CN116947589A - Extraction and purification method for biosynthesis squalene - Google Patents
Extraction and purification method for biosynthesis squalene Download PDFInfo
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- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 title claims abstract description 62
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 title claims abstract description 62
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 title claims abstract description 62
- 229940031439 squalene Drugs 0.000 title claims abstract description 62
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 title claims abstract description 62
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- VWFJDQUYCIWHTN-YFVJMOTDSA-N 2-trans,6-trans-farnesyl diphosphate Chemical group CC(C)=CCC\C(C)=C\CC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-YFVJMOTDSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/005—Processes comprising at least two steps in series
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/04—Purification; Separation; Use of additives by distillation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/10—Purification; Separation; Use of additives by extraction, i.e. purification or separation of liquid hydrocarbons with the aid of liquids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/12—Purification; Separation; Use of additives by adsorption, i.e. purification or separation of hydrocarbons with the aid of solids, e.g. with ion-exchangers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Water Supply & Treatment (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of squalene extraction, and particularly relates to an extraction and purification method of biosynthetic squalene, which comprises the steps of preparation of saccharomyces cerevisiae fermentation liquor, washing thalli after centrifugation, high-pressure homogenization and crushing, two-phase extraction rotary steaming, column chromatography rotary steaming and sterilization filtration, or the steps of two-phase extraction rotary steaming and column chromatography rotary steaming are replaced by the steps of drying thalli, supercritical extraction and molecular distillation. The invention has simple purification process, high recovery rate and higher product purity, the purity can be more than 97 percent, and the purity can also reach 100 percent; and the use of organic reagents can be avoided, the cost is greatly saved, the environment is protected, and industrialization and popularization are easy to realize.
Description
Technical Field
The invention belongs to the technical field of squalene extraction, and particularly relates to an extraction and purification method for biosynthesis of squalene.
Background
Squalene is a triterpene, an intermediate in the synthesis of sterols by plants, animals and humans, and is widely found in plants, animals and humans. At first, vacuum fractionating is carried out on liver oil of deep sea shark to obtain unsaturated hydrocarbon with chemical formula of C 30 H 5 0, the structural formula is as follows:
。
squalene is synthesized by squalene synthetase and converts two units of farnesyl pyrophosphate (the direct precursor of terpenes and steroids) into squalene.
Vitamin D biosynthesis as a secondary steroid is also regulated by squalene. In addition, squalene is an important component of the human body as a precursor to each steroid family. In humans, the concentration of squalene in neonatal blood is highest, but its content suddenly begins to decline between the ages of 30 and 40.
In the human body, squalene is synthesized by the liver and is secreted in large quantities by sebaceous glands, accounting for 12% of the sebaceous gland-secreted lipids, transported in the blood by small-density lipoproteins and very small-density lipoproteins. The highest concentration of squalene in humans occurs at about 500 μg/g in skin lipids and about 300 μg/g in adipose tissue; whereas in organs where active biosynthesis takes place, the concentration is much lower, for example 75. Mu.g/g in the liver or 42. Mu.g/g in the small intestine.
Squalene has wide application: the application of squalene in skin care products is that the stability of squalene to free radicals enables the skin surface of human body to resist lipid peroxidation, and can be used as an antioxidant, an antistatic agent, an emollient and the like in moisturizing cream. The application of squalene in health products is that squalene has relieving and protecting effects on brain workers with excessive and insufficient brain for a long time, people with frequent fatigue, sleepiness, dizziness, memory decline and inattention, fatty liver patients and people who often drink wine and have heavy liver burden. The squalene is used as an intermediate for synthesizing various alkanes and sterols. The application of squalene in disease prevention and treatment has very wide application in disease treatment and prevention. The MF59 adjuvant and AS03 adjuvant prepared by mixing squalene with Tween-80, span 85, tocopherol and the like in proportion are applied to a plurality of approved marketed vaccines, and the safety and effectiveness of a novel adjuvant prepared by squalene are fully proved by large-scale inoculation in the global scope, thus making a great contribution to the disease prevention of human beings.
Squalene can play a certain role in preventing breast cancer, pancreatic cancer, colon cancer and similar tumors related to Ras oncogene mutation. Squalene may play an anticancer role through the following mechanisms:
(1) Indirectly inhibiting HMG-CoA reductase, inhibiting Ras oncoprotein farnesylation, limiting HMG-CoA (3-hydroxy-3-methylglutaryl CoA) conversion to mevalonate necessary for DNA synthesis and cell proliferation through negative feedback regulation of endogenous cholesterol synthesis;
(2) Regulating biosynthesis and function of exogenous metabolic enzymes, thereby altering metabolic activation of the carcinogen;
(3) As a free radical scavenger.
Squalene is considered to be an important factor for the lack of cancer in shark, because shark liver oil contains more than 40% of squalene, and squalene is also considered to be an important factor for the low incidence of cancer in humans in Mediterranean regions, because the diet of people in Mediterranean regions contains a large amount of olives, which are rich in squalene.
The wide application of squalene results in great demand, and the squalene is mainly derived from shark liver, vegetable oil, chemical synthesis, biosynthesis, etc. The extraction and purification method of the squalene mainly comprises the following steps: supercritical extraction, molecular distillation, ion exchange chromatography, reversed phase chromatography, rectification, freeze crystallization, high performance countercurrent chromatography, two-phase extraction, saponification, methyl esterification, reduced pressure distillation, etc.
