JP4915760B2 - Non-mature monoalgal culture, method for producing the same, and algal body in which the same was grown - Google Patents

Description

  The present invention relates to a novel monoalgae culture strain derived from a red seaweed large seaweed that does not mature even when stored or cultured for a long period of time and hardly adheres to other algae, a method for producing the same, and an algal body in which the algae are grown It is about.

  Searching for useful marine resources and making effective use of them is now an important issue in Japan, a resource-reduced country. In particular, large seaweeds that grow in a special environment of the ocean contain special components that are not found in terrestrial organisms, and these special components are used as raw materials for foods and industrial products (Non-patent Document 1). In recent years, several new bioactive substances have been found among the fractions of large seaweed and components separated therefrom, and as a result, large seaweed has attracted attention as a raw material for fine chemicals. (See Non-Patent Documents 3 and 4).

  By the way, components derived from large-scale seaweed are subject to qualitative and quantitative variations depending on the seaweed growth time (see Non-patent Document 5). However, it is difficult to grow large-scale seaweeds in large quantities because of the slow growth rate of seaweeds and large consumption of filtered seawater.

  In other words, for indoor culture of algae, the setting of conditions is important, and a seaweed sample for growth experiments for this condition setting is required. In large seaweed growth evaluation experiments, It is difficult to use as it is.

  The reason for this is that the growth rate of large-sized seaweed-adherent microorganisms is often faster than that of large-sized seaweeds under artificial culture conditions, and the microorganisms grow abnormally and affect the growth of large-scale seaweeds. In order to remove such adherent coexisting microorganisms, a chemical treatment method and a monoalgae culture strain preparation method are known, but the monoalgae culture strain preparation method is the reason why the algal cells are less damaged than the chemical treatment method. preferable.

  By the way, when developing useful components of algae, algae die after maturation, so it is necessary to obtain a single algae culture every year. However, since it does not mature or die, it is not necessary to obtain a fresh culture every year. Of the large seaweeds, for example, inferior-mature seaweeds belonging to the genus Aosa are known for green algae, but this kind of seaweed has never been known for red algae.

  On the other hand, in general, in the previous stage of growing a monoalgae culture strain, a method is known in which a right solid is stored under conditions of slow growth and a single alga culture is grown from this right solid. In order to grow a necessary amount of monoalgal cultures from a straight solid, it usually takes 2 to 4 weeks in the case of seaweeds of the genus Ogonori, so that the experiment is inevitable during that time.

In addition, it is conceivable that the growth of the monoalgae culture strain and the growth of the monoalgae culture strain from a straight solid are performed in parallel to save processing time, but in this case, the operation becomes complicated, There is a disadvantage that the culture equipment and labor increase.
Therefore, in this technical field, there has been a strong demand for the emergence of monoalgal cultures that can be proliferated and cultured immediately when necessary, or can be continuously cultured without maturation.

  Red algae grow quiescent lymphocytes, trigger mitogenic stimuli to proliferate, determine the immunity of patients with various diseases including AIDS, and develop new cancer treatments. In particular, LAK therapy is attracting attention because of its high production of hemagglutinating agents that promote lymphocyte division.

  As a method for artificially cultivating seaweeds, a method for cultivating sterile seaweeds belonging to green algae, for example, Aosa, to purify contaminated sea areas (see Patent Document 1); A method of culturing in a solar dome (see Patent Documents 2 and 3) has been proposed.

However, the green alga Aosa seaweed has a flat shape and a film shape, and has the following disadvantages (1) to (4).
(1) Since it is a film, it cannot be cultured in multiple layers. (2) Algae is weaker than the red alga Ogonori in the form of a cylinder and is easy to tear off. (3) Since the algal bodies are easily broken, they cannot be cultured while fixed on a carrier. It is not easy to collect, and broken algae cause contamination. (4) If the diameter exceeds 30 cm square, dispersion by stirring and stirring will become difficult, resulting in loss of sunlight reception and a decrease in growth speed. Therefore, the growth speed cannot be recovered unless it is collected and cut ( (See Patent Documents 1 and 3).

  In general, green algae need light intensity that is stronger than red algae seaweeds. In order to use the products that accompany the growth of seaweeds and the functions of the growing seaweeds, the use of green algae seaweeds generally requires equipment or conditions that maintain a higher light intensity than the use of red algae seaweeds. Become.

  Sterile Aosa rots, breaks down and disappears, part of it remains, and grows and grows over the next year as nutrients rise. This repetition seems to occur every year. Sterile Aosa actually accumulated on the beach is causing pollution. It has been pointed out that water-containing Aosa is relatively perishable, and, for example, it decays in a day, so that it is necessary to dehydrate and dry immediately after recovery from the medium or seawater (see Patent Documents 1 and 3). ).

