JPH09154591A - Production of ferulic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To effectively obtain a compound useful for medicines, cosmetics, food additives, etc., by bringing a strain having an ability to convert eugenol into the ferulic acid into contact with the eugenol in a resting cell reaction solution containing the eugenol and subsequently collecting the product. SOLUTION: A strain ]e.g. Pseudomonas fluorescens E118 (FERM 9-15185)] having an ability to convert the eugenol into the ferulic acid is brought into contact with the eugenol in a resting cell reaction solution containing one or more kinds of water-insoluble organic solvents such as 6-20C saturated hydrocarbons and toluene and one or more kinds of surfactants to convert the eugenol into the ferulic acid, and the produced ferulic acid is collected. Thus, the objective ferulic acid which is a component constituting the cell walls of plants and which is useful as a raw material for medicines, cosmetics, agrochemicals, food additives, feed additives, liquid crystals, etc., is effectively obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to contacting a eugenol-assimilating strain in a quiescent bacterial cell reaction solution containing eugenol to convert eugenol to ferulic acid.
The present invention relates to a method for producing ferulic acid, which comprises obtaining ferulic acid from a reaction solution of resting cells.

[0002]

BACKGROUND ART Ferulic acid is a constituent component of plant cell walls, and is used as a drug, cosmetic, pesticide, food additive, feed additive,
It is a substance that is expected to have a wide range of uses as a raw material for liquid crystals. It is known that ferulic acid can be produced by the condensation reaction of vanillin and malonic acid (Journal of American Chemical Society, Vol. 74, p. 5346 (1).
952)), but this method is not an industrial method because it takes about 3 weeks to manufacture. A method of obtaining ferulic acid by alkaline hydrolysis of rice bran waste oil and the like is also known (Patent Document 7
-78032 gazette), this manufacturing method is also costly,
Not practical. On the other hand, attempts have been made to produce inexpensive vanillin-related substances by allowing eugenol to act on microorganisms (Japanese Patent Application Laid-Open Nos. 5-227980 and 3-27980).
30683, JP-A-62-190092,
Agricultural Biological Chemistry 4
Vol. 1, pp. 925-929 (1977) and Vol. 47, Vol. 2
639-2640 (1983)) all produce vanillin or vanillic acid, but have not succeeded in efficiently producing ferulic acid. Also, these methods
It is obtained by purifying ferulic acid from the culture solution after culturing the microorganisms, which requires cost and time and is not practical.

[0003]

Although ferulic acid can be expected to have a wide range of uses, its use has been hindered because a practical method for producing ferulic acid has not been found. The present inventors have conducted extensive studies to solve such problems, as a result, search for a microorganism that assimilates eugenol from the soil, and perform a resting microbial cell reaction using eugenol as a substrate using the obtained strain. We have found a method to efficiently convert eugenol in liquid to ferulic acid. That is, an object of the present invention is to provide a strain that can efficiently accumulate ferulic acid using eugenol as a raw material, and a method for efficiently producing ferulic acid using this strain.

[0004]

[Means for Solving the Problems]

(1) A strain having the ability to convert eugenol to ferulic acid is contacted with eugenol in a resting microbial cell reaction solution containing eugenol to convert the eugenol to ferulic acid, which is then collected. Method for producing ferulic acid. (2) A strain having the ability to convert eugenol to ferulic acid is Pseudomonas f.
luorescens) E118 (FERM P-15185). (3) The method for producing ferulic acid according to item 1, wherein the resting microbial cell reaction liquid containing eugenol contains at least one water-insoluble organic solvent. (4) The method for producing ferulic acid according to the above item 3, wherein the water-insoluble organic solvent is a saturated hydrocarbon having 6 to 20 carbon atoms or toluene. (5) The method for producing ferulic acid according to any one of the above items 1, 3, or 4, wherein the resting microbial cell reaction liquid containing eugenol contains at least one surfactant. . (6) Pseudomonas fluorescenes, which have the ability to convert eugenol to ferulic acid
cens) E118 (FERM P-15185).

