CN113604512A

CN113604512A - Method for extracting squalene from schizochytrium limacinum and application of squalene in moisturizing and anti-aging cosmetics

Info

Publication number: CN113604512A
Application number: CN202111006960.5A
Authority: CN
Inventors: 周坤江
Original assignee: Shantou Qiwei Industrial Co ltd
Current assignee: Shantou Qiwei Industrial Co ltd
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2021-11-05
Anticipated expiration: 2041-08-30
Also published as: CN113604512B

Abstract

The invention relates to a method for extracting squalene from schizochytrium limacinum and application of squalene in moisturizing and anti-aging cosmetics, which comprises the following steps: activating schizochytrium limacinum, inoculating to a primary seed culture medium, culturing to obtain a primary seed, and inoculating the primary seed to a secondary seed culture medium, culturing to obtain a secondary seed; inoculating the secondary seeds to a fermentation medium, and performing fermentation culture to obtain a fermentation liquid; adding alkaline protease into the fermentation liquor, and carrying out enzymolysis wall breaking on the schizochytrium; adding absolute ethanol into the bacterial liquid after wall breaking, removing a supernatant water-ethanol phase through oscillation and centrifugal separation, and collecting bottom layer bacterial sludge; adding n-hexane into bottom layer bacterial sludge, oscillating, centrifuging, collecting 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 degumming, deacidifying, bleaching and deodorizing the crude squalene to obtain purified squalene. The invention solves the problems of low yield and high cost of the existing squalene extraction.

Description

Method for extracting squalene from schizochytrium limacinum and application of squalene in moisturizing and anti-aging cosmetics

Technical Field

The invention relates to the technical field of cosmetics, and particularly relates to a method for extracting squalene from schizochytrium limacinum and application of squalene in moisturizing and anti-aging cosmetics.

Background

Squalene is highly unsaturated linear triterpene compound formed by connecting 6 isoprene, and is named as squalene, triacontahexaene, cod liver oil terpene or squalene, and has system name of 2,6,10,15,19, 23-hexamethyl-2, 6,10,14,18, 22-tetracosahexaene and molecular formula of C₃₀H₅₀And is a light yellow or colorless oily liquid at normal temperature. The squalene has biological activity of improving anoxia tolerance, inhibiting microorganism growth, resisting bacteria, relieving inflammation, and regulating cholesterol metabolism. And squalene is a nontoxic enhancer with antiaging and anticancer effects.

The squalene has wide source and exists in microorganisms, plant seeds, microalgae, shark liver and human sebum. At present, squalene is mainly extracted and separated from plant seeds, but the defect is that the extracted amount of squalene is very small, and a large amount of enrichment and purification processes are required, so the cost is high. The schizochytrium limacinum is one kind of marine microalgae, and the schizochytrium limacinum cell grease mainly comprises unsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and also comprises squalene. Although microalgae produce squalene under heterotrophic conditions and can provide high quality squalene by controlled fermentation conditions, which can be purified to meet food, cosmetic and medical needs, the prior art has relatively low yields of squalene extracted from microalgae.

In view of the above, there is a need to provide a new method for extracting squalene from schizochytrium limacinum with high yield.

Disclosure of Invention

The invention mainly aims to provide a method for extracting squalene from schizochytrium limacinum and application of squalene in moisturizing and anti-aging cosmetics, and aims to solve the problems of low yield and high cost of squalene extraction at present.

In order to achieve the above object, the present invention provides, in a first aspect, a method for extracting squalene from schizochytrium limacinum, comprising the steps of:

s1, culturing schizochytrium:

s11, transferring the schizochytrium limacinum to an inclined plane, culturing for 3-4 d at 25 ℃, activating the schizochytrium limacinum, then selecting the activated schizochytrium limacinum by using an inoculating loop, inoculating the activated schizochytrium limacinum to a primary seed culture medium, culturing for 2d at 25 ℃ and 200r/min to obtain primary seeds, finally inoculating the primary seeds to a secondary seed culture medium, and culturing for 2d at 25 ℃ and 200r/min to obtain secondary seeds; wherein the primary seed culture medium and the secondary seed culture medium are both composed of 30g/L glucose, 10g/L yeast extract and 15g/L mineral;

