CN116283476A

CN116283476A - Method for extracting squalene

Info

Publication number: CN116283476A
Application number: CN202310188395.1A
Authority: CN
Inventors: 张志乾; 罗俊; 黄俊远; 邱�益; 王嘉鹏; 吴奕瑞; 王海梅
Original assignee: Tichuang Biotechnology Guangzhou Co ltd; Guangzhou Qianxiang Biotechnology Co Ltd
Current assignee: Tichuang Biotechnology Guangzhou Co ltd; Guangzhou Qianxiang Biotechnology Co Ltd
Priority date: 2023-02-28
Filing date: 2023-02-28
Publication date: 2023-06-23

Abstract

The invention discloses a method for extracting squalene, which comprises the steps of selecting saccharomyces cerevisiae as an extraction raw material of squalene, and adopting a specific thallus drying mode, an extracting agent, a feed-liquid ratio, an extraction temperature and a time to obtain high-yield and high-purity saccharomyces cerevisiae intracellular squalene. The method has the advantages of easily obtained raw materials, low cost, simple and easy steps for extracting the squalene, less time consumption, easy control of the operation process, strong practicability, convenience for large-scale production, and capability of effectively solving the problem of continuously increasing the market demand of the squalene in the prior art, and solves the problems of complex process, lack of raw materials and the like in the traditional method. The squalene obtained by the invention has wide application and can be used for preparing foods, medicines and cosmetics.

Description

Method for extracting squalene

Technical Field

The invention relates to the technical field of squalene extraction, in particular to a method for extracting squalene.

Background

Squalene (SQ), a linear polyunsaturated terpenoid containing 30 carbon atoms, is a biosynthetic precursor of steroid and terpenoid substances, and belongs to an important terpene compound. Squalene is a colorless or yellowish fragrant oily liquid at room temperature, and has chemical formula C ₃₀ H ₅₀ The molecular weight is 410.7, and the structure is shown as the following formula I.

The prior researches show that squalene has various physiological effects, for example, in the cosmetic field, and can be used as an important component in anti-aging face cream and sun cream due to the antioxidant capacity. In addition, squalene has good compatibility with lipid in skin, and can maintain skin softness and smoothness, and can be used as a natural moisturizing agent. In the medical field, squalene is widely used for preparing stable oil-in-water emulsion as a carrier of anticancer, antitumor drugs and vaccines due to its lipophilicity and good surface tension. In the field of health food, the product has effects of improving animal heart function, strengthening body constitution, protecting liver, preventing diabetes, repairing cells, and enhancing resistance. In addition, toxic substances are generally lipid substances, and squalene can be used as an antidote according to the principle of similar compatibility, and can be combined with toxic substances in the body to help the body to remove the toxic substances in the body.

The squalene is mainly extracted from shark liver or plants, and the traditional methods have the problems of complex extraction process, low product yield, serious environmental pollution and the like, damage to wild animal and plant resources and limit the production and use of natural squalene. However, the complexity and instability of the technology for chemically synthesizing squalene are primarily researched and explored based on laboratory pilot scale, and because of various reasons such as severe conditions, high cost, complicated process or no matching equipment, the technology is difficult or impossible to realize real industrial production and cannot meet huge market demands, it is particularly important to find new squalene sources and develop corresponding obtaining methods.

