JPS6024797B2 - Glycolipids and their fermentation production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel glycolipid and its fermentation production method, and more particularly to a glycolipid in which two bran aliphatic groups containing mannose and erythritol are bonded.

BACKGROUND ART Glycolipids are lipids that contain sugars as their constituents and perform various functions within living organisms. Attempts have also been made to produce large amounts of them through fermentation and use them industrially.

For example, Torulopsis bompicola (Mt. Tor ops)
Sophorolipid (J cola) produced by
．． F. T. Spencertal, Ca Misaki DianJ
o Yamawaki lofChemist Hi, 39, 846 (196
1)) or using Q-decenoic acid rhamnose produced by bacteria belonging to the genus Pseudomonas (e.g., JP 52-54083). It has been known. The present inventors discovered Candida sp., which is known as an itaconic acid-producing bacterium. S-10 (Tabuchi et al., AgricBiol.C
hem. 45(2), 475-479 (1 pseudo 1), Nakahara et al. 1981 Japanese Agricultural Chemistry Society Conference Abstracts, 344
When the n-alkanatizing mutant strain (Page 37) was cultured under conditions without the addition of calcium carbonate, an acid neutralizing agent, a novel glycolipid expressed by the following formula was unexpectedly produced in large amounts. I found out that it can be done.

(In the formula, RCO is an aliphatic acyl group having 7 to 20 carbon atoms, and is bonded to any hydroxyl group on mannose among the disaccharides.) The bacteria used in the present invention are the above-mentioned Candida sp. b-1 (KSM-1529) [National Institute of Microbial Technology, Agency of Industrial Science and Technology, Microbial Accession No. 58
No. 84 (FERM1P No. Madaku)), and its mycological properties are
sp. It is exactly the same as S-10.

Sp. The mycological properties of S-10 are as follows:
iol. Chem. 45 (2) 475-479 (1 sea bream 1
)), its contents are as follows.
This bacterium was isolated from the exudate of an unknown tree on Ishigaki Island, Okinawa Prefecture, in 197 B.C. [a} Growth status in each tower area■ After culturing for 3 days in wort tower area 25q0, the cells are oval or spindle-shaped, with a size of 5-10 x 2-4rm, single, double chain, or short. It is mirror-like.

Hyphae with septate walls can be seen. There is no leachate into the medium.
Formation of film and precipitate is observed. ■ After 3 days of streak culture on wort agar medium 2yC, the entire rim becomes flesh-colored and becomes smooth and glossy.

After 10 days, the color of the flesh becomes pale yellow due to the production of carotenoids, the luster is gone, the previously smooth flesh becomes wrinkled, becomes sticky steel-like or filmy, and the periphery becomes filamentous.

■ Bacto yeast morphology observation agar (Difco) and Dalmau made from corn meal agar
Plated true hyphae, pseudohyphae and budding spores are formed.

The budding spores are oval or conical and exist singly or in short chains. No droplet formation is observed in the petite needles. Even after the blastema is separated, the needle remains attached to the mother cell. {b} Ascospore formation V8 - Ascospore formation on agar, carrot pieces, potato pieces, potato dextrose agar, Gorodkova agar, malt agar, and corn mill agar. unacceptable.

(c) Clamp connections are also used in spore formation in wort agar towers.
No other spore formation (ie, resting spores, ejected spores, fisspores, endospores, chlamydospores) is observed.

[d} Physiological properties ■ Optimal growth conditions Temperature 30°C, pH approximately 6 ■ Growth range temperature 10-35°C, pH 3-8 ■ Assimilation of nitrogen compounds Assimilates nitrate and ammonium sulfate, but not nitrite.

■ Break down fat.

■ Decomposes urea.

■ Production of carotenoids Maximum absorption in the intestine, 45 skin (Shoulder absorption at 420, 48 Mm) ■ Production of organic acids Produces itaconic acid.

