JP2021166510A - Euglena culture method - Google Patents

Abstract

To provide a technique for culturing Euglena in large scale culture.SOLUTION: A Euglena culture method of the invention comprises: a conditioning step for preparing a plurality of kinds of conditioning culture media, where the conditioning culture media are formed of low concentration culture medium in which glucose concentration is prescribed concentration from 0% to 2%, and high concentration culture medium in which glucose concentration is greater than 2% and equal to or less than 8%, namely the concentration is prescribed condition, and among the conditioning culture media, glucose concentration is different, and sequentially using the conditioning culture media for culturing Euglena, in an order of having low glucose concentration in the conditioning culture media; and a growth step for culturing the Euglena acquired in the conditioning step, using a growth culture medium in which, the glucose concentration is equal to that of the high concentration culture medium, or the glucose concentration is greater than that of the high concentration culture medium and is equal to or less than 8%.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for culturing Euglena.

Euglena gracilis, a type of microalgae, is about 50 μm long and about 10 μm wide, and is a creature that belongs to both plants and animals. Euglena, also known as Euglena, photosynthesizes organic compounds from water and carbon dioxide to release oxygen.

Euglena is used as a food additive and dietary supplement because of its high nutritional value. In addition, paramylon, which is one of the substances produced by Euglena, is used as a raw material for nanofibers, and wax ester (an ester compound consisting of alcohol and carboxylic acid having more than a dozen carbon atoms) is expected to be used as a fuel. There is.

U.S. Patent Application Publication No. 2003/0180898 JP-A-63-71192

Ogbonna, JC., Tomiyama, S., Tanaka, H., Heterotrophic cultivation of Euglena gracilis Z for efficient production of alpha-tocopherol. Journal of Applied Phycology 10, 67. Doucha, J., Livansky, K., (2011), Production of high-density Chlorella culture grown in fermenters. J Appl Phycol, 24, 35-43. Swaaf, ME., Sijtsma, L., Pronk, JT., (2003), High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii. Biotechnol Bioeng, 81, 666-672. Schmidt, RA., Wiebe, MG., Eriksen, NT., (2005), Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria. Biotechnol Bioeng, 90, 77-84. 29-36 .. Ganuza, E., et al., (2008), High-cell-density cultivation of Schizochytrium sp. In an ammonium / pH-auxostat fed-batch system. Biotechnology Letters. 30, 1559-1564.

In order to realize the industrial and commercial use of the substances produced by Euglena, a technique for stably and mass-culturing Euglena having an excellent ability to produce the target substance is required.

Generally, when culturing a large amount of microorganisms, a high-density culture method is used in which the microorganisms are densely packed in a culture tank and cultured. The culturing ability in high-density culturing can be expressed, for example, by the amount of microorganisms per unit volume that can be cultivated at one time (this is referred to as the maximum biomass yield). The maximum biomass yields of microalgae in high-density culture reported so far are 117.2 g / L for chlorella vulgaris, 109.0 g / L for Cryptecodiniumu cohnii, and Gardieria (Cryptecodiniumu cohnii). Galdieria sulphurariaha) is 116.0 g / L and Thraustochytriales is 221.0 g / L, while Euglena gracilis is 48.2 g / L, which is higher than other microalgae. Cultivation conditions have been sought for which the maximum algae yield is low (Patent Documents 1 and Non-Patent Documents 1 to 4) and the maximum algae yield of Euglena can be increased. Here, the biomass yield is expressed by the dry weight of microalgae per unit volume.

The problem to be solved by the present invention is to provide a technique for mass-culturing Euglena.

The method for culturing Euglena according to the present invention, which was made to solve the above problems, is
A glucose consisting of a low-concentration culture medium having a glucose concentration of a predetermined concentration between 0% and 2% and a high-concentration culture medium having a glucose concentration of more than 2% and a predetermined concentration of 8% or less. An acclimatization step in which multiple types of acclimatization culture solutions having different concentrations are prepared, and Euglena is cultured using the acclimatization culture medium in order from the acclimatization culture medium having the lowest glucose concentration.
It has a growth step of culturing Euglena obtained in the acclimation step using a growth culture solution having the same glucose concentration as the high-concentration culture solution, or higher than the high-concentration culture solution and 8% or less. It is characterized by that.

The method for culturing Euglena according to the present invention, which was made to solve the above problems, is
A low-concentration culture medium having a glucose concentration of a predetermined concentration between 0% and 2%, a high-concentration culture medium having a glucose concentration higher than 2% and a predetermined concentration of 8% or less, and a glucose concentration Prepare a plurality of types of acclimation culture solutions having different glucose concentrations, which consist of an intermediate concentration culture solution having a concentration higher than that of the low-concentration culture solution and a concentration lower than that of the high-concentration culture solution. The acclimation step of culturing Euglena using the acclimation culture solution in order from the solution,
It has a growth step of culturing Euglena obtained in the acclimation step using a growth culture solution having the same glucose concentration as the high-concentration culture solution, or higher than the high-concentration culture solution and 8% or less. It is characterized by that.

In the above method for culturing Euglena, the intermediate concentration culture solution may consist of a plurality of types of culture solutions having different concentrations.

Generally, the higher the concentration of a carbon source such as glucose contained in the culture solution, the higher the density of microalgae can be cultivated, and the higher the biomass yield. However, when euglena is cultivated, the concentration of glycol that can be contained in the culture solution is at most about 2%, and it is known that if the glucose concentration is increased further, the growth rate will decrease or growth inhibition will occur. Was being done. On the other hand, the present inventor gradually increases the glucose concentration contained in the Euglena culture medium, so that Euglena having resistance to the culture medium having a high glucose concentration, that is, to the high-concentration glucose can be obtained. I found it to appear. The present invention has been made based on such findings. In the present invention, making Euglena acquire resistance to high-concentration glucose is referred to as "acclimation".

