JP2008237033A - Method for producing unsaturated fatty acid using microorganism - Google Patents
Method for producing unsaturated fatty acid using microorganism Download PDFInfo
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- JP2008237033A JP2008237033A JP2007078152A JP2007078152A JP2008237033A JP 2008237033 A JP2008237033 A JP 2008237033A JP 2007078152 A JP2007078152 A JP 2007078152A JP 2007078152 A JP2007078152 A JP 2007078152A JP 2008237033 A JP2008237033 A JP 2008237033A
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- Prior art keywords
- lipid
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- acid
- microorganism
- precursor
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 34
- 244000005700 microbiome Species 0.000 title claims abstract description 32
- 235000021122 unsaturated fatty acids Nutrition 0.000 title claims abstract description 10
- 150000004670 unsaturated fatty acids Chemical class 0.000 title claims abstract description 10
- 150000002632 lipids Chemical class 0.000 claims abstract description 75
- 239000002243 precursor Substances 0.000 claims abstract description 41
- 239000004094 surface-active agent Substances 0.000 claims abstract description 18
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 10
- 229930195729 fatty acid Natural products 0.000 claims abstract description 10
- 239000000194 fatty acid Substances 0.000 claims abstract description 10
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims description 20
- 150000003839 salts Chemical class 0.000 claims description 10
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- 150000003431 steroids Chemical class 0.000 claims description 3
- 229910052717 sulfur Inorganic materials 0.000 claims description 3
- 125000004434 sulfur atom Chemical group 0.000 claims description 3
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
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- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- WBWWGRHZICKQGZ-GIHLXUJPSA-N taurocholic acid Chemical compound C([C@@H]1C[C@H]2O)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@H](O)C1 WBWWGRHZICKQGZ-GIHLXUJPSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 description 1
- 229960001661 ursodiol Drugs 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、不飽和脂肪酸などの脂質の製造方法に関する。さらに詳しくは、微生物を使って、本来、疎水性である脂質前駆体から脂質を生産する方法において、予め脂質前駆体を培地に可溶化した後に微生物を培地中で接触させる工程を含む、脂質の高収率な製造方法に関する。 The present invention relates to a method for producing lipids such as unsaturated fatty acids. More specifically, in a method for producing lipid from a lipid precursor that is originally hydrophobic using a microorganism, the method comprises the step of solubilizing the lipid precursor in a medium in advance and then contacting the microorganism in the medium. The present invention relates to a high-yield production method.
近年、不飽和脂肪酸などの脂質が生理活性を有することがわかり、脂質に対して大きく注目され、精力的に研究がおこなわれており、一部実用化されているものもある。
例えば、多価不飽和脂肪酸のアラキドン酸は、胃粘膜保護作用、肝臓保護効果、脂質代謝以上改善、胎児の身体や脳の発育における重要性などが報告されており、エイコサペンタエン酸は、ドコサヘキサエン酸とともに、栽培漁業用の飼料添加物として使用されている。
In recent years, it has been found that lipids such as unsaturated fatty acids have physiological activity, and much attention has been paid to lipids, and intensive research has been conducted, and some have been put into practical use.
For example, arachidonic acid, a polyunsaturated fatty acid, has been reported to protect against gastric mucosa, hepatoprotective effect, improvement over lipid metabolism, importance in fetal body and brain development, etc. Eicosapentaenoic acid is docosahexaenoic acid At the same time, it is used as a feed additive for cultivated fisheries.
このような脂質は、通常、菌体や動物細胞から抽出することによって生産されている。しかしながら、抽出量が非常に少ないことから大量生産に向いておらず、近年の需要に対応できない問題がある。それに加え、コストが非常に高いことが問題となっており、生産プロセスを大量生産可能なプロセスに変更することが望まれていた。発酵法は、大量生産を可能とするため、上記課題を解決するものとして期待されている。 Such lipids are usually produced by extraction from bacterial cells or animal cells. However, since the extraction amount is very small, it is not suitable for mass production, and there is a problem that it cannot respond to recent demand. In addition, it is a problem that the cost is very high, and it has been desired to change the production process to a process capable of mass production. Since the fermentation method enables mass production, it is expected to solve the above problems.
