Porphyridium cruentum encapsulated calcium alginate beads and semi-continuous production method of sulfated polysaccharides using the same

Abstract

The present invention relates to calcium alginate beads supported with Porphyridium cruentum and a semi-continuous method for producing sulfated polysaccharides using the same. Specifically, the present invention comprises: (1) a step of mixing a culture of the microalgae Porphyridium cruentum with a sodium alginate solution; (2) a step of adding the mixture prepared in step (1) to an aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 at a flow rate of 0.2 to 0.6 mL min-1 and stirring to form calcium alginate beads supported with microalgae; and (3) a method for producing calcium alginate beads loaded with porphyridium cruentum, comprising the step of further stirring the calcium alginate beads loaded with microalgae in an aqueous solution containing CaCl2 and then washing with distilled water, spherical calcium alginate beads loaded with porphyridium cruentum produced by the method, a method for fixing culture of porphyridium cruentum using the calcium alginate beads, and a method for mass-producing sulfated polysaccharides or phycobili proteins from porphyridium cruentum using the calcium alginate beads.

Description

Technology Field

[0001] The present invention relates to calcium alginate beads loaded with porphyridium cruentum and a semi-continuous method for producing sulfated polysaccharides using the same. Background Technology

[0002] Sulfated polysaccharides derived from marine microalgae are natural compounds possessing diverse biological activities, including anticancer, antioxidant, antiviral, and immunomodulatory effects. Consequently, methods for applying sulfated polysaccharides to various industries, such as cosmetics, health functional foods, and pharmaceuticals, are being proposed, leading to an increase in their value and demand.

[0003] The red microalgae Porphyridium cruentum ( Porphyridium cruentum Porphyridium cruentum can produce large amounts of phycobili proteins and sulfated polysaccharides, and is characterized by the continuous production and secretion of extracellular macromolecules containing sulfated polysaccharides. Therefore, using Porphyridium cruentum offers the advantage of easily harvesting sulfated polysaccharides from the culture medium without requiring cell disruption, which is a significant difference from methods of harvesting sulfated polysaccharides from other natural sources such as mammals, mushrooms, or macroalgae. Furthermore, the productivity of sulfated polysaccharides per unit area of ​​Porphyridium cruentum is relatively higher than that of other sources, making it a powerful natural source for sulfated polysaccharide production.

[0004] However, the commercialization of sulfated polysaccharides using Porphyridium cruentum is costly and economically problematic, so improvements in production technology are needed to overcome this.

[0005] In particular, to harvest sulfated polysaccharides from Porphyridium cruentum cultures, cells and extracellular macromolecules must first be separated. However, Porphyridium cruentum cells are only 2 to 5 μm in diameter and are encapsulated in a layer of extracellular macromolecules, so they cannot be separated by natural sedimentation alone. Therefore, additional processes such as centrifugation, coagulation, and filtration are required to separate cells and extracellular macromolecules, which consumes energy and reduces economic efficiency.

[0006] Another method developed for isolating Porphyridium cruentum from extracellular macromolecules is cell fixation culture methods. These techniques involve fixing cells to the surface of a specific substrate for culture, which has the advantage of easily separating cells from extracellular macromolecules because only the extracellular macromolecules produced by the cells dissolve into the medium. However, there are issues such as the need for a specialized culture vessel to fix cells outside the substrate, potential cell loss due to the lack of strong binding between the substrate and the cells, and vulnerability to external contaminants (bacteria, fungi, etc.), which necessitates further improvement.

[0007] Therefore, it is necessary to develop a new method that can produce sulfated polysaccharides from Porphyridium cruentum with high efficiency while solving these conventional problems. Prior art literature

[0008] Korean Published Patent 10-2009-0128287 Korean Registered Patent 10-1797356 The problem to be solved

[0009] Accordingly, the inventors developed a method for culturing Porphyridium cruentum by immobilizing it inside calcium alginate beads. By using calcium alginate beads to immobilize Porphyridium cruentum and culturing it semi-continuously, cell growth and the production of sulfated polysaccharides can be enhanced. Furthermore, by easily separating cells from extracellular macromolecules, it was confirmed that sulfated polysaccharides can be continuously mass-produced from Porphyridium cruentum at low cost, thereby completing the present invention.