s12, transferring the secondary seeds into a fermentation medium in a fermentation mechanism according to the inoculation amount of 6% for fermentation culture, wherein the tank pressure is 0.025MPa, the ventilation volume is 5L/min, the stirring speed is 40r/min, and the fermentation is carried out for 6d to obtain fermentation liquor; wherein the fermentation medium consists of 120g/L glucose, 10g/L yeast extract, 30g/L mineral and 0.15g/L vitamin complex; adding glucose in batch and in a flowing manner in the fermentation culture process, wherein the glucose is added once every 8-12 hours, and 30g/L is added every time;

s2, wall breaking of schizochytrium:

transferring the fermentation liquor obtained in the step S12 into an enzymolysis mechanism, adding alkaline protease into the enzymolysis mechanism, and carrying out enzymolysis wall breaking on the schizochytrium limacinum, wherein the enzymolysis temperature is 63 ℃, the pH value is 7.8, and the enzymolysis time is 6 h;

s3, extracting squalene:

s31, adding absolute ethyl alcohol into the bacterial liquid obtained after wall breaking in the step S2, wherein the volume ratio of the bacterial liquid to the absolute ethyl alcohol is 1: 1.2, removing a supernatant water-ethanol phase by oscillating and centrifugal separation of a centrifugal mechanism, and collecting bottom layer bacterial sludge;

s32, adding n-hexane into the bottom layer bacterial sludge, wherein the volume ratio of the bottom layer bacterial sludge to the n-hexane is 1: 1.15, oscillating and centrifuging by a centrifugal mechanism to collect supernatant, repeating the process for a plurality of times until the supernatant is colorless and transparent, finally evaporating to remove n-hexane, and collecting to obtain a squalene crude product;

s4, squalene purification:

and (4) degumming, deacidifying, bleaching and deodorizing the squalene crude product obtained in the step (S3) to obtain purified squalene.

Preferably, in step S11, the mineral substance is composed of 10g/LNaCl, 0.5g/LCaCl₂·2H₂O、1.5g/LMgSO₄·7H₂O、1.25g/LK₂SO₄、0.75g/LKH₂PO₄、0.7g/L(NH₄)2SO₄And 0.3g/LFeSO₄·7H₂And (C) O.

Preferably, in step S12, the mineral substance is composed of 18g/LNaCl, 1.5g/LCaCl₂·2H₂O、3.5g/LMgSO₄·7H₂O、2.25g/LK₂SO4、1.75g/LKH₂PO₄、2g/L(NH₄)2SO₄And 1g/LFeSO₄·7H₂O, and the composite cellulose consists of 60mg/L of vitamin B6, 30mg/L of vitamin B12, 20mg/L of riboflavin, 10mg/L of nicotinic acid and 30mg/L of folic acid.

Preferably, in step S4, the degumming treatment is specifically performed by preheating the crude squalene to 90 ℃, then adding a phosphoric acid solution, stirring uniformly, adding deionized water, stirring until precipitation is separated out, reducing the stirring speed, continuing stirring for 15min, standing for 5min, and performing centrifugal separation to obtain the crude degummed squalene.

Preferably, in step S4, the deacidification treatment is specifically performed by placing the degummed squalene crude product in an alkali refining pot, heating while stirring, spraying a NaOH solution into the degummed squalene crude product after heating to 35 ℃, continuously stirring for 30min, then washing with water, and finally centrifuging and performing oil-water separation to obtain the deacidified squalene crude product.

Preferably, the centrifugal mechanism comprises a support base supported on the ground, an arc-shaped support frame is installed in the middle of the top end of the support base, a centrifugal storage tank is transversely placed on the surface of the top of the arc-shaped support frame, the tank body of the centrifugal storage tank is hollow, a rotating pin shaft is embedded in the middle of the end head of the tank body of the centrifugal storage tank, one end of the rotating pin shaft penetrates through the centrifugal storage tank and is in through connection with the side wall of a placing cover arranged at the end head, and a centrifugal machine is installed inside the centrifugal storage tank;

the top edge of the cover body of the placing cover is provided with a circular groove, the top of the groove of the circular groove is communicated with a conical funnel, a centrifugal rotator for centrifugal processing of schizochytrium limacinum is arranged in the cover body of the placing cover, and one end of the placing cover is provided with a circular discharging groove.

