JP3931219B2 - Process for producing highly unsaturated fatty acid-containing fats and oils - Google Patents

Description

【００２４】
この結果を以下の表１に示す。
【表１】

【００２５】
実施例２ シゾキトリウム属微生物による油脂の製造(２)
シゾキトリウム属ＳＲ２１株について、容量５００ｍｌの三角フラスコに、以下の組成の培地１００ｍｌを仕込み、実施例１と同条件で撹拌培養を行った。
培地組成（ｇ／Ｌ）
１）グルコース：２０
２）ポリペプトン：１０
３）酵母エキス：５
４）５０％人工海水：１Ｌ
５）タンニン酸：０.０５
培養後、実施例１と同様にして脂肪酸メチルエステルを調製した。これを、実施例１と同条件でガスクロマトグラフィーにかけ、脂肪酸組成を分析した。この結果を図１に示す。
結果からわかるように、タンニン酸を添加したときには、対照と比較して、ＥＰＡに相当するピークの減少が認められた。
【００２６】
実施例３ シゾキトリウム属微生物による油脂の製造(３)
シゾキトリウム属ＳＲ２１株について、容量５００ｍｌの三角フラスコに、以下の組成の培地１００ｍｌを仕込み、実施例１と同条件で撹拌培養を行った。
培地組成（ｇ／Ｌ）
１）グルコース：２０
２）ポリペプトン：１０
３）酵母エキス：５
４）５０％人工海水：１Ｌ
５）シアノコバラミン：０.０５
培養後、実施例１と同様にして脂肪酸メチルエステルを調製した。これを、実施例１と同条件でガスクロマトグラフィーにかけ、脂肪酸組成を分析した。この結果を図１に示す。
結果からわかるように、シアノコバラミンを添加したときには、対照と比較して、奇数鎖脂肪酸である１５：０および１７：０の割合が減少し、偶数鎖脂肪酸である１４：０の割合が増加した。これは、シアノコバラミンがＳＲ２１株のアミノ酸代謝に何らかの影響を与え、奇数鎖脂肪酸の前駆体が供給されなくなったためと推定される。
【００２７】
実施例４ シゾキトリウム属微生物由来の油脂のカラム精製
実施例１の方法に従い０.２ｇ／Ｌのｐ−トルイル酸を含む培地で培養して得た菌体を乾燥して乾燥菌体を得た。この乾燥菌体をクロロホルムとメタノールとの混合溶媒中でガラスビーズを用いて破砕し、濾過した後、ヘキサンで抽出することにより油脂を得た。得られた油脂を、ヘキサン中、水酸化カリウムを含むエタノールの添加によってエステル化した。常法に従って後処理することにより、脂肪酸のエステル体を含む油脂混合物を得た。この油脂混合物は、ガスクロマトグラフィーによる分析によれば、ＤＨＡエチルエステルを約５０％、ＤＰＡエチルエステルを約２.４％、ＥＰＡエチルエステルを８.５％含有していた。
【００２８】
この油脂混合物につき、下記条件での液体クロマトグラフィーによりＤＨＡエチルエステル、ＤＰＡエチルエステルおよびＥＰＡエチルエステルを分離した。即ち、粒子径５０μｍ、細孔径１２０オングストロームおよび炭素含有率１７％のオクタデシルシラン(ＯＤＳ)充填剤[YMC＊GEL ODS-AM-120-S50]を充填した内径４００ｍｍおよび長さ２０００ｍｍのカラムを用いてＨＰＬＣに付した。
上記の油脂混合物１.２ｋｇをメタノール中の１０％溶液として上記カラムに導入し、メタノールを使用して４.４リットル／分の流速で溶離し、分画を得た。各分画につき溶媒を留去し、ＤＨＡエチルエステル３３０ｇ、ＤＰＡエチルエステル２ｇ、ＥＰＡエチルエステル８２ｇを得た。
【００２９】
【発明の効果】
本発明に従えば、微生物が産生するＤＨＡ含有油脂に含まれる高度不飽和脂肪酸のうち、例えばＤＰＡやＥＰＡの含有比率を変化させることができるので、ＤＨＡ、ＤＰＡおよびＥＰＡの含有比率の異なる培養菌体または油脂を得ることができ、所望により魚油由来のＤＨＡ含有油脂組成物と同様の油脂組成物を得ることもできる。
また、微生物培養によって得たＤＨＡ含有油脂からＤＨＡを分離および精製するに際し、分離がより困難なＤＰＡ含量を減少させることは、ＤＨＡの分離および精製を容易にするものである。
【図面の簡単な説明】
【図１】 培地に添加物を加えたときに微生物が産生した油脂中の脂肪酸組成をガスクロマトグラフィーで調べた結果を示すチャートである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing docosahexaenoic acid-containing fats and oils by culturing microorganisms. More specifically, the present invention relates to the fats and oils produced by microorganisms produced by culturing microorganisms capable of producing highly unsaturated fatty acids such as docosahexaenoic acid and docosapentaenoic acid to produce docosahexaenoic acid-containing fats and oils. The present invention provides a method for controlling the content ratio of a highly unsaturated fatty acid such as docosahexaenoic acid, docosapentaenoic acid or eicosapentaenoic acid.
[0002]
[Prior art]
