JP3027253B2 - Method for producing cinnamic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention uses a microorganism to convert cinnamic acid, which is useful as a raw material for flavors and flavors, from cinnamic aldehyde, .beta.-methylstyrene and alkylbenzene having a specific n-alkyl group in a side chain. It relates to a method of manufacturing.

[0002]

BACKGROUND ART Conventionally, cinnamic acid is produced from cinnamic aldehyde by an oxidation reaction. (1) A method using an inorganic peroxide such as hydrogen peroxide, selenium dioxide or tellurium dioxide as an oxidizing agent (US Pat. No. 2,744,928) No.)
(2) In addition to a chemical synthesis method such as a method using a silver catalyst together with molecular oxygen (UK Patent No. 784430, JP-A-57-64640), a method using a microorganism (3)
No. 2-248493) is known.

However, many peroxides used in the method (1) are dangerous in handling, and the operation in actual operation becomes extremely complicated. In addition, the silver catalyst used in the method (2) and the cinnamic alcohol used to increase the amount of cinnamic acid generated in the method (3) are expensive, so that the raw material cost rises and the product price naturally increases. Costly and have economic problems.

[0004]

SUMMARY OF THE INVENTION The present invention solves the above problems,
An object of the present invention is to propose an economical method for producing cinnamic acid, which is easy to handle and can keep raw material costs low.

[0005]

SUMMARY OF THE INVENTION The present inventors have conducted studies to achieve the above-mentioned object, and as a result, have found that paraffin-utilizing bacteria, for example, micro-organisms, No. 11753.
And No. 11754, cinnamic acid can be produced more safely and economically than the conventional chemical synthesis method, and can be produced at a higher concentration than the method using a microorganism known at present. In the prior art, only cinnamaldehyde could be used as a raw material, but by using the above microorganisms, not only cinnamaldehyde but also β-methylstyrene and alkylbenzene having a specific n-alkyl group in the side chain were obtained. The inventors have found that cinnamic acid can be produced as a raw material, and have completed the present invention.

That is, the present invention relates to cinnamaldehyde, β
- to summarized as a manufacturing method of cinnamic acid, which comprises oxidizing alkylbenzene with methylstyrene and lower expression n- alkyl group selected from the compounds represented by (a) in the side chain by microorganisms below.

[0007]

Embedded image

[0008] The microorganism used in the present invention, industrial technology Institute of Fermentation Research Institute in Bikoken Yadorikikin No. 11753, 11
No. 754, these are paraffin resources.
It is a fungi. These microorganisms are well cinnamaldehyde, beta-methyl styrene, or by oxidizing alkylbenzene with an odd number n- alkyl group having a carbon number of 5 to 21 carbon atoms represented by formula (a) in a side chain give the cinnamic acid, but it is a microorganism that does not degrade the cinnamic acid.

[0009] Screening of such microorganisms is carried out by culturing the microorganisms in a medium containing nutrients necessary for the growth of general microorganisms, and containing cinnamaldehyde, β-methylstyrene, or the above-mentioned odd-number carbon atoms. It is performed by adding an alkylbenzene having an n-alkyl group in a side chain and confirming whether cinnamic acid is produced.
In this case, in order to efficiently obtain a microorganism having a strong oxidizing power for the alkyl group, n-paraffin is added as a carbon source as an example of the alkyl compound, and its assimilation strength is oxidized for the alkyl group. It can be an indicator of power. In addition, it is preferable to efficiently screen microorganisms having the ability to oxidize the alkyl group but not the ability to assimilate (consume) cinnamic acid from the obtained microorganisms.

One example of screening for microorganisms used in the present invention will be described below. A medium prepared by adding n-paraffin as a carbon source to a medium component necessary for the growth of microorganisms is dispensed into test tubes, sterilized, and then collected soil is added.
Incubate with shaking. This culture solution is added to another test tube containing a medium sterilized as described above, and cultured with shaking. After this,
The culture solution is spread on an agar medium or the like using n-paraffin as a single carbon source and cultured. By picking up the appearing colonies, it is possible to screen for microorganisms that can utilize n-paraffin as a carbon source as described above, that is, have the ability to oxidize alkyl groups.

