JP2003289894A - Method for producing vanillin or its analogue - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing vanillin or a vanillin analogue without needing the addition of a precursor, or the like. <P>SOLUTION: Vanillin or its analogue is produced by subjecting a mold to a liquid culture in a state that culture conditions comprising a nutritive state, temperature, pH, an aeration volume, a stirring rate and a culture time are property controlled, without adding a vanillin precursor, or the like. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing vanillin or an analog thereof.

[0002]

2. Description of the Related Art Vanillin, which is used as a natural aroma substance, has been conventionally obtained by methods such as extraction from vanilla beans, chemical synthesis, and production by microorganisms. Among these methods, vanillin obtained by extraction is very expensive, and chemically synthesized products have problems in use in foods. The microbial production method requires the addition of an expensive precursor represented by ferulic acid as a raw material, and the current situation is that an inexpensive and direct method has not been developed.

[0003]

As described above, any of the methods for synthesizing vanillin and vanillin analogs has problems in economical efficiency, usability, operability, and the like.

[0004]

Means for Solving the Problems As a result of intensive studies in view of the above-mentioned problems of the prior art, a method for obtaining vanillin or an analog thereof by culturing a filamentous fungus without adding a vanillin precursor was found, and the present invention was completed. Let

The present invention has the following configurations (1) to (11). (1) A method for producing vanillin or an analog thereof, which comprises culturing a filamentous fungus in a medium containing no vanillin precursor.

(2) The method for producing vanillin or an analog thereof according to the above item 1, wherein the filamentous fungus is a basidiomycete.

(3) The method for producing vanillin or an analog thereof according to the above item 1, wherein the filamentous fungus is a white-rot fungus having a lignin degrading ability.

(4) The medium contains glucose as a carbon source,
2. The method for producing vanillin or an analog thereof according to the above item 1, which is a medium containing at least one selected from fructose, starch, and molasses.

(5) The method for producing vanillin or an analog thereof according to item 1, wherein the medium contains at least one selected from organic nitrogen and inorganic nitrogen as a nitrogen source.

(6) The vanillin or the analog thereof according to the above item 5, wherein the organic nitrogen is at least one selected from yeast extract, potato extract, peptone, meat extract, fish extract, and soybean peptide. Production method.

(7) Inorganic nitrogen is ammonium nitrate,
7. The method for producing vanillin or an analog thereof according to the above item 5, which is one or more selected from ammonium sulfate and sodium nitrate.

(8) The method for producing vanillin or an analog thereof according to the above item 1, wherein the culture temperature is in the range of 10 to 40 ° C.

(9) The method for producing vanillin or an analog thereof according to the above item 1, wherein the culture time is in the range of 2 to 30 days.

(10) The method for producing vanillin or an analog thereof according to the above-mentioned item 1, which comprises a step of extracting vanillin or an analog thereof from the culture medium after the completion of the culture using an organic solvent.

(11) The method for producing vanillin or an analog thereof according to the above item 10, wherein the organic solvent is at least one selected from ethyl acetate and normal hexane.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is to produce vanillin or its analogs by liquid-culturing a filamentous fungus under appropriate culture conditions described below without adding a vanillin precursor or the like. Vanillin or its analogs produced by filamentous fungi are released and accumulated in the culture medium. The vanillin or its analogs accumulated in this culture medium can be isolated through extraction with an organic solvent. The culture conditions include nutritional status, temperature, pH, aeration rate, stirring speed and culture time. By appropriately controlling these conditions, vanillin or its analog can be efficiently produced.

The filamentous fungus to be used in the present invention is a group of fungi that can be isolated from the soil such as forests and have a vigorous growth of mycelium. Basidiomycetes among these filamentous fungi,
In particular, when a white-rot fungus having a lignin degrading ability is selected, it becomes possible to more efficiently obtain a vanillin analog.

The proper nutritional state means a state in which an appropriate carbon source and nitrogen source are added to the liquid medium. A liquid medium in an appropriate nutritional state is prepared by adding these nutrient sources to pure water and dissolving them.

Various carbohydrates can be used as the carbon source. Of these, glucose, fructose,
When starch and molasses are used, the growth of filamentous fungi is particularly good and the number of culture days can be shortened.

As the nitrogen source, one selected from organic nitrogen and inorganic nitrogen can be added. As the organic nitrogen, various animal and plant extracts can be used. Among these, when yeast extract, potato extract, peptone, meat extract, fish extract, and soybean peptide are used, the growth of filamentous fungi is particularly good and the number of culture days can be shortened. As the inorganic nitrogen, various ammonium salts and nitrates can be used. Among these, when ammonium nitrate, ammonium sulfate, and sodium nitrate are used, the filamentous fungi grow particularly well, and the number of culture days can be shortened.

The temperature for culturing is preferably 10 to 40 ° C.
In particular, when the culture is carried out at 30 to 37 ° C., the growth of filamentous fungi is good and the number of culture days can be shortened.

The pH of the culture solution is not particularly required to be adjusted, but the pH of the culture solution is preferably in the range of 5 to 7.

