JPH0671430B2 - Method for producing β-1,3-glucan by Euglena cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing β-1,3-glucan (paramylon) by Euglena cells.

Conventional technology and its problems In recent years, Euglena cells have been attracting attention from a nutritional viewpoint. That is, the amino acid composition of the protein contained in Eunagel cells is similar to that of milk protein, casein, and has a high digestibility, and the biological value and the net protein utilization efficiency are almost the same as those of casein from the results of experiments by rats. In addition, it is reported that they are superior in value to algae, chlorella, spirulina, etc., which have been conventionally studied (Kitaoka, Hosoya; Agricultural Journal, 51 (8), 477-482 (1977). : ib
id; Agricultural magazine, 51 (8), 483-488 (1977)). In addition, for the production of seedlings in aquaculture, the results of feeding euglena cells to juvenile fish are also being examined. In particular, it has been pointed out that the highly unsaturated fatty acids of the ω3 series found in the vitamins and fatty acid composition contained in Euglena cells at a high content may be useful as a feed for juvenile fish (Sato, Yoshinaka; Aquaculture) , 32 (2), 88-91 (1984)).

However, all of these nutritional studies and studies are focused on proteins and trace components such as vitamins and fatty acids contained in Euglena cells, and β-1, which Euglena cells specifically accumulate, No studies have been done on increasing the yield of 3-glucan.

The physiological usefulness of β-1,3-glucan as a food and the immunological usefulness as an immunostimulator have been reported (Mizuno, Sakamura, Chemistry and Biology, 23 (12). ),
797-802 (1986)), the production method reported in this method uses cells that are difficult to manage and basidiomycetes that require a long period of time for cultivation. Therefore, β-1,3-glucan is produced. The efficiency is low and therefore expensive to manufacture.

In addition, as a polysaccharide that accumulates in Euglena cells, β-1,3-
Although it was known to contain glucan, the conventional culture flask method used to
The content of -1,3-glucan is about 70%, and the production of β-1,3-glucan per medium carbon amount is only 0.83g per 1g carbon.
It was about.

Means for Solving the Problems The inventors of the present invention have conventionally conducted various studies focusing on the usefulness of Euglena cells. As a result, a method for culturing Euglena cells with an increased protein content has been completed, and a patent has already been applied (Japanese Patent Application No. 60-160044). The present inventor, while further researching, the carbon concentration in the medium,
By adjusting the ratio of carbon and nitrogen, pH of the medium, temperature, dissolved oxygen concentration, etc. to specific values, the yield of Euglena cells and the β-1,3-glucan content in Euglena cells are increased, and It was found that the production efficiency of β-1,3-glucan can be dramatically increased. That is, the present invention, the carbon concentration in the initial medium 2-12g /, the atomic weight ratio of carbon and nitrogen (C /
N) 10-30, pH during growth 3-6, temperature 25-34 ℃,
Β-1,3-by Euglena cells characterized by culturing Euglena cells with a dissolved oxygen concentration of 0.1-1 ppm
The present invention relates to a method for producing glucan.

The method of the present invention comprises Euglena gracilis, Euglena
It can be applied to any species of the genus Euglena such as euglenaids such as viridae and euglena intermedia, and wild strains that grow in rivers and marshlands.

The medium components used in the present invention may be ordinary ones, for example, monosaccharides as carbon sources, disaccharides, molasses, corn starch, saccharides such as yeast extract; amino acids, acetic acid, citric acid, succinic acid, malic acid, Organic acids such as lactic acid; alcohols such as methanol and ethanol and carbon dioxide gas are used, and ammonium sulfate, ammonium phosphate, amino acids and the like are used as nitrogen sources. Further, as inorganic salts, MgSO ₄ , MgCO ₃ , MgCl ₂ , CaCO ₃ , KH ₂ PO ₄ , Na ₂ EDTA, F
eSO ₄ (NH ₄ ) ₂ SO ₄ , MnSO ₄ , ZnSO ₄ , (NH ₄ ) ₆ Mo ₇ O ₂₄ , CuS
O ₄ , NH ₄ VO ₃ , CoSO ₄ , H ₃ BO ₃ , NiSO _4, etc. or hydrates of these inorganic salts are used. As the vitamin, cyanocobalamin, thiamine hydrochloric acid and the like are used. A typical example of such a medium is shown in Table 1.