That is, in the present invention, as the conditioned culture medium, a low-concentration culture medium having a glucose concentration of a predetermined concentration between 0% and 2% and a predetermined culture medium having a glucose concentration of more than 2% and 8% or less. Prepare a high-concentration culture solution having a high concentration. Alternatively, as the acclimation culture solution, one or a plurality of types of intermediate concentration cultures, which are a low-concentration culture solution and a high-concentration culture solution, and a glucose concentration higher than that of the low-concentration culture solution and lower than that of the high-concentration culture solution. Prepare the liquid. Then, by culturing Euglena using the acclimation culture solution in order from the acclimation culture solution having the lowest glucose concentration, the glucose concentration becomes higher than 2%, which is the glucose concentration in the culture solution that the conventional Euglena can tolerate. Familiarize Euglena. Euglena obtained in the acclimation step is cultivated in the growth step using a growth culture solution having the same glucose concentration as the high-concentration culture solution, or higher than the high-concentration culture solution and 8% or less. Multiply. In a strict sense, the "predetermined concentration of 8% or less" does not include a concentration exceeding 8%, but a concentration slightly higher than 8% is included as long as it is considered to be substantially the same. do.

For example, by setting the glucose concentration of the low-concentration culture medium to 1.0% and the glucose concentration of the high-concentration culture solution to 2.5%, Euglena acquires resistance to a glucose concentration of at least 2.5%. For example, when the glucose concentration of the low-concentration culture solution is 1.0%, the glucose concentration of the intermediate-concentration culture solution is 2.0%, and the glucose concentration of the high-concentration culture solution is 3.0%, Euglena is resistant to a glucose concentration of at least 3.0%. Acquire. Further, for example, when the glucose concentration of the low-concentration culture solution is 2.0%, the glucose concentration of the intermediate-concentration culture solution is one or more kinds of concentrations of 2.2% to 4.8%, and the glucose concentration of the high-concentration culture solution is 5.0%, Euglena. Acquires resistance to glucose concentrations of at least 5.0%. The applicant refers to Euglena (sugar-tolerant Euglena), which has acquired resistance to a culture medium having a high glucose concentration by the acclimation process, as a "hyper strain". By creating a hyper strain, even if Euglena is cultured in a culture medium having a glucose concentration higher than 2% and 8% or less in the growth step, the growth rate is reduced or the growth is inhibited. Euglena can be grown stably and efficiently without any problem.

The acclimation culture medium used in the acclimation step includes a low-concentration culture solution having a glucose concentration of 2% and a high-concentration culture solution having a glucose concentration of 5%, and a plurality of types of intermediate-concentration culture solutions having a glucose concentration of 2% to 5%. It should consist of liquid. At this time, if the glucose concentration of the intermediate concentration culture solution in which the glucose concentration is between 2% and 5% is set to a value obtained by equally dividing the range from 2% to 5%, the acclimation culture used in the acclimation step is used. It is preferable in that the glucose concentration of the liquid can be increased linearly, but the present invention is not limited to this.

Further, when the glucose concentration of the high-concentration culture solution is 5%, if the glucose concentration of the growth culture solution is a predetermined concentration between 5% and 8%, Euglena can be efficiently grown.

Further, in the present invention, it is preferable to have a selection step for selecting Euglena having a high growth rate from the Euglena obtained in the acclimatization step between the acclimatization step and the proliferation step. This makes it possible to increase the growth rate of Euglena in the growth step.

By the way, in the case of Euglena, which already has resistance to a culture medium having a glucose concentration higher than 2%, a growth culture medium having a glucose concentration higher than 2% is used from the beginning of the culture without going through the acclimation step. Can be cultivated. Therefore, in the method for culturing Euglena according to the present invention, Euglena can be cultured using a growth culture solution having a glucose concentration of more than 2% and 8% or less. The present invention can also be applied to a method for producing Euglena. That is, the method for producing Euglena according to the present invention is characterized in that Euglena is cultured and propagated using a growth culture solution having a glucose concentration of more than 2% and 8% or less. ..

Euglena (sugar-tolerant euglena) having resistance to a culture medium having a glucose concentration of more than 2% can be produced by the method for producing a sugar-tolerant euglena of the present invention. The method for producing a sugar-tolerant Euglena of the present invention comprises a low-concentration culture medium having a glucose concentration of a predetermined concentration between 0% and 2%, and a predetermined concentration having a glucose concentration of more than 2% and 8% or less. It has a step of preparing a plurality of types of acclimation culture broths having different glucose concentrations, which are composed of high-concentration culture broth, and culturing Euglena using the acclimation culture broth in order from the acclimation culture broth having the lowest glucose concentration. Or a low-concentration culture medium having a predetermined concentration of glucose between 0% and 2%, and a high-concentration culture medium having a glucose concentration higher than 2% and a predetermined concentration of 8% or less. , Multiple types of acclimation culture broths having different glucose concentrations, which consist of an intermediate concentration culture broth having a glucose concentration higher than that of the low-concentration culture broth and a concentration lower than that of the high-concentration culture broth, are prepared. It has a step of culturing Euglena using the acclimation culture solution in order from the lowest acclimation culture solution.

In addition to the above, sugar-tolerant euglena may be obtained by selecting and separating euglena that satisfies a predetermined condition from natural euglena collected from natural fresh water or seawater, and the natural euglena may be obtained. Alternatively, it may be a natural variant that appears while continuing the subculture of Euglena obtained from a microbial preservation institution. In addition, sugar-resistant Euglena can be artificially produced by inducing mutations by a known method or by using techniques such as gene recombination and genome editing.

According to the present invention, Euglena can be stably and efficiently mass-cultured.