■ Vitamin requirement: None ■ Production of starch-like substances: Intestinal ■ Urease activity: Yes ■ Extracellular DNAse activity: Yes ■ Degradation of arbutin: Negative or positive {e} Fermentable sugar D-glucose ( 1), D-galactose (1), sucrose (1), maltose (1), lactose (1), raffinose (
1) Sugar assimilation D-glucose (10), D-galactose (10), L-
Sorbose (10), Sucrose (10), Maltose (10), Serpiose (minor), Trehalose (10), Lactose (10)
, melibiose (1), ruffinose (10), melezitose (10), inulin (10), soluble starch (10),
D-xylose (10), L-arabinose (10), D-
Arabinose (1), D-ribose (1), L-rhamnose (1), ethanol (10), glycerin (10), erythritol (10), adonitol (10), dulcitol (1), galaxitol (1), mannitol (1) 10), Q-methyl-D-glucoside (10), salicin (minor), succinic acid (10), citric acid (1), inositol (10), the structure of the glycolipid of the present invention is determined as follows. (The glycolipid for structure determination will be described as an example when cultured using n-hexadecane as a carbon source).

The infrared absorption spectrum of this bran lipid itself shows the presence of hydroxyl groups and carboxylic acid ester bonds on the bran (
(See Figure 1). Also, the sodium complex [M
+Na] In the mass vector of 10, many peaks were observed, including 656 as the main peak and 727, 71 in 699 total 5, 671, etc. as other main peaks (see No. 3). When glycolipids were saponified with alkali and fatty acids were analyzed using gas chromatography as methyl esters,
It was found that the main components are C,2, C8, and C,4, and that it is a straight chain saturated fatty acid from C7 to C,4. On the other hand, sugar obtained by saponification with alkali. The components were further hydrolyzed with acid, and after isolation, it was confirmed that they were ○-mannose and mesoerythritol from various physical and chemical properties, and the bran portion was treated with Q-mannosidase and 8-mannosidase. As a result, it was decomposed into monosaccharides only when treated with 8-mannosidase, so it was confirmed that this sugar bond was an 8-unit bond. Furthermore, this bran was treated with periodic acid (Smith reaction), the aldehyde was reduced to methylol with sodium borohydride, and the ether bond was severed with hydrochloric acid treatment, yielding 1 mole each of glycerin, glycolaldehyde, and ethylene glycol. It was done. From this result, it was confirmed that the sugar moiety was 41○-8-D-mannopyranosyl-mesoerythritol. The molecular weight of this bran part is 284, and the molecular weight of the straight chain saturated fatty acid is C,4, 22&C. 214 in 3, C, 20 or more in 2
、． 180 C,. Considering that the mass vector is 172 and the mass vector is [M + Na] 10,
The 727 peak of the mass vector is C, 2 phantom fatty acids (284 × 228 × 2 × 18 × 2 × 223 = 727), 7
The peak 13 is one each of C,4 and C,3 fatty acids, 69
The peak at 9 is C, 4 and C, 2 fatty acids each, and the peak at 685 is C, 4 and C, . Or two each of C,3 and C,2 fatty acids, the peak of 671 is two C,2 or C,4 and C,
. One fatty acid each, the peaks at 657 are C,2 and C,. It can be identified that each one of the fatty acids is ester-linked. Therefore, it was confirmed that two fatty acid molecules were bound to this sugar moiety. Note that when other hydrocarbons or fats and oils are used as the carbon source instead of n-hexadecane, the composition of fatty acids in the resulting glycolipids changes, but they generally consist of 7 to 20 carbon atoms. Mesoerythritol is the only monosaccharide obtained by hydrolyzing the glycolipid with hydrochloric acid to remove the hydroxyl group attached to the fatty acid, leaving the ester bond intact and cleaving only the ether bond between the disaccharides. Therefore, it was found that it was any two hydroxyl groups on mannose. To produce the glycolipid of the present invention, the mutant strain b-1 is used, and long-chain fatty acids or derivatives thereof, or substances metabolized by them, are added to the medium as a carbon source,
Fermentation may be carried out under normal conditions, except that no acid neutralizer (base such as calcium carbonate) is added.

