Method for separating and purifying squalene from squalene fermentation liquor

Abstract

The invention discloses a method for separating and purifying squalene from squalene fermentation liquor, and belongs to the technical field of squalene extraction. The method comprises the following steps: 1) pretreatment: adding a wall breaking reagent into squalene fermentation liquor for wall breaking, or concentrating the squalene fermentation liquor and then adding the wall breaking reagent for wall breaking; 2) extraction: adding an organic solvent and a demulsifier, layering to obtain a water phase and an organic phase, and collecting the organic phase; 3) impurity removal: adding an aqueous solution, layering to obtain an aqueous phase and an organic phase, and collecting the organic phase; and 4) distilling: distilling to obtain purified squalene. The method is simple to operate, low in equipment investment, simple in post-treatment and low in extraction cost, and the obtained squalene refined product is high in yield, high in purity and suitable for industrialization.

Description

Technical Field

[0001] The invention belongs to the technical field of squalene extraction, and particularly relates to a method for separating and purifying squalene from squalene fermentation liquor. Background Art

[0002] Squalene is a triterpene and an important intermediate in the biosynthesis of sterols. It is widely present in plants, animals and humans. It was originally obtained from the vacuum distillation of liver oil of deep-sea sharks. It is an unsaturated hydrocarbon with a chemical formula of C 30 H 50 After squalene was discovered by a Japanese chemist Tsujimot in 1906, it has attracted widespread attention in the fields of food, medicine, cosmetics, etc. Squalene has significant biological activities such as antioxidant, anti-radiation, oxygen carrying, detoxification, and cholesterol metabolism regulation, and is widely used in cosmetics, health foods, medicine, etc.

[0003]

[0004] At present, the main methods for producing squalene include animal and plant extraction, chemical synthesis, and biosynthesis. Among them, the animal and plant extraction method has problems such as cumbersome extraction steps, long production cycle, large amount of raw materials, and high cost; while the chemical method has a complex synthesis route and harsh reaction conditions, which will cause greater environmental pollution, and is subject to the complex chiral chemical structure of squalene, making chemical synthesis difficult, and the resulting product has low purity and poor biological activity. In comparison, the biosynthesis method has the advantages of short production cycle, green environmental protection, simple extraction, and mild production conditions.

[0005] In production, the fermentation broth containing the product squalene is usually obtained by biosynthesis, but the fermentation broth has complex components and needs further separation and purification to obtain a high-purity squalene product. The specification of Chinese patent application CN113604512A discloses a process for extracting squalene from Schizochytrium fermentation broth, the process of which is to add alkaline protease to the fermentation broth to perform enzymatic cell wall breaking; add anhydrous ethanol to the cell wall-broken cell liquid, separate by oscillation and centrifugation, and collect the bottom bacterial mud; add n-hexane to the bottom bacterial mud, oscillate and centrifuge to collect the supernatant, repeat several times until the supernatant is colorless and transparent, and finally evaporate to remove the n-hexane to collect the crude squalene; degumming, deacidification, bleaching and deodorization of the crude squalene to obtain purified squalene. The specification of Chinese patent application CN116947589A discloses a process for extracting squalene from brewer's yeast fermentation broth, the process sequentially comprises the steps of preparing brewer's yeast fermentation broth, washing the bacteria after centrifugation, high-pressure homogenization and crushing, two-phase extraction and rotary evaporation, column chromatography and rotary evaporation, and sterilization filtration, or replacing the two-phase extraction and rotary evaporation and column chromatography and rotary evaporation steps with drying the bacteria, supercritical extraction and molecular distillation steps, to obtain a squalene product with a purity of >97%.

[0006] Although both of the above two methods can obtain purified squalene products, they have disadvantages such as complicated process steps, long process cycle, high cost, low yield, etc., and cannot be industrialized. Summary of the invention

[0007] In view of the problems existing in the prior art, the object of the present invention is to provide a method for separating and purifying squalene from squalene fermentation broth, which is not only simple to operate, requires less equipment investment, is simple to post-process, and has low extraction cost, but also has high yield and high purity of squalene.