The schematic diagram of the acclimatization process in Example 1 of this invention. The graph which shows the time change of the biomass yield of 6 kinds of Euglena resistant strains and unacclimated strains. The graph which shows the biomass yield and the intracellular composition of 6 kinds of Euglena resistant strains. The graph which shows the biomass yield and the intracellular composition of 6 kinds of Euglena unacclimated strains. The graph which shows the relationship between the culture solution amount and the biomass yield in Example 2. FIG. The graph which shows the influence on the growth of a culture solution volume. The graph which shows the relationship between the pH of a culture solution and a biomass yield. The graph which shows the influence on the growth of the pH of a culture solution. The graph which shows the relationship between the C / N of a culture solution and a biomass yield. The graph which shows the influence on the growth of C / N of a culture solution. The graph which shows the relationship between the glucose concentration in a culture solution and the weight of a dry alga in Example 3. FIG. The graph which shows the influence which the glucose concentration of a culture solution has on growth. The graph which shows the relationship between the turbidity and sugar concentration of the culture solution, and the culture time in Example 4. FIG. Photographs of resistant strains after culturing in dark and light conditions.

The present invention is characterized in that Euglena is cultured using a growth culture medium having a glucose concentration of more than 2% and 8% or less. Specifically, the method for culturing Euglena according to the present invention includes a low-concentration culture medium having a predetermined glucose concentration between 0% and 2%, and a glucose concentration higher than 2% and 8% or less. Prepare a plurality of types of acclimation culture broths having different glucose concentrations, which consist of a high-concentration culture broth having a predetermined concentration of The acclimation step and the Euglena obtained in the acclimation step are cultured using a growth culture medium having the same glucose concentration as the high-concentration culture medium, or higher than the high-concentration culture medium and 8% or less. It has a breeding step.

In addition, the method for culturing Euglena according to the present invention includes a low-concentration culture medium having a glucose concentration of a predetermined concentration between 0% and 2%, and a predetermined culture medium having a glucose concentration of more than 2% and 8% or less. Multiple types of acclimation cultures having different glucose concentrations, consisting of a high-concentration culture medium having a high concentration and an intermediate-concentration culture medium having a glucose concentration higher than that of the low-concentration culture solution and lower than that of the high-concentration culture solution. The acclimation step of preparing a solution and culturing Euglena using the acclimation culture solution in order from the acclimation culture solution having the lowest glucose concentration, and the euglena obtained in the acclimation step are referred to as the high concentration culture solution. It has a growth step of culturing using a growth culture solution which is the same or higher than the high-concentration culture solution and is 8% or less.

The Euglena used in the culture method of the present invention is typically Euglena gracilis of the genus Euglena, but other species may be used as long as it is of the genus Euglena. In addition, Euglena may be collected from natural freshwater or seawater such as lakes, ponds, and paddy fields, or may be obtained from a microbial preservation organization. Furthermore, Euglena collected from natural fresh water or seawater, or Euglena obtained from a microbial preservation institution may be subcultured, and mutations can be induced by known methods, or techniques such as gene recombination and genome editing can be used. You may use the mutants artificially created by using.

As the acclimation culture solution, a culture solution generally used for culturing microalgaes such as Euglena can be used in which an appropriate amount of glucose is added to adjust the glucose concentration.

Hereinafter, examples of the present invention will be described.
[Example 1]
1. 1. The acclimation process FIG. 1 is a schematic view showing the procedure of the acclimation process of this embodiment. In this example, six strains of Euglena gracilis (hereinafter referred to as Euglena) shown in Table 1 below were used.

In the acclimation step, 2N (normal) sodium hydroxide or 2N hydrochloric acid is added to the medium A to adjust the pH to 4.5, and then an appropriate amount of glucose in the range of 20 g / L to 50 g / L is added. A culture medium was used in which the glucose concentration was adjusted to 2.0% to 5.0% by addition. The composition of medium A and the compositions of solution D and solution E contained in medium A are shown in Tables 2, 3 and 4, respectively. As the D solution, a solution to which 5N hydrochloric acid was added until it became transparent was used. In this specification, the glucose concentration (%) means solute weight / volume% (W / V%).

In the acclimation step, six flasks with 100 mL capacity baffles are prepared, 20 mL of the above-mentioned culture solution is put into each, and after sterilization, a predetermined amount of each of the above-mentioned six types of Euglena gracilis strains is inoculated (inoculation). Also called). After culturing this under rotary shaking for 72 hours under the conditions of a temperature of 28 ° C., a stirring speed of 110 rpm, and darkness, 1 mL of a culture solution containing Euglena grown from the culture solution was collected and placed in another flask. Subculture was carried out under the same conditions and at the same time.

In the acclimatization step, the initial glucose concentration was set to 2.0%, and the glucose concentration in the culture solution was increased by 0.2% each time the subculture was performed. Then, when the glucose concentration in the culture solution reached 5.0%, the culture was terminated (that is, if the culture in the same flask was one cycle, the culture was terminated in the 15th cycle). The strain obtained by the above acclimatization step will be referred to as a "resistant strain" below.

On the other hand, as a comparative step, 6 kinds of Euglena strains were cultured under the same conditions and for the same time as the above-mentioned acclimatization step except that a culture solution having a glucose concentration of 2.0% was used. The strains obtained in this comparison step will be referred to as "unconditioned strains" below.

2. Selection of strains with high proliferative capacity
(1) 1 mL each of the culture solution after the acclimation step (that is, the culture solution containing the resistant strain) and the culture solution after the comparative step yield (that is, the culture solution containing the unacclimated strain), 1.5 mL volume tube (Eppendorf) Transferred to (made in Japan) and diluted 100,000 times with physiological saline.
(2) 100 μL of the diluted solution was inoculated on an agar medium (medium A, glucose concentration 5.0%) in a culture dish and spread evenly with a spreader.
(3) One week later, the colonies that appeared on the agar medium were observed, and the colonies with the fastest growth rate were collected, and these were selected strains of 6 types of Euglena resistant strains and unconditioned strains, respectively.

