JPH06113819A - Culture of euglena - Google Patents

Abstract

PURPOSE:To obtain Euglena cell having high protein content. CONSTITUTION:Euglena cells rich in paramylon in the later stage of the logarithmic phase or in the stationary phase is cultured under specific pH condition in the presence of an assimilable nitrogen compound.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing Euglena, and more particularly to a method for culturing Euglena having a high protein content.

[0002]

2. Description of the Related Art Euglena cells contain proteins with excellent amino acid composition and are easily digested by animal digestive enzymes. Further, since it is rich in vitamins A, C, E, etc., it is regarded as promising as SCP (Single Cell Protein) and feed protein. Therefore, various methods of culturing have already been studied and reported. For example, as a method for culturing Euglena, conventionally, a batch culture method in which light is irradiated in a flask or a culture tank or in the dark has been usually used. It is known that the protein contained in Euglena cells is increased by light irradiation, and a culture method assuming light irradiation has been studied so far. (JP-A-61-3
7092, JP-A-61-40785). However, these methods require a device for light irradiation, and a large irradiation cost makes it difficult to supply Euglena inexpensively. Furthermore, the culturing operation such as the control of illuminance was complicated.

[0003]

[Means for Solving the Problem] As a result of the research conducted by the inventors,
Paramylon (β-1,3 glucan) accumulated in the cells of Euglena cells during the late logarithmic growth phase or stationary phase
Is converted into protein, and its conversion efficiency depends on the specific medium pH
In addition, it was found that the conversion of paramylon to protein was efficiently carried out by keeping the nitrogen source in the medium from being depleted in the late logarithmic growth phase to the stationary phase, and completed the present invention. It was done.

The Euglena referred to in the present invention is a protozoan belonging to the genus Euglena (genus Euglena) in terms of zoology, and includes all species, variants and mutants belonging to it. A typical example is Euglena gracilis.
gracilis), Euglena gracilis var. bacillaris, Euglena
Examples include Viglidis (Euglena viridis) and Astasia longa.

Examples of the medium used for culturing these Euglena include Kohren-Hattner medium (Journal of Protozoology, Vol. 14 (1967), Supplement 17, page 17) and Hattner medium (Journal of Protozoology). Orology (Jouenal of Proto
zoology) Volume 6 (1959), page 23) and the like can be used. Glucose, starch hydrolyzate, molasses, glutamic acid, acetic acid, ethanol, etc. are used as carbon sources, and ammonium nitrate, second
Ammonium phosphate, ammonium sulfate, inorganic nitrogen sources such as aqueous ammonia, glutamic acid, amino acids such as aspartic acid, or peptone, casamino acid, yeast extract, organic nitrogen sources such as corn steep liquor are appropriately combined, calcium, Magnesium, manganese,
It is also possible to use a medium in which an inorganic salt such as iron and vitamins B ₁ and B ₁₂ are added in trace amounts. In general, Euglena culture temperature of 20 to 35 ° C. is suitable, and initial pH of 2.0 to 7.5 is suitable, but it is 3.0 for cell growth.
The most preferable range is from 5.0 to 5.0. In addition, it is desirable that shaking is performed 50 to 250 times per minute and appropriate aeration and stirring during culturing.

According to the present invention, Euglena cells having a higher protein than those in the conventional culture under light irradiation can be obtained even in the dark culture, but it is not necessary to limit the presence or absence of the light irradiation. Of course, the present invention can obtain sufficient effects even in culture under light irradiation.

To carry out the present invention, in order to culture Euglena, it is advisable to start the culture at an initial pH of the medium of 2.0 to 7.5, preferably 3.0 to 5.0. After that, when the growth of euglena reaches the late logarithmic growth phase or the stationary phase, euglena cells accumulate a large amount of paramylon in the cell and the protein content is low, but the medium pH is 1.5 to 3 When adjusted to 0.5 or 5.5 to 8.0, and further culturing in the presence of an assimilable nitrogen compound, paramylon in the cell is drastically reduced and the protein content is increased. In that case, the effect can be expected when the culture time after pH adjustment is 3 hours or more, and the culture is preferably continued for 6 to 48 hours. Further, the pH adjustment at this time can be performed using any acid or alkali such as sulfuric acid, hydrochloric acid, a sodium hydroxide solution, a potassium hydroxide solution or aqueous ammonia, as long as it is an acid or alkali that does not adversely affect Euglena. Of course, it may be maintained at a constant pH with a pH controller, etc.
It may be maintained within the range of 1.5 to 3.5 or 5.5 to 8.0.

