JPH06303967A - New microorganism and preparation of nootkatone using the microorganism - Google Patents

Abstract

PURPOSE:To provide a new microbial strain belonging to the genus Rhodococcus, capable of oxidizing allyl group of valencene to form nootkatone, free from pathogenicity and giving a natural flavor substance having characteristic flavor of grapefruit. CONSTITUTION:A spoonful soil collected in Tokushima Prefecture is suspended in 10ml of disinfected and deionized water and properly diluted. The suspension is put into a test tube and cultured by shaking culture at 30 deg.C for 5 days. The cultured product is smeared on a plate medium incorporated with 5% agar and a strain having valencene-assimilating capability is separated. The valencene-assimilating strain is inoculated to a medium in a test tube and cultured at 30 deg.C for 5 days by shaking culture. The cultured product is extracted with ethyl acetate, the ethyl acetate-soluble fraction is subjected to gas chromatography and a fraction confirmed to assimilate valencene and form nootkatone of formula is separated to obtain the new microorganism Rhodococcus sp. KSM-5706 (FERM P-13450) capable of oxidizing the allyl group of valencene and forming nootkatone.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel process for producing Nootkatone, and more particularly to Rhodococcu.
s) A method for producing Nootkatone using a microorganism belonging to the genus.

[0002]

2. Description of the Related Art The following structural formula (I)

[Chemical 1] The nootkatone represented by, for example, is a sesquiterpene ketone that is present in the grapefruit peel, and is a natural fragrance substance in an optically active form (+ form) that has received a great deal of attention because it has the characteristic flavor of grapefruit.

This Noot Katong has a very strong scent and p
Although it can be detected in the order of pb, it is an expensive fragrance that is naturally difficult to obtain because it contains only 1% or less in the grapefruit oil component, and its quantity is limited. Therefore, there is a strong demand for development of a method for economically synthesizing Nootkatone, and various synthetic methods have been conventionally studied.

For example, 4-acetyl-1-ethoxycyclohexene (Che
m. Commun. 1152 tribute, 1968) 4,4-diethoxycarbonylpimelic acid dinitrile (Chem. Commun. 26 tribute, 19
1969), dimethyl 4-oxopimelate (J. Org. Che
m. 36, 594, 1971) and the like are known. However, the total synthesis of Nootkatone has complicated reaction steps, multisteps, and low yields, and although it is of academic interest, it cannot be said to be industrial.

Further, the following structural formula (II) obtained from orange oil or the like

[Chemical 2] A method of synthesizing Nootkatone by oxidation of valencene represented by For example, a method of oxidization using triplet oxygen or singlet oxygen, dehydration of an oxidation product (hydroxyperoxide), and synthesis of nootkatone (Japanese Patent Publication No. Sho 49-35263) and tertiary butyl chromate (J .Food.Sci.30, Item 876, 1965), Chromic anhydride pyridine complex (J.Org.Chem.34, 3587, 1969)
And the like, or a method of using an organic heavy metal catalyst to oxidize with oxygen or an oxygen-containing gas (Japanese Patent Publication No. 59-31728).

However, in these synthetic methods, the intermediate oxidation product (hydroxyperoxide) is unstable, the oxidizing agent itself is expensive, various by-products are produced, and the yield is poor. Therefore, it cannot be said that it is suitable for industrialization.

On the other hand, it is known that when a biocatalyst such as an enzyme or a microbial cell is used for production of a substance, the desired compound can be selectively obtained without causing side reactions as in the chemical synthesis method. ing. Especially when the target compound is an optically active substance like Nootkatone (it is said that the (+) form has an aroma 1000 times higher than that of the (-) form), the production by biocatalysis is a very advantageous method. Can be said.

[0008] In addition, flavors prepared by fermentation with an enzyme or by microorganisms are classified as natural flavors, and there is a great demand from the viewpoint of natural products in recent years.

As an example of the method for converting valencene into nootkatone using such microorganisms or plants,
A method using Enterobacter, which is an intestinal bacterium (Dragoco Rep. Volume 20, 251 tribute, 1974),
Method using citrus (Citrus) (Plant Cell Rep.
3 volumes, 37 tributes, 1984) have been reported.

