JPH03247291A - Production of methyl ketone and/or alcohol corresponding thereto by fermentation method and use of culture solution - Google Patents

Abstract

PURPOSE:To use a fatty acid, salt or ester thereof as a precursor and obtain the subject compound useful as a flavor for dairy products, soaps, etc., according to a fermentation method by using a specific microorganism belonging to the genus Aureobasidium. CONSTITUTION:A microorganism [e.g. Aureobasidium.sp. SM-25 (FERM P-11277)], belonging to the Aureobasidium and having the ability to convert a fatty acid expressed by formula I (A is aliphatic hydrocarbon capable of containing C=C), an ester or salt thereof into a methyl ketone expressed by formula II and/or a secondary alcohol expressed by formula III is cultured in a culture medium containing the compound expressed by formula I, ester or its salt or a mixture thereof to exracellularly produce and accumulate the objective compounds expressed by formulas II and/or III. The resultant compounds are then collected.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides a method for producing methyl ketone and/or its corresponding secondary alcohol from the corresponding fatty acid, its ester or salt, or its mixture by the action of microorganisms, and Concerning the use of culture fluids containing these substances. Methyl ketone and its corresponding secondary alcohol can be used as a flavor for dairy products, soaps, etc., and as a solvent for fragrances, dyes, etc.

[Problems to be solved by conventional techniques and inventions] Conventionally,
A method using microorganisms belonging to Penicillium roqueforti (f?, C, Law) as a method for producing methyl ketone and/or its corresponding secondary alcohol from fats and oils using microorganisms.
Rence et al., J. Gen.

Microbiol, 51.289-302 (I
968), and R, D. King et al., J. Sc.
i, Food Agric, 30. 197
-202 (I979)), a method using microorganisms belonging to Penicillium paritans (P,
W, N, 5tokoe, Biochem, J.

22, 8O-93 (I928) ), to=silium・
A method using microorganisms belonging to caseicolumn (P, caseicolumn) (T, caseicolumn).
et al., Rev, La1tiere Fr, 406 +1
34 (I980)), Penicillium citrinum (
P, citrinum) CMI 298303 and CMI 298309, or Eurotium (Euro
A method using microorganisms belonging to the genus tium) [J, L,
Kinderler et al.

Phytochemistry 23.2847-2
849 (I984)), a method using microorganisms belonging to Aspergillus ruber (A.
ochemistry 26.1417-1420 (
I987)), Aspergillus niger (A,...U)
Or Aspergillus fumigatus (A, Ha1dansai L)
) method using microorganisms belonging to
, Biochem, Z.

Pressure 1, 371-463 (I924)], Trichoderma viride
) (J, Ferment,
Technol, 66403-407.1988),
A method using microorganisms belonging to the genus Trichoderma (Yagi et al.
1985 Japan Fermentation Engineering Conference Abstracts (I0/10
Published) and the conference 11/9-11), Penicillium spp.
A method using microorganisms belonging to the genus Aspergillus, Fusarium, or Trichoderma (Journal of the Japanese Society of Agricultural Chemistry, 6)
3 (3), 350.1989) is known.

Furthermore, a method for producing an oily substance having an apple-like aroma from fats and oils using microorganisms belonging to the genus Rhizopus or the genus Mucor is known (Japanese Unexamined Patent Publication No. 57-208991).

The development of technology to achieve objectives is always required and provides an enrichment of technology.

[Means to solve the problem]

The present inventors have discovered that there are certain microorganisms that produce methyl ketones and their corresponding secondary alcohols using fatty acid esters including fats and oils, fatty acids, or salts thereof as precursors, and have completed the present invention. That is, the present invention belongs to the genus Aureolacidilum, and has the general formula (I) % formula % () (wherein A represents an aliphatic hydrocarbon group which may have a carbon-carbon double bond). A methyl ketone represented by the general formula (II) A CCH3(n) 1 (wherein A has the same meaning as above) and/or a general formula (III) A-CH- CH, (I
II) Cultivating a microorganism capable of converting into a secondary alcohol represented by CH (wherein A has the same meaning as above) in a medium containing the fatty acid, its ester or salt, or a mixture thereof; , contacting a culture of the microorganism or a treated product thereof with the fatty acid, an ester or salt thereof, or a mixture thereof in an aqueous medium to produce and accumulate the methyl ketone and/or the secondary alcohol outside the microbial cell; Next, a method for producing the methyl ketone and/or the secondary alcohol is provided, which comprises collecting them.