[0010] Therefore, the objective of the present invention is to provide a method for manufacturing calcium alginate beads loaded with porphyridium cruentum that can mass-produce sulfated polysaccharides or phycobili proteins from porphyridium cruentum.

[0011] Another objective of the present invention is to provide spherical calcium alginate beads loaded with porphyridium cruentum produced by the method of the present invention.

[0012] Another objective of the present invention is to provide a method for fixing culture of Porphyridium cruentum using spherical calcium alginate beads loaded with Porphyridium cruentum of the present invention.

[0013] Another objective of the present invention is to provide a method for mass-producing sulfated polysaccharides or phycobili proteins from Porphyridium cruentum, comprising the step of culturing using spherical calcium alginate beads loaded with Porphyridium cruentum of the present invention. means of solving the problem

[0014] To achieve the above objectives, the present invention comprises: (1) a microalgae, Porphyridium cruentum ( Porphyridium cruentum ) A step of mixing the culture with a sodium alginate solution; (2) 0.2 to 0.6 mL min of the mixture mixed in step (1); -1The present invention provides a method for manufacturing calcium alginate beads supported with porphyridium cruentum, comprising the steps of: (3) adding to an aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 at a flow rate and stirring to form calcium alginate beads supported with microalgae; and (3) washing the calcium alginate beads supported with microalgae with distilled water after further stirring in an aqueous solution containing CaCl2.

[0015] In one embodiment of the present invention, the porphyridium cruentum culture of step (1) and the sodium alginate solution may be mixed in a volume ratio of 1:2.

[0016] In one embodiment of the present invention, adding the mixture to an aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 may be performed by transferring the mixture with a syringe equipped with a needle and then using a syringe pump.

[0017] In one embodiment of the present invention, the aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 may be an aqueous solution containing 40-50% (w / v) polyethylene glycol (PEG) 6000 and 2-5% (w / v) CaCl2.

[0018] In one embodiment of the present invention, the stirring in step (2) may be performed at 500 to 700 rpm.

[0019] In addition, the present invention provides spherical calcium alginate beads loaded with porphyridium cruentum, manufactured by the method of the present invention.

[0020] In one embodiment of the present invention, the calcium alginate beads may have a diameter of 2 to 5 mm.

[0021] In addition, the present invention provides a method for fixing culture of Porphyridium cruentum using spherical calcium alginate beads loaded with Porphyridium cruentum of the present invention.

[0022] In addition, the present invention provides a method for mass-producing sulfated polysaccharides or phycobili proteins from Porphyridium cruentum, comprising the step of culturing using spherical calcium alginate beads loaded with Porphyridium cruentum of the present invention.

[0023] In one embodiment of the present invention, the culture is carried out at 25°C with stirring at 120 rpm at 60 μmol m⁻¹ -2 s -1 It may be cultured while irradiating with a blue light source at a certain intensity.

[0024] In one embodiment of the present invention, the culture may be performed in a medium deficient in vitamins.

[0025] In one embodiment of the present invention, the culture may be performed by culturing in a vitamin-deficient medium until the 15th day, and then switching to a nitrogen-deficient medium to perform additional culture until the 30th day.

[0026] In one embodiment of the present invention, the sulfide polysaccharide or phycobili protein may be isolated from a culture medium of spherical calcium alginate beads supported with porphyridium cruentum.

[0027] In one embodiment of the present invention, the sulfated polysaccharide or phycobili protein may be separated from the culture medium, and then the spherical calcium alginate beads loaded with Porphyridium cruentum may be transferred to a fresh culture medium and cultured again to continuously induce the production of the sulfated polysaccharide or phycobili protein. Effects of the invention

[0028] When using spherical calcium alginate beads loaded with Porphyridium cruentum according to the present invention, Porphyridium cruentum can be stably immobilized inside the beads, and the production of physiologically active substances such as sulfated polysaccharides and phycobili proteins can be enhanced through the control of culture conditions, and the physiologically active substances can be easily separated from cells and collected, as well as the physiologically active substances can be continuously produced from the spherical calcium alginate beads loaded with Porphyridium cruentum through the replacement of the culture medium. Brief explanation of the drawing