Examples of carbon sources that can be added to the culture medium include long chain fatty acids or their derivatives such as lower alcohol esters (e.g. methyl esters), polyhydric alcohol esters (e.g. triglycerides, fats and oils), and long chain fatty acids. Substances to be metabolized include aliphatic hydrocarbons such as linear alkanes and linear alkenes, as well as higher alcohols. A preferred carbon source is a triglyceride whose constituent components are a straight aliphatic hydrocarbon having 10 to 24 carbon atoms and a straight mirror fatty acid having 10 to 20 carbon atoms. The former includes saturated linear aliphatic hydrocarbons having 10 to 20 carbon atoms (e.g. n-decane, n-bentadecane, n-hexadecane, n-octadecane), Q-olefins having 10 to 20 carbon atoms (
For example, 1-dodeceso, 1-tetradecene, 1-hexadecene), and the latter includes, for example, coconut oil, palm oil, soybean oil, beef tallow, corn oil, and the like. As the carbon source, the carbon source mentioned above may be used as the only carbon source, or glycerin or the like may be added in addition to the carbon source. The medium also contains a suitable nitrogen source, inorganic salts,
Add an organic nutrient source, a nonionic surfactant, etc. as necessary to adjust the pH to 4-8, inoculate the seed culture, and raise the pH to 20-35.
Shake or aerate culture at about 1:00 pm. The production amount of glycolipids can be tracked by extracting glycolipids with methyl ethyl ketone and measuring D-mannose produced after hydrolysis using a reducing sugar colorimetric method or the like. Usually, if the culture is carried out for about 5 to 15 days, about 10 or more of the target product can be obtained per medium. Bran lipids are obtained by removing bacterial cells from a culture solution, extracting with a suitable organic solvent (eg, methyl ethyl ketone, ethyl acetate, ether, etc.), and then purifying the solution. The glycolipids of the present invention have hygroscopicity and hydrophilicity derived from the bran, and lipophilicity derived from the fatty acids, and also have antibacterial properties, and can be used as a hygroscopic oil component, a surfactant component, and an antibacterial component for cleaning. It can be used in agents, waxes, cosmetics, drug bases, etc.

Reference example <mutant strain sp. Method for obtaining b-1> Itaconic acid-producing yeast sp. belonging to the genus Candida. S-
10 was treated with NTG, and a small amount of the washed bacterial cell suspension was taken.
Inoculate into an inorganic salt medium containing 2 to 10% n-alkane,
Shaking culture was performed at 25° C. for several days using an L-shaped tube.

During the culture, bacteria that had gathered in the oil layer or at the oil-water interface were removed with a pipette, transferred to a fresh medium, and cultured. After repeating the same operation, the target mutant strain sp. I got b-1. The medium composition at this time was as follows. n
- Alkane 2-10% (v/v)
NACI O. 1% (w
/v) KH2P04
0.02 〃MgS04・7th 20
0.05 Yeast extract
0.05 Span 80
0.02 Example 1 <Seed culture> Sodium nitrate 0.2%, potassium dihydrogen phosphate 0.0
The mutant strain sp. b-1 (Recruitment Research Institute Deposit Number 588
4) was inoculated and cultured under shaking at 260℃ for 2 days.

<Main culture> Next, sodium nitrate 0.2%, potassium dihydrogen phosphate 0.05%, magnesium sulfate heptahydrate 0.05%, span 800.02% and n-hexadecane 5% (% is n
A culture medium consisting of tap water containing hexadecane (based on weight, excluding volume) was placed in 500 Erlenmeyer flasks, and the top of seed culture 5 was added thereto, and cultured at 25q0 for 8 days.

Paper chromatography of culture solution (developing solvent, n-p. Panol: ethyl acetate: water = 7:1:2 (v/v)
), when color was developed using Yoda's reagent, one spot was observed at an Rf value of approximately 0.82 and one spot was observed at an Rf value of approximately 0.45. A spot with an Rf value of about 0.45 matched that of a standard sample of mesoerithritol. <Separation of Glycolipids> The culture solution was centrifuged to remove bacterial cells gathered in the lower layer, and the upper layer was adjusted to pH 4-5 with 4N hydrochloric acid and extracted twice with methyl ethyl ketone.

The organic phases were combined and the solvent was distilled off to obtain a crude glycolipid as a residue. This is silica gel. Applied to matoography,
Eluted with chloroform and acetone. The chloroform fraction contained n-hexadecane. The acetone fractions were collected and the acetone was removed to obtain purified glycolipids. <Physical properties of glycolipid> The obtained basic lipid was a colorless oily substance.

m (KBr, late-1) 3300-3600 (broad), 302u295ri1
760 (see Figure 1) 13CNMR (CDC13 solvent,
TMS internal standard, chest) 17361, 171.66...
... C of fatty acid ester = 099.15 ...
...Glycosyl bond carbon 79.27 ~ muscle. 67...
・・・・・・Bran carbon 3385～14.16・・・・・・
... Fatty acid carbon (see Figure 2) MS Sodium iodide (Nal) was added and measured as [M+Na] 10.

