Separation and purification method for squalene produced by biosynthesis and fermentation

Abstract

Disclosed is an extraction and purification method for biosynthetic squalene. The present invention pertains to the technical field of squalene extraction. In the present invention, the synergy between antioxidant addition, solvent pretreatment, and process condition control can reduce the peroxide value of the extracted finished product. The peroxide value can be as low as 1 meq / kg or lower, thereby meeting the pharmaceutical-grade peroxide value standard, and both the content and purity can reach 99% or higher. In addition, the use of a single organic solvent for extraction and chromatography avoids the difficult recovery problem with the cross-use of multiple solvents; the single organic solvent can be reused repeatedly. The total extraction yield is as high as 90%. The method is suitable for industrial continuous production.

Description

A method for separating and purifying squalene produced by biosynthetic fermentation Technical Field

[0001] This invention relates to a method for extracting and purifying squalene produced by biosynthesis fermentation, belonging to the field of squalene extraction technology. Background Technology

[0002] Squalene, also known as docosahexaene, is a polyunsaturated hydrocarbon produced during metabolism, such as cholesterol synthesis, in the human body. It contains six isoprene double bonds and belongs to the terpenoid class of compounds. Squalene is found in many foods, but has been primarily extracted from shark liver oil. It is also found in relatively high amounts in a few plant oils, such as olive oil and rice bran oil. Squalene is typically produced by human sebaceous glands, and therefore is commonly used in cosmetics and personal care products for topical skin lubrication and protection. Squalene is also an important component of immune adjuvants administered with vaccines. Adjuvants containing squalene have been shown to enhance the patient's immune response, thereby improving the effectiveness of the corresponding vaccine. In some cases, due to this enhanced response, the amount of antigen in the vaccine can be significantly reduced while still maintaining immune protection.

[0003] Squalene can be prepared through natural biological extraction, chemical synthesis, and microbial synthesis. Natural biological extraction involves extracting squalene from plants and animals, with animal-derived squalene primarily extracted from the liver oil of deep-sea sharks. However, due to the declining numbers of deep-sea sharks, with some species facing extinction, shark liver oil is becoming increasingly scarce and in short supply, and may also carry the risk of pathogen infection. Plant-derived squalene is derived from the fruits, seeds, or oils of plants such as olive, camellia, amaranth, terminalia, monk fruit, and squalene oil, extracted from the deodorized distillate of plant oils (primarily from olive oil deodorized distillate) (CN110283034A). Plant-derived extraction mainly obtains squalene from olive oil deodorized distillate, but this method lacks stable resources, contains many impurities, and results in a lengthy extraction process that consumes large amounts of complex organic reagents. Patent CN101891579A describes a chemical coupling synthesis process for squalene, using dichlorobutane as a raw material. This material reacts with triethyl phosphite to form a phosphate ester, which is then reacted with geranylacetone under alkaline conditions to obtain squalene. However, chemical synthesis methods often utilize toxic chemical reagents, and large-scale industrial production is still some distance away.

[0004] Biosynthetic fermentation for squalene production has become a promising alternative method due to its high efficiency and environmental friendliness. Squalene extraction from *Saccharomyces cerevisiae* and *Schizochytrium* has been reported (CN113604512A, CN116947589A), with biosynthetic fermentation broth yields reaching tens of grams per liter, as exemplified by *Saccharomyces cerevisiae*. Patent CN116947589A proposes two extraction and purification routes, achieving a product purity greater than 97%, but does not mention product content, peroxide value, extraction yield, or other indicators. Content testing ensures that the active ingredient content in the product meets standard requirements. Given the use of squalene in pharmaceutical compositions for human administration, there is a long-term need for improved methods to produce substantially impurity-free squalene. The European Pharmacopoeia stipulates a squalene content of 97-103% and a peroxide value not exceeding 5 meq / kg. Peroxide value is one of the key factors influencing whether squalene can be used as a vaccine adjuvant, representing an indicator of the degree of oxidation of oils and fatty acids. The sources of peroxide value during squalene extraction are mainly of two types. One type is incomplete removal of impurities other than squalene during extraction. These impurities can be certain lipid-soluble components from the culture medium added during upstream fermentation, byproducts generated during biosynthesis and metabolism, or peroxides generated due to improper operating conditions during extraction. The other type is a qualitative change in the squalene product itself during extraction, leading to excessive peroxide value. Therefore, it is crucial to remove excess impurities as much as possible during separation and purification, while minimizing the qualitative changes of the product itself through process control techniques. Thus, there is an urgent need to develop a biosynthetic squalene purification method that can reduce peroxide value while maintaining extraction efficiency, adaptable to scale-up production, and lower extraction costs.

