JPS6232877A - Novel chlorella - Google Patents

Abstract

PURPOSE:To obtain a novel Chlorella capable of producing especially eicosapentaenoic acid in high concentration and having high rate of proliferation, by carrying out the cell-fusion of the protoplast of two kinds of Chlorella. CONSTITUTION:The objective novel Chlorella can be produced by the cell-fucion of (A) the protoplast of a specific Chlorella capable of producing a highly unsaturated fatty acid such as eicosapentaenoic acid and/or a lipid containing said fatty acid as a constituent component and growing in seawater or in a medium containing seawater, e.g. Chlorella minutissima and (B) the protoplast of the other kind of Chlorella having higher rate of proliferation than the Chlorella A and growing in a medium containing fresh water, e.g. Chlorella elipsoidia or Chlorella vlugaris. The obtained Chlorella has eicosapentaenoic acid productivity to >=1/2 of that of the Chlorella A and a rate of proliferation of >=2 times that of the Chlorella A.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel chlorella, particularly a novel chlorella produced by cell fusion of two types of chlorella protoplasts.

As is well known, Chlorella is a type of unicellular green algae that has photosynthetic activity, and naturally lives in both freshwater and seawater, and has also been artificially cultured. Chlorella cells contain protein with a good balance of amino acids, various minerals, vitamins, sugars, lipids, pigments, enzymes, etc., and have high nutritional value. It is used in the field of application.

For example, the fragile SCP (Sing) of Chlorella cells themselves
Ce1l Protein), lipids (including highly unsaturated fatty acids such as eicosapen citric acid), pigments (chlorophyll), etc. are used in health foods, animals, pets, rotifer feed, plant fertilizers, cosmetics, printing, and paints. Used in industrial inks, pharmaceuticals, etc. The present invention therefore relates to a new chlorella that can be used in a wide range of applications.

[Conventional technology]

As mentioned above, since Chlorella is industrially useful, it has been cultivated in various ways. For example, a large amount of chlorella cultured through artificial cultivation is collected, concentrated and sterilized, and then the chlorella cells are subjected to physical treatment such as high-pressure homogenizer, spray dry smoke, and ultrasonic waves to crush the chlorella cells. However, methods are used to extract useful components within cells directly or with water or solvents. However, since Chlorella is a type of algae, its growth rate is extremely slow compared to microorganisms, although there are some differences between species. There were problems in that it required a pool or a tank, and culture had to be continued for a long time.

Therefore, such culture is economically disadvantageous because it increases the cost of utilizing the useful components of Chlorella.

In the above culture, it is common to collect Chlorella in the natural environment, pure culture it by conventional methods, select it into a single strain or a complex strain, and use this. An example of artificially culturing Chlorella without a cell wall by discovering the following (Japanese Unexamined Patent Publication No. 144976/1983)
, an example of studying medium composition and culture method (Japanese Patent Application Laid-open No. 1983-
47476) have been seen only a few times in recent years.

In addition, Chlorella minutissima, a type of chlorella:
There are marine species and freshwater species of C. minutissima (hereinafter referred to as C. minutissima), and among these, marine C. minutissima contains eicosapencitrate in its cells, hereinafter abbreviated as EPA. ) and other highly unsaturated fatty acids and/or lipids containing these as constituent components. EPA is a component of fish lipids, and in recent years it has become clear that it has effects such as preventing arteriosclerosis and dissolving blood clots, and is attracting attention as a raw material for health foods and pharmaceuticals. However, breeding for the purpose of producing useful substances for C. minutissima and other species of Chlorella has not been carried out so far.

On the other hand, with the development of biotechnology, research on cell fusion targeting higher plants or microorganisms has been actively conducted, and methods for breeding useful microorganisms include yeast breeding method (Japanese Patent Application Laid-open No. 163883/1983), amino acid fermentation, etc. Protoplast fusion method of microorganisms for commercial use (8gr, Biol, C
hen+, 43 (5), 1007-1013.
1979, JP-A-56-109587, JP-A-58-
158184, etc.), a method for promoting plant protoplast fusion (Japanese Unexamined Patent Publication No. 120098/1983), etc. have been reported or disclosed. However, regarding cell fusion targeting algae, particularly Chlorella, there is only a method for removing the cell wall of Chlorella (Japanese Patent Application Laid-open No. 181692/1983).

