WO2012128396A1

WO2012128396A1 - Method of producing lauric acid-containing oil or fat and lauric acid or esters thereof

Info

Publication number: WO2012128396A1
Application number: PCT/JP2012/058505
Authority: WO
Inventors: Hiroshi Yoshida; Fumikazu Takahashi; Yasushi Takimura
Original assignee: Kao Corp
Current assignee: Kao Corp
Priority date: 2011-03-24
Filing date: 2012-03-23
Publication date: 2012-09-27
Anticipated expiration: 2013-09-24
Also published as: JP2014508507A; AU2012232064A1; AU2012232064B2; JP5927199B2

Abstract

To provide a method for supplying oil or fat containing lauric acid as a constituent fatty acid and a method for supplying lauric acid or esters thereof using the oil or fat with algae. A method for producing lauric acid or esters thereof, which method includes, culturing at least one species selected from the group consisting of algae in the class Chlorarachniophyceae and algae in the class Cryptophyceae consisting of algae belonging to the genus Rhodomonas and algae belonging to the genus Chroomonas selected from among Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas nordstedtii, and Chroomonas placoidea in a medium; recovering, from the culture product, an oil or fat having a lauric acid content of 3 weight% or higher of the fatty acid composition; and, as needed, esterifying the lauric acid in the recovered oil or fat, followed by separating and recovering the lauric acid or esters thereof.

Description

DESCRIPTION

METHOD OF PRODUCING LAURIC ACID-CONTAINING OIL OR FAT AND LAURIC ACID OR ESTERS THEREOF

[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") and lauric acid or esters

thereof using the oil or fat, the method employing algae.

[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, algae are known to effectively produce an oil or fat, and the productivity per area of the algae is about 10 times that of a plant or the like (Non-Patent

Document 1) . Among algae, 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) (Non-Patent Document 2) .

[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 (Non-Patent

Document 3) , and no algae species has heretofore been known to have higher lauric acid content .

[Non-Patent Document 1] : Biotechnology Advances, (2007) 25, 294-306

[Non-Patent Document 2] : Phytochemistry, (1988) 27, 1679-1683 [Non-Patent Document 3]: J. Ind. Microbiol. Biotechnol .

(2009) 36: 821-826

[Summary of the Invention] [0005]

The present invention provides a method for producing lauric acid or esters thereof, which method including:

culturing at least one species selected from the group consisting of algae in the class Chlorarachniophyceae and algae in the class Cryptophyceae consisting of algae

belonging to the genus Rhodomonas and algae belonging to the genus Chroomonas selected from among Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas nordstedtii , and

Chroomonas placoidea in a medium; recovering, from the culture product, an oil or fat having a lauric acid content of 3 weight% or higher of the fatty acid composition; and, as needed, esterifying the lauric acid in the recovered oil or fat, followed by separating and recovering the lauric acid or esters thereof.

Further, the present invention provides a method for producing an oil or fat containing lauric acid as a

constituent fatty acid, which method including: culturing, in a medium, at least one species of algae belonging to the genus Chroomonas selected from among Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas nordstedtii , and

Chroomonas placoidea; 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.

[Modes for Carrying Out the Invention]

[0006] The present invention relates to provision of a method for supplying oil or fat containing lauric acid as a

constituent fatty acid and a method for supplying lauric acid or esters thereof using the oil or fat, through employment of algae .

[0007]

The present inventors have carried out studies on lauric acid-producing organisms, and have found that among photoautotrophic algae, algae in the class of

Chlorarachniophyceae which are a unicellular algae and algae belonging to the genus Rhodomonas or algae belonging to the genus Chroo onas selected from among Chroomonas diplococca , Chroomonas mesostigmatica , Chroomonas nordstedtii , and

Chroomonas placoidea among algae in the class of

Cryptophyceae have high lauric acid content, and that an oil or fat containing lauric acid as a constituent fatty acid at high content and further lauric acid or esters thereof 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, in a medium, at least one species of algae in the class

Cryptophyceae selected from the group consisting of algae belonging to the genus Rhodomonas and algae belonging to the genus Chroomonas selected from among Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas nordstedtii , and

Chroomonas placoidea and recovering, from the culture product, an oil or fat having a lauric acid content of 3 eight% or higher in the fatty acid composition.