The specification of Chinese patent CN102146014A discloses a process for extracting squalene by taking camellia seeds as raw materials, which comprises the steps of preparing raw materials, crushing, leaching, concentrating, methyl esterification, complexing agent-borax reaction, separating and purifying by macroporous resin chromatography, concentrating, extracting, washing, concentrating to obtain crude squalene oil and performing supercritical carbon dioxide extraction to obtain squalene essential oil with squalene content of more than or equal to 80%. The specification of Chinese patent CN113604512A discloses a process for extracting squalene from schizochytrium, which comprises the steps of adding alkaline protease into fermentation liquor to carry out enzymolysis wall breaking on schizochytrium; adding absolute ethyl alcohol into the bacterial liquid after wall breaking, removing supernatant water-ethanol phase through shaking centrifugal separation, and collecting bottom bacterial mud; adding n-hexane into the bottom bacterial sludge, oscillating and centrifuging to collect supernatant, repeating for several times until the supernatant is colorless and transparent, evaporating to remove n-hexane, and collecting to obtain squalene crude product; and (3) degumming, deacidifying, bleaching and deodorizing the squalene crude product to obtain purified squalene. Both the two methods can obtain squalene products, but the process steps are complicated, the process period is longer, the purity of the obtained squalene is relatively lower, and the cost is higher.
The prior art, although capable of obtaining squalene, has a purity of up to 96% by extraction alone, as disclosed in the above-mentioned patent. However, there is no method for extracting squalene with a purity of 97% or more, and it is difficult to increase the extraction purity from 96% to 97% or more. Because various squalene-derived hosts themselves contain many substances that are very similar in nature to squalene, they are difficult to separate by various methods, are extremely demanding in terms of process conditions, or are extremely costly to produce on a large scale.
Disclosure of Invention
In order to solve the technical problems, the invention provides an extraction and purification method for biosynthesis of squalene, which has the advantages of simple process, high recovery rate, higher product purity which can be more than 97%, and purity which can also reach 100%; and the use of organic reagents is avoided, the cost is greatly saved, the environment is protected, and industrialization and popularization are easy to realize.
The invention provides an extraction and purification method for biosynthesis squalene, which solves the technical problems, and comprises the following steps:
(1) Preparing a saccharomyces cerevisiae fermentation liquor;
(2) Washing the thalli after centrifugation: centrifugally collecting thalli, adding distilled water to resuspend and wash thalli, centrifuging and then collecting sediment;
(3) High-pressure homogenizing and crushing: adding the crushed extracting solution and the precipitate, mixing, and crushing by using a high-pressure homogenizer after uniformly stirring; high-pressure homogenizing, crushing and extracting, good crushing effect and high extraction efficiency.
(4) Two-phase extraction rotary distillation: stirring and extracting by charging nitrogen, filtering liquid phase, standing and layering, then taking organic phase components for reduced pressure distillation, and removing organic solvent to obtain crude extract; adding normal phase equilibrium liquid to dissolve to obtain normal phase sample; the normal phase chromatography has better condition separation effect and higher recovery rate.
(5) Column chromatography and rotary evaporation: normal phase preparation chromatography, loading samples according to the proportion of 5-10% of the volume of a chromatographic column, collecting each component in a segmented way, mixing target components with the detection result meeting the requirements, and removing the organic reagent by rotary evaporation; the detection result meets the requirement that the GC detection purity is more than 97%;
(6) Sterilizing and filtering to obtain the final product.
The mass volume ratio of the precipitation to the crushing extracting solution in the step (3) is 1:1-10.
In the optimization scheme, the mass volume ratio of the precipitation to the crushing extracting solution is 1:1. 1:2. 1: 3. 1:4. 1:5. 1:6. 1: 7. 1:8. 1:9 or 1:10.
the crushed extracting solution in the step (3) is a mixed solution of purified water and an organic reagent, wherein the organic reagent is n-hexane, ethyl acetate, methylene dichloride or petroleum ether, and the purified water and the organic reagent are mixed according to the weight ratio of 1:1 or 1:2 or 1:3 or 1:4 or 1:5 ratio. The crushed extract can effectively separate the target substance from other water-soluble impurities.
The two-phase extraction rotary steaming in the step (4) specifically comprises the following steps: adding nitrogen, stirring, extracting, filtering liquid phase, standing, layering, extracting, centrifuging, adding into rotary steaming bottle, distilling under reduced pressure at 50-60deg.C for 4-6min at 50r/min, and in optimized scheme for 5min at 55 deg.C for 50r/min, and completely extracting organic reagent to obtain crude extract; adding dichloromethane, mixing, adding 90ml of purified water, standing for layering, adding the lower dichloromethane phase into a rotary steaming bottle, distilling under reduced pressure, rotating at 50-60 ℃ for 5min at 50r/min and 55 ℃ for 50r/min in an optimized scheme, adding normal phase equilibrium liquid, mixing, pouring out, adding normal phase equilibrium liquid of the same volume again, mixing, pouring out, and mixing with the previous solution to obtain the normal phase preparation chromatographic sample. Since a part of the water-soluble substances is insoluble in methylene dichloride, a part of impurities can be removed, and the methylene dichloride plays a role in purification.
The column chromatography in the step (5) is specifically as follows: a normal phase is selected for preparing the chromatographic column,
balance: 3ml/min, balancing with normal phase balancing solution 100ml until the ultraviolet reaches 199 nm;
loading: 1ml/min, 1ml was loaded.
