WO2011108755A1

WO2011108755A1 - Method of producing lauric acid-containing oil or fat

Info

Publication number: WO2011108755A1
Application number: PCT/JP2011/055434
Authority: WO
Inventors: Hiroshi Yoshida; Fumikazu Takahashi; Yasushi Takimura
Original assignee: Kao Corp
Current assignee: Kao Corp
Priority date: 2010-03-03
Filing date: 2011-03-02
Publication date: 2011-09-09
Anticipated expiration: 2012-09-03
Also published as: CN102782145B; MY160740A; PH12012501675A1; US20110217743A1; AU2011221769B2; JP5899115B2; CN102782145A; AU2011221769A1; JP2013520959A

Abstract

To provide a method for supplying lauric acid with algae. A method for producing an oil or fat containing 3 weight% or higher lauric acid in the fatty acid composition, by culturing an alga belonging to the genus Symbiodinium in a medium and recovering from the culture.

Description

DESCRIPTION

METHOD OF PRODUCING LAURIC ACID-CONTAINING OIL OR FAT

[Field of the Invention]

[0001]

The present invention relates to a method for producing an oil or fat containing lauric acid as a constituent fatty- acid (hereinafter may also be referred to simply as "lauric acid-containing oil or fat"), the method employing an alga. [Background of the Invention]

[0002]

Lauric acid is a typical fatty acid contained in a large amount in coconut oil and palm kernel oil and is used as a raw material of a variety of surfactants, in foods, and for other materials .

Currently, the supply source of lauric acid is limited to coconut and palm kernels, which are grown in limited areas in the world. Cultivated lands now allocated to production of such lauric acid sources will be shared competitively with areas for bio- fuel for diesel engines and for food production. Excessive land cultivation for the production of lauric acid sources causes destruction of tropical rain forests.

Therefore, there is demand for creating a technique for supplying lauric acid, which technique does not rely on coconut or palm kernels .

[0003] Meanwhile, dinophyceae Crypthecodinium chonii, which grows not via photosynthesis but via heterotrophy, is known to be a lauric acid-producing organism and to have high lauric acid content (15.7%/total lipid) (Phytochemistry,

(1988) 27, 1679-1683) .

[0004]

From the viewpoints of cost for carbon sources and other factors, more preferred are algae species which can grow via photosynthesis (autotrophy) and have higher lauric acid content. However, among such photoautotrophic algae species, only Neochloris oleoabundans, having a lauric acid content of about 1 to 2% at best, is known (J. Ind. Microbiol. Biotechnol. (2009) 36: 821-826), and no algae species has heretofore been known to have higher lauric acid content.

[Summary of the Invention]

[0005]

The present invention relates to a method for producing an oil or fat containing lauric acid as a constituent fatty acid, which method includes culturing an alga belonging to the genus Symbiodinium in a medium and recovering, from the culture product, an oil or fat having a lauric acid content of 3 weight% or higher of the fatty acid composition.

The present invention also relates to a method for producing lauric acid, which method includes separating and recovering lauric acid from the oil or fat.

[Modes for Carrying Out the Invention]

[0006] The present invention provides a method for supplying lauric acid through employment of algae .

[0007]

The present inventors have carried out studies on lauric acid-producing organisms, and have found that algae belonging to the genus Symbiodinium, which are a

photoautotrophic dinophyceae, have high lauric acid content, and that an oil or fat containing lauric acid as a

constituent fatty acid at high content can be efficiently produced by use of the algae .

[0008]

According to the method of the present invention, which employs algae that can readily grow, an oil or fat containing lauric acid as a constituent fatty acid at high content can be efficiently produced, without imposing limitation on the cultivated fields for the growth of coconut and palm kernels or competing in the cultivated land with areas for food production, etc. In addition, according to the method of the present invention, destruction of tropical rain forests can be avoided.

[0009]

The method of the present invention for producing a lauric acid-containing oil or fat includes culturing an alga belonging to the genus Symbiodinium in a medium and

recovering, from the culture product, an oil or fat having a lauric acid content of 3 weight% or higher in the fatty acid composition. The oil or fat has a lauric acid content of 3 weight% or higher of the fatty acid composition. The lauric acid content is preferably 5 to 60 weight%, more preferably 10 to 60 weight¾.

[0010]

The algae employed in the present invention may be any algae strains belonging to genus Symbiodinium in the class Dinophyceae, so long as the strains have an ability to produce an oil or fat having a lauric acid content of 3 weight% or higher in the fatty acid composition.