Further, the method of the present invention for

producing lauric acid or esters thereof includes culturing at least one species selected from the group consisting of algae in the class Chlorarachniophyceae and algae in the class

Cryptophyceae consisting of algae belonging to the genus

Rhodomonas and algae belonging to the genus Chroomonas

selected from among Chroomonas diplococca, Chroomonas

mesostigmatica, Chroomonas nordstedtii , and Chroomonas

placoidea in a medium; recovering, from the culture product, an oil or fat having a lauric acid content of 3 weight% or higher of the fatty acid composition; and, as needed,

esterifying the lauric acid in the recovered oil or fat, followed by separating and recovering the lauric acid or esters thereof.

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 from 3 to 60 weight%, more preferably from 5 to 60 weight%, even more preferably from 6 to 60 weight%, even more preferably from 7 to 60 weight%, even more preferably from 8 to 60 weight%, even more preferably from 9 to 60 weight%, even more preferably 10 to 60 weight%, even more preferably 11 to 5o weight%, and even more preferably 12 to 40 weight%.

[0010]

The algae employed in the present invention may be any algae strains in the class Chlorarachniophyceae, 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 .

The algae in the class Cryptophyceae employed in the present invention may be any algae strains belonging to the genus Rhodomonas or to the genus Chroomonas selected from among Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas nordstedtii , and Chroomonas placoidea, more preferably the algae strains belonging to the genus

Chroomonas selected from among Chroomonas diplococca,

Chroomonas mesostigmatica and Chroomonas nordstedtii , 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 alga 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 weight% or higher based on the total fatty acid in the oil or fat.

[0012]

Examples of algae belonging to the class Chlorarachnion include algae belonging to the genus Chlorarachnion,

Lotharella, Gymnochlora, Cryptochlora, and Bigelowiella .

Among them, the genus Lotharella, Gymnochlora, and

Bigelowiella are preferred.

Examples of more preferred algae in the class

Chlorarachnion include the following algae. Examples of more preferred algae belonging to the genus Lotharella include Lotharella globosa, Lotharella amoebiformis, and Lotharella vacuolata. Examples of more preferred algae belonging to the genus Gymnochlora include Gymnochlora stellata. Examples of more preferred Bigelowiella include Bigelowiella natans.

Among Lotharella globosa strains, Lotharella globosa strain CCMP1729 is more preferred. Among Lotharella amoebiformis strains, Lotharella amoebiformis strain CCMP2058 is more preferred. Among Lotharella vacuolata strains, Lotharella vacuolata strain CCMP240 is preferred. Among Gymnochlora stellata strains, Gymnochlora stellata strain CC P2057 is more preferred. Among Bigelowiella natans strains,

Bigelowiella natans strains CCMP621 and CCMP2757 are more preferred (these strains are available from, for example, the Provasoli-Guillard National Center for Culture of Marine

Phytoplankton (CCMP) ) . Strains having virtually the same phycological properties as those of algae strains are also more preferred. For example, in recent years, Lotharella amoebiformis has been proposed to be assigned to the new genus Amorphochlora amoebiformis . These strains are

considered to have virtually the same phycological properties as those of Lotharella amoebiformis . Among these, Lotharella globosa strains are more preferred and Lotharella globosa strain CCMP1729 or strains having virtually the same

phycological properties as the strain are even more preferred.

[0013]

Examples of the strain having virtually the same

phycological properties as those of Lotharella amoebiformis strain CCMP2058 include Lotharella amoebiformis strain Ryukyu. Examples of the strain having virtually the same phycological properties as those of Lotharella vacuolata strain CCMP240 include Lotharella vacuolata strain FK18G. Examples of the strain having virtually the same phycological properties as those of Gymnochlora stellata strain CCMP2057 include

Gymnochlora stellata strain Guam-1. Examples of the strain having virtually the same phycological properties as those of Bigelowiella natans strains CCMP621 and CC P2757 include Bigelowiella natans strains All, 490, and VA3.

