JP3479103B2 - New microalgae - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel microalga Prasinococcus capsulatus Miyashita e, which has the ability to produce viscous substances.
t Chihara sp. nov., a novel viscous substance PC1 produced by the same, and a method for producing a novel viscous substance PC1 using the novel microalgae.

[0002]

2. Description of the Related Art As a method for producing a viscous substance by a living organism, a method of producing from a bacterium, a method of producing from a macroalgae, and a method of producing from a microalga have been known.
However, all of them are caused by terrestrial organisms and coastal marine organisms, and methods for using organisms that grow in the open ocean have not been known. In addition, a novel viscous substance PC1 and a method for producing the same, and microalgae that produce the same have not been known.

[0003]

Therefore, the object of the present invention is to collect a wide range of seawater in the open ocean of the Pacific Ocean, separate microalgae that grow in seawater, and produce a new microalgae strain that produces viscous substances. To find out and develop a production method for the same viscous substance.

[0004]

[Means for Solving the Problems] The inventors collected seawater in the southern region of the Ogasawara Islands, from which the novel viscous substance PC1 was collected.
A novel microalga Prasinococcus capsulatus with productivity
Miyashita et Chihara sp. Nov. (Hereinafter referred to as this strain)
Was successfully separated, and the present invention was completed. That is, the present invention is a novel microalga Pr having the ability to produce viscous substances.
asinococcus capsulatus and new microalgae belonging to it Prasinococcus capsulatus Miyashita et Chihara
sp. nov. strain.

The present invention also provides a novel microalga Prasinococ.
It is a novel viscous substance PC1 produced by cus capsulatus and having the following physicochemical properties. (1) At least galactose, glucose, xylose, arabinose, and mannose are contained as constituent monosaccharides. (2) A typical composition formula is a sulfur-containing polysaccharide having C _6.3 H _12.3 O _6.12 (SO ₃ ) _0.768 . (3) The molecular weight is in the range of 10 ⁵ to 10 ⁷ . (4) The aqueous solution is colorless and transparent and has no taste or odor. (5) The viscosity of a 1% aqueous solution at 25 ° C. is 1000 to 2500 cP at 30 rpm using a rotary viscometer. Further, the aqueous solution has a structural viscosity (non-Newtonian viscosity). (6) Addition of salts significantly reduces the viscosity.

Furthermore, the present invention is a method for producing a novel viscous substance PC1 characterized by culturing microalgae belonging to the genus Prasinococcus and having the ability to produce a novel viscous substance PC1 and collecting the novel viscous substance PC1 from the culture. Is. In addition,
This strain is preserved and managed under the preservation number 4-0 / 19 at Marine Biotechnology Research Institute.

The present invention will be described in detail below. 1. Selection method for this strain In the waters south of the Ogasawara Islands, surface seawater and seawater according to depth were collected using a bucket and a knee skin water sampler, and 1 liter of this seawater was a membrane filter with a pore diameter of 0.45 μm and a diameter of 47 mm (Toyo Filter Paper ( Company)). This membrane filter was put into a culture solution in which nutrient salts were added to seawater, and it was exposed under fluorescent light to
Static culture was performed at 25 ° C. This sample was observed under a microscope to select this strain producing extracellular viscous substances. 2. Algae properties of this strain A. Morphological properties (1) The cells are spherical and have a diameter of 3.3-5.5 μm (Fig. 1).
. There are rarely 8 μm diameter cells. It is a single colony, has no flagella, and does not exhibit motility.

(2) The cell is surrounded by a viscous substance. The viscous substance has a drop-like shape with a width of 10-15 μm and a length of 12.5-26 μm (Fig. 1). (3) On the cell wall facing the pyrenoid (Fig. 2py), a ring-shaped structure (Fig. 2co) with a diameter of about 1 µm is present in the shape of the bottom of a teacup, and 8-14 holes ( Figure 2h) is present.

