JP2008247845A - Method for producing acid-type sophorose lipid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fermentation production method by which only a chemically stable acid-type sophorose lipid can selectively be produced by one step, in stead of a conventional sophorose lipid fermentation method by which the sophorose lipid is obtained as a mixture of a lactone type and the acid type. <P>SOLUTION: The method for producing the acid-type sophorose lipid includes subjecting Candida floricola of a microorganism selectively producing the acid-type sophorose lipid to liquid culture, and collecting the acid-type sophorose lipid. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a production method for selectively producing only an acid-type sophorose lipid among sophorose lipids which are a kind of biosurfactant.

Glycolipids are substances in which 1 to 10 monosaccharides are bound to lipids, are involved in the transmission of information between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. On the other hand, glycolipids are amphipathic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. being called.
Some microorganisms are known to produce these surfactants efficiently, and this biological surfactant (biosurfactant) is highly safe and has excellent biodegradability with low environmental impact. Research is progressing as an environmentally advanced surfactant. Currently, the surface active substances produced by microorganisms are classified into five groups: glycolipids, acylpeptides, phospholipids, fatty acids, and polymer compounds. For active agents, the most studied and many types of substances produced by bacteria and yeast have been reported.

These biosurfactants such as glycolipids are said to have high biodegradability, low toxicity, environmental friendliness, and novel physiological functions. For this reason, the wide application of these biosurfactants in the food industry, cosmetic industry, pharmaceutical industry, chemical industry, environmental field, etc. has both the realization of a sustainable society and the provision of high-performance products. Meaningful.
Among them, sophorose lipids (also referred to as “sophorolipids”; hereinafter referred to as “SL” means sophorose lipids) can be obtained from a relatively inexpensive raw material in a high production amount. Therefore, it is a kind of typical biosurfactant currently in commercial use. SL is P.I. A. Gorin et al. Was found in the culture solution of Candida (formerly Torulopsis ) bombicola (see non-patent document 1), and then other yeasts such as Candida bogoriensis , Candida magnolie. ( Candida magnoliae ), Candida gropengiseri , and Candida apicola have also been reported to be produced in relatively large amounts in the culture medium (see Non-Patent Document 2). At present, production of 300 g / L or more is possible (see Non-Patent Documents 3 and 4).

酸型 ラクトン型 SL is a glycolipid composed of sophorose or sophorose having a partially acetylated hydroxyl group and a hydroxy fatty acid. Sophorose is a sugar composed of two molecules of glucose linked by β1 → 2, and hydroxy fatty acid is a fatty acid having a hydroxyl group in its structure. SL is also roughly divided into an acid form in which the carboxyl group of hydroxy fatty acid is liberated and a lactone form in which it is bound to sophorose in the molecule (Formula 3). SL obtained by fermentation production is generally obtained as a mixture of a lactone type and an acid type, and usually contains 50% or more of a lactone type.

Acid type Lactone type

Currently, SL is industrially used in a wide range of fields such as detergents, cosmetics, and foods. Use of SL derivatives as cosmetic wetting agents (see Non-Patent Document 5) and gelling agents (see Patent Document 1), and There are reports on the use of SL in the form of a mixture in improving the quality of wheat products (see Patent Document 2), detergents containing SL (see Patent Documents 3 and 4), bleach (see Patent Document 5), and the like. All of these utilize the SL mixture obtained by fermentation production as it is.
On the other hand, SL containing a lactone type is chemically unstable in the lactone portion, and therefore, when used in combination with other chemical substances, there is a risk of causing a decrease in performance. For example, when the SL mixture is formulated as a liquid alkaline detergent, hydrolysis of the lactone moiety occurs. Moreover, also when mix | blending washing | cleaning auxiliary components other than SL, it becomes difficult to maintain a stable performance by reaction of a lactone part, and the use is restrict | limited greatly.
Therefore, as one of these solutions, a method is employed in which a chemically stable acid type SL is produced and used with high purity. As a specific utilization method, there is a report on a detergent containing hydrolyzed lactone portion of SL mixture obtained by fermentation production and acetyl group of sophorose, and blended with high-purity acid type SL produced (see Patent Document 6). ).

Since the main component of SL obtained by fermentation production is a lactone type, there is currently only an effective method for obtaining the acid type SL in a large amount with high purity by alkaline hydrolysis of the SL mixture. However, in this case, after SL is produced and separated by microbial fermentation, a step of performing an alkali treatment again is necessary, and a post-treatment of a used alkali is also necessary. Moreover, since the hydrolysis of the acetyl group of sophorose also occurs at the same time, it is necessary to consider the formation of an acetic acid derivative.
For the above reasons, the industrial use of SL is limited to some applications. However, if a method capable of producing only acid-type SL in one stage is developed, it will have a great influence on the expansion of SL applications. It is expected to lead to the spread of biosurfactants.

Japanese Patent Publication No. 7-17668 JP-A 61-205449 JP 2003-13093 A JP 2006-83238 A JP 2006-274233 A JP 2006-70231 A Pee. A. Gorin, Jay. F. Tee. Spencer (J. F. Spencer), A.M. Pee. T. Tuloch, “Canadean Journal of Chemistry,” Vol. 39, p846-855 (1961). R. R. Hommel, “Biodegradation”, Volume 1, p107-119 (1990). A. M. D. Davila, Earl. R. Marcbal, Jay. Pee. Bandecatele (Appl. Microbiol. Biotechnol.), 47, pp. 506-501 (1997). H. Jay. Daniel (H.-J. Daniel), M.M. M. Reuss, See. C. Syldak, “Biotechnol. Lett.”, 20, p1153-1156 (1998). Yoshimura Kimura, “Oil Chemistry”, 36, p748-753 (1987).

  In view of the above circumstances, in the present invention, a fermentative production method that selectively produces only acid type SL in one step with respect to the conventional SL fermentation production method obtained as a mixture of a lactone type and an acid type. For the purpose of provision.

  As a result of intensive studies to solve the above problems, the present inventor can efficiently produce acid type SL by culturing a microorganism belonging to Candida floricola, which is a kind of Candida genus yeast. Has been found to be possible, and has led to the present invention.