[0014]

The aforementioned algae strains have the following phycological properties. Strains belonging to the same genus as that of the algae strains, and strains having virtually the same mycological properties as those of the algae strains can be identified on the basis of the following properties. <Phycological properties of the algae in the class

Chiorarachnlophyceae>

i) Containing chlorophyll a and b

ii) Chloroplast surrounded by four membranes

iii) Having nucleomorph

iv) Not accumulating starch

v) Presence of amoeba phase and cell-wall-having phase vi) Having no stigma

i) Amoeboid vegetative cell

ii) Nucleomorph in pyrenoid

<Phycological properties of the algae belonging to the genus Cryptochlora> i) Spherical vegetative cell

ii) Pyrenoid structure unknown

i) The pyrenoid matrix was devided into two halves by a slit of the periplastidial compartment.

ii) Nucleomorph present in the periphery of chloroplast in the vicinity of the pyrenoid base

i) Inner membranes of chloroplast envelope invaginating pyrenoid matrix

i) Swarmers asexually proliferating

ii) The pyrenoid matrix was slightly invaded by

periplastidial compartment from the tip of the pyrenoid. iii) Nucleomorph present at the pyrenoid base

<Phycological properties of Lotharella globosa strain

CCMP1729>

i) Spherical vegetative cell, no ameba-like emerging upon proliferation

i) Ameba-like vegetative cell <Phycological properties of Lotharella vacuolata strain CCMP240>

i) Spherical in the main stage of life cycle, ameba- like cells having filopodia observed during the initial to middle culture stage

ii) Having vacuoles larger than those of the other algae belonging to the genus Lotharella

iii) Vegetative proliferation through binary fission of ameba-like cells

i) Star- shape amebic organism having many filopodia not forming network

ii) Cells having cell wall or swarmers are absent throughout the life cycle

i) Swarmers in the vegetative stage, not ameba-like cells

ii) Having two flagella (short and long)

iii) Having no striae

[0015]

As the algae belonging to the genus Chroomonas of the present invention, examples of preferred Chroomonas

diplococca including Chroomonas diplococca strain UTEX

LB2422; examples of preferred Chroomonas mesostigmatica

including Chroomonas mesostigmatica strain NIES1370; examples of preferred Chroomonas nordstedtii including Chroomonas nordstedtii strains NIES707 and NIES710; examples of

preferred Chroomonas placoidea including Chroomonas placoidea strain NIES705 (these strains are available from The culture collection of algae at University of Texas at Austin (UTEX) , National Institute for Environmental Studies (NIES) , etc.); and strains having virtually the same phycological properties as those of algae strains are mentioned.

As the algae belonging to the genus Rhodomonas,

Rhodomonas salina is preferred, with Rhodomonas salina

UTEX1375, Rhodomonas salina CCMP272, and strains having virtually the same phycological properties as those of algae strains being more preferred; and Rhodomonas salina UTEX1375 or strains having virtually the same phycological properties as those of algae strains being even more preferred. These strains are available from UTEX and The Provas li-Guillard National Center for Culture of Marine Phytoplankton (CCMP) .

[0016]

Examples of the strain having virtually the same

phycological properties as those of Chroomonas mesostigmatica strain NIES1370 include Chroomonas mesostigmatica strain TKB- 112. Examples of the strain having virtually the same phycological properties as those of Chroomonas nordstedtii strain NIES707 include Chroomonas nordstedtii strain #00173. Examples of the strain having virtually the same phycological properties as those of Chroomonas nordstedtii strain NIES710 include Chroomonas nordstedtii strain #00331. Examples of the strain having virtually the same phycological properties as those of Chroomonas placoidea strain NIES705 include Chroomonas placoidea strain CCAP 978/8.

Examples of the strain having virtually the same phycological properties as those of Rhodomonas salina strain CCMP272 include Rhodomonas salina strain el-023.