Eluting: 2ml/min, solution A: normal phase equilibrium liquid, method: 0-100min, 100% A, 2ml/min, ultraviolet detection 199nm, and manual collection according to peak shape; sampling each collected component for GC detection;
regeneration: 2ml/min, 200ml of the column was backflushed using n-hexane.
In the optimized scheme, the normal phase equilibrium liquid is 0.1-99.9% of normal hexane-99.9-0.1% of ethyl acetate; in a further optimized scheme, the normal phase equilibrium liquid is 99% normal hexane-1% ethyl acetate.
The contents of the steps (3) to (5) are replaced by a step of drying thalli, supercritical extraction and molecular distillation, and the steps are as follows: adding the crushing liquid and the precipitate, mixing, and crushing by using a high-pressure homogenizer after uniformly stirring; drying the crushed thalli; and (3) sequentially carrying out supercritical carbon dioxide extraction, filtration and molecular distillation on the dried bacteria.
The replaced preparation method has high overall extraction recovery rate, extremely high purity, simple preparation method and no use of organic reagent, thereby greatly reducing the production cost and being a green, ecological and environment-friendly extraction method.
The preparation method after replacement specifically comprises the following steps:
(1) Preparing a saccharomyces cerevisiae fermentation liquor;
(2) Washing the thalli after centrifugation: centrifugally collecting thalli, adding distilled water to resuspend and wash thalli, centrifuging and then collecting sediment;
(3) High-pressure homogenizing and crushing: adding the crushing liquid and the precipitate, mixing, and crushing by using a high-pressure homogenizer after uniformly stirring;
(4) Drying the thalli; drying the crushed thalli; and (3) feeding the complete thalli into a high-pressure homogenizer for homogenizing and crushing, wherein the crushed thalli is obtained after the cell walls are crushed.
(5) The dried bacteria obtained after drying are subjected to supercritical carbon dioxide extraction, filtration and molecular distillation in sequence;
(6) Sterilizing and filtering to obtain the final product.
The drying adopts a vacuum drying, nitrogen charging drying or freeze-drying mode; in the optimized scheme, the drying is vacuum drying, and the crushed thalli are put into a vacuum drying box for drying at the drying temperature of 50-90 ℃ for 5-20 hours.
The thallus is broken and has serious emulsification phenomenon, most of objects and water form oil-in-water, so that the extraction effect is poor.
In a further optimized scheme, the crushing liquid in the step (3) is purified water, and the mass-volume ratio of the sediment to the crushing liquid is 1:2-10.
The supercritical carbon dioxide extraction is carried out by loading all crushed dry bacteria into a filter bag, and then placing into an extraction kettle, wherein the supercritical carbon dioxide extraction pressure is 5-40Mbar, the temperature is 25-60 ℃, and the CO is 2 The flow rate is 1-10ml/min, the extraction time is 1-10 hours, and the extraction components are collected. Extracting for 3 hours at 35 ℃ under 25mpa under CO in the optimized scheme 2 The flow rate was 5ml/min.
The molecular distillation adopts a first-stage molecular distillation for collecting a heavy phase and a second-stage molecular distillation for collecting a light phase. The primary distillation and the secondary molecular distillation are adopted to ensure that the purification effect is better.
The molecular distillation comprises the following steps:
s1, cleaning molecular distillation equipment, and adding liquid nitrogen into a cold trap;
s2: primary molecular distillation: heating the equipment to 90-140 ℃, vacuum 0.1-5pa and 240r/min, adding supercritical extraction components, filtering, slowly opening a liquid inlet, controlling the liquid inlet flow rate to 0.1-1.5ml/min, and collecting heavy phase and light phase respectively; in the optimized scheme, the equipment is heated to 110 ℃ and is subjected to vacuum 2pa and 240r/min, and the flow rate is controlled to be 1ml/min.
S3: secondary molecular distillation: heating the equipment to 160-240 ℃ and vacuum 0.1-5pa and 240r/min, adding the first molecular distillation to collect the heavy phase component, slowly opening a liquid inlet, controlling the liquid inlet flow rate to 0.1-1.5ml/min, and collecting the heavy phase component and the light phase component respectively. In the optimized scheme, the equipment is heated to 210 ℃ and is subjected to vacuum 2pa and 240r/min, and the inflow flow rate is controlled to be 1ml/min.
The Saccharomyces cerevisiae is Saccharomyces cerevisiae BY4742 and Saccharomyces cerevisiae CEN.PK2-1C.
The molecular distillation technology principle in the invention: molecular distillation is a special liquid-liquid separation technology, which is different from traditional distillation by means of the principle of boiling point difference separation, but realizes separation by means of the difference of the moving average free path of molecules of different substances. When the liquid mixture flows along the heating plate and is heated, light and heavy molecules can escape from the liquid surface to enter the gas phase, and due to different free ranges of the light and heavy molecules, the molecules of different substances escape from the liquid surface and have different moving distances, and a condensing plate is arranged, so that the light molecules reach the condensing plate to be condensed and discharged, and the heavy molecules can not reach the condensing plate to be discharged along the mixed liquid, thereby achieving the aim of separating substances.
Supercritical extraction technology principle: the supercritical fluid has special dissolving action on fatty acid, plant alkali, ether, ketone, glyceride and the like, and utilizes the relation between the dissolving capacity of the supercritical fluid and the density thereof, namely the influence of pressure and temperature on the dissolving capacity of the supercritical fluid. In the supercritical state, the supercritical fluid is contacted with the substance to be separated to selectively extract the components with different polarity, boiling point and molecular weight. Of course, the extracts obtained corresponding to the pressure ranges cannot be single, but the conditions can be controlled to obtain the mixed components with the optimal proportion, then the supercritical fluid is changed into common gas by means of decompression and temperature rising, and the extracted substances are separated out, thereby achieving the aim of separation and purification.