[0011]

The algae of the present invention may be selected through, for example, the following screening procedure: i) dispensing a sterilized medium ( A medium (see Table 2) as a fresh water medium or Daigo IMK medium (see Table 3) as a seawater medium) into a culture container;

ii) inoculating an alga strain to the medium and performing stationary culturing at room temperature (22°C to 24°C) under illumination (illuminance: about 3,000 lux, illumination for 12 hours and dark for 12 hours) ;

iii) recovering the produced algae and extracting oil or fat; methyl esterifying the fatty acids; and determining the fatty acid composition, to thereby select an alga strain which can produce a lauric acid-containing oil or fat; and iv) selecting an alga strain having a lauric acid content of 3 weight¾ or higher based on the total fatty acid in the oil or fat. [0012]

Examples of preferred algae species include

Symbiodinium microadriaticum, Symbiodinium goreaui,

Symbiodinium linucheae, Symbiodinium bermudense, Symbiodinium meandrinae, Symbiodinium californium, Symbiodinium kawagutii, Symbiodinium corculorum, Symbiodinium consortia, Symbiodinium muscatinei, Symbiodinium freudenthal , Symbiodinium pulchrorum, Symbiodinium pilosum, and Symbiodinium sp. In some cases, Symbiodinium microadriaticum is called or described as

Zooxanthella microadriatica or Gymnodinium microadriaticum. Similarly, Symbiodinium linuchae is in some cases called or described as Gymnodinium linuchae . Examples of more

preferred Symbiodinium microadriaticum species include

Zooxanthella microadriatica strain LB2281 (available from the Culture Collection of Algae at University of Texas at Austin (UTEX) ) and strains having virtually the same phycological properties as those of strain LB2281. Examples of the

strains having virtually the same phycological properties as those of strain LB2281 include Symbiodinium sp. strain

CCMP2948, Symbiodinium sp. strain CC P 2592, Symbiodinium microadriaticum strain CCMP827, Symbiodinium microadriaticum strain CCMP2458 and the like.

[0013]

The strain LB2281 has the following phycological

properties. Strains belonging to the same genus as that of strain LB2281, and strains having virtually the same

mycological properties as those of strain LB2281 can be identified on the basis of the following properties.

i) symbiotic in other Protista, ullsc, Coelenterata and the like, such as Foraminifera, Radiolaria, shellfish coral and Actiniaria.

ii) having flagella extending from the belly of a cell and cingula; and

iii) cell coat having no platy structure (thecal plate) .

[0014]

The algae of the present invention also encompass

mutants of the aforementioned strain LB2281 or strains having virtually the same mycological properties as those of strain LB2281.

For example, a mutant strain designed so as to produce an oil or fat having a higher lauric acid content as compared with a corresponding wild-type strain is also included in the algae of the present invention.

Furthermore, genes derived from the alga of the present invention belonging to the genus Symbiodinium can be utilized for producing an oil or fat having a high lauric acid content.

[0015]

The algae of the present invention belonging to genus Symbiodinium may be cultured in an appropriate medium

prepared from natural or artificial seawater under

illumination through a cultivation method generally employed in culturing of micro-algae. [0016]

The medium which may be employed in the invention is a known medium which contains natural or artificial seawater as a base, and additives such as a nitrogen source, a phosphorus source, a metal salt, and vitamins.

Examples of the nitrogen source include Na 0₃, KN0₃, Ca(N0₃)₂, H₄NO₃, and ( H₄)₂S0₄. Examples of the phosphorus source include K₂HP0₄, KH₂P0₄, Na₂HP0₄, NaH₂P0 , and sodium glycerophosphate. Examples of the metal salt include NaCl, KC1, CaCl₂, gCl₂, Na₂S0_4f K₂S0₄, MgS0₄, Na₂C0₃, NaHC0₃, Na₂Si0₃, H₃B0₃, MnCl₂, MnS0₄, FeCl₃, FeS0₄, CoCl₂, ZnS0₄, CuS0₄, and

Na₂ o0₄. Examples of the vitamins include biotin, vitamin B12 , thiamine-HCl , nicotinic acid, inositol, folic acid, and thymine .

The aforementioned medium may further contain an

appropriate additive such as a carbon source or a trace metal, in order to promote production of lauric acid-containing oil or fat .

[0017]

Examples of preferred media include Daigo IMK medium, f/2 medium, ESM medium, LI medium, and MNK medium.