[0017]

The aforementioned algae strains have the following phycological properties. Strains belonging to the same genus as that of the algae strains, and strains having virtually the same mycological properties as those of the algae strains can be identified on the basis of the following properties. <Phycological properties of the class Cryptophyceae>

i) Containing phycobilin and chlorophyll c

ii) Chloroplast surrounded by four membranes

iii) Having nucleomorph

iv) Having tubular pleuronematic and tubular unilateral flagella

v) Accumulating a- 1,4 -starch

i) Barrel -form cell with no cingulum

ii) Having two ejectisomes in a row

iii) Blue to green chloroplast

iv) Stigma generally observed centrally in a cell

<Phycological properties of the algae belonging to the genus Rhodomonas> i) Egg- form cell having a short cingulum

ii) Red to reddish brown chloroplast with distinct pyrenoid

iv) Stigma generally observed centrally in a cell

i) Having a large number of lamellar structures in a chloroplast

ii) Having one large pyrenoid with a chloroplast

i) Having no stigma

ii) Having phototaxis with respect to light having a wavelength of 450 nm to 650 nm

i) Having ligules at a flagellum bearing

i) Two flagellua shorter than the cell length arising from a subapical end of the cell

ii) Short sulcus, gullet with ejectisomes in two rows, reaching to the cell center

iii) Having one reddish brown to yellowish orange chloroplast, with one pyrenoid being dorsal and surrounded by marked starch sheath

[0018] The algae of the present invention also encompass mutants of the aforementioned algae strains and strains having virtually the same mycological properties as those of the aforementioned algae strains.

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, a gene derived from the algae in the class Chlorarachniophyceae and a gene derived from the algae in the class Cryptophyceae may be employed to produce an oil or fat having a high lauric acid content.

[0019]

The algae in the class Chlorarachniophyceae and the algae in the class Cryptophyceae of the present invention 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.

[0020]

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 NaN0₃ , K 0₃, Ca(N0₃)_2/ NH₄NO₃, and (NH₄)₂S0₄. Examples of the phosphorus source include K₂HP0₄, KH₂P0₄, Na₂HP0₄, NaH₂P0₄, and sodium glycerophosphate. Examples of the metal salt include NaCl, KC1, CaCl₂, MgCl₂, Na₂S0₄, K₂S0₄, MgS0₄, Na₂C0₃ , NaHC0₃ , Na₂Si0₃, H₃BO₃, MnCl₂, MnS0₄, FeCl₃, FeS0₄, CoCl₂, ZnS0_4; CuS0_4/ and

Na₂Mo0₄. 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 .

[0021]

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

[0022]

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.

[0023]

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.

[0024]

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.

[0025]

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.

[0026]

The pH during culturing is generally 6.5 to 8.5,

preferably 7.0 to 8.0.

[0027]

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 .

[0028]

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

filtration. The thus- separated alga 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.

[0029] When a Gymnochlora stellata strain CC P2057 is used, 100 g of the dry alga contain a lauric acid-containing oil or fat in an amount of about 5 to about 10 g. That is, the amount of lauric acid-containing oil or fat produced in 1 L of medium reaches about 0.02 to about 0.05 g.

In this case, the oil or fat has a lauric acid content as high as 4.0 to 8.5 weight% of the fatty acid composition. Thus, the amount of produced lauric acid in 1 L of medium is as high as about 0.0008 to about 0.0043 g.

[0030]

When a Chroomonas diplococca strain strain UTEX LB2422 is used, 100 g of the dry alga contains a lauric acid- containing oil or fat in an amount of about 3 to about 4 g. That is, the amount of lauric acid- containing oil or fat produced in 1 L of medium reaches about 0.007 to about 0.016 g-

In this case, the oil or fat has a lauric acid content as high as 5.0 to 17.0 weight% of the fatty acid composition. Thus, the amount of produced lauric acid in 1 L of medium is as high as about 0.0004 to about 0.0027 g.

[0031]

Separation and recovery of lauric acid from the lauric acid-containing oil or fat may be carried 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 .

Further, the lauric acid ester can be separated and recovered from the lauric acid-containing oil or fat by esterifying the lauric acid contained in the oil or fat.

For example, the lauric acid-containing oil or fat is reacted with alcohol such as methanol under the presence of alkaline catalyst and esters of lauric acid esters can be separated and recovered from the reaction product.

Here, examples of lauric acid esters include lower alkyl esters such as methyl ester and ethyl ester, and methyl ester is preferred.

Further, lauryl alcohol can be separated and recovered from the lauric acid-containing oil or fat by reducing the lauric acid contained in the oil or fat.

