Method for producing colonies of nostoc cyanobacteria and colony production system

Abstract

To provide a method that enables efficient production of colonies of Nostoc cyanobacteria, and a colony production system.SOLUTION: A method for producing colonies of Nostoc cyanobacteria, the cyanobacteria having, in a life cycle, a phase of releasing hormogonia from a colony and a phase of forming a sheath on an outer side of the hormogonia and proliferating inside the sheath to form daughter colonies, the method comprising: (a) attaching hormogonia released from a parent colony to a mesh-like or thread-like carrier, or to an edible elongated carrier; and (b) culturing the hormogonia attached to the carrier in a culture medium to form daughter colonies.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for producing colonies of cyanobacteria of the genus Nostoc and a system for producing such colonies. [Background technology]

[0002] Nostoc cyanobacteria have long been used as food due to the large number of nutrient-rich species. For example, Nostoc commune (Nostoc commune var. sphaeroides), a species of Nostoc genus, and its subspecies, Nostoc commune var. sphaeroides, are rich in amino acids, vitamins, minerals, polysaccharides, and other nutrients. It has been reported that ingesting them may provide health benefits, such as antibacterial, antioxidant, and anti-inflammatory effects. Therefore, these benefits could be effectively utilized by culturing Nostoc cyanobacteria in large quantities and using them as ingredients or additives in foods and pharmaceuticals. For efficient cultivation of Nostoc cyanobacteria, it is important to avoid predation by natural enemies and infection (contamination or proliferation) by bacteria other than the cyanobacterium in question. For example, Nostoc cells form sheaths on the outside of their single-line filaments and can grow inside them. Growing in this state makes them less susceptible to predation by natural enemies and more resistant to bacterial infection; therefore, the sheath formation rate is thought to be important for efficient cultivation. However, previous attempts to artificially cultivate Nostoc colonies using a suspension of fragments obtained by homogenizing them have resulted in low sheath formation rates and low colony formation rates. Patent Document 1 discloses a method of obtaining daughter colonies from star jelly colonies by immersing them in purified water without homogenizing them, causing the colonies to spontaneously burst, releasing filamentous algal fragments. This method can significantly shorten the culture cycle for obtaining colonies compared to methods that involve homogenization. However, the inventors have experienced that when the procedure described in Patent Document 1 is followed, the reproducibility and yield of sheath and colony formation after obtaining the filamentous algal fragments is poor. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Chinese Patent Application Publication No. 104031865 Summary of the Invention [Problem to be solved by the invention]

[0004] An objective of various embodiments of the present disclosure is to provide a method for efficiently producing colonies of cyanobacteria of the genus Nostoc and a colony production system using the same. [Means for solving the problem]

[0005] The inventors have discovered that culturing Nostoc cyanobacteria attached to a mesh or thread-like carrier enables favorable handling of the cyanobacterial culture and favorable interaction with the medium, and also enables easy medium replacement without centrifugation or other procedures, even when colonies are too large to be visually identified. This reduces contamination and enables efficient and high-yield production of daughter colonies with high sheath formation rates. Furthermore, they have found that culturing in a medium containing almost no inorganic nitrogen also reduces bacterial contamination and significantly improves the sheath formation rate and growth rate of daughter colonies and the hormogonia release rate of parent colonies.

