KR101506554B1 - Extraction method of omega-3 unsaturated fatty acid - Google Patents

Abstract

Provided is a method for extracting omega-3 unsaturated fatty acid comprising the steps of (a) pulverizing a fermented solution of microalgae having omega-3 unsaturated fatty acid production ability, adding an extracted solvent, mixing the same, and obtaining an emulsion; (b) mixing the emulsion obtained in step (a), adding diatomite, and heating the same at 50 to 100°C; and (c) leaving the mixed solution of step (b) without stirring, separating phase, and separating an oil layer of the top floor.

Description

Extraction method of omega-3 unsaturated fatty acid [

The present invention relates to a method for extracting omega-3 unsaturated fatty acids with a high extraction yield using a solvent extraction method. More particularly, the present invention relates to a method for extracting omega-3 unsaturated fatty acids with a high extraction yield by including a step of adding an extraction solvent to form an emulsion and heating the oil in the presence of diatomaceous earth to separate the oil layer by phase separation will be.

Omega-3 unsaturated fatty acids are also referred to as omega-3 highly unsaturated fatty acids or polyunsaturated fatty acids (PUFAs), and typically include docosahexa Docosahexaenoic acid (DHA), Eicosapentaenoic acid (EPA), Docosapentaenoic acid (DPA), and? -Linolenic acid are known. Docosahexaenoic acid and eicosapentaenoic acid are essential fatty acids that are not synthesized in the body and should be ingested mainly through food.

The main source of omega-3 unsaturated fatty acids, including docosahexaenoic acid, is fish oil extracted from fish such as herring, salmon, and tuna. However, the difficulty of supplying fish oil continuously and pollution caused by heavy metals and organic chemicals in fish oil have been studied, and studies on the production method of omega-3 unsaturated fatty acids including docosahexaenoic acid by microbial culture are under way. For example, the preparation of omega-3 unsaturated fatty acids by microorganisms of the genus Thraustochytrium and Schizochytrium , a kind of marine microalgae, is described in Ellenbogen et al. (Ellenbogen BB, S. Aaronson, S. Goldstein and M. Belsky, Comparative Biochemistry and Physiology , 29 , 805-811 (1969)). In addition, microorganisms such as Shizokitrium sp. ATCC 20888 ( Schizochytrium sp . ATCC 20888), Shizokatori sp . ATCC 20889 ( Schizochytrium sp . ATCC 20889), Schizochytrium limacinum SR21) have been developed (U.S. Patent No. 5,130,242, U.S. Patent No. 5,340,742, JP-A-1997-000284, U.S. Patent No. 6,582,941, etc.).

The process of culturing omega-3 unsaturated fatty acid-producing strains and extracting omega-3 unsaturated fatty acids from them usually involves isolation of microorganisms, drying, and extraction of intracellular lipids using a non-polar organic solvent such as hexane. In general, the intracellular lipids of microorganisms are extracted after the microalgae are dried and the dried cells are disrupted. The inventors of the present invention have developed an improved extraction method (Korean Patent Registration No. 10-1115357) that solves the problems of temporal and economic loss due to the microalgae drying process. That is, Korean Patent Registration No. 10-1115357 is a method for extracting crude oil (i.e., omega-3 unsaturated fatty acid) from wet microalgae.

On the other hand, in the case of extracting omega-3 unsaturated fatty acids from a microalgae lysate in a wet state using an extraction solvent (e.g., hexane), not only a physical separation process such as a centrifugation process of an extract is required, The yield of the fermentation broth varies greatly depending on the conditions such as the viscosity and stirring speed of the fermentation broth, and the extraction yield is low, which is unsatisfactory.

The present inventors have conducted various studies to develop a method for extracting omega-3 unsaturated fatty acids from wet microbial algae with high extraction yield. The present inventors have found that when a process of forming an emulsion by adding an extraction solvent to a microalgae lysate in a wet state and separating the oil layer by phase separation by heating in the presence of diatomaceous earth results in a remarkably high yield of omega-3 unsaturated fatty acid Lt; / RTI >

Accordingly, it is an object of the present invention to provide a method for extracting omega-3 unsaturated fatty acids which can achieve a high extraction yield through a combination of an emulsion forming step and a phase separation step.