For example, the lauric acid-containing oil or fat is hydrogenated under the presence of hydrogenation catalyst and lauryl alcohol can be separated and recovered from the reaction product.

[0032]

In accordance with the embodiments mentioned above, the present invention discloses the following [1] method for producing lauric acid or esters thereof and [12] method for producing oil or fat containing lauric acid as a constituent fatty acid, and preferably discloses [2] - [11] methods for producing lauric acid or esters thereof and [12] - [14] methods for producing oil or fat containing lauric acid as a

constituent fatty acid. [1] Method for producing lauric acid or esters thereof, which method including: culturing at least one species

selected from the group consisting of algae in the class

Chlorarachniophyceae and algae in the class Cryptophyceae consisting of algae belonging to the genus Rhodomonas and algae belonging to the genus Chroomonas selected from among Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas nordstedtii, and Chroomonas placoidea in a medium; recovering, from the culture product, an oil or fat having a lauric acid content of 3 weight% or higher of the fatty acid composition; and, as needed, esterifying the lauric acid in the recovered oil or fat, followed by separating and recovering the lauric acid or esters thereof

[2] Method of [1] , in which the algae in the class

Chlorarachniophyceae is those belonging to the genus

Lotharella, Gymnochlora, or Bigelowiella .

[3] Method of [1] or [2] , in which the algae belonging to the genus Lotharella is Lotharella globosa, Lotharella amoebiformis or Lotharella vacuolata, the algae belonging to the genus Gymnochlora is Gymnochlora stellata and the algae belonging to the genus Bigelowiella include Bigelowiella natans .

[4] Method of [3] , in which the algae belonging to the genus Lotharella is Lotharella globosa strain CCMP1729,

Lotharella amoebiformis strain CCMP2058,

Lotharella vacuolata strain CCMP240 or strains having

virtually the same phycological properties as those of the algae strains.

[5] Method of [3] , in which the algae belonging to the genus Gymnochlora is Gymnochlora stellata strain CCMP2057 or strains having virtually the same phycological properties as those of the algae strain.

[6] Method of [3] , in which the algae belonging to the genus Bigelowiella is Bigelowiella natans strains CCMP621, CCMP2757 or strains having virtually the same phycological properties as those of the algae strains.

[7] Method of [1] , in which the algae belonging to the genus Chroomonas is Chroomonas diplococca strain UTEX LB2422, Chroomonas mesostigmatica strain NIES1370, Chroomonas

nordstedtii strains NIES707, Chroomonas nordstedtii strains NIES NIES710, Chroomonas placoidea strain NIES705, or strains having virtually the same phycological properties as those of the algae strains.

[8] Method of [1] , in which the algae belonging to the genus Rhodomonas is Rhodomonas salina.

[9] Method of [8] , in which the Rhodomonas salina is Rhodomonas salina UTEX1375, Rhodomonas salina CCMP272 or strains having virtually the same phycological properties as those of the algae strains.

[10] Method of [1] - [9] , in which the culturing is performed under light irradiation at an illuminance of 300 to 10,000 for 7-120 days.

[11] Method of [1] - [10] , in which the lauric acid ester is methyl laurate. [12] Method for producing an oil or fat containing lauric acid as a constituent fatty acid, which method

including: culturing, in a medium, at least one species of algae belonging to the genus Chroomonas selected from among Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas nordstedtii , and Chroomonas placoidea; 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.

[13] Method of [12] , in which the algae belonging to the genus Chroomonas is Chroomonas diplococca strain UTEX LB2422, Chroomonas mesostigmatica strain NIES1370, Chroomonas nordstedtii strains NIES707, Chroomonas nordstedtii strains NIES710, Chroomonas placoidea strain NIES705, or strains having virtually the same phycological properties as those of the algae strains.

[14] Method of [12] or [13] , in which the culturing is performed under light irradiation at an illuminance of 300 to 10,000 for 7-120 days.

[Examples]

[0033]

Example 1 : Culturing of algae in the class

Chlorarachniophyceae and analysis of fatty acid composition

From "The Provasoli-Guillard National Center for

Culture of Marine Phytoplankton (CCMP) , " the following 6 algae strains were obtained and employed in the experiments.