3. 3. Propagation step Six types of Euglena-resistant strains were selected with a glucose concentration adjusted to 5.0%, and six types of Euglena unconditioned strains were selected with a glucose concentration adjusted to 2.0%. In each case, the culture was continued for 72 hours under the same conditions as the above-mentioned acclimatization step, and the 1.0 mL culture solution collected from the culture solution was subcultured into a new culture solution having the same glucose concentration. At the time of subculture, a predetermined amount of culture solution was collected as a sample from a flask with a baffle, and used for measuring the biomass yield (dry algae weight per 1 L), residual sugar amount, and intracellular component content, which will be described later.

4. Biomass yield measurement
(1) 1.0 mL of the culture solution collected in the growth step was placed in a constant volume of 1.5 mL Eppendorf tube and centrifuged (5000 rpm, 5 minutes).
(2) After centrifugation, the supernatant was removed, 1.0 mL of 0.8% physiological saline was added thereto, and the mixture was stirred using a vortex mixer.
(3) Centrifugation (5000 rpm, 5 minutes) was performed again to remove the supernatant.
(4) The steps (2) and (3) were repeated again.
(5) The Eppendorf tube was heated, and the cells (Euglena cells) in the tube were dried (105 ° C., 12 hours).
(6) After putting the Eppendorf tube in a desiccator and allowing it to cool, the weight of the Eppendorf tube was measured, and the initial weight of the Eppendorf tube was subtracted from the weight to calculate the dry cell weight of the cells.

5. Measurement of residual sugar amount The concentration of glucose contained in 1.5 mL of the culture solution collected at the time of subculture was measured using the Glucose C-II Test Wako Kit (Mutarose / GOD method, manufactured by Wako Pure Chemical Industries, Ltd.). The measurement wavelength was set to 550 nm.

6. Measurement Results of Biomass Yield and Residual Sugar Amount FIG. 2 is a graph showing the temporal changes in biomass yield of 6 types of Euglena resistant strains and 6 types of Euglena unconditioned strains. The horizontal axis of these graphs represents the culture time, and the vertical axis represents the dry weight of the cells.

As can be seen from FIG. 2, it was confirmed that the resistant strains proliferated for a longer time than the unconditioned strains in all 6 types of Euglena, but the growth rate differed greatly depending on the strain type. Specifically, among the resistant strains, 4 strains of NIES48, PO, SM-ZK, and IG reached the stationary phase after 96 hours of culturing, and the biomass yield did not change much after that, whereas NIES47 was cultivated. 120 hours and 144 hours of culture reached the steady phase of NIES49. Among the resistant strains, the three resistant strains NIES48, PO, and SM-ZK showed the same growth rate as the unconditioned strain at 48 hours of culture, but the three strains NIES47, NIES49, and IG showed the same growth rate. The growth rate of the resistant strain was lower than that of the unconditioned strain at 48 hours of culture.

Table 5 shows the biomass yields of the six Euglena resistant strains and the unconditioned strains with respect to sugar. The biomass yield to sugar yield means the ratio of the biomass yield after culturing to the amount of glucose in the culture broth at the start of culturing. When the amount of glucose in 1 L of the culture solution at the start of the culture is 1 g and the biomass yield after the culture is 1 g, the yield to sugar is 100%.
As can be seen from Table 5, no significant difference was confirmed between the resistant strains and the unconditioned strains, but a large difference was observed between the resistant strains and the unconditioned strains. That is, in the unacclimated strain, the sugar yield was 50% or more in all the strains, whereas in the resistant strain, the sugar yield was in the range of 32% to 42%. The yield on sugar was lower than that of the unconditioned strain.

7. Measurement of intracellular components When the growth reached the early stage of the stationary phase or the late stage of the logarithmic growth phase, the culture solution was collected in a 50 mL volume centrifuge tube. The collected culture broth was centrifuged (3000 rpm, 5 min), the supernatant was removed to obtain cells, which were lyophilized and subjected to the analysis of the following intracellular components. For unacclimated strains, the culture broth for 72 hours was collected, and for resistant strains, the growth rate differed depending on the strain, so the recovery time of the culture broth was set as follows. That is, the NIES49-resistant strains were cultured for 144 hours, and the NIES47, NIES48, NIES49, PO, SM-ZK and IG-resistant strains were cultured for 96 hours.

7-1. Measurement of paramylon The content of paramylon contained in 2.0 mL of the culture solution collected at the time of subculture was measured by the following procedure.
A. Purification of paramylon
(1) 2 mL of the culture solution was placed in a 2 mL volume Eppen tube, centrifuged (5000 rpm, 5 min), and the supernatant was removed.
(2) 300 μL of ion-exchanged water was added, and the cells were completely dispersed by stirring with a vortex mixer, and the cells were lightly spun down.
(3) A float was attached to the Eppen tube and floated on an ultrasonic cleaner to crush the cells (5 min).
(4) 1200 μL of acetone was added, and the mixture was sufficiently stirred with a vortex mixer and centrifuged (5000 rpm, 5 min).
(5) The supernatant was carefully removed with Pipetman (registered trademark), 1500 μL of acetone was added, the mixture was sufficiently stirred with a vortex mixer, and the mixture was centrifuged (5000 rpm, 5 min).
(6) Acetone treatment and centrifugation were repeated again.
(7) The supernatant was removed with Pipetman, 1500 μL of a 10% SDS aqueous solution was added, and the cells were sufficiently dispersed with a vortex mixer and lightly spun down.
(8) With the Eppen tube cap open, it was heated for 30 minutes on an aluminum block heater at 100 ° C.
(9) After allowing to cool, the cells were centrifuged (5000 rpm, 5 min), the supernatant was removed with Pipetman, 1500 μL of 0.1% SDS aqueous solution was added, and the cells were sufficiently dispersed with a vortex mixer.
(10) Centrifugation (5000 rpm, 5 min) was performed, the supernatant was carefully removed with Pipetman, 1500 μL of ion-exchanged water was added, and the cells were sufficiently dispersed with a vortex mixer. From the above, paramylon was purified.
(11) 1 mL of 1 N sodium hydroxide solution was added to the purified paramylon and completely dissolved, and this was used as the next sample for quantification of paramylon.