発酵法は、大量生産を可能とする方法であり、アルコール発酵等が広く知られている。従来から脂質の発酵も行なわれてきたが、アルコール発酵の場合と異なり、収率が非常に低いという課題があり、収率の高い発酵プロセスが求められている。
この課題に対して、収率を高くする回収プロセスの開発が精力的に行われ、乾燥工程を必要としない抽出プロセスで、脂質を劣化させずに回収でき、収率を向上させたとの報告がある（特許文献１）。
The fermentation method is a method that enables mass production, and alcohol fermentation and the like are widely known. Conventionally, lipid fermentation has also been performed, but unlike alcohol fermentation, there is a problem that the yield is very low, and a fermentation process with high yield is required.
In response to this issue, a recovery process that increases the yield has been vigorously developed, and it has been reported that the extraction process that does not require a drying step can be recovered without degrading lipids, improving the yield. Yes (Patent Document 1).
しかしながら、依然として収率は低く、根本的な解決には至っていない。また、目的の脂質ごとに特定の菌株を入手する必要があるため、汎用性に欠けるといった問題がある。
本発明の課題は、微生物を使って疎水性である脂質前駆体から不飽和脂肪酸などの脂質を生産する方法において、脂質の収率が極めて高い製造方法を提供することにある。 An object of the present invention is to provide a production method with extremely high lipid yield in a method for producing lipids such as unsaturated fatty acids from a lipid precursor which is hydrophobic using microorganisms.
本発明者らは、上記の目的を達成するべく検討を行った結果、本発明に到達した。
すなわち、本発明は、微生物を使用して脂質前駆体（ａ）から脂質（Ａ）を生産する方法において、界面活性剤（Ｂ）によって予め培地に可溶化された脂質前駆体（ａ）と微生物を培地中で接触させる工程を必須工程として含む脂質の製造方法である。
The inventors of the present invention have reached the present invention as a result of studies to achieve the above object.
That is, the present invention relates to a method of producing a lipid (A) from a lipid precursor (a) using a microorganism, and the lipid precursor (a) solubilized in a medium in advance by a surfactant (B) and the microorganism. It is the manufacturing method of the lipid which includes the process which makes a medium contact in a culture medium as an essential process.
本発明の脂質の製造方法は、脂質前駆体を予め可溶化しているため、微生物で脂質前駆体を発酵させる際に、原料として供する脂質前駆体の量を従来より増加させることができる。その結果として、最終的に生成する脂質の収率を飛躍的に向上させることができる。また、本発明の製造方法によって、目的の脂質ごとに、生産することに最適な微生物を選ばなくても、多種類の脂質を生産することが可能となる。 In the lipid production method of the present invention, since the lipid precursor is solubilized in advance, when the lipid precursor is fermented by a microorganism, the amount of the lipid precursor provided as a raw material can be increased as compared with the conventional method. As a result, the yield of the finally produced lipid can be dramatically improved. In addition, according to the production method of the present invention, it is possible to produce many types of lipids without selecting an optimal microorganism for production for each target lipid.
以下に、本発明に係る脂質の製造方法の実施の形態について説明する。
本発明の脂質の製造方法は、微生物を使用して脂質前駆体（ａ）から脂質（Ａ）を生産する方法において、界面活性剤（Ｂ）によって予め培地に可溶化された脂質前駆体（ａ）と微生物を培地中で接触させる工程を必須工程として含む製造方法である。
本発明の製造方法によれば、界面活性剤などを使用して脂質前駆体（ａ）を予め培地に可溶化することにより、合成反応に供される原料の脂質前駆体の濃度を高くすることができるので、微生物と接触させて脂質を生産する際において、脂質の生産性を飛躍的に向上することができるのが特長である。
Below, embodiment of the manufacturing method of the lipid which concerns on this invention is described.
The method for producing a lipid of the present invention is a method for producing a lipid (A) from a lipid precursor (a) using a microorganism, wherein the lipid precursor (a) previously solubilized in a medium by a surfactant (B) is used. ) And a microorganism in a culture medium.
According to the production method of the present invention, the lipid precursor (a) is solubilized in a medium in advance using a surfactant or the like, thereby increasing the concentration of the raw material lipid precursor used in the synthesis reaction. Therefore, when producing lipids in contact with microorganisms, the productivity of lipids can be dramatically improved.