(4) The cell has a cell wall inside the viscous substance. Inside the cell, the nucleus (Fig. 2n) and chloroplast (Fig. 2c)
h), mitochondria (Fig. 2m), pyrenoids (Fig. 2p)
There is one y), and a Golgi body (Fig. 2go) and the like are recognized. In FIG. 2, s is a pyrenoid starch, v
Indicates a membranous vesicle. (5) The pyrenoid (Fig. 3py) is obovate and covered with a starchy substance (Fig. 3s).

(6) The starch-like substance (Fig. 3s) has a hole on the cytoplasmic side, and the outer membrane of the chloroplast (Fig. 3ch) and the mitochondria (Fig. 3m) are sandwiched by the cytoplasmic substrate from this, and the pyrenoid. Has invaded into the substrate. B. Physiology / Biochemistry (1) Culture solution It can grow in a culture solution containing seawater.

(2) Photosynthetic ability Photoautotrophic growth by photosynthesis is possible. (3) Containing pigments: chlorophyll a, chlorophyll b, praspinoxanthine, uriolide, Mg 2,4-divin
ylphaeoporphyrin a5 monomethylester (hereinafter Mg 2,4-
D) and other carotenoids (4) Anabolic storage substances Starch (5) Growth temperature range 15 ℃ ~ 35 ℃ (optimum temperature 25 ℃) (6) Growth salt concentration range Sea water 1/2 dilution ~ Sea water ( Proper concentration seawater 2/3 dilution (7) Growth pH range pH 6.5 to 9.5 (optimal pH pH 8)
-PH 9) C.I. Reproductive mode (1) This strain grows asexually. No sexual reproduction has been observed.

(2) Two divisions occur, and one of the daughter cells escapes from the cell wall of the mother cell (Fig. 4). At this time, the cell wall is not formed in the daughter cell that escapes at this time, but the cell wall is formed immediately after the escape. The daughter cells remaining in the mother cells grow to the size of the mother cells and use the mother cell wall as it is as the cell wall. The escaped daughter cells form a small amount of extracellular viscous substance with the cell wall, then detach from the viscous substance of the mother cell, and division and separation are completed. 3. Genus and Species Determining the taxonomic characteristics of this strain is pigment composition. This strain is a spherical cell, and the main photosynthetic pigments are chlorophyll a, chlorophyll b, Mg 2,4-D, praspinoxanthine,
Have a Ulioride. This pigment composition is similar to that of the algae of the order Plasmodium, Mamiera. However, the alga of the order Plasinophyceae, Mamiera, has no cell wall, has scales on the cell surface, and has motility with flagella at most stages in the life cycle, whereas this strain Has a cell wall, does not have scales on the cell surface, has extracellular mucilage, and is non-flagellate and non-motile in the entire life cycle. The circular structure of the cell surface and the pore structure around it have not been observed in other known microalgae. The structure in which the chloroplast membrane and mitochondrial membrane penetrate the pyrenoid substrate is similar to that of the two strains of Plasnophyceae. However, these strains also differ in morphological characteristics and pigment composition.

Therefore, this strain is 1) spherical cells surrounded by a viscous substance, 2) without scales, 3) with a characteristic asexual reproduction pattern, and 4) with a characteristic pyrenoid structure. 5) Prasinococcus capsulatus Miyashita et as a new genus strain characterized by having no flagella, 6) having chlorophyll a, chlorophyll b, Mg 2,4-D, prazinoxanthin and uriolide as main photosynthetic pigments.
It was named Chihara sp. Nov. 4. Physicochemical properties of viscous substance produced by this strain (1) The viscous substance produced by this strain contains galactose, glucose, xylose, arabinose and mannose as constituent monosaccharides (Table 4). In addition, elemental analysis (Table 3) and biological material analysis (Table 2) showed that this viscous substance was C _6.3 H _12.3 O
_It has a composition of _6.12 (SO ₃ ) _0.768 , and 3 for 4 monosaccharides.
It is considered to be a sulfur-containing polysaccharide with two sulfate groups.

