JPH06116583A - Aroma substance and its production method - Google Patents

Abstract

(57) [Summary] [Structure] By culturing a Chalara genus microorganism or its teleomorph, and collecting aroma substances from the culture solution,
Produces aroma substances. [Effect] It is possible to produce a fruity aroma substance by using a microorganism of the genus Chalara or its teleomorph, which has hitherto not been recognized as being useful in the perfume industry.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an aromatic substance and a method for producing the aromatic substance using microorganisms.

[0002]

2. Description of the Related Art Chalara is a fungus belonging to the group of incomplete filamentous fungi (Hyphomycetes) taxonomically.
The degree of species is known. As a teleomorph of the genus Chalara (sexual age), the ascomycete Ceratocys
The tis genus and the like are known. For example, a kind of Chalara sp Chalara paradoxa (de Seynes) Sacc. ( Hereinafter also referred to as "the bacterium"), the ascomycetes fungus Ceratocystis Parad
oxa (Dade) C. Moreau's anamorph (asexuality), commonly Ananas , Cocos , Elaeis , Ipomoea , Musa ,
It is known that it grows on plants such as Theobroma and is globally distributed.

In Japan, it was reported in 1957 that this bacterium is a plant pathogen causing black rot of sugar cane. Further, it has been found that cellulase produced by Chalara paradoxa can be used for degrading and saccharifying sago palm starch to produce sugar. However, to date, no usefulness has been found in the perfume industry and industrial use, in which the bacterium is used to produce an aroma substance.

[0004]

The present invention invention is to solve the above is, conventional, Chalara para the utility value on the perfume industry has not been recognized
By utilizing Chalara microorganisms or teleomorph such doxa, and to provide a fruity aroma substances.

[0005]

The present inventors Means for Solving the Problems] are the several and cells in culture studies of their teleomorph cognate heterologous including Chalara paradoxa isolated from Okinawa sugar cane, Chalara paradoxa, Chalara thielavioides and Cer
It was discovered that atocystis paradoxa metabolizes and produces a fragrant aroma (fruity aroma substance) in the medium during culture, and as a result of diligent research, it efficiently produced a large amount of this aroma substance without impairing the aroma substance as it is. It succeeded in collecting and came to complete the present invention.

Namely, the present invention provides aroma substance characterized by belonging to Ceratocystis sp is Chalara genus or a teleomorph, culturing a microorganism having an ability to produce aroma substances, and collecting the aroma substance from its culture broth Production method of
In addition, it is an aroma substance having a fruity aroma, which contains ethanol, phenethyl alcohol, ethyl acetate, isobutyl acetate and isoamyl acetate as main components.

The microorganisms used in the present invention include Chalara
There is no particular limitation as long as it is a microorganism belonging to the genus or the genus Ceratocystis and having an ability to produce an aroma substance, but is preferably
Chalara paradoxa, Chalara thielavioides, Ceratocys
tis paradoxa . Of these, Chalara parado
The xa, for example include Chalara paradoxa 6957 strain, the strain is Agency Fermentation Research has been deposited as Biko Labs bacterium preferred No. 13049 to the laboratory, having bacteriological properties described below. (1) Colony On malt agar medium, the diameter reached about 80 mm in 3 days at room temperature, the aerial mycelium did not develop so much, and initially became white and then black. (2) Conidia stalk bundle Headed, slightly wider toward the top, with conidia mass at the tip and often formed on natural substrates. (3) Conidia peduncle Cylindrical, having 0 to several partition walls, colorless or light brown, and having phialide at the tip. (4) Conidia-forming cells Long-cylindrical or short-cylindrical cells, slightly tapered, with clear or unclear color (fialide), colorless, 10-120 x 4-12 µm. (5) Conidia Single cell, smooth, initially cylindrical / colorless / thin film, gradually barrel-shaped (Fiaro type) to subspherical / dark brown / thick film, often with light-colored germination slits, 3 to 25 × 2 to 10 μm, chain-like or lumpy. (6) Chlamydia spores usually horoblastic / singly apical or symptomatically proliferating / elliptical to subspherical, rarely interstitial / oblong cylindrical to irregularly shaped / single cell, unicellular, smooth, dark brown, Thick film. In addition to the above chlamydospores, dark brown, thick-film phialoid conidia may be included in the chlamydospores. (7) ubiquinone system Q-9 (H ₂₎ (8) Urease + (9) DNase - As the Chalara thielavioides, for example Chalara
Thielavioides strain 1933, which has been deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Microindustrial Research Institute No. 13050, has the following mycological properties. (1) Colony On a potato dextrose agar medium, the diameter reached about 40 mm at 30 ° C for 5 days, and the aerial mycelium did not develop so much, and initially became greenish white, then gray green to dark green brown. (2) Conidia stalk bundles are not formed. (3) Conidia peduncle It is laterally formed from grape hyphae, has septa, is colorless or light brown, and often branches at the tip, forming a phialide at each tip. (4) Conidia-forming cells Long cylindrical shape, slightly tapered, with clear color, colorless, 30 (fialide) ~ 70 x 5-10 µm. (5) Conidia Single cell, smooth, cylindrical, colorless, thin film, chained, 4 to 40 × (fiaro type) 2.5 to 3 μm. (6) Chlamydia spores usually horoblastic ・ Apical or symposium singularly mesogenic ・ Pear-shaped, flat-bottomed pear-shaped, thick film ・ 8 to 20 × 7.5 to 15 μm, rarely between mycelium -Spherical to subspherical-thin film-Diameter 12 to 45 μm, single cell, smooth, colorless to dark greenish brown.