When Euglena is cultivated, the medium pH during culturing
Is reduced. Therefore, even if the initial pH is adjusted to a pH suitable for the growth of Euglena, the initial pH may change during the culture, resulting in a decrease in cell yield. Therefore, it is possible to prevent a decrease in cell yield by maintaining the medium pH in the mid-logarithmic growth phase at 3.0 to 5.0, which is suitable for the growth of Euglena cells. At this time,
It is possible to maintain the pH during the period from the start of the culture until the pH is adjusted to the late logarithmic growth phase or the stationary phase, or only in the mid-logarithmic growth phase where the cells grow most actively.
H may be maintained. Further, for maintaining the pH at this time, any acid or alkali such as sulfuric acid, hydrochloric acid, a sodium hydroxide solution, a potassium hydroxide solution, or ammonia water, which does not adversely affect Euglena, can be used.

[0009] If the vigorous growth is continued in this way, Euglena will easily contain more than 30% paramylon by dry matter weight.

Euglena consumes the nitrogen source in the medium as it grows, and the concentration of the nitrogen source in the medium becomes extremely low in the late logarithmic growth phase to the stationary phase. However, nitrogen is an essential factor for protein synthesis from paramylon,
When paramylon is converted into protein, the conversion to protein cannot be performed efficiently if the nitrogen source in the medium is insufficient. Therefore, in the method of the present invention, the growth of Euglena is carried out in the presence of an assimilable nitrogen source maintained at a constant level. This makes it possible to efficiently obtain Euglena cells having a high protein content. The nitrogen source to be added may be any nitrogen source that can be assimilated without adversely affecting Euglena. Typical examples are inorganic nitrogen sources such as ammonium nitrate, ammonium sulfate, dibasic ammonium phosphate or aqueous ammonia, amino acids such as glutamic acid and aspartic acid, or organic nitrogen such as peptone, casamino acid, yeast extract and corn steep liquor. Source etc. can be used. Of these, ammonia nitrogen and amino acids are preferable because they are likely to be assimilated. If ammonia water is used as an alkaline agent, the level of assimilable nitrogen compounds necessary for practical use can be maintained without measuring the nitrogen concentration in the medium by adding it in proportion to the decrease in pH. It is convenient that the pH can be kept in a range suitable for growth. Euglena seems to take up assimilative nitrogen compounds strongly, and the lower limit of its abundance at which the effect of the present invention is brought about is quite low. That is, the effect of the present invention can be obtained even at 10 ppm or less. However, if it exceeds this, the capture is performed more quickly. Euglena culture example glucose 120 g, ammonium sulfate 42 g, corn steep liquor 30 g, magnesium sulfate 3
g, monopotassium phosphate 3 g, ethylenediaminetetraacetic acid sodium salt 0.3 g, Mohr salt 0.3 g, zinc sulfate 0.15 g, manganese sulfate 0.1 g, vitamin B
₁ 30 mg, vitamin B ₁₂ 60 μg, tap water 6 l
The mixture was dissolved in a jar fermenter and sterilized in an autoclave (120 ° C., 20 minutes).
This was inoculated with 600 ml of a culture solution of Euglena grasilis which had been pre-cultured in the same medium in advance, and subjected to aeration culture at 28 ° C. for 72 hours. At this time, p of the medium
H was maintained at 4.5 with a 2N aqueous sodium hydroxide solution until the end of the culture, using a pH controller. The Euglena used was Euglena gracilis NIES48, which was purchased from the National Institute for Environmental Studies Microorganism Preservation Facility (16-2 Onogawa, Tsukuba, Ibaraki), and the same strain can be obtained upon request.

During the culture, the cells were sampled with time, and the cell density in the culture solution was measured by a Toma hemocytometer. The cell growth reached a stationary phase at 48 hours of culture, and no further growth was observed thereafter. After the completion of the culture, the culture solution was centrifuged to recover the cells, and the dry cell weight, protein content, and paramylon content per liter of the culture solution were measured. As a result, Euglena having a paramylon content of 40.9% was obtained as shown below.
Paramylon was quantified by removing bacterial lipids with acetone and then deproteinizing with sodium dodecyl sulfate aqueous solution (1%).
The obtained glucan was dissolved in sodium hydroxide (2N) and quantified by the phenol-sulfuric acid method, and the protein was quantified by the micro Kjeldahl method.

