JPS608796B2 - Method for producing long chain dicarboxylic acids from fats and oils - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for directly producing the highest chain dicarboxylic acid by microbial means using fats and oils as raw materials.

In recent years, various oil and fat resources have attracted attention as renewable resources, and in particular, palm oil extracted from oil palm is actively researched as a raw material that is inexpensive and can be supplied in large quantities.

Although the use of these oil and fat resources is being considered in the fermentation industry, the current situation is that they can only be used as a substitute for carbon sources. On the other hand, long-chain dicarboxylic acids are compounds useful as raw materials for surfactants, plasticizers, paints, resins, lubricating oils, fragrances, etc. The fermentation method using a fatty acid alkyl ester as a raw material (for example, Hakama Kosho 53-2503) is known as its production method. However, the method using normal paraffin as a raw material ultimately requires the use of petroleum, which is a Tekigata resource, and the method using fatty acid alkyl esters as raw materials requires the use of renewable sources such as fats and oils to obtain those esters. Even if natural resources are used, the problem is that steps such as esterification are required after hydrolyzing the oil resources to obtain fatty acids. In other words, a method for directly producing long mirror dicarboxylic acids from reusable resources such as fats and oils without requiring any pretreatment can be said to be of great significance from the standpoint of effective resource utilization and from an industrial standpoint. The present invention was made in view of the above-mentioned current situation, and an object of the present invention is to provide a method for directly producing long-chain dicarboxylic acids from recyclable oils and fats using microorganisms.

The present invention will be explained in detail below. The feature of the present invention is that the longest-chain dicarboxylic acid-producing bacteria belonging to the genus Candida are cultured or reacted under aerobic conditions in a medium containing fats and oils as a substrate to directly produce long-chain dicarboxylic acids. It lies in producing.

The microorganism used in the present invention is a yeast belonging to the genus Candida, and is Candida tropicalis 1098 (FERMP-32).
91), Quillandida.

Tropicalis MD-10 (BP-100), Candidari Tropicalis BR-254 (FERMP-4664)
) etc. may be exemplified. These strains are characterized by their ability to directly produce the highest chain dicarboxylic acids from fats and oils. In the present invention, in order to efficiently produce the most chain dicarboxylic acid, the producing strain is mutated by a known method,
It is also possible to use a mutant strain that has a reduced ability to decompose fatty acid compounds. In such cases, it is effective to add another carbon source (e.g., sucrose, acetic acid, molasses, etc.) that can be used by the mutant strain to the medium to support the growth of the mutant strain. However, since the strain used in the present invention has the ability to assimilate the glycerin part of fats and oils, it also has the effect of reducing the amount of the auxiliary carbon source added by using fats and oils as a raw material. Below is Candida Tropicalis M plate-105 (
The main mycological properties of BP-100) are shown. '1} Microscopic findings: Cell size and shape: Short ovoid, 4-8 Buddhas x 5-11 Buddhas ■ Findings on the ground of the tower: Glucose - Yeast extract - Beptone - Agar on the ground of the tower Shape: white to cream-colored, soft and smooth.

Glue Maximum growth temperature: 4100 to 44 qo'
4} Fermentability of sugars: glucose + lacto
Sugalactose + melibiosunucrose + raffinose maltose + meletitose cellopiose inulin trehalose + , '6) Assimilation of carbon compounds: glucose + meletitose + galactose + inulin D-ribose - soluble starch + L-rhamnose -D-xylose +L-sorbose +L-arapinose +nuclose +D-arapinose -maltose +etanol +trehalose +glycerol +lactose -erythritol melibio surlipitor + raffinose -galactiol D-mannitol + sarin + D-glucitol + DL-lactic acid + succinic acid + inositol o-methyl-D-decacitric acid + gluconde [61 KN○3 Assimilation ability: None {7} Vitamin requirement: Biotin {8} Vitamin deficiency tower Growth in soil: Weak '91 Salt tolerance: 11-13% W/V (10) Guanosine-cytosine content: 35.3% On the other hand,
The oils and fats used in the present invention include a wide variety of vegetable and animal sources, including coconut oil, palm oil, soybean oil, olive oil, safflower oil, rapeseed oil, corn oil, cottonseed oil, tall oil, and beef tallow. , lard, whale oil, sardine oil, etc.

In the present invention, the strain is inoculated into a medium containing the above-mentioned oil or fat as a substrate and cultured, or the bacterial cells of the strain obtained by culturing in advance in a medium containing a carbon source that can be assimilated by the strain are used. It is possible to use any reaction method in which the two are brought into contact with each other in a medium containing fats and oils. Media components include nitrogen sources that can be used by bacteria (e.g., ammonium sulfate, ammonium chloride, corn staple liquor, etc.), inorganic salts, vitamins, or small amounts of growth-promoting substances. Addition of specific ingredients is not necessarily required. The amount of fats and oils added in the medium is usually 5 to 4% by weight. The culture (or reaction) in the present invention is carried out under aerobic conditions at a temperature of 25 to 35 qo, but the pH of the medium decreases due to the long mirror dicarboxylic acid produced as the culture (or reaction) progresses. Therefore, it is preferable to maintain the pH of the medium at around 6.0 to 7.5 using a neutralizing agent such as sodium hydroxide or potassium hydroxide. To recover long mirror dicarboxylic acid from the culture medium after the completion of the culture (or reaction), the medium is once made alkaline to dissolve the product, and the bacterial cells are separated and removed by a method such as heating or centrifugation. Next, when the disinfectant solution is kept under acidic conditions, long-chain dicarboxylic acids are precipitated. To recover this, a normal solid-liquid separation operation or solvent extraction operation may be applied. The long-chain dicarboxylic acid obtained by the present invention has the same number of carbon atoms as the fatty acid that composes the oil or fat used as the substrate. For example, unsaturated dicarboxylic acid is produced from an unsaturated fatty acid such as oleic acid.