Preferably, fermentation mechanism places in the avris of centrifugal storage tank, fermentation mechanism is including the linear type pipe that is used for the defeated material, the sealed intercommunication of one end of linear type pipe has raw material tanks, the end of linear type pipe is buckled the sealed intercommunication in top and is had high-pressure liquid suction ware, fermentation mechanism is still including placing the reation kettle in centrifuge one side, reation kettle's cauldron body top middle part is provided with constant temperature heating ware, constant temperature heating ware's heating end is connected with reation kettle's lateral wall heat-conduction.

Preferably, a stirring paddle is arranged in the reaction kettle, an extractor is arranged in the middle of the top end of the reaction kettle, the top end of the extractor is hermetically communicated with an arc-shaped bent pipe, one end of the arc-shaped bent pipe is hermetically communicated with a discharge pipe opening, and one end of the discharge pipe opening is hermetically communicated with an extraction pump; a plurality of sliding grooves are sequentially arranged on the surface of the outer wall of the kettle body of the reaction kettle respectively, and a plurality of strip-shaped sliding rails are arranged on the two side walls of the groove positions of the sliding grooves in an embedded mode respectively.

Preferably, the enzymolysis mechanism is communicated with the bottom of the extraction pump in a sealing manner, the enzymolysis mechanism comprises an enzymolysis tank fixedly mounted on the side wall of the kettle body of the reaction kettle, a rotating roll shaft is inserted into the side of the tank body of the enzymolysis tank, one end of the rotating roll shaft is embedded in a shaft coupling, one end of the shaft coupling is in transmission connection with a crushing paddle arranged in the enzymolysis tank, a rotating handle is arranged on the outer side of the rotating roll shaft, one end of the rotating handle is externally connected with a stirring motor, and the bottom of the tank body of the enzymolysis tank is in sealing connection with the circular groove through a hose which is communicated; the bottom fixed mounting of enzymolysis case has the support frame, the PLC controller is installed to the edge of support frame, centrifuge, centrifugal spinner, high-pressure liquid suction ware, constant temperature heater, decimator, extraction pump and agitator motor all with PLC controller electric connection.

In a second aspect, the invention provides an application of squalene extracted by the method in any section of the specification in a moisturizing and anti-aging cosmetic.

Compared with the prior art, the invention at least has the following beneficial effects:

1) in the extraction process, the adopted fermentation medium comprises glucose, yeast extract, mineral substances and vitamin complex, wherein the vitamin complex can promote the growth of schizochytrium limacinum strains, improve the oil content in the strains and increase the yield of squalene. In addition, the glucose is fed-batch in a mode of multiple supplements, so that the inhibition effect of high-concentration substrates on the growth of thalli and the activity of producing squalene is relieved.

2) In the extraction process, the schizochytrium limacinum is subjected to wall breaking treatment by an enzymolysis method, so that the crushing efficiency is high, a solvent is not required, and the potential safety hazard and the recovery cost are reduced.

3) In the extraction process, a fermentation mechanism, an enzymolysis mechanism and a centrifugal mechanism are adopted, schizochytrium to be extracted is pumped into a reaction kettle by a linear guide pipe and a high-pressure liquid extractor, the temperature in the reaction kettle is controlled by a constant-temperature heater, so that conditions are provided for schizochytrium fermentation, the fermented schizochytrium is conveniently pumped into an enzymolysis box for further enzymolysis operation by the aid of the enzymolysis mechanism, the enzymolysis box and a rotating roll shaft, crushing paddles in the enzymolysis box are driven to rotate by an external coupler, enzymolysis stirring and crushing of equipment can be fully realized, preparation for subsequent extraction of a squalene crude product is achieved, and the provided centrifugal storage tank centrifuge is combined with a high-speed rotating centrifuge to extract the squalene crude product at the opposite angle.

4) In the extraction process, the fermentation mechanism can realize fed-batch fermentation and relieve the inhibition effect of high-concentration substrates through the high-pressure liquid extractor, the constant-temperature heater is arranged, so that the relative temperature and humidity can be stably kept, the fermentation can be smoothly realized, the pin shaft, the crushing paddle and the coupler are arranged, schizochytrium inside the enzymolysis tank can be conveniently stirred, cells can be crushed, the crushing efficiency is high, the solvent is not required to be used, and the potential safety hazard and the recovery cost are reduced.

