0069] Comparative Example 2 is essentially the same as Example 3, except that the molar ratio of β-cyclodextrin to cholesterol is adjusted from 1.8:1 to 0.5:1, which is lower than the lower limit of 1.0:1 specified in this invention. The resulting product is denoted as D2.

[0070] Comparative Example 3 was essentially the same as Example 3, except that: the cholesterol enrichment solution after unpacking was added to all water at once, and rapidly cooled and crystallized at a cooling rate of 5°C / min, instead of using a gradient cooling rate of 0.1°C / min-1.0°C / min. The resulting product was designated D3.

[0071] Comparative Example 4 is essentially the same as Example 3, except that: after the saponification reaction, an aqueous sodium chloride solution was not added for salting out, but instead, an extraction solvent was directly added for extraction. The resulting product is denoted as D4.

[0072] Comparative Example 5 was essentially the same as Example 3, except that the saponification reaction temperature was still 62°C, but the reaction time was shortened to 0.5 h. At the end of the reaction, the acid value was 3.8 mg KOH / g, and the saponification rate was 95.6%, which did not meet the endpoint control requirements of an acid value below 1.0 mg KOH / g or a saponification rate above 99%. The resulting product was designated D5.

[0073] Comparative Example 6 is essentially the same as Example 3, except that in step S5, after filtering to obtain the cholesterol-β-cyclodextrin inclusion precipitate, it is not washed with a low-temperature 60% ethanol aqueous solution, but directly subjected to unpacking treatment. The resulting product is denoted as D6.

[0074] Performance testing methods 1. Raw material pretreatment index testing The pretreated fish oils from Examples 1 to 6 were tested for moisture, acid value, and peroxide value. The results are shown in Table 1 below.

[0075] Table 1 Quality Indicators of Pretreated Fish Oil

[0076] As shown in Table 1, the pretreated fish oils of Examples 1 to 6 all meet the control requirements of moisture content not exceeding 0.20 wt%, acid value not exceeding 15 mg KOH / g, and peroxide value not exceeding 10 mmol / kg, indicating that the pretreatment conditions described in this invention can be applied to different fish oil raw materials or by-products.

[0077] 2. Saponification endpoint and process stability test The saponification endpoints of the examples and comparative examples were tested, and the results are shown in Table 2 below.

[0078] Table 2. Saponification endpoint control results

[0079] As shown in Table 2, within the temperature range of 50℃-75℃ and the reaction time range of 1.0h-3.5h specified in this invention, by controlling the reaction endpoint, Examples 1 to 6 all achieved an acid value below 1.0 mg KOH / g and a saponification rate of over 99%. Comparative Example 5 only shortened the saponification time, resulting in insufficient saponification, indicating that the "controlled saponification reaction endpoint" has a direct impact on the purity and fatty acid residue of the subsequent product.

[0080] 3. Finished product quality testing The cholesterol content, other sterol impurities, moisture, total fatty acid residue, organic solvent residue, and cholesterol yield of the cholesterol products prepared in Examples 1 to 6 and Comparative Examples 1 to 6 were tested, and the results are shown in Table 3 below.

[0081] Table 3. Cholesterol Product Quality Test Results

[0082] As shown in Table 3, Examples 1 to 6 all yielded high-purity cholesterol products with a cholesterol content of not less than 98.5 wt%, a total content of other sterol impurities of not more than 0.3 wt%, a total fatty acid residue of not more than 0.5 wt%, a moisture content of not more than 0.5 wt%, and a total organic solvent residue of not more than 5000 ppm. These results support the quality requirements for high-purity cholesterol specified in the product claims of this invention.

[0083] Comparative Example 1, which did not employ selective inclusion with cyclodextrin, had a cholesterol content of only 95.8 wt%, while the total content of other sterol impurities increased to 1.45 wt%, indicating that ordinary crystallization is insufficient to effectively remove structurally similar sterol impurities. Comparative Example 2, although employing cyclodextrin inclusion, used a lower amount of inclusion agent than permitted in this invention, resulting in insufficient cholesterol capture; other sterol impurities remained at 0.78 wt%. This demonstrates the necessity of controlling the molar ratio of cyclodextrin inclusion agent to cholesterol at (1.0-2.5):1.