[Problem that the invention seeks to solve]

Therefore, the present invention is characterized by efficient and economical collection of useful components produced in Chlorella cells, and in particular, a new strain of Chlorella that produces EPλ in high concentration and has a fast proliferation rate. The purpose is to provide.

[Means for solving problems]

The present invention is based on the knowledge that when cell fusion of protoplasts of a specific Chlorella and protoplasts of a different species of Chlorella that have properties different from those of the specific Chlorella, a new Chlorella that has the desirable properties of both is produced. It is based on Furthermore, this was done based on the knowledge that more favorable results could be obtained by repeating the cell fusion described above multiple times.

That is, the present invention provides a novel chlorella obtained by fusing protoplasts of a specific chlorella and protoplasts of a different type of chlorella.

The specific chlorella used in the present invention is one of two types of chlorella cells with different properties used in cell fusion, for example, has 16 to 26 carbon atoms and has 3 or more double bonds in the molecule. Examples include chlorella that produces highly unsaturated fatty acids having a polyunsaturated fatty acid or lipids constituting the fatty acids. Furthermore, ribulose diphosphate carboxylase (an enzyme involved in fixing CD2 in photosynthesis)
Examples include those that produce carbonic anhydrolase (a Co2-wrap enzyme involved in the efficient use of solar energy). Of these, EPA or E
Chlorella, which produces lipids containing PA as a constituent, is preferred. In addition, strains that grow in seawater or a medium containing seawater can be mentioned. In this case, the seawater-containing medium may be any medium containing at least 1% by volume of natural seawater, and may be simply diluted with purified water, or may be a medium containing other nutritional components. The above-mentioned specific Chlorella includes C. minutisima (Chlorellamin), regardless of the ability to produce secondary carotenoid metabolism.
utissima). Chlorella can be easily collected from natural seawater, but it can also be obtained from fisheries experimental stations, cultivation and fisheries centers, universities, and other public institutions in each prefecture. In addition, the specific Chlorella used in the present invention is not limited to C. minutisima that can be prepared manually by the above-mentioned method, but also includes various wild strains, mutant strains, auxotrophic strains,
Drug-resistant strains can also be used.

On the other hand, the different species of chlorella used in the present invention is a chlorella that has different properties from the specific chlorella, and has a faster growth rate than the specific chlorella, preferably has a multiplication power that is twice or more than that of the specific chlorella. Chlorella can be mentioned.

Also, strains that grow in a freshwater medium can be mentioned.

Here, the freshwater medium refers to one that does not contain natural seawater or components obtained therefrom. As the above-mentioned different species of chlorella, Chlorella ellipsobuia (Chlorella
ellipsoidea; r AMC-
27, IAM C-87, etc. (hereinafter referred to as C. ellipsobuia), Chlorella vulgaris (Chlorella vulgaris)
llavulgaris ; IAM C-30,
Examples include IAM C-169 (hereinafter referred to as C. vulgaris). In addition, here IAM C-27
is the number C- kept at the Institute of Applied Microbiology, University of Tokyo.
27 Chlorella strains are shown. Furthermore, in the present invention, strains with large cell sizes, such as Chlorella pyrenoidosa (Chlorella pyrenoidosa), are used as heterologous Chlorella.
dosa: IAM C-28, etc.: Hereinafter,
C.

Pyrenoidosa) and strains that can grow at low or high temperatures can be used. Note that the different species of chlorella used in the present invention are not limited to the above-mentioned chlorella,
Wild strains, mutant strains, auxotrophic strains, and drug-resistant strains belonging to these may be used.

Among the different types of chlorella mentioned above, it is preferable to use chlorella, which has a particularly fast growth rate. It is also preferable to use a cell fusion of a specific Chlorella protoplast and a fast-growing Chlorella protoplast as the heterologous Chlorella.

In the present invention, when performing cell fusion of the above-mentioned specific Chlorella and different species of Chlorella, these protoplasts are adjusted. Chlorella is said to have a harder cell wall than other microorganisms;
``Method for removing cell walls of Chlorella'' described in No. 92'
and Japanese Patent Application No. 59-264428 previously filed by the present inventors.
Protoplasts are prepared based on the method of enzymatically treating Chlorella cell walls described in No. 60-45226.