[0034] [Table 1]

Algae strains

[0035]

Culturing of algae was performed in the following methods. A commercial medium (Daigo IMK medium, product of Nihon Pharmaceutical Co., Ltd.) (composition, see Table 2) was employed as a seawater medium.

[0036]

[Table 2]

Composition of IMK medium

[0037]

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 μΐι (in the case of liquid medium) or 1 platinum loop (in the case of solid medium)) was inoculated to a new 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 algaculture 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 alga, 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 by using a Pasteur pipette.

[0038]

The thus-prepared lipid fraction (about 500 L) was treated with nitrogen to dryness, and 0.5 N potassium hydroxide/methanol solution (700 μΙ_) 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.

[0039]

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 μτη x 0.25 μιτι (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, HP 7890A GC and 5975C MS (products of Agilent); column, DB-1 ms 30 m x 200 μηι x 0.25 μτη (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 alga and multiplying the ratio by 100 was employed as a fatty acid content (%) .

Table 3 shows the fatty acid compositional data of tested algae species.

[0040]

[Table 3]

Fatty acid composition analysis

[0041]

Accumulation of lauric acid (>3% of the total fatty acids) was observed in Lotharella globosa strain CCMP1729, Lotharella amoebiformis strain CCMP2058, Lotharella vacuolata strain CCMP240, Gymnochlora stellata strain CCMP2057, and Bigelowiella natans strains CCMP621 and CC P2757.

Particularly, in Gymnochlora stellata strain CC P2057, high fatty acid productivity and high-level accumulation of lauric acid (about 8.5% of the total fatty acids) were observed.

[0042]

Example 2: Production of lauric acid using algae in the class Chlorarachniophyceae

Gymnochlora stellata CCMP2057 was subjected to

stationary culturing in culture tubes (16 mm x 150 mm, containing IMK medium (10 mL) ) 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 four weeks, to thereby produce a seed culture liquid. The seed culture liquid was inoculated into IMK medium (100 mL) at 2% (v/v) placed in a 200-mL Erlenmeyer flask, 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 31 days. The culture liquid was centrifuged at 3,000 rpm for 30 minutes, to thereby recover cells, which were then washed once with 1% (w/v) aqueous sodium chloride solution.

[0043] The alga which had been recovered from the culture liquid (100 mL) was dried at 80°C for about 16 hours, and chloroform (2 mL) and methanol (4 mL) were added to the dried alga. The mixture was vigorously stirred and then allowed to stand for 30 minutes. Thereafter, chloroform (2 mL) and 1.5% KC1 (2 mL) were added thereto, and the obtained mixture was stirred. The stirred mixture was centrifuged at 3,000 rpm for 15 minutes, and the chloroform layer (lower layer) was collected by using a Pasteur pipette. An aliquot (100 μL) was recovered from the collected chloroform layer and dried to solid through nitrogen gas sprayed thereto. The dried product was dissolved in chloroform (10 μL) . An aliquot (1 L) was sampled from the chloroform solution, and the neutral fat content thereof was determined by means of Iatroscan

(product of Mitsubishi Kagaku Iatron, Inc.). As a result, neutral lipid (1.2 mg) was obtained from the culture liquid

(100 mL) .

[0044]

Through a methyl esterification method similar to that described in Example 1, an aliquot (500 L) of the above- collected chloroform layer was analyzed. As a result, the total amount of the fatty acids obtained from the culture liquid (100 mL) was 6.2 mg, and the lauric acid content of the total fatty acid was 4.9%. That is, lauric acid (0.3 mg) was recovered from the culture liquid (100 mL) .

[0045]

Example 3 : Culturing of algae belonging to the genus Chroomonas and analysis of fatty acid composition

From the Culture Collection of Algae at University of Texas at Austin (UTEX) and National Institute for

Environmental Studies (NIES) , the following 9 algae strains belonging to the genus Chroomonas were obtained and employed in the experiments.

[0046]

[Table 4]

Algae strains

[0047]

Culturing of algae was performed in the following

methods. C medium (composition, see Table 5) and WA medium (composition, see Table 6) were employed as fresh water media, and f/2 medium (composition, see Table 7) and a commercial medium (Daigo IMK medium, product of Nihon Pharmaceutical Co., Ltd.) (composition, see Table 8) were employed as seawater media .