(2) The molecular weight is slightly different depending on the extraction / purification method and has a molecular weight of about 10 ⁵ -10 ⁷ . (3) Aqueous solution is colorless and transparent, tasteless and odorless, and 1% aqueous solution at 25 ° C
The viscosity at is 1 at 30 rpm using a rotary viscometer.
It has a high viscosity of 000 to 2500 cp. The aqueous solution also has structural viscosity (non-Newtonian viscosity).

(4) This substance differs from general polymeric polysaccharides in that the viscosity is remarkably reduced by the addition of salts. Regarding the mechanism of viscosity reduction, it may be related to sulfur-containing polysaccharides, but it is currently unknown. 5. Cultivation conditions for this strain As the culture solution, seawater heated and sterilized and supplemented with nutrient salts, vitamins and trace metal salts shown in Table 1 through a 0.2 μm pore type membrane filter can be used. This strain can be inoculated with this strain and aerated by aerating at 25 ° C. under irradiation of fluorescent light. 6. Means for collecting viscous substance PC1 from the culture The viscous substance can be collected from both the culture supernatant of the culture and the cultured algal cells. 10% of the culture supernatant can be collected
Cetyltrimethylammonium bromide aqueous solution (hereinafter,
(Referred to as CTAB aqueous solution) until no precipitate is formed, the precipitate is recovered, washed with water, and then dissolved in a 0.5 M acetic acid aqueous solution containing 20% sodium chloride. Next, ethanol was added to this so that the final concentration would be 50-80% to produce a white precipitate, which was collected and dried to obtain a white powder of a viscous substance.

The viscous substance can be collected from the cultured algal cells by a method of crushing cells with a French press or a homogenizer or a method of boiling in distilled water. The cell suspension is crushed or boiled, and CTAB aqueous solution is added to the supernatant obtained by centrifugation or filtration until no precipitate is formed, the precipitate is collected, washed with water, and then 0.5 M acetic acid solution containing 20% sodium chloride. Dissolve in. This gives a final concentration of 50-80
When ethanol is added so that the amount becomes 100%, a white precipitate is produced, which is collected and dried to obtain a white powder of a viscous substance. 7. Uses of the viscous substance PC1 The viscous substance PC1 is a thickener, a surfactant, an emulsion stabilizer,
It can be used as an industrial raw material such as a coating agent. It can also be used as dietary fiber or a physiologically active substance.

[0017]

EXAMPLES The present invention will be specifically described below with reference to examples. (Example 1) Seawater collected from Kamaishi Bay, Iwate Prefecture was placed in a flat glass flask and sterilized under pressure. To this, a concentrated solution of the nutrient salt shown in Table 1 was filtered through a membrane filter having a pore size of 0.2 μm and added to obtain a culture solution.

[0018]

[Table 1]

This strain was inoculated with this strain, a breathable stopper was placed, air was aerated in the liquid, and the culture liquid in the flask was stirred at the same time. At this time, the temperature was adjusted to around 25 ° C. by irradiating light from around the flask with a sunlight lamp. Using the turbidity of the culture solution as an indicator of the growth of this strain, the turbidity is measured over time,
In addition, the amount of the viscous substance produced in the culture solution at this time was calculated by glucose conversion, and the change over time was also measured. The results are shown in FIG. In the figure,-○ -indicates the turbidity of the culture solution, and-● -indicates the production amount of the viscous substance.

(Example 2) Seawater collected in Kamaishi Bay, Iwate Prefecture was placed in a polycarbonate tank, sterilized by an autoclave for 30 minutes, and allowed to cool. It was added so that it became 1 and this was made into the culture solution. This strain was added to this culture medium and cultured at room temperature (about 25 ° C.) under aeration of air under the irradiation of light from a fluorescent lamp. After 20-30 days, add approximately 9,000
After centrifugation at g, the culture supernatant and the algal pellet were separated. Extracellular mucilage was obtained from both culture supernatant and algal pellets.