The present invention also provides the use of the above culture medium as described below.

Next, the present invention will be explained in more detail.

The microorganism used in the present invention belongs to the genus Aureobasidium, and any microorganism having the above-mentioned conversion ability may be used. A specific strain is Aureobasidium sp. 5M-25.
(Feikoken Bibori No. 11277).

The taxonomic properties of the new bacterial strain Aureobasidium sp. S25, which the present inventors isolated from the soil of Fukuyama City, Hiroshima Prefecture, are as follows.

1) Growth status on each culture medium Macroscopic and microscopic observations when this strain was inoculated onto each of the agar media shown below and formed into huge colonies were as follows.

2) Morphological characteristics (malt extract agar slide culture) ■ Hyphal width: approximately 2.5 to 4.5 μM. Transparent. There is a bulkhead. As the plant ages, deep green to dark brown, double-walled hyphae are formed, which become chlamydospores. This is also leaving from 12~
15 x 4.5-6.0 μm individual cells (segmented conidia). Spherical chlamydospores average 10.5 x 5 μm, 5
Forms a chain of about 0 μm.

■ Conidiophore: A side-stopping spine-like projection from the hyphae, from which conidia are formed.

■ Conidia: Budding type conidia. 6~12X4.5~6μm
.

Single cell, colorless, smooth surface, with separation marks. Produces secondary conidia by yeast-like budding. Colonies become sticky as conidia form.

3) Physiological properties ■ Growth range pH 2.0-8.0 (MHG'' medium) Temperature 5-33°
C (malt extract agar medium) ■ Optimum growth conditions pH 5.0 to 6.5 (MHG medium) Temperature 20 to 2
5°C (malt extract agar medium) W, B, Cooke+ for the above mycological properties! jycopath1. M
When compared with ycol, appl, n, 1 (I962), it is classified into the genus Aureopacidium.

The production of methyl ketone and/or secondary alcohol in the present invention may be carried out by culturing the above-mentioned microorganism in a medium containing a precursor (hereinafter referred to as the first method), or after culturing the microorganism in a normal medium, Alternatively, the microorganisms may be collected and brought into contact with the precursor in an aqueous medium (hereinafter referred to as the second method).

First, regarding the first method, these microorganisms are treated with an acid of the general formula N) as a carbon source and a precursor, an ester or salt thereof, or a mixture thereof, a nitrogen source, an inorganic substance,
Cultivate in a medium containing micronutrients under aerobic conditions while controlling temperature, pH, etc.

Other carbon sources that are commonly used for culturing filamentous fungi, such as glucose, sucrose, glycerin, dextrin, carboxymethyl cellulose, and kerosene, may also be used in combination. Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium tartrate, sodium nitrate, asparagine, peptone, corn staple liquor, etc. Inorganic substances and micronutrients include magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride,
Sodium chloride, manganese sulfate, zinc sulfate, copper sulfate, ferric chloride, ammonium molybdate, potassium iodide, boric acid, various vitamins (calcium pantothenate,
Inositol, pyridoxine, etc.) are used. Furthermore, in order to avoid contamination due to bacterium during culture, a bactericidal substance such as chloramphenicol may be contained in the medium.

The concentration of the above precursor in the medium is 1 to 500 g/ff
i is appropriate. Note that the precursor concentration is 50 to 500 g/
In the case of using 1, there is an advantage that the target product can be recovered by simple purification means as described later.

The target product-producing strain in the first method may be cultured under aerobic conditions by a liquid culture method, such as a shaking culture method or an aerated agitation culture method. Culture is usually carried out at a temperature of 20-32°C and a pH of 5.
, 0 to 8.5. The culture is preferably carried out until the stationary phase, and the culture time is usually 3 to 14 days. Thereby, the methyl ketone of the general formula (II) and/or the secondary alcohol of the general formula (I[I) can be mainly accumulated outside the bacterial cells.

