JPH10174593A - Production of glycolic acid by yeast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for effectively producing a highly pure glycolic acid by utilizing an yeast. SOLUTION: A strain belonging to one of Pichia, Sporobolomyces, Torulopsis, Rhodotorula and Kluyveromyces is cultured in a culturing medium containing ethylene glycol, and the objective glycolic acid is separated and gathered from the culturing medium in the method for producing a highly pure glycolic acid. A continuous filtering production is facilitated by performing the culturing of the strain in a fermenting vessel 1 having a membrane filtering device 2, separating and gathering the glycolic acid in the culturing medium by the membrane filtering device 2, and continuously charging the ethylene glycol to the culturing medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing glycolic acid by yeast.

[0002]

2. Description of the Related Art In recent years, glycolic acid has been shown to have an effect of preventing wrinkles, and demand for raw materials for cosmetics is expected to increase. Conventionally, glycolic acid has been produced by a chemical synthesis method, but glycolic acid obtained by the chemical synthesis method has a problem that irritants such as formaldehyde remain as contaminants and irritancy remains in cosmetics. Was.

[0003]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned conventional problems, and is intended to efficiently produce high-purity glycolic acid suitable for use as a raw material for cosmetics by utilizing yeast. The purpose of the present invention is to provide a method for producing glycolic acid by a yeast.

[0004]

Means for Solving the Problems A first invention made to solve the above-mentioned problem is that a medium containing Pichia and Rhodotorulum is added to a medium containing ethylene glycol.
a) culturing a strain of one yeast belonging to the genus, genus Sporobolomyces, genus Kluyveromyces, genus Torulopsis,
It is characterized in that glycolic acid is separated and collected from the medium. In the second invention, Pichia naganishii (Pichia naganishii) was added to a medium containing ethylene glycol.
), And glycolic acid is separated and collected from the medium. In this case, the yeast strain is cultured in a fermenter equipped with a membrane filtration device, and glycolic acid is separated and collected from the medium by the membrane filtration device, and ethylene glycol is continuously introduced into the medium,
It is preferable to perform continuous filtration production that repeats production.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, ethylene glycol is contained in a medium as a starting material, glycolic acid is produced using yeast, and separated and collected from the medium. The concentration of ethylene glycol in the medium is preferably from 1 to 20%, and particularly preferably from about 5 to 10% as in Examples described later.

In the present invention, Pichia is used for the above purpose.
a) One yeast strain belonging to the genus, Rhodotorula or Kluyveromyces is used. Examples of the strains of the genus Pichia include Pichia naganishii (IFO 1670) used in the examples,
olymorpha (IFO 0195), Pichia heedii (IFO10019) and the like can be used. Rhodotor is a strain of Rhodotorula.
ula rubra (IFO 0889), Rhodotorula glutinis (IFO 000
3), etc., and as a strain of the genus Torulopsis, Toru
lopsis nitratophila (IFO 10004), Torulopsis candida
(IFO 0405) as a strain of the genus Sporoboromyces,
Sporobolomyces johnsonii (IFO6903), Sporobolomyces
coralliformis (IFO 1032) is available. Furthermore, Kluyveromyces lactis (A
TCC 12426), Kluyveromyces thermotolerans (ATCC 1069
1) etc. are available.

[0007] All of the yeast strains described above are already known per se and can be easily obtained from public depository institutions by the stated deposit numbers. When a strain belonging to any of these genera is cultured in a medium containing ethylene glycol, glycolic acid can be produced more efficiently than when other strains are used. The characteristics of yeasts of each genus are described, for example, in the following documents. Pichia naganishii (IFO 1670) J. Ferment.Technol. 5
2: 1 (1974) Pichia heedii (IFO 10019) Int.J.Syst.Bacterio
l. 28: 326 (1978)

Further, the use of the apparatus shown in FIG. 1 enables continuous filtration and production of glycolic acid. FIG.
, 1 is a fermentation tank, 2 is a membrane filtration device connected to the fermentation tank 1, 3 is an ethylene glycol tank, 4 is an ion exchange column, and 5 is an eluate tank. In the culture medium inside the fermenter 1, yeast of any of the aforementioned strains is cultured, and the culture medium is transferred to the membrane filtration device 2 by the pump 6, where the culture medium and the cells are separated. Returned to 1. On the other hand, ethylene glycol is continuously supplied from the tank 3 to the fermenter 1. The medium containing glycolic acid separated by the membrane filtration device 2 is passed through the ion exchange column 4 to separate and collect glycolic acid. Hereinafter, examples of the present invention will be described.

