JP3442281B2 - Microalgae having novel heteropolysaccharide-producing ability, novel heteropolysaccharide, and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a microalga C belonging to the genus Kokkomika which grows autotrophically by carbon dioxide as a carbon source by photosynthesis and has a novel heteropolysaccharide-producing ability.
occomyxa sp. The present invention relates to NY2F itself, a novel heteropolysaccharide, and a method for producing the novel heteropolysaccharide.

[0002]

2. Description of the Related Art Polysaccharides produced by microorganisms, particularly bacteria, are known to have various functions, and some polysaccharides have been put to practical use. For the functionality of polysaccharides produced by bacteria, see, for example, literature (Hamada, Polymer Processing, Vol. 36, N.
o. 2, P9, 1987), it is known to have various functions. There are also some reports on polysaccharides produced by microalgae. For example, "Ecology of algae" (Akiyama et al., Uchida old crane field, P510, 1
986) briefly summarizes algae species and their polysaccharides produced. There are almost no reports on the knowledge about monosaccharides constituting polysaccharides produced by algae.
G. Report of Moore et al. (SCIENCE, Vol. 1
45,7 Aug. , P586, 1964). Regarding the polysaccharide produced by microalgae,
At present, no studies have been conducted and the polysaccharides produced by microalgae have not been put to practical use.

Polysaccharides have various properties such as thickening, emulsion stability, dispersibility and water retention, and the polysaccharides are utilized in food, general industry, pharmaceuticals, cosmetics, etc. However, it is expected that these applications will be expanded by the development of new polysaccharides. In order to put the polysaccharide produced by microalgae into practical use, it is necessary to develop a novel polysaccharide having properties that have not existed in the past, and to produce it at low cost. Moreover, there is a demand for the search and separation of microalgae having high polysaccharide-producing ability, and the development of a culture method which has high productivity and can be carried out at lower cost.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a novel polysaccharide, a microalgae which produces a novel polysaccharide, and a low-cost and highly productive polysaccharide using the polysaccharide-producing microalgae. It is intended to provide a manufacturing method.

[0005]

In order to provide a novel microalgae that produces a polysaccharide and a method for producing a polysaccharide at low cost using the same, the present inventors have found that carbon dioxide contained in air, exhaust gas, etc. We searched for microalgae that grow autotrophically by photosynthesis with sucrose as a carbon source and produce polysaccharides outside the algal body. As a result, microalgae that produce a novel polysaccharide outside the algal body can be separated from the natural world, and the present invention has been completed.

[0006] That is, the microalgae of the present invention is a microalgae Coc which has the ability to produce a novel heteropolysaccharide, kokkogalactan.
comyxa gloeobotrydiformis
It is NY2F.

The method for producing a novel heteropolysaccharide according to the present invention comprises culturing a microalgae capable of producing a novel heteropolysaccharide belonging to the genus Coccomyxa in a medium and substantially not containing anhydrogalactose as a constituent monosaccharide in the medium.
% Of galactose as the main constituent sugar 30%
The present invention is characterized in that at least 0,000 new heteropolysaccharides are produced outside the algal cells, and the new heteropolysaccharides are collected from the medium.

[0008] A further preferred method for producing the novel heteropolysaccharide of the present invention comprises culturing microalgae capable of producing a novel heteropolysaccharide belonging to the genus Coccomyxa in a medium and containing 60-80% galactose as a constituent monosaccharide. The novel heteropolysaccharide is produced outside the algal body, and the novel heteropolysaccharide having a molecular weight of 1,000,000 or more is collected from the medium.

The novel heteropolysaccharide of the present invention contains 30% or more of galactose, which is substantially free of anhydrogalactose as a constituent monosaccharide, as a main constituent sugar, and has a molecular weight of 100.
It is more than a million heteropolysaccharides. Microheteroalgae Coccomyxa g which has the ability to produce a novel heteropolysaccharide, Koccogalactan
loebotrydiformis NY2F is capable of producing a novel heteropolysaccharide, kokkogalactan, having a molecular weight of 1 million or more, which has galactose as a main constituent sugar at about 70% as a constituent monosaccharide. Since the heteropolysaccharide of the present invention is an extra-algal fermentation product, it does not require a troublesome step of destroying and extracting an alga body such as an intra-algal fermentation product, and is advantageous for industrial production.