[0008] The present invention provides a method for separating and purifying squalene from squalene fermentation broth, comprising the following steps:

[0009] 1) Pretreatment: adding a cell-breaking agent to the squalene fermentation liquid to break the cell walls, or, after concentrating the squalene fermentation liquid, adding a cell-breaking agent to break the cell walls;

[0010] 2) Extraction: adding an organic solvent and a demulsifier, separating the layers to obtain an aqueous phase and an organic phase, and collecting the organic phase;

[0011] 3) Impurity removal: adding an aqueous solution, separating the aqueous phase and the organic phase, and collecting the organic phase;

[0012] 4) Distillation: Distillation to obtain purified squalene.

[0013] Furthermore, in step 1), the concentration multiple is 1 to 6 times, preferably 2 to 4 times, more preferably 2 to 3 times; and / or, in step 1), the cell wall breaking agent is an inorganic base or an inorganic acid.

[0014] Furthermore, in step 1), the inorganic base is sodium hydroxide or potassium hydroxide, and the inorganic acid is sulfuric acid or hydrochloric acid.

[0015] Furthermore, in step 1), the volume mass ratio of the squalene fermentation liquid to the cell wall breaking agent is 1:6-100 L / g, preferably 1:10-40 L / g, and more preferably 1:15-20 L / g; the cell wall breaking time is 0.5-4 h, preferably 1-3 h, and more preferably 2 h, and the temperature is 25°C-100°C, preferably 60°C-90°C, and more preferably 70°C-80°C.

[0016] Furthermore, in step 1), after cell wall breaking, the following step is further included: adding acid to adjust the pH to 9-11, preferably to 9.5-10.5, more preferably to 10.

[0017] Furthermore, the volume ratio of the squalene fermentation liquid in step 1), the organic solvent in step 2), and the demulsifier in step 2) is 1:0.3-10:0.01-0.1, preferably 1:0.5-3:0.02-0.06, and more preferably 1:1-2:0.04;

[0018] And / or, the organic solvent is selected from C4-C12 ether solvents, C4-C12 ester solvents, and C5-C12 alkane solvents, the C4-C12 ether solvent is preferably isopropyl ether, isobutyl ether or methyl tert-butyl ether, the C4-C12 ester solvent is preferably butyl acetate or ethyl acetate, and the C5-C12 alkane solvent is preferably n-heptane, n-hexane, isododecane or n-dodecane;

[0019] And / or, the demulsifier is DP0618, and the concentration of the demulsifier is 10 g / L to 100 g / L, preferably 20 g / L to 60 g / L, and more preferably 50 g / L.

[0020] Furthermore, in step 2), the temperature of the layering is 10-80°C, preferably 20-30°C, more preferably 25°C, and the time is 0.5-6h, preferably 0.5-4h, more preferably 1-2h.

[0021] Furthermore, step 2) further comprises the following steps: adding an organic solvent and a demulsifier to the aqueous phase, separating the layers to obtain an aqueous phase and an organic phase, collecting the organic phase again, and combining the organic phases collected twice.

[0022] Further, the aqueous solution in step 3) is selected from aqueous sodium chloride solution, aqueous calcium chloride solution, aqueous aluminum sulfate solution, aqueous ammonium sulfate solution, aqueous ferric chloride solution, aqueous sodium carbonate solution, aqueous potassium carbonate solution, aqueous sodium hydroxide solution, aqueous potassium hydroxide solution or aqueous ammonia; the concentration of the aqueous solution is 10 g / L to 150 g / L, preferably 20 g / L to 100 g / L, and more preferably 50 g / L to 100 g / L;

[0023] And / or, the volume ratio of the organic phase collected in step 2) to the aqueous solution in step 3) is 1:0.05-2, preferably 0.08-0.5, and more preferably 0.1-0.5.

[0024] Furthermore, step 3) further comprises the following steps: adding activated carbon to the collected organic phase for decolorization, filtering, concentrating the filtrate, adding an aqueous solution, separating the phases to obtain an aqueous phase and an organic phase, and collecting the organic phase;

[0025] Or, step 3) also includes the following steps: adding activated carbon to the collected organic phase for decolorization, filtering, concentrating the filtrate, adding an aqueous solution, layering to obtain an aqueous phase and an organic phase, collecting the organic phase, adding activated carbon for decolorization, filtering, and collecting the filtrate.