The raw materials of the invention are derived from Saccharomyces cerevisiae biosynthesis, the cost of the raw materials is low, the proportion of the dry weight containing target products is high, compared with the extraction of squalene from sharks, the raw material quantity is not limited, and the marine heavy metal and animal source impurities are not contained.
The extraction and purification process is simple, has short process period, high recovery rate and low process cost, and the purity of the final product of the extraction and purification process is high, and the GC detection purity is higher than 99 percent and can reach 100 percent. The extraction and purification process is easy to amplify, and can realize large-scale production. In addition, the process can avoid the use of organic reagent to the maximum extent, thereby greatly reducing the cost.
Drawings
FIG. 1 is a flow chart of the purification process in examples 1-3 of the present invention;
FIG. 2 is a flow chart of the purification process in example 4 of the present invention;
FIG. 3 is a GC detection spectrum of the normal phase preparative chromatography collection component of example 1 of the present invention;
FIG. 4 is a GC detection spectrum of the normal phase preparative chromatography collection component of example 2 of the present invention;
FIG. 5 is a GC detection spectrum of the normal phase preparative chromatography collection fraction of example 3 of the present invention;
FIG. 6 is a GC analysis chart of the light phase collected fraction of the second molecular distillation of example 4 of the present invention.
Detailed Description
The invention is further illustrated in the following description in connection with the specific embodiments in which the Saccharomyces cerevisiae fermentation broth is a conventional product, commercially available from Saccharomyces cerevisiae:
example 1
As shown in FIG. 1, the sample is derived from Saccharomyces cerevisiae CEN.PK2-1C fermentation broth, centrifugated (8000-10000 r,5 min) to collect precipitate, taken out 5g, adding 50ml of crushed extract, homogenizing under high pressure, crushing under 800-1400bar, 3-5 times, stirring under nitrogen filling, extracting for 2 hours, filtering with 0.22um needle filter, pouring into a separating funnel, standing and layering for use. The needle filter is conventional filtering equipment and separates large particles such as thalli from the extracting solution.
The mass volume ratio of the precipitation to the crushing extracting solution is 1:10, crushing the extracting solution to obtain a mixed solution of purified water and an organic reagent, wherein the organic reagent is ethyl acetate, and the purified water and the organic reagent are mixed according to a ratio of 1:2 proportion.
Two-phase extraction rotary distillation:
filtering, standing, adding 250ml rotary steaming bottle into 40ml supernatant, rotating at 55deg.C for 5min at 50r/min, completely spinning ethyl acetate, adding 45ml dichloromethane, mixing, adding purified water 90ml, mixing, standing, and layering. The methylene dichloride plays a role in purification.
Adding the dichloromethane phase into a 250ml rotary steaming bottle, rotating for 5min at 55 ℃ at 50r/min, completely spinning out the dichloromethane, adding 5ml of 99% n-hexane-1% ethyl acetate by volume, uniformly mixing, pouring out, adding 5ml of 99% n-hexane-1% ethyl acetate by volume again, uniformly mixing, pouring out, and mixing uniformly with the previous mixture to obtain the normal phase preparation chromatographic sample.
Normal phase preparation chromatography:
the normal phase preparation chromatographic column (NMSIL 50-100, 10)250mm）；
Balance: 3ml/min, 100ml equilibrated with normal phase equilibration solution (0.1-99.9% normal hexane-99.9-0.1% ethyl acetate) until UV 199nm levels off.
Loading: 1ml/min, 1ml was loaded.
Eluting: 2ml/min, solution A: normal phase equilibrium liquid (0.1-99.9% normal hexane-99.9-0.1% ethyl acetate), method: 0-100min, 100% A, 2ml/min, ultraviolet detection 199nm, manual collection according to peak shape. Sampling each collected component and performing GC detection, sampling each collected component and performing GC detection (using an equipment Agilent 8890 GC System according to an EP.10 GC detection method, wherein the GC detection is performed by using the equipment method unless otherwise specified), mixing components with purity of more than 97%, adding into a 250ml rotary steaming bottle, rotating at 55 ℃ for 20min at 50r/min, and filtering by using a 0.22um filter after the organic reagent is spun out to perform GC detection, as shown in table 1 and fig. 3. Regeneration: 2ml/min, 200ml of the column was backflushed using n-hexane. The recovery rate is more than 90 percent.
Table 1 GC detection of purity of normal phase collected fractions
As is clear from Table 1 above, the purity of the fraction collected by the detection section was 99.35%.
Example 2
The sample is from Saccharomyces cerevisiae BY4742 fermentation broth, centrifugate (8000-10000 r,5 min) to collect precipitate, take out 5g, add crushed extract 40ml, high pressure homogenize and crush for 800-1400bar, 3-5 times, mix with nitrogen and extract for 2 hours, filter with 0.22um needle filter for use.
The mass volume ratio of the precipitation to the crushing extracting solution is 1:8. crushing the extracting solution to obtain a mixed solution of purified water and an organic reagent, wherein the organic reagent is dichloromethane, and the purified water and the organic reagent are prepared according to the following weight ratio of 1:3 proportion.