According to one aspect of the present invention, there is provided a method for producing omega-3 fatty acids, comprising: (a) disrupting a fermentation broth of microalgae having an omega-3 unsaturated fatty acid producing ability, adding an extraction solvent and stirring to form an emulsion; (b) While the emulsion obtained in step (a) is stirred, diatomaceous earth is added and 50 To 100 < 0 >C; And (c) separating the mixed liquid of step (b) by allowing to stand without stirring to separate the oil layer of the uppermost layer, and then extracting the omega-3 unsaturated fatty acid.

The microalgae may be selected from the group consisting of Schizochytrium sp. Strain, Thraustochytrium sp. Strain, Japonochytrium sp. Strain, Ulkenia sp. Strain, Crypthecodinium sp. Strain ≪ / RTI >

In the method for extracting omega-3 unsaturated fatty acids according to the present invention, the extraction solvent of step (a) is selected from the group consisting of pentane, hexane, heptane, cyclohexane, toluene, methylene chloride, methyl acetate, acetone, chloroform, methanol, Mixed solvent, and the stirring of step (a) or step (b) may be carried out at 100 to 500 rpm.

In the method for extracting omega-3 unsaturated fatty acids according to the present invention, the diatomaceous earth may be added in an amount of 0.1 to 10 parts by weight, preferably 0.1 to 5 parts by weight, based on 100 parts by weight of the emulsion obtained in the step (a); The heating temperature was 60 To 80 < 0 > C.

The extraction method according to the present invention is an improved solvent extraction method of omega-3 unsaturated fatty acid, which includes a combination of an emulsion formation process and a phase separation process. The production method of the present invention can maximize the contact area between the extraction solvent and the fermentation broth by minimizing the variation in the extraction yield by including the emulsion formation process. The extraction method according to the present invention can also avoid the physical separation process such as the centrifugation process of the extract by separating the oil layer by phase separation by warming in the presence of diatomite so that the whole extraction process can be simplified. Particularly, the extraction method according to the present invention can achieve a remarkably high extraction yield (i.e., an extraction yield improved by about 40%) as compared with the extraction method using the conventional solvent extraction method.

1 is a photograph showing an emulsion formed by adding hexane to a microalgae lump in accordance with the present invention and stirring.
2 is a photograph showing a bubble generated when diatomite is added to the emulsion obtained according to the present invention and heated
Fig. 3 is a photograph showing that the lowest layer (including water layer and sediment), the middle layer (including cell debris), and the oil layer are layered by the extraction method of the present invention.

As used herein, the term " omega-3 unsaturated fatty acid "refers to an omega-3 highly unsaturated fatty acid or polyunsaturated fatty acid (PUFA) Free acid forms and acyl ester forms in which the terminal carboxylic acid of a fatty acid forms an ester bond with a C ₁ -C ₄ alcohol or the like. Specifically, docosahexaenoic acid (Docosahexaenoic acid, DHA), eicosapentaenoic acid (Eicosapentaenoic aicd, EPA), Toko Inc. yen acid penta (Docosapentaenoic aicd, DPA), and α- linolenic acid, etc. and their C ₁ ~ C ₄ alcohol, and the like, preferably DHA or EPA.

The term "omega-3 unsaturated fatty acid-producing strain" refers to a microalgae or the like having omega-3 unsaturated fatty acid producing ability. The strain is selected from the group consisting of Schizochytrium sp. Strain, Thraustochytrium sp. Strain, Japonochytrium sp. Strain, Ulkenia sp. Strain, Crypthecodinium sp. Strain For example, a strain selected from the group consisting of Shizokatori sp . ATCC 20888 ( Schizochytrium sp . ATCC 20888), Shizokatori sp . ATCC 20889 ( Schizochytrium sp . ATCC 20889), Schizochytrium limacinum SR21), Schizochytrium sp . RT0100P1 ( Schizochytrium sp. RT0100P1) (KCTC 10937BP), Schizochytrium sp . SEK-228 ( Schizochytrium sp . RT0100P1) (KCTC 10938BP), Shizokitriium sp . CC44 ( Schizochytrium sp . CC44) (KCTC 11566BP), and the like. Culturing of the omega-3 unsaturated fatty acid-producing strains can be carried out according to a known method (U.S. Patent No. 5,130,242, U.S. Patent No. 5,340,742, Japanese Patent Application No. 1997-000284, U.S. Patent No. 6,582,941, Patent Registration No. 10-0700486, Korean Patent Registration No. 10-1068520, etc.).

