JPS62175189A - Production of oligosaccharide ester of fatty acid by fermentation method - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as a surfactant, immunotherapeutic agent for cancer, etc., by a fermentation method, by cultivating a specific actinomyces belonging to the genus Micropolyspora in a nutrient culture medium. CONSTITUTION:An actinomyces, e.g. Micropolyspora sp. NS-1085 strain (FERM-P No.8508), belonging to the genus Micropolyspora and having the ability to produce an oligosaccharide ester of a fatty acid is cultivated in a nutrient culture medium, preferably at 28-32 deg.C and about neutral pH for 30-40 days to afford the aimed compound.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing oligosaccharide fatty acid esters by fermentation. The oligosaccharide fatty acid ester obtained by the present invention is expected to have physiological effects such as use as a surfactant and as an immunotherapeutic agent for cancer.

[Conventional technology]

Pseudomonas aeruginosa (P. aeruginosa) is a glycolipid biosurfactant produced by microorganisms.
ramtulipid CF produced by S. inosa),
G. Jarvis et al. + Journal
of American Chemical 5o
sophorolipids CF, J, T produced by Torulopsis bombicola (T, bombicola), etc.

5pencer et al, Canadian
Journal of Chemi-stry+competition,
846 (1961)) and the like are known. Additionally, microorganisms belonging to the genus Nocardia and Mycobacterium have been found to produce fructose and sucrose fatty acid esters (
(Special Publication No. 54-14666).

[Problem that the invention seeks to solve]

However, the yield of glycolipids produced by microorganisms is generally low, and their recovery and purification are difficult.

[Means for solving problems]

As a result of intensive research in order to solve the above-mentioned conventional problems, the present inventors have found that by using specific bacterial cells, it is possible to precipitate crystals of oligosaccharide fatty acid esters in the medium. We have discovered that oligosaccharide fatty acid esters can be recovered in a simple manner, and have completed the present invention.

That is, the present invention involves culturing actinomycetes belonging to the genus Micropolisvora and having the ability to produce oligosaccharides and fatty acid esters in a nutrient medium, producing oligosaccharide fatty acid esters in the bacterial cells or the medium, and collecting the actinomycetes. The present invention provides a method for producing oligosaccharide fatty acid esters by a fermentation method, which is characterized by:

Unlike conventional substances, the oligosaccharide fatty acid ester of the present invention has a lipid bonded to the oligosaccharide, and is expected to have new performance.

The actinomycetes belonging to the genus Micropolisbora and having the ability to produce oligosaccharide fatty acid esters used in the present invention include Micropolysvora sp.
ora Sp,) NS-1085.

The mycological properties are shown below.

(1) Morphology This strain is a Durham-positive, aerobic actinomycete that grows well in various organic and inorganic media. Aerial hyphae and basal hyphae are relatively long with a width of 0.4 to 0.6 trm, and form spore chains on both threads.

That is, on the aerial hyphae, 5 to 15 spore chains are formed in a looped or loose spiral shape on a sporophyte branched into papillae.

In the subsoil hyphae, several to several dozen approximately spherical spores form chains or sometimes clusters. Spores on aerial hyphae are oblong in shape, 0.4-0.9 X O, 8-1.5
At ρ, the surface exhibits a phase-like shape. Spores on substrate hyphae are 0.4
~0.13 X O, 6X 1.01 degrees, almost spherical, and the surface is not clear because it is a sticky substance. Both of the above spores are not motile. In addition, special forms such as ascospores, whorled filaments, and sclerotia are not observed.

(2) Growth conditions on various media The properties of various agar media are as shown below. Unless otherwise specified, the cells were cultured at 28°C for 21 days and observed according to conventional methods. For description of color tone, please refer to the color label.
48 (Japan Color Research Institute).

In addition, the symbols in the table mean G: degree of growth, A: attachment of aerial mycelium and its hue, R: hue of the back surface, and S: soluble pigment.

(3) Physiological properties Growth temperature range: 15-37°C Optimum growth temperature: 28°C Liquefied gelatin Coagulation of skim milk Positive Nitrate reducing action of skim milk Positive
Hydrolysis of negative starch The growth temperature for the production of negative melanin-like pigments is at various temperatures (5, 10, 1, 5, 20, 25, 2
8,30°33.37,40,45,50°C) 7~
Observation results up to the 21st day. The action against milkum is 37
Observation results from 3 to 21 days at °C. Unless otherwise specified, the results are shown after 2 weeks at 28°C.