Docosahexaenoic acid (hereinafter also referred to as “DHA”) is contained in fish oils belonging to blue fish, and in particular, it is contained in about 20% in oils derived from sardines and tuna. In recent years, it has been discovered that tuna orbital fat contains a high concentration of DHA among fish oils. In addition, due to the development of advanced fatty acid purification technology, research on the bioactive function of DHA has progressed, and cholesterol lowering action, anticoagulant action, anticancer action, etc. have been clarified. Furthermore, it has been revealed that it is effective in improving memory learning ability, prevention of senile dementia, treatment of Alzheimer's disease, etc. in relation to the brain metabolic system. It has also been revealed that it is a growth essential fatty acid for fry. For this reason, DHA is used in various foods, feeds or feeds.
[0003]
Docosapentaenoic acid (hereinafter also referred to as “DPA”) is also known to be contained in fish oil, but the amount is negligible. Further, the physiologically active function of DPA still has many unclear points, and only a few are known to function as a carrier that facilitates transport of drugs to the brain [Japanese Patent Laid-Open No. 61-204136]. However, it is known that DPA increases as a compensation for the deficiency of DHA in animals [Hometown et al., J. Neurochem., Vol. 51, p. 45 (1988); Hamm et al., Biochem. 245, p.907 (1987); and Rebhung et al., Biosci. Biotech. Biochem., Vol. 58, p. 314 (1994)], that DPA has some physiological role in the animal body. It is suggested.
[0004]
When such DHA or DPA is to be obtained from fish oil, its content is low, and it is difficult for fish oil to be a stable supply source in terms of the migratory properties of fish, and there are also disadvantages such as a strange odor peculiar to fish oil. There is.
[0005]
Examples of the supply source of DHA and DPA other than fish oil include fats and oils (microbe oil) accumulated in cultured cells of microorganisms having DHA and DPA production ability. These microorganisms capable of producing DHA and DPA include Vibrio marinus ATCC15381 isolated from the deep sea, Vibrio bacteria isolated from the intestines of deep sea fish, and Thraustochytrium aureus which is a flagellate fungus. aureum ATCC 34304, Thraustochytrium sp. ATCC 28211 and ATCC 20891, Schizochytrium sp. ATCC 20888 and ATCC 20899 [US Pat. Chem. Soc., Vol. 73, p. 1421 (1996)], Japonochytrium sp. ATCC 28207 [JP-A-1-199588], Cyclotella cryptica, a microalgae, Kripte Kodiniu Known are Cryptocodenium cohnii (JP-A-5-503425), Emilia-Ania (JP-A-5-308978), and the like.