  On the other hand, red algae, for example, ogonori, have strong algal bodies and are difficult to cut, so they can be fixed on a carrier and cultured in large quantities, and are easy to manage and collect. It is suitable for large-scale indoor culture because it does not easily occur, grows even in low light, does not spoil, does not cause environmental pollution, and can be cultured even if the algal bodies are filamentous and overlapped.

Satoshi Tokuda, Masao Ohno, Hisauro Ogawa, "Seaweed Resource Aquaculture", Midori Shobo, 1987, p. 35-66 “Food Development”, 1984, Vol. 19, p. 43-48 “Monthly Ocean”, 1995, Vol. 27, p. 13-21 “Monthly Ocean”, 1995, Vol. 27, p. 34-39 “Hydrobiology”, 1993, 260/261, p. 541-547 Japanese Patent Laid-Open No. 2000-254685 (Claims and others) JP-A-11-289894 (Claims and others) JP-A-2004-97003 (Claims and others)

  Under such circumstances, the present invention has the property that red algae macroalgae are immature, can be stored for a long period of time, can be cultured, and have a high production amount of physiologically active substances. It was made for the purpose of providing a novel monoalgal culture strain having at least one of the properties of high growth rate and high ability of absorbing nutrient salts, and having high culture efficiency. is there.

  The present inventors have conducted various studies on monoalgae culture strains from red seaweed large seaweed, and as a result, the female gametophyte is not detected as a natural mature body, and only a tetraspore mature body is detected. In addition, monoalgae cultures derived from large red seaweeds grown in fresh seawater-containing natural seawater do not mature over a long period of time, and other algae are extremely difficult to attach even after continuous culture over a long period of time. The present invention has been made based on this finding.

  That is, the present invention is characterized in that a female gametophyte is not detected as a mature body in nature, and only a tetraspore body mature body is detected, and a red alga (Gracilaria sp) that breeds in freshwater-mixed natural seawater. .) Derived non-mature monoalgal cultures, which are characterized by the fact that the female gametophyte is not detected as a natural mature body, and only the tetraspore-like mature body is detected, The adult spores of Gracilaria sp. Are collected, and the spores are cut and allowed to stand to release spores. The released spores are cultured, and a solid body grows from the germinated spores. The present invention also provides a method for producing a non-mature monoalgae culture strain characterized in that the non-mature monoalgae culture strain is proliferated and cultured, and an algal body in which the above-described non-mature monoalgae culture strain is propagated.

  Here, a non-mature monoalgal culture does not mature even if it is continuously cultured for 3 years or more under culture conditions, and produces the same physiologically active substance from seaweed as the culture immediately after the creation of the monoalgal culture. Means what to do. In addition, even if the monoalgae culture strain is preserved for 3 years or more under non-growth culture conditions such as low nutrients, low temperature or low light intensity, and then brought to the culture conditions, it is continuously cultured under the culture conditions for 3 years or more. Even though it does not mature, it has the same properties as the culture strain immediately after the creation of the monoalgae culture strain from seaweed, that is, the property that the production amount of physiologically active substances is high, the property that the growth rate of algal bodies is high, and the ability to absorb nutrients Means at least one of the properties of high.

Next, the present invention will be described in detail.
The immature monoalgae culture strain of the present invention is a natural matured adult female gametophyte in a freshwater-mixed natural seawater area, particularly a seawater area with a salinity of 1.0% by mass or less, such as an estuary where river water flows into the ocean. It is not detected and has a feature that only a tetraspore-like matured body is detected, and it can be produced using a breeding red algae large seaweed as a raw material.

  In the present invention, the red seaweed large seaweed belongs to the red seaweed net of the plant classification system, refers to a large one, has chlorophyll a and phycobilin as main synthetic pigments, and generates and stores fluroside and red seaweed starch by photosynthesis. It has the feature of doing. Among these, there are tengusa, ogonoris, cedars, hornworts, and seaweeds. Species are preferred.

  In the present invention, the genus Red seaweed (Gracilaria sp.) Is (1) a seaweed classified as a seaweed genus (Gracilaria sp.), Or (2) Gracilaria spis sp. Or (3) Gracilariopsis sp. Includes seaweeds classified in the past.

  For example, in Japanese seaweeds, Gracilaria sp. Is a genus of red seaweeds (Gracilaria sp.) In “New Japan Seaweed Magazine, Japanese Seaweeds Review, Tadao Yoshida, published by Uchida Otsutsuru, 1998”. It includes seaweeds that are classified in the family of the genus Lariares (Gracilariaceae). Although these red algae are also present in the cold sea, they are particularly abundant in the warm sea, are distributed in almost all coastal areas in Japan, and are used for agar agar and sashimi.