[0026] Further, in step 4), the distillation is molecular distillation; preferably, the temperature of the molecular distillation is 100-200°C, preferably 130-170°C, more preferably 150-170°C, most preferably 160-170°C, and the pressure is 50-150Pa, preferably 55-120Pa, more preferably 60-90Pa, most preferably 90Pa.

[0027] The present invention has achieved the following beneficial effects:

[0028] The present invention provides a method for separating and purifying squalene from squalene fermentation broth by optimizing the separation and purification process steps and process parameters of each step. The method of the present invention is simple to operate, has low equipment investment, high extraction rate, simple post-processing, low extraction cost, and the recovered solvent can be applied to the next batch, which can further reduce the production cost and is suitable for industrialization.

[0029] In addition to the above advantages, in the preferred embodiment of the present invention, the yield of the separated and purified squalene is more than 65%. In the most preferred embodiment of the present invention, the yield of the separated and purified squalene is as high as 70%, the gas phase content is as high as 991.35 ug / mg, and the purity is as high as 99.5%.

[0030] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0031] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the gas chromatogram of squalene standard.

[0033] Figure 2 This is the gas chromatogram of the squalene product separated and purified in Example 8. DETAILED DESCRIPTION

[0034] Unless otherwise specified, the raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0035] The method for separating and purifying squalene of the present invention is applicable to any fermentation liquid containing squalene obtained by a fermentation production process known in the prior art.

[0036] The following examples provide a method for separating and purifying squalene using a fermented broth of Saccharomyces cerevisiae containing the product squalene as an example. The preparation method of the fermented broth of Saccharomyces cerevisiae containing the product squalene used in the following examples comprises the following steps:

[0037] (1) Preparation of squalene fermentation medium:

[0038] Formula: soy peptone Y005T 30g / L, yeast powder 10g / L, corn steep liquor powder 20g / L, magnesium sulfate heptahydrate 6g / L, potassium dihydrogen phosphate 3g / L, defoamer 0.3g / L, and the rest is water.

[0039] Preparation method: Dissolve the components except the defoamer in water, then add the defoamer and sterilize at 121-123°C for 30 minutes.

[0040] (2) Production of squalene: Take a strain of Saccharomyces cerevisiae and culture it at a temperature of 30°C and a rotation speed of 200 rpm for about 24 hours for activation, inoculate the activated seed liquid into a squalene seed culture medium and culture it for about 48 hours, and transplant the seed liquid that reaches the transplant index into the squalene fermentation culture medium described in step (1) for fermentation for 100 hours to obtain a squalene fermentation liquid. The formula of the squalene seed culture medium is: 2% glucose, 2% soy peptone, 1% yeast extract powder, and the rest is water. The preparation method is: mix the ingredients according to the formula, adjust the pH to 6.5, and sterilize at 121°C for 30 minutes.

[0041] The concentration of the obtained squalene fermentation liquid was 40 g / L.

[0042] The demulsifier used in the following examples is DP0618 (type B).

[0043] Example 1: A method for separating and purifying squalene from squalene fermentation broth