（１）
（式中、Ｒ₁およびＲ₂は、それぞれ独立して水素（−Ｈ）又はアセチル基（−ＣＯＣＨ₃）であり、ｎは９〜２０の数であり、

で表される基は、飽和脂肪族炭化水素鎖又は二重結合を少なくとも１個有する不飽和脂肪族炭化水素鎖である。）
〔２〕生産される酸型ＳＬが、以下の式（２）で表される６’，６’’−ジアセチル化物を９０質量％以上含有することを特徴とする、〔１〕に記載の方法：

（２）
（式中、ｎは９〜２０の数であり、

で表される基は飽和脂肪族炭化水素鎖又は二重結合を少なくとも１個有する不飽和脂肪族炭化水素鎖である。）
〔３〕キャンディダ（Ｃａｎｄｉｄａ）属に属する微生物がキャンディダ・フロリコーラ（Ｃａｎｄｉｄａ ｆｌｏｒｉｃｏｌａ）に属する微生物である、〔１〕又は〔２〕に記載の方法。
〔４〕キャンディダ（Ｃａｎｄｉｄａ）属に属する微生物がキャンディダ・フロリコーラ（Ｃａｎｄｉｄａ ｆｌｏｒｉｃｏｌａ）ＺＭ−１５０２株（ＦＥＲＭ Ｐ−２１１３３）、ＺＭ−１５１７株（ＦＥＲＭ Ｐ−２１１５３）、ＺＭ−０１３２株（ＦＥＲＭ Ｐ−２１１５２）のいずれかである、〔１〕から〔３〕のいずれかに記載の方法。
〔５〕グリセロールを含有する液体培地を用いて該微生物を培養することを特徴とする、〔１〕から〔４〕のいずれかに記載の方法。
〔６〕前記グリセロールが、植物油の分解又はエステル交換の際の副生成物である、〔５〕に記載の方法。
〔７〕以下の構造を有する酸型のＳＬを９０質量％以上含有する、酸型ＳＬ：

（２）
（式中、ｎは９〜２０の数であり、

で表される基は飽和脂肪族炭化水素鎖又は二重結合を少なくとも１個有する不飽和脂肪族炭化水素鎖である。） That is, the present invention is shown in the following [1] to [7].
[1] A method of culturing a microorganism belonging to the genus Candida having a 26S rDNA-D1 / D2 region of a ribosomal RNA gene containing a base sequence homologous to 95% or more of the base sequence shown in SEQ ID NO: 1 in a liquid medium. A method for selectively producing acid type SL represented by the following formula (1):

(1)
Wherein R ₁ and R ₂ are each independently hydrogen (—H) or an acetyl group (—COCH ₃ ), and n is a number from 9 to 20,

Is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond. )
[2] The method according to [1], wherein the produced acid form SL contains 90% by mass or more of 6 ′, 6 ″ -diacetylated product represented by the following formula (2): :

(2)
(Wherein n is a number from 9 to 20,

Is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond. )
[3] The method according to [1] or [2], wherein the microorganism belonging to the genus Candida is a microorganism belonging to Candida floricola.
[4] Microorganisms belonging to the genus Candida are Candida floricola ZM-1502 strain (FERM P-21133), ZM-1517 strain (FERM P-21153), ZM-0132 strain (FERM P -21152), the method according to any one of [1] to [3].
[5] The method according to any one of [1] to [4], wherein the microorganism is cultured using a liquid medium containing glycerol.
[6] The method according to [5], wherein the glycerol is a by-product during decomposition or transesterification of vegetable oil.
[7] Acid type SL containing 90% by mass or more of acid type SL having the following structure:

(2)
(Wherein n is a number from 9 to 20,

Is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond. )

With respect to the fermentative production of SL, the lactone type and the acid type cannot be made separately by the conventional method, and the product was obtained as a mixture of both, but according to the present invention, only the acid type SL is produced in one step. It becomes possible.
In contrast to a mixture with lactone type SL, which is chemically unstable and has a very limited application, acid type SL as a single product is highly polar and soluble in water, which is chemically stable in liquids. It has characteristics such as high derivatization and ease of derivatization using the reactivity of carboxyl groups, etc. Compared with a mixture in which more than 10 kinds of structural analogs existed, there are few variations in structure and physical properties, and the intended use The performance can be easily adjusted according to
So far, acid type SL has been obtained only by precise separation and purification from the mixture, alkaline hydrolysis of lactone type SL, or extremely difficult total synthesis by chemical synthesis. The ability to easily collect can be an extremely effective means for expanding the use of SL.

In particular, according to the production method of the present invention, it is possible to obtain SL in which 90% by mass or more of the obtained acid type SL is composed of 6 ′, 6 ″ -diacetylated product. In the production method of acid type SL, most of the acetyl groups are also hydrolyzed at the same time, so that it is extremely difficult to produce a large amount of this compound by ordinary chemical synthesis methods. Compared with the acid type SL, the acid type SL, which is a diacetylated product obtained in the present invention, has a low critical micelle concentration (cmc) and is expected to have the same performance even when used in a small amount. Expression of different physiological activities is expected.
Furthermore, when producing biodiesel or the like, glycerol produced as a by-product in the transesterification reaction is not currently used as surplus biomass, and its utilization method and application development have been problems, but the production method of the present invention Can produce the acid form SL from glycerol, particularly glycerol as a by-product generated in the decomposition or transesterification of vegetable oil, and therefore, effective utilization of surplus glycerol can be expected.
As described above, the use of the present invention is expected to expand the use of SL in the fields of pharmaceuticals, foods, cosmetics and the like that are required to maintain stable performance in aqueous solutions, including the use of detergents.

The contents of the present invention will be described in detail below. Hereinafter, unless otherwise specified, g / l means the mass of a substance contained in 1 L of the medium.
<Production method of acid type SL>
Microorganism to be used The microorganism of the present invention comprises a ribosomal RNA gene comprising a base sequence shown in SEQ ID NO: 1 that is 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more. It is a microorganism belonging to the genus Candida.
As an example of such a microorganism, the ZM-1502 strain isolated by the present inventors from a plant (leaf) sample collected in Okayama Prefecture can be mentioned. This strain is cultured on a YM agar medium at 25 ° C. for 2 days to form colonies having a diameter of about 2 to 3 mm (shape: circular, raised state: cushion shape, peripheral edge: whole edge, surface shape: smooth, Color: white to creamy, gloss and properties: buttery and wet). In addition, the formation of sexual reproductive organs and pseudohyphae is not observed.

In the ZM-1502 strain, the nucleotide sequence (rDNA sequence) of the 26S rDNA-D1 / D2 region of the ribosomal RNA gene was determined, the DNA database (DDBJ) was accessed, and the FASTA program (http://www.ddbj.nig.ac). .Jp / search / fasta-j.html), the homology search was 100% identical to the rDNA sequence of Candida floricola. As a result of the physiological property test of ZM-1502 strain, this strain was named Candida floricola ZM-1502 strain. The results of the physiological property test are as shown in Table 1. This strain was deposited under the accession number FERM P-21133 at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary (IPOD) (1-1-3 East Tsukuba, Ibaraki) on December 13, 2006. Has been.