Drawings

FIG. 1 is a schematic view of the fermentation mechanism, the enzymolysis mechanism and the centrifugal mechanism;

FIG. 2 is a schematic view of the structure of a fermentation mechanism according to the present invention;

FIG. 3 is a schematic structural view of the coupling of the present invention;

fig. 4 is a schematic view showing the construction of the suction pump of the present invention.

In the figure: 1. a centrifugal mechanism; 11. a support base; 12. an arc-shaped support frame; 13. a centrifugal storage tank; 14. rotating the pin shaft; 15. placing a cover; 16. circular slotting; 17. circular discharging and grooving; 2. a fermentation mechanism; 21. a linear conduit; 22. a high-pressure liquid extractor; 23. a reaction kettle; 24. a constant temperature heater; 25. an extractor; 26. an arc-shaped bent pipe; 27. a pump; 28. sliding and slotting; 29. a strip-shaped slide rail; 291. a triangular bracket; 3. an enzymolysis mechanism; 31. an enzymolysis box; 32. rotating the roll shaft; 33. a coupling; 34. the handle is rotated.

Detailed Description

The technical solutions in the embodiments of the present invention are described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Examples

A method for extracting squalene from Schizochytrium limacinum comprises the following steps:

s1, culturing schizochytrium:

s11, transferring the schizochytrium limacinum to an inclined plane, culturing for 3-4 d at 25 ℃, activating the schizochytrium limacinum, then selecting the activated schizochytrium limacinum by using an inoculating loop, inoculating the activated schizochytrium limacinum to a primary seed culture medium, culturing for 2d at 25 ℃ and 200r/min to obtain primary seeds, finally inoculating the primary seeds to a secondary seed culture medium, and culturing for 2d at 25 ℃ and 200r/min to obtain secondary seeds; wherein the first-stage seed culture medium and the second-stage seed culture medium are respectively composed of 30g/L glucose, 10g/L yeast extract and 15g/L mineral substance; the minerals are composed of 10g/LNaCl, 0.5g/LCaCl2 & 2H2O, 1.5g/LMgSO4 & 7H2O, 1.25g/LK2SO4, 0.75g/LKH2PO4, 0.7g/L (NH4)2SO4 and 0.3g/LFeSO4 & 7H 2O;

s12, transferring the secondary seeds into a fermentation medium in a fermentation mechanism according to the inoculation amount of 6% for fermentation culture, wherein the tank pressure is 0.025MPa, the ventilation volume is 5L/min, the stirring speed is 40r/min, and the fermentation is carried out for 6d to obtain fermentation liquor; wherein the fermentation medium comprises 120g/L glucose, 10g/L yeast extract, 30g/L minerals and 0.15g/L vitamin complex, and the minerals comprise 18g/L NaCl and 1.5g/L NaCl₂·2H₂O、3.5g/LMgSO₄·7H₂O、2.25g/LK₂SO₄、1.75g/LKH₂PO₄、2g/L(NH₄)₂SO₄And 1g/LFeSO₄·7H₂O, wherein the composite cellulose consists of 60mg/L vitamin B6, 30mg/L vitamin B12, 20mg/L riboflavin, 10mg/L nicotinic acid and 30mg/L folic acid; in the process of fermentation cultureAdding glucose in batches, wherein the glucose is added once every 8-12 hours, and 30g/L is added every time;

s2, wall breaking of schizochytrium:

s3, extracting squalene:

s4, squalene purification:

Comparative example 1

In contrast to the examples:

the fermentation medium of this comparative example consisted of 120g/L glucose, 10g/L yeast extract, 30g/L minerals, and contained no complex cellulose.

The rest of the embodiments are the same and are not described in detail here.

Comparative example 2

In contrast to the examples:

the fermentation medium of this comparative example consisted of 120g/L glucose, 10g/L yeast extract, 30g/L minerals and 150mg/L vitamin B6.

The rest of the embodiments are the same and are not described in detail here.

Comparative example 3

In contrast to the examples:

the composite cellulose of this comparative example consisted of 100mg/L vitamin B6 and 50mg/L vitamin B12.

The rest of the embodiments are the same and are not described in detail here.

