JPH0532019B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for continuous culturing of Euglena. Conventional techniques and problems Euglena cells are unicellular eukaryotes,
In terms of biological taxonomy, it belongs to both the animal and plant categories. The most distinctive feature of cellular components is that they have both a plant-like aspect, in which they have chloroplasts, and an animal-like aspect, in that they are covered with a soft cell membrane. The amino acids that make up cell proteins are similar to animal proteins, so they are highly nutritious.Algae such as Chlorella have cell walls made of cellulose, whereas Euglena cells have cell membranes that are composed of cellulose as a tissue component. Since it does not contain cellulose and is composed of soft proteins and phospholipids, it is easily digested, and it also contains large amounts of vitamins A, C, and E compared to other algae. There is. Therefore, Euglena is seen as a promising future edible protein and vitamin source. The conventional method for culturing Euglena cells is a closed batch method. In this method, the nutrients required for culture are first dissolved, and a predetermined amount is fed into a culture tank. After sterilization using steam, a heating heater, etc., the solution is cooled to culture temperature using tap water, construction water, etc. do. Next, seed cells previously cultured in another culture tank are inoculated, and after culturing under predetermined conditions until the maximum number of cells is reached, the cell suspension is centrifuged to harvest the cells. In this batch culture method, Euglena cells are
After a lag phase, which is a period of medium adaptation, cells reach a logarithmic growth phase in which cells divide exponentially, and then reach a stable phase in which the rate of proliferation and death are equal, reaching the maximum cell number. This period is approximately 4-5 days. Considering sterilization, harvesting, cleaning, etc. before and after cultivation, it takes 6 to 7 days from seeding to harvesting, and after harvesting, it is necessary to introduce a new medium again and repeat the above steps from sterilizing the medium. is necessary. For this reason, the batch culture method has the disadvantage that the cell yield is low for the culture period. Means for Solving the Problems As a result of various studies in view of the above points, the present invention has been developed by separately sterilizing a culture medium, which is a nutrient solution, and continuously flowing it into a Euglena culture tank. Euglena cells can be harvested continuously over a long period of time by retaining them for a certain period of time and then draining them as a cell suspension, and the cell concentration at this time is close to the maximum cell concentration in the batch culture method. I found that. The present invention is based on this knowledge. That is, in the present invention, the cell concentration is 5×10 ⁶ cells/ml.
Within the period from the time when the growth stabilized to the second day, the carbon concentration was 1.2 to 8.0 g/, and the nitrogen concentration was 0.07 to 4.5.
The present invention relates to a method for continuous culturing Euglena, which is characterized in that a medium of 1.5 g/g/g of a medium is fed to a culture tank so that the average residence time is 5 to 96 hours. The method of the present invention is applicable to any species of the genus Euglena, such as euglenoids such as Euglena gracilis, Euglena viride, and Euglena intamideia, and wild strains growing in rivers, lakes, and marshes. In the present invention, it is desirable to use pure cultured Euglena cells as seed cells. The culture medium used in the present invention may be the same as that used in the batch method, for example, monosaccharides as the carbon source,
Disaccharides, polysaccharides; organic acids such as acetic acid, citric acid, succinic acid, malic acid, lactic acid; alcohols such as methanol and ethanol; various amino acids, carbon dioxide gas, etc. are used, and as nitrogen sources, amnonium sulfate, phosphoric acid, etc. Ammonium, ammonia, various amino acids, etc. are used. In addition, as inorganic salts,
_MgSO4 , _CaCO3 , _KH2PO4 , _{Na2EDTA ,}
FeSO ₄ (NH ₄ ) ₂ SO ₄ , MnSO ₄ , ZnSO ₄ ,
(NR ₄ ) ₆ Mo ₇ O ₂₄ , CuSO ₄ , NH ₄ VO ₃ , CoSO ₄ ,
H ₃ BO ₃ , NiSO ₄ , etc., or hydrates thereof are used, and cyanocobalamin, thiamine hydrochloride, etc. are used as vitamins. Representative examples of such media are shown in Table 1.