[0048] [Table 5]

Composition of C medium

[Table 6]

Composition of WA medium

[0050]

[Table 7]

Composition of f/2 medium

[0051]

[Table 8]

Composition of IMK medium

[0052]

Through centrifugation of the alga 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 alga, 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 μΐ 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% KCl (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 by using a Pasteur pipette.

[0053]

The thus-prepared lipid fraction (about 500 L) was treated with nitrogen to dryness, and 0.5 N potassium

hydroxide/methanol solution (700 L) 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 .

[0054]

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 μπι x 0.25 μτη (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 : 1) , 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, HP 7890A GC and 5975C MS (products of Agilent); column, DB-1 ms 30 m x 200 μτη x 0.25 μτη (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 (g) . The fatty acid productivity (g/L or mg/L) was obtained by dividing the total fatty acid amount by the volume of culture liquid (L) . The value obtained by dividing the amount of each fatty acid by the total amount of the fatty acids and multiplying the ratio by 100 was employed as a fatty acid content (%) .

Table 9 shows the fatty acid compositional data of tested algae species.

[0055]

[Table 9]

Fatty acid composition analysis

[0056]

Accumulation of lauric acid (>3% of the total fatty acids) was observed in Chroomonas diplococca strain UTEX

LB2422, Chroomonas mesostigmatica strain NIES1370, Chroomonas nordstedtii strain NIES707, Chroomonas nordstedtii strain NIES710, and Chroomonas placoidea strain NIES705.

Particularly, in Chroomonas diplococca strain UTEX LB2422, a very high-level accumulation of lauric acid (about 17% of the total fatty acids) was observed.

[0057]

Example 4: Production of lauric acid using algae belonging to the genus Chromonas

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

Chroomonas diplococca (strain LB2422) was subjected to stationary culturing in culture tubes (16 mm x 150 mm, containing IMK medium (10 mL) ) 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 four weeks, to thereby produce a seed culture liquid. The seed culture liquid was inoculated into IMK medium (100 mL) at 2% (v/v) placed in a 200-mL Erlenmeyer flask, and stationary culturing was performed at room temperature (22°C to 24°C) under illumination (illuminance: about 3,000 lux,

[0058]

The alga which had been recovered from the culture liquid (100 mL) was dried at 80°C for about 16 hours, and chloroform (2 mL) and methanol (4 mL) were added to the dried alga. The mixture was vigorously stirred and then allowed to stand for 30 minutes. Thereafter, chloroform (2 mL) and 1.5% KC1 (2 mL) were added thereto, and the obtained mixture was stirred. The stirred mixture was centrifuged at 3,000 rpm for 15 minutes, and the chloroform layer (lower layer) was collected by using Pasteur pipette. An aliquot (100 μΐ,) was recovered from the collected chloroform layer and dried to solid through nitrogen gas sprayed thereto. The dried product was dissolved in chloroform (10 μΐ.) . An aliquot (1 μΐι) was sampled from the chloroform solution, and the neutral fat content thereof was determined by means of Iatroscan (product of Mitsubishi Kagaku Iatron, Inc.). As a result, neutral lipid (0.64 mg) was obtained from the culture liquid (100 mL) .

[0059]

Through a methyl esterification method similar to that described in Example 3, an aliquot (500 μΐι) of the above- collected chloroform layer was analyzed. As a result, the total amount of the fatty acids obtained from the culture liquid (100 mL) was 3.5 mg, and the lauric acid content of the fatty acids was 5.7%. That is, lauric acid (0.2 mg) was recovered from the culture liquid (100 mL) .

[0060]

Example 5 : Culturing of algae belonging to the genus

Rhodomonas and analysis of fatty acid composition

As algae belonging to the genus Rhodomonas, Rhodomonas salina UTEX1375 and Rhodomonas salina CCMP272 were purchased from The culture collection of algae at University of Texas at Austin (UTEX) and The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) , and these algae strains were tested by using an IMK medium through the method similar to that employed in Example 3. The test procedure of Example 3 was repeated, except that the culture times

described in Table 9 were employed. The total fatty acid productivity and the ratio of each fatty acid were determined. The test has revealed that both algae strains had lauric acid contents of 9.4% and 8.8% respectively, which are higher than 3%.