Further, since the ascomycete Ceratocystis paradoxa also produces an aroma substance in the same manner as the above-mentioned microorganism, the aroma substance can also be produced using the microorganism. Ceratocystis
Examples of paradoxa include Ceratocystis paradoxa 696
1 strain, which has been deposited at the Institute of Microbial Science and Technology, Institute of Industrial Science and Technology, as Micro-industrial Research Institute No. 13051, and has the potential for producing ascospores and ascospores on natural substrates. Except for the formation of reproductive organs, the mycological properties on medium are Chalara.
Similar to the paradoxa 6957 strain.

Culturing of the above-mentioned microorganism can be carried out, for example, as follows. From the slant medium
M medium is inoculated to form a seed, and this seed is subjected to large-scale culture in a jar fermenter. The seed medium includes YM medium and potato sucrose (P
S) medium, potato dextrose (PD) medium, and the like commonly used medium may be used.

When the seed amount is inoculated at a ratio of 100 ml with respect to 20 L of a medium suitable for large-scale culture of jar fermenters, aroma substances are rapidly produced. Suitable mediums for large-scale culture include YM medium, which is generally used for yeast culture, and various mediums as well as seed medium, but Chalara, which has a high saccharide assimilation ability.
In the paradoxa 6957 strain, the composition of sugars added to the medium has a large effect on large-scale culture.

Sugars that can be used for large-scale culture include glucose, fructose, sucrose, rhamnose,
Fucose, arabinose, xylose, mannose, galactose, ribose, etc., but Chalara paradoxa 6
Among them, glucose, fructose, sucrose, xylose, and galactose are preferably used in order to obtain a large amount of fruity aroma substances using the strain 957.

[0012] In general, the amount of the medium of saccharides case of a microorganism is of the order of 0.1 ~0.7% (W / W) , C
In order to obtain aroma substances with the halara paradoxa 6957 strain, 1 to 15% (W / W) of the sugars preferable for the above-mentioned large-scale culture is used.
When it is added to the medium, the yield of the cells of this bacterium and the production of good fruity aroma substances are increased. Preferably, if these sugars (glucose, fructose, saccharose, xylose, galactose) are used alone or in combination, the amount of the compound in the medium is set to 3 to 7% (W / W), and large-scale culture is performed. The most efficient and good aroma substance can be obtained in a short period of time.

The production amount can be increased by further adding an amino acid. As for the types of amino acids,
Examples thereof include tryptophan, leucine, lysine, valine, threonine, phenylalanine, methionine, isoleucine, asparagine and glutamine, and lysine and asparagine are particularly preferable. The content of amino acid in the medium is preferably 0 to 1.5% (W / W).