表中の「＋」は反応が陽性、「−」は反応が陰性、「Ｗ (weak)」は弱い陽性反応、
「S (slow)」は試験開始後２週間から３週間にかけて徐々に陽性反応が認められたこと、「Ｌ (latent)」は試験開始後２週間以降に急速に陽性反応が認められたことを示す。

In the table, “+” indicates a positive reaction, “−” indicates a negative reaction, “W (weak)” indicates a weak positive reaction,
“S (slow)” indicates that a positive reaction was gradually observed from 2 to 3 weeks after the start of the test, and “L (latent)” indicates that a positive reaction was rapidly observed after 2 weeks after the start of the test. Show.

The microorganism of the present invention includes related microorganisms in addition to the above-mentioned ZM-1502 strain. Examples of related microorganisms of strain ZM-1502 include, for example, ribosomal RNA containing a nucleotide sequence of 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more homologous to the nucleotide sequence of SEQ ID NO: 1. A microorganism having a gene. Examples of the related microorganisms include Candida floricola ZM-1517 strain (FERM P-21153), ZM-0132 strain (FERM P-21152), and the like. Here, the acid type SL is SL having the structure represented by the formula (1).
Moreover, in this invention, it is preferable to use the microorganisms which have acid type SL high productivity among the said microorganisms. Here, “acid type SL high productivity” means the ability to selectively and efficiently produce only the acid type SL, specifically, 100 g / L glucose, 50 g / L oleic acid, 50 g / L Soybean oil, 1 g / L yeast extract (Bacto Yeast Extract (Becton, Dickinson and Company)), 3 g / L urea, 1 g / L potassium dihydrogen phosphate, and 1 g / L magnesium disulfate, 0.4 g When a microorganism is cultured at 28 ° C. for 7 days in a medium containing 1 / L sodium sulfate, SL is produced at 1 g / l or more, preferably 2 g / l or more, and 90% by mass or more of the produced SL, preferably It means that 95 mass% or more is acid type SL. Examples of the fungus having such performance include the above-mentioned Candida floricola, ZM-1502 strain (FERM P-21133), ZM-1517 strain (FERM P-21153), ZM-0132 strain (FERM P- 21152).

A related microorganism of strain ZM-1502 can be isolated using the base sequence described in SEQ ID NO: 1 as an index. Samples collected from nature (eg soil, river / lake water, sap, flowers, leaves, fruits, etc.) are cultured in a medium in which microorganisms can grow at 26-30 ° C. for 3-7 days. GC / MS analysis is performed to select a sample in which the production of acid type SL is recognized (primary screening). Next, the culture solution of the selected sample is cultured on a plate, a colony is formed to isolate a microorganism, the isolated microorganism is further cultured in a medium in which the microorganism can grow, and GC / MS analysis is performed to determine the acid. A microorganism in which type SL production was observed was selected (secondary screening), and DDBJ's FASTA program (http://www.ddbj.nig.ac.jp/search/) was used for the nucleotide sequence of the ribosomal RNA gene of this microorganism strain. (Fasta-j. html) base sequence database.
Examples of the medium used for the screening include 200 g / L glycerol and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L. Liquid medium.

  In the present invention, acid-type SL can be produced using the microorganisms as described above. Among them, ZM-1502, ZM-1517 and ZM-0132 strains are acid-type SL, especially total acid-type SL. The acid type SL containing 90% by mass or more, preferably 95% by mass or more, more preferably 97% by mass or more of the diacetylated product of the above formula (2) can be produced, so that it can be particularly preferably used.

Medium Composition The medium used for the culture is not particularly limited, and a medium generally used for microorganisms (particularly yeast) such as YPD medium (10 g yeast extract, 20 g polypeptone, and 20 g glucose) may be used. it can.
As components to be added to the medium, carbon sources, nitrogen sources, inorganic salts, and necessary nutrient sources that are usually used in the art can be used alone or in combination. Any carbon source may be used as long as it can be assimilated by the microorganism, and includes carbohydrates (monosaccharides such as glucose, mannose, glycerol, and mannitol, oligosaccharides such as sucrose, maltose, and lactose, starch), organic acids (acetic acid) Propionic acid, maleic acid, fumaric acid, malic acid, oleic acid, etc.), alcohols (ethanol, propanol, etc.), medium chain aliphatic hydrocarbons (hexadecane, tetradecane, etc.) are used. As a nitrogen source, ammonium salts of inorganic acid or organic acid such as ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate, sodium nitrate, urea, peptone, yeast extract, malt extract, meat extract, corn steep liquor, casamino acid, etc. These nitrogen-containing compounds are used. Inorganic salts include monobasic potassium phosphate, dibasic potassium phosphate, magnesium phosphate, potassium dihydrogen phosphate, sodium sulfate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. Is used.

  In particular, in order to efficiently produce acid SL using the microorganism of the present invention, fats and oils such as soybean oil, rapeseed oil, cottonseed oil, sunflower oil, kapok oil, sesame oil, corn oil, rice oil, peanut are used as the carbon source. Oil, safflower oil, olive oil, linseed oil, tung oil, castor oil, palm oil, palm kernel oil, coconut oil and the like can be used alone or in combination of two or more thereof. Further, in order to further improve the production efficiency, it is preferable to use an organic acid together with the oil, such as caproic acid, caprylic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, Myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, behenic acid, lignoceric acid, tosuccinic acid, lindelic acid, tuzuic acid, myristoleic acid, palmitoleic acid, petroseric acid, oleic acid, Elaidic acid, vaccenic acid, erucic acid, sorbic acid, linoleic acid, linoleic acid, γ-linolenic acid, linolenic acid and arachidonic acid can be used alone or in combination of two or more thereof. When using the said fats and oils together, it is preferable to use 0-500 masses, preferably 50-100 mass parts of organic acids with respect to 100 mass parts of fats and oils. The concentration of fats and oils in the medium is preferably 0 g / l to 400 g / l, preferably 10 g / l to 200 g / l, more preferably 50 to 100 g / l. The concentration of the organic acid in the medium is preferably 0 to 400 g / l, preferably 10 to 200 g / l, and more preferably 50 to 100 g / l. The fats and oils and organic acids can be used together with the other carbon sources or in place of the other carbon sources, and can be used in appropriate combination with the nitrogen source and inorganic salt.

