JPH0767620A - Fed batch culture of euglena - Google Patents

Abstract

PURPOSE:To effect fed batch culture of Euglena useful as a raw material for food products with its phospholipid content increased in a high yield by initiating the culture in a medium containing carbon source, etc., and adding carbon source in the way of culture. CONSTITUTION:The culture of Euglena is started in a medium containing one or more carbon compounds selected from glucose, fructose, galactose and xylose and a nitrogen source containing acidic amino acid, ammonium phosphate, etc., and continued while the carbon and nitrogen sources are added whereby the fed batch culture of Euglena is conducted.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for culturing Euglena. According to the method of the present invention, the yield of euglena can be increased and the phospholipid content therein can be improved.

[0002]

BACKGROUND OF THE INVENTION Euglena is a unicellular eukaryotic organism and is a unique organism that is a taxonomically classified plant of the Euglena plant genus and an animal classified as the Protozoan phylum. . This euglena is widely distributed in freshwater, and most of them live in still water such as ponds and swamps, but it is known that they can grow in seawater or sewage. Euglena is useful as a material organism for comparative nutrition, and many studies have been conducted on its physiology [Euglena-Physiology and Biochemistry, edited by Shozaburo Kitaoka, Academic Society Publishing Center, 1989]. Euglena is also known to produce highly unsaturated fatty acids such as arachidonic acid and eicosapentaenoic acid, vitamins such as vitamins C, E, and β-carotene, and useful substances such as high-quality proteins and polysaccharides. Has been.

In recent years, many researchers have conducted various studies on the culture method of Euglena for the purpose of utilizing those components produced by Euglena. For example, by aeration of high concentration oxygen, the production of waxy material and mucilage,
1.5 to 10 times the amount of Euglena in the same culture time as the culture method of suppressing secretion and promoting the production of unsaturated fatty acids and vitamin E (Japanese Patent Publication No. 4-4865) and the conventional batch culture method. A continuous culture method (Japanese Patent Application Laid-Open No. 61-37091) capable of obtaining the above is known, and further, a culture method in which culture in a closed dark environment and culture in an open light irradiation are combined ( Japanese Patent Laid-Open No. 61-40785) has also been proposed. The inventors of the present invention have a history of previously developing a Euglena culture device (Japanese Patent Laid-Open No. 98574/1993) that reduces shear stress and is excellent in stirring power.

Conventionally, as a method for culturing Euglena,
The batch culture method is used. In this method, after sterilizing a fermenter in which nutrients necessary for cultivation are sterilized, cells precultured in advance are inoculated, and Euglena is cultured until the number of cells and the cell yield reach a predetermined level (Japanese Patent Publication No. −
4865, JP-A-61-40785). However, when cultured by this method, there is a problem that the cell yield obtained during a normal culture period is at most about 10 g / l, which is extremely low. A continuous culture method has also been proposed in which sterilized nutrients are continuously fed into a fermenter to be retained in the fermentor for a certain period of time and a part of the cell suspension is collected (Japanese Patent Laid-Open No. 61-61). -37091). However, when this method is used, it is difficult to obtain a desired product in a high content because the physiological state of cells is changed during the culture, and further, since the culture is performed for a long time, there is a risk of being contaminated with various bacteria. There is a problem of increase. There is also a problem that the collection of the useful substance is complicated several times, or if the collection work is carried out in order to avoid it, the cell components change during storage.

On the other hand, phospholipids are constituents of biological membranes, and it is known that the amount taken from foods is usually sufficient. It has also become clear that organisms can biosynthesize phospholipids in proportion to the required amount. However, in recent years, it has become clear that membrane phospholipids in vivo play various roles such as serum cholesterol lowering action, blood pressure lowering action, and neurotransmission system activating action, and the physiological activity of phospholipids has been reviewed. It is in the current situation. Under such circumstances, it is known that the amount of phospholipid produced by Euglena may account for 70% or more of the total amount of lipid. Most of fatty acids are contained in this phospholipid fraction, and the tendency of polyunsaturated fatty acids is particularly remarkable. Therefore, eulipena which has a high polyunsaturated fatty acid content and a specific physiological activity is euglena. Is also expected to produce.

[0006]

In view of the above-mentioned problems, the present inventors have increased the yield of Euglena, and
In order to cultivate Euglena with a high phospholipid content, we conducted extensive studies on the method and found that the nutrient components were added at any time during the culturing to the fermentation tank that had been cultivated by the method similar to the batch culturing method. The present inventors have completed the present invention by finding that euglena yield can be increased and euglena with a high phospholipid content can be obtained by culturing until a constant cell number and cell yield are achieved. Therefore, it is an object of the present invention to provide a Euglena culture method for increasing the yield of Euglena and increasing the phospholipid content therein.