In recent years, the production of squalene by utilizing microorganisms is rising, and the production of squalene by adopting a microbial fermentation method is gradually becoming a hot research direction at home and abroad compared with the traditional squalene production because of the advantages of sustainable squalene production, simple operation, high economic benefit and no limitation of raw materials and environment. Coli can synthesize squalene through the MEP pathway, however, the MEP pathway is inefficient in supplying IPP and DMAPP for product synthesis, and the MEP pathway lacks post-transcriptional modification mechanisms, limiting squalene synthesis. In contrast, yeast not only has an endogenous mevalonate pathway and a sterol synthesis pathway, has sufficient precursors supplied to squalene synthesis, but also has an intact intracellular membrane system, is very easy to synthesize squalene, so that yeast has a wide application prospect in squalene biosynthesis as a main host. At present, high-pressure crushing or ultrasonic crushing thalli are selected preferentially for extracting squalene from saccharomyces cerevisiae cells, but the two methods have the defects of high difficulty and incomplete crushing of the saccharomyces cerevisiae, so that the efficiency of extracting squalene by a subsequent organic solvent is low; the existing organic solvent for extracting squalene mainly comprises n-hexane, ethyl acetate, petroleum ether and the like, is applied to the industries of cosmetics and medicines, has large residual hazard and unsafe, does not have dominant price, and certainly increases the production cost; in the aspects of bacterial cell crushing and organic solvent, the production difficulty is increased for industrialized squalene extraction.

Disclosure of Invention

The present invention aims to solve at least one of the above technical problems in the prior art. Therefore, the invention aims to improve the extraction rate and purity of squalene and solve the problems of difficult process and high process cost of microorganism production and squalene extraction.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect the invention provides a method for extracting squalene.

In a second aspect the invention provides squalene extracted intracellularly from Saccharomyces cerevisiae.

In a third aspect the invention provides the use of squalene extracted intracellularly from Saccharomyces cerevisiae.

According to a first aspect of the present invention, there is provided a method of extracting squalene, comprising the steps of:

collecting fermented Saccharomyces cerevisiae thallus, lyophilizing, pulverizing, and extracting with anhydrous ethanol.

In the invention, ethanol is used as a solvent, ethanol is a water-soluble solvent and has certain oil solubility, especially when the temperature is increased, the ethanol has the further great advantage of forming an azeotrope with water, and in the solvent distillation step, the characteristic of simplifying the removal of alcohols, ensuring that the alcohols are cheaper and easy to recycle, and compared with normal hexane organic solvents, the alcohols have less environmental pollution and are not easy to damage human bodies.

In some embodiments of the invention, the freeze-drying temperature is-80 ℃ to-100 ℃ for 24 hours to 48 hours.

In some embodiments of the invention, the ratio of the feed solution extracted by the absolute ethanol is (0.2-2) g to 20mL, namely 1% -10% (w/V).

In some embodiments of the invention, the temperature of the absolute ethanol extraction is 70 ℃ to 90 ℃.

In some embodiments of the invention, the absolute ethanol extraction time is 60min to 120min.

In some embodiments of the invention, the method further comprises centrifuging, collecting supernatant, filtering, and collecting filtrate after the extraction with absolute ethanol; the filtration uses a 0.2 μm to 0.3 μm filter membrane.

In some preferred embodiments of the invention, the fermentation culture comprises: activating Saccharomyces cerevisiae, culturing the seed solution, inoculating to fermentation medium, and fermenting.

In some preferred embodiments of the invention, the time of activation is from 2 to 4 days.

In some preferred embodiments of the invention, the seed solution is incubated for a period of time ranging from 16 hours to 24 hours at a temperature ranging from 30 ℃ to 37 ℃.

In some preferred embodiments of the invention, the ratio by volume of seed liquid to medium inoculated is (1-4): 100.

in some preferred embodiments of the invention, the fermentation time is 90h to 100h and the temperature is 30 ℃ to 37 ℃.

In some more preferred embodiments of the present invention, the collecting the Saccharomyces cerevisiae cells after fermentation culture comprises centrifuging at 8000rpm to 12000rpm for 8min to 10min to collect the precipitate.

According to a second aspect of the present invention there is provided squalene extracted intracellularly from Saccharomyces cerevisiae, said squalene being obtainable using the extraction process according to the first aspect.

According to a third aspect of the present invention, there is provided the use of squalene extracted from Saccharomyces cerevisiae cells according to the second aspect for the preparation of food, pharmaceutical, cosmetic products.