As a method for obtaining a bacterium having the ability to convert eugenol to ferulic acid from soil, it is preferable to carry out an integrated culture by a known method using a culture solution containing eugenol as a single carbon source. For example, 2 g of a soil sample is placed in 50 mL of concentrated culture medium and cultured at 28 ° C. for 7 to 10 days. One drop of the obtained culture medium is transferred to a newly prepared culture medium of the same composition and cultured for 7 to 10 days. The same operation is repeated three times, and ferulic acid in the final culture solution is measured by a method such as thin layer chromatography. From the culture solution, a bacterium capable of detecting ferulic acid, that is, a bacterium capable of converting eugenol into ferulic acid is obtained.

Although any microorganism can be used in the present invention as long as it has the ability to convert eugenol to ferulic acid, the E118 strain having particularly high activity is preferable. Strain E118 is a Gram-negative bacillus that is motile and does not produce spores. When the colonies were cultured on ordinary broth agar plate medium at 30 ° C. for 72 hours, the colonies had a diameter of 3 to 4 mm and were dull yellow, circular, full-edged, translucent and low convex, and produced a fluorescent dye. It did not grow at 37 and 41 ° C., both catalase and oxidase activity were positive, and glucose OF test was negative. From these results, the E118 strain was identified as Pseudomonas fluorescens and deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology. The deposit number is FERM P-15185.

In the present invention, ferulic acid is manufactured as follows. First, in order to obtain cells to be used in the resting cell reaction, eugenol-utilizing bacteria are aerobically cultured in a culture solution containing eugenol. Any culture medium may be used as long as it contains eugenol so that the bacterium can grow, but preferably, eugenol is 0.1 to 0.4% (v / v) and sucrose is 0.5 to 3%. (W /
v), peptone 0.05 to 0.2% (w / v), magnesium sulfate heptahydrate 0.01 to 0.1% (w / v),
Consisting of yeast extract 0.1 to 1% (w / v) and metal salt mixed solution 0.05 to 0.2% (v / v), pH 6.5
To 8.0 are used. The metal salt mixture is 0.4 g of calcium chloride dihydrate, 0.3 g of boric acid, 0.04 g of copper sulfate pentahydrate, 0.1 g of potassium iodide, 0.2 g of ferrous sulfate heptadhydrate in 1 L of deionized water. , Manganese sulfate 7
Hydrochloride 0.4g, Sodium molybdate dihydrate 0.2g
It is preferable to use a solution in which is dissolved. Culture is 30 ℃
Culture with aeration and stirring for 18 to 48 to 72 hours. After culturing, the culture solution is centrifuged or filtered to obtain bacterial cells, and the suspension is suspended at a ratio of 1 mL of 1 M potassium phosphate buffer (pH 7.0) to 0.5 g (wet weight) of the obtained bacterial cells. It becomes cloudy and a cell suspension is prepared. The reaction solution for the resting cell reaction is
Any composition may be used as long as it contains the bacterial cell suspension, a buffer solution near neutrality, and eugenol as a reaction substrate. The concentration of eugenol in the reaction solution is preferably about 5 to 10 mM, which does not show toxicity to bacterial cells. When eugenol is consumed from the reaction solution and disappears, it may be sequentially added to the reaction solution.

When a surfactant or a water-insoluble organic solvent is added to the reaction solution, the resting microbial cell reaction is promoted, and eugenol having a higher concentration of 20 to 80 mM can be converted to ferulic acid. Any surfactant may be used as long as it has no toxicity to the cells and activates the resting cell reaction, but polyoxyethylene sorbitan fatty acid ester, deoxycholic acid, sodium dodecyl sulfate, cetyltrimethyl bromide. Ammonium, dodecyltrimethylammonium chloride, 3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonic acid,
It is preferable to add any one of n-octyl glucopyranoside, polyoxyethylene alkylphenyl ether, and alkoxypolyoxyethylene phosphoric acid ester to the reaction solution at a concentration of 0.1 to 5% (w / w). .
As the water-insoluble organic solvent, either a linear saturated hydrocarbon having 6 to 20 carbon atoms or toluene is added to the reaction solution in an amount of 2
It is preferable to add it at a concentration between 3 and 30% (w / w).

