JP2997767B2 - Production method of highly functional euglena using ethanol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an Euglena cell body enriched with vitamins, docosahexaenoic acid (DHA), squalene, a superoxide-eliminating active substance and the like, and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, as a medium for culturing Euglena, for example, Koren-Hattner medium (Journal of Protozoology)
14, p. 17 (1967)) or Hattner's medium (Journal of Protozoology, Vol. 6, 2
3 (1959)), glucose, fructose, starch hydrolyzate, acetic acid and the like are used as carbon sources, and glutamic acid, aspartic acid, arginine, glycine, various ammonium salts and the like are used as nitrogen sources. Was.

[0003] Cell bodies obtained by growing in such a medium are not nutritionally sufficient. For example, feeds such as artemia and rotifer necessary for the production of fish fry, or feeds for small hatched larvae are required. When used as EP, separately prepared EP
A (eicosapentaenoic acid), DHA or vitamins were added and absorbed and incorporated into Euglena.

[0004]

As described above, it has been customary to add separately prepared nutrients to Euglena for the production of fry feed, but this is complicated in the production process and cost. It was disadvantageous from the aspect. Accordingly, the present invention provides a method for obtaining highly functional cells only by culturing Euglena, and a cell obtained by the method.

[0005]

Means for Solving the Problems The present inventors have intensively studied a method of culturing Euglena, and surprisingly, by adding ethanol appropriately during the culture, various highly functional substances can be added to the cell body. It has been found that sexual substances are produced and accumulated. Thus, the present invention relates to Euglen in a medium in which some or all of the carbon source is ethanol.
a gracilis), and a method for producing a functionally enhanced Euglena cell body.

[0006] The present invention also includes, as functional enhancement material of Euglena cell bodies, carotene, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K _1, vitamin B _12, pantothenic acid, docosahexaenoic acid (DHA), arachidonic acid , Eucosapentaenoic acid, squalene and / or a superoxide scavenging active substance. According to the present invention, when Euglena is used for fry feed, animal feed or food, the Euglena cell body can be effectively used without adding a separately prepared nutrient enhancer or by greatly reducing the additives compared to the prior art. Can be used.

[0007]

DETAILED DESCRIPTION OF THE INVENTION The Euglena according to the present invention has the above-mentioned features, and its components are described in detail below. In other words, Euglena cell bodies obtained by culturing with the addition of ethanol contain normal Koren-Hattner medium (J. Protozool., 14, Suppl., 17 (1967)
) Or Hattner's medium (Methods Enzymol, 23, 14
3-162 (1971)) as compared to culturing and obtained cell bodies by, crude protein, sterols, squalene, carotenes, vitamin A, tocopherol, vitamin K _1, pantothenic acid and / or superoxide scavenging activity, in particular DHA content Was increasing.

As described above, the Euglena according to the present invention is:
Compared with the conventional medium, it has excellent nutritional properties and can be widely used in fish farms, animal feeds and foods. Euglena according to the present invention can be effectively produced by culturing with the initial addition or successive addition of ethanol to the nutrient medium, while squalene can be produced more efficiently by simultaneously adding an inhibitor of the sterol metabolism system. It can be produced effectively.

The Euglena used in the present invention is widely distributed in fresh water such as ponds and swamps, and may be used separately from these. Alternatively, any Euglena already isolated may be used. You may. As the Euglena of the present invention, species such as Euglena gracilis, Euglena gracilis Krebs, Euglena grasilis barbatilas and the like can be used. Particularly preferred Euglena strain is Euglena gracilis Z strain or a mutant strain thereof. It is SM-ZK strain (chloroplast-deficient strain). The method of obtaining the Euglena gracilis strain Z (SM-ZK strain) is described in, for example, "Euglena, Physiology and Biochemistry" by Shozaburo Kitaoka (published by Gakkai Shuppan Center, 239-241).
P.).