本発明は、脂質前駆体を予め界面活性剤を用いて培地に可溶化し、その培地溶液と、別途に微生物を培養させた培養液とを接触させて発酵をおこなう方法である。
接触方法は、脂質前駆体を可溶化した培地に微生物培養液を加える方法でも、微生物培養液に脂質前駆体を可溶化した培地を加える方法でもよい。あるいは、脂質前駆体を可溶化させた同じ培地中で微生物を培養させてもよい。
The present invention is a method in which a lipid precursor is previously solubilized in a medium using a surfactant, and the medium solution is separately brought into contact with a culture solution in which microorganisms are cultured to perform fermentation.
The contact method may be a method of adding a microorganism culture solution to a medium in which a lipid precursor is solubilized or a method of adding a medium in which a lipid precursor is solubilized to a microorganism culture solution. Alternatively, the microorganism may be cultured in the same medium in which the lipid precursor is solubilized.
本発明において脂質前駆体（ａ）とは、脂質（Ａ）の前駆体であり、微生物によって脂質（Ａ）に変換される前駆体のことを指し、例えば、脂肪酸（ａ’）に適用した場合に本発明の方法は有効である。
脂質前駆体（ａ）としての脂肪酸（ａ’）としては、ステアリン酸などの飽和高級脂肪酸；オレイン酸、パルミチン酸、リノール酸等の不飽和高級脂肪酸が挙げられる。
In the present invention, the lipid precursor (a) is a precursor of lipid (A) and refers to a precursor that is converted into lipid (A) by a microorganism. For example, when applied to fatty acid (a ′) In addition, the method of the present invention is effective.
Examples of the fatty acid (a ′) as the lipid precursor (a) include saturated higher fatty acids such as stearic acid; unsaturated higher fatty acids such as oleic acid, palmitic acid, and linoleic acid.
本発明における目的物質の脂質（Ａ）とは、不飽和高級脂肪酸（Ａ’）が挙げられ、具体的に、オレイン酸、リノール酸、アルファ−リノレン酸、ガンマ−リノレン酸、ジ−ホモリノレン酸、ミード酸、アラキドン酸、エイコサテトラエン酸、エイコサペンタエン酸、ドコサヘキサエン酸などが挙げられる。 Examples of the target substance lipid (A) in the present invention include unsaturated higher fatty acids (A ′). Specific examples include oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, and di-homolinolenic acid. , Mead acid, arachidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosahexaenoic acid and the like.
例えば、オレイン酸に枯草菌を作用させた場合、リノール酸、アルファ−リノレン酸、ガンマ−リノレン酸、ジ−ホモリノレン酸、ミード酸、アラキドン酸、エイコサテトラエン酸、エイコサペンタエン酸、ドコサヘキサエン酸などが産生される。
また、ステアリン酸にＭ．Ａｌｐｉｎａ １Ｓ−４株を作用させた場合、オレイン酸および上記の不飽和高級脂肪酸が産生される。
For example, when Bacillus subtilis is allowed to act on oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, di-homolinolenic acid, mead acid, arachidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosahexaenoic acid Etc. are produced.
In addition, M.I. When Alpina 1S-4 strain is allowed to act, oleic acid and the above unsaturated higher fatty acids are produced.
脂質前駆体の培地への可溶化は、脂質前駆体を可溶化できる方法、手段であれば、特に限定するものではなく、界面活性剤による可溶化、塩（塩化ナトリウムなどの無機電解質）による可溶化、機械的せん断力による可溶化などが挙げられる。可溶化しやすさの観点から界面活性剤による可溶化が好ましい。 The solubilization of the lipid precursor in the medium is not particularly limited as long as it is a method and means capable of solubilizing the lipid precursor. Solubilization with a surfactant or salt (inorganic electrolyte such as sodium chloride) is possible. Examples include solubilization and solubilization by mechanical shearing force. Solubilization with a surfactant is preferred from the viewpoint of ease of solubilization.
本発明における界面活性剤（Ｂ）とは、前駆体（Ａ）を可溶化できる界面活性剤であれば、その種類は限定されないが、脂質前駆体の可溶化しやすさの観点で、下記のリン脂質またはその誘導体（B１）、ステロイド誘導体の金属塩（Ｂ２）、硫黄原子含有アニオン性界面活性剤（Ｂ３）などの界面活性剤が挙げられる。 The surfactant (B) in the present invention is not limited as long as it is a surfactant that can solubilize the precursor (A), but from the viewpoint of ease of solubilization of the lipid precursor, Surfactants such as phospholipids or derivatives thereof (B1), metal salts of steroid derivatives (B2), sulfur atom-containing anionic surfactants (B3) and the like can be mentioned.
