JPH0398574A - Production of euglena and device therefor - Google Patents

Abstract

PURPOSE:To reduce shear stress and to improve yield of algae by spraying and feeding a gaseous component required for culture toward the periphery of the bottom of a culture tank at the lower part of the culture tank to culture euglenas and stirring the culture solution in such a way that the culture tank is rotated along the inner wall of the culture tank. CONSTITUTION:A ring-shaped gas spray pipe 2 having plural nozzle holes to spray and feed a gaseous component (e.g. air) required for culture toward the periphery of bottom of a culture tank 1 is equipped at the lower part of the culture tank of euglenas. Then the culture tank 1 is charged with a culture solution and euglenas, culture is carried out while a gaseous component required for culture is sprayed and fed from a gas spray pipe 2 toward the periphery of the bottom of the culture tank 1 and the culture solution is stirred in such a way that the culture solution is rotated along the inner wall of the culture tank by a stirring rotary blade 9. Consequently, culture can be carried out by dispersing algae by spraying power of gas while collecting the algae at the periphery of the bottom of the culture tank and uniformly dispersing the algae by stirring rotary blade 9.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention uses a large amount of Euglena which produces highly unsaturated fatty acids such as arachidonic acid and EPA, vitamins C and E, β-carotene, proteins and polysaccharides. The present invention relates to a method for cultivating Euglena and an apparatus therefor for culturing Euglena and using these useful components as food materials, feed materials, pharmaceutical materials, etc.

(Prior Art) Euglena is a plant classified into the phylum Euglena, and also an animal belonging to the phylum Protozoa and the subfamily Phenoflagellate, and is a protist that belongs to both animals and plants.

In addition, Euglena grows under a variety of growth conditions and is useful as a material organism for comparative nutrition studies. Attempts are being made to cultivate the plant for its use.

Cultivation of Euglena is old, and was introduced by Professor Kitaoka et al. in the Journal of the Japanese Society of Agricultural Chemistry, Noka, Vol. Can be seen.

Specifically, Japanese Patent Application Laid-Open No. 196184/1984 discloses that by aerating high-concentration oxygen during the culture of the protist Euglena, the production or secretion of waxy substances, mucilage substances, etc. is suppressed.
A method for cultivating Euglena has been proposed for the purpose of promoting the concentration of nutritional components such as unsaturated fatty acids and vitamin E.

In addition, Japanese Patent Application Laid-open No. 61-37091 discloses that by continuously culturing Euglena cells while adjusting and supplying carbon and nitrogen concentrations, the same culture time can be achieved compared to the conventional batch culture method. It has been shown that 1.5 to 10 times as many Euglena cells can be changed using this method, and that the intracellular protein content of Euglena cells can be changed to the same level as conventional ones.

Furthermore, Japanese Patent Application Laid-Open No. 61-40785 discloses a method for culturing Euglena cells that combines culturing in a closed system in the dark and culturing in an open system under light irradiation.

(Problem to be solved by the invention) According to the knowledge of Professor Kitaoka et al., the yield of Euglena algae in batch culture using a jar fermenter is about 8 g as a dry weight per 12 medium, It is predicted that growth was poor due to shear stress.

As described above, there is a problem in that in the culture tank, Euglena is subjected to shear stress due to collisions with the stirrer itself and turbulence of the culture solution caused by the stirrer, causing shear stress, which inhibits growth and makes it impossible to increase yield.

Furthermore, all of the conventional techniques cited in JP-A-60-196184, JP-A-61-37091, and JP-A-61-40785 solve growth inhibition of Euglena due to shear stress. There is no mention of.

However, in order to achieve stable cultivation of Euglena, a solution is needed to eliminate the above-mentioned shear stress and to achieve an average distribution of bacterial cells in the culture medium in order to provide even nutritional support. Therefore, it is necessary to set the conditions for large-scale cultivation of the culture tank, including the stirring device and gas supply method.
The shape of the stirring blade, its rotation speed, the aeration method, the amount of aeration, etc. must be designed in consideration of the influence on the growth rate, algae yield, and products.