[0005] Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides an extraction and purification method for biosynthetic squalene, which aims to solve the technical problem that the prior art lacks a method for purifying biosynthetic squalene that can reduce peroxide value while maintaining extraction rate.

[0007] The first technical solution provided by this invention is a method for extracting and purifying squalene through biosynthesis, comprising the following steps:

[0008] (1) Membrane separation: The pretreated yeast fermentation broth is filtered through a membrane to remove the supernatant, the cells are collected, and then washed to obtain a concentrated cell solution.

[0009] (2) High pressure homogenization: Add antioxidant to the bacterial cell concentrate in step (1), and then perform cell disruption and homogenization to obtain homogenized liquid;

[0010] (3) Extraction: Add an extractant to the homogenized liquid from step (2) to extract the extract;

[0011] (4) First concentration: The extract from step (3) is concentrated for the first time to obtain crude squalene;

[0012] (5) Decolorization: The crude squalene from step (4) is added to a decolorizing agent for decolorization to obtain a decolorized solution;

[0013] (6) Chromatography: The decolorizing solution from step (5) is subjected to chromatography, and the sample eluent and elution are collected;

[0014] (7) Second concentration: The sample effluent and eluent from step (6) are concentrated a second time to obtain pure squalene.

[0015] In some embodiments, in step (1), the yeast is Saccharomyces cerevisiae ySC382, Saccharomyces cerevisiae CEN.PK2-1c, Saccharomyces cerevisiae BY4741, etc.

[0016] In some embodiments, the pretreatment method for yeast fermentation broth in step (1) is as follows: adjust the pH of yeast fermentation broth to 10-11, raise the temperature to 50-70℃, and keep it warm for 1.0-6.0h.

[0017] In some implementations, a ceramic membrane is selected for membrane filtration in step (1).

[0018] In some embodiments, in step (2), the amount of antioxidant added is 50-500 ppm of the weight of squalene in the bacterial concentrate.

[0019] In some embodiments, in step (2), the antioxidant is one or more of resveratrol, vitamin E, tert-butylcatechol, β-carotene, lycopene, and ergothioneine.

[0020] In some implementations, the parameters of the high-pressure homogenization in step (2) are as follows: pressure 500-1500 bar, temperature 5-15 degrees Celsius, and 3-5 cycles.

[0021] In some embodiments, in step (3), the extractant is added at a ratio of 50-200% of the volume of the homogenized liquid.

[0022] In some embodiments, in step (3), the extractant is any one of n-hexane, cyclohexane, petroleum ether, pentane, heptane, and isooctane.

[0023] In some embodiments, in step (3), the extraction is carried out as follows: stirring at room temperature for 0.5-1.0 h, centrifuging to obtain the organic phase; the extraction is performed 1 to 2 times.

[0024] In some embodiments, in step (4), a first concentration is performed by vacuum distillation at a temperature not exceeding 60°C.

[0025] In some embodiments, in step (5), the amount of the decolorizing agent added is 5-20% of the crude squalene mass.

[0026] In some embodiments, in step (5), the decolorizing agent is one or more of the following: white clay, diatomaceous earth, activated carbon, silica gel sand, and resin.

[0027] In some implementations, the chromatography in step (6) is column chromatography.

[0028] Furthermore, in column chromatography, the packing material is 200-600 mesh spherical silica gel, the sample loading amount is 10%-150% of the silica gel packing amount, and after loading, it is eluted with the same reagent as the extractant in step (3) for 5-10 BV.

[0029] In some embodiments, in step (7), a second concentration is performed by vacuum distillation and thin-film evaporator treatment.

[0030] Furthermore, in step (7), the temperature of the vacuum distillation does not exceed 60°C.

[0031] Furthermore, in step (7), the parameters of the thin film evaporator are as follows: vacuum degree below 10Pa, scraper speed 50-300rpm, and temperature 50-90℃.

[0032] The second technical solution provided by this invention is the application of the method described in the first technical solution in the preparation of squalene-containing products.

[0033] In some embodiments, the product includes daily chemical products, food, pharmaceuticals, and health products.