[0006] The present disclosure includes the following embodiments. [1] A method for producing colonies of cyanobacteria of the genus Nostoc, whose life cycle consists of a phase in which hormogonia are released from the colony and a phase in which the hormogonia form sheaths outside the hormogonia and grow inside the sheaths to form daughter colonies, the method comprising: (a) attaching the hormogonia released from the parent colony to a mesh-like or thread-like carrier or an edible elongated carrier; and (b) culturing the hormogonia attached to the carrier in a medium to form daughter colonies. [2] The method according to [1], wherein the culture medium has a dissolved inorganic nitrogen concentration of 0.05 mM or less. [3] The method according to [1] or [2], wherein in (b), the medium is changed at least once every five days. [4] The method according to any one of [1] to [3], which comprises, prior to (a), a step of immersing the parent colony in pure water for two or more days to release hormogonia. [5] A method for producing colonies of cyanobacteria of the genus Nostoc, whose life cycle consists of a phase in which hormogonia are released from the colony and a phase in which the hormogonia form sheaths outside the hormogonia and grow inside the sheaths to form daughter colonies, the method comprising culturing the hormogonia released from the parent colony in a medium to form daughter colonies, the medium having a dissolved inorganic nitrogen content of 0.05 mM or less, and changing the medium at least once every five days during the formation of the daughter colonies. [6] The method described in [5], which includes a step of releasing hormogonia by immersing the parent colony in pure water for two or more days before culturing the hormogonia released from the parent colony in a medium. [7] A colony production system for Nostoc cyanobacteria, whose life cycle consists of a phase in which hormogonia are released from the colony and a phase in which the colony forms a sheath around the hormogonia and grows inside the sheath to form daughter colonies, comprising a culture medium having a dissolved inorganic nitrogen concentration of 0.05 mM or less, a mesh-like or thread-like carrier or an edible elongated carrier immersed in the culture medium, and a plurality of hormogonia and / or colonies attached to the carrier. [8] the phase of releasing hormogonia from the colony; a phase in which the hormogonia form a sheath outside and grow inside the sheath to form daughter colonies; multiple colonies of Nostoc cyanobacteria having the following life cycle: an edible elongated carrier to which the plurality of colonies are attached in clusters; A food composition comprising: [Brief explanation of the drawings]

[0007] [Figure 1] Figure 1 shows an overview of the typical life cycle of Nostoc commune. [Figure 2] FIG. 2 is a schematic diagram of a method for culturing Nostoc commune according to one embodiment. [Figure 3] Figure 3 shows a micrograph of Nostoc commune attached to a mesh support. The arrow points to a hormogonia. [Figure 4] FIG. 4 shows the change in phosphate concentration in the medium over time associated with the growth of hormogonia and / or daughter colonies in an example of culture. [Figure 5] FIG. 5 shows the change in the rate of sheath formation when hormogonia were cultured in media with different inorganic nitrogen contents. DETAILED DESCRIPTION OF THE INVENTION

[0008] Hereinafter, embodiments of the present disclosure will be described in detail, but these are merely examples and the present invention is not limited to these examples.

[0009] Nostoc cyanobacteria are cyanobacteria that can form colonies by embedding numerous cell filaments consisting of string-like cells in a common agar-like substrate. Species that belong to the Nostoc genus include Nostoc commune, Nostoc commune var. sphaeroides, Nostoc verrucosm, and Nostoc flagelliforme. Nostoc cyanobacteria can grow in freshwater or on land and include species capable of nitrogen fixation, such as Nostoc jelly. Therefore, in an embodiment of the present disclosure, the Nostoc cyanobacteria can be a cyanobacterium capable of nitrogen fixation, such as Nostoc commune.

[0010] As an example of a typical life cycle of cyanobacteria in the genus Nostoc, the typical life cycle of Nostoc commune will be described. As shown in Figure 1, Nostoc commune is known to have a cyclical life cycle that includes at least four phases: (1) parent colony → (2) hormogonia → (3) sheath formation → (4) daughter colony. More specifically, (1) a portion of the parent colony first differentiates into (2) hormogonia, specialized cells specialized for movement, which are then released from the parent colony in a filamentous form. (3) The hormogonia then form a sheath composed primarily of polysaccharides and proliferate within the sheath (this process may involve cellular dedifferentiation and differentiation), and (4) ultimately grow into daughter colonies. As mentioned in Patent Document 1, daughter colonies can also be formed, albeit with low efficiency, from parent colonies fragmented into filaments by mechanical means such as homogenization.

[0011] In this disclosure, the term "colony" of Nostoc cyanobacteria refers to a visible mass of cell filaments, corresponding to phases (1) and (4) of the life cycle, as understood by those skilled in the art. The size, morphology, and color of a colony can vary greatly depending on the species and individual specimens within the Nostoc genus. For example, star jelly can form spherical colonies measuring approximately 0.1 mm to several centimeters in diameter, typically ranging in color from yellow-green to reddish-brown or dark green to black. In this disclosure, the terms "parent colony" and "daughter colony" are relative terms used to refer to a colony of a given generation during the life cycle and a subsequent generation colony formed by the proliferation of some cells separated from the colony, respectively. It should be understood that the resulting daughter colony can become a new parent colony.