The number of days for culturing depends on other culturing conditions, but is preferably about 2 to 30 days. In the culture in which other culture conditions are optimized, about 2 to 15 days are preferable. The end of the culture is determined by indirectly measuring the yield of the vanillin analog.

A flask such as an Erlenmeyer flask or a Sakaguchi flask or a jar fermenter is used as a culture container. When using a flask, culture by shaking culture. If using a jar fermenter, vent and agitate as needed.

After culturing the filamentous fungus, the culture solution is taken out,
The filamentous fungus is removed by filtration using a centrifugal separator or a membrane filter having a pore size of 0.45 μm or less. Then, vanillin or its analog is extracted from the culture broth from which the filamentous fungus has been removed, using an organic solvent. As the organic solvent, any non-aqueous solvent can be used, but particularly when ethyl acetate and normal hexane are used, it is possible to efficiently extract vanillin or its analog from the culture medium without insufficient extraction or extraction of impurities. I can.

[0026]

The present invention will be described in detail with reference to the following examples. Example 1 Cultivation of Basidiomycetes and Isolation of Vanillin-Related Compounds from the Culture Solution Basidiomycetes YL185 (18 independently isolated from the soil of forest
Assigned from the SrRNA gene nucleotide sequence and identified as Basidiomycetes subretinal anamorph fungi) on a PDA agar petri medium (D
-Glucose 20g, potato exudate powder 4g, agar
One platinum loop was inoculated into 15 g of deionized water (1 L) and cultured at 30 ° C. for 5 days. Subsequently, 8 cells having a diameter of 12 mm were punched out together with the culture medium, which had spread on the petri dish, and were autoclaved at 121 ° C. in a PMY liquid medium (D-glucose 30 g, peptone 10 g, malt extract 10 g,
Yeast extract 4g, ion exchanged water 1L) 150ml
Add to the added Sakaguchi flask, 30 ℃ × 120rpm
The cells were shaken overnight under the above shaking conditions to uniformly disperse the bacterial cells.
The whole amount of the bacterial cells dispersed in a jar fermenter (volume 5 L) added with 4 L of the same PMY medium was aseptically inoculated.
Culture in a jar fermenter at 30 ° C x 120 rpm
Was aerated at a rate of 2 L / min for 5 days.

After the culture with the jar fermenter was completed, the cells were separated by filtration, 1.3 L of ethyl acetate was added to the culture solution, and extraction was performed with a separating funnel. This extraction operation was carried out 5 times in total, and then ethyl acetate was removed by an evaporator to obtain 440 m of ethyl acetate extract from the basidiomycete culture solution.
g was obtained. This extract was analyzed by NMR and FABMS, and it was confirmed to be an aromatic aldehyde having a vanillin-like partial structure in which a hydroxyl group and a methoxy group were bonded to the benzene ring.

[0028]

EFFECT OF THE INVENTION By using the method for producing vanillin or vanillin analog by culturing filamentous fungus of the present invention, vanillin or vanillin analog can be produced without adding a precursor. Also, unlike the method by synthesis, it can be used for foods.

Continued front page    (72) Inventor Mototake Tsutsui             5-1, Okawa, Kanazawa-ku, Yokohama-shi, Kanagawa             Chisso Corporation Yokohama Research Center (72) Inventor Ikuo Sato             5-1, Okawa, Kanazawa-ku, Yokohama-shi, Kanagawa             Chisso Corporation Yokohama Research Center F term (reference) 4B064 AC26 CA07 CD09 CE08 DA10

Claims (11)

[Claims] 1. A method for producing vanillin or an analog thereof, which comprises culturing a filamentous fungus in a medium containing no vanillin precursor. 2. The method for producing vanillin or an analog thereof according to claim 1, wherein the filamentous fungus is a basidiomycete. 3. The method for producing vanillin or an analog thereof according to claim 1, wherein the filamentous fungus is a white-rot fungus having a lignin degrading ability. 4. The method for producing vanillin or an analog thereof according to claim 1, wherein the medium contains at least one selected from glucose, fructose, starch and molasses as a carbon source. 5. The method for producing vanillin or an analog thereof according to claim 1, wherein the medium contains at least one selected from organic nitrogen and inorganic nitrogen as a nitrogen source. 6. The method for producing vanillin or an analog thereof according to claim 5, wherein the organic nitrogen is at least one selected from yeast extract, potato extract, peptone, meat extract, fish extract, and soybean peptide. . 7. The method for producing vanillin or an analog thereof according to claim 5, wherein the inorganic nitrogen is at least one selected from ammonium nitrate, ammonium sulfate, and sodium nitrate. 8. The method for producing vanillin or an analog thereof according to claim 1, wherein the culture temperature is in the range of 10 to 40 ° C. 9. The method for producing vanillin or an analog thereof according to claim 1, wherein the culture time is in the range of 2 to 30 days. 10. The method for producing vanillin or an analogue thereof according to claim 1, which comprises a step of extracting vanillin or an analogue thereof from the culture medium after completion of the culture using an organic solvent. 11. The method for producing vanillin or an analog thereof according to claim 10, wherein the organic solvent is at least one selected from ethyl acetate and normal hexane.