JP2006000058A - Method for proliferating algae and the resultant cultured algae - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method enabling macroalgae, especially red algae, to be preserved in a non-matured condition over a long period and to be cultured, and also designed to efficiently culture a new unialgal strain having at least one property among the following:(1) high productivity of physiologically active substances, (2) high rate of growing algal bodies and (3) high ability to absorb nutritive salts, and to provide a large quantity of algae cultured by such a method. <P>SOLUTION: The method for proliferating algae comprises making a nutritive culture of pieces of an artificial culture strain of the algae, preferably Gracilaria verrucosa. The resultant cultured algae are also provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an algae growth method and an algae culture obtained by the method.

  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. As a method for removing such coexisting microorganisms, a chemical treatment method and a monoalgae culture strain preparation method are known, but the monoalgae culture strain preparation method is preferable in that the algal body is less damaged.

  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. Such a non-mature culture strain is known to be, for example, a hardly mature seaweed belonging to the genus Aosa in green algae, but this kind of single-algae culture strain has never been known so far for red algae. There wasn't.

  In general, in the previous stage of growing the monoalgal culture, the straight solid is stored under conditions of slow growth, and the monoalgal culture is proliferated and cultured from this straight 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.

  For this reason, it is considered 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. Furthermore, there is a disadvantage that the culture equipment and labor increase.

  Therefore, in this technical field, there has been a strong emergence of a method for efficiently growing algae using a single algae culture that can be grown and cultured immediately when necessary, or can be continuously cultured without maturation. It was requested.

  And this can cause mitogenic stimulation that triggers the proliferation and proliferation of lymphocytes in the stationary phase, determines the immune capacity of patients with various diseases including AIDS, and therapies for new cancers Particularly, there is a great demand for red algae having a high production amount of hemagglutinating agent for promoting lymphocyte division in LAK therapy.

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

  Under such circumstances, the present invention is immature, can be stored for a long period of time, and can be cultured for macroalgae, particularly red algae, and (1) the production amount of physiologically active substances is high. (2) A novel monoalgal culture having a high growth rate of algal bodies, (3) a high ability to absorb nutrient salts, and having at least one of the above properties (1) to (3) The object of the present invention is to provide a method for efficiently cultivating a strain and a large amount of algae cultured by such a method.

  The present inventors have made various studies on a monoalgal culture strain from the large seaweed Gracilaria sp., And the female gametophyte was not detected as a matured body in nature, and only tetraspores were detected. As a result of further research, we found that a monoalgae culture strain derived from the red seaweed of the genus Ogonori that has the characteristics of detecting the mature body of the genus and breeds in natural seawater mixed with fresh water does not mature over a long period of time. It has been found that the algae wet mass can be efficiently increased by cutting the algae obtained by artificial culture as described above and culturing them into algal body slices, and the present invention is made based on this finding. It came to.

  That is, the present invention provides an algae growth method characterized by nutritionally cultivating a section of an artificial culture strain of algae and an algae culture cultured in this manner.

  In the method of the present invention, the algae used as a raw material must be a monoalgal culture strain obtained by artificial culture. In the case of a naturally grown strain, smooth growth by nutrient culture is hindered by the influence of organisms that coexist and coexist, and smooth growth is not performed.

  Here, the monoalgal culture strain refers to artificial seawater obtained by collecting mature seaweeds from natural sea areas, washing and cutting mature alga bodies, releasing spores in sterilized artificial seawater, and sterilizing the released spores. It is a cultured strain of algae that has been cultivated and purified as algae.

The algae culture strain may be a culture strain of algae that is easily matured, but a culture strain that is immature, that is, difficult to mature, particularly a culture strain that does not mature.
The form of seaweed that can be used may be any of a Hempei type, a filamentous type, etc., but a filamentous type of seaweed such as ogonori, ooogonori, tsurushiramo is preferred.

In seaweeds, there is only one growth end on the branch of seaweed, so the conventional algae growth method using only seaweed growth ends has poor culture efficiency. However, in the method of the present invention, an intermediate section that can be collected from a lot of seaweed branches can be used in the same manner as the growth end, so that the culture efficiency can be remarkably improved.
Among the seaweeds, the red seaweeds of the genus Ogonori have a large regenerative power in the intermediate section and show the same growth as the growth edge.

  The efficiency is better when the length of the section is as short as possible. For example, when the length is 100 mm or more, the culture efficiency is low, but when the length is 5 mm or less, particularly 3 mm or less, the culture efficiency is remarkably improved.