(4) Assimilation of carbon sources (Pridham-Godelive agar medium, cultured at 28°C) L-arabinose + D-xylose + D-glucose + D-fructose + sucrose + inositol + rough-1-nose ± D-mannitol ＋ Starch ＋ Note) ＋; Grows using starch.

±: Usability is questionable.

(5) Bacterial cell components The method of Becker et al.
B etal, 8pp1. Microbio
l,,↓yo2. 421-423 (1964); 8pp
1. Microbiol, 13. 236-
243 (1965)) and the method of Rushharie et al.
Lechevalier, Mpet al, + p.
227-238 in Dietz, A et al.
,,ed.

Actinomycete Taxonomy, SI
M 5special public-cation No.
, 6 (1980)), bacterial cell components were analyzed. The results are as follows.

■ Cell wall type ■ Total cell sugar component ■ Mycolic acid was not detected. Based on these facts, the cell wall type of this strain was determined to be type ■.

The characteristics of the strain of NS-1085 mentioned above are described in Bergey's Manual of Determination.
(minative Bacteriology) 8th edition,
1974 and other documents. This strain is: (1) A Durham-positive aerobic actinomycete that forms a relatively small number of spore chains on aerial and basal hyphae.

(2) Aerial hyphae and real hyphae hardly break.

(3) The cell wall type is ■-type, which characteristically contains galactose and arabinose as whole-cell sugars.

Moreover, nocardomicolic acid was not detected.

Based on these properties and the other properties mentioned above, this strain was determined to belong to the genus Micropolyspora, and was classified as Micropolyspora s.
p, NS-1085 was designated as strain. This strain was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on November 2, 1985.
It has been deposited as FERM P-8508.

In general, Micropolyspora bacteria are easily variable in their properties and can mutate naturally or by mutagens. For example x'+? , many mutant strains obtained by irradiation with radiation such as gamma rays and ultraviolet rays, isolation of spores, artificial mutation in media containing various drugs, or naturally obtained mutant strains. , which are not substantially different from each other in comparison with the mycological properties described above or the mycological properties shown below, and which also have the property of producing the oligosaccharide fatty acid ester, can be used in the method of the present invention. It can be used.

The medium used for culturing the substance-producing fungi can be either liquid or solid as long as it contains a nutrient source that the wood fungi can use, but it is more appropriate to use a liquid medium for large-scale processing. be. The medium contains a carbon source that can be assimilated by the substance-producing bacteria, a nitrogen source that can be digested, inorganic substances, and micronutrients.

Examples of carbon sources include glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol,
Sorbitol, fatty acids, fats and oils, n-paraffin, etc. Nitrogen sources include meat extract, yeast extract, dried yeast, soy flour, corn steep liquor, peptone, cottonseed meal, urea, ammonium salts (e.g. ammonium sulfate,
Ammonium chloride, ammonium nitrate, ammonium acetate), and others are used. Furthermore, salts containing sodium, potassium, calcium, magnesium, etc., metal salts such as iron, manganese, zinc, cobalt, nickel, etc., salts such as phosphoric acid, boric acid, and salts of organic acids such as acetic acid, propionic acid, etc. may be used as appropriate. It will be done. In addition, amino acids (e.g. glutamic acid, aspartic acid, alanine, lysine, methionine, proline, etc.), peptides (e.g. dipeptides, tripeptides, etc.), vitamins (e.g. vitamin Bl+ 821 B1210, nicotinic acid, etc.),
Nucleic acids (eg, purines, pyrimidines, other derivatives, etc.) may also be included. In addition, acids, alkalis, buffers, etc. may be added to adjust the pH of the medium, or appropriate amounts of oils and fats, surfactants, etc. may be added to prevent foaming.

The culture may be carried out by static culture, shaking culture, or aerated agitation culture, but it is preferable to use liquid static culture to produce the substance.