Comparative example 4

In contrast to the examples:

the composite cellulose of this comparative example consisted of 82mg/L vitamin B6, 41mg/L vitamin B12, and 27mg/L riboflavin.

The rest of the embodiments are the same and are not described in detail here.

Comparative example 5

In contrast to the examples:

the composite cellulose of this comparative example consisted of 75mg/L vitamin B6, 37.5mg/L vitamin B12, 25mg/L riboflavin, 12.5mg/L niacin.

The rest of the embodiments are the same and are not described in detail here.

Comparative example 6

In contrast to the examples:

the comparative example was fed glucose directly in one portion, without batch feeding.

The rest of the embodiments are the same and are not described in detail here.

Experimental example 1

The squalene content extracted in the above examples and comparative examples was measured, and the measurement results are shown in table 1 below.

TABLE 1 Squalene content measurement results

As can be seen from Table 1, the fermentation broths of the examples had a high content of both biomass and squalene obtained by extraction. Wherein, the fermentation medium of comparative example 1 is not added with vitamin complex, and the squalene content is obviously lower, while in comparative examples 2-5, when a plurality of different vitamins are adopted for compounding, the squalene content is improved, but still slightly lower than that of the examples. Further, it is understood from examples and comparative example 6 that when the glucose was fed in batch, the squalene content was increased to some extent, because the problem of inhibition of cell growth due to an excessively high substrate concentration could be avoided by feeding glucose in batch, and the squalene production could be increased. In conclusion, the best results are achieved if and only if the extraction method of the invention is used, where the fermentation medium is supplemented with multivitamins and glucose is fed in batch.

Experimental example 2

The squalene extracted above is used in a moisturizing and anti-aging cosmetic, wherein the moisturizing and anti-aging cosmetic of the embodiment is prepared from the following raw materials in percentage by mass: 0.8% of cosmetic active substance, 5% of emulsifier, 18% of grease, 6% of humectant, 0.4% of squalene extracted in the embodiment and the balance of deionized water. The blank control differs from the examples in that no squalene is added. The moisturizing and anti-aging effects of the cosmetics are detected, and the specific test results are shown in the following table 2.

Table 2 moisturizing anti-aging test results

As can be seen from Table 2, the squalene extracted by the invention has good moisturizing effect and anti-aging effect when used in cosmetics. Specifically, the skin moisture content value is higher and the change value is reduced slowly by adding 0.4% of squalene in the embodiment, so that the moisturizing effect is better; in addition, the embodiment has higher free radical clearance rate, which shows that the antioxidant and anti-aging effects are better. Therefore, the squalene extracted from schizochytrium limacinum has good moisturizing and anti-aging effects when added into cosmetics.

In addition, the existing squalene crude product still has the following defects in the production and preparation process:

1) the raw materials for preparing the squalene crude product are expensive, so that the stability and the purity of continuous extraction in the extraction process need to be ensured, most of the existing extraction steps aiming at the squalene crude product are manually carried out according to the work in the extraction aspect, the waste in the raw material extraction aspect is caused, and the excessive time is consumed;

2) the extraction steps are complicated, so that the subsequent extraction operation is not favorable for batch operation.

The present invention therefore provides the following means to solve the above-mentioned drawbacks:

referring to fig. 1, a centrifugal mechanism 1 includes a support base 11 supported on the ground, an arc support frame 12 is installed in the middle of the top end of the support base 11, a centrifugal storage tank 13 is transversely placed on the top surface of the arc support frame 12, the tank body of the centrifugal storage tank 13 is hollow, a rotating pin 14 is embedded in the middle of the end head of the tank body of the centrifugal storage tank 13, one end of the rotating pin 14 penetrates through the centrifugal storage tank 13 and is connected with the side wall of a placing cover 15 arranged at the end head in a through manner, and a centrifugal machine is installed inside the centrifugal storage tank 13;

place the cover body top edge of cover 15 and seted up circular fluting 16, conical hopper has been placed to the trench top intercommunication of circular fluting 16, places the internal centrifugal rotator that is used for schizochytrium centrifugal processing that is provided with of cover 15, places the one end of cover 15 and has seted up circular ejection of compact fluting 17.