Further, among the microorganisms obtained as described above, in order to efficiently obtain a microorganism having the ability to oxidize an alkyl group but not the ability to assimilate cinnamic acid, cinnamic aldehyde, The screened microorganism is inoculated into the above-mentioned liquid medium to which β-methylstyrene or alkylbenzene having an n-alkyl group represented by the formula (a) in the side chain is added, and cultured with shaking. The supernatant of this culture solution is analyzed by, for example, high performance liquid chromatography, and strains with high cinnamic acid production are selected. A strain that does not grow on a medium containing only cinnamic acid as a carbon source is selected from the strains thus obtained. Additional preferred microorganisms are finely Industrial Research bacterium obtained by the screening 11
No. 753 and No. 11754, which have the following mycological properties.

(A) Nuclear Staining The above-mentioned two strains were stained with a nucleus, but no nuclear membrane was observed. Both strains are prokaryotes. (B) Observation of colonies For both of the above strains, pock formation was observed in the colony group on the ISP2 agar medium. (C) Form ISP2 agar medium: filamentous fungi having a thickness of 2 to 3 μm for both strains inorganic agar medium *: filamentous fungi having a thickness of 1 to 2 μm for both strains (* an inorganic agar medium having a composition shown in Table 2 of Example 1) (D) Gram staining Both strains are positive (E) Analysis of isoprenoid quinone Both strains have menaquinone type (MK) isoprenoid quinone.

[0013] Based on the above mycological properties, both strains are considered to be actinomycetes. Furthermore, in order to identify both microorganisms,
Further, the results of examination of the mycological properties of both microorganisms are shown below.

(A) Morphology Branching method and morphology of spore-forming hyphae Branching method: Simple branching of both strains Morphology: Number of straight spores of both strains: Linked spores of about 4 to 5 spores for both strains・ Surface structure: oval for both strains ・ Size: 0.5-1.0 μm × 1.5-2.0 μm for both strains Presence of flagellar spores: not observed for both strains Presence: Neither strain is observed

(B) Growth state in each medium Sucrose / nitrate agar medium Growth state: No. 11753; medium growth, yellow, flat No. 11754, slightly weak; Growth, white, planar ・ Color of the back of the colony: No. 11753, slightly brown; No. 11754, light brown; Formation of aerial hyphae: No. 11753, slightly; Formation, cream color No. 11754; poor diffusible dye: No. 1753; no production No. 11754; no production

Glucose / asparagine agar medium • Growth state: Microtechnical Laboratories No. 11753; Medium growth, cream color, raised growth Microtechnical Laboratories No. 11754; Weak growth, white, flat • Colony Color of the back surface: Microtechnical Laboratories No. 11753; light brown Microtechnical Laboratories No. 11754; white ・ Formation of aerial hyphae: Microtechnical Laboratories No. 11753; Poor ・ Diffusible dye: No. 11753; No production No. 11754; No production

Glycerin-asparagine agar medium ・ Growth state: Microtechnical Laboratories No. 11753; weak growth, white, flat, Microtechnical Laboratories No. 11754; weak growth, white, planar ・ Color of back of colony: No. 11753, white; No. 11754, white; white-formation of aerial hyphae: no. 11753, slightly formed, white No. 11754, white; slightly Formed, white ・ Diffusible pigment: No. 11753; No production No. 11754, No production

Starch agar medium ・ Growth state: Microtechnical Laboratories No. 11753; weak growth, cream color, navel state Microtechnological Laboratories No. 11754; weak growth, white, heady ・ Color of the back of colony: microscopic Koken No. 11175; light brown Fine Koken No. 11754; white ・ Formation of aerial mycelium: Microkoken No. 11754; poor Microkoken No. 11754; poor ・ Diffusible dye No: No. 11753; No production No. 11754; No production

Tyrosine agar medium ・ Growth state: Microtechnical Laboratories No. 11753; medium growth, white, flat Microtechnological Laboratories No. 11754; Medium growth, white, raised growth ・ Backside of colony Color: No. 11753, Microtechnical Laboratory; White No. 11754, Microtechnological Laboratory; Formation of aerial hyphae: No. 1753, Microtechnological Laboratory; Good, white No. 11754, Microtechnological Laboratory; Good, white ・ Diffusible pigment: No. 11753, No production No. 11754, No production