  Nutrient culture can be performed indoors or outdoors, but indoor culture is preferable because environmental factors that affect seaweed growth such as light intensity and water temperature can be controlled. Cultivation can be performed by static culture, shaking culture, aeration culture by aeration, or the like. Considering the growth rate of algae, shaking culture or aeration culture by aeration is preferable.

As the culture medium, artificial seawater, natural seawater, filtered natural seawater, sterilized natural seawater, or seawater derived therefrom can be used, but artificial seawater is preferably used.
When artificial seawater is used, both sodium chloride and hardly soluble substance formation-preventing sodium chloride can be used as sodium chloride for preparing artificial seawater, but it is preferable to use hardly soluble substance formation-preventing sodium chloride.

  In particular, when a 20% strength aqueous solution is used, water based on water containing 20 to 40 g / liter of low magnesium sodium chloride having a magnesium ion concentration of 10 ppm or less is preferable.

  Next, the method of the present invention will be described in detail using an immature monoalgae culture strain as an example. However, the method of the present invention is not limited to this and can be used for the growth of artificial culture strains of other algae.

  In non-mature monoalgal cultures, naturally occurring female gametophytes are not detected as mature adults in freshwater-contaminated natural seawater areas, for example, estuaries where river water flows into the ocean, but only tetraspore mature bodies are detected. It can be produced using the breeding red seaweed large seaweed as a raw material. As the red seaweed large seaweed, ogonyori (Gracilaria verrucosa), tsurushiraamo (Gracilaria chorda), and their subspecies are preferable.

  The above-mentioned red seaweeds (Gracilaria sp.) Are (1) seaweeds classified into the seaweeds (Gracilaria sp.), Or (2) Gracilaria spis sp. Or (3) Gracilariopsis sp. Includes seaweeds classified in the past.

  For example, in the Japanese seaweed, the genus Gracilaria sp. Is referred to as “Shin Nihon Seaweed Magazine Japanese Seaweed Overview, Tadao Yoshida, published by Uchida Otsukaku, 1998”. Seaweeds classified in the family (Lariares) (Gracilariaceae) are included. 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 monoalgal culture from this genus Red algae, for example, there is a feature that a male and female gametophyte is not detected as a natural mature body, but a mature body of only tetraspore is detected. The mature sporophytes of the genus Rhizophorus genus breeding in freshwater-mixed natural seawater are cut into 2-5 cm, preferably 3-4 cm lengths, 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.

Thus, the algal body in which the right solid is grown by the growth culture is referred to as a monoalgae culture strain.
This straight three-dimensional or single-algae culture strain is capable of suppressing algal body growth rate by being placed under non-growth culture conditions such as low nutrition, low temperature, or low light intensity, and can be preserved or low-growth culture. Storage and low-growth culture are convenient when there is no plan to use a straight solid or monoalgal culture, or when the amount of algal growth is adjusted.

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 concentration and ammonia nitrogen concentration of 3 μM or less, phosphate ion concentration of 1 μM or less, 2) 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). .

In the method of the present invention, a section of this monoalgal culture is used as a raw material. That is, an algal body of a straight solid or a monoalgae culture strain is cut to a length of 10 mm or less, preferably 5 mm or less, and this seaweed slice (intercalary fragment) is taken for 10 to 15 hours under light irradiation and in the dark. Repeat alternately and aerating, changing the nutrient medium in a timely manner, and statically culture for 30-50 days. In this case, as the intercalary fragment, it is preferable to use a branch of an algal body between the growth end and the main axis other than the main axis of seaweed. Further, instead of the intercalary fragment, a growth end fragment (apical fragment) of a previously prepared immature monoalgal culture strain cut to a length of 10 mm or less may be used.
In this way, it is possible to obtain a non-mature monoalgal culture that does not mature even if it is continuously cultured for 3 years or more under culture conditions.

The nutrient culture conditions for the slices in the method of the present invention are, for example, a temperature of 16 to 30 ° C., a light intensity of 50 to 120 μmol / m ² sec, and a photoperiod of 8 hours light period-16 hours dark period to 24 hours light period-0. A time dark period is mentioned. 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.

  The non-mature monoalgal culture obtained by the method of the present invention does not mature even if it is continuously cultured for 3 years or more under the culture conditions, and (1) the production amount of the physiologically active substance is high. (2) Algae A long-lasting growth rate of the body, (3) a high ability to absorb nutrients, and a long-lasting red alga having at least one of the properties (1) to (3) Can be cultured or stored without ripening.