[0084] Comparative Example 3 did not employ antisolvent gradient crystallization but instead underwent rapid cooling crystallization, resulting in increased levels of other sterol impurities and fatty acid residues. This indicates that gradient addition of antisolvents and control of the cooling rate can reduce impurity encapsulation. Comparative Example 4 did not undergo salting-out separation, resulting in a fatty acid residue of 1.46 wt% and a significant decrease in cholesterol yield. This demonstrates that salting-out separation can reduce the entrainment of emulsification and saponification byproducts. Comparative Example 5 did not reach the saponification endpoint, resulting in a total fatty acid residue as high as 1.85 wt%. This indicates that controlling the reaction endpoint plays a crucial role in releasing cholesterol and reducing fatty acid residues. Comparative Example 6 did not undergo low-temperature alcohol-water washing. Although the cholesterol yield was high, other sterol impurities and total fatty acid residues exceeded the product specifications of this invention. This indicates that the inclusion precipitation washing step makes a practical contribution to improving the quality of the finished product.

[0085] 4. Stability test for the recycling and reuse of cyclodextrin Based on the process of Example 3, the β-cyclodextrin recovered by filtration was washed with water, dried and reused in step S5 for 5 consecutive batches. The purity of cholesterol products and the recovery rate of cyclodextrin were tested, and the results are shown in Table 4 below.

[0086] Table 4. Stability of β-cyclodextrin with repeated use

[0087] As shown in Table 4, after recycling and reusing 5 batches of cyclodextrin inclusion agents, the cholesterol product still maintained a purity of over 99.0 wt%, and the total content of other sterol impurities was less than 0.3 wt%, indicating that the process of the present invention has good economic efficiency and feasibility for continuous production.

[0088] 5. Structural integrity test before and after cyclodextrin recovery To further verify the structural stability of cyclodextrin inclusion agents during recycling and reuse, Fourier transform infrared spectroscopy and X-ray powder diffraction tests were performed on β-cyclodextrin before its first use and after five batches of reuse in Example 3. The test results are shown in Table 5 below.

[0089] Table 5. Structural characterization results of β-cyclodextrin before and after recovery.

[0090] As shown in Table 5, the recovered β-cyclodextrin, after being reused for 5 batches, still retained typical infrared characteristic absorption peaks such as OH, CH, COC, and CO, and the peak positions did not shift significantly. The positions of its main XRD diffraction peaks were basically consistent with those of fresh β-cyclodextrin, indicating that the main framework and crystal structure of β-cyclodextrin were not significantly damaged during the recovery process. Combined with the product purity and yield data after reuse in Table 4, it can be seen that cyclodextrin inclusion agents can maintain their selective inclusion of cholesterol even after repeated use following washing and drying.

[0091] Results Analysis As can be seen from the comprehensive embodiments and comparative examples, the technical progress of the present invention is mainly reflected in the following aspects: First, the controlled saponification reaction, through the coordinated control of temperature, time, alkali dosage, and endpoint indicators, ensures the full saponification of cholesterol esters and related lipid components, while preventing oxidative degradation of cholesterol at excessively high temperatures or for excessively long reaction times. Examples 1, 2, and 3 cover the low-end, high-end, and intermediate regions, respectively, and all achieve the endpoint requirements of an acid value below 1.0 mg KOH / g or a saponification rate of over 99%, demonstrating the feasibility and stability of the scope defined in the claims.

[0092] Secondly, salting out can effectively reduce the entrainment of saponified products and fatty acids into the unsaponified phase. In Comparative Example 4, which did not undergo salting out, the residual fatty acid content in the final product was significantly increased, and the cholesterol yield was reduced. This indicates that salting out is not a simple auxiliary step, but a key process step to ensure the stability of subsequent extraction and refining.

[0093] Third, the selective inclusion of cyclodextrins is a key technical feature that distinguishes this invention from conventional solvent extraction and recrystallization. In Comparative Example 1, without cyclodextrin inclusion, the total content of other sterol impurities was significantly increased; in Comparative Example 2, when the amount of inclusion agent was insufficient, the cholesterol capture and impurity separation effects decreased. This demonstrates that cyclodextrin inclusion agents, in appropriate molar ratios and alcohol-water systems, can increase the enrichment ratio of cholesterol in the inclusion precipitate and reduce the residue of other unsaponifiable impurities in the final product, thereby effectively removing other unsaponifiable impurities, especially structurally similar sterol impurities.

[0094] Fourth, antisolvent gradient crystallization can further improve the purity of cholesterol crystals. Comparative Example 3 used rapid cooling crystallization, which led to increased impurity encapsulation and decreased product purity; in the examples, the gradual addition of water and a cooling rate of 0.1℃ / min-1.0℃ / min resulted in a more stable appearance and higher purity crystalline powder.

[0095] Fifth, cyclodextrin-based inclusion agents exhibit good stability during recycling. After five consecutive batches of reuse, β-cyclodextrin still maintains a cholesterol product purity above 99.0 wt% and the total content of other sterol impurities below 0.3 wt%. Simultaneously, FTIR and XRD characterization results show no significant changes in the main infrared characteristic absorption peaks and crystal form characteristic peaks of the recovered β-cyclodextrin, indicating that its main structure remains intact. Therefore, this invention not only improves product purity but also reduces inclusion agent consumption, enhancing economic efficiency during industrial scale-up.