リン脂質またはその誘導体（Ｂ１）としては、グリセロリン脂質、スフィンゴリン脂質が挙げられる。グリセロリン脂質としては、ホスファチジルコリン（レシチン）、
ホスファチジルエタノールアミン、ホスファチジルイノシトール、ホスファチジルセリン、
ホスファチジルグリセロール、またはジホスファチジルグリセロールなどが挙げられる。スフィンゴリン脂質としては、スフィンゴミエリンなどが挙げられる。
入手しやすさの観点で好ましくは、ホスファジルコリン（レシチン）である。
Examples of the phospholipid or derivative (B1) include glycerophospholipid and sphingophospholipid. As glycerophospholipid, phosphatidylcholine (lecithin),
Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine,
Examples thereof include phosphatidylglycerol and diphosphatidylglycerol. Examples of sphingophospholipids include sphingomyelin.
From the viewpoint of availability, phosphadylcholine (lecithin) is preferable.
カルボキシル基含有ステロイド誘導体の金属塩（Ｂ２）としては、コール酸、デオキシコール酸、ケノデオキシコール酸、コラン酸、リトコール酸、ヒオデオキシコール酸、ウルソデオキシコール酸、アポコール酸、デヒドロコール酸、グリココール酸、またはタウロコール酸およびそれらの金属塩が挙げられる。金属塩としてはナトリウム、カリウム等のアルカリ金属塩が挙げられる。
入手しやすさの観点で好ましくは、コール酸、デオキシコール酸、ケノデオキシコール酸のナトリウム塩である。
Examples of the metal salt (B2) of the carboxyl group-containing steroid derivative include cholic acid, deoxycholic acid, chenodeoxycholic acid, cholanic acid, lithocholic acid, hyodeoxycholic acid, ursodeoxycholic acid, apocholic acid, dehydrocholic acid, glycocholic acid Or taurocholic acid and their metal salts. Examples of the metal salt include alkali metal salts such as sodium and potassium.
From the viewpoint of availability, cholic acid, deoxycholic acid, and sodium salt of chenodeoxycholic acid are preferable.
硫黄原子含有アニオン性界面活性剤（Ｂ３）としてはアルキル硫酸塩（Ｂ３１）またはアルキルスルホン酸塩（Ｂ３２）が挙げられる。
アルキル硫酸塩（Ｂ３１）としては、通常、炭素数８〜２４のアルキル硫酸塩が挙げられる。炭素数８〜２４のアルキル基としては、オクチル基、ノニル基、デシル基、ウンデシル基、ドデシル基（ラウリル基）、ドデシルベンジル基、ステアリル基、ベヘニル基などが挙げられる。塩としては、アルカリ金属塩、アルカリ土類金属塩が挙げられる。入手しやすさの観点からラウリル硫酸ナトリウムなどが好ましい。
アルキルスルホン酸塩（Ｂ３２）としては、通常、炭素数８〜２４のアルキルスルホン酸塩が挙げられる。炭素数８〜２４のアルキル基としては、オクチル基、ノニル基、デシル基、ウンデシル基、ドデシル基（ラウリル基）、ドデシルベンジル基、ステアリル基、ベヘニル基などが挙げられる。塩としては、アルカリ金属塩、アルカリ土類金属塩が挙げられる。入手しやすさの観点からドデシルベンゼンスルホン酸ナトリウムなどが好ましい。
Examples of the sulfur atom-containing anionic surfactant (B3) include alkyl sulfate (B31) and alkyl sulfonate (B32).
Examples of the alkyl sulfate (B31) usually include alkyl sulfates having 8 to 24 carbon atoms. Examples of the alkyl group having 8 to 24 carbon atoms include octyl group, nonyl group, decyl group, undecyl group, dodecyl group (lauryl group), dodecylbenzyl group, stearyl group, and behenyl group. Examples of the salt include alkali metal salts and alkaline earth metal salts. From the viewpoint of availability, sodium lauryl sulfate and the like are preferable.