[0010] The microalga Coccomyxa gloe having the ability to produce the novel heteropolysaccharide of the present invention, Koccogalactan.
obotrydiformis NY2F is a microalgae (hereinafter referred to as the algae) separated from nature,
It was separated from the soil of an oil mine in Niigata Prefecture.
The algaeological properties of this alga are as follows.

1. Morphological Properties (1) The algal cells are single cells, and the cells have an elliptic shape with a length of 4 to 6 μm or a spherical shape close to an ellipse. FIG. 1 shows an electron micrograph of a cross section of the alga.

(2) The cell has one chloroplast, which is cup-shaped or plate-shaped and has no pyrenoid.

(3) The algal cells form four, sometimes eight autospores and do not form zoospores.

(4) The cell wall is single-layered, secretes agar-like polysaccharides around the cells, and the alga body is colonized as a whole.

2. Physiological properties (1) Photosynthetic ability: An autotrophic microorganism that grows by photosynthesis.

(2) Containing pigment system: having chlorophyll a and chlorophyll b.

(3) Growth temperature: optimum growth temperature 25 to 30 ° C. growth temperature range 15 to 35 ° C. (4) Growth pH: optimum growth pH 4 to 8 growth pH range 3 to 10 (5) Growth CO ₂ concentration : the determination of the position on the classification based on the nature of the optimum CO ₂ concentration from 0.03 to 5 percent growth CO ₂ concentration from 0.03 to 40% present algae was performed as follows.

The characteristics of the NY2F strain, which has a single cell of green color, green and chlorophyll a and b, and which is autotrophic, indicate that the microalgae are Chlorophyceae, a green alga net, and Chlorococcales of the order Chlorococcum (Kazutoshi Nishizawa, Chihara). Edited by Mitsuo, Algae Research, 197
9).

Komaek et al. (J. Komaek &
B. Fott, Das Phytoplankton
According to the classification of des Suswasers, 1983), among the chlorococcales, algae that secrete agar around the cells and grow in autospores belong to the Radiococcaceae family Radiococcaceae. The characteristics of (3) and (4) in the column of "1. Morphological properties" are in agreement with these, and it is considered that the NY2F strain is a member of the Radiococcidae. In addition, Komaek et al. Changed the Radiococcidae to Radiococideae, Di.
sporoideae, Dictyochlorell
It is classified into four subfamilies, Oideae and Palmodictyoideae. The latter, Sanya family, has filamentous agar, etc., or has the characteristic that cells are evenly arranged in the agar, which does not match that of the NY2F strain. This is consistent with the characteristic of the Radiococcoidae, a subfamily of the Radiococcus, that cells are unevenly arranged in the agar. The fact that the cells have chloroplasts, that there is no concentric pattern in the agar, that there are no pyrenoids in the cells, and that 2 to 4 autospores are formed diagonally or distorted in the mother cells It shows that the strain is of the genus Coccomyxa.

Nakano (Edited by Teruzo Hori, Life history collection of algae 1,
1994), a species of the genus Kokkomika C. gloe
In obotrydiformis Reisigl,
Autogenous spores are formed in vegetative cells, and after they are released, they repeat division, and at the same time secrete the agar substance to the outside of the cells, and the whole becomes colony-like. From the above, the NY2F strain was identified as Coccomyxa sp. Can be identified as

This species is also known in Central Europe and according to Komaek et al.
, C. Gloobotrydiform
is is a cell whose cells are slightly long and irregular and have a spherical shape,
The size is 5.5 to 6.5 μm × 2.8 to 5.0 μm,
It is said to form 2 to 8 autospores. The NY2F strain is a C.I. Consistent with the characteristics of gloeobotrydiformis .

From the above, the inventors of the present invention selected the NY2F strain as Coccomyxa gloeobotrydifo.
was identified as rmis, Coccomyxa gloeob
It was named trydiformis NY2F and used in the following experiments.

Further, it was confirmed by the phenol-sulfuric acid method and HPLC analysis that the agar substance secreted by the algae around the alga was a polysaccharide.

As described above, since the present algae produce polysaccharides outside the algal body, the algae are crushed and the polysaccharides are taken out of the cell as compared with the extraction of the polysaccharides from the algae that produce the polysaccharides inside the algal body. Since it does not require a step, it has an advantage that the polysaccharide can be easily extracted and energy and cost for separating and purifying the polysaccharide from the culture solution can be small.