Among these, the cell wall of Chlorella is treated with one or more cell wall-degrading enzymes of cellulase, hemicellulase, pectinase, chitinase, β-glucuronidase, and agarase in the presence of an osmotic pressure regulator to remove the cell wall. It is preferable to use a method in which the cell wall of chlorella is removed using a plankton extract.

Next, the protoplasts obtained by the above method are subjected to cell fusion treatment. In the present invention, as a cell fusion method, a water-soluble polymer aqueous solution such as polyethylene glycol, polyvinyl alcohol, or polyvinylpyrrolidone contains calcium ions such as calcium chloride or calcium sulfate, and a polymer prepared with sorbitol, mannitol, sucrose, magnesium sulfate, etc. The so-called polymer agent fusion method, electric fusion method, liposome fusion method, etc., which perform fusion in a hypertonic solution at pH, can be used. Among such methods, for example, in the polymer agent fusion method, the fusion treatment can be performed by the following procedure.

That is, the specific Chlorella protoplasts obtained by the above method and the target for cell fusion (heavy Chlorella protoplasts) are mixed in approximately equal proportions and centrifuged to obtain a protoplast mixture. Suspend it in a hypertonic solution (PH8-13) containing calcium ions, add a 10-50% polyethylene glycol (PEG 1540.4000.6000, etc.) solution to it, and hold it at 20-40°C for 10 minutes to 2 hours. Fuse. Protoplasts fused in this way are
The above-mentioned fusion treatment agent and the like are washed away by centrifugation, and the cell wall is regenerated by the method described below. For example, cells with regenerated cell walls are obtained by culturing for 1 to 2 days in a chlorella culture solution or solid medium containing an osmotic pressure regulator such as sorbitol, mannitol, or sucrose. The culture medium here may be a natural medium using seawater, a freshwater medium, or a nitrogen source such as ammonium chloride, ammonium sulfate, urea, nitrates, carbonates such as baking soda, carbonates such as lactic acid, acetic acid, citric acid, glucose, etc. Artificial products containing metals such as potassium, calcium, magnesium, iron, boron, manganese, zinc, copper, and molybdenum in the form of chlorides, nitrates, sulfates, and complexes, as well as various vitamins and amino acids. Nutrient media can be used. Then, under natural or artificial lighting or in a dark place, 10 to 60°C, preferably 20 to 45°C.
The cell walls are regenerated by culturing at a temperature of 0.degree. C. with air, carbon dioxide gas, or a mixture thereof, with or without aeration, and with stirring or standing still.

Next, to obtain the desired fused cells, a combination of a seawater-based medium and a freshwater medium can be used.

For example, the cell walls of fused cells are regenerated in seawater or a medium containing seawater components, and the colonies that appear after culturing for several days are transplanted to a freshwater medium, and the colonies that appear after culturing for several more days, that is, the desired fused cells, are collected. .

In the present invention, the above-described cell fusion is performed once, but it is possible to proceed further and perform cell fusion two or more times. Specifically, a specific Chlorella protoplast and a heterologous Chlorella protoplast are fused once using the method described above, the cell wall of the fused protoplast is regenerated, new Chlorella is selected, this is cultured, and then the new Chlorella is recombined with the heterologous one. Chlorella and performs cell fusion with a specific chlorella.

More specifically, C. growing in a medium containing seawater.

A fusion strain of protoplasts of C. minutisima (a strain distributed from the Kanagawa Prefectural Fisheries Experiment Station) and protoplasts of C. C growing in protoplasts of Minutisima (strain distributed from the Nagasaki Fisheries Experiment Station) or C growing in freshwater culture medium;
A second generation fused cell can be obtained by performing fusion treatment with protoplasts of Ellipsobuia (JAM C-87). Furthermore, by repeating this operation, third or higher generation fused cells can be obtained. That is, a new chlorella obtained by repeating fusion multiple times is preferable because it has both higher proliferation ability and higher EPA production ability than a new chlorella obtained by a single fusion.