As a culture nutrient medium for the present euglena, any medium containing a carbon source, a nitrogen source, a yeast extract, inorganic salts, and vitamins capable of growing general microalgae and protozoa can be used. For example, Kramer-Myers medium (Arch, Mikrobio)
l., 17, 384-402 (1952)), Hutner medium (Methods Enzymol., 23, 143-162 (1971)), Koren-Hutner medium (J. Protozool.,
14, Suppl., 17 (1967)). Normally, molasses hydrolyzate, acetic acid, ethanol, and fatty acids (C3 to C18) can be used as a carbon source, but is characterized by containing ethanol as all or at least a part of the carbon source.

[0011] Ethanol can be added to the initial culture medium only once, or can be added intermittently or continuously during the culture. When added to the initial medium only once, the amount of ethanol added is 0.1 to 2 per medium.
% Is preferred. In the case of adding during the culture, ethanol is added to the initial medium, and it can be added during the culture with the consumption thereof, or the addition of the ethanol can be started in the middle of the culture. The amount of ethanol added to the initial medium is 0.1 to 2%. During the culture, the consumed ethanol is intermittently or continuously measured while measuring the ethanol concentration in the medium according to a conventional method, for example, by gas chromatography. It can be compensated for by adding it. Thus, a total of 1 to 5% of ethanol can be added per medium.

As the nitrogen source, various ammonium salts, amino acids such as glutamic acid, aspartic acid, arginine and glycine, or organic nitrogen sources such as yeast extract, peptone and corn steep liquor (CSL) can be used. Further, it is appropriate to add trace nutrients such as metal salts (iron, manganese, zinc, cobalt, nickel, copper, sodium) and vitamins (B ₁ , B ₁₂ ). In particular, it is effective to add ethanol in a preferred embodiment. Glucose is 1-3%, amino acids are 0.05-0.4%,
The ammonium salt can be used at 0.025 to 0.05%, and the metal salt can be used at 0.5 to 50 ppm. Trace elements such as vitamins can be added at 0.005 to 2.5 ppm.

The cultivation is usually carried out for 1 to 8 days under aerobic conditions such as shaking culturing in a flask and deep aeration and stirring in a jar fermenter and tank. The culture temperature is 20-30
C., preferably 25-30 ° C., and the pH is preferably 2-8, preferably 2-5.

In order to increase the yield of squalene itself,
Under aerobic conditions, a carbon source such as ethanol is added, and at the same time, squalene epoxidase inhibitor Talnaftate, cholesterol synthesis inhibitor N-lauryl-dimethylamino-N-oxide and ergosterol synthesis inhibitor miconazole (Miconazole) and the like, and surfactants used in foods such as sucrose ester, monoglyceride / sodium oleate, and deoxycholic acid may be added to suppress the conversion of squalene to sterols. After completion of the culture, the cells are collected by centrifugation, membrane filtration, etc., washed, and then subjected to vacuum freeze-drying, spray impression, heating and vacuum drying, etc., to provide the Euglena cell body according to the present invention.

[0015]

EXAMPLES The present invention will be described in more detail with reference to Examples. Example 1: Influence of carbon source 100 ml of KH medium shown in Table 1 was placed in a 500 ml Sakaguchi flask, and Euglena Grasilis SK-ZK strain was inoculated.
After shaking culture at 30 ° C. for 2 days, a seed culture was prepared. 1 ml of this culture was inoculated into a KH medium or a medium obtained by adding 0.2% ethanol to the KH medium, and cultured at 30 ° C. and 100 rpm (reciprocal shaking) for 120 hours. The culture was centrifuged (5,000 rpm, 15 minutes), and the precipitated cell bodies were shaken and extracted with chloroform: methanol = 2: 1 to perform saponification.

