JPH02119786A - Production of l-carnitine - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful for preventing and treating hyperlipemia and cardiopathy without carrying out troublesome operation, such as separation of the L-isomer from the DL-isomer, by culturing a specific microorganism belonging to the genus Rhizopus, etc. CONSTITUTION:At least one microorganism, belonging to the genus Rhizopus, Mucor, Actinomyces, Neurospora or Penicillium and having the ability to produce L-carnitine is cultured to collect the objective L-carnitine from the resultant culture containing the produced and accumulated L-carnitine.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing lunitin, which is useful for the prevention and treatment of hyperlipidemia and heart disease.

[Prior Art] L-carnitine is a substance that functions as a carrier that allows activated long-chain free fatty acids to pass through the mitochondrial membrane, and is also called hitamine BT.

Carnitine present in natural products is levorotatory L-carnitine, and D-carnitine exhibits competitive inhibition.

Conventionally, racemic carnitine has been used as an appetite stimulant, and when racemic carnitine is administered for a long period of time, side effects on the myocardium have been observed, so it has not been recommended for therapeutic use in cardiovascular diseases, etc. It has become clear that using only L-carnitine is more effective.

Is it already known that carnitine can be produced by chemical synthesis?According to chemical synthesis, a racemic form is produced, which requires a process for chemical resolution, making the production process complicated.

3-dehydrocarnitine, γ-butyrohetaine, crotonobetaine, O-acylcarnitine as a precursor,
L- by microorganisms or enzymes produced by the microorganisms
Methods for producing carnitine are also known, but all of them have problems such as the precursor is unstable, the precursor or coenzyme is expensive, and it is difficult to separate the precursor and I5-carnitine. have.

In addition, -carnitine, which is produced using a chemical synthesis method or a semi-synthetic method including a conversion process using microorganisms or enzymes, is a synthetic product and is not approved for non-medicinal use.

A known method for preparing carnidine other than synthetic products is to separate it from milk or dairy products (Japanese Patent Application Laid-open No. 62-63553), but milk contains a large amount of inorganic salts. Therefore, the purification process to remove it becomes complicated.

In addition, a method for producing L-lunithine using microorganisms is also known (Japanese Patent Application Laid-Open No. 60-214890).

[Problems to be Solved by the Invention] However, in the above production method using microorganisms, the microorganisms used are microorganisms belonging to the genus Emericella, and are not microorganisms used in traditional fermented foods. When using produced carnidine for other purposes, there are safety concerns.

The inventor of the tree made earnest efforts to find a method to advantageously produce L-carnitine, which can be used for non-medicinal purposes, by fermentation, and as a result, he succeeded in producing yeast, kahi, etc., which have traditionally been used in fermented foods. The present invention was completed by obtaining a high L-carnitine producing strain from among the microorganisms.

The object of the invention is to provide a method for producing L-carnitine by a fermentation method using microorganisms such as yeast and Kahi that have been conventionally used in fermented foods.

[Means for solving the problem] The method for producing I,-carnitine according to the wood invention is based on Rhizopus
(Rhizopus) genus, Mucor (Mucor) genus,
L-carnitine is produced and accumulated by culturing at least one type of microorganism belonging to the genus Actinomucor, the genus Neurospora, or the genus Penicillium and having the ability to produce L-carnidine. It is used to collect carnitine.

The microorganisms used in the present invention include Rhizopus (Rhi
zopus, Mucor, Actinomucor, Neurospora and Penicillium (PC).
Any species can be used as long as it belongs to at least one of the five genera selected from the genus nicilium) and has the ability to produce L-carnitine.

An example of this is Rhizopus oligosporus (
Rhizopus oligoSporus), Mucor° Himalis f.
malis fhiemalis), Actinomucor elegans (8Ctinomucor elega)
ns), Neurospora sitophila (Neurospo
ra 5itophila), Penicillium caseicolum (Penicillium 9 caseicolum)
n) , Penicillium 10
illium roqueforti).