[0012]

[Table 1] Table 1 ─────────────────────────── Cell yield 8.2 g / l Protein 45.2% Paramylon 40.9% (Dry basis) ─────────────────────────── Example 1 In the above Euglena culture example, further operation was continued after 48 hours of culture. It was That is, first, Euglena was cultured as in the above culture example, and the pH of the medium was changed to 1.0 to 8.5 48 hours after the start of the culture using a 2N sodium hydroxide aqueous solution or 2N sulfuric acid. After 48 hours of culture, the pH of the medium was controlled to each pH, and the cells were collected 72 hours after the start of culture to recover the protein content,
The paramylon content was measured. The results are shown below.

[0013]

[Table 2] Table 2 ─────────────────────────────────── pH after 48 hours of protein content Paramylon Content ─────────────────────────────────── 1.0 44.9 41.2 1.5 55. 8 30.9 2.0 56.9 29.1 2.5 55.0 29.8 3.0 53.1 32.6 3.5 53.9 33.1 4.0 49.0 38.3 4 .5 45.1 40.2 5.0 43.9 43.2 5.5 51.0 34.2 6.0 55.4 29.7 6.5 59.0 24.1 7.0 59.3. 24.3 8.0 58.6 25.0 8.5 45.0 40.6 (%) ─────────────────────────── ───────── As mentioned above, Kicking the medium pH is 1.5 to 3.
Euglena cultivated at 5, or 5.5 to 8.0 had lower paramylon and higher protein than those shown in the culture example.

Example 2 Euglena was cultured in substantially the same manner as in the culture example. However, the medium pH during the culture was 4.5 until 48 hours and 6.5 after 48 hours. Then, during the culturing after the 48th hour, a nitrogen source was added at the following settings.

[0015]

[Table 3] Table 3 ─────────────────────────────────── Addition nitrogen source Addition time Addition amount pH control ─────────────────────────────────── 1st section Potassium nitrate 48 hours 0.5% sodium hydroxide 2nd section Ammonium sulfate 48 Time 0.5% 〃 3 wards Ammonium chloride 48 hours 0.5% 〃 4 wards Sodium glutamate 48 hours 0.5% 〃 5 wards − − 〃 6 wards − − Ammonia water ─────────────── ─────────────────────

[0016]

[Table 4] Table 4 ─────────────────────────── Protein content Paramylon content (%) ─────────── ─────────────── 1st ward 48.4 34.9 2nd ward 68.6 15.5 3rd ward 66.9 18.0 4th ward 65.7 19.7 5th ward 47 1 35.6 6 wards 74.1 11.8 ────────────────────────── As mentioned above, the addition of nitrogen source makes paramylon more effective. It was possible to obtain cells with a low protein content but a high protein content. In particular, by performing pH control with ammonia water during the culture, high protein cells can be obtained without adding a separate nitrogen source. This is a result of the fact that ammonia water added for the purpose of pH control acts as a nitrogen source. Further, it is understood that the effect of the present invention cannot be sufficiently expected when the added nitrogen source is nitrate nitrogen, probably because Euglena cannot utilize nitrate nitrogen as a nitrogen source. In addition, in this example, ammonia nitrogen was not detected in the medium at the 48th hour except for the 6th section. In the medium of the 6th section, the ammonia nitrogen concentration was almost constant during the culture period.

[0017]

As is apparent from the above examples, the method for culturing Euglena according to the present invention can provide Euglena cells with high protein simply and efficiently as compared with the conventional culture methods. Further, cells having a protein content of 60% or more, which are difficult to obtain by the conventional method, can be obtained without requiring any special device.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shozaburo Kitaoka 17-11 Oya Funakanakamachi, Kawachinagano City, Osaka Prefecture

Claims (3)

[Claims] 1. A pH of the medium is 1.5 to 3.5 or 5.5 to 8 while contacting Euglena cells containing paramylon in an amount of 30% or more (dry matter weight ratio) with assimilable nitrogen compounds in the medium. A method for culturing Euglena, characterized in that it is carried out in the range of 0.0. 2. The method according to claim 1, wherein the assimilable nitrogen compound is contained in the medium in an amount of at least 10 ppm or more. 3. The method according to claim 2, wherein the assimilable nitrogen compound is either an ammoniacal nitrogen compound or an amino acid.