[0009]

Example 1 Example 1 Pichia obtained by previously culturing in 300 ml of a medium having a composition of 0.8% Nutrient broth (DIFCO), 1% propylene glycol, and pH 7.0 on the same slant medium at 28 ° C. for 2 days. Strains belonging to the genus
One platinum loop was inoculated from a seed culture of Pichia naganishii (IFO 1670), and shake culture was performed at 28 ° C. for 2 days. After completion of the culture, the cells were collected by centrifugation (8000 rpm, 20 min), and washed with physiological saline. 250 mM, phosphate buffer (pH 7.0) containing 10% ethylene glycol in the obtained cells
Was added to 50 ml, and the reaction was carried out at 30 ° C. for 30 hours. After completion of the reaction, the cells were removed by centrifugation, and the supernatant was separated by HPL.
C was analyzed under the following conditions. As a result, 35.3 g / L of glycolic acid was obtained. (Analysis conditions) Column: QAE-2SW (4.6 × 250 mm) Mobile phase: 1/15 M KPB (pH 6.4) Detection: UV 210 nm Flow rate: 1 ml / min

Example 2 Glycolic acid was produced for other strains under the same conditions as in Example 1.
Table 1 shows the results. As shown in Table 1, according to the present invention, when a yeast strain belonging to any of the genus Pichia, Rhodotorula, Saccharomyces, or Kleberomyces was used, the strain was compared with that of other strains of the genus Hansenula or Candida. Thus, it was shown that the productivity of glycolic acid was good, and that the genus Piscia was particularly suitable for the production of glycolic acid.

[Table 1]

[Example 3] Nutrient broth (DIFCO) 0.8
%, Ethylene glycol 5%, pH 7.0, medium 3L
A seed culture of Pichianaganishii (IFO 1670) obtained by shaking culture in 100 ml of the same medium at 28 ° C for 2 nights was inoculated in advance, and the pH was adjusted to 7.0 with 0.5 M NaOH at 28 ° C.
Incubated for 40 hours. After completion of the culture, the cells were removed by centrifugation, and the supernatant was analyzed by HPLC. As a result, the glycolic acid concentration was 28.7 g / L. The resulting supernatant is
After charging to an anion exchange column (Diaion PA308), washing and elution with 1 M NaCl, the glycolic acid solution was recovered, desalted and concentrated, and dried to obtain 68.8 g of powdered glycolic acid. The identity of this substance as glycolic acid was determined by H
This was based on good agreement with standard glycolic acid in retention time in PLC, infrared absorption spectrum, and nuclear magnetic resonance spectrum. When the formaldehyde content of the obtained glycolic acid powder was measured, it was not detected at all.

Example 4 Pichia naganishi in the same manner as in Example 3 using a fermenter (FIG. 1) equipped with a membrane filtration device.
i (IFO 1670) in a medium having a composition of 0.8% Nutrient broth (DIFCO), 5% ethylene glycol, pH 7.0, 0.5M N
The cells were cultured at 28 ° C. for 40 hours while adjusting the pH to 7.0 with aOH. Filtration was started after 40 hours, and the cells were treated with MF membrane (Cefilt).
0.2 μm: made by Nippon Insulator), and the produced glycolic acid flows out of the system through the MF membrane and is connected to the anion exchange column (Diaion PA308).
Was adsorbed. After washing, the adsorbed glycolic acid becomes 1
Eluted with M NaCl aqueous solution and collected. On the other hand, 27.4 g / L of glycolic acid could be continuously produced by using the cells remaining in the system and continuously adding ethylene glycol.

[0013]

As described above, according to the method for producing glycolic acid by yeast of the present invention, high-purity glycolic acid can be efficiently produced. Moreover, glycolic acid produced by yeast in this way is different from glycolic acid produced by conventional chemical synthesis,
Since impurities such as formaldehyde do not remain, there is an advantage that there is no irritation when used as a raw material for cosmetics.

[Brief description of the drawings]

FIG. 1 is a block diagram illustrating a continuous filtration production device.

[Explanation of symbols]

1. Fermentation tank, 2. Membrane filtration device, 3. Ethylene glycol tank, 4. Ion exchange column, 5. Eluate tank, 6. Pump

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:84) (C12P 7/42 C12R 1: 645) (C12P 7/42 C12R 1:88) (72) Inventor Mitsuo Kawase Inside Insulators Co., Ltd., 2-56, Suda-cho, Mizuho-ku, Nagoya-shi, Aichi (72) Inventor Yuji Kawase Inside Insulators Co., Ltd. 2-56, Suda-cho, Mizuho-ku, Nagoya, Aichi, Japan 4-24-9 Biwajima, Nishi-ku, Nagoya City, Aichi Prefecture

Claims (3)

[Claims] 1. A medium containing ethylene glycol, a genus of Pichia, a genus of Rhodotorula, a genus of Sporobolomyces, a genus of Kluyveromyces, and a tolulopsis.
A method for producing glycolic acid by yeast, comprising culturing a strain of one yeast belonging to the genus, and separating and collecting glycolic acid from the medium. 2. A method for preparing a medium containing ethylene glycol, comprising:
A method for producing glycolic acid by yeast, comprising culturing Pichia naganishii and separating and collecting glycolic acid from the medium. 3. The yeast strain is cultured in a fermenter equipped with a membrane filtration device, and glycolic acid is separated and collected from the medium by the membrane filtration device, and ethylene glycol is continuously introduced into the medium. 2. The method for producing glycolic acid by yeast according to claim 1, wherein continuous filtration production is repeated.