  In the production method of the acid type SL in the present invention, in order to efficiently produce the acid type SL more efficiently, in particular, the acid type SL as the diacetylated product represented by the above formula (2), for example, Among them, carbohydrate is 0 to 400 g / l, preferably 50 to 200 g / l, organic acid is 0 to 400 g / l, preferably 20 to 100 g / l, fat and oil is 0 to 400 g / l, preferably 20 to 100 g / l, It is more preferable to use a medium containing 0.5 to 100 g / l of nitrogen source, preferably 1 to 5 g / l, and 0 to 100 g / l, preferably 0.5 to 5 g / l of inorganic salts.

  The acid form SL of the present invention can be produced from various carbon sources, but using glycerol as a by-product in the degradation or transesterification of vegetable oil, in place of or in combination with the carbon source. Can also be produced. At that time, it is preferable from the viewpoint of productivity of the acid type SL that glycerol as a by-product is treated with activated carbon and used for culture. In the activated carbon treatment, for example, 10 to 300 g of activated carbon is added to 1 L of glycerol as a by-product (including water, impurities, and the like), and this is stirred for 1 to 12 hours. This can be done by removing. It is considered that the activated carbon treatment can remove substances that inhibit the growth of microorganisms contained in glycerol, which is a by-product, and can improve the productivity of acid type SL. When the glycerol is used as a carbon source, it is preferably contained in a medium of 10 to 500 g / l, preferably 50 to 300 g / l, more preferably 100 to 250 g / l as a pure glycerol content.

Culturing conditions for production The acid form SL in the present invention can be produced by culturing the microorganism using the medium.
General culture conditions are as follows. The culture temperature is 20-30 ° C, preferably 26-30 ° C. The culture days may be appropriately set according to the acid-type SL production amount, but for example, 3 to 20 days is preferable. As a culture method, known general microorganism culture methods such as shaking culture and aeration stirring culture can be applied. For good microbial growth and acid-type SL production, it is preferable to supply oxygen to the culture solution. For this purpose, when using a jar fermenter, stirring or shaking is performed with aeration of air. What is necessary is just to culture.

  When the acid type SL is selectively produced using the above-mentioned microorganism, the amount of acid type SL produced per unit culture solution and unit time is higher when a strain having a high growth ability is used. It is preferable to activate the body and inoculate it into the medium for main culture. For example, it is preferable to scale up in the order of seed culture, main culture, and acid-type SL production culture. Examples of specific medium composition and culture conditions in these cultures are as follows.

Glucose 5-20 g / L, yeast extract 0.1-2 g / L, sodium nitrate 0.1-5 g / L, potassium dihydrogen phosphate 0.1-2 g / L, and magnesium sulfate 0.1-1 g / L One platinum loop of the stock strain is inoculated into a test tube containing 4 mL of a liquid medium having the composition of (1), and cultured with shaking at 26-30 ° C. for 1 to 3 days (seed culture). Next, the culture which performed said seed culture to the Sakaguchi flask containing 50-100 mL of liquid culture medium of the same composition as the above which added fats and oils, such as vegetable fats and oils of 20-300 g / L, preferably 40-100 g / L The solution is inoculated and cultured with shaking at 26-30 ° C. for 1-7 days (main culture). Further, a jar fermenter containing 1 to 2 L of liquid medium having the same composition as that of the main culture is inoculated with the culture solution obtained by performing the main culture, and an aeration rate of 1 to 2 L / min at 26 to 30 ° C. Culture is performed at a stirring speed of 600 to 1000 rpm (acid type SL production culture). The culture temperature at this time is 20 to 30 ° C., preferably 26 to 30 ° C., and the culture time is preferably 3 to 20 days.
Inoculation into the medium used for acid type SL production culture of a microorganism strain is, for example, an amount containing 100 to 200 ml, preferably 50 to 100 ml, of seed culture solution or main culture solution in 1 L of medium used for acid type SL production culture. It can be done by adding so that.

The culture in the present invention can be terminated when the desired amount of SL produced in the culture reaches the maximum, so that the acid SL in the culture can be subjected to high performance liquid chromatography, thin layer chromatography, etc. It is preferable to carry out the measurement while measuring by a known method.
In the above culture, the form of the microorganism is not particularly limited, and refers to a microorganism cell, a treated cell of the microorganism (for example, a disrupted cell).
The microbial cells or the processed microbial cells can be immobilized and used. Examples of the immobilization method include conventionally known methods such as a carrier binding method, a crosslinking method, and a comprehensive method. In the carrier binding method, the cells are immobilized on the carrier, and the immobilization may be any of physical adsorption, ionic bond, and covalent bond. As the carrier, polysaccharides (acetylcellulose, agarose), inorganic substances (porous glass, metal oxide), synthetic polymers (polyacrylamide, polystyrene) and the like are used. In the crosslinking method, the cells are immobilized by crosslinking and bonding with each other using a bifunctional reagent such as glutaraldehyde. In the inclusion method, the cells are immobilized by wrapping the cells in a lattice of a polymer gel such as polysaccharide (alginate, carrageenan), polyacrylamide, collagen, polyurethane, or a semipermeable membrane capsule.

  The acid form SL accumulated in the culture solution can be obtained by extracting the culture solution after completion of the culture. Extraction is performed using an organic solvent such as ethyl acetate. Extraction may be performed according to a method usually performed in the field, for example, by performing extraction operation once or several times using about 0.5 to 1.5 L of an organic solvent per 1 L of a culture solution, and combining the organic solvent phase with a solvent. To obtain a lipid component. The obtained lipid component is treated as follows to recover and purify the acid SL.

<Recovery and purification of acid type SL composition>
After completion of the culture, the obtained lipid component is purified to obtain acid form SL. As a purification method, a known purification method can be used without any particular limitation. In the present invention, for example, the following method can be used. The lipid component dissolved in ethyl acetate is subjected to silica gel chromatography, and eluted in the order of chloroform: acetone (10: 0, 9: 1, 8: 2, 5: 5, 2: 8, 1: 9, 0:10). . Each solution is charged on a thin layer chromatography (TLC) plate and developed with chloroform: methanol = 8: 2 (volume ratio). After completion of the development, the presence of glycolipid is confirmed with an anthrone sulfate reagent. The eluate containing the glycolipid is collected and the solvent is distilled off to obtain a glycolipid component. Among these, since acid type SL is a highly polar component, it is contained in a large amount in the eluate of chloroform: acetone = 2: 8, so that a single component can be recovered by recovering these and further purifying them.