Cultivation containing a good aroma component becomes possible 36 to 52 hours after seed inoculation, so it is preferable to finish the culture in 40 to 44 hours and collect the aroma substances. As a method for collecting aroma substances from the culture solution, extraction is preferable in order to obtain good aroma substances, but other methods, for example, low temperature decompression as long as it is a method capable of preventing changes due to heating of aroma substances It may be carried out by distillation.

The extraction is either gas / liquid extraction or liquid / liquid extraction.
However, alcohols such as ethanol and other
Extraction with organic solvents such as tons and ethers is preferred
Yes. After extraction with these organic solvents, the concentration was increased to 5 to 10 times.
Compressed extract or extract with solvent completely removed
The aroma during feeding is reproduced very well. Besides this, super
Extraction using a critical gas or the like is also possible. For this extraction,
Carbonic acid (CO ₂) Gas, nitrogen gas, etc. can be used
But CO₂Gas is preferred. CO₂When using gas,
CO₂The critical point temperature of 20 ~ 50 ℃, preferably 35 ~ 45 ℃
And pressure 100-250kg / cm², Preferably 120-220kg /
cm²Done in.

The aroma substance thus obtained has a fruity aroma such as banana-like, melon-like, pineapple-like, apple-like, etc., and its composition is ethanol, phenethyl alcohol, ethyl acetate, isobutyl acetate, isoamyl acetate. Etc. are the main components. The content of the above main components is
Ethanol 3-10%, phenethyl alcohol 1-10%,
40-80% ethyl acetate, 1-5% isobutyl acetate, 0.9-5% isoamyl acetate, and some other unidentified substances.

[0017]

EXAMPLES The present invention will be described in more detail with reference to examples below, but these examples do not limit the scope of the present invention. And (Example 1) (1) Chalara paradoxa 6957 strain 2L Sakaguchi flask (medium 500 ml), 67 hours with shaking cultured at 25 ° C. (2% seed).

The following YM medium was used as the medium. YM medium anhydrous crystalline glucose (0.5-15% as shown in Table 1),
Polypeptone (Nippon Pharmaceutical) 0.5%, yeast extract (DIFC
O) 0.3%, malt extract (Far East Pharmaceuticals) 0.3%, 120 ° C 2
Sterilization for 0 minutes (2) The culture solution was centrifuged to remove bacterial cells, and the supernatant was extracted twice with the same amount of ether. Anhydrous magnesium sulfate was added to dehydrate and then concentrated. The volatile components of the concentrate were identified by gas chromatography and gas chromatography / mass spectrometry.

The analysis conditions of gas chromatography are as follows. Column: PEG-20M Bonded df = 0.3 μm 0.25 mm I.D. × 25 m Column temperature: 55 ° C. (5 minutes) to 210 ° C., 5 ° C./minute Flow rate: He 1.0 ml / minute The results are shown in Table 1.

[0020]

[Table 1]

Further, a sensory evaluation test for the extracted substances according to the amount of saccharides in the above-mentioned medium was carried out by using 5 panelists. The results are shown in Table 2. In Table 2, ⊚ is “very good”, ◯ is “good”, Δ is “neither”, and x is “bad”.
Represents

[0022]

[Table 2]

From Table 2, it can be seen from the sensory evaluation that the content of saccharides in the medium is preferably 2 to 8%. Next, among the YM media used in (1), a YM medium containing 3% anhydrous crystalline glucose was used, and a sensory evaluation test was conducted by 5 panelists while changing the culture time. The results are shown in Table 3. In Table 3, ⊚ represents “very good”, ◯ represents “good”, Δ represents “neither”, and x represents “bad”.

[0024]

[Table 3]

From Table 3, the sensory evaluation revealed that the culture time was 36 to 52.
It can be seen that time is preferred and 40 to 48 hours is more preferred. And (Example 2) (1) Chalara paradoxa 6957 strain 2L Sakaguchi flask (medium 500 ml), 67 hours with shaking cultured at 25 ° C. (2% seed).

As the medium, YM medium and Ap shown below are used.
ple medium was used. YM medium Anhydrous crystalline glucose 3%, Polypeptone (Nippon Pharmaceutical) 0.
5%, yeast extract (DIFCO) 0.3%, malt extract (Kyokuto Pharmaceutical Co., Ltd.) 0.3%, 120 ° C 20 minutes sterilized Apple medium concentrated apple juice (1/5) 10%, yeast extract (DIFCO) 0.
Sterilization at 3%, 100 ° C for 15 minutes (2) The culture solution was centrifuged to remove bacterial cells, and the supernatant was extracted twice with the same amount of ether. Anhydrous magnesium sulfate was added to dehydrate and then concentrated. The volatile components of the concentrate were identified by gas chromatography and gas chromatography / mass spectrometry.