  In order to produce a non-mature monoalgae culture strain from this red algae large seaweed, for example, there is a feature that a female gametophyte is not detected naturally as a mature body, but a mature body of only tetraspores is detected. The mature spores of red algae large seaweed that are breeding in fresh seawater-mixed natural seawater are cut into 2-5 cm, preferably 3-4 cm long, washed with sterilized water or seawater, and then sterilized seawater. Leave in for 6-15 hours to release spores.

Next, the released spores are separated, transplanted to a container containing a culture solution, and statically cultured alternately at a temperature of 10 to 30 ° C. for 10 to 15 hours under exposure and in a dark place. As the culture solution at this time, for example, a sterilized seawater to which a normal seawater-enriched nutrient is added is used.
In this way, after a static culture for 15 to 25 days, a thick solid solid color solid is selected from the straight seaweed solids that have sprouted and grown, and when the static culture is continued for 50 to 80 days, Grows to 10 mm.
The straight solid is removed from the bottom of the culture vessel with tweezers, transplanted to a flask, and cultured under storage culture conditions, whereby algal bodies grow, and as a result, a monoalgae culture strain of a certain amount or more can be obtained.

Examples of the culture conditions include a temperature of 15 to 30 ° C., a light intensity of 50 to 120 μmol / m ² sec, and a light cycle of 8 hours light period-16 hours dark period to 24 hours light period-0 hours dark period. . If necessary, shaking (about 50 to 200 rpm) or aeration may be performed. The culture solution may be natural seawater or artificial seawater. In some cases, the culture medium contains Provasoli's seawater supplemented nutrients [Kazutoshi Nishizawa, Mitsuo Chihara, Algae Research Method, Kyoritsu Shuppan, Tokyo (1979), pp. 281-305] may be added.

In the present invention, an algal body in which a solid body is grown by growth culture is referred to as a monoalgae culture strain.
In addition, a straight three-dimensional or single-algae culture strain can be controlled for storage and low-growth culture by placing it under non-growth culture conditions such as low nutrition, low temperature or low light intensity. Storage and low-growth culture are convenient when there is no plan to use a straight solid or monoalgae culture strain, or when it is desired to adjust the algal growth amount.

Non-growth culture conditions such as low nutrition or low temperature or low light intensity are, for example, (1) nutrient salt concentration conditions such as nitrate nitrogen and ammonia nitrogen concentrations of 3 μM or less, phosphate ion concentrations of 1 μM or less, (2 Examples include low temperature conditions where the temperature is 5 to 14 ° C., (3) low light intensity conditions where the light intensity is 20 to 40 μmol / m ² sec, and combinations of (4) and (1) to (3).

  The non-mature monoalgal culture of the present invention does not mature even if it is continuously cultured for 3 years or more under culture conditions, and attached algae hardly grow. In general, when the number of attached algae increases, the seaweed ingests nutrients in the medium to the attached algae that grow faster than the seaweed, and the growth is inhibited. In the worst case, the algae die.

  The immature monoalgal culture of the present invention can be stored for a long period of 3 years or longer because adherent algae hardly adhere. In addition, rapid culture using a medium is possible, and after storage, proliferation can be started quickly at a desired time.

  The immature monoalgae culture strain of the present invention has an advantage that impurities and toxic components derived from the attached algae are not mixed when recovering useful substances from the algal bodies after the growth because the attached algae hardly adhere. Moreover, (1) the production amount of physiologically active substances is high, (2) the growth rate of algal bodies is fast, (3) the ability to absorb nutrients is high, and at least one of the properties (1) to (3) above It is a red seaweed large seaweed having the above properties, and can be cultured or stored for a long period of time without ripening.

  Next, the best mode for carrying out the present invention will be described by way of examples.

Selection of red seaweeds As an example of red seaweeds (Gracilaria sp.), Seaweeds from 3 April 1998 through 3 March 2001 at three locations for the red seaweeds, Gracilaria chorda The appearance amount (growth) and the maturity survey were conducted once a month.

The survey site A was selected in the Katsuura River at the mouth of the Katsuura River in Tokushima City, Tokushima Prefecture. The seaweed growing at the survey point A is hereinafter referred to as Katsuura River Ogonori seaweed. In this place, at the time of low tide, all or a part of the seaweed seaweed (Tsursilamo) community from Katsuura River was dried out.
The seaweed growing at this survey point A is hereinafter referred to as Katsuura River seaweed genus seaweed (Katsuura River Tsurusilamo).

As the survey point B, we chose the coast of Tochishima City, Tochishima City (the coast adjacent to the mouth of the Yoshino River, which is a first-class river. Adaptability to brackish water is lower than that of the genus Ogonori from Katsuura River). The seaweed growing at the survey point B is hereinafter referred to as the seaweed genus seaweed [or Tsurusiramo (produced in the Yoshino River estuary area, Tokushima Prefecture)] off Tokushima City, Tokushima Prefecture.
We selected the Seto Inland Sea off Wadajima, Komatsushima City, Tokushima Prefecture as the survey point C. The seaweed growing at this survey point C is hereinafter referred to as Komatsushima offshore tsurusilamo.