[0007]

The present invention relates to a fed-batch culture method of euglena, which comprises performing fed-batch culture while adding a carbon source and a nitrogen source using Euglena as a fed-batch substrate. The carbon source and the nitrogen source in the present invention are not particularly limited, but a carbon source and a nitrogen source which are commonly used for culturing Euglena and other microorganisms, cells and the like are used. Examples of such carbon sources include sugars such as glucose, fructose, galactose, xylose and the like, and examples of nitrogen sources include ammonium salts and amino acids. Examples of the ammonium salt include ammonium phosphate, monoammonium hydrogenphosphate, diammonium hydrogenphosphate and the like, and of these, diammonium hydrogenphosphate is particularly preferable. Examples of the amino acid include isoleucine, lysine, valine, aspartic acid, glutamic acid, and the like, which are ordinary amino acids. Of these, particularly acidic amino acids,
Glutamic acid is especially preferred. Further, in the present invention, water-soluble salts such as sodium salts of these amino acids can also be used. In particular, it is preferable to use glutamic acid and diammonium hydrogen phosphate in combination as the nitrogen source from the viewpoint of the Euglena yield and the phospholipid content. Hereinafter, in the present invention, glucose will be mainly described as the carbon source, and the case where glutamic acid and diammonium hydrogen phosphate are used as the nitrogen source will be mainly described, but the carbon source and the nitrogen source of the present invention are selected from a wide range as described above. And should not be construed as limited to these. The present invention can increase the yield of Euglena and can obtain Euglena having a high phospholipid content as compared with the various conventional methods mentioned above. Fed-batch culture method of the present invention, in a fermenter that started the culture in the same manner as the conventional batch culture method,
By adding nutrients at any time during the culture and culturing until a constant cell number and cell yield are achieved, it is possible to increase the yield of euglena and to culture euglena with a high phospholipid content. .

The present invention can be applied to all species belonging to the genus Euglena. Typical Euglena include Euglena gracilis , Eugl
ena gracilis var. bacillar
is , Euglena viridis and the like are known, and mutants thereof can also be used.

In the present invention, the medium used for culturing Euglena is not particularly limited, and is usually
The composition used for culturing Euglena may be used.
Further, it may be a combination of various compositions such as a carbon source, a nitrogen source, an inorganic compound and vitamins, including a known medium composition known in the literature. For example, Hatner's medium [Journal of Protozoology, Volume 6, 2
Known media such as Koren-Hatner medium [Journal of Protozoology, Vol. 14, p. 17, 1967] can be used. The Hatner medium was 2 g of malic acid, 5 g of sodium glutamate, 0.4 g of potassium dihydrogen phosphate, 0.2 g of diammonium hydrogen phosphate per liter of the medium.
0.5 g of magnesium sulfate, 0.2 g of calcium carbonate,
Zinc sulfate 22 mg, manganese sulfate 5.8 mg, ferrous ammonium sulfate 5.7 mg, ammonium molybdate 1.5 mg, copper sulfate 1.6 mg, cobalt sulfate 1.9 m
g, boric acid 11.4 mg, EDTA ・ 2 sodium 50
mg and vitamin B ₁ 2.5 mg, vitamin B ₁₂ 0.0
It contains 2 mg and has a pH of 3.3.

In the present invention, glucose as a carbon source is 10 to 25 g / l, preferably 13 in the medium at the start of the culture.
˜20 g / l, glutamic acid and diammonium hydrogen phosphate as nitrogen sources 1 to 10 g / l and 0.1, respectively.
~ 5 g / l, preferably 2-7 g / l and 0.2-3 g
/ L is added to start the culture. At this time, the pH of the medium is adjusted to 2.5 to 8.0, preferably 3.0 to 4.5, and the culture temperature is adjusted to 10 to 35 ° C, preferably 25 to 32 ° C, and the medium is exposed to light or in the dark. 2 to 10 days, preferably 5
Culture for ~ 7 days. It should be noted that there is no particular problem with the apparatus used for the culture as long as the fed-batch substrate can be aseptically added during the culture, but an apparatus suitable for the culture of Euglena proposed by the present inventors 3-98574). The fed-batch substrate consumes almost 5 to 20 g / l of glucose as a carbon source at any time during the culture, preferably on the 3rd to 5th day of the culture, since the carbon source and the nitrogen source are almost consumed. Preferably 8 to 13 g
/ L, glutamic acid and diammonium hydrogen phosphate as nitrogen sources are 1 to 8 g / l and 0.5 to 5 g / l, respectively.
1, preferably 3 to 5 g / l and 0.7 to 2 g / l are added and the culture is continued. It is not particularly necessary to add other trace inorganic compounds to the fed-batch substrate. Immediately after the carbon source and the nitrogen source are added, the dissolved oxygen concentration in the medium is remarkably reduced, so it is desirable to increase the aeration rate. In the fed-batch culture method of the present invention, the yield of euglena can be increased by supplementing glucose and glutamic acid that disappear from the medium around the third day of the culture in the ordinary batch culture method as a fed-batch substrate. It is possible to obtain Euglena having a high lipid content.