Nutrient agar medium ・ Growth state: fine engineering laboratory No. 11753; good growth, cream color,
Raised growth Microtechnological Laboratory No. 11754; good growth, cream color,
Raised growth ・ Color of the back of the colony: No. 11753, White; No. 11754, White; White ・ Formation of aerial hyphae: No. 11753, No. 117, slightly formed, white Koken Bacteria No. 11754; Slightly formed, white ・ Diffusible dyes: Micro Koken Bacteria No. 11753; Not produced Micro Koken Bacteria No. 11754; Not produced

Yeast / malt agar medium • Growth state: Microtechnical Laboratories No. 11753; Medium growth, cream color, flat surface Microscopic Research Laboratories No. 11754; Medium growth, cream color, planar status・ Color of the back of the colony: No. 1753, P.K .; light brown No. 11754, P. K .; formation of aerial hyphae: No. 1753, K.K. No. 11754; Poor ・ Diffusible dye: No. 11753; No production No. 11754; No production

Oatmeal agar medium ・ Growth state: P.K. No. 11753; Medium growth P.K. No. 11754; Good growth ・ Color of the back of the colony: P.K. No. 11753; White No. 11754; light brown-Formation of aerial mycelium: No. 11753; poor No. 11754; bad Diffusible dye: No. 11753 No. Not produced No. 11754

(C) Physiological properties Growth temperature range: 11-36 ° C., micro-organisms, No. 11754; 10-36 ° C. Gelatin liquefaction (on glucose-peptone gelatin medium) No. 11753; liquefaction; liquefaction; No. 11754; liquefaction. Starch hydrolysis (on starch agar medium): No. 11753; positivity. No. 11754; Coagulation and peptonization of positive skimmed milk: No. 11753; No coagulation, peptone No. 11754; No coagulation, weak peptone formation of melanin-like pigment (tyrosine On agar medium and peptone yeast iron agar medium): No. 11753, micro-organism, no generation No. 11754, micro-organism, no generation

(D) Assimilation of each carbon source (on Prideham-Godlieve agar medium) L-arabinose: Microtechnical Laboratories No. 11753; used, but weakly growing Microtechnical Laboratories No. 11753; But weak growth D-xylose: Microtechnical Laboratories No. 11753; used but weak growth Microtechnological Laboratories No. 1753; used but weak growth D-glucose: Microtechnological Laboratories No. 11753 Used, but weakly grown Microtechnological Laboratories No. 11753; used but weakly growing D-fructose: Microtechnological Laboratories No. 11753; used, but weakly grown Microtechnological Laboratories No. 11753; Sucrose to be used but weakly growing: Microtechnical Laboratories No. 11753; To be used but to be weakly grown Microtechnological Laboratories No. 11753; Profit L-rhamnose L-rhamnose: use, but growth is weak L-rhamnose: Microworking L. No. 11753; use, but growth is weak L-rhamnose: No. 11753; use But weak growth Raffinose: Microtechnical Laboratories No. 11753; used but weak growth Microtechnological Laboratories No. 11753; used but weak growth D-mannit: Microtechnological Laboratories No. 11753; Use, but growth is weak Microtechnological Laboratory No. 11753; Use, but growth is weak

(E) Analysis of Sugar Glucose: Microtechnical Laboratories No. 11753; Existing Microtechnological Laboratories No. 11754; Existing Mannose: Microtechnical Laboratories No. 11753; Existing Microtechnical Laboratories No. 11754; Riboses present: Microtechnical Laboratories No. 11753; not present Microtechnical Laboratories No. 11754; non-existent rhamnose: Microtechnical Laboratories No. 11753; No. 11754; non-existent galactose: PMK No. 11753; exists PMK No. 11754; exists arabinose: PMK No. 11175; exists microkink No. 11754 Non-existent xylose: microtechnological bacterium No. 11753; non-existent microtechnical bacterium No. 11754; non-existent majulose: microtechnical bacterium No. 11753; non-existent FERM No. 11754; does not exist