[0096] Sixth, there is a clear correspondence between the product indicators and the method steps of this invention. The cholesterol content of not less than 98.5 wt% is mainly achieved through controlled saponification, inclusion separation, and gradient crystallization; the total content of other sterol impurities of not more than 0.3 wt% is mainly achieved through selective inclusion with cyclodextrin; the total fatty acid residue of not more than 0.5 wt% is mainly achieved through controlled saponification, salting-out layering, and washing; and the moisture content and residual solvent indicators are achieved through vacuum drying and solvent control. Therefore, the limitations in the claims are clearly supported by the specification.

[0097] Figure 1 In the figure, the horizontal axis represents retention time, and the vertical axis represents detector response value. The main peak at 12.48 min is the cholesterol peak, while impurity peaks A, B, and C are trace sterol impurity peaks. As can be seen from the figure, the cholesterol main peak is symmetrical and has a high response intensity, while the impurity peaks are relatively weak, indicating that the product obtained in Example 3 has high purity, with a cholesterol content of 99.5 wt% and a total content of other sterol impurities of 0.12 wt%.

[0098] Figure 2 In the figure, the horizontal axis represents retention time, and the vertical axis represents detector response value. The cholesterol peak is located at 12.50 min, while impurity peaks A, B, C, and D represent other sterol impurities and trace amounts of unsaponifiable matter. As can be seen from the figure, Comparative Example 1, which did not undergo cyclodextrin selective inclusion treatment, showed more prominent impurity peaks on both sides of the main peak, with a cholesterol content of 95.8 wt% and a total content of other sterol impurities of 1.45 wt%.

[0099] Figure 3In the figure, the horizontal axis represents retention time, and the vertical axis represents total ion current intensity. The main cholesterol peak is located at 21.35 min. A, B, C, D, and E represent 7-dehydrocholesterol, cholesterol, campesterol, stigmasterol, and β-sitosterol, respectively. As can be seen from the figure, the response of each impurity peak is low, indicating that the product obtained in Example 3 has few other sterol impurities remaining, with a total content of 0.12 wt%.

[0100] Figure 4 In the study, scanning electron microscopy was used to observe the microstructure of the samples. The image shows that the obtained cholesterol crystals have a regular flaky structure with clear crystal edges, a relatively smooth surface, and good particle dispersion, with no obvious amorphous agglomeration areas observed. This figure indicates that antisolvent gradient crystallization treatment can promote the formation of a regular crystal structure in cholesterol, which is beneficial to improving product purity and stability.

[0101] In summary, this invention achieves the goal of preparing high-purity cholesterol from fish oil and its refining byproducts through a combined process of controlled saponification, salting-out layering, selective inclusion and de-inclusion of cyclodextrin, and antisolvent gradient crystallization. Test data from examples and comparative studies demonstrate that, compared to conventional extraction crystallization, low inclusion agent dosage, no salting-out treatment, insufficient saponification, or rapid crystallization processes, this invention significantly improves cholesterol purity, reduces other sterol impurities and fatty acid residues, and maintains a high yield, exhibiting significant inventiveness and industrial application value.

Claims

1. A method for extracting cholesterol from fish oil, characterized in that, Includes the following steps: S1. The fish oil raw material is pretreated by filtration, dehydration and degumming to obtain pretreated fish oil; S2. The pretreated fish oil is mixed with an alcohol solvent, and an alkaline solution is added under inert gas protection to carry out a controlled saponification reaction, so that the glycerides, cholesterol esters and fatty acid esters in the fish oil are converted into saponification products, and cholesterol enters the saponification system in a free state. S3. Add a saline phase to the saponification system to perform salting out and separation, obtaining a saponified aqueous phase and a cholesterol-containing non-saponifiable phase; S4. Extract the cholesterol-containing unsaponifiable phase using a non-polar or moderately polar organic solvent, combine the organic phases, wash with water, dry and concentrate under reduced pressure to obtain crude cholesterol extract; S5. Dissolve the crude cholesterol extract in an alcohol-water mixed solvent, add a cyclodextrin inclusion agent for selective inclusion, so that cholesterol is enriched in the inclusion precipitate and some other unsaponifiable impurities are retained in the mother liquor, forming a cholesterol-cyclodextrin inclusion precipitate. Separate the inclusion precipitate and wash it with a low-temperature alcohol-water mixed solvent. S6. The cholesterol-cyclodextrin inclusion precipitate is placed in an inclusion-uninclusion solvent for inclusion-uninclusion treatment to release cholesterol from the inclusion precipitate. After separating the cyclodextrin inclusion agent, a cholesterol enrichment solution is obtained. S7. The cholesterol enrichment solution is subjected to antisolvent gradient crystallization, the crystals are separated and vacuum dried to obtain high-purity cholesterol; The controlled saponification reaction is carried out at a temperature of 50℃-75℃ for 1.0h-3.5h; the selective inclusion reaction is carried out at a temperature of 35℃-60℃, and after inclusion, the mixture is cooled to 0℃-15℃ and kept at this temperature to precipitate the inclusion precipitate; the antisolvent gradient crystallization involves gradually adding water as an antisolvent to the cholesterol enrichment solution in at least two stages, and cooling the solution at a cooling rate of 0.1℃ / min-1.0℃ / min for crystallization.