B. Quantification of paramylon (phenol sulfuric acid method)
(1) 900 μL of ion-exchanged water was added to 100 μL of the above-mentioned quantitative sample for dilution.
(2) 750 μL of ion-exchanged water was added to 250 μL of a glucose solution of 200 μg / mL to prepare a standard sample having a glucose concentration of 50 μg / mL.
(3) 25 μL of 80% phenol solution was added to the diluted quantitative sample and standard sample, and the mixture was sufficiently stirred with a vortex mixer.
(4) 2.5 mL of concentrated sulfuric acid was added in the draft, and the mixture was sufficiently stirred with a vortex mixer.
(5) After allowing to cool, the absorbance was measured at an absorption wavelength of 490 nm.

C. Calculation method of paramylon content The paramylon content (μg) was calculated from the following formula (1). In equation (1), E represents the absorbance of the sample and E0 represents the absorbance of the standard sample. In formula (1), "180.16" is the molecular weight of glucose contained in the standard sample, and "162.14" is the molecular weight of paramylon, which is a polysaccharide obtained by dehydration condensation of glucose and β-1,3 binding.
Paramylon content (μg) = E / E 0 x 50 (μg / mL) x 1 (mL) / (180.16 / 162.14) x dilution ratio
… (1)

7-2. Quantification of other components The Kjeldahl method was used for the quantification of proteins (Patent Document 2). As the lipid, the revised Folch method (Non-Patent Document 5) was used.

8. Measurement results of intracellular components 8-1. Paramylon content-to-sugar yield Table 6 shows the paramylon-to-sugar yields of the six Euglena-resistant strains and unconditioned strains. The paramylon content-to-sugar yield means the ratio of the paramylon content after culturing to the amount of glucose in the culture broth at the start of culturing. When the amount of glucose in the culture solution at the start of culture is 1 g and the paramylon content after culture is 1 g, the yield to sugar is 100%.
As can be seen from Table 6, the yield of sugar to sugar in the paramylon content was higher in the resistant strain than in the unconditioned strain in the two strains of PO and SM-ZK, but the three strains of NIES47, NIES48 and NIES49. Showed the same value in the resistant strain and the unconditioned strain. In addition, the paramylon content of IG, which had the lowest biomass yield to sugar in the resistant strain, showed a lower value in the resistant strain than in the unacclimated strain.

8-2. Ratio of each component to biomass yield FIGS. 3 and 4 show the maximum biomass yield and intracellular composition of 6 types of Euglena resistant strains and unconditioned strains. The horizontal axis of FIGS. 3 and 4 shows the name of the type of Euglena, and the vertical axis shows the value of the intracellular composition and the maximum biomass yield (g / L). That is, the height of each bar graph represents the maximum biomass yield of each type.

As can be seen from the comparison between FIGS. 3 and 4, the maximum biomass yield of the resistant strain was about 1.5 to 2.0 times the maximum biomass yield of the unacclimated strain in all six types of Euglena. In addition, the NIES48-resistant strain showed a maximum biomass yield of 20 g / L or more, and Tukey's multiple comparison test showed a significantly higher value than IG (p <0.05), but with other strains. No significant difference was observed.

Regarding the paramylon content and the paramylon content, the resistant strains showed higher values than the unacclimated strains in all six types of Euglena. In particular, among the 6 types of Euglena, the PO and SM-ZK resistant strains had a paramylon content of 50% or more, which was significantly higher than that of the other 4 types of resistant strains (p <0.05). ..
From the above results, it can be said that the culture method of this example is useful as a method for culturing Euglena for the purpose of obtaining paramylon.

In all six Euglena, the protein content of the resistant strain was higher than that of the unconditioned strain, but the protein content of the resistant strain was lower than that of the unconditioned strain. On the other hand, regarding the lipid content, the resistant strain was higher than that of the unacclimated strain, but regarding the lipid content, no significant difference could be confirmed between the resistant strain and the unacclimated strain.

In this example, the culture solution having a glucose concentration of 2.0% used in the acclimation step was used as the low-concentration culture solution of the present invention, and the culture solution having a glucose concentration of 2.2% to 4.8% was used as the intermediate-concentration culture solution. A culture medium having a glucose concentration of 5.0% corresponds to a high-concentration culture medium, but from a different point of view, a culture medium having a glucose concentration of 2.0% should be regarded as a low-concentration culture medium, and a culture medium having a glucose concentration of 2.2% should be regarded as a high-concentration culture medium. Can be done. In this case, the culture medium having a glucose concentration of 2.2% or 2.4% corresponds to the growth culture medium. If the culture medium having a glucose concentration of 2.0% is a low-concentration culture medium, the culture medium having a glucose concentration of 4.0% is a high-concentration culture solution, and the culture medium having a glucose concentration of 2.2% to 3.8% is an intermediate-concentration culture solution, the glucose concentration is 3.8% or 4.0. % Corresponds to the culture medium for growth. That is, from the results of this example, it can be seen that Euglena having a glucose concentration higher than 2% and resistance to a culture solution of 5% or less can be produced.