[0012] Hormogonia are motile, filamentous cell groups found in cyanobacteria of the genus Nostoc. They are known to be induced and released by changes in the physical environment or the presence of symbiotic plants. Here, "filamentous" refers to the linear connection of multiple cells. For example, the hormogonia of Star Jelly are typically filamentous, consisting of a dozen or so cells. Hormogonia preferably form a sheath on the outside and form daughter colonies that grow within the sheath, but they can also grow without sheathing.

[0013] In cyanobacteria, cells arranged in a continuous, thread-like pattern are generally called trichomes. Cyanobacteria of the genus Nostoc, such as star jelly, typically form a sac-like space (sheath) on the outside of the trichome and grow inside it to form colonies. Trichomes covered in a sheath are sometimes called filaments. The sheath typically contains polysaccharides as its main component. Because the sheath can function as a defense mechanism against external enemies and contamination by other bacteria, improving the efficiency of sheath formation in artificial cultures could lead to the efficient mass production of colonies.

[0014] [Method for producing Nostoc cyanobacterial colonies] An outline of a method for producing colonies of cyanobacteria of the genus Nostoc (typically Nostoc commune) according to this embodiment is shown in Figure 2. In this embodiment, hormogonia and colonies are cultured with cell aggregates attached to a mesh-like or thread-like carrier. The medium is preferably changed at least once every three days, but this is not a limitation.

[0015] Parent colonies of cyanobacteria of the genus Nostoc, such as star jelly, can be collected from natural environments. Colonies from which impurities such as stones and plant fragments attached to the colony have been removed may be used as parent colonies, or colonies that have been pretreated by wetting, washing, drying, rewetting, or the like may be used as parent colonies. The method of the present disclosure can also be carried out by using daughter colonies obtained by the colony production method described herein or by other methods as new parent colonies.

[0016] After harvesting, the parent colony may be washed by immersing it in pure water or other aqueous liquid to remove contaminants. The wet parent colony may also be dried. The dried parent colony may be rewetted with pure water or other aqueous liquid. The inventors have found that drying the parent colony can improve the sheath formation rate in the daughter colony formation process. Without being bound by any particular theory, one possible reason for this may be that the drying process reduces the number of surviving contaminating bacteria and their adverse effects. It is known that colonies of Nostoc cyanobacteria can be repeatedly dried and rewetted, even in natural environments.

[0017] Hormogonia can be released by soaking the parent colony in pure water for a certain period of time. Homogenization of the parent colony is not necessary; however, parent colonies that have been macroscopically fragmented to some extent from a large colony may be used. The soaking period in pure water is preferably 2 to 7 days, more preferably 3 to 7 days, and even more preferably 3 to 4 days. While the above period is insufficient for the significant proliferation of contaminating bacteria, it ensures sufficient time for the hormogonia to be released. Hormogonia release may be performed on a culture plate or the like, but is preferably performed on a mesh-like, thread-like, or edible elongated carrier, as described below. Releasing hormogonia on these carriers allows for a more convenient transition from hormogonia release to colony cultivation.

[0018] In this embodiment, examples of the pure water include ion-exchanged water, distilled water, and ultrapure water.

[0019] In this embodiment, a "mesh" support refers to a mesh-like support in which linear structures are connected at angles to each other to form a porous structure, while a "thread" support refers to a support consisting of individual linear structures that can form such a mesh-like support but are not necessarily connected to each other. A support is an artificially provided culture substrate. Embodiments of the present disclosure are based, at least in part, on the discovery that hormogonia and initial colonies attach more easily to thread-like or mesh-like supports than to flat structures such as the bottom of a culture plate, thereby significantly simplifying medium replacement and colony harvesting. Such attachment is also thought to facilitate the flow of fluids and solutes surrounding the hormogonia and colonies. This favorable interaction between cyanobacteria and the culture medium promotes growth and relatively suppresses contamination. The mesh opening (or maximum pore diameter) of the mesh support is, for example, 500 μm or less or 200 μm or less, preferably 150 μm or less, more preferably 100 μm or less, and even more preferably 75 μm or less. The mesh opening (or maximum diameter of the holes) of a mesh-like carrier is usually 20 μm or more. Because the average length of hormogonia is about 50 μm, if the mesh opening is larger than 500 μm, the probability that hormogonia will slip through the mesh and fail to attach to the carrier increases. The diameter of the thread-like carrier (or the individual linear structures forming the mesh-like carrier) is, for example, 5 to 1000 μm, preferably 10 to 500 μm, and more preferably 20 to 200 μm. In the thread-like carrier, multiple linear structures may be provided, for example, regularly arranged side by side. When multiple linear structures are provided side by side in the same direction, the spacing between adjacent linear structures may be, for example, 500 μm or less or 200 μm or less, preferably 150 μm or less, more preferably 100 μm or less, and even more preferably 75 μm or less. The spacing is usually 20 μm or more. In some embodiments of the thread-like carrier, the linear structures are provided in an irregular arrangement, for example, intertwined. In some other embodiments of the thread-like carrier, the linear structure is provided in a three-dimensional structure, for example in a three-dimensional network structure.The material of the mesh or thread carrier is preferably a water-insoluble carrier such as, but not limited to, nylon, polyester, polyethylene, polypropylene, and mixtures of any of these.