In addition, in order to obtain such a repeated fusion strain, if you fuse seawater Chlorella with a fusion strain of seawater Chlorella and freshwater Chlorella, the fused strain will grow in a medium with a high seawater concentration depending on the number of fusions. Based on the knowledge that the fusion strain will be able to grow, the seawater concentration will be increased sequentially and the fused strain will be selected.

Next, the novel chlorella of the present invention is illustrated in Table-1.

In addition, PC■ in the table indicates Packed Ce1l Volume
It is an abbreviation of me (m42/β) and indicates the sedimentation volume of Chlorella cells that were sedimented when Chlorella culture solution 11 was centrifuged at 300 Orpm for 10 minutes. 1.The table lists the PCM values when Chlorella with a PCV of 1.0 at the start of culture was cultured for 7 days.
The growth rate of chlorella is expressed by 萩.

Furthermore, the EPA content in the table was determined by crushing chlorella cells by a conventional method, extracting the oil with a solvent, and determining the fatty acid composition by chemical analysis such as GLC.

The mycological properties of the above fusion strain (new Chlorella) are as follows.

(2) Morphological properties Cells were cultured for 7 days at 21° C. under 5000 lux illumination using a 25% seawater-containing medium and were observed using an optical microscope to determine the shape and size of the cells. The results are shown in Table-2.

Table 2 [Effects of the invention] According to the present invention, the characteristics of conventional chlorella, which produce useful substances within cells but have a slow growth rate, which was a major drawback in industrial use, have been changed. New chlorella is provided. Although it is known that industrially useful substances exist within cells, the conventional inefficient and uneconomical productivity can be greatly improved. Therefore, according to the present invention, E produced by specific chlorella
A novel chlorella having a productivity that is more than half of the PΔ production ability and a growth rate that is twice or more than that of a specific chlorella is provided.

New Chlorella obtained by repeating the fusion two or more times is particularly preferable in terms of EPA production ability and proliferation ability. In addition, one fusion strain (N0M10065 strain, NO
o 12038 strain), the PCV was 5 to 5 in the first culture.
6, and the EPA content in the total fatty acids is 28.8-30.5
%, which has good EPA production ability, but when subcultured, E.
l) The A content decreases, which means that the EPA-producing ability of the first fusion strain is very unstable, but the new Chlorella obtained by fusing the K strain several times does not increase even after subculture. E
It has the advantage that PA production capacity does not decrease.

Hereinafter, the present invention will be explained in more detail in Examples.

[Example] Table 3 shows the composition of the medium used to culture Chlorella in the Example. In addition, in the Examples, the confirmation of protoplast production and cell wall regeneration was carried out by observing the presence or absence of cell walls using a fluorescence microscope using Calcofluor White) staining, and by examining viable protoplasts using a neutral light reagent. .

Example 1 Saltwater Chlorella C, Munitisima (K) (strain obtained from the Kanayo Prefectural Fisheries Experiment Station) and freshwater Chlorella C
, Ellipsoidea (IAM C-27) were cultured in artificial seawater medium and MC medium, respectively, and patent application 1986. -0
Protoplasts were formed using the method described in No. 45226, that is, using a cell wall lytic enzyme extracted from plankton. 0
．． 0.05 M containing 6 M mannitol/sorbitol (1:1)! J acid buffer (pH 6,0) (hereinafter referred to as
After washing with an isotonic solution and concentrating by centrifugation,
Mix equal amounts of both and add 40% polyethylene glycol 4
000,100m M Ca CII 2.0.3
M-' Isotonic solution containing disodium haltaate (pH 8,5>
In addition, cell fusion was performed for 1 hour. Next, the above mixture was diluted to 2 times by isotonic dilution, left to stand for 15 minutes, centrifuged to remove the supernatant, and the same dilution-centrifugation operation was repeated several times to obtain a fusion mixture. Ta. This mixture was inoculated into a 50% artificial seawater medium containing 0.6M mannitol/sorbitol (1:1) to regenerate cell walls, and cell wall regeneration was observed 1 to 2 days later. Next, a portion of this culture solution was transferred to MC medium containing 1.5% agar, cultured under light for several days, and colonies were collected. Further, these colonies were spread on a 50% artificial seawater medium containing 1.5% agar, and after culturing for several days, the resulting colonies were collected and fused strains were selected. 50 selected fusion strains
% artificial seawater medium, and the growth rate and the EPA content in the total fatty acids constituting the lipids were determined. A typical example is shown below. The main fatty acid constituting the fusion strain NOM 10065 is C10. (15,2%), c, 6. .