The analysis conditions of gas chromatography are as follows. Column: PEG-20M Bonded df = 0.3 μm 0.25 mm I.D. × 25 m Column temperature: 55 ° C (5 min) to 210 ° C, 5 ° C / min Flow rate: He 1.0 ml / min (3) Extraction with YM medium The product had a pineapple-like fruity aroma, and the extract in the case of Apple medium had an apple-like fruity aroma. The results of each analysis are shown in Tables 4 and 5. These aromas were not due to the specific components, but due to the balance of ester, alcohol and acid.

[0028]

[Table 4]

[0029]

[Table 5]

(Example 3) (1) Recovery by distillation a) Normal pressure distillation About 20% of the culture solution of YM medium (anhydrous crystalline glucose 3%) was taken by normal pressure distillation, and this was redistilled again. Then, the redistant was separated. From the strength and aroma balance, 0.6-0.7 of the culture solution
% The top part was taken.

(B) Low temperature vacuum distillation Distilled at 50 ° C. and 100 mmHg, and 0.6 to 0.7% of the culture solution was trapped at −60 ° C. The aroma of this product was fruitier than that of a) and more closely reproduced the aroma of the culture solution. The fruity aroma produced by the main culture is considered to be changed by heating. (2) Recovery by extraction (a) Since the product obtained by culturing is water-soluble, it was extracted with an oily flavor to make it an oil-soluble flavor.

A) A culture solution of YM medium (anhydrous crystalline glucose 3%) was extracted with an oily flavor and concentrated to a volume equivalent to 10 times the culture fluid. B) An atmospheric distillation product (about 150 times) was extracted with an oily flavor. (1 to 1) The same fruity aroma was reproduced in both a) and b). Although triethyl citrate was used as the oily flavor here, a melon-like blended flavor can be used if the flavor is melon-like, and a pineapple-like blended flavor can be used if the flavor is pineapple-like.

(B) YM medium (anhydrous crystalline glucose 3%)
When the culture solution was extracted with CO ₂ gas as a supercritical gas at a temperature of 40 ° C. and a pressure of 200 kg / cm ² , a fruity and sweet aroma was reproduced.

[0034]

EFFECTS OF THE INVENTION According to the present invention, it is possible to produce a fruity aroma substance by using a microorganism of the genus Chalara or its teleomorph, which has not been recognized as a valuable value in the perfume industry. The industrial value of inventions is great.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display location // (C12S 13/00 C12R 1: 645) (72) Inventor Mitsuo Tsunakawa 392 Tsukiji Tsukiji, Fujieda City, Shizuoka Prefecture Tsumura Co., Ltd. (72) Inventor Yoshimi Kawasaki 392 Tsukiji, Fujieda City, Shizuoka Prefecture Tsumura Co., Ltd.

Claims (6)

[Claims] 1. A culture of a microorganism belonging to the genus Chalara or the genus Ceratocystis and capable of producing an aroma substance,
A method for producing an aroma substance, which comprises collecting an aroma substance from the culture solution. 2. A microorganism belonging to the genus Chalara or the genus Ceratocystis and having the ability to produce aroma substances is Chalara.
paradoxa, Chalara thielavioides or Ceratocystis p
The production method according to claim 1, which is aradoxa . 3. The production method according to claim 1, wherein the aroma substance is collected by extracting the culture solution. 4. The production method according to claim 1, wherein the culturing is carried out in a medium having a sugar content of 1 to 15% (W / W). 5. An aroma substance having a fruity aroma, which contains ethanol, phenethyl alcohol, ethyl acetate, isobutyl acetate and isoamyl acetate as main components. 6. The content of the main component is ethanol 3 to 10%,
Phenethyl alcohol 1-10%, ethyl acetate 40-80%,
The fragrance substance according to claim 5, which comprises 1 to 5% isobutyl acetate and 0.9 to 5% isoamyl acetate.