The resting cell reaction is carried out by shaking or stirring the reaction solution at a temperature between 25 and 35 ° C. until the eugenol is no longer consumed. Although this time varies depending on the volume of the reactor, the amount of bacterial cells in the reaction solution, and the amount of eugenol, it is preferably shaken or stirred for 2 to 18 hours. The same amount of methanol as the reaction solution is added to the reaction solution to stop the resting cell reaction. When the hydrocarbon is added, leave it still and remove the hydrocarbon by decantation or the like. Then, the cells are removed from the reaction solution by centrifugation or filtration to obtain a supernatant. Next, ferulic acid is separated from the supernatant and purified by a known method. That is, hydrochloric acid is added dropwise to the supernatant to obtain crude crystals of ferulic acid. The crude crystal is dissolved in a small amount of hot water and cooled to obtain recrystallization. Recrystallization is repeated to obtain purified ferulic acid. The purification of ferulic acid from the resting cell reaction of the present invention, unlike the purification from the culture supernatant of the prior application, has very few medium components and metabolites,
It requires no chromatography and is very easy.

[0010]

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

Ferulic acid was identified by the following method. 1) Identification by thin layer chromatography The Rf value of the spot of thin layer chromatography developed with benzene-dioxane-acetic acid (90: 25: 4, v / v) of the supernatant prepared in the example was measured to obtain a standard Rf value. It was compared with 0.52 which is the Rf value of ferulic acid. 2) Identification by Nuclear Magnetic Resonance Spectrum The sample obtained in the example was dissolved in dimethyl sulfoxide, trimethylsilylated, and then measured by nuclear magnetic resonance spectroscopy, which was the standard δ value of ferulic acid.
0, 6.28 (J = 16Hz), 6.73 (J = 8H
z), 7.20, 7.44 (J = 16 Hz) and 9.4.
Compared to 6 ppm. 3) Identification by mass spectrometry spectrum The parent peak of the mass spectrometry spectrum of the sample was measured and compared with the standard parent peak of ferulic acid, 194 m / z. 4) Identification by infrared absorption spectrum The infrared absorption spectrum of the sample was compared with the infrared absorption spectrum of standard ferulic acid.

Example 1 Pseudomonas fluorescens E118 FERM P-15185
Eugenol 0.15% (V / V), sucrose 1% (W
/ V), peptone 0.1% (W / V), dipotassium phosphate 0.2% (W / V), magnesium sulfate heptahydrate 0.0
The pH consisting of 5% (W / V), yeast extract 0.03% (W / V) and metal salt mixed solution 0.1% (V / V) was 7.0.
Aeration-stirring culture was carried out for 24 hours in 1 L of the culture solution prepared above.
As a metal salt mixed solution, calcium chloride dihydrate 0.4 g, boric acid 0.3 g, copper sulfate pentahydrate 0.0 in 1 L of deionized water
4 g, potassium iodide 0.1 g, ferrous sulfate heptahydrate 0.
A solution containing 2 g, 0.4 g of manganese sulfate heptahydrate and 0.2 g of sodium molybdate dihydrate was used. The microbial cells obtained by centrifuging the culture solution after culturing were washed with physiological saline and suspended in physiological saline to obtain a microbial cell suspension. The concentration of the obtained bacterial cell suspension was 0.5 g (wet weight) / mL. 0.2 mL of 1M potassium phosphate buffer (pH 7.0) and 10 mM of eugenol were added to 1 mL of this cell suspension.
The reaction solution was added so that it became 2 mL with deionized water, and was shaken at 30 ° C. for 2 hours. After that, 2 mL of methanol was added to this reaction solution to stop the reaction, centrifugation was performed to remove the bacterial cells, and ferulic acid was identified in the supernatant by the above method, and ferulic acid was analyzed by high performance liquid chromatography. Quantitative analysis of acid was performed. High Performance Liquid Chromatography uses ODS C of size 4.6 x 150 mm
18 columns using methanol: deionized water: acetic acid = 4
The eluate having a component ratio of 5: 52: 3 was caused to flow at a flow rate of 1.0 mL / min for development, and the absorbance at 280 nm was detected. The peak around 3.4 minutes, which is the retention time of standard ferulic acid, was designated as ferulic acid. As a result, 9.8 mM ferulic acid was detected, and the conversion rate was 98%.