The beneficial effects of the invention are as follows:

1. the invention adopts an industrially feasible mode to extract the squalene from the squalene producing saccharomyces cerevisiae intracellular, gets rid of the problems of complex process, lack of raw materials and the like of the traditional method, is convenient for large-scale production, and can effectively solve the problem of continuously increasing the market demand of the squalene at present.

2. The invention has the advantages of easily obtained raw materials, low cost, simple and easy squalene extraction, low cost, easy control of the operation process, realization of industrialized mass production and strong practicability.

Drawings

FIG. 1 is a graph showing squalene yield by different drying modes according to example 1 and comparative examples 7 to 8 of the present invention;

FIG. 2 is a graph showing the yields of squalene extracted by different organic solvents in example 1 and comparative examples 1 to 4 of the present invention;

FIG. 3 is a graph showing the results of various extraction times versus squalene yield for examples 1, 4, 5 and comparative example 5 of the present invention;

FIG. 4 is a graph showing the results of various extraction temperatures and squalene production amounts in examples 1, 6 and 7 and comparative example 6 according to the present invention;

FIG. 5 is a graph showing the results of various feed ratios and squalene yields in examples 1 to 3 and comparative examples 9 to 11 according to the present invention;

FIG. 6 is a high performance liquid chromatography detection chart of squalene obtained in example 1.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The starting materials, reagents or apparatus used in the examples and comparative examples were either commercially available from conventional sources or may be obtained by prior art methods unless specifically indicated. Unless otherwise indicated, assays or testing methods are routine in the art.

The following examples 1 to 7 and comparative examples 1 to 6 were prepared by the following methods:

activating Saccharomyces cerevisiae Y6 preserved by glycerol at-80 ℃ in a solid culture medium at 30 ℃ for 3-4 days until large and round colonies grow, picking single colonies, inoculating the single colonies in a liquid culture medium at 30 ℃ and 220rpm for culture for 24 hours, inoculating the seed solution in a fermentation culture medium according to the volume ratio of the seed solution to the culture medium of 2:100, culturing for 96 hours at 220rpm at 30 ℃, collecting bacterial liquid, centrifuging for 10 minutes at 8000r/min, removing the supernatant, washing for 3 times by using sterilizing water, freeze-drying at-100 ℃ for 60 hours, and then crushing and preserving dry bacterial at room temperature.

Example 1

The embodiment extracts squalene from saccharomyces cerevisiae cells, and the specific process is as follows:

preparing squalene extracting solution: 1g of dry thalli of saccharomyces cerevisiae is weighed, 20mL of absolute ethanol solution is added, at the moment, the feed-liquid ratio is 1g to 20mL (namely 5% w/V), the mixture is uniformly mixed, the mixture is heated in a water bath at 80 ℃ for 90min, the mixture is uniformly mixed every 10min, the mixture is centrifuged at 8000rpm for 10min at room temperature after the heating is finished, the supernatant is reserved, and a filter membrane with the thickness of 0.22 mu m is used for collecting filtrate, so that the extract squalene is obtained.

The squalene yield of this example was 98.9mg/g DCW; squalene purity 95.91%.

Example 2

preparing squalene extracting solution: weighing 0.2g of dried thalli of saccharomyces cerevisiae, adding 20mL of absolute ethanol solution, wherein the feed-liquid ratio is 0.2g to 20mL (namely 1% w/V), uniformly mixing, heating in a water bath at 80 ℃ for 90min, uniformly mixing every 10min, centrifuging at 8000rpm for 10min at room temperature after heating, reserving the supernatant, and collecting filtrate by using a 0.22 mu m filter membrane to obtain the extract squalene.

The squalene yield of this example was 74.16mg/g DCW; squalene purity was 95%.