2. The method for extracting cholesterol from fish oil according to claim 1, characterized in that, The fish oil raw material is crude fish oil, refined fish oil, deodorized fish oil distillate, fish oil refining by-products, or a mixture thereof; the moisture content of the pretreated fish oil is controlled below 0.20 wt%, the acid value is controlled below 15 mg KOH / g, and the peroxide value is controlled below 10 mmol / kg.

3. The method for extracting cholesterol from fish oil according to claim 1, characterized in that, In step S2, the alcohol solvent is ethanol, isopropanol, or a mixture of ethanol and isopropanol; the mass-volume ratio of the pretreated fish oil to the alcohol solvent is 1g:(3mL-8mL).

4. The method for extracting cholesterol from fish oil according to claim 1, characterized in that, In step S2, the alkaline solution is a potassium hydroxide ethanol solution, a sodium hydroxide ethanol solution, a potassium hydroxide isopropanol solution, or a sodium hydroxide isopropanol solution, and the concentration of the alkaline solution is 0.8 mol / L-2.5 mol / L. The amount of alkali used is 1.05-1.50 times the theoretical amount required for the saponification value of fish oil. In step S2, the endpoint of the controlled saponification reaction is marked by the acid value of the reaction system dropping below 1.0 mg KOH / g or the saponification rate reaching 99% or more.

5. The method for extracting cholesterol from fish oil according to claim 1, characterized in that, In step S2, before the controlled saponification reaction, an antioxidant is added to the reaction system. The antioxidant is one or more of tocopherol, ascorbyl palmitate, and tert-butylhydroquinone, and the amount added is 0.01%-0.10% of the mass of the pretreated fish oil. The inert gas is nitrogen or argon.

6. The method for extracting cholesterol from fish oil according to claim 1, characterized in that, In step S3, the saline phase is a sodium chloride aqueous solution with a mass concentration of 5%-20%, and the amount added is 0.3-1.2 times the volume of the saponification system; the system temperature is controlled at 35℃-55℃ during salting-out and the pH of the cholesterol-containing non-saponifiable phase after separation is controlled at 8.0-10.

5.

7. The method for extracting cholesterol from fish oil according to claim 1, characterized in that, In step S4, the organic solvent is one or more of n-hexane, petroleum ether, ethyl acetate, and methyl tert-butyl ether; the extraction is performed 2-4 times, and the volume ratio of the organic solvent to the cholesterol-containing unsaponifiable phase used in each extraction is (0.8-2.0):1; the combined organic phase is washed with water until the pH of the washing solution is 6.5-7.5, and then dried with anhydrous sodium sulfate or molecular sieve.

8. The method for extracting cholesterol from fish oil according to claim 1, characterized in that, In step S5, the cyclodextrin inclusion agent is β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or methyl-β-cyclodextrin; the molar ratio of the cyclodextrin inclusion agent to cholesterol in the crude cholesterol extract is (1.0-2.5):1; the volume fraction of alcohol in the alcohol-water mixed solvent is 40%-75%; and the inclusion reaction time is 0.5h-2.5h.

9. The method for extracting cholesterol from fish oil according to claim 1, characterized in that, In step S6, the unpacking solvent is an aqueous solution of ethanol, an aqueous solution of isopropanol, or a mixed solvent of ethyl acetate and ethanol with a volume fraction of 75%-95%. The unpacking temperature is 45℃-70℃, and the unpacking time is 0.5h-2.0h. The separated cyclodextrin-type inclusion agent is washed with water and dried before being returned to step S5 for reuse.

10. A high-purity cholesterol, characterized in that, The high-purity cholesterol is prepared by the extraction method of cholesterol from fish oil according to any one of claims 1-9; its crystals are white to off-white crystalline powder, with a cholesterol content of not less than 98.5 wt%, a cholesterol moisture content of not more than 0.5 wt%, a total fatty acid residue of not more than 0.5 wt%, a total organic solvent residue of not more than 5000 ppm, and a total content of other sterol impurities of not more than 0.3 wt%.

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