[0034] The technical effects of this invention are as follows:

[0035] This invention provides a method for separating and purifying biosynthesized squalene. This method can reduce the peroxide value of the extracted product by adding antioxidants, solvent pretreatment, and process condition control in a synergistic manner. The peroxide value can be as low as below 1 meq / kg, thus meeting the pharmaceutical grade peroxide value standard. The content and purity can reach more than 99%. Furthermore, a single organic solvent is used for extraction and chromatography, avoiding the difficulties of recycling multiple solvents. The solvents can be reused repeatedly, and the total extraction yield is as high as 90%, making it suitable for continuous industrial production. Attached Figure Description

[0036] Figure 1 is a flowchart of the process for separating and purifying squalene produced by biosynthesis fermentation according to the present invention.

[0037] Figure 2 is a purity detection spectrum of the squalene obtained in Example 1 of the present invention. Detailed Implementation

[0038] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0039] Technical terms used in the embodiments:

[0040] Squalene: The term "squalene" refers to a naturally occurring organic compound with the chemical formula C6H2O. 30 H 50 It is a colorless to pale yellow oily liquid, an unsaturated triterpenoid compound composed of six isoprene units linked together.

[0041] Fermentation broth: The term "fermentation broth" refers to a preparation produced by cell fermentation that is either unrecovered or recovered and / or purified. For example, fermentation broth is produced when a microbial culture is incubated to saturation under carbon-limited conditions that allow protein synthesis (e.g., expression of enzymes by the host cell) and secretion of proteins into the cell culture medium. The fermentation broth may contain the contents of the fermentation material obtained at the end of fermentation. For example, the fermentation broth may contain culture medium components utilized by the microorganisms, as well as cell debris remaining after the microbial cells can be removed by centrifugation.

[0042] Antioxidants: The term "antioxidant" refers to a chemical substance that can prevent or slow down the oxidation process, including but not limited to resveratrol, vitamin E, tert-butylcatechol, beta-carotene, lycopene, and ergothioneine.

[0043] High-pressure homogenization: The term "high-pressure homogenization" refers to a physical processing technique that homogenizes liquid materials by exposing them to extremely high pressure for a very short period of time and then rapidly releasing the pressure.

[0044] Extractant: The term "extractant" refers to a chemical substance used to extract a specific component from a mixture, including but not limited to n-hexane, cyclohexane, petroleum ether, pentane, heptane, and isooctane. Extraction processes typically involve two distinct phases: a mixture containing the target component and the extractant itself. The choice of extractant depends on its affinity for the target component and its interaction with other components in the mixture.

[0045] Decolorizing agent: The term "decolorizing agent" refers to a chemical reagent or natural substance used to remove color from a material, including but not limited to bleaching clay, diatomaceous earth, activated carbon, silica gel sand, and resin. Decolorizing agents are used to purify or alter the appearance of products, making them lighter or colorless. The mechanisms of action of decolorizing agents may include adsorption, chemical binding, and redox reactions.

[0046] Column chromatography: The term "column chromatography" refers to a separation technique used to separate different components in a mixture based on molecular size, charge, polarity, or specific affinity. This process typically involves a column containing a stationary phase through which a mobile phase (usually a liquid or gas) carries the components of the mixture upwards or through the column.

[0047] Vacuum distillation: The term "vacuum distillation" refers to a distillation process conducted at sub-atmospheric pressure. By reducing the system pressure, the boiling points of the components in the mixture can be lowered, thus allowing separation at lower temperatures. This method is particularly suitable for substances that may decompose at high temperatures or for which high-temperature operation is undesirable.

[0048] Aqueous soluble impurities: The term "aqueous soluble impurities" refers to substances that can dissolve in water. These substances can be organic or inorganic, natural or synthetic. They can originate from various sources, such as raw material contamination, chemical reaction byproducts, equipment corrosion products, and microbial metabolic products.

[0049] Test method:

[0050] 1. Squalene content detection:

[0051] 1.1 Reagent: n-Heptane (GC, >99%).

[0052] 1.2 Instruments: Analytical balance, gas chromatograph.

[0053] 1.3 Chromatographic conditions:

[0054] Chromatographic column: Agilent 19091J-413, HP-5, 30m × 0.32mm × 0.25μm, or equivalent column.

[0055] Heating program:

[0056] Column temperature: 100℃, carrier gas flow rate: 2.1mL / min, injection volume: 1μL, injection port temperature: 250℃, injection mode: split injection, split ratio 10:1, carrier gas saving: off, detector temperature: 280℃, hydrogen flow rate: 30mL / min, air flow rate: 400mL / min, make-up gas flow rate: 25mL / min, constant make-up gas + fuel gas flow rate.