[0020] The carrier to which hormogonia and / or colonies are attached can also be an edible elongated carrier. In this embodiment, "edible elongated carrier" refers to a non-cellular culture substrate (e.g., konjac threads, which are plant-derived but in which the cells are substantially destroyed) that is artificially provided and is made of an elongated material known to be suitable for human consumption. The "elongated material" has a length at least 10 times greater than its thickness. The diameter of the edible elongated carrier is, for example, 500 to 7000 μm, preferably 1000 to 6000 μm, and more preferably 2000 to 5000 μm. In the edible elongated carrier, multiple elongated structures may be provided, for example, in a regular arrangement, or in an irregular arrangement, for example, intertwined. Examples of edible elongated carriers include, but are not limited to, carriers that are insoluble at room temperature, such as polysaccharide gel-based noodles, gelatin-based noodles, and mixtures thereof, including thread agar, jelly, thread konjac, vermicelli, kudzu noodles, and starch noodles. The use of edible elongated carriers allows multiple colonies to attach and grow in clusters around the edible elongated carrier. This provides advantages similar to those of the mesh-like and thread-like carriers described above, significantly simplifying medium replacement and colony collection. Such an attachment state is also thought to promote the circulation of fluids and solutes surrounding the hormogonia and colonies. Furthermore, because the carriers are edible as a whole, there is no need to separate the colonies from the carrier when shipping them for consumption; they can be shipped together. Edible elongated carriers, typically composed primarily of polysaccharides, can provide stronger adhesion to not only hormogonia but also grown colonies than, for example, nylon-based meshes and threads. For example, colonies of star jelly or the like attached to agar thread carriers can have a texture similar to that of sea grapes, making them an attractive new edible product.

[0021] Thus, in one aspect, the present disclosure provides a food composition comprising a plurality of colonies of cyanobacteria of the genus Nostoc and an edible elongated carrier to which the plurality of colonies are attached in the form of clusters. The diameter of these colonies may be, for example, 1 to 10 mm. In the "clusters," the plurality of colonies are each attached to a common edible elongated carrier.

[0022] In this embodiment, "attached" refers to a state in which a portion or the entirety of a cell aggregate, such as a hormogonia or colony, is detachably (i.e., reversibly) fixed to a mesh-like, thread-like, or edible elongated carrier. An example of an adherent cell state is shown in Figure 3. An adherent state may refer to a state in which a portion or the entirety of a cell aggregate is entangled with the carrier, or a state in which some or all of the cells of the cell aggregate are adhered to the carrier, or a state in which some or all of the cells of the cell aggregate are caught on the carrier. Culturing cell aggregates while they are attached to the carrier allows for easy medium replacement without centrifugation or other procedures, even for colonies that are difficult to visually identify. Furthermore, medium replacement can be performed even in the early stages when hormogonia are present. This allows for frequent medium replacement. For example, a new culture vessel containing fresh medium can be prepared and the cyanobacteria and carriers can be transferred to the new culture vessel. Alternatively, the old medium can be removed by suction or pouring, leaving the cyanobacteria and carriers in the culture vessel, and fresh medium can be replenished. In this new environment, young colonies can gain access to fresh medium and solutes from multiple directions while remaining fixed to the mesh or thread-like support or edible elongated support. When cultured on a flat container bottom as in the past, access to fresh substances from the bottom does not occur, so it is presumed that the colonies had no choice but to detach from the substrate and roll in order to gain access from multiple directions. When colonies grow large enough to approach a spherical shape and reach the harvestable stage, they become easily detached from the mesh or thread-like support, making colony recovery more efficient.