(15,3%”) ,C,, (8,6%),C,,
,,(4,9%),C1e:2(12,0%),C,
,,3(5,3%) ,C20:4(5,0%),C2
゜, 5 (28.8%).

The experiment was conducted five times, and the analysis results of the growth rate (PCV) and EPA content of representative fusion strains that were tested for each are shown in the table.
4.

Table-4 Example 2 C. minutissima (K) and C. vulgaris (IA
MC-169) were cultured in artificial seawater and DM medium, respectively, and converted into protoplasts in the same manner as in Example 1. In this example, 0.5M mannitol was used as the isotonic agent.
07M) US hydrochloric acid buffer (pH 7.0) was used. After washing each protoplast with an isotonic solution, centrifuging and concentrating, equal amounts of each were mixed and mixed with 30% polyethylene glycol 1450.0, IMCaCj! Fusion was carried out in the same manner as in Example 1 using an isotonic solution (pH 9,0) containing . The cell walls of the fusion mixture were regenerated on an agar medium of 25% artificial seawater containing 0.5 M mannitol, and fast-growing colonies were collected and diluted with isotonic dilution. Next, this was added to a 50% artificial seawater culture medium (raw salt concentration 13.35 g/1
) by growing C. vulgaris (IAMC
-169) strain, and then the fused strain was selected on a DM agar medium, excluding the C. minuteisima (K) strain. The experiment was conducted five times, and the analysis results of the growth rate and EPA content of representative fusion strains obtained for each are shown in Table 5.

Table 5 Example 3 The fusion strain NOM 10065 obtained in Example 1 was cultured in a 50% artificial seawater medium, and C. minutisima (K) were cultured in an artificial seawater medium, and protoplasts were produced in the same manner as in Example 1. The NOV 10065 strain was removed by culturing in 100% seawater medium, and the strains with high BPA content, except for the NOV 10065 strain, were grown in freshwater medium (MC). Double fusion strain N0M200
I chose 78. N0M20078 and C. minutisima (N) (strains distributed by the Nagasaki Prefectural Fisheries Research Institute) were cultured in 25% artificial seawater culture medium and 100% artificial seawater culture medium, respectively, according to the method of Japanese Patent Application No. 59-264428. Protoplasts were made into protoplasts, and the fusion treatment was performed in the same manner as in Example 1.
The fused strain was selected by culturing at ~29g/j and using MC agar medium. In other words, the N0M20078 strain was removed by growing it in a 101-108% artificial seawater medium (raw salt concentration 27-29 g/l), and then in a freshwater medium (
MC), the N0M31050 strain was selected by excluding the N strain. Table 6 shows the analysis results of the growth rate and EPA content of representative fusion strains.

Table 6 Example 4 Fusion strains NOM 20078 and C. minutissima (K), which were subjected to fusion treatment twice, were cultured using a 25% artificial seawater medium and an artificial seawater medium, respectively, in the same manner as in Example 1. After protoplastization using the method of 35% polyvinylpyrrolidone 100' OO/polyethylene glycol 3
Fusion processing was performed using 000 (=1:1). Next, cell walls were regenerated in the same manner as in Example 1, and then 10
1-108% artificial seawater culture medium (raw salt concentration 27-29g/β
), the N0V20078 strain was removed, and the 3-fold fusion strain N0M30013, which had a fast growth rate and high EPA content, was selected by growing it in a freshwater medium (MC). NOM30013
and C. minutissima (N) were cultured in 25% artificial seawater medium and 100% artificial seawater medium, respectively, and treated in the same manner as in Example 3 to fuse and regenerate the cell wall. Seawater culture medium (raw salt concentration 29 ~
31g/β), N0V3001
After removing 3 strains, the N0V41007 strain, which is a 4-times fusion strain, was selected by growing the cells in a freshwater medium (MC) and removing the N strain. Table 7 shows the analysis results of the growth rate and EPA content of the representative fusion strain.