(See Figure 3) Solubility in solvents Easy solubility: acetone, ethyl alcohol, methyl ethyl ketone Solubility: chloroform Scalability: benzene, ether, water, insoluble: hexane <Structure determination of glycolipids> [1 'Purified glycolipids were saponified using caustic soda to obtain fatty acid sodium salts and oligosaccharides.

Furthermore, this oligosaccharide was hydrolyzed with hydrochloric acid to obtain a mixture of sugars. When a mixture of oligosaccharides and monosaccharides was subjected to paper chromatography (developing solvent, n-propanol: ethyl acetate: water = 7:1:2 (v/v)), one oligosaccharide had an Rf value of around 0.20. For the monosaccharide mixture, the spots are around Rf value 0.5 and around 0.4M, respectively.
One spot was observed. The spot of monosaccharide mixture is
It was consistent with that of standard samples of D-mannose and mesoelythritol. After developing the monosaccharide mixture by paper chromatography, cut out the corresponding spot,
Each was extracted with water. The extract containing a monosaccharide thought to be mesoelythritol was concentrated to dryness, then hot ethanol was added to dissolve it, and monosaccharide crystals were obtained from the ethanol solution. On the other hand D-
After concentrating the extract containing a single component thought to be mannose, ethanol was added to the bound syrup and reacted with phenylhydrazine to obtain crystals as phenylhydrazone. The melting point of each crystalline substance was measured and found to match that of the standard sample, and as a result of a waster test with the standard sample, it was confirmed that the melting point did not decrease. Furthermore, each IR was measured and confirmed to match that of the standard sample. Therefore, it was confirmed that the bran lipids were two brans consisting of D-mannose and soeristritorini. '21 When the above oligosaccharide was treated with Q-mannosidase and 8-mannosidase and subjected to paper chromatography under the same conditions as '1', the oligosaccharide treated with Q-mannosidase showed no change at all. On the other hand, in those treated with 8-mannosidase, spots corresponding to D-mannose and mesoerythritol were observed.

From this result, it was found that D-mannose and mesoerithritol were bonded through an 8-bond. '
31 Two of the above oligosaccharides were treated with five portions of 20M sodium periodate for 400.9 hours, and then 200.000.9 hours of sodium borohydride was added and treated for 400.6 hours to reduce the aldehyde.

After decomposing excess sodium borohydride with acetic acid, it was desalted with AmPmte (Niju, OH-). When this was further treated with 1N hydrochloric acid at 100°C for 2 hours and subjected to gas chromatography, compounds identified as glycerin, glycolaldehyde, and ethylene glycol were obtained in a molar ratio of 1:1:1. Ta. This result is interpreted as follows.

First, the C of mesoelythritol was removed by periodic acid treatment.
2-C3 bond and C2-C3 bond of D-mannose and C
3-C was cut.

The two ether bonds were then hydrolyzed. From the results of Chikugami, it was confirmed that the bran portion of the glycolipid of the present invention is 410181D-mannopyranosyl mesoerythritol.

'41 The fatty acid sodium salt obtained in the above [1'] was acidified with hydrochloric acid and extracted with ether to obtain a fatty acid.

Methanol was added to this, and the mixture was reacted with a sulfuric acid catalyst at 70° C. for 1 hour to obtain a fatty acid methyl ester. From the results of gas chromatography of this product, n-dodecanoic acid (C
, 2) Methyl about 52%, n-octanoate (C8) methyl about 27%, n-tetradecanoate (C,4) methyl about 10
% (% is based on weight as carboxylic acid) was found to be a mixture of straight chain saturated fatty acid methyl esters having 7 to 14 carbon atoms. '5- Refined glycolipids 30/9
0.0 CI2' was added on the same day, and after reacting at 100°C for 2 hours, chloroform 2' was added for extraction, and the aqueous and chloroform layers were subjected to paper chromatography (developing solvent,
n-propanol: ethyl acetate: water = 7:1:2 (v'
v)) and color development using Yoda's reagent revealed spots identified as mesoerythritol and oligosaccharides in the aqueous layer.