[0006]
[Problems to be solved by the invention]
Oils and fats produced by the microorganism as a source of DHA or DPA contain highly unsaturated fatty acids such as DHA, DPA, and eicosapentaenoic acid (hereinafter also referred to as “EPA”). The content ratio and production amount of these highly unsaturated fatty acids vary depending on the type of microorganism, and it is difficult to obtain fats and oils having a desired content ratio in good yield. On the other hand, if the content ratio of highly unsaturated fatty acids such as DHA, DPA and EPA can be changed, it will not only be closer to the composition of natural fish oil, but it will be easier to separate and purify individual highly unsaturated fatty acids. It becomes.
[0007]
[Means for Solving the Problems]
As a result of studying 155 kinds of compounds to solve the above-mentioned problems, the present inventors cultured microorganisms having docosahexaenoic acid and docosapentaenoic acid producing ability in the presence of p-toluic acid, tannic acid or cyanocobalamin. Thus, the knowledge that the DPA and EPA contents in the DHA-containing oil produced by the microorganism can be changed was obtained, and the present invention was completed.
That is, the present invention is selected from the group consisting of p-toluic acid, tannic acid, and cyanocobalamin in a method for producing highly unsaturated fatty acid-containing fats and oils by culturing microorganisms capable of producing docosahexaenoic acid and docosapentaenoic acid. The present invention provides a method for producing a highly unsaturated fatty acid-containing fat and oil characterized by culturing the microorganism in the presence of an additive.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
As described herein, “docosahexaenoic acid (DHA)” refers to (n-3) -based DHA, and “docosapentaenoic acid (DPA)” refers to (n-3) -based and / or (n -6) Refers to DPA of the system. In the present specification, the terms “oil” and “lipid” and “oil” are used interchangeably.
[0009]
Examples of the microorganism having the ability to produce docosahexaenoic acid and docosapentaenoic acid in the present invention include microorganisms belonging to the genus Schizochytrium or Thraustochytrium. Among these microorganisms, the most preferable one is Schizochytrium SR21 strain (deposited with the name of “marine fungus SR21 strain” at the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as of March 6, 1995. FERM BP-5034).
[0010]
The amount of p-toluic acid, tannic acid or cyanocobalamin added to the medium may be 1.0 g / L or less, and is preferably in the range of 0.02 to 0.2 g / L.
[0011]
In order to culture the microorganisms in the present invention, a preculture solution obtained by culturing the strain in advance is inoculated into a liquid medium or a solid medium and cultured. The medium may be natural seawater or artificial seawater, or a prepared synthetic medium.
[0012]
Carbon sources added to the medium include carbohydrates such as glucose, fructose, xylose, saccharose, maltose, soluble starch, fucose, glucosamine, dextran, fats and oils such as oleic acid and soybean oil, glutamic acid, molasses, glycerol, Commonly used materials such as mannitol and sodium acetate can be used, but are not limited thereto.
[0013]
Nitrogen sources include natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid, corn steep liquor, organic nitrogen sources such as sodium glutamate, urea, or ammonium acetate, ammonium chloride, ammonium nitrate, sodium sulfate An inorganic nitrogen source such as can be used.
[0014]
In addition, mineral salts and vitamins such as phosphates such as potassium phosphate and potassium dihydrogen phosphate, ammonium sulfate, sodium sulfate, magnesium sulfate, iron sulfate, copper sulfate, and calcium chloride are also available as trace nutrients as needed. Can be used.
[0015]
These medium components are not particularly limited as long as they do not harm the growth of microorganisms. In general, the carbon source may be used at a concentration of 0.6 to 60 g per liter of medium. The nitrogen source is desirably increased as the carbon source increases.
[0016]
Cultivation can be carried out using a liquid medium in a shake culture in a flask or aeration and agitation culture. A normal stirred fermenter or bubble column type culture apparatus can also be used. The culture temperature may be in the range of 20 to 40 ° C., and the culture period is preferably 3 to 5 days.