Examples of the alkyl sulfonate (B32) usually include alkyl sulfonates having 8 to 24 carbon atoms. Examples of the alkyl group having 8 to 24 carbon atoms include octyl group, nonyl group, decyl group, undecyl group, dodecyl group (lauryl group), dodecylbenzyl group, stearyl group, and behenyl group. Examples of the salt include alkali metal salts and alkaline earth metal salts. From the viewpoint of availability, sodium dodecylbenzenesulfonate is preferred.
これらの界面活性剤（Ｂ）としては、微生物に対して発酵阻害をしにくいという観点からリン脂質またはその誘導体（Ｂ１）が好ましい。
一部の界面活性剤には、微生物に対し、細胞壁または細胞膜を破壊するものが存在する。微生物は細胞膜または細胞壁を破壊されると発酵しにくくなるため、結果として発酵を阻害することとなる。したがって、脂質前駆体を培地に可溶化することができても、微生物の発酵を阻害しにくい界面活性剤が適している。
As these surfactants (B), phospholipids or derivatives thereof (B1) are preferable from the viewpoint that fermentation inhibition is difficult for microorganisms.
Some surfactants destroy the cell wall or cell membrane against microorganisms. Microorganisms are difficult to ferment when their cell membranes or cell walls are destroyed, resulting in inhibition of fermentation. Therefore, even if the lipid precursor can be solubilized in the medium, a surfactant that hardly inhibits the fermentation of microorganisms is suitable.
本発明において使用する界面活性剤（Ｂ）の濃度は、微生物培養液を含む培地全体に対し、通常０．０５〜２０重量％であり、好ましくは０．１〜１０重量％である。０．１重量％未満の場合、脂質前駆体を培地に可溶化することが困難となるので適さない。２０重量％より高い場合、粘度が高くなり、脂質の生産性が低下するので適さない。 The concentration of the surfactant (B) used in the present invention is usually 0.05 to 20% by weight, preferably 0.1 to 10% by weight, based on the whole medium containing the microorganism culture solution. If it is less than 0.1% by weight, it is difficult to solubilize the lipid precursor in the medium, which is not suitable. If it is higher than 20% by weight, the viscosity becomes high and the productivity of lipid is lowered, which is not suitable.
本発明の製造方法における培地は、微生物を用いて脂質前駆体（ａ）を脂質に変換することが可能な培地であれば特に限定するものではなく、ＳＣＤ培地などの市販の培地を使用することができる。 The medium in the production method of the present invention is not particularly limited as long as it is a medium capable of converting the lipid precursor (a) into lipid using microorganisms, and a commercially available medium such as an SCD medium should be used. Can do.
本発明において、培地中に可溶化された脂質前駆体の濃度は、通常０．０５〜２０重量％濃度であり、脂質の生産性の観点から好ましくは０．１〜１０重量％である。０．０５重量％未満の場合、生産量が非常に低く実用的でない。２０重量％より高い場合は、粘度が高すぎて生産性が低下するので好ましくない。 In the present invention, the concentration of the lipid precursor solubilized in the medium is usually 0.05 to 20% by weight, and preferably 0.1 to 10% by weight from the viewpoint of lipid productivity. If it is less than 0.05% by weight, the production amount is very low and impractical. If it is higher than 20% by weight, the viscosity is too high and the productivity is lowered, which is not preferable.
本発明において使用される微生物は、特に限定するものではなく、枯草菌等の入手しやすい菌でも良いが、Ｍｏｒｔｉｅｒｅｌｌａ属、Ｍｕｃｏｒ属、Ｓａｐｒｏｌｅｇｎｉａ属、Ｐｙｔｈｉｕｍ属などの糸状菌類が菌体内の油脂含量が多いことから好ましい。 The microorganism used in the present invention is not particularly limited and may be an easily available bacterium such as Bacillus subtilis. It is preferable because there are many.
本発明の実施態様の概要の１例を以下に具体的に説明する。
（１）脂質前駆体の培地への可溶化
市販の培地（ＳＣＤ培地など）をイオン交換水に溶解させ、この培地にオレイン酸およびレシチンを加えて撹拌し、オレイン酸を可溶化させる。その後、オートクレーブ滅菌をおこなう。
（２）微生物の培養
別途、市販の培地（ＳＣＤ培地など）をイオン交換水に溶解させ、オートクレーブ滅菌（１２０℃、１時間）する。この培地にシャーレ培養した枯草菌を植菌し、３７℃の振とう培養機で１日培養する。
（３）脂質前駆体の発酵
上記（２）の微生物を培養した培養液をガラス試験管に移し、これを上記（１）の培地に添加し、微生物と脂質前駆体を接触させる。３７℃の振とう培養機に入れ、好気的に発酵をおこなう。
One example of the outline of the embodiment of the present invention will be specifically described below.