[Example 2]
In order to examine the culture conditions in the growth step of the present invention, the amount of the culture solution, the pH of the medium, and the ratio (C / N) of the carbon source (C) and the nitrogen source (N) contained in the medium were different. Resistant strains were cultivated using a plurality of types of culture media. As the resistant strain, a resistant strain (selected strain) of NIES48 obtained in the acclimation step of Example 1 was used.
1. 1. Effect of liquid volume In the growth step of Example 1, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, and 30 mL of the culture solution having the same composition as the culture solution used for the resistant strain were placed in separate flasks with baffles. A resistant strain of NIES48 was cultured under the same conditions as the growth step of Example 1. After continuing the culture for 24 hours, the work of substituting 1.0 mL of the culture solution collected from the culture solution into a new culture solution of the same amount (passage) was repeated. At the time of subculture, each culture solution was collected as a sample and used for _{measuring the biomass yield (dry algae weight per 1 L) and turbidity (OD 660} ). The turbidity is an index showing the growth rate, and the larger the turbidity, the more the resistant strain has grown (the higher the growth rate).

FIG. 5 shows the relationship between the amount of culture medium and the biomass yield. As can be seen from the figure, the highest biomass yield was shown in the test plot with a culture solution volume of 5 mL, which was significantly higher than the other test plots as a result of Tukey's multiple comparison method (p). <0.05). On the other hand, the test group with the culture solution volume of 30 mL showed the lowest biomass yield, and no significant difference was observed between the test group with the culture solution volume of 25 mL, but the other test groups. The value was significantly lower than that of (p <0.05). In other words, the smaller the amount of culture medium, the higher the biomass yield tended to be. It is considered that the smaller the amount of the culture solution, the larger the amount of air supplied into the culture solution, and as a result, the biomass yield increased.

In addition, FIG. 6 shows the result of _{OD 660 measured every 24 hours.} As can be seen from the figure, _{it was confirmed that OD 660} increased in almost the same manner in all the test plots and proliferated in the same manner up to 48 hours of culture. _{After 72 hours of culturing, the OD 660} values tended to level off and growth stagnated in the test plots with 25 mL and 30 mL of culture solution _{, but in the other test plots, OD 660} was still present after 72 hours of culturing. It showed a tendency to increase. From the above, it was suggested that the smaller the amount of the culture solution, the better the growth in the late stage of the culture.

2. Effect of pH In the growth step of Example 1, the pH of the culture solution having the same composition as the culture solution used for the resistant strain was adjusted to 2.5, 3.5, 4.5, 5.5, 6.5 and 7.5, respectively, and each culture solution was baffled. The strain was placed in a flask and a resistant strain of NIES48 was cultured under the same conditions as in the growth step of Example 1. After continuing the culture for 24 hours, the work of substituting 1.0 mL of the culture solution collected from the culture solution into a new culture solution of the same amount was repeated, and at the time of substituting, 5 mL of the culture solution was collected as a sample, and the biomass yield was obtained. , Used for measurement of turbidity (OD _660). In addition, 5 mL of the culture solution after culturing for 72 hours, 96 hours, and 120 hours was collected, centrifuged (6000 rpm, 5 min), respectively, and then the supernatant was collected and the pH was measured.

FIG. 7 shows the relationship between the pH of the culture solution and the biomass yield. As can be seen from the figure, the highest biomass yield was shown in the test plot with a pH of 7.5. As a result of Tukey's multiple comparison method, there was no significant difference between the test group with pH 7.5 and the test group with pH 6.5, but there was a significant difference between the other test groups. (P <0.05). In addition, no significant difference was observed between the test groups having a pH of 3.5 and 4.5, and the test group having a pH of 2.5 showed a significantly lower value than the other test groups. From the above, it was suggested that the higher the pH of the test plot, the higher the biomass yield tended to be.

In addition, FIG. 8 shows the result of _{OD 660 measured every 24 hours.} As can be seen from the figure, in the test plots having pHs of 3.5, 4.5, 5.5, 6.5 and 7.5, the steady state was reached and the growth stagnated after 96 hours of culturing. The test group having a pH of 2.5 had a slower growth rate than the other test groups, and tended to grow even after reaching 120 hours of culturing.
Table 7 shows the pH of the initial culture solution and the pH of the culture solution after culturing. As can be seen from this table, in all the test groups in which the pH of the initial culture solution was 2.5 to 6.5, the pH after culturing was lowered to 3.0 or less. In addition, the pH after culturing decreased to 3.59 in the test group having a pH of 7.5 in the initial culture solution, and the pH after culturing decreased to 1.77 in the test group having a pH of 2.5. From the above, it was shown that the pH after culturing tended to be biased toward acidity in all the test plots.

3. 3. Effect of C / N In the growth step of Example 1, polypeptone was added as a nitrogen source to the culture solution having the same composition as the culture solution used for the resistant strain without changing the amount of glucose, and C / N was added to 10 and 20, respectively. , 30, 40, 50 and the culture test was carried out. In the growth step of Example 1, the C / N of the culture solution used for the resistant strain was 22.9.

FIG. 9 shows the relationship between the C / N of the culture solution and the biomass yield. As a result of Tukey's multiple comparison, the C / N40 test group showing the highest biomass yield was not significantly different from the C / N50 test group, but the other test groups (C). A significant difference was observed as compared with / N10, 20, 30). In addition, the higher the C / N of the test plot, the higher the biomass yield tended to be (p <0.05).

In addition, regarding the paramylon content and content of each test group, as a result of Tukey's multiple comparison, a significant difference was observed among all test groups, and the higher the C / N of the test group, the higher the paramylon content tended to be. (P <0.05).