(F) Analysis of diaminopimelic acid (DAP) LL-DAP: No. 11753 of Microtechnical Research Laboratories; No existence No. 11754 of Microtechnical Research Laboratories; None present meso-DAP: No. 17175 No .: Existing Microtechnical Laboratories No. 11754; Existing (g) Analysis of Mycolic Acid Mycolic Acid: Microtechnical Laboratories No. 11753; Absent Microtechnical Laboratories No. 11754; Absent (h) GC (guanine / cytosine) content GC content: No. 1153; 60%; No. 11754; No. 61; 61%

[0027] From the above mycological properties, it was found that the Burges Manual of Systematic Bacteriology (B
ergey's manual of Systemma
tic Bacteriology), it was found that Nocardia ( N.
ocardia) and Actinosynnema (Actinosy
nnema ) was found. These properties are shown in Table 1 in comparison. Note that in Table 1, Microtechnical Laboratories No. 11753 is designated as H-503, and Microtechnical Laboratories No. 11754 is designated as H-5.
09.

[0028]

[Table 1]

As can be seen from Table 1, the micro-organisms No. 117
No. 53, 11754 is a genus distinctly different from the similar genera Nocardia and Actino cinema. Also, morphologically, no filamentous fungi having a thickness of 2 to 3 μm have been reported on ISP2 agar medium, and these two strains are new bacteria and do not belong to any currently known genera. Is determined. From the above mycological properties, both of the above bacteria were recognized as novel microorganisms, and were sent to the Microbial Industry Research Institute of the National Institute of Advanced Industrial Science and Technology by Microtechnical Laboratory No. 11753 (FERM P-11).
753), Microtechnical Research Bacteria No. 11754 (FERM P-
11754).

The medium used for culturing these microorganisms for the purpose of growth may be any medium as long as the microorganisms can be sufficiently grown, and alkyl benzene which is a raw material of the production method of the present invention may be used as a carbon source. Although it is possible, in order to improve the productivity of cinnamic acid, it is preferable to use sugars such as glucose or alcohols such as ethanol as the carbon source, and it is more preferable to use n-paraffin such as n-hexadecane. Examples of the nitrogen source include ammonia nitrogen compounds such as ammonium sulfate and ammonium salt, inorganic nitrogen sources such as nitrate nitrogen compounds such as sodium nitrate, urea, and the like.
Organic nitrogen compounds such as polypeptone and yeast extract may be used. Further, it is desirable to add trace components such as inorganic salts as appropriate.

The microorganism used in the production method of the present invention is 10
Culture may be performed under aerobic conditions at 〜40 ° C., and the pH of the medium is preferably 4 to 7. The microorganism may be either a growing cell or a quiescent cell, and can be used as it is in the oxidation reaction.However, the microorganism is collected by an apparatus such as centrifugation and resuspended in an appropriate solution such as a phosphate buffer. For example, the bacteria concentration can be increased.

In the oxidation reaction in the production method of the present invention, the starting materials such as cinnamaldehyde, β-methylstyrene, and the above-mentioned alkylbenzene having an odd-numbered carbon n-alkyl group as a side chain are reacted with the culture solution or suspension of the microorganism. 0-4 with turbid liquid
It proceeds by contacting at 5 ° C, preferably at 20 to 40 ° C. The above-mentioned oxidation reaction is carried out simultaneously with the culturing of the microorganism or after culturing the microorganism in advance. At the time of this reaction, the pH in the medium is lowered by the cinnamic acid generated. Therefore, it is desirable to adjust the pH with an alkaline solution such as sodium hydroxide, ammonia, potassium hydroxide and the like.

[0033]

【Example】

Example 1 10 ml of a medium having the composition shown in Table 2 was dispensed into a 21 mmφ test tube, sterilized at 121 ° C. for 15 minutes, cooled, and added with 100 μl of n-hexadecane as a carbon source.
One platinum loop of a paraffin-assimilating bacterium (Microtechnical Laboratories No. 11753) cultured on an SP2 agar medium at 30 ° C. for 24 hours was inoculated. After this was cultured at 30 ° C. for 24 hours, 30 μl of cinnamaldehyde was added as a raw material to this culture solution, followed by shaking culture at 30 ° C. Thereafter, the reaction solution obtained by removing the bacteria was analyzed by high performance liquid chromatography, and it was found that 570 mg / l cinnamic acid was produced.