(A) disrupting a fermentation broth of microalgae having omega-3 unsaturated fatty acid producing ability, adding an extraction solvent and stirring to form an emulsion; (b) While the emulsion obtained in step (a) is stirred, diatomaceous earth is added and 50 To 100 < 0 >C; And (c) allowing the mixed solution of step (b) to stand in the absence of stirring to effect phase separation, and then separating the oil layer of the uppermost layer of the omega-3 unsaturated fatty acid.

In step (a), the crushing of the fermentation broth of the microalgae can be used without limitation as long as it is a crushing method capable of breaking down the cell walls of microalgae in a wet state. For example, disruption of the fermentation broth of the microalgae can be accomplished by enzymatic shredding; Chemical method; Mechanical methods such as heat treatment, French press, compactor, colloid mill, bead mill; And a combination method thereof.

The extraction solvent used in step (a) may be selected from the conventional extraction solvents known in the art, such as (i) water, (ii) anhydrous or hydric alcohol having 1-4 carbon atoms, such as methanol, ethanol, propanol, (Iii) a mixed solvent of the lower alcohol and water, (iv) acetone, (v) ethyl acetate, (vi) chloroform, (vi) 1, 3-butylene glycol, (vii) hexane, (viii) diethyl ether, or (ix) butyl acetate, preferably lower alcohol or hexane, more preferably hexane.

The stirring of step (a) may be carried out under conditions that allow the formation of an emulsion, for example, at 100 to 500 rpm, preferably 200 to 300 rpm. By performing the emulsion forming process as described above, not only the extraction yield can be increased by maximizing the contact area between the extraction solvent and the fermentation broth, but also the deviation of the extraction yield can be minimized.

The stirring in step (b) is not particularly limited. Considering that step (b) is carried out on the emulsion obtained in step (a), that is, taking into consideration that it is carried out through a continuous process, it is preferable to keep the stirring condition of step (a) Do. Thus, the stirring in step (b) may be performed at 100 to 500 rpm, preferably 200 to 300 rpm, but is not limited thereto.

In step (b), the diatomaceous earth may be added in an amount of 0.1 to 10 parts by weight, preferably 0.1 to 5 parts by weight, based on 100 parts by weight of the emulsion obtained in step (a). Further, To 100 < 0 > C, preferably from 60 to 80 < 0 > C. When diatomaceous earth is added and heated, the air existing in the pores of the diatomaceous earth expands and bubbles are generated to effectively phase-separate the emulsion (see FIG. 2), and thus a physical separation process such as centrifugation Performance can be avoided.

Step (c) is performed by phase separation by leaving the mixed solution of step (b) without agitation, and then separating the uppermost oil layer. When the mixed solution of the step (b) is left without stirring, it is layered into the lowest layer (including water layer and sediment), the middle layer (including cell debris), and the oil layer (see FIG. The setting may be carried out at ambient conditions (for example, room temperature of 20 to 26 DEG C) for about 10 to 60 minutes, preferably for 20 to 30 minutes.

The separated oil layer is subjected to removal of the extraction solvent by a method such as concentration under reduced pressure to obtain omega-3 unsaturated fatty acid in a crude oil state (crude oil state). The obtained omega-3 unsaturated fatty acid can be purified according to a known method. For example, it is possible to carry out a process for removing saturated fatty acids by performing a winterization process according to US Pat. No. 6,812,009, and the like. Also, according to U.S. Patent No. 6,750,048 and the like, maintenance such as defection, neutralization, By performing the manufacturing process, it is possible to remove phospholipids, free fatty acids, pigments, remaining saponification substances, and low-molecular volatiles that cause odors.

Example 1.