Note that the pFl range that provides the optimum yield differs somewhat between methyl ketone and secondary alcohol, with the former being 7 to 8, particularly about 7.7, and the latter being 6 to 7.5.

Next, regarding the second method, first, the microorganism used in the present invention is cultured in a medium containing appropriate amounts of carbon source, nitrogen source, micronutrients, etc. under aerobic conditions while controlling temperature, pH, etc.

Carbon sources include glucose, sucrose, glycerin,
Dextrin, carboxymethyl cellulose, soybean oil,
In addition to carbon sources commonly used for culturing filamentous fungi such as palm kernel oil and kerosene, an acid of formula (I), an ester or salt thereof, or a mixture thereof is used. nitrogen source,
The same inorganic substances and micronutrients as in the first method are used. Bactericidal substances similar to those in the first method may also be used.

Cultivation in the second method can be performed by a liquid culture method, and the temperature and pH conditions may be the same as in the first method.

Culture is preferably carried out from the latter half of the logarithmic growth phase to the stationary phase, and the culture time is usually 3 to 7 days.

The microorganism culture thus obtained is brought into contact with the fatty acid of general formula (I), its ester or salt, or a mixture thereof as a precursor, or a treated product obtained by various treatments of the culture. As processed materials,
Examples include concentrates of cultures, bacterial cells obtained by subjecting cultures to centrifugation, immobilized bacterial cells, and enzyme preparations extracted from bacterial cells.

The contact reaction can be carried out in any aqueous medium, but it is preferable to use a culture solution of the bacteria to be used, its concentrate, bacterial collection condensate, or a medium from which a carbon source has been removed (other than the one used in the previous step except for the lack of a carbon source). A fatty acid of general formula (I) or an ester or salt thereof, or a mixture thereof is present in a suspension in a medium similar to that used in culture, and p
1 at 20-32°C while adjusting H to 5.0-9.0.
Methyl ketone of general formula (n) and/or general formula (
The secondary alcohol of DI) can be mainly accumulated outside the bacterial cells. The concentration of the above precursor in the reaction solution is 1
~500g#! is appropriate. In addition, considering the purification aspect, for the same reason as in the first method, 50 to 500 g/
1 is preferred.

The second method can also be carried out by continuously converting the precursor into the target product using a single bioreactor system using immobilized bacterial cells obtained by immobilizing the bacteria used in the present invention by a conventional method.

As mentioned above, the compound used as a precursor of the target compound in the present invention is a fatty acid represented by the general formula (I), an ester or salt thereof, or a mixture thereof. In the general formula (I), A is an aliphatic hydrocarbon group which may have a carbon-carbon double bond as described above, and is usually a linear or branched alkyl, alkenyl, alkagenyl, alkagenyl, etc. The number of carbon atoms is usually 1 to 15.
Considering the conversion efficiency, it is preferably 3 to 13, more preferably 3 to 9. Specific examples of A include methyl group, ethyl group, n-propyl group, isopropyl group, n
-butyl group, isobutyl group, n-pentyl group, n-hexyl group, n-heptyl group, n-octyl group, n-nonyl group, n-undecyl group, n-)lysosyl group, n-pentadecyl group, 6- Examples thereof include a pentadecenyl group, a 6.9-pentadecagenyl group, a 6.9.12-pentadetrienyl group, and the like. The fatty acid represented by general formula (I) typically includes fatty acids that form fats and oils, and the number of carbon atoms thereof is usually 4 to 18, preferably 6 to 16, and more preferably 6 to 18.
12, and may be not only linear but also branched. Specific examples of such fatty acids include butyric acid, pentanoic acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, lylunic acid, and the like.

The ester of the acid of general formula (I) is not particularly limited as long as it is an ester that can be hydrolyzed by the microorganism in its metabolic system to produce the compound of general formula (I), but glycerin ester is most commonly used. These may be not only so-called triglycerides, but also diglycerides and monoglycerides.