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

Reference Example (1) Spore collection and spore planting for preparing monoalgal cultures;
As a raw material, the natural gametophyte is not detected as a mature body, and the only quasi-spore mature body is detected. The mature spores of the large seaweed Gracilaria chorda collected at 5% by mass) were used.

The mature part of the mature spore thus obtained 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 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).

  The seawater medium used at this time was 1/10 volume of distilled water after filtering seawater collected at a depth of about 1.5 m in Yashima Bay, Takamatsu City, Kagawa Prefecture with a 0.20 μm cellulose acetate membrane filter (Advantech Toyo Co., Ltd.). After the addition and mixing, the mixture was sterilized at 100 ° C. for 30 minutes and prepared by adding a pre-sterilized Provasoli seawater reinforcing nutrient.

(2) Straight solid selection;
Among the experimental groups in which spore germination was observed at the time of stationary culture for 21 days, the experimental conditions were such that the solid solid was thick, the red pigment was vivid, and there were no suspended solids in the culture solution, that is, the temperature was 18 ° C. A straight solid germinated under the conditions of light intensity of 40 μmol / m ² sec was selected.

  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.

  A salt composition for artificial seawater for growing algae was prepared using sodium chloride, which is a hardly soluble substance, as sodium chloride. The salt composition for artificial seawater for growing algae is 548 g of sodium chloride, 250 g of magnesium chloride hexahydrate, 92.5 g of sodium sulfate, 35.0 g of calcium chloride dihydrate, 35.0 g of potassium chloride for preparation of artificial seawater for 25 liters. 15.8 g, sodium bicarbonate 4.5 g, potassium bromide 2.25 g, orthoboric acid 0.75 g, strontium chloride 0.25 g, iron chloride hexahydrate 0.13 mg, sodium glycerophosphate pentahydrate 8.75 mg Then, 4.0 mg of sodium nitrate was mixed, sealed in a laminate bag, and stored in a constant temperature room at 20 ° C.

  Artificial seawater was prepared by dissolving 1 bag of a salt composition for artificial seawater for algae growth using the above-mentioned hardly soluble substance-preventing sodium chloride in 25 liters of distilled water.

  Seaweed slices of the following four types (A), (B), (C), and (D) from monoalgal cultures of red seaweed seaweed Culsilamo [scientific name: Gracilariaceae Gracilaria chorda] obtained in Reference Example Was prepared.

  (A) is a 5 mm long growth end (referred to as an apical fragment), (B) is a 10 mm long growth end to an intermediate section (intercalary fragment) obtained by removing 5 mm from the growth end, (C ) Is an intermediate section obtained by cutting out a length of 3 mm from the growth end from an intermediate section (intercalary fragment) obtained by removing a length of 10 mm from the growth end and 5 mm from the growth end, and (D) is a growth of 10 mm in length. This is an intermediate section obtained by cutting out a length of 2 mm from the growth end from an intermediate section (intercalary fragment) excluding the length of 5 mm from the growth end.

Six pieces of various seaweed slices were added to each Erlenmeyer flask containing 400 ml of artificial seawater. The culture conditions were a temperature of 20 ° C., a light intensity of 60 μmol / cm ² s, and a photoperiod of 14 hours light period and 10 hours dark period. Exchange of artificial seawater as a culture solution and wet mass measurement of seaweed slices in each Erlenmeyer flask were performed every week, and the flask was stirred at a rate of 100 rpm during the culture.
The results are shown in Table 1. This value is an average value of five experiments.

  As can be seen from this table, the wet mass of the seaweed slices increases with the incubation time.

  And in the seaweed growth end sample (A), the wet mass of the six growth ends before the start of the culture is 1.4 mg, whereas the wet mass of the six growth ends in the third week of culture is 43. 94 mg and the wet mass increased 31.4 times at the start of the culture, and the wet mass at the 6th growth end at the 7th week of culture increased to 120.22 mg and the wet mass increased to 85.9 times the start of the culture. ing. The daily growth rate was as high as 11.5% / day between the second and third weeks.

  In the seaweed intermediate section sample (B) having a length of 5 mm, the wet mass at the six growth ends before the start of the culture is 2.40 mg, whereas the wet mass at the six growth ends at the third week of culture is 44.80 mg and the wet mass increased 18.7 times at the start of the culture, and the wet mass at the 6th growth end at the 7th week of culture was 114.04 mg, and the wet mass was 47.5 times the start of the culture. It has increased. The daily growth rate was high at 10.2% / day between the second and third weeks.