[0018]

Preferably, the pH of the thus-prepared medium is

adjusted to fall within a range of 7.0 to 8.0 through

addition of an appropriate acid or base, and is sterilized in an autoclave before use .

[0019] In culturing, no particular limitation is imposed on the amount of algae inoculated to the culture medium.

However, the amount is preferably 1.0 to 10.0% (vol/vol), more preferably 1.0 to 5.0% (vol/vol) , with respect to the amount of culturing medium.

[0020]

No particular limitation is imposed on the culture temperature, so long as the growth of the algae of the present invention is not adversely affected. Generally, the culturing is preferably performed at 10 to 30°C, more

preferably 15 to 25°C.

[0021]

Light irradiation may be performed under any conditions, so long as photosynthesis can be performed. Needless to say, either artificial light or sunlight may be employed.

The illuminance preferably falls within a range of 100 to 50,000 lux, more preferably 300 to 10,000 lux.

[0022]

The pH during culturing is generally 6.5 to 8.5,

preferably 7.0 to 8.0.

[0023]

Culturing is performed so that an alga is grown in a high density. For example, the culturing period is 7 to 120 days, preferably 7 to 30 days. Any of aeration and agitation culturing, shake culturing, and stationary culturing may be employed.

[0024] After completion of culturing, an alga is separated through a customary method such as centrifugation or

filtration. The thus-separated algae mass as is, or a broken product thereof obtained through sonication, by means of Dyno Mill or by other means is subjected to solvent extraction with organic solvent such as chloroform, hexane, butanol, methanol, or ethyl acetate, whereby lauric-acid-containing oil or fat can be recovered.

[0025]

When strain LB2281 is used, 100 g of the dry algae contain a lauric acid-containing oil or fat in an amount of about 10 to about 20 g. That is, the amount of lauric acid- containing oil or fat produced in 1 L of medium reaches about 0.05 to about 0.1 g.

In this case, the oil or fat has a lauric acid content as high as 6.0 to 15.0 weight% of the fatty acid composition. Thus, the amount of produced lauric acid-containing oil or fat in 1 L of medium is as high as about 0.003 to about 0.015 g-

[0026]

Lauric acid may be separated from the lauric acid- containing oil or fat by transforming the oil or fat into a fatty acid mixture or an ester of a fatty acid through a known method; and recovering high concentration of lauric acid through the urea addition method, cooling separation, HPLC, supercritical liquid chromatography, etc.

[Examples] [0027]

Referential Example 1: Culturing of algae and analysis of fatty acid composition

From the Culture Collection of Algae at University of Texas at Austin (UTEX) , the following 20 algae strains were obtained.

[0028]

[Table 1]

Algae strains and culture media

[0029]

Culturing of algae was performed in the following methods. WA medium (composition, see Table 2) and a commercial medium (Daigo IMK medium, product of Nihon

Pharmaceutical Co., Ltd.) (composition, see Table 3) were employed as a fresh water medium and a seawater medium, respectively.

[0030]

[Table 2]

Composition of WA medium

[0031]

[Table 3]

Composition of IMK medium

[0032]

Sterilized culture tubes (16 mm x 150 mm) (product of VWR) each plugged with a sponge stopper (60882-167, product of VWR) were used, and a sterilized medium (10 mL/tube) was dispensed to the tubes. Each alga strain (100 μ]_ι (in the case of liquid medium) or 1 platinum loop (in the case of solid medium) ) was inoculated to the culture medium.

Stationary culturing was performed at room temperature (22°C to 24°C) under a fluorescent lamp (illuminance: about 3,000 lux, illumination for 12 hours and dark for 12 hours) .

Through centrifugation of the algae culture at 3,000 rpm for 30 minutes, an alga pellet was obtained. The alga pellet was dried at 80°C for about 3 hours to about 16 hours, to thereby obtain dry algae, and the weight of the dry

product was measured. The dry product was suspended in 1% saline (0.5 mL) , and 5 mg/mL 7-pentadecanone (10 μΐι) was added as an internal standard to the suspension.

Subsequently, chloroform (0.5 mL) and methanol (1 mL) were added to the suspension, and the mixture was vigorously stirred and then allowed to stand for 30 minutes. Thereafter, chloroform (0.5 mL) and 1.5% KC1 (0.5 mL) were added to the mixture and stirred, followed by centrifugation at 3,000 rpm for 15 minutes. The formed chloroform layer (lower layer) was recovered.