More specifically, Rhizopus oligosporus rhinoceros1
~-IF08631, Actinomucor erecaras I
FO6408, Neurospora sitophila IFO459
6. Penicillium caseicolumn heinier IFO5
840, Penicillium roqueforti tom IFO
4622, and these are provided by the Fermentation Research Institute (TP).
O) Publication List of Saibu Caldiers 7th Edition, 198
4th year (In5tituto For Fermenta)
tion 0saka Li5tof Culture
s, +984. Edition).

Muco Himaris F.
r hiemalis f, hiemalis) S
I, which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 9966.

Regarding Mucor hemaris F. hemaris S, the wood fungus is a filamentous fungus isolated from fermented foods. The hyphae do not have septa and form ascospores. The spores are multivesicular and have a diameter of 10-25 μm. Ascospores are about 3
It has an elliptical shape with a size of 45 μm and a smooth surface. Aerial hyphae are colorless in the early stages of growth and turn pale yellowish brown in the later stages. No protuberances are observed on the spore stalk, and it rarely branches at a position relatively distant from the spore stalk. Growth is good at 25-29℃,
No growth was observed at 40°C. These characteristics were reported in Studies in Mycologia No. 4, 1973.
YNo.

4.1973) inside zipper (M8, A 5hipp)
er), A Study on Valiability
In Mucor Hemaris and Relateds
y in Mucorhiemalis and re
rated 5 pecies) and Tamsh (
Conbenzium on Soil Fansia, edited by K. H. Domsch et al., Volume 1, 1980 (C.
ompendium of 5oil Fungi, v
As a result of comparing the properties with those described in 1980), the filamentous fungus isolated from the fermented food was identified as Mucor hemalis F. himalis.

Bacteria belonging to the genus Limbus, Mucor, Actinomucor, Neurospora, or Penicillium can mutate naturally or by mutagens as a general property of microorganisms.

Therefore, productivity can be further improved by mutating the microorganisms used by commonly used applied microbial means.

Examples include irradiation with radiation such as X-rays, comma rays, and ultraviolet rays, as well as monospore isolation, treatment with various drugs, cultivation in media containing drugs, and many other methods of mutation. Any mutant or naturally obtained mutant that has the property of producing L-carnitine can be used in the method of the present invention.

The medium used for culturing in the method of the present invention may be either liquid or solid as long as it contains nutrients that can be utilized by the strain used. In addition, a natural medium or a synthetic medium may be used, and the required carbon source, nitrogen source, inorganic substances, and trace nutrients may be appropriately added to the medium. Of course, inorganic or organic acids, alkalis, buffers, etc. are added for the purpose of adjusting the pH, and appropriate amounts of oils and fats, surfactants, etc. are added for the purpose of defoaming.

The culture may be carried out by fixed culture, stationary culture, shaking culture, or aerated agitation culture.

It is natural that the culture conditions vary depending on the state and composition of the medium, the type of strain, the method of culturing, etc., but in the case of strains belonging to the genus used in the present invention, it is usually carried out at a temperature of 15°C to 37°C. It is preferable to select an initial pH of around 3 to 8. Preferably 20°C to 30°C and initial p114 to 6. The culture period may also vary depending on the conditions mentioned above, or the culture may be carried out until the concentration of lunitine reaches its maximum. The time required for this is about 2 to 10 days when using a solid medium, and usually about 3 to 12 days when using shaking culture or aerated agitation culture using a liquid medium.

In the case of liquid culture, the produced carnidine is mainly present in the culture filtrate, so it is advantageous to separate the culture into a supernatant and bacterial cells by centrifugation or filtration, and then purify from the supernatant. It is. However, it is also possible to purify directly from culture.

On the other hand, in the case of solid culture, L-carnitine produced in the medium is extracted with water, the supernatant liquid is separated from insoluble matter or proteins by centrifugation or filtration, and then purified from the supernatant liquid, or A method in which L-carnitine is extracted using an acidic protein denaturant, neutralized with a basic substance, centrifuged or filtered to separate the supernatant and insoluble matter, and purified from the supernatant. However, in this case, it is preferable to use 1 to 5% perchloric acid as the acidic protein denaturant and potassium hydroxide as the basic substance.