  In the seaweed intermediate section sample (C) having a length of 3 mm, the wet mass at the six growth ends before the start of the culture is 1.36 mg, whereas the wet mass at the six growth ends at the third week of culture is 26.10 mg and the wet mass increased 19.2 times from the start of the culture, and the wet mass at the end of the six growths at the 7th week of culture was 92.44 mg, and the wet mass was 68.0 times the start of the culture. It has increased. The daily growth rate was as high as 10.7% / day between the second and third weeks.

  In the seaweed intermediate section sample (D) having a length of 2 mm, the wet mass of the six growth ends before the start of the culture is 0.88 mg, whereas the wet mass of the six growth ends in the third week of culture is 17.20 mg and the wet mass increased 19.6 times at the start of the culture, and the wet mass at the end of 6 growth at the 7th week of culture was 83.84 mg, and the wet mass was 95.3 times the start of the culture. It has increased. The daily growth rate was as high as 10.9% / day between the second and third weeks.

It can be seen that among the four types of seaweed slices, the sample (D) which is the shortest slice has the highest rate of increase in wet mass.
In the growth end sample (A) and the seaweed intermediate section sample (B) of the same length, the wet mass increase rate was higher in the growth end sample (A).
In the growth end sample (A) having a length of about 5 mm and a seaweed intermediate section sample (C) having a length of 3 mm, the growth rate of wet mass was higher in the growth end sample (A).

  Nutrient culture of seaweed in the same manner as in Example 1 except that instead of the samples (A), (B), (C) and (D) in Example 1, one growth end having a length of 100 mm was used. Went. As a result, the wet mass at one growth end before the start of the culture was 7.84 mg, whereas the wet mass at one growth end at the third week of culture was 17.60 mg, indicating an increase in the wet mass. Stayed 2.2 times. The daily growth rate was 2.1% / day between the second and third weeks. From this, when using the short seaweed growth end having a length of 5 mm in Example 1 rather than using the seaweed growth end having a length of 100 mm, the wet mass increase rate and the daily growth rate are higher, and the algae is more efficient. You can see that it can grow.

Comparative Example In place of the artificial culture strain samples (A), (B), (C) and (D) in Example 1, the red seaweed seaweed seaweed Tsurusilamo [Scientific name: Gracilariaceae Gracilaria chorda: Gracilariaces gracilaria corda ], The nutrient culture was carried out in the same manner as in Example 1 except that a 5 mm long growth end sample was prepared and used.

  Two weeks after the start of the culture, the microalgae grew in the flask containing the seaweed slices, the increase in the wet weight of the seaweed was reduced, and the wet weight of the seaweed at the third week of the culture was lower than the wet weight of the second week. .

  From these results, when seaweed slices from naturally collected seaweeds are artificially grown, the cultivation of seaweeds can be continued by the growth of other organisms such as microalgae that are attached to or coexist with seaweeds. You can see it disappears.

  According to the present invention, a non-mature monoalgal culture strain useful for production of a physiologically active substance such as a hemagglutinating agent can be efficiently propagated.

Claims (11)

  A method for growing algae, comprising culturing a slice of an artificial culture strain of algae.   2. The method of algae growth according to claim 1, wherein the algae is a red alga of the genus Ogonori.   The method of algae growth according to claim 2, wherein the red alga of the genus Ogonori is Ogonori (Gracilaria verrucosa) or Tsurusiramo (Gracilaria chorda).   4. The algal growth method according to claim 1, 2 or 3, wherein the artificial culture of algae is a single algae culture.   The algal growth method according to claim 4, wherein the monoalgal culture is an immature culture.   The method of algae growth according to claim 5, wherein the non-mature culture is derived from a red alga belonging to the genus Ogonori that propagates in natural water mixed with fresh water.   The algal growth method according to any one of claims 1 to 6, wherein the algal slice is an intercalary fragment.   The algal growth method according to any one of claims 1 to 6, wherein the algal slice is an apical fragment.   The method of algae growth according to any one of claims 1 to 8, wherein artificial seawater is used as the culture solution.   The algae according to claim 9, wherein when artificial seawater is made into an aqueous solution with a concentration of 20%, water containing 20 to 40 g / liter of low magnesium sodium chloride having a magnesium ion concentration of 10 ppm or less is used as a base. Proliferation method. An algal culture obtained by the method according to any one of claims 1 to 10.