[0033]

The thus-prepared lipid fraction (about 500 μL) was treated with nitrogen to dryness, and 0.5 N potassium hydroxide/methanol solution (700 ΐ;) was added to the dried fraction, and then incubated at 80°C for 30 minutes.

Subsequently, 14% boron trifluoride solution (product of

SIGMA) (1 mL) was added to the fraction, and then incubated at 80°C for 20 minutes. Then, hexane (1 mL) and saturated saline (1 mL) were added to the above mixture, and the

mixture was allowed to stand at room temperature for 30 minutes. The thus-obtained hexane layer (upper layer) was recovered and analyzed by GC .

[0034]

The GC analysis was performed under the following

conditions: chromatograph, HP 7890A GC-FID (product of

Agilent); column, DB-1 MS 30 m x 200 μιη x 0.25 μπι (product of J&W scientific) ,- mobile phase, high-purity helium,- flow rate,

1 mL/min; and temperature elevation, 100°C (1 minute) ,

5°C/min, and 280°C (20 minutes) . As saturated fatty acid controls, the following commercial products (all produced from SIGMA) were purchased and analyzed: methyl laurate (C12) , methyl myristate (C14) , methyl palmitate (C16) , and methyl stearate (C18) . As unsaturated fatty acid controls, the following commercial products (all produced from SIGMA) were purchased and analyzed: methyl palmitoleate (C16:l), methyl oleate (C18:l), methyl linoleate (C18:2), methyl linolenate (C18:3), methyl eicosapentaenoate (C20:5), and methyl

docosahexaenoate (C22:6). Identification of fatty acids was performed on the basis of coincidence in retention time between the fatty acid analyte and the corresponding standard. Laurie acid was also identified by GC-MS. C16 multi- unsaturated fatty acids were estimated from the GC-MS

analytical results and are represented by C16:x (x is 2 or 3, wherein x represents the number of unsaturated bonds in fatty acid) . The GC-MS analysis was performed under the following conditions: chromatograph, HP7890A GC and 5975C MS (products of Agilent); column, DB-1 ms 30 m x 200 μπι x 0.25 μιη (product of J&W scientific) ; mobile phase, high-purity helium,- flow rate, 1 mL/min; and temperature elevation, 100°C (1 minute) , 5°C/min, and 280°C (20 minutes) . The amount of a fatty acid ester detected through GC analysis was calculated with reference to the internal standard, and the sum of the amounts of fatty acids was employed as the total fatty acid amount . The value obtained by dividing the total fatty acid amount by the amount of dry algae and multiplying the ratio by 100 was employed as a fatty acid content (%) .

Table 4 shows the fatty acid compositional data of tested algae species .

[0035]

[Table 4]

Fatty acid composition analysis

[0036]

The fatty acid composition of the lipids produced by various algae species was analyzed. However, no algae species in which lauric acid was accumulated was found.

[0037]

Example 1

A Zooxanthella microa.dria.tica (in class Dinophyceae) strain LB2281 obtained from UTEX was employed in the

experiment .

The microorganisms were cultured in a Daigo IMK medium.

Sterilized culture tubes (16 mm x 150 mm) (product of VWR) each plugged with a sponge stopper (60882-167, product of VWR) were used as culture containers, and a sterilized medium (10 mL/tube) was dispensed to the tubes. 100 μΐι of algal culture was inoculated to a new culture medium. Culturing was performed at room temperature (22°C to 24°C) under a fluorescent lamp (illuminance: about 3,000 lux, illumination for 12 hours and dark for 12 hours) for 59 days.

In a manner similar to that employed in Referential Example 1, recovery of algae, extraction of lipid, methyl esterification, and GC analysis were performed, to thereby analyze the fatty acid composition. Table 5 shows the results .

[0038] [Table 5]

Fatty acid composition of Z. icroadriatica (strain LB2281)

[0039]

In Zooxanthella microadriatica (strain LB2281) ,

accumulation of lauric acid was observed that it content reached 13.2% of total fatty acid amount.

[0040]

Example 2 : Production of algae oil or fat having high lauric acid content

[0041]

An oil or fat having high lauric acid content was produced in the following manner.

Zooxanthella microadriatica (strain LB2281) was

cultivated in a 500-mL Sakaguchi flask that contained 200 mL of IMK Medium, and stationary culturing was performed at room temperature (22°C to 24°C) under illumination (illuminance: about 3,000 lux, illumination for 12 hours and dark for 12 hours) for 38 days. The culture liquid was centrifuged at 3,000 rpm for 30 minutes, to thereby recover cells.