Specific examples of such glycerin esters include tributyrin (triglyceride of butyric acid), tricaprone (triglyceride of caproic acid), tricaprylin (triglyceride of caprylic acid), tricaprine (triglyceride of capric acid), trilaurin (triglyceride of lauric acid), and trimyristin. (myristic acid triglyceride), tripalmitin (valmitic acid triglyceride)
, tristearin (triglyceride of stearic acid),
Examples include mono- or cicaprin (mono- or diglyceride of capric acid), mono- or dilaurin (mono- or diglyceride of lauric acid), and the like. General formula (
Esters of the acids of I) can also be alkyl esters, in particular straight or branched alkyl esters having 1 to 6 carbon atoms (
For example, methyl ester, ethyl ester, etc.), benzyl ester, etc. may be used. Specific examples of such esters include methylcaprin (methyl caprate), ethylcaprin (ethyl caprate), methyllauric (methyl laurate), and ethyllauric (ethyl laurate).

As the salt of the acid of general formula (I), salts with alkali metals such as sodium salts, potassium salts, salts with alkaline earth metals such as calcium salts, ammonium salts, salts with amines, etc. are used.

The precursor used in the present invention may be a mixture of any two or more acids of general formula (I), esters or salts thereof. That is, it may be a combination of acids, esters, salts, acids and esters, esters and salts, or a combination of acids, esters, and salts. The most common darning mixture used in the present invention is oil, which may be either vegetable oil or animal fat. Vegetable oils include coconut oil, palm oil, palm kernel oil, sunflower oil, camellia oil, olive oil, castor oil,
Sesame oil, rapeseed oil, cottonseed oil, soybean oil, corn oil,
Peanut oil, safflower oil, oleic oil, grapeseed oil, etc. are used, and animal fats include milk fat (butter fat).
, lard, tallow beef bran oil, etc. are used.

In both the first method and the second method, the target substance can be isolated and purified from the conversion reaction completed liquid by a conventional method. That is, the target product is extracted from the conversion reaction completed liquid or its bacterial cell removal liquid with a solvent such as chloroform, hexane, petroleum ether, etc., and the extract is subjected to a column separation operation such as liquid chromatography or silica gel column chromatography. The desired product can then be obtained by distillation (methyl ketone and secondary alcohol are obtained individually).

Furthermore, steam distillation may be incorporated into the initial stage of purification by taking advantage of the volatility of the target compound.

Note that even when the amount of precursor is increased and oil droplets are present in a layered manner on the surface of the culture solution, methyl ketone can be sufficiently produced. Adopting such conditions is advantageous because the product can be recovered by easier means such as centrifugation rather than by post-cultivation solvent extraction. As mentioned above, an advantageous precursor loading of <50 to 500 g/l is suitable.

The present invention also relates to the use of the culture solution itself in which the methyl ketone and/or secondary alcohol of the present invention has been produced and accumulated, or the solution from which bacterial cells have been removed, for flavoring various substances. That is, when producing foods such as mayonnaise and dressings, fermented foods such as wine, whiskey, liquor, miso, bread, shampoos, cosmetics, etc., the bacterial cells used in the present invention and the fatty acid of general formula (I) or its ester or It is also possible to add flavoring by adding salt or a mixture thereof, such as palm kernel oil, as a precursor.Flavoring can also be done by adding salt or a mixture thereof, such as palm kernel oil, as a precursor, and flavoring can be achieved by adding the strain used in the present invention and the precursor to some of these food ingredients. It can also be used as a seed. For example, by applying wine yeast to the juice of apples, grapes, etc., and once it has fermented to some extent, adding the strain used in the present invention and the precursor to further fermentation can produce a distinctive wine containing the flavor of the produced methyl ketone. Can be manufactured.

〔Example〕

Next, examples of the present invention will be shown.

500 d of Aureobasidium sp. 5M-25 (Feikoken Bacteria No. 11277) in a Yosaka Lof flask.
00 d, inoculated into the following 1% palm kernel oil medium whose initial pH was changed as shown in Table 1 (5 x 10 h cells), and cultured with reciprocating shaking at 28 °C for 6 days (I20 times/min, 7
cm) I did.