Example 3

preparing squalene extracting solution: weighing 2g of dry thalli of saccharomyces cerevisiae, adding 20mL of absolute ethanol solution, mixing uniformly at the time when the feed liquid ratio is 2g to 20mL (namely 10% w/V), heating in a water bath at 80 ℃ for 90min, uniformly mixing every 10min, centrifuging at room temperature for 10min at 8000rpm after heating, retaining the supernatant, and collecting filtrate by using a 0.22 mu m filter membrane to obtain the extract squalene.

The squalene yield of this example was 73.71mg/g DCW; squalene purity 96.07%.

Example 4

preparing squalene extracting solution: 1g of dry thalli of saccharomyces cerevisiae is weighed, 20mL of absolute ethanol solution is added, at the moment, the feed-liquid ratio is 1g to 20mL, the mixture is uniformly mixed, the mixture is heated in a water bath at 80 ℃ for 60min, the mixture is uniformly mixed every 10min, centrifugation is carried out for 10min at 8000rpm at room temperature after the heating is finished, the supernatant is reserved, and a filter membrane with the thickness of 0.22 mu m is used for collecting filtrate, thus obtaining the extract squalene.

The squalene yield of this example was 81.77mg/g DCW; squalene purity 95.55%.

Example 5

preparing squalene extracting solution: 1g of dry thalli of saccharomyces cerevisiae is weighed, 20mL of absolute ethanol solution is added, at the moment, the feed-liquid ratio is 1g to 20mL, the mixture is uniformly mixed, the mixture is heated in a water bath at 80 ℃ for 120min, the mixture is uniformly mixed every 10min, centrifugation is carried out for 10min at 8000rpm at room temperature after the heating is finished, the supernatant is reserved, and a filter membrane with the thickness of 0.22 mu m is used for collecting filtrate, thus obtaining the extract squalene.

The squalene yield of this example was 91.72mg/g DCW; squalene purity 95.52%.

Example 6

preparing squalene extracting solution: 1g of dry thalli of saccharomyces cerevisiae is weighed, 20mL of absolute ethanol solution is added, at the moment, the feed-liquid ratio is 1g to 20mL, the mixture is uniformly mixed, the mixture is heated in a water bath at 70 ℃ for 90min, the mixture is uniformly mixed every 10min, centrifugation is carried out for 10min at 8000rpm at room temperature after the heating is finished, the supernatant is reserved, and a filter membrane with the thickness of 0.22 mu m is used for collecting filtrate, thus obtaining the extract squalene.

Squalene yield 77.05mg/g DCW in this example; squalene purity 96.44%.

Example 7

preparing squalene extracting solution: 1g of dry thalli of saccharomyces cerevisiae is weighed, 20mL of absolute ethanol solution is added, at the moment, the feed-liquid ratio is 1g to 20mL, the mixture is uniformly mixed, the mixture is heated in a water bath at 90 ℃ for 90min, the mixture is uniformly mixed every 10min, centrifugation is carried out for 10min at 8000rpm at room temperature after the heating is finished, the supernatant is reserved, and a filter membrane with the thickness of 0.22 mu m is used for collecting filtrate, thus obtaining the extract squalene.

Squalene yield 74.5mg/g DCW in this example; squalene purity 96.44%.

Comparative example 1

This comparative example shows the intracellular extraction of squalene from Saccharomyces cerevisiae, differing from example 1 in that the extraction was carried out using 95% (V/V) ethanol solution, and the specific procedure was carried out with reference to example 1.

The squalene yield of this comparative example was 33.8mg/g DCW; squalene purity 92.31%.

Comparative example 2

This comparative example shows that squalene is extracted from Saccharomyces cerevisiae intracellular, and differs from example 1 in that the extraction is performed using a methanol solution, and the specific procedure is performed with reference to example 1.

The squalene yield of this comparative example was 50mg/g DCW; squalene purity 93.09%.

Comparative example 3

This comparative example shows the intracellular extraction of squalene from Saccharomyces cerevisiae, differing from example 1 in that the extraction was carried out using isopropanol solution, and the specific procedure was carried out with reference to example 1.