The fermentation mechanism 2 is arranged at one side of the centrifugal storage tank 13. Fermentation mechanism 2 is including the linear type pipe 21 that is used for the defeated material, and the sealed intercommunication of the one end of linear type pipe 21 has raw material tanks, and the sealed intercommunication in top that buckles of the end of linear type pipe 21 has high-pressure liquid suction ware 22, and fermentation mechanism 2 is still including placing reation kettle 23 in centrifuge one side, and reation kettle 23's cauldron body top middle part is provided with constant temperature heater 24, and constant temperature heater 24's heating end is connected with reation kettle 23's lateral wall heat-conduction.

The constant temperature heater 24 who is equipped with guarantees 23 internal temperature's of reation kettle constancy of cauldron, combines temperature and humidity detector in time to detect the internal temperature of cauldron.

Be provided with the stirring rake in reation kettle 23, reation kettle 23's top middle part is provided with extractor 25, and the sealed intercommunication in top of extractor 25 has arc return bend 26, and the sealed intercommunication of the one end of arc return bend 26 has the ejection of compact mouth of pipe, and the sealed intercommunication of the orificial one end of ejection of compact has extraction pump 27.

Through the extractor 25, the arc-shaped bent pipe 26 and the extraction pump 27 which are arranged, the squalene crude product fermented by the reaction kettle 23 is conveniently pumped into the enzymolysis mechanism 3 by combining the extraction pump 27 and the extractor 25, the composite conveying of semi-finished products is ensured, and the processing efficiency of subsequent finished products is improved.

As shown in fig. 2: a plurality of sliding grooves 28 are sequentially arranged on the surface of the outer wall of the kettle body of the reaction kettle 23, a plurality of strip-shaped slide rails 29 are respectively embedded and arranged on two side walls of the groove positions of the plurality of sliding grooves 28, triangular supports 291 are supported at the bottoms of the strip-shaped slide rails 29, and the bottoms of the triangular supports 291 are supported and fixed with the ground through damping bases.

Through reation kettle 23, slip fluting 28 and a plurality of strip slide rail 29 that are equipped with, the support of guaranteeing the outside can be smooth to the firm support of top reation kettle 23, with the help of the stability advantage that tripod 291 supported, does certain adjustment through rising at reation kettle 23 inside temperature, can avoid producing regional rocking of a great extent in equipment operation process by utmost.

As shown in fig. 3-4: the enzymolysis mechanism 3 is communicated with the bottom of the extraction pump 27 in a sealing way. Enzymolysis mechanism 3 includes the enzymolysis tank 31 with reation kettle 23's cauldron body lateral wall fixed mounting, the box side interlude of enzymolysis tank 31 has the rotation roller axle 32, the one end scarf joint of rotation roller axle 32 has shaft coupling 33, the one end and the built-in broken oar transmission in enzymolysis tank 31 of shaft coupling 33 are connected, the outside of rotation roller axle 32 is provided with rotates hand (hold) 34, the one end of rotating hand (hold) 34 is external to have agitator motor, sealing connection between the bottom half of enzymolysis tank 31 and circular fluting 16 through the hose that the intercommunication set up.

Through enzymolysis case 31, live-rollers axle 32 and shaft coupling 33 that are equipped with under the agitator motor circular telegram circumstances, it is rotatory to drive the shaft coupling 33 of output, combines shaft coupling 33 to drive the inside broken oar of enzymolysis case 31 rotatory, carries out the breakage to the cell of suction enzymolysis incasement, and crushing efficiency is high, and need not to use the solvent, has reduced potential safety hazard and recovery cost.

The bottom fixed mounting of enzymolysis case 31 has the support frame, and the PLC controller is installed to the edge of support frame, and centrifuge, centrifugal spinner, high-pressure liquid suction ware 22, constant temperature heater 24, temperature and humidity detector, extractor 25, extraction pump 27 and agitator motor all with PLC controller electric connection.

The PLC is used for facilitating the electrification control of personnel on each electric device, and automatic operation is realized.