Further, FIG. 10 shows the result of _{OD 660 measured every 24 hours.} As can be seen from the figure, at 48 hours of culture, _{the value of OD 660} was lower in the C / N10 test group than in the other test groups, but at 96 hours of culture, the C / N10 test was performed. The value of OD ₆₆₀ was the highest in the plot, and _{the value of OD 660} was the lowest in the C / N 50 test plot. Therefore, in the latter half of the culture, the lower the C / N, the higher the value of _{OD 660 tended to be.}

Table 8 shows the relationship between the amount of residual sugar contained in the supernatant after culturing and C / N. As can be seen from this table, glucose remained in the culture medium in all test groups at 106 hours of culture. In addition, at 120 hours of culturing, the glucose concentration was only about 1% in the test groups having C / N of 10, 20, and 30, but the glucose concentration was 3% in the test group of C / N 40, and C / It was confirmed that 7% of the glucose concentration remained in the test group having N of 50. Furthermore, at 144 hours of culturing, glucose in the culture broth was depleted in the test plots other than C / N50. From the above, it was shown that the lower the C / N in the test group, the faster the sugar consumption, and the higher the C / N, the slower the sugar consumption.

Table 9 shows the quantitative results of nitrogen and phosphorus contained in the supernatant of the C / N40 test group, which showed the highest biomass yield. From this table, it was found that the supernatant contained 0.13 mg / ml of nitrogen and 0.15 mg / ml of phosphorus at 106 hours of culturing. This corresponds to 26.0% and 55.6% of the nitrogen and phosphorus contents contained in the initial culture medium.

[Example 3]
The resistant strains in the growth step were cultured under the same conditions as in Example 1 except that the glucose concentration contained in the culture solution was adjusted to 6%, 7%, 8%, 9%, and 10%. As the resistant strain, a resistant strain (selected strain) of NIES48 obtained in the acclimation step of Example 1 was used.

Similar to Examples 1 and 2, after continuing the culture for 24 hours, the work of substituting 1.0 mL of the culture solution collected from the culture solution into a new culture solution of the same amount is repeated, and the culture solution is collected at the time of substituting. Then, the weight of the dried algae was measured. The culture was terminated when the glucose in the culture solution was depleted.

FIG. 11 shows a graph showing the relationship between the glucose concentration and the dry algae weight per 1 L (biomass yield (g / L)), and FIG. 12 shows the temporal change of _{OD 660.} As can be seen from FIG. 11, the test group having the highest glucose concentration showed the highest value in the dry algae weight, and a significant difference was observed from the other test groups. The test group with the lowest glucose concentration showed the lowest dry algae weight, and no significant difference was observed between the test groups with glucose concentrations of 8% and 10%, but other groups. There was a significant difference from the test plot.

FIG. 12 is a growth curve represented by a value of _{OD 660.} FIG. 12 also shows a growth curve at a glucose concentration of 2% for comparison of growth rates. From FIG. 12, when the glucose concentration was 6%, the growth rate was almost the same as that of the culture solution having a glucose concentration of 2%, and when the glucose concentration was 7%, the growth rate was slightly slower than that of the culture solution having a glucose concentration of 2%. .. In addition, there was almost no growth in the test group having a glucose concentration of 8 to 10%.

[Example 4]
The NIES48 resistant strain (selected strain) obtained in the acclimation step of Example 1 was subjected to a growth step of culturing using a culture solution having a glucose concentration adjusted to 8%. The composition of the culture broth adjusted to 8% glucose concentration is shown in Table 10 below.

In this example, 50 mL of the above culture solution was placed in a flask with a baffle having a capacity of 100 mL, and about 1 mL of the culture solution containing the NIES48 resistant strain obtained in the acclimation step of Example 1 was placed therein (the glucose concentration was 8%). 2% equivalent amount of the culture solution L) was added, and rotary shaking culture was carried out under the conditions of a temperature of 28 ° C., a stirring speed of 100 rpm and a dark condition for 458 hours. After starting the culture, collect the culture solution at an appropriate timing to determine its turbidity (OD ₆₆₀ ), residual sugar amount (sugar concentration), biomass yield (g / L) (dry algae weight per 1 L). It was measured. The method for measuring the residual sugar amount and the biomass yield is as described in Example 1.

Table 11 shows the turbidity (OD ₆₆₀ ), sugar concentration, biomass yield (g / L), and sugar yield (%) of the culture solution together with the culture time. In addition, FIG. 13 is _{a graph showing the turbidity (OD 660} ) of the culture solution and the temporal change of the sugar concentration.

As can be seen from Table 11 and FIG. 13, almost no change in turbidity was observed up to 95 hours of culture, but the turbidity began to gradually increase after 95 hours of culture, and became turbid after 212 hours of culture. The degree increased significantly, and proliferation of resistant strains was observed. That is, in Example 3, the growth of the resistant strain in the test group of the culture solution having a glucose concentration of 8% was not observed until the culture time was 216 hours, but in this example, the culture time exceeded 95 hours. Growth of resistant strains was observed from the above, and the growth rate increased especially after 216 hours of culturing. In addition, the sugar concentrations at 361 hours, 406 hours, and 458 hours of culture were 7.32%, 6.37%, and 6.07%, respectively, and the consumption of glucose in the culture solution also supported the growth of resistant strains. From the above results, it was confirmed that resistant strains proliferate even when a culture medium having a glucose concentration of 8% or more is used. In addition, when using a culture medium having a glucose concentration of 8% or more, it is speculated that a longer growth step is more effective in growing resistant strains than when using a culture medium having a glucose concentration of less than 8%. Was done.

On the other hand, the biomass yields of 406 hours of culture and 458 hours of culture were both 10.0 g / L, and the yields with respect to sugar were 59.6% and 50.6%. From this result, it was considered that the growth of the resistant strain reached almost the stationary phase in about 406 hours of culture.