As described above, according to the present invention, it is possible to advantageously produce long-chain dicarboxylic acids directly from fats and oils using microorganisms.

The present invention will be explained in more detail with reference to examples below.Example 1: Preparation of inoculum solution: Potato dextrose agar slant tower ground Candida.
Tropicalis MD-105 (BP-100) cells were scored on a maltoextract agar slant medium and cultured at 30°C for 2 hours. Three loopfuls of the cells were added to a medium 50' with the composition shown in Table 1. The inoculum was inoculated into a 5001z' Erlenmeyer flask containing 3000ml, and cultured in a rotary shaking moat for 24 hours at a shaking speed of 20 pm to prepare an inoculum solution.

Table 1 Syucrose 30th evening C
H3COONa・3rd 20
10ta NAC〆 4th evening KH2P04
2nd evening M cough S〇4th and 7th 20th
0.6taFeS.

4th and 7th 20, 1
9MnS of 0.

4.QH20 8 evening 2
nS.

4-7 days 20 8 9 piotin 5 overnight Add 1 tsp of distilled water to the above composition and adjust the pH to 6.5.

Cultivation: Inoculate 1 of the above inoculum solution into a 500-ml shoulder flask containing 3.0 ml of crude coconut oil and 20 ml of a medium with the composition shown in Table 2, and inoculate 1 of the above seed culture solution at 30°C every 96 hours. Reciprocating shaking culture was performed at a shaking distance of 155 times per minute.

Table 2 L-Asparagine 6t KH200
4 2.7ta K2HP04 1
3.9taMgS.

4th and 7th 20 0.
6 QFeS0417th 20th
10 female MS04・ro20
8-3ZnS04・7th 20
8 of 9 pyotine
5 Muta yeast extract
Add distilled water to the above composition for 2 nights, adjust the temperature to 6.5, and leave the whole thing for 1 night.

Confirmation of long-chain dicarboxylic acids in the culture solution: After culturing, add potassium hydroxide particles to the culture solution, adjust the pH to 10 to dissolve the product, and add 1 of the above culture solution to [ was collected.

Methyl pentadecanoate ester 30 Yanagi was added to this as an internal standard, and extracted with diethyl ether under acidic sulfuric acid (pH 4 or less). The obtained ether extract was methylesterified using diazomethane, and the product was analyzed by gas chromatography. analyzed. As a result, the culture solution contained dodecanedioic acid 9.8 t/s, tetradecanedioic acid 4.0 t/s,
Hexadecanedioic acid 0.9 y/h, octadecanedioic acid 0.
The formation of octadecenedioic acid and 0.2 ta of octadecenedioic acid was confirmed. In the above gas chromatography analysis, a 30mm capillary column with silicone OVIOI as the fixed liquid phase was used as the separation column, and temperature-rising analysis was performed in the range of 90 to 240 °C at a rate of 5 °C per minute. A hydrogen flame ionization detector was used as the detector. Identification of each peak on the gas chromatogram is performed by analyzing the relative retention time with the internal standard and, if necessary, the mass spectrum using a gas chromatography-direct mass spectrometer.The concentration of the product is determined by the internal standard methyl pentadecanoate. It was calculated from the area ratio of the peak of the product. The crude coconut oil used in this example was oKOH' having a saponification value of 257.8, and its fatty acid composition was as follows.

Caprylic acid 8.5 (wt%) Capric acid 6.5 Lauric acid
48.7 myristic acid
17.6 palmitic acid
2.5 stearic acid
2.5 oleic acid
5.1 Linoleic acid 1
．． 1 Example 2 Palm oil as the oil used as the substrate 3
．． Culture was carried out in the same manner as described in Example 1, except that 0.0% of the solution was used, and the culture solution was analyzed. It was confirmed that 3.2% of octadecenedioic acid and 3.2% of octadecenedioic acid were produced.

The saponification value of the palm oil used was 197.8, 2KOH/day, and the fatty acid composition was as follows. Myristic acid 1.0 (wt%) Palmitic acid
44.0 Stearic acid
4.7 Oleic acid
38.8 linoleic acid
9.8 Example 3 Culture was carried out in the same manner as described in Example 1, except that 3.0 g of beef tallow was used as the fat and oil used as the substrate, and the culture solution was analyzed. .9/so, octadecanedioic acid 1.8 ta'
It was confirmed that 2.2% of octadecenedioic acid and 2.2% of octadecenedioic acid were produced.

Claims (1)

[Scope of Claims] 1. A long-chain dicarboxylic acid-producing bacterium belonging to the genus Candeida is cultured in a medium containing fats and oils as a substrate, or cells of the bacterium are grown in advance on a carbon source that can be assimilated by the bacterium. A method for producing a long-chain dicarboxylic acid, which comprises reacting in a medium containing oil and fat to produce a long-chain dicarboxylic acid, and coloring the long-chain dicarboxylic acid. 2. The production method according to claim 1, wherein the long-chain dicarboxylic acid-producing bacterium is Candida tropicalis.