[0061]

[Table 10]

Fatty acid composition analysis

Claims

[Claim 1]

A method for producing lauric acid or esters thereof, which method comprises, culturing at least one species

selected from the group consisting of algae in the class

Chlorarachniophyceae and algae in the class Cryptophyceae consisting of algae belonging to the genus Rhodomonas and algae belonging to the genus Chroomonas selected from among Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas nordstedtii , and Chroomonas placoidea in a medium; recovering, from the culture product, an oil or fat having a lauric acid content of 3 weight% or higher of the fatty acid composition; and, as needed, esterifying the lauric acid in the recovered oil or fat, followed by separating and recovering the lauric acid or esters thereof .

[Claim 2]

The method according to claim 1, wherein the algae in the class Chlorarachniophyceae are algae belonging to the genus Lotharella, Gymnochlora, or Bigelowiella.

[Claim 3]

The method according to claim 1 or 2 , wherein the algae belonging to the genus Lotharella is Lotharella globosa,

Lotharella amoebiformis, or Lotharella vacuolata, the algae belonging to the genus Gymnochlora is Gymnochlora stellata, and the algae belonging to the genus Bigelowiella is

Bigelowiella natans .

[Claim 4] The method according to claim 3 , wherein the algae belonging to the genus Lotharella is a Lotharella globosa strain CCMP1729, a Lotharella amoebiformis strain CC P2058, a Lotharella vacuolata strain CCMP240, or an algae strain having virtually the same phycological properties as those of the algae strains .

[Claim 5]

The method according to claim 3, wherein the algae belonging to the genus Gymnochlora is a Gymnochlora stellata strain CCMP2057 or an alga strain having virtually the same phycological properties as those of the algae strains.

[Claim 6]

The method according to claim 3, wherein the algae belonging to the genus Bigelowiella is a Bigelowiella natans strain CCMP621 , a Bigelowiella natans strain CC P2757, or an alga strain having virtually the same phycological properties as those of the algae strains .

[Claim 7]

The method according to claim 1, wherein the alga belonging to the genus Chroomonas is a Chroomonas diplococca strain UTEX LB2422, a Chroomonas mesostigmatica strain

NIES1370, a Chroomonas nordstedtii strains NIES707, a

Chroomonas nordstedtii strains NIES710, a Chroomonas

placoidea strain NIES705, or an alga strain having virtually the same phycological properties as those of the algae strains .

[Claim 8] The method according to claim 1, wherein the algae belonging to the genus Rhodomonas is Rhodomonas salina.

[Claim 9]

The method according to claim 8, wherein the Rhodomonas salina is a Rhodomonas salina UTEX1375, a Rhodomonas salina CCMP272, or an alga strain having virtually the same

phycological properties as those of the algae strains.

[Claim 10]

The method according to any one of claims 1 to 9, wherein culturing is performed for 7 to 120 days under light irradiation at an illuminance of 300 to 10,000 lux.

[Claim 11]

The method according to any one of claims 1 to 10, wherein the lauric acid ester is methyl laurate .

[Claim 12]

A method for producing an oil or fat comprising lauric acid as a constituent fatty acid, which method comprises culturing, in a medium, at least one species of algae

belonging to the genus Chroomonas selected from among

Chroomonas diplococca, Chroomonas mesostigmatica , Chroomonas nordstedtii , and Chroomonas placoidea and recovering, from the culture product, an oil or fat comprising a lauric acid content of 3 weight% or higher of the fatty acid composition.

[Claim 13]

The method according to claim 12, wherein the alga belonging to the genus Chroomonas is a Chroomonas diplococca strain UTEX LB2422, a Chroomonas mesostigmatica strain NIES1370, a Chroomonas nordstedtii strains NIES707, a

Chroomonas nordstedtii strains NIES710, a Chroomonas placoidea strain NIES705, or an alga strain having virtually the same phycological properties as those of the algae strains .

[Claim 14]

The method according to Claim 12 or 13, wherein culturing is performed for 7 to 120 days under light

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