[0057] 1.4 Standard Solution

[0058] 1.4.1 Squalene standard: 98% purity.

[0059] 1.4.2 Methyl laurate standard: purity 99.5%.

[0060] 1.4.3 Solvent: Weigh approximately 1368 g (2 L) of n-heptane solution, add 4 mL of methyl lauryl ester, shake well, and obtain an n-heptane solution containing the internal standard.

[0061] 1.4.4 Squalene Standard Stock Solution

[0062] Accurately weigh 0.100 g (accurate to 0.0001 g) of squalene standard into a 20 mL volumetric flask, add an appropriate amount of the above solvent to dissolve it, and then dilute to the mark with the solvent. Shake well to obtain a squalene standard stock solution of about 5000 μg / mL.

[0063] 1.4.5 Standard series solutions (i.e., standard curves)

[0064] 1.4.6 Accurately measure a certain amount of squalene standard stock solution, add the corresponding solvent, and prepare a series of standard solutions of about 500 to 5000 μg / mL. Take 150 μL of each standard solution and place them in a sample vial with an inner tube for later use.

[0065] 1.5 Analysis Steps

[0066] 1.5.1 Sample Preparation

[0067] Accurately weigh 0.100 g of the product (accurate to 0.0001 g) into a 20 mL volumetric flask, add an appropriate amount of solvent to dissolve, and then dilute to the mark with solvent and mix well. Transfer 150 μL to a sample vial with an inner tube for later use. (Note: The concentration of the prepared sample should be close to, but not exceed, the highest concentration of the standard curve; otherwise, the accuracy of the measurement results will be affected.)

[0068] 1.5.2 Sample Determination

[0069] 1.5.3.1 Inject the standard series solutions and the sample solution to be tested into the gas chromatograph for analysis, and record the peak areas of squalene and methyl lauryl ester.

[0070] 1.5.3.2 A standard curve was obtained by performing a linear regression on the squalene concentration using the ratio of the squalene peak area to the methyl laurate peak area in the standard solution. The ratio of the squalene peak area to the methyl laurate peak area in the sample solution was substituted into the standard curve to calculate the squalene concentration C (μg / mL) in the sample solution. If the concentration of the analyte in the sample solution exceeded the detection range, it was diluted with solvent before measurement, and the concentration was multiplied by the dilution factor during calculation.

[0071] 1.6 Results

[0072] The squalene content (X) in the sample is calculated using the following formula:

[0073] In the formula:

[0074] X — Squalene content in the sample, %;

[0075] C—The concentration of squalene in the sample solution, in μg / mL;

[0076] f—purity of the standard substance;

[0077] V—The volume of the sample after final volume adjustment, in mL;

[0078] D—Sample dilution factor;

[0079] m — Sample mass, in grams.

[0080] If the relative standard deviation of the parallel sample test results is ≤5%, the results are expressed as the average value; otherwise, resampling and testing are performed.

[0081] 2. Squalene yield = mass of 004 ​​in the extracted product / total mass of squalene in the untreated fermentation broth * 100%; In the following examples, the total squalene extraction yield was calculated by multiplying the crude product extraction yield by the decolorization to distillation extraction yield.

[0082] 3. Purity of squalene:

[0083] 3.1 Reagent: Dichloromethane (GC, >99%).

[0084] 3.2 Instrument: Gas chromatograph.

[0085] 3.3 Chromatographic column: HP-5 or equivalent chromatographic column.

[0086] Temperature gradient:

[0087] Initial column temperature: 100℃, injection volume: 0.5μL, split ratio: 50:1

[0088] 3.4 Sample preparation: The sample was purified. After diluting 20 times with dichloromethane, the sample was filtered through an organic membrane, and 200 μL of the filtrate was transferred to a vial for analysis.

[0089] 3.5 Sample Determination:

[0090] The sample solution to be tested is injected into a gas chromatograph for analysis, and the peak areas are recorded. All peaks in the chromatogram are integrated, and the sum of all peak areas is calculated. The purity of the target analyte is the ratio of the target analyte peak area to the total peak area.