<Structure determination of acid type SL composition>
The structure of the glycolipid component obtained as described above is determined as follows (Candida floricola ZM-1502 strain (FERM P-21133) using glucose, oleic acid, and soybean oil as a carbon source. This will be described below by taking as an example the structure determination method of acid type SL obtained by culturing).
The isolated glycolipid component can be judged to be a glycolipid component by being colored blue-green with an anthrone sulfate reagent on a TLC plate. This glycolipid was subjected to ¹ H, ¹³ C, and two-dimensional NMR analysis, and the obtained spectrum and data of acid type SL compound group having a known structure (KS Bisht et al., J. Org. Chem., 64, p780-789 (1999), SK Singh et al., J. Org. Chem., 68, p5466-5477 (2003), etc.) are used for structural analysis.

1) Analysis of sugar composition Since analysis of sugar composition is difficult by NMR spectroscopy, it is determined by high performance liquid chromatography analysis (HPLC). After hydrolyzing glycolipids into monosaccharides and lipid parts with acid, the monosaccharide solution obtained by purifying from the aqueous phase containing monosaccharides is used in the order using a column with silica gel bonded with carbamoyl groups. Analyze by phase HPLC method. By comparing with the elution time of the standard sample, the sugar component contained in the monosaccharide solution can be determined.

2) Analysis of lipid composition On the other hand, the composition of the lipid part is determined by GC / MS analysis of fatty acid methyl ester obtained by methanolysis of the obtained glycolipid with methanolic methanol and extraction with hexane.

3) Structural analysis
^{In the 1} H-NMR spectrum, protons other than the reducing end on the sugar alcohol are generally detected around 3.5 ppm, but when the hydroxyl hydrogen is ester-bonded to the acyl group, the α-proton signal is low in the magnetic field. It is known to shift to The ¹ H-NMR spectrum of this glycolipid is compared with that of known acid type SL, and the chemical shift of the peak derived from each proton is assigned. Further, a signal derived from an acetyl group (—COCH ₃ ) observed in the vicinity of 2.1 ppm is confirmed, and it is inferred how many molecular acetyl groups are bound to which hydroxyl group.
In the ¹³ C-NMR spectrum, confirm the signal derived from the carbonyl carbon (C═O) of the acyl group observed at around 170 ppm and the signal derived from the acetyl group carbon (—COCH ₃ ) observed at around 20 ppm. However, the presence of an acetyl group in the molecule can be confirmed.

^{Then, 1 H- 1 H COSY (Correlation} Spectroscopy), HMQC (Heteronuclear Multiple Quantum Coherence), performed HMBC (Heteronuclear Multiple Bond Coherence) spectral analysis, based on the results of ¹ H, ¹³ C-NMR analysis of the Complete assignment of the sugar skeleton. The chemical shifts of carbon and hydrogen are completely assigned by the COSY and HMQC spectra, respectively. Further, it can be proved in which hydroxyl group an acetyl group exists in the HMBC spectrum.
The resulting glycolipid is recovered as a mixture of components having different fatty acid chain lengths in the lipid portion, and this is a compound having a single fatty acid by performing HPLC analysis using a reverse phase column (ODS column). Can be easily separated. The structure of the main component is determined by performing MS analysis on each separated peak compound.
In addition, in the compound group produced by these cells, several other trace spots have been detected on TLC, and there are components with different numbers of acetyl groups on the sugar skeleton and their binding positions. It is recognized that

（１）
（式中、Ｒ₁およびＲ₂は、それぞれ独立して水素（−Ｈ）又はアセチル基（−ＣＯＣＨ₃）であり、ｎは一般に９〜２０、より通常には１１〜１７、さらにより通常には１３〜１５の数であり、

基は飽和脂肪族炭化水素鎖または二重結合を少なくとも１個有する不飽和脂肪族炭化水素鎖である。） <SL obtained by the present invention>
The acid type SL obtained by carrying out the production method of the present invention as described above is represented by the following formula (1), and the acid type of SL that was produced only as a mixture with the lactone type by the conventional method: However, the major feature is that it is selectively produced only in the process of culturing microorganisms.

(1)
Wherein R ₁ and R ₂ are each independently hydrogen (—H) or acetyl group (—COCH ₃ ), and n is generally 9-20, more usually 11-17, and even more usually Is a number from 13 to 15,

The group is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond. )

（２）
（式中、ｎは一般に９〜２０、より通常には１１〜１７、さらにより通常には１３〜１５であり、

基は飽和脂肪族炭化水素鎖または二重結合を少なくとも１個有する不飽和脂肪族炭化水素鎖である。） In particular, in the present invention, an acid type SL containing 90% by mass or more, preferably 95% by mass or more, more preferably 97% by mass or more of the 6 ′, 6 ″ -diacetylated product represented by the formula (2) is obtained. be able to.

(2)
Wherein n is generally 9-20, more usually 11-17, even more usually 13-15,

The group is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond. )

In general, in order to obtain a large amount of acid type SL, a method of once producing a lactone type / acid type SL mixture by a fermentation production method and then hydrolyzing an ester bond with an acid or an alkali is employed. At that time, since the acetyl groups present in R ₁ and R ₂ in the formula (2) are also hydrolyzed, most of the obtained acid form SL is a deacetylated compound. Therefore, the 6 ′, 6 ″ -diacetylated acid type SL composition is selectively produced only by the fermentation production method of the present invention.

  The acid form SL obtained by carrying out the present invention has a critical micelle concentration of 1 mg / ml or less, preferably 0.5 mg / ml or less, more preferably 0.3 mg / ml or less. Therefore, it is possible to exhibit a surface-active effect at a low concentration as compared with lactone-type and acid-type SL mixtures obtained using conventional microorganisms and acid-type SL from which acetyl groups have been removed. Therefore, the acid form SL of the present invention can be suitably used in a wide range of fields such as detergents, cosmetics and foods.

Example 1
(Isolation and identification of ZM-1502 strain)
The homology search by the nucleotide sequence (rDNA sequence) of the 26S rDNA-D1 / D2 region of the ribosomal RNA gene of the ZM-1502 strain isolated from the plant (leaf) sample collected in Okayama Prefecture was performed as follows. GenTLE (manufactured by TaKaRa) was used for genome extraction, and the operation followed the protocol of TaKaRa. Using the extracted genomic DNA as a template, a primer A (5′-gcatatcaatagcgggagaag-3 ′: SEQ ID NO: 2) and a primer B (5′-ggtccgtgtttcaagagggg-3 ′: SEQ ID NO: 3) specific for the ribosomal RNA gene (rDNA) The base sequence of the 26S rDNA-D1 / D2 region was amplified by PCR. Thereafter, the base sequence of the amplified region was determined. The determined base sequence is shown in SEQ ID NO: 1. The obtained rDNA sequence was accessed to the DNA database (DDBJ), and homology search was performed using the FASTA program (http://www.ddbj.nig.ac.jp/search/fasta-j.html) However, it was 100% identical with the rDNA sequence of the corresponding region of Candida floricola . Further, as a result of molecular phylogenetic analysis by NJ method after multiple alignment of base sequences of 26S rDNA-D1 / D2 region of ribosomal RNA gene of Candida genus and representative yeast species obtained from DNA database, The position of the molecular phylogenetic tree of this strain coincided with Candida floricola (FIG. 1). Furthermore, as a result of combining the results of the physiological property test described in Table 1, the ZM-1502 strain was classified as Candida floricola . The ZM-1502 strain was registered with the Patent Organism Depositary (IPOD) (1-1-3 Higashi 1-1-3, Tsukuba, Ibaraki) on December 13, 2006, with the accession number FERM P-21133. Has been deposited.