Two-phase extraction rotary distillation:
filtering, standing, adding 250ml rotary steaming bottle into 40ml supernatant, rotating at 55deg.C for 5min at 50r/min, completely spinning out dichloromethane, adding 45ml dichloromethane, mixing, adding purified water 90ml, mixing, standing, and layering.
Adding the dichloromethane phase into a 250ml rotary steaming bottle, rotating for 5min at 55 ℃ at 50r/min, completely spinning out the dichloromethane, adding 5ml of 99% n-hexane-1% ethyl acetate by volume, uniformly mixing, pouring out, adding 5ml of 99% n-hexane-1% ethyl acetate by volume again, uniformly mixing, pouring out, and mixing uniformly with the previous mixture to obtain the normal phase preparation chromatographic sample.
Normal phase preparation chromatography:
the normal phase preparation chromatographic column (NMSIL 50-100, 10)250mm）
Balance: 3ml/min, 100ml equilibrated with normal phase equilibration solution (0.1-99.9% normal hexane-99.9-0.1% ethyl acetate) until UV 199nm levels off.
Loading: 1ml/min, 1ml was loaded.
Eluting: 2ml/min, solution A: normal phase equilibrium liquid (0.1-99.9% normal hexane-99.9-0.1% ethyl acetate), method: 0-100min, 100% A, 2ml/min, ultraviolet detection 199nm, manual collection according to peak shape, and recovery rate more than 90%. The fractions were collected and sampled for GC detection, the fractions with a purity of more than 97% were combined and then added to a 250ml rotary evaporator, the mixture was spun at 55℃and 50r/min for 20min, and after the organic reagent was spun out, the mixture was filtered using a 0.22um filter and sampled for GC detection, as shown in Table 2 and FIG. 4. Regeneration: 2ml/min, 200ml of the column was backflushed using n-hexane.
Table 2 GC detection of purity of normal phase collected fractions
As is clear from Table 2 above, the purity of the fraction collected by the detection section was 99.11%.
Example 3
The sample is from Saccharomyces cerevisiae BY4742 fermentation broth, centrifugate (8000-10000 r,5 min) to collect precipitate, take out 5g, add 45ml of broken extract, break under high pressure and homogenizing for 800-1400bar, 3-5 times, stir and extract for 2 hours with nitrogen, and filter with 0.22um needle filter for use.
The mass volume ratio of the precipitation to the crushing extracting solution is 1:9, crushing the mixed solution of purified water and an organic reagent, wherein the organic reagent is petroleum ether, and the purified water and the organic reagent are prepared according to the following steps of 1:4 proportion.
Two-phase extraction rotary distillation:
filtering, standing, adding 250ml rotary steaming bottle into 40ml supernatant, rotating at 55deg.C for 5min at 50r/min, completely spinning out petroleum ether, adding 45ml dichloromethane, mixing, adding purified water 90ml, mixing, standing, and layering.
Adding the dichloromethane phase into a 250ml rotary steaming bottle, rotating for 5min at 55 ℃ at 50r/min, completely spinning out the dichloromethane, adding 5ml of 99% n-hexane-1% ethyl acetate by volume, uniformly mixing, pouring out, adding 5ml of 99% n-hexane-1% ethyl acetate by volume again, uniformly mixing, pouring out, and mixing uniformly with the previous mixture to obtain the normal phase preparation chromatographic sample.
Normal phase preparation chromatography:
the NMSIL100-200 normal phase preparation chromatographic column (NMSIL 50-100, 10)250mm）：
Balance: 3ml/min, 100ml equilibrated with normal phase equilibration solution (99.9% n-hexane-0.1% ethyl acetate) until UV 199nm level.
Loading: 1ml/min, 1ml was loaded.
Eluting: 2ml/min, solution A: normal phase equilibrium liquid (0.1-99.9% normal hexane-99.9-0.1% ethyl acetate), method: 0-100min, 100% A, 2ml/min, ultraviolet detection 199nm, manual collection according to peak shape, and recovery rate more than 90%. Each fraction collected was sampled for GC detection, the fractions with purity greater than 97% were combined and added to a 250ml rotary evaporator, spun at 55 ℃ for 20min at 50r/min, after which the organic reagent was spun off, filtered using a 0.22um filter and sampled for GC detection, as shown in table 3 and fig. 5. Regeneration: 2ml/min, 200ml of the column was backflushed using n-hexane.
Table 3 GC detection of purity of normal phase collected fractions
As is clear from Table 3 above, the purity of the fraction collected by the detection section was 99.10%.
Example 4
As shown in FIG. 2, the sample is derived from Saccharomyces cerevisiae CEN.PK2-1C fermentation broth, the thalli are collected by centrifugation, the thalli are washed by adding distilled water in a resuspension manner, the precipitate is collected again by centrifugation (8000-10000 r,5 min), 150g of the precipitate is taken out, purified water is added and mixed, and after uniform stirring, the mixture is homogenized and crushed at high pressure for 800-1400bar for 3-5 times, and 54g of crushed dry bacteria are obtained.
The mass volume ratio of the sediment to the purified water is 1:5. and (5) putting the crushed thalli into a drying box for drying, wherein the drying temperature is 70 ℃, and the drying time is 12 hours.
CO 2 Supercritical extraction:
all 54g of crushed dry bacteria are put into a filter bag and then put into an extraction kettle for extraction for 3 hours at the temperature of 35 ℃ and the pressure of 25mpa and CO 2 The flow rate is 5ml/min, 21g of extraction component is collected, the content of squalene is 85.98% by sampling and detection, and the extraction rate of squalene is more than 95% by detection. The extracted components are filtered for later use.