[0091] 3.6 Calculation of Results

[0092] In the formula:

[0093] A 总 The sum of the areas of all peaks, A 目 Target peak area

[0094] 4. Peroxide value detection:

[0095] 4.1 Instruments and equipment: analytical balance, conical flasks (250mL*2), burette, iron stand, butterfly clamp, graduated cylinder (100mL)

[0096] 4.2 Reagents: Glacial acetic acid, chloroform, potassium iodide, soluble starch, sodium thiosulfate standard solution (0.1 mol / L)

[0097] 4.3 Reagent Preparation

[0098] 4.3.1 Chloroform-Glacial Acetic Acid Mixed Solution: Measure 40 mL of chloroform and 60 mL of glacial acetic acid, mix them, and set aside for later use;

[0099] 4.3.2 Saturated potassium iodide solution: Weigh 20g of potassium iodide, add 10mL of freshly boiled and cooled water, shake well and store in a brown bottle in a dark place for later use, ensuring that potassium iodide crystals precipitate in the solution.

[0100] 4.3.3 Starch indicator: Weigh 0.5g of soluble starch, add a small amount of water to make a paste, and pour in 50mL of boiling water while stirring. Boil and mix well. After the solution is clear, let it cool and use it for later use. Prepare it just before use.

[0101] 4.4 Analysis Steps

[0102] 4.4.1 Weigh 5g of sample (accurate to 0.0001g) and place it in a 250mL Erlenmeyer flask. Add 30mL of chloroform-glacial acetic acid mixture and gently shake to completely dissolve the sample.

[0103] 4.4.2 Add 0.5 mL of saturated potassium iodide solution, shake gently for 1 min, then add 30 mL of water and titrate with 0.01 M sodium thiosulfate standard solution, shaking the conical flask while titrating, until the solution turns pale yellow;

[0104] 4.4.3 Add another 5 mL of starch indicator; the solution will now turn blue. Continue adding 0.01 M sodium thiosulfate standard solution until the blue color disappears, at which point the experiment is complete.

[0105] 4.4.4 At the same time, add the same mass of purified water to another conical flask and perform a blank test according to the above steps.

[0106] 4.5 Result Calculation

[0107] In the formula:

[0108] X — Peroxide value of the sample (meq / kg);

[0109] m — Sample mass (g);

[0110] n1—The volume (mL) of sodium thiosulfate standard solution consumed by the sample;

[0111] n2 — Volume (mL) of sodium thiosulfate standard solution consumed in the blank.

[0112] 4.6 Precision

[0113] The absolute difference between two independent measurements obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

[0114] Raw materials used in the examples:

[0115] 1. Saccharomyces cerevisiae ySC382 is derived from a gene-edited strain independently developed by Senruis Biotechnology Co., Ltd. Its starting strain is Saccharomyces cerevisiae CEN.PK2-1c, which has been disclosed in the patent with publication number CN116635527A.

[0116] 2. Fermentation broth of Saccharomyces cerevisiae ySC382: The ySC382 strain was inoculated into a 10L fermenter and fermented continuously for 120 hours. The culture medium was YPD with an initial volume of 5L. Glucose was added starting after 12 hours of fermentation at a rate of 3g / L / h. The fermentation control conditions were: 30℃, 0.05MPa, rotation speed 300 to 600rpm, aeration rate 1vvm, and the correlation between rotation speed and aeration rate DO ≥ 30%.

[0117] Example 1

[0118] A method for extracting and purifying biosynthesized squalene, as shown in Figure 1, includes the following steps:

[0119] (1) Membrane separation: 6L of Saccharomyces cerevisiae ySC382 fermentation broth was subjected to alkaline heat treatment, and the pH of the fermentation broth was adjusted to 10.3 with sodium hydroxide. The broth was then heated to 60℃ and kept at that temperature for 2 hours. The broth was then concentrated to 2L by filtration through a ceramic membrane with a pore size of 50-200nm. Pure water was added for top washing to remove residual aqueous soluble impurities, and the concentrated cell solution was collected. The squalene content in the concentrated cell solution was determined to be 98.3g / L.

[0120] (2) High-pressure homogenization: Add 150 ppm of vitamin E (based on the weight of squalene in the bacterial concentrate) to a high-pressure homogenizer for cell disruption. The parameters of the high-pressure homogenizer are as follows: pressure 800-900 bar, temperature 5-15 degrees Celsius, 5 cycles to obtain a homogenized solution.

[0121] (3) Extraction: n-hexane is degassed and pretreated by a deoxygenation system. 1L of n-hexane is added to the homogenate for extraction. The mixture is stirred for 15-20 minutes and then centrifuged to obtain the n-hexane phase. After two extractions, 1.95L of extract is obtained.

[0122] (4) First concentration: The extract was vacuum distilled (reduced pressure distillation) at 60°C to obtain crude squalene with a content of 83% and a crude product extraction yield of 95.8%.