[0023] The light conditions during culture can be appropriately determined by those skilled in the art based on their general knowledge. For example, white LED light can be irradiated under a 12-hour light-dark cycle program. The irradiation is 50 to 300 μmol photons m -2 sec -1 (420 nm photon equivalent), but 200 μmol photon m -2 sec -1 Preferably less than 100 μmol photon m -2 sec -1 Less than 50 μmol photons m -2 sec -1 More preferably, it is 300 μmol photons m -2 sec -1 Light intensities above this level may have a negative impact on pod formation and survival rate due to damage to photosynthetic function, etc.

[0024] The temperature conditions during cultivation can also be appropriately determined by those skilled in the art based on their general knowledge. Culturing can be carried out in a temperature-controlled room or incubator that can maintain a constant temperature. Culturing can be carried out, for example, at 15°C to 35°C, preferably 20°C to 30°C, and more preferably 25°C to 30°C. Maintaining the temperature within these ranges makes it easier to ensure efficient growth and sheath formation.

[0025] Those skilled in the art can prepare a suitable medium capable of supporting the cultivation of algae based on their general knowledge or select a suitable medium from known media. The medium is preferably a liquid medium. BG11 medium and BG11 00 Media based on BG11 medium can be used, but are not limited to BG11 medium and BG11 00Representative compositions of the media are shown in Tables 1 and 2, respectively. These media can be prepared by adding all reagents except FeCl3·6H2O to pure water, stirring thoroughly, and autoclaving (120°C, 20 min) (A), followed by the addition of filter-sterilized FeCl3·6H2O. Agar media corresponding to each liquid medium can be prepared, for example, by adding agar powder to the above A to make a 1.5% (w / v) suspension before autoclaving, then autoclaving and adding filter-sterilized FeCl3·6H2O. The media are BG11 00 It is preferable to use a medium containing 17.6 mM of dissolved inorganic nitrogen in BG11 medium. 00 The medium is characterized by having dissolved inorganic nitrogen levels that are 200 times lower than those of BG11. 00 It has been found that culturing from hormogonia to the formation and growth of daughter colonies in a medium with a low inorganic nitrogen content, such as a medium containing nitrite, can significantly improve the ability to maintain colonies with a high sheath formation rate. While culturing using mesh or thread-like carriers or edible elongated carriers can be even more advantageous due to the synergistic effect, it is preferable to use these media with low inorganic nitrogen content even in other cultures. Cultivation using the medium described in this disclosure can be continued (including medium replacement) from the hormogonia stage for, for example, two weeks or more, for two weeks to two months, or until the maximum diameter of the daughter colonies exceeds 1 mm or 5 mm. As will be understood by those skilled in the art, inorganic nitrogen refers to any or all of ammonium nitrogen, nitrite nitrogen, and nitrate nitrogen, which can be dissolved in an aqueous environment in the form of ions and serve as a nitrogen source that can be readily utilized by microorganisms for assimilation. The amount of inorganic nitrogen in the medium during the culture from hormogonia to the formation and growth of daughter colonies is preferably 0 mM or more (e.g., more than 0 mM, 0.01 μM or more) and 0.05 mM or less, more preferably 0.01 mM or less, and even more preferably 0.001 mM or less. The amount of inorganic nitrogen in the medium may be 0 mM to 0.0001 mM.

[0026] BG11 00The medium may be diluted with pure water and used as 1 / 2BG11, which is half the concentration of the medium components other than pure water. 00 Medium is preferred, 1 / 4 BG11 00 A medium containing cyanobacteria is more preferable. Diluting the concentrations of the medium components as described above can further improve the sheath formation rate. It should be understood that the medium composition discussed in this disclosure refers to the composition of the environment to which the cyanobacteria are exposed at the time of starting culture in a new medium, and that the environmental composition may change from the initial composition over the course of culture time.