[0044] 30.82 L of squalene fermentation liquid was measured and placed in a ceramic membrane, concentrated to 13.5 L to obtain a concentrated solution, 525 g of sodium hydroxide was added to the concentrated solution and stirred at 60° C. for 2 h to break the wall, and then methyl tert-butyl ether (2 times the volume of the concentrated solution) and 4% of the volume of the concentrated solution (demulsifier concentration is 50 g / L) were added, stirred at 25° C. for 2 h, stirring was stopped, the mixture was allowed to stand for stratification, the organic phase and the aqueous phase were separated, and the organic phase was collected, and methyl tert-butyl ether (1 times the volume of the original concentrated solution) and 4% of the volume of the original concentrated solution (demulsifier concentration is 50 g / L) were added to the aqueous phase, stirred at 25° C. for 2 h, stirring was stopped, the mixture was allowed to stand for stratification, the organic phase and the aqueous phase were separated, and the organic phase was collected. Combine the two extracted organic phases, then add 0.3BV of 50g / L sodium hydroxide aqueous solution, stir for 30min, let stand for stratification, collect the organic phase, and perform vacuum distillation at 40-60°C to obtain 631.6g of a crude product. Add 0.3BV of 30g / L sodium carbonate aqueous solution to the crude product for washing for 30min, layer the product, collect the organic phase, add 3g of activated carbon to decolorize the organic phase for 30min, filter, and perform molecular distillation of the filtrate at 155°C and 70Pa to obtain a purified squalene product. The product is a light yellow liquid with a slight odor. The product yield, gas phase content and purity are 55%, 955.98ug / mg and 98.02%, respectively.

[0045] Example 2: A method for separating and purifying squalene from squalene fermentation broth

[0046] Measure 23.56L of squalene fermentation liquid and place it in a ceramic membrane, concentrate it to 11.2L, and obtain a concentrated solution. Add 467g of sodium hydroxide to the concentrated solution and stir it at 80℃ for 2h to break the wall. Add 20% sulfuric acid solution to the concentrated solution after breaking the wall to adjust the pH to 10, then add ethyl acetate twice the volume of the concentrated solution and 4% demulsifier (demulsifier concentration is 50g / L) of the concentrated solution, and start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, collect the organic phase, add ethyl acetate twice the volume of the original concentrated solution and 4% demulsifier (demulsifier concentration is 50g / L) of the original concentrated solution to the aqueous phase, and start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, and collect the organic phase. Combine the organic phases of the two extractions, then add 0.5BV of a 50g / L sodium carbonate aqueous solution, stir for 30 minutes, let stand and separate, collect the organic phase, add 150g of activated carbon to the organic phase for decolorization for 40 minutes, filter, and distill the filtrate under reduced pressure at 40-60°C to obtain 448.32g of a crude product, add 0.5BV of an ammonium sulfate aqueous solution (50g / L) to the crude product and wash for 30 minutes, separate, and collect the organic phase, i.e. the washed crude product, which is molecularly distilled at 160°C and 90Pa to obtain a purified squalene product, which is a colorless liquid, and the product yield, gas phase content and purity are 68%, 977.96ug / mg and 98.36%, respectively.

[0047] Example 3: A method for separating and purifying squalene from squalene fermentation broth

[0048] Measure 23.56L of squalene fermentation liquid and place it in a ceramic membrane, concentrate it to 9.34L, and obtain a concentrated solution. Add 467g of sodium hydroxide to the concentrated solution and stir it at 70℃ for 2h to break the wall. Add 20% sulfuric acid solution to the concentrated solution after breaking the wall to adjust the pH to 10, then add ethyl acetate 1.5 times the volume of the concentrated solution and 4% of the demulsifier (demulsifier concentration is 50g / L) of the concentrated solution, and start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, collect the organic phase, add ethyl acetate equal to the original concentrated solution volume and 4% of the demulsifier (demulsifier concentration is 50g / L) of the original concentrated solution to the aqueous phase, and start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, and collect the organic phase. The organic phases of the two extractions were combined, 0.1 BV of a 100 g / L sodium chloride aqueous solution was added to the organic phase, stirred for 30 min, allowed to stand and stratified, the organic phase was collected, and vacuum distillation was performed at 40-60° C. to obtain 375.7 g of a crude product. The crude product was molecularly distilled at 165° C. and 90 Pa to obtain a purified squalene product, which was a colorless liquid. The product yield, gas phase content and purity were 65%, 970.66 ug / mg and 99.64%, respectively.