In the present invention, the carbon concentration in the initial medium in the batch system is 2 to 12 g /, and the atomic weight ratio of carbon and nitrogen (C / N) is 10 to 30.
The concentrations of the other components are the same as those of the medium that is usually used, and after sterilization, seed cells of Euglena are seeded and the culture is started. The carbon concentration in the initial medium is
It is less than 2 g /, is insufficient carbon amount, the cell concentration in the culture stable period does not reach 15 to 20 × 16 ⁶ cells / ml. on the other hand,
If it exceeds 12 g /, denaturation of the medium components after sterilization is observed, and cell growth is significantly inhibited. When the C / N ratio is less than 10, the cumulative content of β-1,3-glucan is low, and when it exceeds 30, the cell proliferation is decreased. The concentration of cells to be inoculated is about 1 × 10 ⁵ to 1 × 10 ⁶ cells / ml, and a stable period is reached about 4 to 5 days after the inoculation. As the number of cells to be seeded increases, the stable period is reached within a short time. The cell type is β
As long as it has a metabolic system that accumulates -1,3-glucan, it does not matter whether it is a wild strain or a mutant strain. The pH of the medium in the culture is about 3-6. When the pH is less than 3, the proliferation of cells decreases and the content of β-1,3-glucan decreases, and when the pH exceeds 6, the content of β-1,3-glucan increases, Poor cell growth. The pH is adjusted with an acid such as hydrochloric acid or sulfuric acid or an alkaline agent such as sodium hydroxide, potassium hydroxide, calcium hydroxide or aqueous ammonia. The temperature during culture is 24 to 34 ° C.
When the temperature is outside the range of 24 to 34 ° C, the growth of Euglena cells is also inhibited. A culture temperature of about 30 ° C. is particularly suitable. The oxygen concentration in the culture (hereinafter referred to as DO) needs to be in the range of 0.1 to 1 ppm. DO
Is less than 0.1 ppm, Euglena cells are hardly proliferated due to a decrease in oxygen concentration that affects respiratory action,
On the other hand, when it exceeds 1 ppm, intracellular β-1,3-glucan production decreases and protein production increases. Oxygen is supplied by aerating air or oxygen gas.

It is desirable to carry out the culture in the darkest possible state.
When the light illuminance increases, the photosynthetic function becomes active, the amount of protein produced increases, and the amount of β-1,3-glucan produced decreases relatively.

EFFECTS OF THE INVENTION According to the present invention, 75-83% on the basis of dry Euglena cells
The degree of β-1,3-glucan was obtained, and the amount of carbon in the medium was
The amount of β-1,3-glucan produced per gram reaches about 1.15 to 1.25 g.

Examples Hereinafter, examples and will be shown to further clarify the features of the present invention.

Example 1 Euglena gracilis was cultured in the dark under the conditions shown in Table 2 below. The number of seeded cells is 1 x 10
It was ⁵ / ml. The results 4 days after planting are shown in Table 3.

In Tables 2 and 3, No. 1 is the result of using a Streptomycin-treated Cogrena gracilis mutant strain (Kitaoka, Hosoya; Agricultural Journal, 51 (8), 477-482 (197).
7): ibid; Agricultural Journal, 51 (8), 483 to 488 (1977)), which is a method that is said to show the highest β-1,3-glucan production efficiency in the past. Also, No. 3, 5, 7, 9, 10
And 11 are methods in which any one of the culture conditions is outside the scope of the present invention. It is clear that excellent effects are obtained in the methods Nos. 2, 4, 6 and 8 according to the present invention.

Example 2 In the method of No. 4 in Table 2 of Example 1, culture was performed under the same conditions except that DO was changed.

The results are as shown in FIG. In FIG. 1, the curve (A) shows the amount of β-1,3-glucan produced, and the curve (B) shows
Represents the amount of protein produced, and curve (C) represents the amount of lipid produced. The production amount of β-1,3-glucan is particularly large in the DO range of 0.1 to 1 ppm, and the amount thereof rapidly decreases as the DO amount increases.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the amount of dissolved oxygen in the medium and the amounts of β-1,3 and glucan produced.

Claims (1)

[Claims] 1. The initial concentration of carbon in the medium is 2 to 12 g /, the atomic ratio of carbon and nitrogen (C / N) is 10 to 30, and the pH during growth is 3
-6, the temperature is 25 to 34 ° C, the dissolved oxygen concentration is 0.1 to 1 ppm, and Euglena cells are cultured, and a method for producing β-1,3-glucan by Euglena cells.