In light of the above, an object of the present invention is to provide a method and apparatus for culturing Euglena which can be cultured in a stable state without shear stress.

(Means for Solving the Problems) In order to achieve the above objects, the present invention provides the following Euglena culturing method and apparatus.

That is, a method for culturing Euglena characterized by supplying a certain amount of gas necessary for culture by jetting it toward the bottom of the culture tank below the culture tank, and stirring the culture solution so as to rotate it along the inner wall of the culture tank. The device for carrying out this method consists of an annular gas ejection pipe provided with a plurality of nozzle holes provided below the culture tank to eject a certain amount of gas necessary for culture toward the bottom of the culture tank, and a culture solution placed on the inner wall of the culture tank. This is a Euglena culture device equipped with a rotating stirring blade that rotates along the following directions.

In addition, in such a device, the stirring rotary blade has a radial blade protruding from the rotating shaft in a radial direction, and a radial blade at the tip of the radial blade.
It is a Euglena culture device that is composed of inclined blades inclined at an angle of 5 to 55 degrees, with the radial blades having a small area and the inclined blades having a large area.

Furthermore, in the above device, the rotation speed of the stirring device is set to 50 to 1.
00rplI+, and the gas supply amount is 0.2 to 1. Ovv
This is a culturing device for Euglena m.

(Function) According to the present invention, the flow of the culture solution rises in a swirling manner on the inner wall surface of the culture tank by the stirring rotary blade, and descends along the circumference of the rotation axis, which naturally creates vertical agitation. be done.

In addition, the algae that tend to accumulate around the bottom of the culture tank are dispersed by the gas ejection force, so they can be dispersed without applying mechanical shear stress.

The algae dispersed by the gas jet force are rotated by the rotating blades, and are smoothly spread throughout the tank by rising along the inner wall of the tank and descending near the axis.
Unbalanced consumption of nutrients can be avoided.

(Example) Hereinafter, the present invention will be explained based on embodiments shown in the drawings.

In Figure 1, (1) is a culture tank, and this culture tank (
Below 1), there is an annular gas ejection pipe (2) that ejects and supplies a certain amount of gas necessary for culture towards the bottom of the culture tank, and from the air bomb (3) to the pipe (4) and the flow meter (5).
Air is jetted through a pipe (7) with a filter (8) through a nozzle hole opening around the annular gas jet pipe (2).

The nozzle hole is opened facing the bottom of the tank, and the gas ejected from the nozzle hole can disperse the algae that tend to accumulate around the bottom of the culture tank.

The culture tank (1) also has rotating blades (9) for stirring that rotate the culture solution along the inner wall of the culture tank (1a).
) located nearby. The stirring rotary blade (9) is rotated from the stirrer (10) via its shaft (10a).

In Fig. 1, (11) is an air vent pipe, and there is a filter (12) in this pipe. And filters (8) (12)
Both are sterilizing filters for preventing bacterial contamination.

The specific structure of the stirring rotary blade (9) according to the present invention is shown in FIGS. 2 and 5 (a).

That is, the stirring rotary blade (9) includes a radial blade (9a) projecting in the radial direction from the rotating shaft (10a) and an inclined blade (9b) inclined at an angle of 35 to 55 degrees with the radial blade at the tip thereof. The radiating blade (9a) has a small area and the inclined blade (9b) has a large area.

Since the stirring rotary blade (9) is configured as described above, when the blade rotates in the direction of the arrow in Fig. 5 (opening), the inclined blade (9b) consisting of a large area part will rotate against the stirring direction of the culture solution. It comes into contact with the culture solution at an angle, and the culture solution flows into the culture tank (
1) The shearing force between the culture solution and the stirring blade can be reduced by acting so as to be pushed toward the peripheral wall.

Further, since the rotating blade (9b) has an angle, it is possible to configure the algae to slide on the blade surface without contacting or colliding with the blade surface from a perpendicular direction.