Acetone was added to the obtained unsaponifiable substance to remove polar lipids, and nonpolar lipids containing hydrocarbons were packed in a silica gel column and eluted in the order of heptane, benzene, chloroform and methanol. Each fraction was evaporated to dryness to obtain a sample, which was then subjected to gas chromatography (detection: FID,
Key agent: SE-30, Initial temperature: 240 ° C, Final temperature: 27
(0 ° C., heating rate: 2 ° C./min, injection temperature: 280 ° C.).
g / l and 270 μg / l in the control KH medium. Also,
The amount of squalene produced when other alcohols were added instead of the above ethanol is shown in parentheses for each of the following alcohols. Myristyl alcohol (365 μg / l), palmityl alcohol (310 μg / l), oleyl alcohol (315 μg / l)

[0017]

[Table 1]

Example 2: Comparison of cell components with culture medium 100 ml of the liquid medium shown in Table 1 was placed in a 500 ml Sakaguchi flask, inoculated with Euglena gracilis SM-ZK strain, cultured for 2 days, and cultivated in seed culture. Liquid. 5 ml of this culture was placed in a 3 L mini-jar fermenter containing 1 L of the medium shown in Table 1 at 30 ° C. for 144 hours (aeration culture 0.5 vvm).
And cultured. After the completion of the culture, the cell bodies were collected by centrifugation and freeze-dried in a vacuum. The dried protein was measured for crude protein, lipid components, vitamin and superoxide elimination activity.

The crude protein is prepared by the Kjeldahl method using nitrogen
The protein conversion coefficient used was 6.25. Docosahexaenoic acid, sterol composition and squalene were determined by gas chromatography. Vitamin B ₁₂ is, Lactobacillus de
lbrueckii subsp.lactis (L. leichmannii) ATCC 783
0 microbial quantification method, pantothenic acid is Lactobacillus
It was performed by a microorganism quantification method using plantarum ATCC8014.
Total ascorbic acid was quantified by high performance liquid chromatography after derivatization with hydrazine. Other vitamins were quantified by high performance liquid chromatography. Superoxide scavenging activity was determined by electron spin resonance (ESR). JM McCo
Units defined by rd and I. Fridovich (Journal of Biological Chemistry 224, 6049
Page (1969)). The results of these analyzes are shown in Table 2.

[0020]

[Table 2]

According to the results, by adding ethanol as a carbon source, crude protein, docosahexaenoic acid, ergosterol, squalene, carotene, vitamin A, vitamin D, α-tocopherol, vitamin K1,
It is clear that the activity of scavenging pantothenic acid and superoxide is significantly increased.

Example 3 Glucose medium and ethanol medium
Inoculating the cell body different storage medium (KH medium plus 1.5% agar) to grown Euglena gracilis SK-ZK pre-culture medium (KH medium) 150 ml of 500ml Sakaguchi flask containing ingredients in the land After shaking at 25 ° C and 100 rpm for 2 days. 10 ml of this culture solution was added to a 3 L mini jar containing 800 ml of KH medium, and stirred at 25 ° C., 70 rpm, and aeration rate of 0.5.
Cultured in vvm. In the case of adding ethanol, 240 mg of LDAO was added after culturing for 72 hours (about 8 × 10 ⁶ cells / ml).
Dissolve in ml of ethanol, add the whole amount to a mini jar,
Culture was continued for another 24 hours. After completion of the culture, the cell bodies were collected by centrifugation and vacuum-dried.

Β-carotene and vitamin E in the dried cell body were analyzed according to a vitamin analysis method (edited by The Vitamin Society of Japan [Chemical Doujinshi], pp. 10-11 and 32-34). The results are shown in Table 3 below.

[0024]

[Table 3]

[0025]

According to the present invention, by adding ethanol or ethanol and a cholesterol inhibitor to the medium in the culture of Euglena, proteins, vitamins, lipids, fatty acids, superoxide-eliminating active substances, etc. are added to the Euglena cell body. Can be increased, and an effective response to fry feed, fish feed, animal feed, health food, etc. can be achieved.

Claims (3)

(57) [Claims] 1. A method for producing an Euglena cell body, comprising culturing Euglena gracilis in a culture medium containing a sterol metabolic system inhibitor in which part or all of a carbon source is ethanol. 2. The method for producing Euglena cell bodies according to claim 1, wherein the nutrient medium for Euglena culture comprises a carbon source, a nitrogen source, metal salts and vitamins. 3. An Euglena cell body produced by the method according to claim 1 or 2.