Further, since carbon dioxide has a property of autotrophically growing by photosynthesis using carbon dioxide as a carbon source to produce a polysaccharide, there is an advantage that air can be used as a carbon source. Furthermore, the range of growth temperature and growth pH is wide, and the culture is easy. The concentration of carbon dioxide that can grow is relatively high, and for example, carbon dioxide in exhaust gas can be used as a carbon source to produce a polysaccharide, and the production cost of the polysaccharide can be reduced. In addition, this alga has a high polysaccharide-producing ability, as will be described later.
The concentration of polysaccharide in the culture solution for a day is about 1 g / L.

The polysaccharide produced by the present alga (hereinafter referred to as the present polysaccharide) will be described below. This polysaccharide is a heteropolysaccharide whose main constituent is galactose. According to the analysis value, the composition ratio of monosaccharides constituting the present polysaccharide is 70% galactose,
Mannose 13%, xylose 10%, arabinose 3
%, Glucose 2% and rhamnose 2%. Also,
The elemental composition of this polysaccharide is as follows: carbon 42%, hydrogen 7%, oxygen 50
%, Nitrogen is 1% or less, and sulfur and phosphorus are 0.3% or less.

At present, no heteropolysaccharide containing 70% of galactose has been reported. Further, as a heteropolysaccharide having galactose as a main constituent monosaccharide, only agarose (the composition ratio of galactose in agarose is 50%) is known. However, although agarose contains galactose and anhydrogalactose in almost the same ratio, this polysaccharide is not agarose because it does not contain anhydrogalactose. Also, an agarose aqueous solution (agarose concentration 1
% Or more) gel at room temperature, but an aqueous solution of the present polysaccharide does not gel at room temperature. From the above, the present polysaccharide is considered to be a novel heteropolysaccharide, and thus the one having the above-mentioned sugar composition ratio was designated as Kokkogalactan.

Kokko galactan is hot water (100 ° C. or higher)
It has the characteristics that it dissolves only in, is hardly hydrolyzed by an acid or an alkali, and is extremely difficult to decompose. In addition, Kokkogalactan is used for high performance liquid chromatography (HPL
According to C), the molecular weight is 1,000,000 or more (pullulan conversion)
Is an ultra-polymer, and its aqueous solution has a high viscosity of 0.5%.
It has about 1000 mPa · s (25 ° C.) of clay.

Due to the above characteristics, it is highly possible that coccogalactan can be used as, for example, a thickener, a suspending agent, and more specifically, a suspending agent for paints.

The method for producing kokkogalactan by the present alga will be described below. Culturing in the present invention, usually a liquid medium is used to blow air or a gas containing carbon dioxide,
It is preferable to carry out the culture by stirring while supplying light into the culture tank. Carbon dioxide is used as the carbon source of the liquid culture medium. As the carbon dioxide source, for example, air,
Exhaust gas, or synthetic gas in which carbon dioxide gas is mixed with air can be used. The concentration is CO ₂ 0.03
-20% is preferable. As the nitrogen source, nitrate nitrogen compounds such as calcium nitrate, potassium nitrate and sodium nitrate are used. Nitrogen concentration is 25 ~ 250mg
/ L (nitrogen equivalent) is preferable.

The inorganic salts added to the medium include, for example, potassium hydrogen phosphate, potassium dihydrogen phosphate, disodium glycerophosphate, magnesium sulfate, ferric chloride, iron sulfate, manganese chloride, manganese sulfate, zinc chloride,
Zinc sulfate, cobalt chloride, calcium chloride, sodium chloride, sodium carbonate, copper sulfate, sodium molybdate, potassium iodide and the like are used. Furthermore, if necessary, vitamins such as vitamins B ₁ , B ₁₂ and biotin, and organic acids such as citric acid, iron citrate, ammonia citric acid, etc. are added to the medium as a micronutrient source,
Better growth is obtained.

The culture is carried out by directly inoculating the liquid medium having the above-mentioned composition with algae or separately inoculating the seed culture solution obtained by the pre-culture. This seed culture solution is prepared, for example, by adding 1
It is carried out by inoculating platinum loops and culturing at 30 ° C. for 3 to 7 days.