The Euglena thus cultivated is
It can be recovered from the culture broth using a technique such as centrifugation. The cultured euglena is extracted as it is or by subjecting it to a drying means such as freeze-drying or aeration drying. Lipids are extracted from the recovered Euglena using an organic solvent such as hexane, chloroform, diethyl ether, acetone, methanol, and ethanol which is usually used for lipid extraction. The extraction operation is dipping,
Shaking or stirring is sufficient. After the lipid extraction, the pressure is reduced and the solvent is distilled off from the extract at a temperature at which the contained components are not decomposed. Then, the extracted lipid can be fractionated by a method such as silica gel column chromatography to collect the phospholipid fraction.

Next, the present invention will be described in detail with reference to examples.

Example 1 A fermenter having a capacity of 2 liters was charged with 1.3 liters of modified Hatner's medium containing glucose (18 g / l), glutamic acid (3 g / l) and diammonium hydrogen phosphate (2.5 g / l) instead of malic acid. Euglen pre-cultured in medium
a. gracilis SM-ZK strain 5% was inoculated and fed-batch culture was performed. The culture device is disclosed in Japanese Patent Laid-Open No. 3-98
The one disclosed in Japanese Patent No. 574 was used. The culture was carried out at 25 ° C. under stirring at 60 rpm with an aeration rate of 0.32 l / min. 1.3l / for 3-5 days
min. , 5 to 7 days, 1.95 l / min. Aerate with
Aeration-agitation culture was performed. Then, as a fed-batch substrate, glucose 13 g / l, glutamic acid 3 g / l, diammonium hydrogen phosphate 1.5 g / l on the 3rd and 5th day of culture.
Were added respectively. After culturing, centrifuge (3,000 x
Euglena was recovered by freeze-drying. The yield of Euglena obtained was 30 g / l.

On the other hand, glucose 3
Eugl prefilled with 1.3 liters of modified Hatner's medium containing 0 g / l and 5 g / l of glutamic acid and pre-cultured in the same medium
ena gracilis SM-ZK strain 5% was inoculated and batch culture was performed. Incidentally, as the culture device, the one disclosed in Japanese Patent Application Laid-Open No. 3-98574 is also used,
Under the conditions of 25 ° C. and 60 rpm, the aeration rate was 0 to 4 for culture.
The day is 0.32 l / min. 0.65l / m for 4-7 days
in. And After culturing, centrifuge (3,000 xg, 1
Euglena was collected by performing 0 minutes) and lyophilized. The yield of Euglena obtained was 16.7 g / l.

[0014]

Example 2 Phospholipids were extracted from each Euglena obtained in Example 1. Chloroform: methanol = 2: 1 was used as a solvent, and the shaking extraction operation was performed 10 times, and then the obtained extract was kept under reduced pressure at 35 ° C. to distill off the solvent. Next, after the solvent was distilled off and the extract was adsorbed on silica gel column chromatography, 10% diethyl ether ( n -hexane), 20% diethyl ether ( n -hexane), 1% methanol (chloroform),
Eluted with a solvent of 10% methanol (chloroform) and 100% methanol successively, and fractions obtained by each elution are wax ester fraction, triglyceride fraction, free fatty acid fraction, and other fractions containing a small amount of phospholipid. And phospholipid fraction. Then, the obtained phospholipid fraction was subjected to thin layer chromatography [adsorbent: HPTLC plate,
Silica gel (Merck), developing solvent: chloroform:
n -propanol: ethyl acetate: methanol: 0.25
% Potassium chloride aqueous solution = 23: 25: 25: 15: 9]
The Dittmer-Lester reagent positive spot was subjected to a dual wavelength chromatography scanner (manufactured by Shimadzu Corporation).
Was quantified by. For each phospholipid, a substance showing the same Rf value as the standard product was identified as the substance, and spots that did not match the standard product were quantified as unknown phospholipids. As a result of the quantification, the total phospholipid amount was 37.98 μmol / g of the freeze-dried product in the Euglena obtained by the fed-batch culture, whereas it was 18.58 μmol / g of the freeze-dried product in the Euglena obtained by the batch culture. It was the total amount of phospholipids.

[0015]

By culturing Euglena using the fed-batch culture method of the present invention, the Euglena yield can be increased and Euglena having a high phospholipid content can be obtained as compared with the conventional batch culture method. be able to. The phospholipids produced by Euglena have a high content of polyunsaturated fatty acids and are expected to have specific physiological activities.
Although it is useful as a material for medicines, foods, etc., the present invention can provide a large amount of the material.

Claims (2)

[Claims] 1. When culturing Euglena, it is characterized by carrying out fed-batch culture while adding a carbon source and a nitrogen source as a fed-batch substrate to increase the yield of Euglena and the phospholipid content therein. Fed-batch culture of Euglena. 2. The fed-batch culture method according to claim 1, wherein at least one selected from the group consisting of glucose, fructose, galactose and xylose is used as a carbon source, and an acidic amino acid and an ammonium phosphate salt are used as a nitrogen source.