[0027] [Table 1]

[0028] [Table 2]

[0029] The culture medium may be changed during the culture period. This not only provides fresh nutrients but also suppresses the growth of contaminating bacteria. The medium may be changed by removing the existing medium and adding new medium, or by transferring hormogonia and / or daughter colonies attached to a mesh-like or thread-like carrier or an edible elongated carrier to a petri dish containing new medium. The carrier with hormogonia and / or daughter colonies attached may be washed during the medium change. When changing the medium, it is preferable to change at least 80% of the old medium, more preferably at least 90%, and even more preferably the entire amount. While Patent Document 1 teaches changing the medium every 10 to 20 days, the present disclosure has found that it is preferable to change the medium at least every 5 days during the culture from hormogonia to the formation of daughter colonies. "At least once every 5 days" means that the interval is 5 days or less. Such frequent medium changes are particularly preferable and easy to achieve when using mesh-like or thread-like carriers or edible elongated carriers. However, frequent medium changes are also preferable when culturing without these carriers. Medium changes are preferably performed at least once every three days, more preferably at least once every two days, and even more preferably at least once per day. In particular, from the second week after the start of culture from hormogonia, the frequency of medium changes is preferably increased to at least once per day. Figure 3 shows an example of the actual measured values ​​for changes in phosphate ion concentration in the medium following medium changes. In the specific embodiment shown in Figure 3, medium changes were performed on days 10, 14, 16, and 18 after the start of culture, and the phosphate concentration was measured before and after the medium changes on the day of the medium changes. The above-mentioned frequency of medium changes ensures sufficient supply of nutrients according to the colony size, leading to further colony growth. As an alternative to determining the frequency of medium changes, for example, medium changes may be performed when the phosphate ion concentration in the medium falls to 1 ppm or less.

[0030] In another aspect, the present disclosure provides a colony production system for the cyanobacterium Nostoc genus described above. This production system corresponds to the embodiment of the colony production method described above, and it should be understood that the colony production method can be suitably carried out using this production system. Accordingly, this production system includes a culture medium having a dissolved inorganic nitrogen concentration of 0.05 mM or less, a mesh-like or thread-like carrier or an edible elongated carrier immersed in the culture medium, and a plurality of hormogonia and / or colonies attached to the carrier. This system not only improves the sheath formation rate compared to conventional systems, but also allows for easy medium replacement, allows daughter colonies to be obtained in a relatively short culture period, and is easy to harvest, making it suitable for mass production of colonies. [Example]

[0031] Specific embodiments will be described in more detail below with reference to examples, but these are merely illustrative and the embodiments of the present disclosure are not limited to these specific examples. In particular, the cyanobacteria of the genus Nostoc are not limited to the Nostoc commune exemplified herein, and other species such as Nostoc commune var. sphaeroides, Nostoc verrucosm, and Nostoc flagelliforme can also be used.

[0032] Example 1. Sheath formation rate in cultures using media with different dissolved inorganic nitrogen contents In this example, we used dried natural colonies of Nostoc commune collected in Ina City, Nagano Prefecture. The colonies were immersed in ion-exchange water for 3 to 7 days to release hormogonia. Two types of colonies were prepared: "unwashed colonies," which were collected without washing except for removing large stones and plant fragments measuring approximately 5 mm or more on a side; and "washed and dried colonies," which were dried, washed with water containing low concentrations of hypochlorous acid (1.5 ppm) to remove contaminating bacteria, and then rewetted with ion-exchange water.

[0033] Hormone was sucked up with a pipette and BG11, BG11 00 , or 1 / 4 BG11 00 96-well plates containing 10–100 hormogonia / well were seeded at 50 μmol photons m -2 s -1 The cells were cultured at 25°C under a light intensity of 1000 kJ / s, and the sheath formation rate was counted at different times after the start of culture. The results are shown in Table 3. The medium was not changed during this experiment.

[0034] [Table 3]

[0035] In hormogonia prepared from unwashed colonies, the sheath formation rate in BG11 medium peaked at 86% after 3 days, then decreased to 4% after 8 days, and to 0% after 18 days. This suggests that the sheaths formed were degraded over time. In contrast, in BG11 00 Medium and 1 / 4BG11 00 In the culture medium, the sheath formation rate was high at 58% and 85%, respectively, even after 18 days. In the hormogonia prepared from the washed and dried colonies, the sheath formation rate was 0% after 18 days in BG11, but 00 Medium and 1 / 4BG11 00 In BG11 medium, the sheath formation rates were 48 and 60%, respectively, even after 18 days. Figure 5 shows a graph showing the progression of sheath formation rates for three different isolates (#2, #7, and #18) in essentially the same experiment. While sheath-forming colonies may be maintained in some cases in BG11 medium, it is clear that the overall culture efficiency may be significantly reduced.