Example 2 1 mL of eugenol and various surfactants were added to 1 mL of the bacterial cell suspension obtained according to Example 1 and 0.2 mL of 1M potassium phosphate buffer (pH 7.0). The reaction mixture was added with eugenol at 40 mM and the surfactant at 2% (W / W) to 2 mL with deionized water.
Shake time. However, 0.25% (w / w) of dodecyltrimethylammonium chloride was added. The resting bacterial cell reaction solution containing no surfactant was also performed in parallel at the same time. After 2 mL of methanol was added to stop the reaction, centrifugation was performed to remove bacterial cells, and the supernatant was used in Example 1
The analysis was performed by high performance liquid chromatography according to.
As a result, as shown in Table 1, various surfactants used in the experiment were confirmed to have an effect of promoting the production of Ferula by resting bacterial cells.

[0014]

[Table 1]

Example 3 To 1 mL of the cell suspension obtained according to Example 1, 0.2 mL of 1M potassium phosphate buffer (pH 7.0), eugenol and various water-insoluble organic solvents were added, Eugenol 40 mM and various water-insoluble organic solvents 20% (W /
W) to 30 mL of the reaction mixture made up to 2 mL with deionized water
Shake at 20 ° C. for 20 minutes. The resting bacterial cell reaction solution containing no organic solvent was also run in parallel at the same time. After 2 mL of methanol was added to stop the reaction, the mixture was left standing for 5 minutes.
The lower layer of the resting bacterial cell reaction solution was taken out, centrifuged to remove bacterial cells, and the supernatant was analyzed by high performance liquid chromatography in accordance with Example 1. As a result, Table 2
As shown in Fig. 5, the promoting effect of the ferulic acid formation reaction of the resting cells was confirmed by the various water-insoluble organic solvents used in the experiment.

[0016]

[Table 2]

Example 4 2 mL of 1M potassium phosphate buffer (pH 7.0), eugenol and Tween 20 were added to 10 mL of the cell suspension obtained according to Example 1 to obtain 20 mM eugenol.
And the reaction liquid made into 20 mL with deionized water so that Tween 20 might be 2% (W / W) was shaken at 30 degreeC. After 2 hours, when the eugenol concentration in the reaction solution was measured,
Since there was no eugenol, eugenol was added to the reaction solution so that the eugenol concentration was 20 mM. 4 from the start of the reaction
Similarly, since eugenol had disappeared in the reaction solution after the lapse of time and after 7 hours, eugenol was sequentially added so as to be 20 mM. After reacting for 12 hours, 20 mL of methanol was added to stop the reaction, followed by centrifugation to remove bacterial cells and obtain a supernatant. Add hydrochloric acid dropwise to the supernatant to adjust the pH to 4
Then, the mixture was allowed to stand overnight to obtain crude crystals of ferulic acid. The crude crystals were dissolved in 1 mL of hot water, hydrochloric acid was added dropwise to adjust the pH to 4, and cooled to obtain crystals again. The same operation was repeated once again to obtain 0.2 g of purified ferulic acid. The identification of ferulic acid was performed by the above method.

[0018]

According to the present invention, it is possible to efficiently produce a large amount of ferulic acid using eugenol as a raw material.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display C12R 1:39)

Claims (6)

[Claims] 1. A strain having the ability to convert eugenol to ferulic acid is brought into contact with eugenol in a reaction solution of resting microbial cells containing eugenol to convert the eugenol to ferulic acid, which is then collected. Characteristic method for producing ferulic acid. 2. A strain having the ability to convert eugenol to ferulic acid is Pseudomonas fluorescens (Pseudom).
onas fluorescens) E118 (FERM P-15185). 3. The resting microbial cell reaction liquid containing eugenol contains at least one water-insoluble organic solvent.
The method for producing ferulic acid described. 4. The water-insoluble organic solvent has a carbon number of 6 to 20.
The method for producing ferulic acid according to claim 3, wherein the saturated hydrocarbon or toluene is up to. 5. The production of ferulic acid according to claim 1, wherein the quiescent bacterial cell reaction liquid containing eugenol contains at least one surfactant. Law. 6. Pseudomonas f (Pseudomonas f) having the ability to convert eugenol to ferulic acid.
luorescens) E118 (FERM P-15185).