Molecular distillation:
the molecular distillation equipment is cleaned, and liquid nitrogen is added into the cold trap.
Primary molecular distillation: heating the equipment to 110 ℃, vacuum 2pa and 240r/min, adding 20g of filtered sample of supercritical extraction component, slowly opening a liquid inlet, controlling the liquid inlet flow rate to be 1ml/min, and respectively collecting 18g of heavy phase and 1g of light phase.
Secondary molecular distillation: heating the equipment to 210 ℃ and vacuum 2pa and 240r/min, adding 18g of first molecular distillation to collect heavy phase components, slowly opening a liquid inlet, controlling the liquid inlet flow rate to be 1ml/min, and respectively collecting 4g of heavy phase components and 13.5g of light phase components. The bacteria were removed and filtered using a 0.22um/0.45um filter. The recovery of molecular distilled squalene was greater than 85.62% (there was a small amount of residue in the pipes, vessels, etc. during the small test, resulting in reduced recovery, actual recovery was greater than 90%, and GC purity of the two-stage molecular distilled light phase component was 99.96%, as shown in table 4, table 5, and fig. 6.
Table 4 GC detection of purity of the second molecular weight distilled light phase component
TABLE 5 squalene content of the respective collection components of example 4
The method in the embodiment has extremely high overall extraction recovery rate, no organic reagent is used, and the production cost is extremely low.
Example 5
Other matters are as in example 4, wherein the mass volume ratio of the precipitation to the purified water is 1:2. and (5) putting the crushed thalli into a drying box for drying, wherein the drying temperature is 50 ℃, and the drying time is 20 hours.
CO 2 Supercritical extraction:
all crushed dry bacteria are put into a filter bag and then put into an extraction kettle for extraction for 1 hour, the temperature is 30 ℃, the pressure is 10mpa, and CO 2 The flow rate was 1ml/min and the extract fraction was collected. The extracted components are filtered for later use.
Molecular distillation:
the molecular distillation equipment is cleaned, and liquid nitrogen is added into the cold trap.
Primary molecular distillation: heating the equipment to 90 ℃ and vacuum of 0.1pa and 240r/min, adding the filtered sample of the supercritical extraction component, slowly opening a liquid inlet, controlling the liquid inlet flow rate to be 0.1ml/min, and respectively collecting the heavy phase and the light phase.
Secondary molecular distillation: heating the equipment to 160 ℃, vacuum 0.1pa and 240r/min, adding the first molecular distillation to collect the heavy phase component, slowly opening a liquid inlet, controlling the liquid inlet flow rate to be 0.1ml/min, and collecting the heavy phase component and the light phase component respectively.
Example 6
Other matters are as in example 4, wherein the mass volume ratio of the precipitation to the purified water is 1:10. and (5) putting the crushed thalli into a drying box for drying, wherein the drying temperature is 90 ℃, and the drying time is 5 hours.
CO 2 Supercritical extraction:
all crushed dry bacteria are put into a filter bag and then put into an extraction kettle for extraction for 5 hours at 40 ℃ under 40mpa under CO 2 The flow rate was 10ml/min and the extract fraction was collected. The extracted components are filtered for later use.
Molecular distillation:
the molecular distillation equipment is cleaned, and liquid nitrogen is added into the cold trap.
Primary molecular distillation: heating the equipment to 140 ℃, vacuum 5pa and 240r/min, adding the filtered sample of the supercritical extraction component, slowly opening a liquid inlet, controlling the liquid inlet flow rate to be 1.5ml/min, and collecting the heavy phase and the light phase respectively.
Secondary molecular distillation: heating the equipment to 240 ℃ and vacuum 5pa and 240r/min, adding first molecular distillation to collect heavy phase components, slowly opening a liquid inlet, controlling the liquid inlet flow rate to be 1.5ml/min, and collecting heavy phase components and light phase components respectively.
Example 7
Other matters are as in example 4, wherein the mass volume ratio of the precipitation to the purified water is 1:2-10. And (3) putting the crushed thalli into a drying box for drying, wherein the drying temperature is 50-90 ℃ and the drying time is 5-20h.
CO 2 Supercritical extraction:
all crushed dry bacteria are put into a filter bag and then put into an extraction kettle for extraction for 1-5 hours at the temperature of 30-40 ℃ and the pressure of 10-40mpa and CO 2 The flow rate is 1-10ml/min, and the extraction components are collected. The extracted components are filtered for later use.
Molecular distillation:
the molecular distillation equipment is cleaned, and liquid nitrogen is added into the cold trap.
Primary molecular distillation: heating the equipment to 90-140 deg.C, vacuum 0.1-5pa and 240r/min, adding the filtered sample of supercritical extraction component, slowly opening the liquid inlet, controlling the liquid inlet flow rate to 0.1-1.5ml/min, and collecting heavy phase and light phase respectively.
Secondary molecular distillation: heating the equipment to 160-240 ℃ and vacuum 0.1-5pa and 240r/min, adding the first molecular distillation to collect the heavy phase component, slowly opening a liquid inlet, controlling the liquid inlet flow rate to 0.1-1.5ml/min, and collecting the heavy phase component and the light phase component respectively.