  The change in wet mass of vine sorghum per unit volume in the tsurusilamo community inhabiting the flat tide of the intertidal zone at each survey point (the estuary that rises at low tide for survey point A), and the number of mature individuals among all tsurusilamo Examined. At this time, the number of rectangular frames 20 cm in length and breadth in the Tsurushiramo community was set to 4 times each time, and the average value of 4 times was set as the average.

  Determination of maturation and non-maturity of the collected algal bodies was observed using a stereomicroscope, and was judged by whether or not a tetraspore or sac was formed in the algal bodies. The alga body in which formation of a tetraspore sac was detected by observation was determined to be a mature tetraspore body, while the alga body observed to form a sac was determined to be a mature female gametophyte. From this observation result, the number of mature tetraspore bodies relative to the total number of tsurusilamo individuals was determined as a percentage (%) of mature tetraspore bodies. In addition, the number of mature female gametophytes relative to the total number of Culsilamo individuals was determined as a percentage (%) of mature female gametophytes. By comparing the results at each survey point, it is possible to select a red alga belonging to the genus Ogonori, which has a characteristic that a female gametophyte is not detected in nature as a mature but only a tetraspore is detected.

  Table 1 shows the results of research on mature culm silos from April 1998 to March 1999, Table 2 shows results of research on mature citrus moths from April 1999 to March 2000, April 2000 to 2001 Table 3 shows the results of the survey of mature tsurusilamo individuals up to March 2012. The numerical values in each table are the average values of four square 20 cm vertical and horizontal frames installed in the tsurusilamo community.

  According to Tables 1 to 3, “Katsuura River is breeding at the survey site A as a red alga belonging to the genus Ogonori, which has a characteristic that a natural gametophyte is not detected as a mature body and only a tetraspore body is detected. "Ogonori seaweed (Katsuuragawa tsurusilamo)" can be selected.

(1) Spore collection and spore planting for preparing monoalgal cultures;
As a raw material, the female gametophyte is not detected as a natural matured body, but a spotted algae with a characteristic that only a tetraspore is detected as a red alga, which is a survey point A, that is, the Katsuura River estuary in Tokushima City Mature spores of the large seaweed large seaweed Gracilaria chorda collected at a salt concentration of 0.5% by mass were used.

The mature part of the mature spore body was cut into a length of 30 mm, washed with sterilized seawater, and then left overnight in sterilized seawater to release spores. The released spores were sucked up with a sterilized Pasteur pipette, separated into a screw tube containing 30 ml of a culture medium for preservation culture, and subjected to static culture by applying light at a cycle of 14 hours light period and 10 hours dark period. . Twenty spores were planted in one screw tube. A total of 1000 screw tubes were used. In static culture, (i) a light intensity of 60 μmol / m ² sec under a constant condition, a temperature of 6 conditions (4 ° C. fluctuation from 10 ° C. to 30 ° C.), and (ii) a light intensity of 5 conditions (20 μmol / m ² ). The test was carried out under a total of 10 conditions from m ² sec to 100 μmol / m ² sec (20 μmol / m ² sec variation).

  In this seawater medium, seawater collected at a depth of approximately 1.5m in Yashima Bay, Takamatsu City, Kagawa Prefecture is filtered through a 0.20μm cellulose acetate membrane filter (manufactured by Advantech Toyo Co., Ltd.), then 1/10 volume of distilled water is added and mixed. After that, it was sterilized at 100 ° C. for 30 minutes, and pre-sterilized Provasoli seawater supplemented nutrients were added.

(2) Straight solid selection;
From the experimental group in which spore germination was observed after 21 days of static culture, the experimental conditions were selected, where the solid solid was thick, the red pigment was vivid, and there was no suspended matter in the culture. In Example 2, a straight solid germinated under the conditions of “temperature 18 ° C., light intensity 40 μmol / m ² sec” was selected as an experimental material.

  The selected solid is continuously cultured until the length of the solid is 10 mm by stationary culture. At this time, the medium was exchanged once every 4 weeks. Thus, a straight solid having a length of 10 mm was obtained in about 70 days.

(3) Right-dimensional growth culture;
The straight solid grown to about 10 mm was removed from the bottom of the screw tube with tweezers and transplanted to a flask to carry out a solid growth culture. Right-angle growth culture is aerated in a 1 liter round bottom flask containing 1 liter of culture solution at a temperature of 16 ° C. and a light intensity of 40 μmol / m ² sec (14 hours light period, 10 hours dark period photoperiod). I went there. The culture medium was exchanged once every two weeks. Proliferation culture was performed for 70 days to proliferate the vertical solid. Since this process can also be applied to preservation of a right solid, it is also called a right three-dimensional preservation culture process. By dividing a straight solid grown in one round bottom flask into a 1 liter round bottom flask containing 1 liter of several culture solutions, the preservation culture process period can be extended.