However, none of these methods is industrial because the initial concentration of valencene is low. Also,
The method using enterobacteria such as Enterobacter has a drawback in that it may cause pathological concerns and may hinder its use as a flavor.

[0011]

Therefore, the development of a method which is capable of selectively converting valencene to nootkatone using a biocatalyst and has no problem when using the obtained nootkatone as a flavor. Was required.

[0012]

[Means for Solving the Problems] As a result of searching the properties and actions of various microorganisms isolated from nature, the present inventors have found that microorganisms belonging to the genus Rhodococcus obtained from soil collected in Tokushima Prefecture. Found the conversion of Valensen to Nootkatone and completed the present invention.

That is, the present invention relates to Rhodococcus (Rhodoc).
The present invention provides a method for producing Nootkatone characterized by allowing a microorganism belonging to the genus occus) to act on valencene.

The microorganism of the genus Rhodococcus of the present invention has the ability to oxidize valene at the allylic position, and its representative microorganism, Rhodococcus sp.
p.) KSM-5706 strain has the following mycological properties.

Mycological properties: (1) Morphological properties Bacteria having a size of 0.8 to 1 μm × 1 to 10 μm and polymorphism. That is, hyphae are formed at the initial stage of culture, and thereafter, the hyphae are ruptured to become short rod-shaped. The strain is non-motile and has no flagella. No spores were found, gram positive and no acid resistance.

(2) Culture properties (a) Flat culture of broth agar The plant grows well and forms a milky white opaque, smooth and glossy surface, and a conical ridge-like colony. The shape of the settlement is circular. (B) Meat broth agar slope culture It grows well and exhibits milky white color. (C) Meat juice liquid culture Both upper and lower layers of the surface grow. (D) Meat broth gelatin stab culture It grew well and liquefied gelatin. (E) Litmus milk Change the litmus dye from purple to blue. No change in milk was observed.

(3) Physiological properties (a) Reduction of nitrate; Positive (b) Denitrification reaction; Negative (c) MR test; Negative (d) VP test; Negative (e) Indole formation; Negative (f) Generation of hydrogen sulfide; Negative (g) Starch hydrolysis reaction; Positive (h) Action of citric acid; Simmons medium; Positive Christensen medium; Positive

(I) Utilization of nitrate; Positive (j) Utilization of ammonium salt; Positive (k) Dye formation; King A medium; Negative King B medium; Negative (l) Urease; Positive (m) Oxidase; Positive ( n) catalase; positive

(O) Growth pH range; Growth pH 4.5-10 Optimum pH 5-7 (p) Growth temperature range; Growth temperature 6-37 ° C. Optimum temperature 20-30 ° C. (q) Attitude toward enzymes; It is aerobic, but it can grow well even under static conditions. (R) OF test; no reaction (no reaction with BTB indicator) (s) Growth in medium containing NaCl;
It grows in both% and 7%.

(T) Availability of carbon source; L-arabinose-D-xylose-D-glucose + D-mannose-D-fructose + D-galactose-maltose + (weak) sucrose +

Lactose-trehalose + D-sorbitol + D-mannitol + inositol + glycerol + starch + (weak) D-ribose + However, + is used but-is not used.

(4) Chemotaxonomic properties (a) Glycolate test (glycolyl type) (b) Cell wall cross-linking amino acid meso (meso) -2,6-diaminopimelic acid (c) Arabinose and galactose are detected, but xylose is detected. Is not detected. (D) menaquinone system MK-8 (H ₂₎

The above-mentioned mycological properties can be obtained by using the Vergi's Manual of Systematic Bacteriology (Be
rgey's Manual of Systematic Bacteriology), Volume 2 (1986), the taxonomic position of this strain was examined, and it was found that KSM-5706 was a microorganism belonging to the genus Rhodococcus.