Furthermore, in order to collect this L-lunithine, it is possible to appropriately utilize the means normally used for collecting metabolites produced by microorganisms. For example, after removing bacterial cells by centrifugation, effective substances are generally separated, collected, and purified from the filtrate.

i.e. solubility and solubility differences in appropriate solvents,
Differences in precipitation methods and precipitation rates from solutions, differences in various adsorption affinities, ion exchange chromatography using ion exchangers, vacuum concentration, freeze drying, crystallization, recrystallization,
Means such as drying may be used alone, in combination in any order, or repeatedly.

An example of this is as follows. That is, after the completion of the culture, the supernatant obtained by centrifuging the culture solution or culture extract is treated with a strongly basic ion exchange resin, such as Diaion 5KTB (H"") (manufactured by Hiki Rensui Co., Ltd.). Leads to the column. In order to elute the adsorbate from the resin, it is possible to use aqueous solutions of ammonia water, alkaline water, mineral acids or inorganic salts. In order to simplify subsequent operations, it is desirable to use ammonia water. After concentrating the eluate containing L-carnitine, it is passed through a strongly acidic anion exchange resin such as Diaion SAI OA (○H-) to recover L-carnitine in the non-adsorbed fraction. If pigments are mixed in, they are removed by adsorption on activated carbon to obtain a crudely purified product of (1),-carnitine. In order to further purify this, a strong basic ion exchange resin such as Taiai Ion 5KIB is used at pl+3.5~
43, preferably equilibrated with a p++4.0 buffer. A solution of the crude product is passed through a column, the adsorbed matter is eluted with a buffer solution of pH 50 or higher, preferably pH 5.5, and the portion containing L-carnitine is concentrated. Select a salt that can be removed during vacuum concentration. This eliminates the need for subsequent draft removal operations. L-carnitine can be crystallized from alcohol or alcohol-acetone solutions.

[Effect] In the present invention, it is obtained in this way.

Carnitine is a treatment for heart disease (Basic and Clinical Studies Vol. 18, No. 6, 14)
7 pages 1984 1nt, J, Ti5s, 1leac,
11, p. 175, 1982), improvement of lipid metabolism ([, an
cet volume 2, page 805 1978 J o h n s
tl o p k i n s M e d , , 1
150, p. 51, 1976).

In addition, high-calorie infusions (Surgery and Metabolic Nutrition Vol. 17, No. 1, 82)
Page 1983-Japanese Unexamined Patent Publication No. 54-154512).

"Example" An example of the present invention will be specifically described below.

Example 1 Tap water containing 50 g of soluble starch, 10 g of sodium nitrate, and 10 g of potassium phosphate at 1°C and I kg of wheat bran
Rhizopus orychosvorus was added to the bran medium mixed with
Site-IF08631, Actinomucor elegans T F O64'08, Mucor heemalis F. heemalis S1 (Feikoken Bacterial Serial No. 9966), Neurospora ci[-fici IFO4596] were inoculated and placed in a cage. They were incubated at 25°C for 7 days, 4 days, 6 days, and 4 days, respectively. 3.5 after completion of culture, C
Extraction was performed twice with 2% 11 CIO, and the extract was collected by gauze filtration. After neutralizing the filtrate by adding potassium hydroxide, it was centrifuged at 3,000 x g for 15 minutes to obtain a supernatant at 6°C.

”Climb the second tier with Aion 5KIB (H”) (φ40x
After washing with water and eluting with 1.5 N ammonia water, the eluted portion containing (1),-carnidine was collected.

The L-carnitine content was measured using the DTNB method (Metb,
in En7ymol, issue 14, page 612 +96
9), and commercially available carnidine after recrystallization was used as the internal standard. To divide the concentration, use the molecular extinction coefficient of 13600
M-/cm- was adopted.