(4) Preculture of monoalgal cultures;
The straight solid grown in the previous step was heated at a temperature of 18 ° C. and a light intensity of 40 μmol / m ² sec (14 hours light period, 10 hours dark period photoperiod) in a 1 liter round bottom flask containing 1 liter of culture solution. It was performed while aeration was performed under conditions. The culture medium was exchanged once every two weeks. Pre-culture was performed for 35 days to obtain a monoalgal culture.

(5) Maturity evaluation and growth rate evaluation of monoalgal cultures;
Using algae culture tester capable of temperature control (temperature distribution ± 0.5 ° C.), light intensity control (non-conformal gradation light), day long time control, etc., the maturity of monoalgal cultures was evaluated. In addition, this apparatus can culture 50 500 ml Erlenmeyer flasks simultaneously (in-tank dimensions 1250 W × 720 D × 900 Hmm). A 4 mm long apical fragment was prepared from a single-algae culture strain of the large seaweed Culsilamo, and 6 fragments were added per Erlenmeyer flask containing 400 ml of cultured seawater. Irradiation was performed under conditions of 14 hours light period and 10 hours dark period, and culture medium exchange was performed every week. The number of experimental points under the same culture conditions was 5 points.

Subsequently, maturity evaluation of the monoalgae culture strain was carried out by (i) temperature 6 conditions (4 ° C. fluctuation from 10 ° C. to 30 ° C.) under constant conditions of light intensity of 60 μmol / m ² sec, and (ii) temperature 22 ± 0.5. The test was carried out while aeration was performed under a total of 10 conditions of 5 light intensities ( ²⁰ μmol / m ² sec variation from ²⁰ μmol / m ² sec to 100 μmol / m ² sec) under the condition of ° C.

  Moreover, culture solution exchange and seaweed wet mass measurement were performed in the clean booth. In this manner, the wet mass of seaweed per flask is recorded, and the presence or absence of reproductive organs such as cysts, quadrant spores or sperm cysts on the surface of the seaweed is observed with a microscope. The presence or absence of was judged.

  As a result, no mature experimental plot was observed even after 12 weeks of culture. When the wet mass of seaweed in one 400 ml Erlenmeyer flask reached 0.2 g, culturing was continued by thinning out to 0.02 g, but 3 years have passed since the start of cultivation [(5) process start]. Even did not mature.

Growth rate Relative growth rate (RGR) is represented as R. Seaweed wet mass at the start of cultivation W _0, the seaweed wet mass after cultivation day t and _{W t, R = (lnW t} -lnW 0) / t by the relative growth rate is obtained. The growth rate (% / day) was calculated by multiplying R by 100.
The growth rate of Tsurusiramo (Katsuura River Kawaguchi) monoalgae culture strain from 2 to 3 weeks in culture is the maximum growth rate under the conditions of a temperature of 22 ° C. and light intensity of 60 μmol / m ² sec. The value was 14.4% / day.

Growth and maturation evaluation in 20 liter culture solution Culsilamo (Katsuuragawa Kawaguchi) monoalgal culture is cultured in 10 1 liter flat bottom flasks and grown to a wet mass of 4 g or more. At this time, the conditions under which the maximum growth rate was obtained in a 400 ml scale culture were “temperature 22 ° C., light intensity 60 μmol / m ² sec, photoperiod 14 hours light period-10 hours dark period, all-day aeration, medium change every week” The culture conditions were set as follows. This culture condition is referred to as a growth culture condition.

  The culture solution (seawater medium) was 1/10 volume of distilled water after filtering seawater collected at a depth of 1.5 m in Yashima Bay, Takamatsu City, Kagawa Prefecture through a 0.20 μm cellulose acetate membrane filter (Advantech Toyo Co., Ltd.). After mixing and mixing, the mixture was sterilized at 100 ° C. for 30 minutes, and pre-sterilized Provasoli seawater supplemented nutrients were added. Hereinafter, this culture solution (seawater medium) is referred to as growth culture seawater.

4 g of Culsilamo (Katsuura River Kawaguchi) monoalgae culture strain obtained by growth culture was transplanted to a 30 liter culture vessel containing 20 liters of seawater for growth culture, and cultured under growth culture conditions for 4 weeks. After 4 weeks, the seaweed wet mass increased by about 12 times to about 47 g.
There was no mature experimental plot even after 12 weeks of culture. Thereafter, when the wet mass of seaweed in the 20 liter culture solution containing 20 liters of growth cultured seawater reached 300 g, culturing was continued by thinning out to 10 g. Even after 3 years from the start of culture, it did not mature. Table 4 shows the growth rate, seaweed yield, and maturation of monoalgal cultures in 400 ml culture solution and 20 liter culture solution.