Since the above-mentioned mycological properties of the KSM-5706 strain are different from any of the known microorganisms of the genus Rhodococcus, the present inventors determined that this was a novel strain, and determined that the strain was a novel strain. It was deposited in the laboratory (accession number FERM P-13450).

The Rhodococcus genus microorganism of the present invention obtained as described above has the ability to specifically oxidize the allylic position of valencene and convert it to nootkatone. Therefore, it is advantageous to allow the microorganism to act on valencene. You can get the Noot Katong.

In the present invention, there is no particular limitation on the method of action of the microorganism of the genus Rhodococcus on valencene, a method of culturing the microorganism in a medium containing valencene, a method of adding valencene to cultured cells of the microorganism, Nootkatone can be obtained by contacting valence cells with resting cells, immobilized cells, crushed cells, etc. of the microorganism.

The medium used for culturing the microorganism of the present invention includes fructose, maltose, trehalose, sorbitol, mannitol, glycerol, glucose, sucrose, ribose and inositol as carbon sources, peptone as a nitrogen source, yeast extract, Urea, sodium nitrate, ammonium sulfate, amino acids, etc., inorganic salts such as potassium phosphate, magnesium sulfate, calcium chloride, and, if necessary, Mn ²⁺ , Z
Metal salts such as n ²⁺ and Ni ²⁺ , vitamins such as biotin and thiamine, etc. are appropriately added and used.

The culture conditions are not particularly limited, but generally, it may be carried out at a temperature of about 20 to 30 ° C. and a pH of about 5 to 7 with shaking or aeration stirring.

As a method for using the thus obtained cultured bacterial cells of the microorganism of the present invention as resting bacterial cells, an ordinary method may be used. For example, the cultured bacterial cells may be treated with an appropriate buffer or deionized water. Examples of the method include washing, and specifically, a method in which cells separated from the culture solution by centrifugation or filtration are resuspended in a buffer solution or deionized water.

The microbial cells obtained as described above can be allowed to act on valencene by adding valencene to the culture medium in which the number of cells has been increased, or by resting the microbial cells in an appropriate buffer or deionization. After the suspension in water and the addition of valencene thereto, the conversion reaction may be carried out by shaking stirring or aeration stirring.

The number of cells used in the conversion reaction is not limited, but generally, the concentration of the cell suspension is 600 nm.
It may be adjusted so that the absorbance at about 20-80. The stirring in this reaction is 100 to 4
The reaction may be performed for about 72 to 120 hours at about 00 rpm and the temperature of the suspension at about 20 to 30 ° C.

In order to separate and collect the desired product Nootkatone from the conversion reaction product obtained as described above, known purification means, for example, column chromatography, high performance liquid chromatography and the like, may be used alone or in combination. You can use it.

[0033]

INDUSTRIAL APPLICABILITY According to the present invention, nootkatone can be selectively produced from valencene without causing various side reactions as seen in the chemical synthesis reaction.
In addition, unlike the conversion reaction of enterobacteria into nootkatone that has been reported so far, it is possible to produce nootkaton without concern about pathogenicity.

[0034]

The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 A spoonful (about 0.5 to 1 g) of the soil of Tokushima Prefecture is taken, suspended in 10 ml of sterile deionized water, and then appropriately diluted. This dilution was added to a large-sized test tube containing 10 ml of the medium A described later, and culture was carried out by shaking at 30 ° C. for 5 days to carry out accumulation culture. This culture was applied to a plate medium prepared by adding 5% agar to the medium A to isolate a strain having a valencene modifying ability.

The isolated valencenogenic strain was inoculated again into a large test tube containing 10 ml of the following medium A, and the temperature was 30 ° C.
Shaking culture was carried out for 5 days. The obtained culture broth was extracted with ethyl acetate, and the ethyl acetate-soluble fraction was subjected to gas chromatography to quantify the product (FIG. 1).
As a result, KSM-5706 strain was obtained as a strain that assimilates valencene and produces Nootkatone.

(Cultivation site A) FeCl ₃ 6 mg MgCl ₂ 80 mg CaCl ₂ 75 mg NH ₄ NO ₃ 3.5 mg Na ₂ SO ₄ 0.71 mg thiamine hydrochloride 1 mg biotin 0.01 mg valencene 10 g 50 mM phosphoric acid Bring the total volume to 1 liter with buffer (pH 7).