Furthermore, since the radial blade (9a) with a small area constitutes the rotating stirring blade (9) together with the above-mentioned inclined blade (9b), the flow of the culture solution rises to swirl around the inner wall surface of the culture tank. However, the vertical agitation that descends along the rotating shaft (10a) is created naturally, and nutrients can be evenly supplied to the algae.

The culture tank according to the present invention is capable of culturing a large amount of Euglena which produces highly unsaturated fatty acids such as arachidonic acid and EPA, vitamins C and E, β-carotene, proteins and polysaccharides, Incidentally, the rotation speed of the stirring device is 50 to 100 rpm+, and the gas supply amount is 0.2 to 100 rpm.
1. It is cultivated as Ovvm.

According to the present invention, the algae that tend to accumulate around the bottom of the culture tank can be dispersed by the gas jet force, and the rotating blades do not contact or collide with the algae from the right angle direction, and the algae Because it is made to slide,
It can reduce the shear stress of Euglena and double the algal yield compared to conventional culture tanks.

Now, with the same culture period, medium, rotational speed of the blade, and gas supply amount, the flat blade shown in Figure 3 (a) (opening) and the flat blade shown in Figure 4 (a)
(a) according to the present invention.
When I compared it with the special feather shown in (mouth), I got the following results.

Table 1 shows the results of 1.5 liters of 0.1% xanthan gum solution filled into a 2 liter transparent culture tank and the stirring abilities of flat blades, slanted blades, and special blades.

[Table 1] Time until the pigment becomes uniform Modified Hutn with 0.5% monosodium glutamate
Filled with 1.52ml of er medium, inoculated with 5% of the pre-cultured Euglena gracilis Z strain, and at an initial pH of 3.3.
Dark place 25°CS stirring number 6Orpm, ventilation amount 0.25V
Tables 2 and 3 show the number of Euglena algae counted using a hematometer after culturing in vm for 7 days.

0.1% xanthan gum t volume 1.51/21! Fermenter, 2d addition of dye, 60 rpm or more Table 1 shows the agitation capacity of flat blades, slanted flat blades, and special blades in the main culture tank, in each case, when adding dye to xanthan gum solution, the dye becomes uniform. This is a comparison table showing the results of measuring the time taken. From this table, it can be seen that the time required for the dye to become uniform is extremely short in the case of the present invention. Incidentally, the rotation speed of each blade is the same, and the total amount of solution is also the same.

3% glucose in a transparent container with culture tank 21, Table 2 shows flat blade, inclined flat blade, with constant linear velocity applied to the culture solution.
This is a comparison of the number of algae bodies and the yield of algae bodies for each case of special blades.

Here, it can be seen that the yield of algae bodies is greatly different between the flat blade 1 (Fig. 3) and the special blade (Fig. 5), and it can also be seen that there is no effect even if the flat blade is tilted (Fig. 4).

Furthermore, it can be predicted that flat blades are likely to give shear stress to the algae and have little effect on the destruction of the algae in the culture solution.

Table 3 shows that installing flat blades in multiple stages not only has no effect but also reduces yield.

Therefore, it can be seen that by attaching a plurality of rotating blades to the rotating shaft, the culture solution does not move in the upper and lower parts of the culture tank, and stirring in the inner cylindrical shape has no effect, and stirring is easier with one stage of flat blades.

In addition, when the special blade rotates and stirs, the culture medium inside the blade flows toward the inner wall of the culture tank, causing the culture medium to rise, and a downward flow is generated around the rotating shaft, causing vertical agitation. It has been demonstrated that this method can evenly disperse the algae in the culture solution, increasing the number of algae and the yield of algae.

FIG. 6 is a diagram representing Table 2, with the yield of Euglena algae on the horizontal axis and the number of Euglena algae on the vertical axis.

Below, in Figures 6, 7, and 8, ○ indicates a flat blade, △ indicates an angled flat roof, and the mouth indicates a special blade.

Compared to the case of flat blades, the number of algae on special blades is about five times higher, and the yield of algae is about twice as high.

The results show that the difference in the number of algae bodies is larger than the difference in the yield of algae bodies, which is thought to reflect the greater effect of reducing shear stress.