[0123] (5) Decolorization: Add 15% by mass of white clay to crude squalene, stir at room temperature for 20 minutes, filter, and purge with nitrogen to obtain decolorized liquid.

[0124] (6) Chromatography: The decolorized solution was subjected to column chromatography using 200-300 mesh spherical silica gel. Hexane was used for elution. The hexane was pretreated by degassing through an oxygen removal system. The silica gel packing amount was 0.8 times the crude product mass, the height-to-diameter ratio was 4.5:1, and the elution volume was 5 BV. The sample effluent and elution were collected.

[0125] (7) Second concentration: The sample eluent and cyclohexane eluent were combined and distilled under reduced pressure. Most of the solvent was first evaporated under negative pressure, and then treated with a thin-film evaporator to obtain a colorless, odorless, transparent oily liquid at a temperature of 60°C, a scraper speed of 250 rpm, and a vacuum degree of 25 Pa. The final product had a purity of 99.67% (Figure 2), a content of 99.5%, a solvent residue reduced to 58 ppm, and a peroxide value of 0.27 meq / kg. The yield of the product obtained by decolorization and distillation was 94.5%, and the total extraction yield was 90.53%.

[0126] Example 2

[0127] A method for extracting and purifying biosynthesized squalene, comprising the following steps:

[0128] (1) Membrane separation: 6L of Saccharomyces cerevisiae ySC382 fermentation broth was subjected to alkaline heat treatment, and the pH of the fermentation broth was adjusted to 10.39 with sodium hydroxide. The broth was then heated to 60℃ and kept at that temperature for 2 hours. The broth was then concentrated to 2.1L by filtration through a ceramic membrane. Pure water was added for top washing to remove residual aqueous soluble impurities, and the concentrated cell solution was collected. The squalene content in the concentrated cell solution was determined to be 100.5g / L.

[0129] (2) High pressure homogenization: Add 170 ppm of resveratrol based on the weight of squalene in the bacterial concentrate, and perform cell disruption treatment in a high pressure homogenizer. The parameters of the high pressure homogenizer are as follows: pressure 900-1000 bar, temperature 5-15 degrees Celsius, 5 cycles to obtain homogenized liquid.

[0130] (3) Extraction: n-hexane is degassed and pretreated by a deoxygenation system. 1L of n-hexane is added to the homogenate for extraction. The mixture is stirred for 15-20 minutes and then centrifuged to obtain the n-hexane phase. After two extractions, 1.95L of extract is obtained.

[0131] (4) First concentration: The extract was vacuum distilled at 60°C to obtain crude squalene with a content of 84.3% and a crude product extraction yield of 96.4%.

[0132] (5) Decolorization: Add 12% activated carbon to crude squalene, stir at room temperature for 20 minutes, filter, and purge with nitrogen to obtain decolorized solution.

[0133] (6) Chromatography: The decolorized solution was subjected to column chromatography using 300-400 mesh spherical silica gel. Hexane was used for elution. The hexane was pretreated by degassing through a deoxygenation system. The silica gel packing amount was 1.0 times the crude product mass, the height-to-diameter ratio was 4.7:1, and the elution volume was 4.7 BV. The sample effluent and elution were collected.

[0134] (7) Second concentration: The sample eluent and cyclohexane eluent were combined and distilled under reduced pressure. Most of the solvent was first evaporated under negative pressure, and then treated with a thin-film evaporator to obtain a colorless, odorless, transparent oily liquid at 60°C, 250 rpm, and 25 Pa vacuum. The final product had a purity of 99.56%, a content of 99.23%, a solvent residue of 70 ppm, and a peroxide value of 0.73 meq / kg. The yield of the product obtained by decolorization and distillation was 94.2%, and the total extraction yield was 90.8%.

[0135] Example 3

[0136] A method for extracting and purifying biosynthesized squalene, comprising the following steps:

[0137] (1) Membrane separation: 6L of Saccharomyces cerevisiae ySC382 fermentation broth was subjected to alkaline heat treatment, the pH of the fermentation broth was adjusted to 10.7 with sodium hydroxide, and the temperature was raised to 60℃ and kept at 2h. Then, it was concentrated to 2.1L by ceramic membrane filtration, and pure water was added for top washing to remove residual aqueous soluble impurities. The concentrated cell solution was collected. The squalene content in the concentrated cell solution was detected to be 97.9g / L.