(1) Solubilization of lipid precursor in medium A commercially available medium (such as SCD medium) is dissolved in ion-exchanged water, and oleic acid and lecithin are added to the medium and stirred to solubilize oleic acid. Then, autoclave sterilization is performed.
(2) Microbial culture Separately, a commercially available medium (such as SCD medium) is dissolved in ion-exchanged water, and autoclaved (120 ° C., 1 hour). Bacillus subtilis cultured in a petri dish is inoculated into this medium and cultured for 1 day in a 37 ° C. shaker.
(3) Fermentation of lipid precursor The culture solution obtained by culturing the microorganism of (2) above is transferred to a glass test tube, which is added to the medium of (1) above, and the microorganism and lipid precursor are brought into contact with each other. Place in a 37 ° C shaking incubator and ferment aerobically.
以下に、本発明を実施例によって具体的に説明するが、本発明は実施例に限定されるものではない。なお、特記しない限り、部は重量部、％は重量％を意味する。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples. Unless otherwise specified, “part” means “part by weight” and “%” means “% by weight”.
＜実施例１＞ （レシチン）
ＳＣＤ培地（日本製薬社製）３０ｇをイオン交換水１０００ｇに溶解させた。この培地水溶液４５ｇにレシチン（東京化成製）０．４５ｇと、オレイン酸５．０ｇを加え、室温で１時間超音波照射し懸濁溶解させ、オートクレーブ滅菌（１２０℃、１時間）した。
これに、前もって別途、ＳＣＤ培地で培養しておいた枯草菌（Ｂａｃｉｌｌｕｓ Ｓａｂｔｉｌｉｕｓ）懸濁液１ｇを加え、３７℃で７日間好気的に培養した。培養後、菌体を遠心分離した。回収した上澄（７日後の培養液）および発酵直前の培養液のそれぞれ１８倍希釈溶液の吸光度を測定した。
<Example 1> (Lecithin)
30 g of SCD medium (Nippon Pharmaceutical Co., Ltd.) was dissolved in 1000 g of ion-exchanged water. To 45 g of this medium aqueous solution, 0.45 g of lecithin (manufactured by Tokyo Chemical Industry) and 5.0 g of oleic acid were added, suspended by ultrasonic irradiation for 1 hour at room temperature, and autoclaved (120 ° C., 1 hour).
To this, 1 g of Bacillus Sabtilius suspension separately cultured in advance in SCD medium was added and cultured aerobically at 37 ° C. for 7 days. After culturing, the cells were centrifuged. The absorbance of each of the collected supernatant (7 days later culture solution) and the 18-fold diluted solution of the culture solution immediately before fermentation was measured.
＜実施例２＞ （コール酸Ｎａ）
レシチンをコール酸ナトリウム（東京化成製）０．４５ｇに変える以外は実施例１と同様に行った。
<Example 2> (Na cholate)
The same procedure as in Example 1 was conducted except that lecithin was changed to 0.45 g of sodium cholate (manufactured by Tokyo Chemical Industry).
＜実施例３＞ （ＳＤＳ）
レシチンをドデシルベンゼンスルホン酸ナトリウム０．４５ｇに変える以外は実施例１と同様に行った。
<Example 3> (SDS)
The same procedure as in Example 1 was conducted except that lecithin was changed to 0.45 g of sodium dodecylbenzenesulfonate.
＜比較例１＞ （水）
レシチンを使わず、イオン水０．４５ｇのみに変える以外は実施例１と同様に行った。
<Comparative Example 1> (Water)
The same procedure as in Example 1 was performed except that lecithin was not used and only the ion water was changed to 0.45 g.