Seawater of Kamaishi Bay, which had been previously filtered with a membrane filter having a pore size of 0.2 μm, was added to the recovered algal cells in an amount about 3 times the volume of the recovered algal cells and suspended, and then the algal cells were manufactured by Odake French Press. It was crushed using. The algal cell lysate was centrifuged at about 20,000 × g, and the supernatant was collected. To this, 10% CTAB aqueous solution was added until no insoluble precipitate was formed. After overnight standing at room temperature, centrifuge (approx.
The precipitate was collected by xg). The precipitate was washed twice with deionized water and then dissolved in a 0.5M acetic acid aqueous solution containing 20% sodium chloride. Ethanol was added to this solution to a final concentration of 50% and left overnight at 4 ° C, followed by centrifugation (about
2,000 × g) to obtain a white precipitate. Dissolve the obtained precipitate in distilled water, add ethanol to a final concentration of 80%, leave at 4 ° C overnight, and then centrifuge (about 2,000 xg).
And a white precipitate was obtained. After the above operation was further repeated, the precipitate obtained was washed several times with ethanol and suction dried to obtain a white powder. The biological substance composition and elemental composition of this white powder are shown in Tables 2 and 3.

[0022]

[Table 2]

[0023]

[Table 3]

On the other hand, the extraction of the mucilage substance from the centrifugal supernatant of the culture broth was carried out after the 10% CTAB aqueous solution was added to the supernatant.
A white powder was obtained in the same manner as the extraction from the algal sediment. This powder was acid-hydrolyzed in a solution containing 5% hydrochloric acid in anhydrous methanol, dried under reduced pressure, and then subjected to TMS treatment with a solution of pyridine, trimethylchlorosilane and hexamethyldisilazane mixed at 5: 1: 1. The composition of monosaccharides was analyzed by using capillary gas chromatography.
The results are shown in Table 4. The composition ratio is shown as a peak area ratio of each substance obtained by capillary gas chromatography (GC-17A manufactured by Shimadzu Corporation).

[0025]

[Table 4]

When the supernatant of the algal culture solution centrifuged and the white powder obtained from the algal precipitation were dissolved in distilled water, a colorless transparent, tasteless and odorless viscous solution was obtained. The viscosity of a 1% aqueous solution at 25 ° C. was measured with a No. 3 rotor of BL type viscometer manufactured by Tokyo Keiki Co. at 30 rpm. As a result, the viscosity of this aqueous solution was 1000-2500 cp. To investigate the effect of the substance concentration on the viscosity of the aqueous solution of this substance, dissolve the viscous substance in distilled water, change the substance concentration, and
Viscosity at 30 ℃, B type viscometer (Tokyo Keiki Co., Ltd.)
Was measured using. The result is shown in FIG.

In order to investigate the effect of temperature on the viscosity of the aqueous solution of this substance, the viscous substance was dissolved in distilled water, the temperature was changed, and the viscosity at 30 rpm was measured using a B-type viscometer (Tokyo Keiki Co., Ltd.). It was measured. The result is shown in FIG. 7. The value on the vertical axis in the graph is 2 for a 0.1% substance concentration aqueous solution.
The relative viscosity when the viscosity at 5 ° C. is 100 is shown. In addition, in order to investigate the effect of pH on the viscosity of the aqueous solution of this substance, a viscous substance was dissolved in distilled water, the pH was changed, and the viscosity at a temperature of 25 ° C and 30 rpm was measured using a B-type viscometer (manufactured by Tokyo Keiki Co., Ltd.). Measured. The result is shown in FIG. The value on the vertical axis in the graph is the p of the 0.1% aqueous solution of the substance.
The relative viscosity when the viscosity at H7 is 100 is shown.

Further, in order to investigate the influence of the coexisting substance on the viscosity of the aqueous solution of this substance, the viscous substance and the coexisting substance were dissolved in distilled water, and the viscosity at a temperature of 25 ° C. and 30 rpm was measured with a B type viscometer (manufactured by Tokyo Keiki Co., Ltd.). Was measured using. The results are shown in Table 5. The value in the table is 0.02 that does not include coexisting substances.
% (W / V) Viscosity of viscous substance aqueous solution (at this time, 30.6 cp) 10
The relative viscosity when 0 is shown. The additive substance concentration is
It is 0.01 (mol / l).