Example 2
(Production of acid type SL)
(1) Candida floricola ( Candida floricola ) ZM-1502 culture (a) Seed culture Preservation medium (malt extract 3 g / L (Malt extract (Becton, Dickinson and Company)), yeast extract 3 g / L (Bacto Yeast Extract (manufactured by Becton, Dickinson and Company)), peptone 5 g / L, glucose 10 g / L, agar 30 g / L), and the above-mentioned Candida floricola ZM-1502 strain, 20 g / L, yeast extract 1 g / L (Bacto Yeast Extract (manufactured by Becton, Dickinson and Company)), sodium nitrate 1 g / L, dihydrogen phosphate Um 0.5 g / L, and was carried out one day shaking culture at 1 platinum loop inoculated 26 ° C. in a test tube containing the liquid medium 2mL of the composition of magnesium sulfate 0.5 g / L.
(B) Acid-type SL production culture 100 g / L glucose, 50 g / L oleic acid, 50 g / L soybean oil, 1 g / L yeast extract (Bacto Yeast) Extract (manufactured by Becton, Dickinson and Company)) 30 mL of liquid medium having a composition of 3 g / L urea, 1 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 0.4 g / L sodium sulfate 2 ml was inoculated into an Erlenmeyer flask containing, and shaking culture was performed at 28 ° C. for 7 days.

(2) Confirmation of acid type SL production ability of ZM-1502 strain The culture solution after completion of the culture in (1) above was collected, and the acid form SL production ability of ZM-1502 strain was used for thin layer chromatography ( TLC) (FIG. 2, lane A). As a standard of SL, Candida bombicola NBRC 10243T strain was cultured under the same conditions as described above, and an ethyl acetate extract fraction was used (FIG. 2, lane B). Furthermore, conventional acid type SL (deacetylated product) obtained by alkaline hydrolysis of this was also spotted (FIG. 2, lane C). In addition, Silica gel 60F (made by Wako) was used for the thin layer chromatography, and the developing solvent was chloroform: methanol = 8: 2. For detection, anthrone reagent (0.2% anthrone / 75% sulfuric acid aqueous solution) was sprayed on the developed plate and heated at 110 ° C. for 5 minutes.

As shown in FIG. 2, C.I. Compared to the SL standard (lane B) obtained by the conventional method using the bombicola strain, the glycolipid mainly produced by the ZM-1502 strain is the acid type SL having a higher polarity than the lactone type, and the lactone It was confirmed that there was no type SL (lane A). Also, the number of spots was small, and it was expected that there were few kinds of mixtures. Further, compared with the conventional acid type SL (lane C) that has been deacetylated, a major feature is that the glycolipid having high hydrophobicity is the main component.

Example 3
(Isolation and purification of acid form SL)
The purification of SL obtained in Example 2 was separated by known purification techniques. That is, the ethyl acetate extract was applied to a silica gel column and purified by column chromatography using a mixed solution of chloroform and acetone as a developing solvent. The acid type SL was separated and recovered in a fraction having a ratio of chloroform and acetone of 2: 8, and then components having higher polarity were separated and recovered with acetone. And each collect | recovered fraction was used for the said TLC (FIG. 3). According to this, it turned out that a main component (a) and a trace component (b) exist. Furthermore, a single component was obtained by repeating these purifications.

Example 4
(Structural analysis of acid type SL)
1) Analysis of sugar composition 1 mL of 3N hydrochloric acid was added to 10 mg of the acid type SL (a) obtained in Example 3, and heated at 90 ° C. for 3 hours to completely decompose into a lipid part and a sugar. The lipid portion was removed by extraction into 2 mL of hexane, and the aqueous solution portion was desalted with a desalting resin (Amberlite IRA-410) and then analyzed by high performance liquid chromatography (HPLC). As an HPLC analysis column, an Amide 80 column (manufactured by Tosoh Corporation) was used, acetone: water = 85: 15 as an eluent, and an evaporative light scattering detector was used for detection. An HPLC chart is shown in FIG. As a result of comparing the elution time of HPLC with the elution time of the standard substance, it was shown that the sugar part of the acid type SL (a) obtained in Example 3 was composed entirely of glucose.

2) Analysis of lipid composition The composition of the lipid part of acid type SL (a) obtained in Example 3 was determined by GC / MS analysis of fatty acid methyl ester obtained by extraction with hexane from hydrochloric acid methanol hydrolyzate. It was. The analysis results are shown in Table 2. In this glycolipid, medium-chain straight-chain saturated and unsaturated hydroxy fatty acids (C16 to C18) having one hydroxyl group in the molecular structure are detected, and in particular, it is shown that C18 unsaturated hydroxy fatty acids are the main components. It was. Moreover, it was shown that the unsaturated fatty acid is contained about 1/2 of the whole.

3) Complete assignment of sugar skeleton The structure of the acid type SL (a) obtained in Example 3 was identified by NMR analysis using deuterated methanol (CD ₃ OD) as a solvent. By performing each NMR spectrum analysis of ¹ H, ¹³ C, ¹ H- ¹ H COSY, HMQC, and HMBC, the structure of the glycolipid was completely assigned. The ¹ H-NMR spectrum of this glycolipid is shown in FIG. According to this, in this glycolipid, two α-proton signals shifted by a low magnetic field due to the ester bond with the acyl group were observed in the vicinity of 4.2 ppm and 4.35 ppm, which were 6 ′ and 6 ′ on sophorose, respectively. It was confirmed that the protons were distinguished at 6 ″ position (6′-a, 6 ″ -a, and 6′-b, 6 ″ -b). In addition, acetyl groups were present at 2.046 ppm and 2.058 ppm. signals for methyl protons of (-COC H ₃₎ were detected two.
Furthermore, in the ¹³ C-NMR spectrum, there are two peaks derived from acetyl groups each (signals derived from carbonyl group carbon (C═O) are 171.46 ppm and 171.48 ppm, methyl carbon (—CO 2 C ₃ H ₃ )). This was also confirmed from the fact that the signals derived from 1958 ppm and 19.75 ppm were observed.
The above results indicate that the acid type SL obtained in the present invention is a diacetylated product in which two acetyl groups are ester-bonded to the 6 ′ and 6 ″ positions of sophorose.