[Table] In the culture method according to the present invention, the cell number concentration is 5 × 10 ⁶ Cells/at the intermediate stage of the logarithmic growth phase of Euglena.
ml, the carbon concentration will increase within the period of 2 days after reaching the stable phase when the cell number reaches its maximum.
A previously sterilized culture medium having a nitrogen concentration of 1.2 to 8.0 g/ and a nitrogen concentration of 0.07 to 4.50 g/ is continuously fed into the culture tank using a liquid pump or pressurized outflow method. At this time, Euglena cells can be continuously harvested by keeping the average residence time of the medium in the culture tank within the range of 5 to 96 hours. If the carbon concentration is less than 1.2 g/2, the carbon source necessary for cell division will be insufficient, and the number of cells in the culture tank will rapidly decrease from the first to second day after the start of continuous culture. Carbon concentration is 8
When the concentration exceeds g/g, cells rapidly accumulate carbon sources and convert them into the storage polysaccharide paramylon, but on the other hand, division is severely inhibited, and the carbon sources remain in the effluent, reducing the cost of the medium. Undesirable due to gender.
In addition, if the average residence time is less than 5 hours, most of the cells will flow out from the culture tank, and if it exceeds 96 hours, the Euglena cells will enter a decay period and the cell number concentration in the tank will be 5 to 50%.
Since the amount is very small at 6×10 ⁶ cells/ml, the cell yield obtained is also comparable to that obtained by the batch culture method, so there is no merit. In the method of the present invention, the average residence time of the culture medium is 5 to
By culturing continuously for 96 hours, it is possible to increase the productivity of the batch culture method, which conventionally takes 6 to 7 days from seeding to harvest.
5 to 5 times the same amount of Euglena cells as the harvest amount
It can be harvested continuously in a 96 hour cycle. Therefore, compared to the conventional batch method, it is possible to obtain 1.5 to 10 times the amount of Euglena cells in the same culture time, and this intracellular protein content is
It is about the same level as the conventional batch method. In the present invention, the other conditions are not limiting factors for continuous culture, so normal culture conditions may be used. Preferred conditions are PH2-5, temperature 10-34℃,
The dissolved oxygen temperature of the culture solution is 1 ppm or higher, and stirring is done to the extent that the cells in the tank do not settle to the bottom.
If stirring by aeration is sufficient, mechanical stirring may not be necessary. Effects of the invention By culturing Euglena cells using the continuous culture method of the present invention, compared to the case of culturing using the conventional batch culture method,
1.5-10 times more Euglena cells will be obtained. Furthermore, the intracellular protein content of the obtained Euglena cells is comparable to that of conventional Euglena cells. EXAMPLES Next, the present invention will be explained in more detail with reference to Examples. Example 1 In 10 glass culture tanks, glucose 10g/(carbon concentration 4g/), ammonium sulfate 2g/(nitrogen concentration 0.2g/
), the other ingredients are the same as those in Table 1,
Continuous culture was carried out under the environmental conditions shown in Table 2. The average residence time of the culture medium in the culture tank is 3~
It was varied over a period of 120 hours.

[Table] Figure 1 shows the relationship between the number of culture days and the cell yield per culture tank. In the figure, average residence time is 9 hours and 10 hours, average residence time is 15 hours, average residence time is 20 hours, average residence time is 30 hours,
represents the average residence time of 40 hours, and represents the cases where the average residence time was 5 hours and 96 hours, respectively. Also,
The broken line shows the relationship between the number of culture days and cell yield using the batch method. In addition, in the case of the batch method, it is a straight line connecting the sum of the cell numbers obtained every 4 days. As a result, when the average residence time of the medium was 5 to 96 hours, the yield in the same number of culture days was about 1.5 to 10 times as much as the cell yield obtained by the batch culture method. In addition, the protein content in the cells was 50-65%, which was comparable to that of cells obtained by conventional batch culture. Example 2 Continuous culture was carried out for 4 days in 10 glass culture vessels using the same medium as shown in Table 1, with glucose concentration and ammonium sulfate concentration as variables, and other medium components as shown in Table 1. The average residence time of the culture solution was 20 hours, and the environmental conditions were as shown in Table 2. Carbon concentration in the medium and culture tank 1
The relationship with the cell yield per unit is shown in Figure 2. In the figure, A is the case where the nitrogen concentration is 0.07 to 4.5 g/, B is the case where the nitrogen concentration is 0.05 g/, C is the case where the nitrogen concentration is 5 g/, and the broken line is the case where the nitrogen concentration was 0.04 to 5 g/ and cultured by the batch method. Indicate the case. From FIG. 2, it is clear that when cultured according to the method of the present invention, the cell yield is higher than when cultured by the batch method. Also, the protein content in cells is 50-65
% was comparable to that of cells grown using the conventional batch culture method.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between the number of culture days and the cell yield per culture tank. FIG. 2 is a graph showing the relationship between carbon concentration in the medium and cell yield per culture tank. In Figure 1, the average residence time is 9 hours and 10 hours, the average residence time is 15 hours, the average residence time is 20 hours, the average residence time is 30 hours, the average residence time is 40 hours,
show the cases where the average residence time was 5 hours and 96 hours, respectively. Furthermore, the broken line indicates the case using the batch culture method. In Figure 2, A has a nitrogen concentration of 0.07 to 4.5.
g/B is when the nitrogen concentration is 0.05g/, C is when the nitrogen concentration is 5g/, and the broken line is when the nitrogen concentration is 0.04
This shows the case of culturing by batch method at ~5g/.

Claims (1)

[Claims] 1. Within the period from the time when the cell concentration reaches 5 × 10 ⁶ cells/ml to the second day of the stable growth phase, the carbon concentration
A continuous culturing method for Euglena, which comprises feeding a culture medium with a nitrogen concentration of 1.2 to 8.0 g/, and a nitrogen concentration of 0.07 to 4.5 g/ to a culture tank such that the average residence time is 5 to 96 hours.