＜オレイン酸消費量の評価方法＞
脂質前駆体であるオレイン酸の消費量を、ガスクロマトグラフィーによって分析し、評価した。すなわち、発酵が進みオレイン酸の消費が進むと、全脂質および脂質前駆体中のオレイン酸の占める割合が低下する。そのため、培養液中のオレイン酸の含量を測定することで、発酵の進行具合を評価することができる。
枯草菌による発酵前と７日後の培地にジエチルエーテルを加え、水層と分液し、エーテル溶液をガスクロマトグラフィー測定を行う。クロマトグラムの全面積中のオレイン酸の面積の割合から、脂質前駆体を含む全脂質中のオレイン酸の含量（％）を算出することができる。
<Evaluation method of oleic acid consumption>
The consumption of oleic acid, a lipid precursor, was analyzed and evaluated by gas chromatography. That is, as fermentation progresses and consumption of oleic acid proceeds, the proportion of oleic acid in the total lipid and lipid precursor decreases. Therefore, the progress of fermentation can be evaluated by measuring the content of oleic acid in the culture solution.
Diethyl ether is added to the medium before and 7 days after fermentation with Bacillus subtilis, and it is separated from the aqueous layer, and the ether solution is subjected to gas chromatography measurement. From the ratio of the area of oleic acid in the total area of the chromatogram, the content (%) of oleic acid in the total lipid including the lipid precursor can be calculated.
オレイン酸の消費量を以下の式で定義する。
オレイン酸消費量（％）＝発酵前のオレイン酸含量（％）−７日後のオレイン酸含量（％）
The consumption of oleic acid is defined by the following formula.
Oleic acid consumption (%) = Oleic acid content before fermentation (%) − Oleic acid content after 7 days (%)
このオレイン酸の消費量が大きいと、オレイン酸が微生物によって変換された量が多いことを表す。オレイン酸消費量を下記の尺度で評価した。オレイン酸消費量を表１に示す。
○：消費量が１．０％以上
△：消費量が０．２％以上１．０％未満
×：消費量が０．２％未満
A large consumption of oleic acid indicates that the amount of oleic acid converted by the microorganism is large. Oleic acid consumption was evaluated on the following scale. Table 1 shows the oleic acid consumption.
○: Consumption is 1.0% or more △: Consumption is 0.2% or more and less than 1.0% ×: Consumption is less than 0.2%
表１から明らかなように、予め脂質前駆体を培地に可溶化しておくとオレイン酸消費量が多いことがわかる。よって不飽和脂肪酸の生産方法として生産性が非常に高いと言える。 As is clear from Table 1, it can be seen that when the lipid precursor is solubilized in advance in the medium, the amount of oleic acid consumed is large. Therefore, it can be said that productivity is very high as a production method of unsaturated fatty acid.
本発明の脂質の製造方法は、脂質前駆体を予め可溶化してあるので、微生物による発酵が極めて効率的に起こり、生産性に優れている。特に、不飽和脂肪酸のような難水溶性物質の発酵に適しており、生産性が飛躍的に向上する。
また、この方法によって得られた不飽和脂肪酸は医薬品、化粧品、健康食品として使用することができ、この製造方法は有益である。
In the lipid production method of the present invention, since the lipid precursor is solubilized in advance, fermentation by microorganisms occurs extremely efficiently, and is excellent in productivity. In particular, it is suitable for fermentation of poorly water-soluble substances such as unsaturated fatty acids, and productivity is dramatically improved.
Moreover, the unsaturated fatty acid obtained by this method can be used as a pharmaceutical, cosmetics and health food, and this production method is beneficial.
Claims (7)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015104381A (en) * | 2013-12-03 | 2015-06-08 | 花王株式会社 | Method of producing sophorolipid |
| KR101531842B1 (en) * | 2014-04-15 | 2015-06-29 | 한국에너지기술연구원 | High-yield extraction of free fatty acid using acid catalyst and surfactant from microorganism |
| JP2017075280A (en) * | 2015-10-16 | 2017-04-20 | コリア インスティチュート オブ エナジー リサーチ | Method for producing biodiesel from microorganism with high efficiency |
-
2007
- 2007-03-26 JP JP2007078152A patent/JP2008237033A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015104381A (en) * | 2013-12-03 | 2015-06-08 | 花王株式会社 | Method of producing sophorolipid |
| KR101531842B1 (en) * | 2014-04-15 | 2015-06-29 | 한국에너지기술연구원 | High-yield extraction of free fatty acid using acid catalyst and surfactant from microorganism |
| JP2017075280A (en) * | 2015-10-16 | 2017-04-20 | コリア インスティチュート オブ エナジー リサーチ | Method for producing biodiesel from microorganism with high efficiency |
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