The pH of the medium is in the range of 3 to 10, preferably 4 to 8, but pH control is not particularly required during culturing.

The culture temperature is 15 to 35 ° C., which is a growth range, and preferably 25 to 30 ° C.

The irradiation light intensity is preferably low when the algal cell concentration is low at the initial stage of culture and increased as the algal cell concentration increases during the culture, but a constant intensity of light may be applied during the culture. . In this case, it is important that the light intensity is not too strong. For example, 50 to 250 μE / m ² /
Culture is performed at a light intensity of s. The culture period varies depending on the culture conditions, but is usually 20 to 30 days. The polysaccharide concentration in the culture solution can be measured by the phenol-sulfuric acid method.

The heteropolysaccharide accumulated in the culture medium can be separated and extracted by a conventional method. That is, the culture solution can be diluted and heated, and after separating algal cells by centrifugation or the like, ethyl alcohol or the like is added to precipitate crystals of the heteropolysaccharide, and the heteropolysaccharide can be separated by filtration or the like.

As described above, according to the present invention, a novel heteropolysaccharide containing galactose as a main constituent monosaccharide can be easily obtained with high productivity. Further, since carbon dioxide is used as a carbon source, air, and in some cases, exhaust gas can be used, and production can be performed at low cost.

[0038]

Example: Calcium sulfate tetrahydrate 150 mg, potassium nitrate 100 mg, magnesium sulfate heptahydrate 40 m
g, β-glycerophosphate disodium 50 mg, trishydroxyaminomethane 500 mg, ferric chloride hexahydrate 0.588 mg, manganese chloride tetrahydrate 0.108 m
g, zinc chloride 0.0315 mg, cobalt chloride hexahydrate 0.012 mg, sodium molybdate dihydrate 0.
0075 mg, disodium ethylenediaminetetraacetate 3
After adjusting the medium containing 1 mg of L to pH 7.5,
The mixture was sterilized at 20 ° C. for 20 minutes and placed in a 20 L internal irradiation stirring type culture tank that had been steam sterilized in advance.

After controlling the temperature in the bath at 30 ° C., Coccomyxa was precultured in the same medium as above for 7 days.
Gloeobotrydiformis NY2F was inoculated and cultivated by agitating at 70 rpm while aerating air containing 5% CO ₂ concentration at 4 L / min. Light was transmitted from a xenon light source to an illuminator also functioning as a stirring blade inside the reactor using an optical fiber, and the light was radiated and supplied from the illuminator. The light intensity on the surface of the illuminant is 57 μE / m at the beginning of culture.
² / s, gradually increasing the intensity of the light, and finally 2
Irradiation was performed at 15 μE / m ² / s.

The concentrations of algal cells and heteropolysaccharides were measured by the dry weight method and the phenol-sulfuric acid method, respectively, and changes over time in the amounts of algal cells and polysaccharides were examined. A graph showing the result of the culture at this time is shown in FIG. Algal bodies and heteropolysaccharides increased steadily,
The ultimate concentration of heteropolysaccharide after 546 hr culture was 1.1 g /
It was L. The main culture solution was heated at 90 ° C. for 1 hour and then immediately centrifuged at 7,000 rpm for 15 minutes to collect a supernatant. In this supernatant, absorption showing a polysaccharide was observed at an absorbance of 490 nm.

Next, 4 times the amount of ethanol was added to this supernatant and the mixture was left to cool overnight and the produced crystals were filtered through a 40 μm mesh and collected. After further drying at 60 ° C. and pulverizing, 22 g of a powdery heteropolysaccharide was obtained.

The obtained heteropolysaccharide was prepared into a monosaccharide by acidolysis, and the constituent sugars were examined by HPLC analysis. HPLC
3 is shown in FIG. 3, and the result of the quantitative calculation is shown in Table 1 below.

[0043]

[Table 1]

Two heteropolysaccharide samples obtained by carrying out the above-mentioned embodiment a plurality of times (each heteropolysaccharide of sample 1 and sample 2)
Was subjected to organic elemental analysis. The results are shown in Table 2 below.
Shown in. The molecular weight distribution curve of the obtained heteropolysaccharide is shown in FIG.

[0045]

[Table 2]

From these results, the weight average molecular weight was converted into pullulan to be 1,000,000.