Next, HMQC and HMBC spectra were measured. From the obtained spectra, the chemical shifts of the carbon and hydrogen of the hydroxy fatty acid bonded by the sugar skeleton and the ether bond were completely assigned, and the acid type SL obtained in the present invention was 6 It was proved to be ', 6 "-diacetylated acid type SL. An enlarged view of the HMBC spectrum showing the correlation between the carbonyl carbon of the acetyl group and the sugar skeleton is shown (FIG. 6). Shown together.
¹ H NMR (CD ₃ OD, δ): 1.20 (d, CH ₃ (H-18)), 1.26-1.44 (br, CH ₂ (H-4-7, 12-15)), 1.55-1.64 (m , CH ₂ (H-3, 16)), 2.0-2.08 (m, -C H ₂ CH = CH- (H-8, 11)), 2.046, 2.048 (Ac × 2), 2.27 (t, -C H ₂ COOH (H-2)), 3.2-3.7 (m, C H OH (H-2 'to 5', 2 '' to 5 '')), 3.75 (m, CH (H-17)), 4.20 ( dd, CH (H-6'a, 6``a)), 4.36 (dd, CH (H-6'b, 6''b)), 4.45 (d, CH (H-1 ')), 4.55 (d , CH (H-1 ”)), 5.35 (m, -CH = CH- (H-9, 10))
¹³ C NMR (CD ₃ OD, δ): 19.58, 19.75 (CH ₃ × 2 (Ac)), 20.67 (CH ₃ (C-18)), 23.05, 25.0, 29.0-29.8 (CH ₂ (C-4 ~ 7, 12-15)), 24.92 (CH ₂ (C-3, 16)), 26.99 (CH ₂ (C-8, 11)), 33.77 ( -C H ₂ COOH (C-2)), 63.57, 63.74 (C-6 ', 6 "), 70.2, 70.3 (C-4', 4"), 73.68 (C-5 '), 74.39 (C-2 "), 74.90 (C-5"), 76.34, 76.63 (C-3 ', 3 "), 77.37 (CH (C-17)), 82.60 (C-2'), 101.40 (C-1 '), 104.48 (C-1"), 129.6 (CH = CH (C-9, 10)), 171.48 (C = O (Ac)), 176.5 (COOH (C-1))

Furthermore, in this acid type SL, pseudomolecular ions [M + Na] ⁺ m / z 730.0 and 704.0 were detected in MALDI-TOF / MS analysis (FIG. 7). Therefore, the present acid type SL is mainly composed of 6 ′, 6 ″ -diacetylated acid type SL to which a linear unsaturated hydroxy fatty acid having one unsaturated double bond is present at C18, and subsequently C16. It is considered that a lot of 6 ′, 6 ″ -diacetylated acid type SL to which a linear saturated hydroxy fatty acid is bonded is contained. Further to them, the peak of compound 6 ′, 6 ″ -diacetylated acid form SL to which C18 saturated and C16 unsaturated hydroxy fatty acids were bound was also detected. These results are the results of fatty acid composition analysis (Table In the above formula (1) or (2), C16 corresponds to the structure of n = 13, and C18 corresponds to the structure of n = 15.
In addition, as a result of conducting the same analysis on the other component (b) separated in Example 3, it was found that the monoacetylated acid type SL lacking one acetyl group at the 6′-position or 6 ″ -position on sophorose. Therefore, the analysis of components (a) and (b) confirmed that SL produced by the method of the present invention does not contain a lactone type, in other words, 100% by mass of SL produced. It became clear that it was an acid type.

Example 5
(Determination of SL production of ZM-1502 by HPLC analysis)
About the ethyl acetate extract fraction of the culture solution of ZM-1502 strain obtained in Example 2 and the culture solution of Candida bombicola NBRC 10243T strain cultured under the same conditions as in Example 2 for comparison, silica gel column (Inertosyl Sil-100A ) Was used to analyze the components of the product. The eluent was a chloroform / methanol mixed solvent and eluted with a gradient system set so that the mixing ratio was changed from 10: 0 to 0:10 at a flow rate of 1 mL / min. For detection, an evaporative light scattering detector (ELSD-LT, manufactured by Shimadzu Corporation) was used. As a result of measurement of each sample (FIG. 8), an almost single peak product was obtained from the ZM-1502 strain (FIG. 8a). About various SL fraction refine | purified by the method similar to Example 3, as a result of producing a calibration curve by changing a density | concentration, the product of the single peak obtained from ZM-1502 strain is diacetylated acid type SL. The production amount was 42 g / L. Furthermore, as a result of calculating the composition ratio of the acid type SL produced by the ZM-1502 strain from the peak area, the diacetylated acid type SL was 98% by mass or more and the monoacetylated acid type SL was less than 2% by mass. On the other hand, C.I. A plurality of components were produced from Bombicola NBRC 10243T strain, and it was confirmed that the lactone type was the main component (FIG. 8b). C. The production amount of all SL of bombicola NBRC 10243T is 56 g / L, of which lactone type SL is 66% by mass, acid type is 34% by mass, and 50% by mass of acid type SL is diacetylated acid type SL Met. The above results were in good agreement with the results of TLC analysis in FIG.

Example 6
(Production of acid type SL using glycerol as a raw material)
1) Activated carbon treatment of glycerol 50 g of activated carbon was added to 1 L of 50% by mass of glycerol aqueous solution by-produced by transesterification of vegetable oils and stirred for 1 hour. The solution was No. It filtered with the filter paper of 5, and activated carbon was removed. The filtrate was activated carbon-treated glycerol.

2) Production of acid type SL using activated carbon-treated glycerol as a carbon source
ZM-1502 is treated with activated charcoal glycerol 200 g / L (glycerol pure), yeast extract 1 g / L, urea 3 g / L, potassium dihydrogen phosphate 1 g / L, and magnesium sulfate 1 g / L The Erlenmeyer flask containing 30 mL of the liquid medium containing the composition was inoculated and cultured with shaking at 28 ° C. for 7 days. As a control experiment, instead of the activated carbon-treated glycerol, a medium containing 200 g / L (glycerol pure) of glycerol as a by-product without activated carbon treatment, and 0.4 g / L instead of the activated carbon-treated glycerol. ZM-1502 strain was cultured under the same conditions using a medium containing 200 g / L of reagent-grade sodium sulfate and 200 g / L of reagent grade glycerol (purified glycerol).