[0034]

[Table 2]

Example 2 The same procedure as in Example 1 was carried out except that β-methylstyrene was added as a raw material, and the result was 62 mg / l.
Of cinnamic acid had been produced.

Example 3 The same procedure as in Example 1 was carried out except that n-undecylbenzene was added as a raw material.
/ L of cinnamic acid was produced.

Example 4 The same procedure as in Example 1 was carried out except that n-tridecylbenzene was added as a raw material, and the result of analysis was 61 mg.
/ L of cinnamic acid was produced.

Example 5 The same procedure was followed as in Example 1 except that the microorganism used was changed to No. 11754, and analyzed.
700 mg / l of cinnamic acid was produced.

Example 6 The same procedures as in Example 2 were carried out except that the microorganism used was changed to No. 11754, and analyzed.
52 mg / l cinnamic acid was produced.

Example 7 The same procedure as in Example 3 was carried out except that the microorganism used was changed to No. 11754, and analyzed.
93 mg / l of cinnamic acid was produced.

Example 8 The same procedure as in Example 4 was carried out except that the microorganism used was changed to No. 11754, and analyzed.
42 mg / l of cinnamic acid was produced.

Example 9 n-undecylbenzene 100 μm as a carbon source and a raw material
Except that 1/10 ml was added all at once at the start of the culture, it was carried out and analyzed in the same manner as in Example 1.
/ L of cinnamic acid was produced.

Example 10 The same procedure as in Example 9 was carried out except that n-tridecylbenzene was used instead of n-undecylbenzene, and it was found that 32 mg / l of cinnamic acid was produced. .

Example 11 The same procedure as in Example 9 was carried out except that the microorganism used was changed to No. 11754, and analyzed.
13 mg / l of cinnamic acid was produced.

Example 12 The same procedure as in Example 10 was carried out except that the microorganism used was changed to Microtechnological Laboratory No. 11754, and as a result, 21 mg / l cinnamic acid was produced.

Example 13 The procedure of Example 5 was repeated, except that 0.1 ml of a paraffin mixture having the composition shown in Table 3 was used as a growing carbon source. As a result, 1260 mg / l of cinnamic acid was obtained. Was done.

Example 14 The same procedure as in Example 5 was carried out except that 0.1 ml of ethanol was used as a carbon source for growth, and the result of analysis was 55
0 mg / l cinnamic acid was obtained.

Example 15 One liter of a medium having the same composition as in Example 1 was placed in a 2 liter fermenter and sterilized at 121 ° C. for 15 minutes.
Cooled to room temperature. This was inoculated with No. 11754, and 5 ml of n-hexadecane was added thereto.
Culturing was performed at 1 rpm / v / v / min (in the same amount of air as the medium was fed into the tank in one minute). PH of culture solution
Was adjusted to 6.5 with a 2N NaOH solution. After 24 hours had elapsed, 500 μl of cinnamaldehyde was added every 12 hours, and 3 ml of n-hexadecane was added every 36 hours. One week later, 3500 mg / l cinnamic acid was obtained.

[0049]

[Table 3]

[0050]

As described in detail above, the manufacturing method of the present invention has the following effects. (1) cinnamaldehyde, β-methylstyrene, formula (a)
The cinnamic acid can be produced from an alkylbenzene having an n-alkyl group represented by the following formula as a raw material. (2) Cinnamic acid can be produced under mild conditions to utilize the alkyl oxidizing ability of microorganisms that cannot assimilate cinnamic acid, and the cinnamic acid produced does not disappear. Can be. (3) Compared with conventional methods, there is no need for dangerous peroxides, expensive silver catalysts or cinnamon alcohol, etc.
Cinnamic acid useful as a raw material for flavors and flavors can be produced safely and economically.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Koji Hotta, Inventor 2-598-1, Kushibiki-cho, Omiya-shi, Saitama (56) References JP-A-62-248493 (JP, A) US Patent 3,301,766 (US, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 7/40 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A cinnamic aldehyde, beta-methyl styrene and the following formula alkylbenzene having a side chain an n- alkyl group selected from the compounds represented by (a), AIST microorganism
No. 11753, No. 11
Method for producing cinnamic acid, which comprises oxidizing by the microorganism deposited as No. 754. Embedded image