Composition of medium used: Palm kernel oil (produced in Malaysia) 1.0 dNa
N030.2g (NH4) 2SO40,2g KZHPO4-0,7g K)IZPO,0,2g MgSO4・7)1z0 0.0
6gCaC1z ・28z0 0
．． 01g NaCl Vitamin Preservation Solution Mineral Preservation Solution 1% Chloramphenicol 0.05g 0.1d O,ll11 0.2d 100 in deionized water Composition of Vitamin Preservation Solution (mg/l) Biotin Calcium Pantothenate Folate Inositol Niacin p-Amino Pyridoxine benzoate hydrochloride Riboflavin thiamine hydrochloride Composition of mineral preservation solution (mg/l) MnSO4'
4 to 51 (20 ZnSOa 7HzO CuSO4 58zO FeC1:+ 6[1z0 d00 000 00 00 00 00 00 0 00 0 50)1. Bo,
60(NH4)4 MO? Ova・41b0
On 25■
The 100-cultured solution was extracted twice with 20 m of chloroform each, and the solutions were combined and subjected to gas chromatography (column: 5% 0V-17,2
Peng glass column Column temperature near 0-170°C, 5
°C/win temperature increase inlet detector temperature: 230 °C Detector:
PID carrier gas: The amount of production of the target products, methyl ketone and secondary alcohol, was measured by subjecting it to N rinsing. The results are shown in Table 1.

Table 1 5.3 0.61 6.2 1.11 7.2 1.50 7.7 2.39 8.3 3.21 3.82 0.65 3.60 5.56 0.83 5.47 7.80 2.23 6.77 11.39 0.91 0.91 0.10 0.70 0.5B 0.75 0.57 0°76 0.67 1.39 2.82 3.50 0. 69 1.49 4.10 4.82 2.12 Generated methyl ketone C7 Methyl amyl ketone C, methyl hebutyl ketone C
0 Methyl nonyl ketone producing secondary alcohol c, 2-heptatool C92-nonanol C
++ 2-undecanol fruit 11% 1.0m of palm kernel oil replaced with triglyceride 1 shown in Table 2
．． The same operation as in Example 1 was performed using the same medium as in Example 1 except that 0- was used.

The results are shown in Table 2.

Table 2 Product (mg) Carbon source Methyl ketone Secondary alcohol Lyrin methyl ketone JL [vomit 1 Tricaprin i, oj! 1! Glucose 2.0 instead of
Aureobasidium sp. 5M-25 was inoculated into 100 m of the same medium as in Example 1 except that it contained 28°
The cells were cultured with shaking at C for 4 days and used as a seed mother.

Tricabroin 1 instead of Tricaprin 1.0III
Medium 32, which was the same as in Example 1 except that it contained 0-, was placed in a 52-capacity culture tank and inoculated with seeds. 6 at an incubation temperature of 28°C.
Culture was carried out for 1 day at an aeration rate of 0.512/+in and stirring at 250 times/min.

After culturing, the mixture was centrifuged to obtain a 94 g oil layer. Distill this to obtain 22g of fraction A at 100-110°C, 115-125
2 g of fraction B at °C was obtained. Fraction A was methylpropyl ketone with a purity of 92%, and fraction B was 2-pentanol with a purity of 90%.

〔Effect of the invention〕

Until now, the production of methyl ketones of general formula (II) and/or secondary alcohols of general formula (I [I) using fatty acids of general formula (I), esters or salts thereof, or mixtures thereof as precursors has been difficult. It is carried out by a previously unknown Aureobasidium bacterium.

Claims (1)

[Claims] 1. Belongs to the genus Aureobasidium and has the general formula (I) ▲
There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) (In the formula, A represents an aliphatic hydrocarbon group that may have a carbon-carbon double bond) General formula (II) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) Methyl ketone represented by (in the formula, A has the same meaning as above) and/or general formula (III) ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (III) (wherein A has the same meaning as above) A microorganism having the ability to convert into a secondary alcohol is placed in a medium containing the fatty acid, its ester or salt, or a mixture thereof. The methyl ketone and/or the secondary alcohol are produced outside the microbial cell by culturing or contacting a culture of the microorganism or a treated product thereof with the fatty acid, its ester or salt, or a mixture thereof in an aqueous medium. A method for producing methyl ketone and/or secondary alcohol, which comprises accumulating and then collecting the methyl ketone and/or secondary alcohol. 2. Use of the culture solution itself in which methyl ketone and/or secondary alcohol have been produced and accumulated according to claim 1, or the solution from which bacterial cells have been removed, for flavoring various substances.