Next, the obtained heteropolysaccharide was subjected to IR analysis as follows. Sample 0.5 mg to KBr 200 m
Tablets were formed by mixing with g. Using the formed tablets I
The R spectrum was measured. The measurement conditions are shown below.

Apparatus: Impact 400D (Nicolet) Resolution: 2 cm ^-1 Number of integrations: 64 Detector: DTGS KRS5 Measurement method: Transmission method (KBr tablet method) The results are shown as IR spectrum in FIG. It is shown in Table 3 below.

[0049]

[Table 3]

[0050]

Industrial Applicability According to the present invention, a novel heteropolysaccharide having industrial utility can be provided.

According to the present invention, it is possible to provide a microalga belonging to the genus Coccomyxa having the ability to produce a heteropolysaccharide.

According to the present invention, by culturing a microalgae belonging to the genus Coccomyxa capable of producing a heteropolysaccharide using carbon dioxide contained in air, exhaust gas, etc. as a carbon source, the novel heteropolysaccharide is highly productive and at low cost. Can be produced in.

Since the novel heteropolysaccharide of the present invention is produced outside the algal cells, it can be extracted easily and can be produced at low cost, so that it has high productivity.

Since the novel heteropolysaccharide of the present invention has a high viscosity, it can be used as a thickener and a dispersant in various industries.

[Brief description of drawings]

FIG. 1 Coccomyxa gloeobotryd
It is an electron micrograph of ifomis NY2F.

FIG. 2 Coccomyxa gloeobotryd
It is a graph which shows the time-dependent change of the amount of algal cells and the amount of polysaccharides when culturing iformis NY2F.

FIG. 3 shows a chart obtained by HPLC analysis of the heteropolysaccharide obtained in Example prepared into a monosaccharide by acidolysis.

FIG. 4 is a graph showing a distribution curve of the molecular weight of the heteropolysaccharide obtained in the example.

FIG. 5 shows an IR spectrum of the heteropolysaccharide obtained in the example.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12R 1:89) (72) Inventor Kenji Yamamura 2-8-11 Nishishimbashi, Minato-ku, Tokyo 7th Toyo Kaiji Building 8th floor Global Environment Industry Foundation Technical Research Institute (72) Inventor, Masao Karube 1-13-16 Higashiarima, Miyamae-ku, Kawasaki-shi, Kanagawa (56) Reference JP-A-7-102069 (JP, A) Algae (1989), Vol. 37, No. 4, p. 253-262 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 1/00 C12P 1 / 00-19 / 00 CA (STN) JISST file (JOIS) BIOSIS / MEDLINE / WPID S (STN)

Claims (5)

(57) [Claims] 1. Anhydrogalactose as a constituent monosaccharide.
The main constituent sugar is galactose that does not substantially contain
Is it a constituent monosaccharide within the allowable range of the analytical value error of the following ratio
Rannahli, dissolved in hot water (100 ° C. or higher), the aqueous solution is gelled at room temperature
Without being easily hydrolyzed by acid or alkali,
It is degradable , has a molecular weight of 1 million or more, and has the ability to produce heteropolysaccharides belonging to the genus Coccomyxa.
Characterized by being collected from a culture medium of a novel microalgae
Heteropolysaccharide Coccogalactan : Galactose 70%; Mannose 13%; Xylose 10%; Arabinose 3%; Glucose 2%; Rhamnose 2%. 2. The heteropolysaccharide Kokkogaraku according to claim 1.
Microalgae Coccomyxa gl having tan production ability
eobotrydiformis NY2F. 3. The growth temperature range of the microalgae is 15 to
The temperature is 35 ° C, and the concentration of growing carbon dioxide is 0.03 to 40%.
The microalgae Coccomyxa according to claim 2.
gleobotrydiformis NY2F. 4. A culture medium containing a contractant belonging to the genus Coccomyxa
It has the ability to produce the heteropolysaccharide Kokkogalactan according to claim 1.
The microalgae according to claim 1, which is cultured in a medium.
The telopolysaccharide kokkogalactan is produced outside the algal body and
From the ground, the heteropolysaccharide Kokkogalactan according to claim 1
A method for producing a heteropolysaccharide, which comprises collecting the heteropolysaccharide. 5. A gas containing carbon dioxide is introduced into the medium.
And cultivate it to utilize carbon dioxide as a carbon source
The method for producing a heteropolysaccharide according to claim 4, characterized in that