[0042]

The alga was dried at 80°C for about 16 hours, and chloroform/methanol (C/M) (1:1) (4 mL) was added to the dried algae. Under sonication, oil or fat was extracted at 40°C for 30 minutes. In a similar manner, extraction with C/M (1:1) (4 mL) was performed again. The thus-recovered

supernatant (about 8 mL) was filtered through a membrane filter (product of Millipore, Millex-LH, Hydrophilic PTFE,

0.45 μπι, φ25 mm) . The filtrate was dried by means of a centrifugal evaporator, to thereby obtain 19.4 mg of an oil or fat fraction of the strain LB2281.

[0043]

Through a method similar to that described in Example 1, the oil or fat composition was analyzed. The fatty acid content of the oil or fat fraction was 34.4%, and the lauric acid content of the total fatty acid amount was 7.8%. That is, lauric acid (0.518 mg) was recovered from the oil or fat fraction (19.4 mg) .

[0044]

Example 3 : Accumulation of lauric acid in dinophyceae

A Zooxanthella microadriatica (in class Dinophyceae) strain LB2282 obtained from UTEX and Symbiodinium sp.

CCMP2948 obtained from the Proyasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) were employed in the experiment .

The alga was cultured in a Daigo IMK medium. Sterilized culture tubes (16 mm x 150 mm) (product of VWR) each plugged with a sponge stopper (60882-167, product of VWR) were used, and a sterilized medium (10 mL/tube) was dispensed to the tubes. 100 μΐι of each alga culture was inoculated to the culture medium. And each of the algae was cultured at room temperature (20°C) under a fluorescent lamp (illuminance:

about 3,000 lux, illumination for 12 hours and dark for 12 hours) for about two months .

In a manner similar to that employed in Referential Example 1, recovery of algae, extraction of lipid, methyl esterification, and GC analysis were performed, to thereby analyze the fatty acid composition. Table 6 shows the results .

[0045]

[Table 6]

Fatty acid composition of genus Symhiodinium {Zooxanthella)

[0046]

In genus Symbiodinium (Zooxanthella) , accumulation of lauric acid was observed with 3 weight% or higher of the total fatty acid amount .

[0047]

Example 4 : Accumulation of lauric acid in dinophyceae A Symbiodinium sp. CCMP2592 and Symbiodinium

microadriaticum CC P827 and CCMP2458 obtained from the CCMP. were employed in the experiment .

The alga was cultured in a Daigo IMK medium.

Sterilized culture tubes (16 mm x 150 mm) (product of VWR) each plugged with a sponge stopper (60882-167, product of VWR) were used, and a sterilized medium (10 mL/tube) was dispensed to the tubes. 100 μΐι of each alga culture was inoculated to the culture medium. And each of the algae was cultured at room temperature (20°C) under a fluorescent lamp (illuminance: about 3,000 lux, illumination for 12 hours and dark for 12 hours) for about 42 days.

In a manner similar to that employed in Referential Example 1, recovery of algae, extraction of lipid, methyl esterification, and GC analysis were performed, to thereby analyze the fatty acid composition. Table 7 shows the results .

[0048] [Table 7]

Fatty acid composition of genus Symbiodinium

[ 0049]

In genus Symbiodinium, accumulation of lauric acid was observed with 3 weight% or higher of the total fatty acid amount .

Claims

[Claim 1]

A method for producing an oil or fat containing lauric acid as a constituent fatty acid, which method comprises culturing an alga belonging to the genus Symbiodinium in a medium and recovering, from the culture product, an oil or fat having a lauric acid content of 3 weight% or higher of the fatty acid composition.

[Claim 2]

A method according to claim 1, wherein the alga belonging to the genus Symbiodinium is Symbiodinium

microadriaticum.

[Claim 3]

A method according to claim 2 , wherein the alga belonging to the genus Symbiodinium is a Zooxanthella microadriatica strain LB2281, or an alga strain having virtually the same mycological properties as those of strain LB2281.

[Claim 4]

A method according to any one of claims 1 to 3 , wherein culturing is performed for 7 to 120 days under light

irradiation at an illuminance of 300 to 10,000 lux.

[Claim 5]

A method for producing lauric acid, wherein the method comprises separating and recovering lauric acid from an oil or fat produced through a method as recited in claim 1.