[0049] Example 4: A method for separating and purifying squalene from squalene fermentation broth

[0050] Measure 31.3L of squalene fermentation liquid and place it in a reactor, add 1565g of sodium hydroxide and stir at 80℃ for 2h to break the cell wall. After breaking the cell wall, add 0.5BV of ethyl acetate and 4% of the fermentation liquid volume of demulsifier (demulsifier concentration is 20g / L) to the fermentation liquid, and start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, collect the organic phase, add 0.5 times the original fermentation liquid volume of ethyl acetate and 4% of the original fermentation liquid volume of demulsifier (demulsifier concentration is 20g / L) to the aqueous phase, start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, and collect the organic phase. The extraction was repeated three times in total, and the organic phases extracted three times were combined. Then 0.5BV of purified water was added, stirred for 30 minutes, and the mixture was allowed to stand for stratification. The organic phase was collected and distilled under reduced pressure at 40-60°C to obtain 254g of a crude product. 0.5BV of an aqueous solution of ammonium sulfate (concentration of 50g / L) was added to the crude product and washed for 30 minutes. The product was separated and the organic phase was collected, i.e., the washed crude product. The washed crude product was molecularly distilled at 160°C and 90Pa to obtain a purified squalene product. The product was a light yellow liquid with an odor. The product yield, gas phase content and purity were 35%, 980.33ug / mg and 98%, respectively.

[0051] Example 5: A method for separating and purifying squalene from squalene fermentation broth

[0052] Measure 35.52L of squalene fermentation liquid and place it in a reactor, add 1400g of sodium hydroxide, 0.5BV of methyl tert-butyl ether and 4% of the fermentation liquid volume demulsifier (demulsifier concentration is 20g / L), and start stirring to mix it evenly. Stir at 25°C for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, collect the organic phase, add 0.5 times the original fermentation liquid volume of methyl tert-butyl ether to the aqueous phase, start stirring to mix it evenly. Stir at 25°C for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, and collect the organic phase. The extraction was repeated three times in total, and the organic phases extracted three times were combined. 0.5BV of an aqueous solution of aluminum sulfate with a concentration of 40g / L was added to the organic phase, stirred for 30min, allowed to stand for stratification, the organic phase was collected, and vacuum distillation was performed at 40-60°C to obtain 254g of a crude product. 0.5BV of an aqueous solution of sodium carbonate (with a concentration of 50g / L) was added to the crude product, stirred for 30min, and the organic phase was separated, i.e. the washed crude product. The washed crude product was molecularly distilled at 160°C and 80Pa to obtain a purified squalene product, which was a light yellow liquid. The product yield, gas phase content and purity were 50%, 970ug / mg and 98.8%, respectively.

[0053] Example 6: A method for separating and purifying squalene from squalene fermentation broth

[0054] Measure 30L of squalene fermentation liquid and place it in a reactor, add 1500g of sodium hydroxide, 0.5BV of n-heptane of the fermentation liquid volume and 4% of the demulsifier of the fermentation liquid volume (demulsifier concentration is 20g / L), and start stirring to mix it evenly. Stir at 25°C for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, collect the organic phase, add 0.5 times the original volume of n-heptane to the aqueous phase, start stirring to mix it evenly. Stir at 25°C for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, and collect the organic phase. The extraction was repeated three times in total, and the organic phases extracted three times were combined. 0.5BV of sodium hydroxide aqueous solution (concentration of 80g / L) was added to the organic phase, stirred for 30min, allowed to stand for stratification, the organic phase was collected, and vacuum distillation was performed at 40-60°C to obtain 230g of a crude product. 0.5BV of sodium carbonate aqueous solution (concentration of 50g / L) was added to the crude product, stirred for 30min, and the organic phase was separated, i.e. the washed crude product. The washed crude product was molecularly distilled at 160°C and 90Pa to obtain a purified squalene product, which was a light yellow liquid. The product yield, gas phase content and purity were 25%, 980ug / mg and 98.8%, respectively.