When the device is used specifically, firstly, a user sucks schizochytrium to be extracted into the reaction kettle 23 by means of a linear guide pipe and a high-pressure liquid extractor, the temperature inside the reaction kettle 23 is ensured to be suitable for fermentation by the detection effect of a temperature and humidity sensor of the reaction kettle 23, the proper conditions are provided, the mass propagation and fermentation of flora inside the reaction kettle 23 are ensured, the corresponding semi-finished schizochytrium is sucked into the enzymolysis box 31 from the arc-shaped bent pipe 26 at the top by means of the extraction pump 27, and the crushing paddle arranged in the enzymolysis box 31 is driven to rotate by the coupler 33 outside the enzymolysis box 31, so that the enzymolysis efficiency can be accelerated;

open centrifuge through the PLC controller, the centrifuge output rotates and drives the rotation round pin axle 14 of end department rotatory, it is rotatory when rotating round pin axle 14, the end drives the centrifugal spinner circular telegram that sets up, the schizochytrium after the zymohydrolysis of this moment gets into centrifuge from circular fluting 16's position through the hose that the bottom half intercommunication of enzymolysis case 31 set up, need be provided with the pump body on the hose here and draw, utilize inside centrifugal force, thereby separate out the higher squalene crude product of purity, surround reation kettle 23's fermentation all the time, the purification of the broken enzymolysis processing of enzymolysis case 31 up to last centrifuge, a series of processing has ensured the programmed standardized production of diagonal squalene crude product.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the present specification and directly/indirectly applied to other related technical fields within the spirit of the present invention are included in the scope of the present invention.

Claims

1. A method for extracting squalene from schizochytrium limacinum is characterized by comprising the following steps:

s1, culturing schizochytrium:

s12, transferring the secondary seeds into a fermentation medium in a fermentation mechanism (2) according to the inoculation amount of 6% for fermentation culture, wherein the tank pressure is 0.025MPa, the ventilation volume is 5L/min, the stirring speed is 40r/min, and the fermentation is carried out for 6d to obtain fermentation liquor; wherein the fermentation medium consists of 120g/L glucose, 10g/L yeast extract, 30g/L mineral and 0.15g/L vitamin complex; adding glucose in batch and in a flowing manner in the fermentation culture process, wherein the glucose is added once every 8-12 hours, and 30g/L is added every time;

s2, wall breaking of schizochytrium:

transferring the fermentation liquor obtained in the step S12 into an enzymolysis mechanism (3), adding alkaline protease into the fermentation liquor, and performing enzymolysis wall breaking on the schizochytrium limacinum, wherein the enzymolysis temperature is 63 ℃, the pH value is 7.8, and the enzymolysis time is 6 hours;

s3, extracting squalene:

s31, adding absolute ethyl alcohol into the bacterial liquid obtained after wall breaking in the step S2, wherein the volume ratio of the bacterial liquid to the absolute ethyl alcohol is 1: 1.2, removing a supernatant water-ethanol phase by oscillating and centrifugal separation of a centrifugal mechanism (1), and collecting bottom layer bacterial sludge;

s32, adding n-hexane into the bottom layer bacterial sludge, wherein the volume ratio of the bottom layer bacterial sludge to the n-hexane is 1: 1.15, oscillating and centrifuging by a centrifugal mechanism (1) to collect supernatant, repeating the process for a plurality of times until the supernatant is colorless and transparent, and finally evaporating to remove n-hexane to obtain a squalene crude product;

s4, squalene purification:

2. The method of claim 1, wherein the minerals are selected from the group consisting of 10g/L NaCl, 0.5g/L CaCl in step S11₂·2H₂O、1.5g/LMgSO₄·7H₂O、1.25g/LK₂SO₄、0.75g/LKH₂PO₄、0.7g/L(NH₄)₂SO₄And 0.3g/LFeSO₄·7H₂And (C) O.

3. The method of claim 1, wherein the mineral is selected from the group consisting of 18g/L NaCl, 1.5g/L CaCl and Squalene in step S12₂·2H₂O、3.5g/LMgSO₄·7H₂O、2.25g/LK₂SO₄、1.75g/LKH₂PO₄、2g/L(NH₄)₂SO₄And 1g/LFeSO₄·7H₂O, and the composite cellulose consists of 60mg/L of vitamin B6, 30mg/L of vitamin B12, 20mg/L of riboflavin, 10mg/L of nicotinic acid and 30mg/L of folic acid.

4. The method for extracting squalene from schizochytrium limacinum according to claim 1, wherein in step S4, the degumming treatment is carried out by preheating the crude squalene at 90 ℃, adding phosphoric acid solution, stirring uniformly, adding deionized water, stirring until precipitation is separated out, reducing stirring speed, continuing stirring for 15min, standing for 5min, and centrifuging to obtain the degummed crude squalene.