[0138] (2) High pressure homogenization: Add 100 ppm tert-butylcatechol based on the weight of squalene in the bacterial concentrate, and perform cell disruption treatment in a high pressure homogenizer. The parameters of the high pressure homogenizer are as follows: pressure 900-1000 bar, temperature 5-15 degrees Celsius, 5 cycles to obtain homogenized liquid.

[0139] (3) Extraction: n-hexane was degassed and pretreated by a deoxygenation system. 1.2L of n-hexane was added to the homogenate for extraction. The mixture was stirred for 15-20 minutes and then centrifuged to obtain the n-hexane phase. After two extractions, 2.3L of extract was obtained.

[0140] (4) First concentration: The extract was vacuum distilled at 60°C to obtain crude squalene with a content of 83% and a crude product extraction yield of 97.2%.

[0141] (5) Decolorization: Add 15% by mass of white clay to crude squalene, stir at room temperature for 20 minutes, filter, and purge with nitrogen to obtain decolorized liquid.

[0142] (6) Chromatography: The crude squalene in the decolorized solution was subjected to column chromatography using 300-400 mesh spherical silica gel packed into a column and eluted with n-hexane. The n-hexane was pretreated by degassing through an oxygen removal system. The silica gel packing amount was 1.2 times the mass of the crude product, the height-to-diameter ratio was 5:1, and the elution volume was 5.2 BV. The sample effluent and the eluent were collected.

[0143] (7) Second concentration: The sample eluent and cyclohexane eluent were combined and distilled under reduced pressure. Most of the solvent was first evaporated under negative pressure, and then treated with a thin-film evaporator to obtain a colorless, odorless, transparent oily liquid at a temperature of 60°C, a scraper speed of 250 rpm, and a vacuum of 20 Pa. The final product had a purity of 99.7%, a content of 99.5%, a solvent residue reduced to 49 ppm, and a peroxide value of 0.54 meq / kg. The yield of the product obtained by decolorization and distillation was 94.6%, and the total extraction yield was 91.95%.

[0144] Example 4

[0145] The squalene obtained in Examples 1-3 can be used as a moisturizer in cosmetics and has the functions of an antioxidant and a free radical scavenger. It can be used in products such as creams, lotions, hair oils, hair creams, lipsticks, fragrance oils and powders.

[0146] The squalene obtained in Examples 1-3 can also be added to pharmaceuticals for oral administration to treat various diseases, such as hypertension, hypotension, anemia, diabetes, cirrhosis, and cancer. For external application, it can be used to treat tonsillitis, asthma, bronchitis, gastric ulcers, rheumatism, and neuralgia.

[0147] The squalene obtained in Examples 1-3 can also be added to food or health products. Squalene can promote hepatocyte regeneration and protect hepatocytes, thereby improving liver function.

[0148] Comparative Example 1

[0149] In Example 1, the amount of antioxidant vitamin E added was 5 ppm of the weight of squalene in the bacterial concentrate. Other conditions or parameters were the same as in Example 1. The extracted squalene was a colorless, transparent, oily liquid with a purity of 98.7%, a content of 98.9%, a solvent residue reduced to 55 ppm, and a peroxide value of 6.08 meq / kg.

[0150] Comparative Example 2

[0151] The decolorization step in Example 1 was omitted, and other conditions or parameters were the same as in Example 1. The extracted squalene was a pale yellow oily liquid with a purity of 98.1%, a content of 97.9%, a solvent residue reduced to 65 ppm, and a peroxide value of 6.3 meq / kg.

[0152] Comparative Example 3

[0153] In Example 1, the vacuum distillation temperature was changed to 90°C, while other conditions or parameters remained the same as in Example 1. The extracted squalene was a colorless, transparent, oily liquid with a purity of 98.07%, a content of 98.78%, a solvent residue reduced to 109 ppm, and a peroxide value of 4.9 meq / kg.

[0154] Comparative Example 4

[0155] In Example 1, the final step of the thin-film evaporator was replaced with a vacuum distillation column to further reduce the solvent content. The vacuum degree was 230-250 Pa, the magnetic stirring speed at the bottom of the column was 1020 rpm, the heating temperature was 70℃, and the heating treatment time was maintained for 1.5 h. Other conditions or parameters were the same as in Example 1. The extracted squalene was a colorless, transparent, oily liquid with a purity of 98.34% and a content of 98.27%. The solvent residue was reduced to 753 ppm, and the peroxide value was 15.05 meq / kg.