According to the method disclosed in Korean Patent Registration No. 10-1068520, Shizokatorium sp . Flask fermentation of CC44 ( Schizochytrium sp . CC44) (KCTC 11566BP) was performed. After the fermentation broth was subjected to a strain breaking process using a bead mill, hexane was added to about 200 parts by weight of hexane to 100 parts by weight of the fermentation broth and stirred at about 300 rpm to form an emulsion (see FIG. 1). Diatomaceous earth was added to about 100 parts by weight of the emulsion while stirring the emulsion continuously, and the mixture was heated to about 70 캜 (see FIG. 2). The mixed solution was allowed to stand at room temperature for 60 minutes without stirring to separate into the lowest layer (including water layer and sediment), the middle layer (including cell debris), and the oil layer. The oil layer was separated and concentrated under reduced pressure to remove the hexane. Thereafter, the omega-3 unsaturated fatty acids were purified by performing a dipping and maintenance manufacturing process according to U.S. Patent No. 6,812,009 and U.S. Patent No. 6,750,048. The amount of crude oil in the reference microalgae was measured in accordance with the Food and Drug Administration's Standard Test Method 1.1.5.1 Food and Drug Administration Test Method 1.1.5.1, and the extraction yield was determined by measuring the crude oil of the microalgae The amount of contrast extracted oil was calculated. The extraction yield thus calculated was about 92%.

Example 2.

The omega-3 unsaturated fatty acid was obtained in the same manner as in Example 1, except that hexane was added in an amount of about 300 parts by weight per 100 parts by weight of the fermentation broth. The extraction yield was about 92% as measured by the same method as in Example 1, and no substantial change in the yield was found with increasing extraction solvent.

Comparative Example 1

According to the method disclosed in Korean Patent Registration No. 10-1068520, Shizokatorium sp . Flask fermentation of CC44 ( Schizochytrium sp . CC44) (KCTC 11566BP) was performed. After the fermentation broth was subjected to the strain crushing process using a bead mill, hexane was added so as to be about 200 parts by weight of hexane to 100 parts by weight of the fermentation broth, and the mixture was extracted with stirring at about 50 rpm for 60 minutes. The extract was centrifuged to separate the oil layer and concentrated under reduced pressure to remove the hexane. Thereafter, the omega-3 unsaturated fatty acids were purified by performing a dewaxing and maintenance manufacturing process according to U.S. Patent No. 6,812,009 and U.S. Patent No. 6,750,048. The extraction yield was measured in the same manner as in Example 1, and the extraction yield was about 52%.

Comparative Example 2

According to the method disclosed in Korean Patent Registration No. 10-1068520, Shizokatorium sp . Flask fermentation of CC44 ( Schizochytrium sp . CC44) (KCTC 11566BP) was performed. The fermentation broth was subjected to a strain breaking process using a bead mill, and then hexane was added thereto so that about 200 parts by weight of hexane was added to 100 parts by weight of the fermentation broth and stirred at about 300 rpm to form an emulsion. The obtained emulsion was continuously stirred for 30 minutes to perform extraction. The extract was centrifuged to separate the oil layer and concentrated under reduced pressure to remove the hexane. Thereafter, the omega-3 unsaturated fatty acids were purified by performing a dewaxing and maintenance manufacturing process according to U.S. Patent No. 6,812,009 and U.S. Patent No. 6,750,048. The extraction yield was measured in the same manner as in Example 1, and the extraction yield was about 77%.

Claims (7)

(a) disrupting a fermentation broth of microalgae having omega-3 unsaturated fatty acid producing ability, adding an extraction solvent and stirring to form an emulsion;
(b) While the emulsion obtained in step (a) is stirred, diatomaceous earth is added and 50 To 100 < 0 >C; And
(c) leaving the mixed solution of step (b) without stirring and phase separation, and separating the uppermost oil layer
Of the omega-3 unsaturated fatty acid. The microalgae of claim 1, wherein the microalgae are selected from the group consisting of Schizochytrium sp., Thraustochytrium sp., Japonochytrium sp., Ulkenia sp. Wherein the strain is selected from the group consisting of strains belonging to the genus Crypthecodinium . The method of claim 1, wherein the extraction solvent of step (a) is selected from the group consisting of pentane, hexane, heptane, cyclohexane, toluene, methylene chloride, methyl acetate, acetone, chloroform, methanol, A method of extracting omega-3 unsaturated fatty acids. 2. The method according to claim 1, wherein the stirring of step (a) or step (b) is carried out at 100 to 500 rpm. The method for extracting omega-3 unsaturated fatty acids according to claim 1, wherein diatomite is added in an amount of 0.1 to 10 parts by weight based on 100 parts by weight of the emulsion obtained in step (a). The method for extracting omega-3 unsaturated fatty acids according to claim 5, wherein the diatomaceous earth is added in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the emulsion obtained in step (a). The method according to claim 1, To < RTI ID = 0.0 > 80 C. < / RTI >

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