Fig. 7 is a diagram showing algae yield on the horizontal axis and time on the vertical axis.The yield differs in each culture device at the same culture time, and those using the stirring blade (special blade) of the present invention are The yield was higher than the other two examples with feathers, and the stirring conditions of the culture solution were good, indicating that nutrients and algae were in sufficient contact.

Figure 8 is a diagram showing time on the horizontal axis and the number of Euglena algae on the vertical axis.Although the difference in the number of algae in each culture device seems small, the vertical axis is Log (or logarithmic value).
So the reality is very different. It can be seen here that the situation of reducing sharing stress is good.

Although the present invention is applicable to all species belonging to the genus Euglena according to the zoological classification, the representative species is Euglena gracilis + varbac.
Examples include P. illaris and P. viridis.

Furthermore, in the industrial scale of the culture according to the present invention, it is conceivable to perform fed-batch culture, supply oxygen, and add glucose, monosodium glutamate, inorganic salts, vitamins, etc. alone or in combination as a medium. Furthermore, in this example, batch culture is the cheapest method, and this method has been used to promote mass culture, but the method and device of the present invention can also be used in other culture methods, etc. with the basic configuration. By doing the same, you can eliminate shearing stress and improve culture.

(Effects of the Invention) According to the present invention, the stirring flow forms a laminar flow state along the wall surface of the culture device, the shearing force is reduced, and the algal bodies are precipitated.
Moreover, the algal bodies are prevented from accumulating around the bottom of the culture tank and are evenly dispersed in the culture solution.

In addition, algae that tend to accumulate around the bottom of the culture tank are dispersed by the gas ejection force, so they can be dispersed without applying mechanical shear stress.

Furthermore, since the large area portion of the stirring blade comes into contact with the culture solution at an angle to the stirring direction of the culture solution, and the culture solution is pushed out toward the peripheral wall of the culture tank,
The shearing force between the culture solution and the stirring blade can be reduced, and since the rotating blade has an angle, the algae can be configured to slide on the blade surface without contacting and colliding from the direction perpendicular to the blade surface. .

Therefore, sharing stress can be reduced.

The flow of the culture solution rises in a swirling manner on the inner wall surface of the culture tank and descends along the circumference of the rotation axis, creating a natural vertical agitation system, which has the effect of evenly supplying nutrients. Coupled with the above-described reduction in shear stress, the algae yield can be doubled compared to conventional culture tanks.

[Brief explanation of drawings]

Figure 1 is an overall view of the apparatus for implementing the method of the present invention, Figure 2 is a sectional view of the culture tank, Figures 3 (a) and (b) are slope views and plan views of the flat blades, and Figure 4 (a). (Opening) is a slope view and a plan view of the inclined flat blade. Figure 5 (A) (Opening) is a slope view and a top view of the stirring blade according to the present invention. Figure 6 is the number of Euglena algae bodies and the yield of algae bodies. Figure 7 is a diagram showing the yield of Euglena algae, and Figure 8 is a diagram showing the number of Euglena algae. (1)・ (2)・ (3)・ (5)・ (9)・ (9a)・ (9b)・ (10)・

Claims (4)

[Claims] (1) A method for cultivating Euglena characterized by supplying gaseous components necessary for culture by jetting them toward the bottom of the culture tank at the bottom of the culture tank, and stirring the culture solution so as to rotate along the inner wall of the culture tank. . (2) An annular gas ejection tube provided with a plurality of nozzle holes below the culture tank to eject gas components necessary for culture toward the bottom of the culture tank and stir the culture solution so as to rotate along the inner wall of the culture tank. Euglena cultivation device equipped with rotating blades for stirring. (3) The stirring rotary blade consists of a radial blade that protrudes radially from the rotation axis and an inclined blade that is inclined at an angle of 35 to 55 degrees with the radial blade at the tip of the radial blade. A claim consisting of a large area (2
) Euglena culturing device described. (4) Claim (2)(
The Euglena culturing device according to any one of 3).