[0055] Example 7: A method for separating and purifying squalene from squalene fermentation broth

[0056] Measure 25L of squalene fermentation liquid and place it in a ceramic membrane, concentrate it to 8.5L, and obtain a concentrated solution. Add 425g of sodium hydroxide to the concentrated solution and stir it at 80℃ for 2h to break the wall. Add 3BV of n-dodecane and 4% of the volume of the concentrated solution by demulsifier (demulsifier concentration is 20g / L) to the concentrated solution after breaking the wall, and start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, collect the organic phase, add 1BV times the volume of the original fermentation liquid to the aqueous phase, and start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, let stand and separate the organic phase and the aqueous phase, and collect the organic phase. Combine the organic phases of the two extractions, add 0.5BV of sodium carbonate aqueous solution (concentration of 50g / L), stir for 30min, let stand for stratification, collect the organic phase, and perform molecular distillation at 70-80°C to obtain 100g of a crude product. Add 0.5BV of sodium carbonate aqueous solution (concentration of 50g / L) to the crude product, stir for 30min, separate the organic phase, i.e. the washed crude product, and perform molecular distillation at 160°C and 90Pa to obtain a purified squalene product, which is a light yellow liquid. The product yield, gas phase content and purity are 30%, 970ug / mg and 98.7%, respectively.

[0057] Example 8: A method for separating and purifying squalene from squalene fermentation broth

[0058] Measure 22L of squalene fermentation liquid and place it in a ceramic membrane, concentrate it to 8L, and obtain a concentrated solution. Add 365g of sodium hydroxide to the concentrated solution and stir it at 60℃ for 2h to break the wall. Add 20% sulfuric acid solution to the concentrated solution after breaking the wall to adjust the pH to 10, then add ethyl acetate twice the volume of the concentrated solution and 4% demulsifier (demulsifier concentration is 50g / L) of the concentrated solution, and start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, stand for stratification, separate the organic phase and the aqueous phase, collect the organic phase, add ethyl acetate equal to the original concentrated solution volume and 4% demulsifier (demulsifier concentration is 50g / L) of the original concentrated solution to the aqueous phase, and start stirring to mix it evenly. Stir at 25℃ for 2h, stop stirring, stand for stratification, separate the organic phase and the aqueous phase, and collect the organic phase. Combine the two extracted organic phases, then add 0.1 times the volume (i.e., 0.1BV) of a 100g / L sodium hydroxide aqueous solution, stir for 30min, stand for phase separation, collect the organic phase, add 120g of activated carbon to the organic phase for decolorization for 40min, filter, and distill the filtrate under reduced pressure at 40-60°C to obtain 375.7g of a crude product, add 0.5BV of a 50g / L ammonium sulfate aqueous solution to the crude product, stir for 30min, stand for stratification, collect the organic phase, add 1.8g of activated carbon to the organic phase for decolorization for 30min, filter, and molecularly distill the filtrate at 160°C and 90Pa to obtain a purified squalene product, which is a colorless liquid, and the product yield, gas phase content and purity are 70%, 991.35ug / mg, and 99.5%, respectively.

[0059] The products obtained in the above embodiments are all verified to be squalene by gas chromatography test. Taking the product of Example 8 as an example, the test results are as follows: Figure 2 shown. Figure 1 This is the gas phase diagram of squalene standard. Figure 2 Squalene products and Figure 1 The response time of the squalene standard was consistent.

[0060] Comparing Examples 1-8, it can be seen that the yield of squalene products obtained by the methods of Examples 1, 2, 3, 5, and 8 is higher than that of Examples 4, 6, and 7. It should be particularly pointed out that the yield of squalene products obtained by the methods of Examples 2, 3, and 8 is more than 65% after the cell wall is broken by first adding acid to adjust the pH and then extracting. Among them, the method of Example 8 is the best, with a product yield of up to 70%, a gas phase content of up to 991.35ug / mg, and a purity of up to 99.5%.

[0061] In summary, the present invention provides a method for separating and purifying squalene from squalene fermentation broth, which method is not only simple to operate, requires less equipment investment, is simple to post-process, and has low extraction cost, but also has high yield and high purity of the obtained squalene product, and is suitable for industrialization.

Claims

1. A method for separating and purifying squalene from squalene fermentation broth, characterized in that: The following steps are involved: 1) Pretreatment: adding a cell wall breaking agent to the squalene fermentation liquid to break the cell wall, or, after concentrating the squalene fermentation liquid, adding a cell wall breaking agent to break the cell wall; 2) Extraction: adding an organic solvent and a demulsifier, separating the layers to obtain an aqueous phase and an organic phase, and collecting the organic phase; 3) Impurity removal: adding an aqueous solution, separating the aqueous phase and the organic phase, and collecting the organic phase; 4) Distillation: Distillation to obtain purified squalene.