In the chloroform layer, in addition to spots identified as mesoerythritol and raw material glycolipids, another spot was observed in the middle. Spots with only D-mannose are in the water layer. Since it is not observed in any of the chloroform layers, it is thought that this spot in the middle of the chloroform layer is an ester bond of a fatty acid to a hydroxyl group on D-mannose. Example 2 Glycolipid 1 was fermented in the same manner as in Example 1 except that n-bentadecane was used instead of n-hexadecane in the main culture.
Obtained 0 evening/Z.

When the constituent fatty acids were analyzed using gas chromatography as methyl esters, n-tridecanoic acid (C,
3) Methyl about 43%, n-undecanoate (C,.) methyl about 16%, n-octanoate (C8) methyl about 15%,
It was found to be a mixture of straight chain saturated fatty acid methyl esters having 7 to 13 carbon atoms, the main component being about 12% methyl n-/nananoate (C9). Example 3 Glycolipid 8 was fermented in the same manner as in Example 1 except that 1-hexadecane was used instead of n-hexadecane in the main culture.
I got Ta'Z.

Similarly, when the constituent fatty acids were analyzed using gas chromatography as methyl esters, 11 dodecenoic acid (C8
F,) methyl about 25%, (C,2) methyl 13-dodecanoate about 18%, n-tridecanoate (C,3) methyl about 1
2%, (C,4F,)methyl n-tetradecenoate approx. 13
% as the main component, saturated and unsaturated (with 8 to 14 carbon atoms)
It was found to be a mixture of straight chain fatty acid methyl esters (saturated:unsaturated = approximately 45:55). Example 4 Fermentation was carried out in the same manner as in Example 1 except that the carbon source and temperature in the main culture were changed as shown in the table below, and the produced linoleum lipids were extracted with methyl ethyl ketone. After hydrolysis, the resulting D-mannose was converted to a reduction ratio. The production amount of glycolipids was calculated by measuring by color quantitative method.

The results are shown in the table below. Reference Example 2 The antibacterial properties of the glycolipids obtained in Example 1 were evaluated by Kato et al.
3, 793 (1975)) at 30°C.

The results are shown in the table below. MC: Minimum inhibitory concentration

[Brief explanation of drawings]

Figure 1 shows the infrared absorption spectrum of the glycolipid of the present invention.
FIG. 2 shows its 13C NMR spectrum, and FIG. 3 shows its mass spectrometry spectrum. Figure 1 Figure 2 Figure 3

Claims (1)

[Claims] 1. A glycolipid represented by the following formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, RCO is an aliphatic acyl group with 7 to 20 carbon atoms, and is ester bonded to any hydroxyl group on mannose among disaccharides) 2 Formula (I ), RCO has 7 or more carbon atoms
The glycolipid according to claim 1, which is a mixture of 18 linear saturated aliphatic acyl groups. 3. The glycolipid according to claim 2, wherein in formula (I), a linear saturated aliphatic acyl group having 8 to 14 carbon atoms is contained in an amount of 50 mol% or more in the acyl group. 4. The glycolipid according to claim 1, wherein in formula (I), RCO is a mixture of linear saturated and unsaturated aliphatic acyl groups having 8 to 14 carbon atoms. 5 sp. belonging to the genus Candida. Mutant strain b- of S-10
A method for producing a glycolipid represented by the following formula (I), which comprises culturing 1 and collecting the glycolipid from the culture. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, RCO is an aliphatic acyl group with 7 to 20 carbon atoms, and is ester bonded to any hydroxyl group on mannose among disaccharides) 6 As a carbon source The number of carbon atoms in the medium is 10 or more
Claim 5 adding 24 straight chain aliphatic hydrocarbons
Method for producing glycolipids as described in section. 7. The method for producing glycolipids according to claim 6, wherein the aliphatic hydrocarbon is a saturated straight-chain aliphatic hydrocarbon having 10 to 20 carbon atoms. 8. The method for producing glycolipids according to claim 6, wherein the aliphatic hydrocarbon is an α-olefin having 10 to 20 carbon atoms. 9. The method for producing glycolipids according to claim 5, wherein a triglyceride comprising a linear aliphatic component having 10 to 20 carbon atoms is added to the medium as a carbon source.