Table 7 Example 5 In this example, chlorella protoplast fusion was carried out by electrofusion method. First, C. minutissima (K) and C. ellipsobuia (IAM C-87) were made into protoplasts in the same manner as in Example 1.

In addition, C, ellipsobuia (IAM,, C, -87
) was used in MBM medium. 0.05 MCa
An isotonic suspension containing Cβ2 (pH 9,0) was prepared so that the cell number of both protoplasts was 5 x 10' cells/ml or more, and platinum parallel electrodes of an electrofusion device (2 x 2
++onXlOmm) between 0.05 and 0.0.
The protoplasts were bonded by placing the tube under an electric field of 8.0 mm and 80 KHz for 30 seconds to 1 minute. Next, the transmembrane voltage of the cell was adjusted to 120■, 80Hz, so that it became 1 to 2■.
Pulses were applied to the cells several times with a pulse width of 50 μs, and the cells were fused over a period of several minutes to several tens of minutes. Next, the mixture was diluted using an isotonic solution, and the supernatant was removed by centrifugation. This dilution-centrifugation operation was repeated several times to obtain a fusion mixture. As in Example 1, the cell wall of the fusion product was regenerated using 50% artificial seawater medium and MBM medium, and the fusion strain was further selected. Among the selected fusion strains, the preferred one in terms of growth rate and EPA content is PCV = 5.7, EPA content = 28.5
%Met.

Procedural amendment 1. Display of case 1985 patent application No. 172721
No. 2, Title of the invention: New Chlorella 3, Relationship with the case of the person making the amendment: Name of applicant: Nisshin Oil Co., Ltd. 4, Agent 5, Date of official order: Initiator 1, Page 9, line 1 of the specification; Insert the following text between the second line.

``In addition, regarding the classification of Chlorella that grows in seawater or a medium containing seawater, it is classified as Nanochlorobutis.
There is also an announcement that it belongs to the genus Chloropsis, but currently it generally includes what is called Chlorella minutissima as mentioned above (Bullet
in of the Japanese 5ociety
of 5 scientific Fisheries, Vol. 44, No. 10, pp. 1109-1114 (1978),
Vol. 45, No. 7, pp. 883-889 (1979), Vol. 45, No. 8, pp. 955-959 (1979), Oil Chemistry, Vol. 31, No. 2, pp. 77-90 (1982)) ”2
, Replace "*1: M, solution" in the lower column of Table-3 on page 23 of the same book with "*
2: Corrected to "M, solution".

3. Same book, p. 24. Correct “munity sima” in the second line to “minutisima”.

4. Correct "IMM C-27" in Table-4 on page 26 of the same book to rIAM C-27J.

Claims (11)

[Claims] (1) A novel chlorella obtained by fusing protoplasts of a specific chlorella and protoplasts of a different species of chlorella. (2) The chlorella according to claim (1), wherein the specific chlorella is a strain that produces highly unsaturated fatty acids and/or lipids containing the fatty acids as constituent components. (3) Chlorella according to claim (1), wherein the highly unsaturated fatty acid is eicosapentaenoic acid. (4) The chlorella according to claim (2), wherein the specific chlorella is a strain that grows in seawater or a medium containing seawater components. (5) The chlorella according to claim (2), wherein the specific chlorella is Chlorella minutissima. (6) The chlorella according to claim (1), wherein the different species of chlorella is a chlorella whose growth rate is faster than that of the specific chlorella. (7) Claims that use a cell fusion of Chlorella protoplasts that produce highly unsaturated fatty acids and/or lipids containing the fatty acids as constituent components, and Chlorella protoplasts that proliferate faster than the Chlorella, as a heterologous Chlorella. Chlorella described in paragraph (6). (8) The chlorella according to claim (6), wherein the heterologous chlorella is a strain that grows in a freshwater medium. (9) Claim No. 1 in which the different species of Chlorella is a strain to which Chlorella ellipsoidia or Chlorella vulgaris belongs (
Chlorella described in section 6). (10) The chlorella according to claim (1), wherein the specific chlorella is a wild type or a mutant type of chlorella. (11) The amount of eicosapentaenoic acid produced by the new chlorella is 1/2 or more of the amount produced by the specific chlorella,
The Chlorella according to claim (1), which has a growth rate that is twice or more that of a specific Chlorella.