(6) Evaluation of the amount of physiologically active substance in monoalgal cultures;
(A) Extraction of water-soluble fraction;
After washing with 0.15M sodium chloride aqueous solution, 25 g wet mass of Tsurushiramo (Katsuura River Kawaguchi) obtained in the 4th week of culture was frozen at -30 ° C. Finely ground frozen seaweed (Tsursilamo) using 0.5M Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.2) containing 30 mM potassium chloride, 3 μM zinc sulfate, 5 mM 2-mercaptoethanol as the extraction buffer. After adding 40 ml of extraction buffer to the wet mass (equivalent to 500 g) and homogenizing the mixture, the homogenized solution was allowed to stand at 4 ° C. for 6 hours and then centrifuged to obtain a crude crude extract.

  Subsequently, ammonium sulfate was added to the crude extract so as to obtain a saturated solution having a final concentration of 35% by mass, and the first salting out was performed. After completion of the addition of ammonium sulfate, the mixture was allowed to stand at 4 ° C. for 1 hour, and the produced precipitate was removed by centrifugation. By this operation, contaminants such as pigment were removed as a precipitate fraction. Next, ammonium sulfate was added to the supernatant obtained by centrifugation so as to be a saturated solution having a final concentration of 70% by mass, and the mixture was allowed to stand at 4 ° C. overnight, and then the produced precipitate was separated by centrifugation. The fractionated precipitate was redissolved with 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride, and then dialyzed against 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride. A crude active fraction was obtained. The hemagglutination activity of the obtained crude active fraction on rabbit erythrocytes was 512 units, and the specific activity was 6948 units / mg protein. Here, the unit of the agglutinating activity was defined as the reciprocal of the maximum dilution rate of the sample where the agglutinating activity can be detected.

  25 g wet mass obtained from the third year of cultivation (from Katsuuragawa Kawaguchi) was washed with 0.15 M aqueous sodium chloride solution and frozen at -30 ° C. Finely ground frozen seaweed (Tsursilamo) using 0.5 M Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.2) containing 30 mM potassium chloride, 3 μM zinc sulfate, and 5 mM 2-mercaptoethanol as the extraction buffer. After adding 40 ml of extraction buffer to the wet mass (equivalent to 500 g) and homogenizing the mixture, the homogenized solution was allowed to stand at 4 ° C. for 6 hours and then centrifuged to obtain a crude crude extract.

  Subsequently, ammonium sulfate was added to the crude extract so as to obtain a saturated solution having a final concentration of 35% by mass, and the first salting out was performed. After completion of the addition of ammonium sulfate, the mixture was allowed to stand at 4 ° C. for 1 hour, and the produced precipitate was removed by centrifugation. By this operation, contaminants such as pigment were removed as a precipitate fraction. Next, ammonium sulfate was added to the supernatant obtained by centrifugation so as to be a saturated solution having a final concentration of 70% by mass, and the mixture was allowed to stand at 4 ° C. overnight, and then the produced precipitate was separated by centrifugation. The fractionated precipitate was redissolved with 100 M phosphate buffer (pH 6.9) containing 0.15 sodium chloride, and then dialyzed against 100 mM phosphate buffer (pH 6.9) containing 0.15 M sodium chloride. A crude active fraction was obtained. The hemagglutination activity of the obtained crude active fraction on rabbit erythrocytes was 512 units, and the specific activity was 6810 units / mg protein. The results are shown in Table 5.

  Mitogenic activity was measured for the crude active fraction. A human lymphocyte blastogenesis test was performed.

Next, a human lymphocyte rejuvenation test was conducted by ³ H-thymidine incorporation, and the mitogenic activity of the crudely active fraction was measured for a purified preparation. In this case, all the cell culture materials, eg microplate, cell harvester, glass fiber filter, counting vial, ³ H-thymidine, toluene scintillator (POPO 0.1 g + PPO 5 g / liter toluene), liquid scintillation counter preparation and All operations performed using these were performed aseptically.

Next, an aqueous solution in which RPM1 1640 1.05 g, NaHCO ₃ 0.2 g, penicillin 10000 Unit, streptomycin 10 mg, and fetal bovine serum 10 ml are prepared with respect to 100 ml pure as a culture solution is used after filtration sterilization with a filter. According to the amount, it was packed in a small bottle, sealed, and stored at -20 ° C. In this state, storage was possible for 2 months. When used, it was closed and used up, and freeze-thawing was not repeated.