Cultivation conditions vary depending on the state and composition of the medium, the type of strain, and the means of cultivation, but it is usually 25 to 38°C, preferably 28 to 32°C, and the initial ρII should be selected to be near neutral. The culture period also varies depending on the conditions mentioned above, but 20
~50 days, preferably 30-40 days.

To collect the substances produced in the culture medium, conventional methods for isolation from microbial cultures are applied. Since the target substance is mainly contained in the bacterial cells and the medium, the target substance is extracted after filtering the culture solution. That is, general methods that utilize differences in solubility and solubility in appropriate solvents, differences in precipitation properties and precipitation rates from solutions, differences in adsorption affinity for various adsorbents, and differences in distribution between two liquid phases, etc. separated, collected, and purified by means used in These methods may be used alone, or may be combined or repeated in any order as necessary.

The substance thus obtained was found to be an oligosaccharide fatty acid ester from the results of thin layer chromatography, infrared absorption spectroscopy, gas chromatography, nuclear magnetic resonance absorption spectroscopy, and mass spectrometry.

Example 1 Micropolyspora sp.
-ora sp,) Using the strain NS-108!5, sucrose 30g/IV, Nisshin 0r) O-P (Nissin Oil ■
) 30g/β, polybentone 2g/j! , and was statically cultured at 28° C. for 35 days in a culture medium having a composition of 0.5 g of sodium chloride/β. At the end of the culture, 1.5 l of the culture solution was filtered through a filter paper, and the produced substance was extracted from the obtained solid fraction using 450 ml of methanol at room temperature. Furthermore, n-hexane 15
After removing the oil phase component using 0 ml, the solvent was distilled off using a rotary evaporator to obtain 22.5 g of product material.

The obtained substance was a white crystal, and was purified by silica gel thin layer chromatography using Hensen; ethyl ether: methanol (80 N+10)/Soybean liquid. f-0,
15 was shown. In addition, it was soluble in various solvents such as methanol, ethanol, chloroporum, and terahydrofuran at room temperature, but was insoluble in water.

The infrared absorption spectrum of this substance is not shown in Figure 2.

This substance reacts with Anthrone reagent to give a bluish-green color. When the aqueous layer after saponification with IN caustic soda was examined by silica gel thin layer chromatography, it was found that it was more lactic than sucrose. ,
Because spots are detected in areas with low f-numbers,
It was proven that it is an oligosaccharide fatty acid ester. The fact that the bond between the fatty acid and oligosaccharide of this substance is an ester bond means that the infrared absorption spectrum of this substance is 1710 cm.
This is confirmed by the absorption at -' and the fact that this substance easily liberates fatty acids in alkali.

Example 2 Culture was carried out in the same manner as in Example 1, except that Nissin 000-P in the medium of Example 1 was replaced with oleic acid, and 50 g/l of starch was used.

Culture solution 1 and 2 at the end of culture! was extracted with a chloroform:methyl alcohol (1:1) solvent, and the solvent was distilled off using a rotary evaporator. Obtained ') Matsuma 13.2
g to chloroporum 50ml? After dissolving and adsorbing on a silica gel column, the mixture was recovered with chloroform:methyl alcohol (7:3) to obtain 8 g of the desired oligosaccharide fatty acid ester 1].

Example 3 Olive oil 10g/β, soluble starch 10g/β, polypeptone 2g/β, salt 0.5g#! , Agar 10g/7!
The cells were cultured in the same manner as in Example 1, except that they were cultured in a culture medium having the following composition.

At the end of the culture, 1.51 liters of the culture solution was centrifuged, and 66.
5g (wet weight) was obtained. Next, the bacterial cells were washed with ethanol and the produced substances were collected. The ethanol solution was concentrated, water was added to form crystals, and the crystals were collected by filtration to obtain 14.5 g of the target oligosaccharide fatty acid ester. .

[Brief explanation of drawings]

FIG. 1 is an infrared absorption spectrum of the product obtained in Example 1.

Claims (1)

[Claims] Cultivating actinomycetes belonging to the genus Micropolyspora and having the ability to produce oligosaccharide fatty acid esters in a nutrient medium, producing oligosaccharide fatty acid esters in the bacterial cells or the medium,
A method for producing oligosaccharide fatty acid ester by a fermentation method, which is characterized by collecting this.