In addition, the same NIES48-resistant strain used for the above-mentioned rotary shaking culture was planted on an agar medium having a glucose concentration adjusted to 8% and cultured under dark and light conditions, and all of them grew. Observed. Furthermore, the greening function (photosynthetic function) of the resistant strain was not lost in either the dark condition or the light condition. FIG. 14 shows resistant strains after culturing under dark and light conditions.

From the above results, it was found that the glucose concentration of the culture solution of the resistant strain is preferably 5 to 8%. In addition, it is possible to produce Euglena having resistance to a culture solution having a glucose concentration of 5% or more and 8% or less, which is the same as or higher than the glucose concentration (5%) of the high-concentration culture solution used in the acclimation step. I understand. Further, although the detailed reason is unknown, as can be seen from the results of Examples 3 and 4, even if the culture medium has the same glucose concentration, the growth start time and the growth rate of the resistant strain may vary depending on the conditions of the growth step. It makes a difference. For this reason, when actually culturing Euglena in large quantities, it is desirable to use a content solution having an appropriate glucose concentration according to the culture conditions such as temperature and medium composition.

Although the embodiments of the present invention have been described in detail with reference to specific examples, the present invention is not limited to the above-described examples, and various modifications are possible. For example, in Example 1, Euglena is acclimated to a culture solution having a glucose concentration of 5% or more by culturing Euglena in a plurality of types of acclimation culture solutions having a glucose concentration of 2% to 5% by an acclimatization step. However, if the euglena (sugar-tolerant euglena) has resistance to a culture medium having a glucose concentration of 5% or more, the acclimation step can be omitted. Sugar-tolerant Euglena is produced by a production method comprising a step of sequentially culturing Euglena in a plurality of types of acclimation culture mediums ranging from 2% to 5%, which comprises a culture solution having a glucose concentration of 2% and a culture solution having a glucose concentration of 5%. Can be manufactured. In addition, sugar-tolerant euglena may be obtained by selecting and separating euglena that meets predetermined conditions from natural euglena collected from natural fresh water or seawater, and is obtained by natural euglena or a microbial preservation institution. It may be a natural variant that appeared while continuing the subculture of Euglena obtained from. In addition, sugar-resistant Euglena can be artificially produced by inducing mutations by a known method or by using techniques such as gene recombination and genome editing.

Claims (10)

A glucose consisting of a low-concentration culture medium having a glucose concentration of a predetermined concentration between 0% and 2% and a high-concentration culture medium having a glucose concentration of more than 2% and a predetermined concentration of 8% or less. An acclimatization step in which multiple types of acclimatization culture solutions having different concentrations are prepared, and Euglena is cultured using the acclimatization culture medium in order from the acclimatization culture medium having the lowest glucose concentration.
It has a growth step of culturing Euglena obtained in the acclimation step using a growth culture solution having the same glucose concentration as the high-concentration culture solution, or higher than the high-concentration culture solution and 8% or less. , Euglena culture method. A low-concentration culture medium having a glucose concentration of a predetermined concentration between 0% and 2%, a high-concentration culture medium having a glucose concentration higher than 2% and a predetermined concentration of 8% or less, and a glucose concentration Prepare a plurality of types of acclimation culture solutions having different glucose concentrations, which consist of an intermediate concentration culture solution having a concentration higher than that of the low-concentration culture solution and a concentration lower than that of the high-concentration culture solution. The acclimation step of culturing Euglena using the acclimation culture solution in order from the solution,
It has a growth step of culturing Euglena obtained in the acclimation step using a growth culture solution having the same glucose concentration as the high-concentration culture solution, or higher than the high-concentration culture solution and 8% or less. , Euglena culture method. In the method for culturing Euglena according to claim 2,
A method for culturing Euglena, wherein the intermediate concentration culture solution comprises a plurality of types of culture solutions having different concentrations. In the method for culturing Euglena according to claim 1,
The culturing solution for acclimation is a low-concentration culture solution having a glucose concentration of 2%, a high-concentration culture solution having a glucose concentration of 5%, and a plurality of types of intermediate-concentration culture solutions having a glucose concentration of 2% to 5%. A method for culturing Euglena, including. In the method for culturing Euglena according to claim 4,
A method for culturing Euglena, wherein the glucose concentration of the plurality of types of intermediate concentration culture solutions is set to a value obtained by equally dividing the range from 2% to 5%. In the method for culturing Euglena according to claim 4 or 5.
A method for culturing Euglena, wherein the glucose concentration of the growth culture solution is a predetermined concentration between 5% and 8%. In the method for culturing Euglena according to any one of claims 1 to 6.
A method for culturing Euglena, which comprises a selection step of selecting Euglena having a high growth rate from Euglena obtained in the acclimatization step between the acclimatization step and the growth step. A method for producing Euglena, which comprises culturing and growing Euglena using a growth culture medium having a glucose concentration of more than 2% and 8% or less. A glucose consisting of a low-concentration culture medium having a glucose concentration of a predetermined concentration between 0% and 2% and a high-concentration culture medium having a glucose concentration of more than 2% and a predetermined concentration of 8% or less. A method for producing sugar-tolerant euglena, which comprises a step of preparing a plurality of types of acclimation culture solutions having different concentrations and culturing euglena using the acclimation culture solution in order from the acclimation culture solution having the lowest glucose concentration. A low-concentration culture medium having a glucose concentration of a predetermined concentration between 0% and 2%, a high-concentration culture medium having a glucose concentration higher than 2% and a predetermined concentration of 8% or less, and a glucose concentration Prepare a plurality of types of acclimation culture solutions having different glucose concentrations, which consist of an intermediate concentration culture solution having a concentration higher than that of the low-concentration culture solution and a concentration lower than that of the high-concentration culture solution. A method for producing glucose-tolerant euglena, which comprises a step of culturing euglena using the conditioned culture solution in order from the solution.

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