5. The method for extracting squalene from schizochytrium limacinum according to claim 4, wherein in step S4, the deacidification treatment is carried out by putting the degummed squalene crude product into an alkali refining pot, heating while stirring, spraying NaOH solution into the degummed squalene crude product after heating to 35 ℃, continuing stirring for 30min, then washing with water, finally centrifuging and carrying out oil-water separation to obtain the deacidified squalene crude product.

6. The method for extracting squalene from schizochytrium limacinum according to claim 1, wherein the centrifugal mechanism (1) comprises a support base (11) supported with the ground, an arc-shaped support frame (12) is installed in the middle of the top end of the support base (11), a centrifugal storage tank (13) is transversely placed on the top surface of the arc-shaped support frame (12), the tank body of the centrifugal storage tank (13) is hollow, a rotating pin shaft (14) is embedded in the middle of the end of the tank body of the centrifugal storage tank (13), one end of the rotating pin shaft (14) penetrates through the centrifugal storage tank (13) and is connected with the side wall of a placing cover (15) arranged at the end in a penetrating manner, and a centrifuge is installed inside the centrifugal storage tank (13);

place the cover body top edge of cover (15) and seted up circular fluting (16), the trench top intercommunication of circular fluting (16) has placed conical hopper, the internal centrifugal rotator that is used for schizochytrium centrifugal processing that is provided with of cover of placing cover (15), the one end of placing cover (15) has seted up circular ejection of compact fluting (17).

7. The method for extracting squalene from schizochytrium limacinum according to claim 6, wherein the fermentation mechanism (2) is placed at the side of the centrifugal storage tank (13), the fermentation mechanism (2) comprises a linear conduit (21) for conveying materials, one end of the linear conduit (21) is hermetically communicated with a raw material tank, the bent end of the linear conduit (21) is hermetically communicated with a high-pressure liquid extractor (22), the fermentation mechanism (2) further comprises a reaction kettle (23) placed at one side of the centrifuge, a constant-temperature heater (24) is arranged in the middle of the top end of the kettle body of the reaction kettle (23), and the heating end of the constant-temperature heater (24) is in heat conduction connection with the side wall of the reaction kettle (23).

8. The method for extracting squalene from schizochytrium limacinum according to claim 7, wherein a stirring paddle is arranged in the reaction kettle (23), an extractor (25) is arranged in the middle of the top end of the reaction kettle (23), the top end of the extractor (25) is hermetically communicated with an arc-shaped bent pipe (26), one end of the arc-shaped bent pipe (26) is hermetically communicated with a discharge pipe opening, and one end of the discharge pipe opening is hermetically communicated with an extraction pump (27); a plurality of sliding grooves (28) are sequentially arranged on the surface of the outer wall of the kettle body of the reaction kettle (23), and a plurality of strip-shaped sliding rails (29) are respectively embedded and arranged on the two side walls of the groove position of each sliding groove (28).

9. The method for extracting squalene from schizochytrium limacinum according to claim 8, wherein the enzymolysis mechanism (3) is in sealed communication with the bottom of the extraction pump (27), the enzymolysis mechanism (3) comprises an enzymolysis tank (31) fixedly mounted with the side wall of the kettle body of the reaction kettle (23), a rotating roll shaft (32) is inserted into the side of the tank body of the enzymolysis tank (31), a coupling (33) is embedded at one end of the rotating roll shaft (32), one end of the coupling (33) is in transmission connection with a crushing paddle built in the enzymolysis tank (31), a rotating handle (34) is arranged outside the rotating roll shaft (32), a stirring motor is externally connected at one end of the rotating handle (34), and the bottom of the tank body of the enzymolysis tank (31) is in sealed connection with the circular slot (16) through a hose which is in communication; the bottom fixed mounting of enzymolysis case (31) has the support frame, the PLC controller is installed to the edge of support frame, centrifuge, centrifugal spinner, high-pressure liquid extractor (22), constant temperature heating ware (24), decimator (25), extraction pump (27) and agitator motor all with PLC controller electric connection.

10. Use of squalene in a moisturizing and anti-aging cosmetic, wherein the squalene is extracted by the method of any one of claims 1 to 9.