Lymphocytes were separated from heparinized blood by Ficoll Conlay method. Next, after washing the third floor with CMF-PBS (pH 7.0), the suspension was suspended in 1 ml of the culture solution, and the lymphocyte count was calculated. Subsequently, the culture solution was adjusted to 5 × 10 ⁵ cells / ml.

The lymphocytes were cultured by dispensing 200 μl of lymphocyte suspension into each well of the microplate. Next, the microplate containing lymphocytes was allowed to stand in a clean booth for 30 minutes, and then 20 μl of a crude active fraction and phosphate buffer (PES) were dispensed into each well as a mitogen solution. For the crude active fraction, a diluted solution (diluted from 10 times to 320 times) diluted with a buffer solution was prepared and used for the experiment. The amount of ³ H-thymidine incorporation (cpm) in the crude active fraction was determined by calculating the value converted to the stock solution by multiplying the measured value in the diluted solution by the dilution factor.
Subsequently, it was cultured in a humidified state of 37 ° C. in air containing 5% CO _{2 for} 3 days. Next, ³ H-thymidine was dispensed into each well so that the final concentration per culture broth was 1 μCi / ml 8 hours before the end of the culture.

  The activity was measured as follows. While harvesting the inside of the well with saline using Labo-MASH or the like, the cells were collected on a glass fiber filter, and this was continuously aspirated to wash the cells on the filter (approximately 20 seconds for about 1 saline solution). 5 ml). Next, the cell fixing part on the glass filter was peeled off and placed in a counting vial. Next, after sufficiently drying, 5 ml of liquid scintillator was dispensed into each vial using a dispenser, and measured with a scintillation counter. Evaluation of the crude active fraction obtained from the monoalgae culture strain at 4 weeks in culture was performed using lymphocytes from three specimens (hereinafter referred to as specimen I, specimen II and specimen III). The number of experiments under a certain experimental condition is three times, and the average indicates the average value of three measurements. The results are shown in Table 6. In addition, the evaluation of the crude active fraction obtained from the monoalgae culture strain in the third year of culture was performed using lymphocytes from three specimens (hereinafter referred to as specimen IV, specimen V, and specimen VI). The number of experiments under a certain experimental condition is three times, and the average indicates the average value of three measurements. The results are shown in Table 7.

Large seaweed is capable of absorbing nutrients. Nutrient salts include nitrate nitrogen, phosphate ions, ammonium ions (nitrogen), and the like. The maximum amount of nitrate nitrogen absorbed per day was evaluated as the nutrient absorption capacity of the monoalgal culture.
The maximum load of nitrate ion per unit wet mass for 4 weeks of culture of monoalgal cultures prepared from spores of the seaweeds from Katsuura River (Tsursilamo from Katsuura River) is about 0.4 mg nitrogen / seaweed wet mass g · day Met. The results are shown in Table 8. The maximum daily nitrate ion load per unit wet mass in the third year of culture was also about 0.4 mg nitrogen / seaweed wet mass g · day.

Comparative Example 1
Instead of the large seaweed genus seaweed (Gracilaria chorda) “Katsuuragawa seaweed (Katsuura River tsurusilamo)” collected in the Katsuura River estuary brackish water area (salt concentration 0.5 mass%) in Tokushima City, Tokushima Prefecture, A monoalgae culture strain was obtained in the same manner as in Example 2 except that Tsurusilamo (produced in the Yoshino River estuary) was used.

  As a result of the evaluation of the maturity and the growth rate of the monoalgal culture strain prepared from Tsurusilamo (Yoshinogawa estuary), maturation was observed in 12 weeks in both 400 ml culture and 20 liter culture. Also, the growth rate is 8.2% / day, and the mass after 4 weeks of cultivation of 4 g of seaweed is 12 g, and an immature monoalga prepared from “Katsuura River Ogonori seaweed (Katsuura River tsurusilamo)” It was lower than the culture strain (Table 4). The hemagglutinating activity contained in the crude active fraction is 256 units, the specific activity is 3204 units / mg protein, and lower than that of the immature monoalgae culture strain prepared from “Katsuuragawa Ogonori genus seaweed (Katsuuragawa tsurusilamo)” (Table 5). Mitogen activity was lower in all three specimens than the immature monoalgae cultures prepared from “Konuragawa-born seaweeds (Katsuuragawa tsurusilamo)” (Table 6). The daily maximum nitrogen load tolerance is 0.2 mg of nitrogen / seaweed wet mass g · day and half of the immature monoalgae culture strain prepared from “Katsuura River Ogonori genus seaweed (Katsuura River tsurusilamo)” Value (Table 7).

Comparative Example 2
Instead of the large seaweed genus seaweed (Gracilaria chorda) “Katsuuragawa seaweed (Katsuura River tsurusilamo)” collected in the Katsuura River estuary brackish water area (salt concentration 0.5 mass%) in Tokushima City, Tokushima Prefecture, A monoalgae culture strain was obtained in synchronism with Example 2 except for using Tsurusilamo off Komatsushima.