Example 2 The product obtained in Example 1 was subjected to solvent extraction, followed by analysis and identification, and this was determined to be Nootkatone. Among the analytical data, the GC-MS spectrum is shown in FIG. 2, and the MS spectrum comparison between the product and Nootkatone is shown in FIG.

Example 3 The KSM-5706 strain obtained in Example 1 was cultured at 30 ° C. for 3 days in the agar slant medium of the following medium B. This was inoculated into a large test tube containing 10 ml of the above medium A. Three
Culture was carried out at 0 ° C. for 2 days with shaking, and this was used as a preculture liquid. 0.5 ml of this pre-culture solution was added to 50 ml of the above-mentioned medium A.
A 0 ml Sakaguchi flask was inoculated and shake culture was performed at 30 ° C. for 4 days. The obtained culture solution was extracted with ethyl acetate, and the ethyl acetate-soluble fraction was quantified by gas chromatography. As a result, 0.5 mg of Nootkatone was obtained.

(Cultivation B) Polypeptone (casein peptone) 17 g Polypeptone S (soybean peptone) 3 g K ₂ HPO ₄ 2.5 g Glucose 2.5 g NaCl 5 g Agar 15 g The total volume is 1 liter with purified water.

Example 4 KSM-5706 strain cultured at 30 ° C. for 3 days in the agar slant medium of the above-mentioned medium B was inoculated into a large test tube containing 10 ml of the following medium C. The culture was carried out at 30 ° C. for 2 days with shaking, and this was used as a preculture liquid. This preculture solution (1.0 ml) was inoculated into two 500 ml Sakaguchi flasks each containing 100 ml of the medium C described below, and the mixture was cultured at 30 ° C. for 2 days with shaking.

200 ml of the obtained culture solution was centrifuged (1
2,000 xg for 10 minutes) and the resulting cells are 50m
It was washed twice with M phosphate buffer (pH 7.0) and suspended in 50 ml of the same buffer to give a resting cell suspension. 50 ml of this resting cell suspension was placed in a 500 ml Erlenmeyer flask, 500 mg of valencene was added thereto, and shake culture was carried out at 30 ° C. for 4 days on a rotary shaker. The obtained culture solution was extracted with 50 ml of ethyl acetate, and the ethyl acetate-soluble fraction was quantified by gas chromatography.
2.5 mg of Nootkatone was obtained (Fig. 4).

(Cultivation site C) Polypeptone (casein peptone) 17 g Polypeptone S (soybean peptone) 3 g K ₂ HPO ₄ 2.5 g Glucose 2.5 g NaCl 5 g The total amount is 1 liter with purified water.

[Brief description of drawings]

FIG. 1 is a drawing showing gas chromatography (GC) of the product of Example 1.

2 is a drawing showing a GC-MS spectrum of a fraction containing Nootkatone in the product of Example 1. FIG.

FIG. 3 is a drawing showing a comparison of the MS spectra of the fraction containing Nootkaton in the product of Example 1 and the Nootkaton standard.

FIG. 4 is a drawing showing GC of the product of Example 4. that's all

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Katsuko Oshima 4-10-25-108 Takahama, Mihama-ku, Chiba City, Chiba Prefecture (72) Inventor Susumu Ito 3441-64 Higashimine Town, Utsunomiya City, Tochigi Prefecture

Claims (4)

[Claims] 1. A microorganism belonging to the genus Rhodo-coccus, which has the ability to oxidize allene position of valencene. 2. Rhodococcus sp. KSM-5706 having the ability to oxidize the allylic position of valencene and produce nootkatone. 3. A process for producing Nootkatone characterized in that a microorganism belonging to the genus Rhodococcus is allowed to act on valencene. 4. The method for producing Nootkatone according to claim 3, wherein the microorganism belonging to the genus Rhodococcus is Rhodococcus sp. KSM-5706 strain.