[0017]
After completion of the culture, cultured cells are obtained from the culture solution using conventional solid-liquid separation means such as centrifugation and filtration. The cells are thoroughly washed with water and preferably dried. Drying can be performed by freeze drying or air drying.
[0018]
The cultured cells obtained in this way can be used as they are as animal feed or fish feed. Moreover, the fats and oils containing DHA, DPA, EPA, etc. can be extract | collected from this microbial cell as follows. In addition, DHA, DPA or EPA can be isolated if desired.
[0019]
The oil and fat is collected by crushing the dried cells with, for example, dynomill or ultrasonic waves, and then extracting with an organic solvent, preferably under a nitrogen stream. As the organic solvent, ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, or the like can be used. Good results can also be obtained by alternate extraction of methanol and petroleum ether or extraction using a one-layer solvent of chloroform-methanol-water. By distilling off the organic solvent from the extract under reduced pressure, oils and fats containing high concentrations of DHA, DPA and EPA are obtained.
Moreover, it can replace with said method and can extract using a wet cell. In this case, a mixed solvent of water and an organic solvent compatible with water such as methanol or ethanol is used. Other procedures are the same as described above.
[0020]
In the fats and oils thus obtained, highly unsaturated fatty acids such as DHA, DPA and EPA contain neutral lipids (eg triglycerides) or polar lipids (eg phosphatidylcholine, phosphatidylethanolamine, phosphatidase). It exists in the form of fatidylinositol. Purification of DHA, DPA, and EPA-containing triglycerides from DHA, DPA, and EPA-containing fats and oils collected from cultured cells can be performed, for example, by a cooling separation method or a column chromatography method.
[0021]
Separation and purification of DHA, DPA, and EPA from DHA, DPA, and EPA-containing fats and oils can be carried out by hydrolyzing the fats and oils and the resulting mixed fatty acids as they are or mixed fatty acid esters, followed by conventional methods (eg, urea addition method, cooling Separation method, column chromatography method and the like).
[0022]
【Example】
Next, the present invention will be described more specifically with reference to examples.
Example 1 Production of fats and oils by Schizochytrium microorganisms (1)
For Schizochytrium SR21 strain, a 500 ml Erlenmeyer flask was charged with 100 ml of the medium having the following composition and stirred and cultured under the following conditions.
(1) Medium composition (g / L)
1) Glucose: 20
2) Polypeptone: 10
3) Yeast extract: 5
4) 50% artificial seawater: 1L
5) p-Toluic acid: 0, 0.02, 0.05, 0.1, 0.2
(2) Culture conditions 1) Culture temperature (° C): 28
2) Number of stirring (rpm): 100
[0023]
After cultivation, the cells were collected by centrifugation and dried at 105 ° C. for 3 hours. Fatty acid methyl ester was prepared by dissolving 10 mg of dried microbial cells in an equal volume mixture of a methanol solution containing 10% HCl and dichloromethane and heat treating at 50 ° C. for 3 hours. This was subjected to gas chromatography to analyze the fatty acid composition. The separation conditions for gas chromatography are shown below.

[0024]
The results are shown in Table 1 below.
[Table 1]

[0025]
Example 2 Production of fats and oils by Schizochytrium microorganisms (2)
About Schizochytrium SR21 strain | stump | stock, 100 ml of culture media of the following compositions were prepared to the Erlenmeyer flask with a capacity | capacitance of 500 ml, and stirring culture was performed on the same conditions as Example 1. FIG.
Medium composition (g / L)
1) Glucose: 20
2) Polypeptone: 10
3) Yeast extract: 5
4) 50% artificial seawater: 1L
5) Tannic acid: 0.05
After the cultivation, fatty acid methyl ester was prepared in the same manner as in Example 1. This was subjected to gas chromatography under the same conditions as in Example 1 to analyze the fatty acid composition. The result is shown in FIG.
As can be seen from the results, when tannic acid was added, a decrease in the peak corresponding to EPA was observed compared to the control.