Approximately 15 eluates containing L-lunithine were concentrated under reduced pressure to remove ammonia, and then made into a 100 ml aqueous solution.

This m night is Thai Aion SA 10A (OH-)(
4.5 x 39 cm) and washed with water, L-carnitine was recovered in the area that passed through. After collection, about 450 ml was dried under reduced pressure and dissolved in 100 ml of 0.1N ammonium acetate buffer (p++4.o). 2 g of activated carbon was added to this to obtain a stirred solution.

This first consideration? Night 0. Tiaiion 5KIB (
φ2, Ox 30 cm), washed with 400 ml of equilibration buffer, and then washed with 400 ml of 0. L-carnitine was eluted with IN acetate-ammonium buffer (pu5.s).

The eluate containing L-carnitine was dried under reduced pressure and further dried under vacuum to obtain (1)--carnidine. The amount of L-carnitine not obtained is shown in Table 1.

Table 1 The purity of carnidine produced by microorganisms was examined using silica gel thin layer chromatography (developing solvent: methanol/28% aqueous ammonia - 75/25, detection: iodine, methanol sulfate, ninhydrin, etc.) No impurities were observed.

Furthermore, L-carnitine was crystallized from ethanol-acetone.

When mass spectrometry (secondary ion mass spectrometer) was performed using crystals of L-carnitine produced by Mucor hemaris F. hemaris S1, the results shown in Figure 1 were obtained (the spectrum is secondary Because it was measured by ion mass spectrometry (SIMS), the molecular weight peak is indicated by molecule ■+1).

As shown in the figure, the molecular weight was 161, confirming that it was carnitine.

When the optical rotation of the same sample was measured, it was [α] fathom -211° (cm0.2, water), confirming that the sample had levorotation and was integral.

Similar results were obtained with samples of L-carnitine produced by other strains.

Example 2 Rhizopus oligosporus cyto-IFO8631
120 g of potato dextrose, 1 portion of ammonium sulfate
A medium containing 5 g of potassium phosphate and 10 g of potassium phosphate was inoculated at 5°C, adjusted to p) 15.0, and cultured with aeration at 25°C for 170 hours. The culture solution was subjected to case filtration, and the resulting filtrate was purified by the purification process described in Example 1 to obtain 14.2 mg of L-carnitine. The optical rotation was measured and found to be [α]2o 20.5 (c = 0.5 water).

Example 3 Penicillium caseicolumn Beiniel IF05840 or Penicillium roqueforti tom IFO4622 was cultured for 7 days and 5 days, respectively, using the method shown in Example 1.
Peiniel IFO5840 contains 12 mg/Kg wheat bran, Penicillium roqueforti tom IFO
4622 produced 28 mg/Kg wheat bran carnidine.

[Effects of the Invention] As explained above, the present invention enables the production of L-carnitine by conventional chemical synthesis.
It is possible to easily extract carnitine, which is contained in the culture, without performing complicated operations to separate L-integrate from L-integrate.

The medium components used can be selected from natural products or food additives, and the bacterial strains used can be selected from strains conventionally used in fermented foods. Unlike monocarnidine, it is obtained by the biochemical synthesis method used, and there is no need to purify pure L-carnitine, and it has the advantage that it can be easily applied to foods and cosmetics as a natural hitamine BT.

Furthermore, when L-carnitine is produced by fermentation, productivity can be expected to be further improved by mutating the microorganisms used by commonly used applied microbial means.

[Brief explanation of the drawing]

Figure 1 is Mucor Himaris F Himaris S1
FIG. 2 is a diagram showing the mass spectrum of L-carnitine crystals produced by. Agent Patent Attorney Masatoshi Sato

Claims (1)

[Claims] Rhizopus genus, Mucor
r) Genus, Actinomucor
L- belonging to the genus Neurospora or Penicillium
A method for producing L-carnitine, which comprises culturing at least one type of microorganism capable of producing carnitine, and collecting L-carnitine from the culture in which L-carnitine is produced and accumulated.