2. The method according to claim 1, characterized in that: In step 1), the concentration multiple is 1 to 6 times, preferably 2 to 4 times, more preferably 2 to 3 times; and / or, in step 1), the cell wall breaking agent is an inorganic base or an inorganic acid.

3. The method according to claim 2, characterized in that: In step 1), the inorganic base is sodium hydroxide or potassium hydroxide, and the inorganic acid is sulfuric acid or hydrochloric acid.

4. The method according to claim 1, characterized in that: Step 1) The volume mass ratio of the squalene fermentation broth to the cell wall breaking agent is 1:6-100 L / g, preferably 1:10-40 L / g, and more preferably 1:15-20 L / g; the cell wall breaking time is 0.5-4 h, preferably 1-3 h, and more preferably 2 h, and the temperature is 25°C-100°C, preferably 60°C-90°C, and more preferably 70°C-80°C.

5. The method according to claim 1, characterized in that: In step 1), after cell wall breaking, the following steps are further included: adding acid to adjust the pH to 9-11, preferably to 9.5-10.5, more preferably to 10.

6. The method according to claim 1, characterized in that: The volume ratio of the squalene fermentation liquid in step 1), the organic solvent in step 2), and the demulsifier in step 2) is 1:0.3-10:0.01-0.1, preferably 1:0.5-3:0.02-0.06, and more preferably 1:1-2:0.04; And / or, the organic solvent is selected from C4-C12 ether solvents, C4-C12 ester solvents, and C5-C12 alkane solvents, the C4-C12 ether solvent is preferably isopropyl ether, isobutyl ether or methyl tert-butyl ether, the C4-C12 ester solvent is preferably butyl acetate or ethyl acetate, and the C5-C12 alkane solvent is preferably n-heptane, n-hexane, isododecane or n-dodecane; And / or, the demulsifier is DP0618, and the concentration of the demulsifier is 10 g / L to 100 g / L, preferably 20 g / L to 60 g / L, and more preferably 50 g / L.

7. The method according to claim 1, characterized in that: Step 2) also includes the following steps: adding an organic solvent and a demulsifier to the aqueous phase, separating the layers to obtain an aqueous phase and an organic phase, collecting the organic phase again, and combining the organic phases collected twice.

8. The method according to claim 1, characterized in that: The aqueous solution in step 3) is selected from aqueous sodium chloride solution, aqueous calcium chloride solution, aqueous aluminum sulfate solution, aqueous ammonium sulfate solution, aqueous ferric chloride solution, aqueous sodium carbonate solution, aqueous potassium carbonate solution, aqueous sodium hydroxide solution, aqueous potassium hydroxide solution or aqueous ammonia; the concentration of the aqueous solution is 10 g / L to 150 g / L, preferably 20 g / L to 100 g / L, more preferably 50 g / L to 100 g / L; And / or, the volume ratio of the organic phase collected in step 2) to the aqueous solution in step 3) is 1:0.05-2, preferably 0.08-0.5, and more preferably 0.1-0.

5.

9. The method according to claim 1, characterized in that: Step 3) further comprises the following steps: adding activated carbon to the collected organic phase for decolorization, filtering, concentrating the filtrate, adding an aqueous solution, separating the layers to obtain an aqueous phase and an organic phase, and collecting the organic phase; Or, step 3) also includes the following steps: adding activated carbon to the collected organic phase for decolorization, filtering, concentrating the filtrate, adding an aqueous solution, layering to obtain an aqueous phase and an organic phase, collecting the organic phase, adding activated carbon for decolorization, filtering, and collecting the filtrate.

10. The method according to any one of claims 1 to 9, characterized in that: In step 4), the distillation is molecular distillation; preferably, the temperature of the molecular distillation is 100-200°C, preferably 130-170°C, more preferably 150-170°C, and the pressure is 50-150Pa, preferably 55-120Pa, more preferably 60-90Pa.

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