[0026]
Example 3 Production of fats and oils by Schizochytrium microorganisms (3)
About Schizochytrium SR21 strain | stump | stock, 100 ml of culture media of the following compositions were prepared to the Erlenmeyer flask with a capacity | capacitance of 500 ml, and stirring culture was performed on the same conditions as Example 1. FIG.
Medium composition (g / L)
1) Glucose: 20
2) Polypeptone: 10
3) Yeast extract: 5
4) 50% artificial seawater: 1L
5) Cyanocobalamin: 0.05
After the cultivation, fatty acid methyl ester was prepared in the same manner as in Example 1. This was subjected to gas chromatography under the same conditions as in Example 1 to analyze the fatty acid composition. The result is shown in FIG.
As can be seen from the results, when cyanocobalamin was added, the ratio of odd chain fatty acids 15: 0 and 17: 0 decreased and the ratio of even chain fatty acids 14: 0 increased compared to the control. This is presumably because cyanocobalamin had some influence on the amino acid metabolism of the SR21 strain, and the precursor of odd-chain fatty acids was not supplied.
[0027]
Example 4 Column purification of fats and oils derived from Schizochytrium microorganisms According to the method of Example 1, cells obtained by culturing in a medium containing 0.2 g / L of p-toluic acid were dried to obtain dry cells. The dried cells were crushed using glass beads in a mixed solvent of chloroform and methanol, filtered, and extracted with hexane to obtain fats and oils. The resulting fat was esterified by the addition of ethanol containing potassium hydroxide in hexane. By post-processing according to a conventional method, an oil and fat mixture containing an ester of fatty acid was obtained. This oil / fat mixture, according to analysis by gas chromatography, contained about 50% DHA ethyl ester, about 2.4% DPA ethyl ester and 8.5% EPA ethyl ester.
[0028]
From this oil / fat mixture, DHA ethyl ester, DPA ethyl ester and EPA ethyl ester were separated by liquid chromatography under the following conditions. That is, using a column having an inner diameter of 400 mm and a length of 2000 mm packed with an octadecylsilane (ODS) filler [YMC * GEL ODS-AM-120-S50] having a particle size of 50 μm, a pore size of 120 Å and a carbon content of 17%. Subjected to HPLC.
1.2 kg of the above oil / fat mixture was introduced into the column as a 10% solution in methanol and eluted at a flow rate of 4.4 liters / minute using methanol to obtain fractions. The solvent was distilled off for each fraction to obtain 330 g of DHA ethyl ester, 2 g of DPA ethyl ester, and 82 g of EPA ethyl ester.
[0029]
【The invention's effect】
According to the present invention, among polyunsaturated fatty acids contained in DHA-containing fats and oils produced by microorganisms, for example, the content ratio of DPA and EPA can be changed, so that cultured bacteria having different content ratios of DHA, DPA and EPA A body or fat can be obtained, and if desired, a fat composition similar to a DHA-containing fat composition derived from fish oil can be obtained.
Further, when separating and purifying DHA from DHA-containing fats and oils obtained by microbial culture, reducing the DPA content, which is more difficult to separate, facilitates the separation and purification of DHA.
[Brief description of the drawings]
FIG. 1 is a chart showing the results of gas chromatographic investigation of the fatty acid composition in fats and oils produced by microorganisms when additives are added to the medium.

Claims (2)

An additive selected from the group consisting of p-toluic acid, tannic acid, and cyanocobalamin in a method for producing highly unsaturated fatty acid-containing fats and oils by culturing microorganisms belonging to the genus Schizochytrium having the ability to produce docosahexaenoic acid and docosapentaenoic acid A method for producing highly unsaturated fatty acid-containing fats and oils, comprising culturing the microorganism in the presence of.   The production method according to claim 1, wherein the microorganism capable of producing docosahexaenoic acid and docosapentaenoic acid is Schizochytrium SR21 strain (FERM BP-5034).

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