[0029]

[Table 5]

[0030]

According to the present invention, it is possible to produce a novel viscous substance PC1. In addition, since the viscous substance PC1 is colorless and transparent and has a viscous property, it can be used as an industrial raw material for thickeners, surfactants, emulsion stabilizers, coatings, etc., and is also used as dietary fiber. It can also be applied to the fields of food and medicine, such as use as a bioactive substance.

[Brief description of drawings]

[Figure 1] Phase contrast micrograph of this strain A. Algae in culture B. Algae when India ink is added to the culture solution

FIG. 2 Schematic diagram of cells A. Figure B. When viewed from the side where the chloroplast is cracked with the annular structure facing up. Diagram when viewed from the side of the annular structure

Fig. 3 Structure of pyrenoid

FIG. 4 Schematic diagram of cell division The division occurs in the order of A to H and becomes A again.

[Fig. 5] Growth of this strain and total sugar production

FIG. 6: Viscosity at each viscous substance concentration

FIG. 7: Relative viscosity of viscous substance aqueous solution at various temperatures

FIG. 8: Effect of pH on viscosity of aqueous viscous substance

[Explanation of symbols]

ch ... chloroplast, co ... annular structure, go ... Golgi body, h
… Pore, m… mitochondria, n… nucleus, py… pyrenoid, s… pyrenoid starch, v… membrane vesicle

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12R 1:89) C12R 1:89 (C12P 1/00 C12P 1/00 C12R 1:89) 19/04 (C12P 19/04 C12R 1:89) (56) Reference Journal of Experimental Marine Marine Biology and Ecology, 1992, Vol. 161, No. 1, p. 91-113 J Phycol, 1991, Vol. 27, 3 suppl. , P. 52 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 1/12 C07G 17/00 C12P 1/00 C12P 19/04 BIOSIS / WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A novel microalga, Prasinococcus capsulatus, which has the following physicochemical properties and is capable of producing a viscous substance.
s ). (1) At least galactose and gluco as constituent monosaccharides
Sucrose, xylose, arabinose , mannose. (2) As a typical composition formula, C6.3H12.3O6.1
It is a sulfur-containing polysaccharide having 2 (SO3) 0.768 . (3) The molecular weight is in the range of 105 to 107. (4) The aqueous solution is colorless and transparent and has no taste or odor. (5) The viscosity of a 1% aqueous solution at 25 ° C is the rotational viscosity.
1,000 to 2500 cP at 30 rpm using a meter
Is. Also, the aqueous solution has a structural viscosity (non-Newtonian viscosity).
Have. (6) Addition of salts significantly reduces the viscosity. 2. A novel microalgal plug Sino Lactococcus Cap Sula task viscous substance producing ability Miyashita et chestnut Hara sp knob (Prasinococcuscapsulat
us Miyashitaet Chiharasp. no
v. )stock. 3. A novel microalgal plug Sino Lactococcus capsule Sula task (Prasinococ cus capsulatu
s ) produced a novel viscous substance PC1 having the following physicochemical properties. (1) At least galactose, glucose, xylose, arabinose, and mannose are contained as constituent monosaccharides. (2) As a typical composition formula, C _6.3 H _12.3 O
It is a sulfur-containing polysaccharide having _6.12 (SO ₃ ) _0.768 . (3) The molecular weight is in the range of 10 ⁵ to 10 ⁷ . (4) The aqueous solution is colorless and transparent and has no taste or odor. (5) The viscosity of a 1% aqueous solution at 25 ° C. is 1000 to 2500 cP at 30 rpm using a rotary viscometer.
Is. Also, the aqueous solution has a structural viscosity (non-Newtonian viscosity).
Have. (6) Addition of salts significantly reduces the viscosity. 4. A prasinococcus ( Prasinoco)
ccus ) and the novel viscous substance P according to claim 3.
Culturing microalgae capable of producing C1 and claiming from the culture
Method for producing a novel gum PC1 according to claim 3, and collecting the novel gum PC1 according to claim 3.