Example 8
The other content is as in example 1, wherein the mass-volume ratio of the precipitation and crushing extract is 1:1. crushing the extracting solution to obtain a mixed solution of purified water and an organic reagent, wherein the organic reagent is n-hexane, and the purified water and the organic reagent are mixed according to a ratio of 1:1 proportion. The normal phase equilibrium solution used (99% n-hexane-1% ethyl acetate).
The two-phase extraction rotary steaming specifically comprises the following steps: adding nitrogen, stirring, extracting, filtering liquid phase, standing, layering, extracting, centrifuging, adding supernatant into rotary steaming bottle, distilling under reduced pressure, rotating at 58 deg.C for 5min at 50r/min, and completely spinning out organic reagent to obtain crude extract; adding dichloromethane, mixing, adding 90ml of purified water, standing for layering, adding the lower dichloromethane phase into a rotary steaming bottle, distilling under reduced pressure, rotating at 58 ℃ for 5min at 50r/min, completely spinning out the dichloromethane, adding normal phase equilibrium liquid, mixing, pouring out, adding normal phase equilibrium liquid with the same volume, mixing, pouring out, and mixing with the previous normal phase equilibrium liquid to obtain the normal phase preparation chromatographic sample.
Example 9
The other content is as in example 1, wherein the mass-volume ratio of the precipitation and crushing extract is 1:5 or 1:6. crushing the extracting solution to obtain a mixed solution of purified water and an organic reagent, wherein the organic reagent is ethyl acetate, and the purified water and the organic reagent are mixed according to the following ratio of 1:2 proportion. The normal phase equilibrium solution used (99.9% n-hexane-0.1% ethyl acetate).
The two-phase extraction rotary steaming specifically comprises the following steps: adding nitrogen, stirring, extracting, filtering liquid phase, standing, layering, extracting, centrifuging, adding supernatant into rotary steaming bottle, distilling under reduced pressure, rotating at 60deg.C for 4min at 50r/min, and completely spinning out organic reagent to obtain crude extract; adding dichloromethane, mixing, adding 90ml of purified water, standing for layering, adding the lower dichloromethane phase into a rotary steaming bottle, distilling under reduced pressure, rotating at 60 ℃ for 4min at 50r/min, completely spinning out the dichloromethane, adding normal phase equilibrium liquid, mixing, pouring out, adding normal phase equilibrium liquid with the same volume, mixing, pouring out, and mixing with the previous normal phase equilibrium liquid to obtain the normal phase preparation chromatographic sample.
Example 10
The other content is as in example 1, wherein the mass-volume ratio of the precipitation and crushing extract is 1:3 or 1:4. crushing the extracting solution to obtain a mixed solution of purified water and an organic reagent, wherein the organic reagent is ethyl acetate, and the purified water and the organic reagent are mixed according to the following ratio of 1:3 proportion. The normal phase equilibrium liquid used was 0.1% n-hexane-99.9% ethyl acetate.
The two-phase extraction rotary steaming specifically comprises the following steps: adding nitrogen, stirring, extracting, filtering liquid phase, standing, layering, extracting, centrifuging, adding supernatant into rotary steaming bottle, distilling under reduced pressure, rotating at 50deg.C for 6min at 50r/min, and completely spinning out organic reagent to obtain crude extract; adding dichloromethane, mixing, adding 90ml of purified water, standing for layering, adding the lower dichloromethane phase into a rotary steaming bottle, distilling under reduced pressure at 50 ℃ for 6min at 50r/min, completely spinning out the dichloromethane, adding normal phase equilibrium liquid, mixing, pouring out, adding normal phase equilibrium liquid with the same volume, mixing, pouring out, and mixing with the previous normal phase equilibrium liquid to obtain the normal phase preparation chromatographic sample.
The above examples/experiments are only examples for clarity of illustration and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. A method for extracting and purifying biosynthetic squalene is characterized in that: the method comprises the following steps:
(1) Preparing a saccharomyces cerevisiae fermentation liquor;
(2) Washing the thalli after centrifugation: centrifugally collecting thalli, adding distilled water to resuspend and wash thalli, and centrifugally collecting sediment;
(3) High-pressure homogenizing and crushing: adding the crushed extracting solution and the precipitate, mixing, charging nitrogen, stirring uniformly, and crushing by a high-pressure homogenizer;
(4) Two-phase extraction rotary distillation: stirring and extracting by charging nitrogen, filtering liquid phase, standing for layering, taking organic phase component for reduced pressure distillation, and removing organic solvent to obtain crude extract; then adding normal phase equilibrium liquid to dissolve to obtain normal phase sample;
(5) Column chromatography and rotary evaporation: preparing chromatography by normal phase, loading samples according to the proportion of 5% -10% of the volume of the chromatographic column, collecting each component in a segmented way, mixing the components with the detection result meeting the requirements, and removing the organic reagent by rotary evaporation;
(6) Sterilizing and filtering to obtain the final product.
2. The method for extracting and purifying the biosynthetic squalene according to claim 1, wherein the method comprises the following steps: the mass volume ratio of the precipitation to the crushing extracting solution in the step (3) is 1:1-10; in the optimization scheme, the mass volume ratio of the precipitation to the crushing extracting solution is 1:1. 1:2. 1: 3. 1:4. 1:5. 1:6. 1: 7. 1:8. 1:9 or 1:10.
3. the method for extracting and purifying the biosynthetic squalene according to claim 1 or 2, wherein: the broken extracting solution is a mixed solution of purified water and an organic reagent, wherein the organic reagent is n-hexane, ethyl acetate, dichloromethane or petroleum ether; in the optimized scheme, purified water and organic reagent are mixed according to the following ratio of 1:1 or 1:2 or 1:3 or 1:4 or 1:5 ratio.