The squalene yield of this comparative example was 36.05mg/g DCW; squalene purity 94.61%.

Comparative example 4

This comparative example shows the intracellular extraction of squalene from Saccharomyces cerevisiae, differing from example 1 in that the extraction was carried out using 80% (V/V) ethanol solution, and the specific procedure was carried out with reference to example 1.

The squalene yield of this comparative example was 3.4mg/g DCW; squalene purity 68.83%.

Comparative example 5

The comparative example is a method for extracting squalene from Saccharomyces cerevisiae, which is different from example 1 in that the water bath heating time is 30min during the extraction process, and the specific process is performed according to example 1.

The squalene yield of this comparative example was 73.35mg/g DCW; squalene purity 95.05%.

Comparative example 6

This comparative example shows that squalene is extracted from Saccharomyces cerevisiae intracellular, and differs from example 1 in that the water bath heating temperature during the extraction process is 60℃and the specific process is performed with reference to example 1.

The squalene yield of this comparative example was 43.95mg/g DCW; squalene purity 95.88%.

Comparative example 7

The comparative example has squalene extracted from Saccharomyces cerevisiae cells, and is different from example 1 in that the Saccharomyces cerevisiae cells are collected by adopting drying instead of freeze-drying, and the specific process is as follows:

activating Saccharomyces cerevisiae preserved by glycerol at-80 ℃ in a solid culture medium at 30 ℃ for 3-4 days until large and round colonies grow, picking single colonies, inoculating the single colonies in a liquid culture medium at 30 ℃ and 220rpm for culturing for 24 hours, inoculating seed liquid in a fermentation culture medium according to the volume ratio of the seed liquid to the culture medium of 2:100, culturing for 96 hours at 220rpm at 30 ℃, collecting bacterial liquid, centrifuging for 10 minutes at 8000r/min, removing supernatant, washing with sterilized water for 3 times, drying at 60 ℃, pulverizing, and preserving dry bacterial cells at room temperature.

Preparing squalene extracting solution: weighing 1g of dried thallus of the saccharomyces cerevisiae, adding 20mL of absolute ethanol solution, mixing uniformly at the time when the feed-liquid ratio is 1g to 20mL, heating in a water bath at 80 ℃ for 90min, uniformly mixing every 10min, centrifuging at 8000rpm at room temperature for 10min after heating, retaining the supernatant, and collecting filtrate by using a 0.22 mu m filter membrane to obtain the extract squalene.

The squalene yield of this comparative example was 21.3mg/g DCW; squalene purity 89.76%.

Comparative example 8

The comparative example has squalene extracted from Saccharomyces cerevisiae cells, and is different from example 1 in that spray drying is adopted instead of freeze drying when collecting Saccharomyces cerevisiae cells, and the specific process is as follows:

activating Saccharomyces cerevisiae preserved with glycerol at-80deg.C in solid culture medium at 30deg.C for 3-4 days until large round colony grows, selecting single colony, inoculating to liquid culture medium, culturing at 30deg.C and 220rpm for 24 hr, inoculating to fermentation culture medium according to volume ratio of seed liquid to culture medium of 2:100, collecting bacterial liquid, centrifuging at 8000r/min for 10min, removing supernatant, washing with sterilized water for 3 times, spray drying (outlet temperature of 90deg.C, vermicular pattern)The rotating speed of the dynamic pump is 10r/min and the air flow is 85m ³ And/h) crushing and preserving the dry bacterial cells at room temperature.

Preparing squalene extracting solution: 1g of dry thalli of saccharomyces cerevisiae is weighed, 20mL of absolute ethanol solution is added, at the moment, the feed-liquid ratio is 1g to 20mL, the mixture is uniformly mixed, the mixture is heated in a water bath at 80 ℃ for 90min, the mixture is uniformly mixed every 10min, centrifugation is carried out for 10min at 8000rpm at room temperature after the heating is finished, the supernatant is reserved, and a filter membrane with the thickness of 0.22 mu m is used for collecting filtrate, thus obtaining the extract squalene.