[0156] In summary, this invention provides a method for the separation and purification of biosynthesized squalene. Squalene is an intracellular product, an acyclic triterpenoid composed of six isoprene bonds. Due to its six double bonds, it is extremely unstable and easily oxidized. The separation and purification process first removes water-soluble impurities from the fermentation broth, enriches the bacterial cells, and then extracts a crude product rich in squalene through cell disruption. During this process, antioxidants are added to inhibit squalene oxidation. The extraction process also extracts some lipid-soluble impurities, including dextran, glycoproteins, fatty acids, phospholipids, and pigments. These impurities are removed through adsorption in a subsequent decolorization step before column chromatography. This increases product content, reduces peroxide value, improves the color and oxidation stability of the crude product, and prevents these relatively polar impurities from clogging the chromatography column. This allows for increased sample loading in the purification process, significantly improving processing efficiency and scale-up production capacity. Furthermore, the solvent removal process in the crude and finished product distillation steps is best performed under vacuum at low temperatures, resulting in a shorter residence time in the evaporator and reducing the likelihood of squalene deterioration.

[0157] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for the extraction and purification of biosynthetically produced squalene, characterized in that, Includes the following steps: (1) Membrane separation: The pretreated yeast fermentation broth is filtered through a membrane to remove the supernatant, the cells are collected, and then washed to obtain a concentrated cell solution. (2) High pressure homogenization: Add antioxidant to the bacterial cell concentrate in step (1), and then perform cell disruption and homogenization to obtain homogenized liquid; (3) Extraction: Add an extractant to the homogenized liquid from step (2) to extract the extract; (4) First concentration: The extract from step (3) is concentrated for the first time to obtain crude squalene; (5) Decolorization: The crude squalene from step (4) is added to a decolorizing agent for decolorization to obtain a decolorized solution; (6) Chromatography: The decolorizing solution from step (5) is subjected to chromatography, and the sample eluent and elution are collected; (7) Second concentration: The sample effluent and eluent from step (6) are concentrated a second time to obtain pure squalene.

2. According to the method of claim 1, in step (1), the yeast is Saccharomyces cerevisiae ySC382, Saccharomyces cerevisiae CEN.PK2-1c or Saccharomyces cerevisiae BY4741; 3. The method of claim 1, wherein, In step (1), the pretreatment method of yeast fermentation broth is as follows: adjust the pH of yeast fermentation broth to 10-11, raise the temperature to 50-70℃, and keep it warm for 1.0-6.0h.

4. The method of claim 1, wherein, In step (2), the amount of antioxidant added is 50-500 ppm of the weight of squalene in the bacterial cell concentrate; The antioxidant is one or more of the following: resveratrol, vitamin E, tert-butylcatechol, beta-carotene, lycopene, and ergothioneine; 5. The method of claim 1, wherein, In step (2), the parameters of the high-pressure homogenization are as follows: pressure 500-1500 bar, temperature 5-15 degrees Celsius, and 3-5 cycles.

6. The method according to claim 1, characterized in that, In step (3), the proportion of the extractant added is 50-200% of the volume of the homogenized liquid; The extractant is any one of n-hexane, cyclohexane, petroleum ether, pentane, heptane, and isooctane; The extraction method is as follows: stir at room temperature for 0.5-1.0 h, centrifuge to obtain the organic phase; the extraction is performed 1-2 times.

7. The method according to claim 1, characterized in that, In step (4), the first concentration is carried out by vacuum distillation, and the temperature of the vacuum distillation does not exceed 60°C.

8. The method according to claim 1, characterized in that, In step (5), the amount of decolorizing agent added is 5-20% of the crude squalene mass; The decolorizing agent is one or more of the following: kaolin, diatomaceous earth, activated carbon, silica gel sand, and resin.

9. The method according to claim 1, characterized in that, In step (6), the chromatography is column chromatography. In the column chromatography, the packing material is 200-600 mesh spherical silica gel, the sample loading amount is 10%-150% of the silica gel packing amount, and after loading, the sample is eluted with the same reagent as the extractant in step (3) for 5-10 BV.

10. The method according to claim 1, characterized in that, In step (7), a second concentration is carried out by vacuum distillation and thin-film evaporator treatment.

11. The method according to claim 11, characterized in that, The temperature of the vacuum distillation shall not exceed 60°C; The parameters of the thin-film evaporator are as follows: vacuum degree below 10Pa, scraper speed 50-300rpm, temperature 50-90℃.

12. The use of the method according to any one of claims 1 to 11 in the preparation of squalene-containing products.

13. The application according to claim 12, wherein the product includes daily chemical products, food, pharmaceuticals, and health products.

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