  As a result of evaluating the maturity and measuring the growth rate of the monoalgae culture strain prepared from Tsurusilamo from off Komatsushima, maturation was observed in 11 weeks in both 400 ml culture and 20 liter culture. The growth rate is 7.7% / day, and the mass after 4 weeks of cultivation of 4 g of seaweed is 11 g, which is an immature monoalga prepared from “Konuragawa Ogonori genus seaweed (Katsuuragawa tsurusilamo)” It was lower than the culture strain (Table 4). The hemagglutination activity contained in the crude active fraction was 256 units, specific activity 3063 units / mg protein and lower than that of the immature monoalgae culture strain prepared from “Katsuuragawa Ogonori seaweed (Katsuuragawa Tsurusilamo)” (Table 5). Mitogen activity was lower in all three specimens than the immature monoalgae cultures prepared from “Konuragawa-born seaweeds (Katsuuragawa tsurusilamo)” (Table 6). The maximum daily nitrogen load tolerance is 0.1 mg nitrogen / wet mass of seaweed g · day and a quarter of the immature monoalgae culture strain prepared from “Konuragawa Ogonori seaweed (Katsuuragawa Tsurusilamo)” Value (Table 7).

  From each result, the monoalgae culture strain prepared from the spores of the seaweed genus Seaweed from Katsuuragawa (Tsurushiramo from Katsuuragawa) does not mature even if cultured continuously for more than 3 years under the culture conditions. High production amount of physiologically active substance, (2) High growth rate of algal bodies, (3) High ability to absorb nutrients, At least one of the above properties (1) to (3) It can be seen that it is a red alga belonging to the genus Ogonori.

  Monoalgal cultures prepared from spores of the seaweeds of the genus Ogonori (Katsuuragawa) from Katsuura River were prepared from spores of Tsurusilamo (from the Yoshinogawa estuary) or spores of Koshikojima offshore Komatsushima It is superior to the monoalgal culture strain in the following points. (1) Not mature. (2) Growth is high. (3) High bioactive substance content. (4) High nutrient absorption capacity. These advantages are industrially advantageous.

  In the same manner as in Example 2, a non-mature monoalgal culture was prepared from spores of kuluramo from Katsuuragawa Kawaguchi, and this was continuously cultured for 5 years. When the number of other algae adhering to the surface of this cultured strain was measured by microscopic observation, it was less than 10 cells per 400 mg of the cultured strain wet mass.

For comparison, tsurusilamo off Komatsushima was collected from natural waters, washed three times with the seawater medium described in Example 2, and measured for other algae adhering to the surface by microscopic observation. About 70000 cells were attached per 400 mg of mass. Further, this natural algal body off Komatsushima was washed 10 times with the seawater medium described in Example 2, and then the alga body was cut into 3 cm, and further washed 10 times with the seawater medium described in Example 2. When a slice was obtained and the culture of the washed slice in the seawater medium described in Example 2 was started, the growth of microalgae was conspicuous in the flask containing the seaweed slice on day 14 of the culture, and the increase in seaweed wet mass was reduced. The seaweed wet mass of the 21st culture was lower than the seaweed wet mass of the 14th day.
From this, it can be seen that the immature monoalgae culture strain of the present invention has the property that it is difficult to grow attached algae.

  The immature monoalgal culture of the present invention is suitably used for the production of a physiologically active substance such as a hemagglutinating agent.

Claims (6)

Non-mature derived from Gracilaria chorda , which is characterized by the fact that it does not detect female gametophytes in nature and only matures of tetraspores are detected, and breeds in natural seawater mixed with fresh water. Sex algae cultures. After three years continuous culture, adhere algae per wet weight 400mg is less than 10 cells according to claim 1 Symbol non maturity unialgal cultures of the mounting. The maturation of the genus Red- breasted algae Gracilaria chorda , which is characterized by the fact that it does not detect the female gametophyte as a naturally occurring mature body, but detects only the tetraspore mature body, and breeds in freshwater-mixed natural seawater. The spores are collected, and the spores are cut and allowed to stand to release the spores, the released spores are cultured, and after the germinated spores have grown, a solid culture is grown and cultured. A method for producing a mature monoalgal culture. The method for producing an immature monoalgal culture according to claim 3, wherein the freshwater-mixed natural seawater area is a seawater area having a salinity of 1.0% by mass or less. The method for producing an immature monoalgae culture strain according to claim 3 or 4, wherein the obtained nonmature monoalgae culture strain has less than 10 cells of attached algae per 400 mg of wet mass after continuous cultivation for 3 years. An algal body in which the immature monoalgae culture strain according to claim 1 or 2 is grown.