The squalene yield of this comparative example was 18mg/g DCW; squalene purity 88.87%.

Comparative example 9

preparing squalene extracting solution: 1g of dry thalli of saccharomyces cerevisiae is weighed, 20mL of absolute ethanol solution is added, at the moment, the feed-liquid ratio is 3 g/20 mL (namely 15% w/V), the mixture is uniformly mixed, the mixture is heated in a water bath at 80 ℃ for 90min, the mixture is uniformly mixed every 10min, centrifugation is carried out for 10min at 8000rpm at room temperature after the heating is finished, the supernatant is reserved, and a filter membrane with the thickness of 0.22 mu m is used for collecting filtrate, thus obtaining the extract squalene.

Squalene yield 56.68mg/g DCW in this example; squalene purity 96.28%.

Comparative example 10

preparing squalene extracting solution: 1g of dry thalli of saccharomyces cerevisiae is weighed, 20mL of absolute ethanol solution is added, at the moment, the feed liquid ratio is 4g to 20mL (namely 20%w/V), the mixture is uniformly mixed, the mixture is heated in a water bath at 80 ℃ for 90min, the mixture is uniformly mixed every 10min, centrifugation is carried out for 10min at 8000rpm at room temperature after the heating is finished, the supernatant is reserved, and a filter membrane with the thickness of 0.22 mu m is used for collecting filtrate, so that the extract squalene is obtained.

The squalene yield of this example was 48mg/g DCW; squalene purity 96.49%.

Comparative example 11

preparing squalene extracting solution: 1g of dry thalli of saccharomyces cerevisiae is weighed, 20mL of absolute ethanol solution is added, at the moment, the feed liquid ratio is 5g to 20mL (namely 25% w/V), the mixture is uniformly mixed, the mixture is heated in a water bath at 80 ℃ for 90min, the mixture is uniformly mixed every 10min, centrifugation is carried out for 10min at 8000rpm at room temperature after the heating is finished, the supernatant is reserved, and a filter membrane with the thickness of 0.22 mu m is used for collecting filtrate, so that the extract squalene is obtained.

Squalene yield 49.25mg/g DCW in this example; squalene purity 96.07%.

Test examples

Establishing a squalene standard curve: ethanol is used as a solvent to prepare squalene standard solutions with the concentration of 0.54g/L,0.27g/L,0.135g/L,0.0675g/L and 0.03375g/L respectively; the standard solution was tested using a high performance liquid chromatograph. High performance liquid chromatography detection conditions: an Agilent 1260 high performance liquid chromatograph is adopted, and the detector is an ultraviolet detector; the column was C18 (5 μm 4.6X1250 mm); column temperature: 40 ℃; mobile phase: acetonitrile; flow rate: 1.6mL/min; detection wavelength: 195nm; sample injection amount: 10. Mu.L;

taking squalene concentration g/L as an abscissa and peak area UV.S as an ordinate to obtain a linear regression equation of squalene concentration and peak area; the regression equation is: y=50046.0915x+558.6249 (R ² =0.9948), a standard curve is established;

the squalene obtained in examples 1 to 7 and comparative examples 1 to 11 were each examined under the same high performance liquid chromatography conditions as those used for the above standard solutions to obtain the peak areas of the response, and the peak areas were substituted into the above standard curves, respectively, the squalene yield was calculated by the formula, and the purity was obtained by curve integration, as shown in Table 1 below. Wherein, the calculation formula of squalene yield is: n=x×y×z ^-1 The method comprises the steps of carrying out a first treatment on the surface of the N is squalene yield (i.e., weight mg squalene per g dry cell weight DCW), in mg/g DCW; x is squalene extraction concentration obtained according to a standard curve, and the unit is mg/mL; y is the volume of the extracting solution, and the unit is mL; z is dry weight of the extracted packed bacterial cells, and the unit is g.