[0036] Example 2. Cultivation of hormogonia to daughter colonies on a mesh carrier A nylon mesh with a catalog-standard mesh size of 149 μm (the diameter of each thread forming the mesh is approximately 50 μm) was placed so that it covered the entire bottom of a 90 mm diameter Petri dish. A parent colony immersed in pure water was placed on the mesh and left to stand for 3-4 days to release hormogonia and allow them to adhere to the carrier. The mesh with hormogonia attached was then placed in a 1 / 4 BG11 00 The cells were transferred to a petri dish containing medium and cultured for at least approximately 18 days. By transferring the mesh with hormogonia or colonies still attached to a petri dish containing fresh medium every few days, medium changes were significantly simplified while minimizing the loss or damage of hormogonia / colonies. Furthermore, colonies could be efficiently harvested by simply scooping them onto the mesh. Experiments were also conducted using thread agar as an edible elongated carrier instead of nylon mesh, and similar results were obtained, with multiple colonies growing efficiently in clusters.

[0037] The phosphate concentration was also measured over time. In this experiment, the medium was changed on days 10, 14, 16, and 18 after the start of culture, and the phosphate concentration was measured before and after the medium change. The changes in phosphate concentration are shown in Figure 4. The results in Figure 4 show that 1 / 4 BG11 00 This suggests that most of the phosphate is consumed within the first 10 days, and then the rate of phosphate consumption accelerates as the colony grows. Therefore, it is understood that colony growth can be further promoted by increasing the frequency of medium changes and / or increasing the concentration of medium components (especially phosphate) after, for example, 5 days, 1 week, or 10 days from the start of hormogonia culture.

[0038] Compared with the case where the medium was not changed for more than six days, the more frequent medium changes promoted colony growth, resulting in an increase in carbon and nitrogen fixation. In addition, the number of colonies increased because colony bursting due to nutrient deficiency was suppressed.

Claims

1. the phase of releasing hormogonia from the colony; a phase in which the hormogonia form a sheath outside and grow inside the sheath to form daughter colonies; in their life cycle, 1. A method for producing colonies of Nostoc cyanobacteria, comprising: (a) attaching hormogonia released from a parent colony to a mesh or thread-like carrier or an edible elongated carrier; (b) culturing the hormogonia attached to the carrier in a medium to form daughter colonies; A method comprising:

2. The method according to claim 1, wherein the culture medium has a dissolved inorganic nitrogen content of 0.05 mM or less.

3. The method according to claim 1 or 2, wherein in (b), the medium is replaced at least once every five days.

4. The method according to claim 1 or 2, further comprising, prior to (a), a step of immersing the parent colony in pure water for two or more days to release the hormogonia.

5. the phase of releasing hormogonia from the colony; a phase in which the hormogonia form a sheath outside and grow inside the sheath to form daughter colonies; in their life cycle, 1. A method for producing colonies of Nostoc cyanobacteria, comprising: culturing hormogonia released from the parent colony in a medium to form daughter colonies; The medium has a dissolved inorganic nitrogen content of 0.05 mM or less, A method in which the medium is changed at least once every five days during the formation of the daughter colonies.

6. The method of claim 5, further comprising the step of releasing the hormogonia by immersing the parent colony in pure water for two or more days before culturing the hormogonia released from the parent colony in a culture medium.

7. the phase of releasing hormogonia from the colony; a phase in which the hormogonia form a sheath outside and grow inside the sheath to form daughter colonies; in their life cycle, A colony production system for Nostoc cyanobacteria, comprising: A system comprising a culture medium having a dissolved inorganic nitrogen concentration of 0.05 mM or less, a mesh-like or thread-like carrier or an edible elongated carrier immersed in the culture medium, and a plurality of hormogonia and / or colonies attached to the carrier.

8. the phase of releasing hormogonia from the colony; a phase in which the hormogonia form a sheath outside and grow inside the sheath to form daughter colonies; multiple colonies of Nostoc cyanobacteria having the following life cycle: an edible elongated carrier to which the plurality of colonies are attached in clusters; A food composition comprising:

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