4. The method for extracting and purifying the biosynthetic squalene according to claim 1, wherein: the normal phase equilibrium liquid in the step (4) is 0.1-99.9% of normal hexane-99.9-0.1% of ethyl acetate.
5. The method for extracting and purifying biosynthetic squalene according to claim 4, wherein: the normal phase equilibrium liquid is 99% normal hexane-1% ethyl acetate.
6. The method for extracting and purifying the biosynthetic squalene according to claim 1, wherein the method comprises the following steps: the contents of the step (3) and the step (5) are replaced by a step of drying thalli, supercritical extraction and molecular distillation, specifically, crushing liquid is added and mixed with sediment, and the mixture is uniformly stirred and then crushed by a high-pressure homogenizer; drying the crushed thalli; and (3) sequentially carrying out supercritical carbon dioxide extraction, filtration and molecular distillation on the dried bacteria.
7. The method for extracting and purifying the biosynthetic squalene according to claim 6, wherein the method comprises the steps of: the drying adopts a vacuum drying, nitrogen charging drying or freeze-drying mode; in the optimized scheme, the drying is vacuum drying, and the crushed thalli are put into a vacuum drying box for drying, wherein the drying temperature is 40-90 ℃ and the drying time is 5-20h.
8. The method for extracting and purifying the biosynthetic squalene according to claim 5, wherein the method comprises the steps of: the crushing liquid is purified water, and the mass volume ratio of the sediment to the crushing liquid is 1:2-10.
9. The method for extracting and purifying the biosynthetic squalene according to claim 6, wherein the method comprises the steps of: the supercritical carbon dioxide extraction is carried out by loading all crushed dry bacteria into a filter bag, and then placing into an extraction kettle, wherein the supercritical carbon dioxide extraction pressure is 5-40Mbar, the temperature is 25-60 ℃, and the CO is 2 The flow rate is 1-10ml/min, the extraction time is 1-10 hours, and the extraction components are collected; extracting for 3 hours at 35 ℃ under 25mpa under CO in the optimized scheme 2 The flow rate was 5ml/min.
10. The method for extracting and purifying the biosynthetic squalene according to claim 6, wherein the method comprises the steps of: the molecular distillation adopts primary molecular distillation and secondary molecular distillation, wherein the primary molecular distillation is used for collecting a heavy phase, and the secondary molecular distillation is used for collecting a light phase; in an optimized scheme, the molecular distillation comprises the following steps:
s1, cleaning molecular distillation equipment, and adding liquid nitrogen into a cold trap;
s2: primary molecular distillation: heating the equipment to 90-140 ℃, vacuum 0.1-5pa and 240r/min, adding supercritical extraction components, filtering, slowly opening a liquid inlet, controlling the liquid inlet flow rate to 0.1-1.5ml/min, and collecting heavy phase and light phase respectively; in the optimized scheme, the equipment is heated to 110 ℃ and is subjected to vacuum 2pa and 240r/min, and the flow rate is controlled to be 1ml/min;
s3: secondary molecular distillation: heating the equipment to 160-240 ℃ and vacuum 0.1-5pa and 240r/min, adding first molecular distillation to collect heavy phase components, slowly opening a liquid inlet, controlling the liquid inlet flow rate to be 0.1-1.5ml/min, and collecting heavy phase components and light phase components respectively; in the optimized scheme, the equipment is heated to 210 ℃ and is subjected to vacuum 2pa and 240r/min, and the inflow flow rate is controlled to be 1ml/min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597204B (en) * | 2009-07-09 | 2013-03-13 | 天津大学 | Method for extracting high-purity squalene by taking olive oil as raw material |
| FR2989373A1 (en) * | 2012-04-16 | 2013-10-18 | Roquette Freres | Preparing squalene composition used in e.g. cosmetic field by fermentation of microorganisms, involves purifying squalene by extracting supercritical carbon dioxide on multistage fractionation column and molecular distillation column |
| CN105016956A (en) * | 2014-04-23 | 2015-11-04 | 中粮营养健康研究院有限公司 | Squalene extracting method |
| CN115010572A (en) * | 2022-07-07 | 2022-09-06 | 广州美林生态科技有限公司 | Method for extracting squalene and vitamin E |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597204B (en) * | 2009-07-09 | 2013-03-13 | 天津大学 | Method for extracting high-purity squalene by taking olive oil as raw material |
| FR2989373A1 (en) * | 2012-04-16 | 2013-10-18 | Roquette Freres | Preparing squalene composition used in e.g. cosmetic field by fermentation of microorganisms, involves purifying squalene by extracting supercritical carbon dioxide on multistage fractionation column and molecular distillation column |
| CN105016956A (en) * | 2014-04-23 | 2015-11-04 | 中粮营养健康研究院有限公司 | Squalene extracting method |
| CN115010572A (en) * | 2022-07-07 | 2022-09-06 | 广州美林生态科技有限公司 | Method for extracting squalene and vitamin E |
Non-Patent Citations (2)
| Title |
|---|
| P. BHATTACHARJEE: "Extraction of squalene from yeast by supercritical carbon dioxide", 《WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY》, vol. 2018, pages 605 - 608 * |
| 康彦芳主编: "《中药化学成分提取分离技术与方法》", vol. 2021, 中央广播电视大学出版社, pages: 391 - 393 * |
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