3) Production amount of acid type SL The production amount of acid type SL was performed by HPLC using the silica gel column and the evaporative light scattering detector. In the glycerol treated with activated carbon, 4.6 g / L of acid type SL was produced. On the other hand, a medium containing glycerol not treated with activated charcoal has 1.9 g / L acid type SL, and a medium containing 0.4 g / L sodium sulfate and 200 g / L reagent grade glycerol has 3.5 g / L acid. A mold SL was produced.

Example 7
(Production of acid type SL using ZM-1517 strain and ZM-0132 strain)
Using the ZM-1517 strain and the ZM-0132 strain, culturing was performed in the same manner as in Example 6 (a method of culturing using a medium containing activated carbon-treated glycerol). The culture solution after completion of the culture was collected, and the glycolipid production ability of the above two strains was confirmed by thin layer chromatography using this (FIG. 9). As with the ZM-1502 strain, the above two strains were in acid form. It was confirmed that only SL was produced selectively. From the spot of thin layer chromatography, it is judged that the production amount of acid type SL of ZM-1502 strain, ZM-1517 strain and ZM-0132 strain is almost equal.

Example 8
(Comparison of acid-type SL production ability among Candida yeast strains)
Candida floricola NBRC10700T strain (homology with the nucleotide sequence shown in SEQ ID NO: 1: 99% <), and existing SL producing strain Candida bombicola NBRC 10243T strain with respect to ZM-1502 strain Was cultured in the same manner as in Example 6 (method of culturing using a medium containing activated carbon-treated glycerol). The culture solution after completion of the culture was collected, and the glycolipid production ability of the above two strains was confirmed by thin layer chromatography using this (FIG. 10). As with the ZM-1502 strain, the Candida florica NBRC10700T strain was also acid. It was confirmed that only the mold SL was selectively produced. On the other hand, it was confirmed that the existing Candida bombicola NBRC 10243T strain that produces an SL mixture having a lactone type as a main product mainly produces a lactone type. For production of SL, the TLC spot, although production of ZM-1502 strain and Candida bombicola NBRC 10243T strain is almost the same, production of Candida floricola NBRC10700T strain, compared with ZM-1502 strain A small amount was confirmed.

Example 9
(Surfactant ability of acid type SL)
Next, the surface activity ability of the acid type SL corresponding to “component (a) + (b)” produced in Example 2 and having only the raw material oil or the like removed in the purification process of Example 3 was examined. FIG. 11 shows the results obtained by measuring the surface tension of acid-type SL aqueous solutions having various concentrations using a drop master DM500 manufactured by Kyowa Interface Science. As is apparent from the figure, the surface tension value decreased as the surfactant concentration increased, and eventually became constant when the critical micelle concentration (CMC) was reached. As is clear from the figure, the CMC of the acid form SL was 0.2 mg / ml, and the rCMC was 38.92 mN / m, and it was found that the surface activity was excellent from an extremely low concentration. The CMC of the conventional deacetylated acid type SL is 0.30 mg / ml, and the rCMC is 35.33 mN / m. The acid type SL of the present invention expresses surface activity from a lower concentration. It was confirmed.

The molecular phylogenetic tree created based on the base sequence obtained from Candida floricola ZM-1502 strain is shown. The analysis result by the thin layer chromatography (TLC) of the culture solution of Candida floricola (Candida floricola) ZM-1502 strain is shown. (Lane A: ZM-1502 strain culture solution, B: C. bobmicola NBRC 10243T strain culture solution, C: Existing acid type SL obtained by alkaline hydrolysis of Lane B) In Example 3, the TLC chart of the acid type SL separated and produced is shown. In Example 4, the HPLC chart which analyzed the sugar composition of acid type SL (a) is shown. In Example 4, it shows the ¹ H NMR spectrum was subjected to structural analysis of acid-form SL (a). In Example 4, the enlarged view which shows the correlation with a carbonyl carbon and sugar skeleton about the HMBC spectrum utilized for determination of the coupling | bonding position of an acetyl group, when assigning the structure of acid type SL (a) completely is shown. In Example 4, the MALDI-TOF / MS spectrum which measured the molecular weight of acid type SL (a) is shown. In Example 5, the HPLC chart which analyzed the ethyl acetate extraction fraction of each culture solution of ZM-1502 strain and Candida bombicola NBRC 10243T strain is shown. In Example 7, the TLC chart of the various strain culture solution obtained by culture | cultivation which uses glycerol as a carbon source is shown. (Lane 1: ZM-1502 strain culture solution, 2: ZM-1517 strain culture solution, 3: ZM-0132 strain culture solution) In Example 8, the TLC chart of the various strain culture solution obtained by the culture | cultivation which uses glycerol as a carbon source is shown. (Lane 1: ZM-1502 strain culture solution, 2: Candida floricola NBRC10700T strain culture solution, 3: Candida bombicola NBRC 10243T strain culture solution) In Example 9, it is a graph which shows the relationship between the density | concentration obtained from the measurement result of surface active ability of acid type SL (a) + (b), and a surface tension value.

Claims (7)

Culturing a microorganism belonging to the genus Candida having a 26S rDNA-D1 / D2 region of a ribosomal RNA gene containing a base sequence of 95% or more homologous to the base sequence shown in SEQ ID NO: 1 in a liquid medium, A method for selectively producing an acid-form sophorose lipid represented by the following formula (1):

(1)
Wherein R ₁ and R ₂ are each independently hydrogen (—H) or an acetyl group (—COCH ₃ ), n is a number from 9 to 20,

Is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond. ) The method according to claim 1, wherein the produced acid-type sophorose lipid contains 90% by mass or more of 6 ′, 6 ″ -diacetylated product represented by the following formula (2):

(2)
(Wherein n is a number from 9 to 20,

Is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond. )   The method according to claim 1 or 2, wherein the microorganism belonging to the genus Candida is a microorganism belonging to Candida floricola.   The microorganisms belonging to the genus Candida are Candida floricola ZM-1502 (FERM P-21133), ZM-1517 (FERM P-21153), and ZM-0132 (FERM P-21152). The method according to claim 1, wherein the method is any one of the following.   The method according to any one of claims 1 to 4, wherein the microorganism is cultured using a liquid medium containing glycerol.   6. The method of claim 5, wherein the glycerol is a by-product during vegetable oil degradation or transesterification. Acid-type sophorose lipid containing 90% by mass or more of acid-type sophorose lipid having the following structure:

(2)
(Wherein n is a number from 9 to 20,

Is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond. )

Images