TABLE 1 production and purity of squalene obtained in examples 1 to 7 and comparative examples 1 to 11

FIG. 1 is a graph showing squalene yield by different drying modes according to example 1 and comparative examples 7 to 8 of the present invention; from fig. 1, it can be seen that the yield of squalene extracted after lyophilization of yeast is highest among the three drying modes, indicating that lyophilization is more suitable as a way of drying yeast cells. FIG. 2 is a graph showing the yields of squalene extracted by different organic solvents in example 1 and comparative examples 1 to 4 of the present invention; from fig. 2, it can be seen that the yield of squalene extracted with absolute ethanol is highest among the five extraction solvents, indicating that absolute ethanol is more suitable for extracting squalene from yeast intracellular corners. FIG. 3 is a graph showing the results of various extraction times versus squalene yield for examples 1, 4, 5 and comparative example 5 of the present invention; from FIG. 3, it can be seen that the squalene yield is higher at 60-120 min than at 30min, indicating that the time for extracting squalene from intracellular of yeast can be selected from 60-120 min. FIG. 4 is a graph showing the results of various extraction temperatures and squalene production amounts in examples 1, 6 and 7 and comparative example 6 according to the present invention; as can be seen from FIG. 4, the squalene yield is higher at 70-90℃than at 60℃showing that the temperature for extracting squalene from the intracellular of yeast can be 70-90 ℃. FIG. 5 is a graph showing the results of various feed ratios and squalene yields in examples 1 to 3 and comparative examples 9 to 11 according to the present invention; as can be seen from FIG. 5, when the extraction liquid is (0.2-2) g, namely, 20mL, namely, 1-10% (w/V), the squalene yield is high, and when the extraction liquid is (3-5) g, namely, 20mL, namely, 15-25% (w/V), the squalene yield is reduced by less than 70mg/g DCW; FIG. 6 is a high performance liquid chromatography test chart of squalene obtained in example 1, and it can be seen from FIG. 6 that the impurity peaks are very few except for the solvent peaks, indicating that high purity squalene is obtained in example 1.

In conclusion, the yield and purity of squalene can be obviously improved under the synergistic effect of all conditions in the technical scheme of the invention, and all conditions in the technical scheme of the invention are indispensable.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A method for extracting squalene, comprising the steps of:

2. The method for extracting squalene according to claim 1, wherein the ratio of the liquid to the solid extracted with absolute ethanol is (0.2-2) g/20 mL.

3. The method for extracting squalene according to claim 1, wherein the temperature of the extraction with absolute ethanol is 70-90 ℃.

4. The method for extracting squalene according to claim 1, wherein the time of the extraction with absolute ethanol is 60min to 120min.

5. The method for extracting squalene according to claim 1, wherein the freeze drying temperature is-80 ℃ to-100 ℃ for 24h to 48h.

6. The method for extracting squalene according to claim 1, wherein the saccharomyces cerevisiae is selected from any one strain of Z103, Y6, che 4.

7. The method for extracting squalene according to claim 1, wherein the collecting the Saccharomyces cerevisiae cells after fermentation culture comprises centrifugation at 8000rpm to 12000rpm for 8min to 10min.

8. The method for extracting squalene according to claim 1, wherein the fermentation culture comprises: activating Saccharomyces cerevisiae, culturing the seed solution, inoculating to a fermentation medium, and fermenting; the volume ratio of the seed liquid to the culture medium of the inoculation is (1-4): 100; the fermentation time is 90-100 h, and the temperature is 30-37 ℃.

9. Squalene extracted from saccharomyces cerevisiae intracellular, characterized in that it is extracted using the method according to any one of claims 1 to 8.

10. Use of squalene extracted from Saccharomyces cerevisiae intracellular according to claim 9 for the preparation of food, pharmaceutical, cosmetic.