[0029] Figure 1 shows a schematic diagram of the culture of Porphyridium cruentum using the calcium alginate beads of the present invention. Figure 2 shows the results of analyzing the wear resistance according to the diameter size of the calcium alginate beads of the present invention over time. FIG. 3 shows the results of measuring (a) cell growth curve, (b) daily sulfide polysaccharide productivity, (c) phycobiliprotein production, and (d) intracellular phycobiliprotein content of Porphyridium cruentum according to the use of calcium alginate beads of the present invention and irradiation with a blue light source, wherein the control group represents a group in which cells were cultured without the use of beads, the bead group represents a group in which cells were fixed and cultured with the calcium alginate beads of the present invention, and the bead(blue) group represents a group in which calcium alginate beads were used and simultaneously irradiated with a blue light source. Figure 4 shows the elemental analysis of extracellular polymeric substances harvested on days 15 and 30 of culture of Porphyridium cruentum using the calcium alginate beads of the present invention and irradiation with a blue light source (a and b), and the results of the analysis of the physiological activity of the extracellular polymeric substances (c and d). Figure 5 shows the results of analyzing (a) cell growth curve, (b) daily sulfated polysaccharide productivity, (c) phycobiliprotein production, and (d) intracellular phycobiliprotein content of Porphyridium cruentum under the use of calcium alginate beads of the present invention and nutrient deficiency conditions (nitrogen deficiency: -N, vitamin deficiency: -V, nitrogen and vitamin simultaneous deficiency: -NV). Specific details for implementing the invention

[0030] The present invention is characterized by providing calcium alginate beads encapsulated with porphyridium cruentum and uses thereof, which enable the mass production of useful physiologically active substances from porphyridium cruentum.

[0031] While researching a method to obtain useful bioactive substances from Porphyridium cruentum semi-continuously and easily and inexpensively compared to conventional microalgae culture methods, the inventors confirmed that using calcium alginate beads allows for effective immobilization and culture of Porphyridium cruentum, and enables easy acquisition of bioactive substances without performing additional processes such as coagulation or filtration for separating microalgae and bioactive substances after culture.

[0032] In addition, optimal culture conditions capable of enhancing the production of physiologically active substances from Porphyridium cruentum were established using the calcium alginate beads of the present invention.

[0033] Therefore, the present invention can provide a method for manufacturing calcium alginate beads supported with porphyridium cruentum, wherein the method comprises (1) porphyridium cruentum, which is a microalgae ( Porphyridium cruentum ) A step of mixing the culture with a sodium alginate solution; (2) 0.2 to 0.6 mL min of the mixture mixed in step (1); -1The method comprises the steps of: (3) adding to an aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 at a flow rate and stirring to form calcium alginate beads supported with microalgae; and (3) washing the calcium alginate beads supported with microalgae with distilled water after further stirring in an aqueous solution containing CaCl2.

[0034] To explain the above method in detail, first, the microalgae Porphyridium cruentum ( Porphyridium cruentum ) The culture is mixed with a sodium alginate solution to prepare a mixture.

[0035] At this time, the sodium alginate solution may be a solution containing 1.5 to 2% (w / v) of sodium alginate, and a solution containing Tween 80 may be used.

[0036] In addition, the above-mentioned Porphyridium cruentum culture and sodium alginate solution can be mixed in a volume ratio of 1:2, taking into account the viscosity of the solution, the size of the beads, the thickness of the outer wall of the beads, and durability, to prepare a mixture.

[0037] Next, the above mixture is pumped into an aqueous solution containing polyethylene glycol and calcium chloride to form calcium alginate beads supported with microalgae. Specifically, the mixture is 0.2 to 0.6 mL min -1 Calcium alginate beads supported with microalgae are formed by adding to an aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 at a flow rate and stirring.

[0038] The above aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 may be an aqueous solution containing 40-50% (w / v) polyethylene glycol (PEG) 6000 and 2-5% (w / v) CaCl2, and the aqueous solution is contained in a beaker with an inner diameter of 60-100 mm.

[0039] In the present invention, a beaker with an inner diameter of 60 to 100 mm was used. This is because, under the conditions for manufacturing calcium alginate beads of the present invention, if the inner diameter of the manufacturing container is less than 60 mm, there is a risk that the beads will clump together, and if it exceeds 100 mm, the shape of the beads may not be uniform. Preferably, an inner diameter of 85 mm can be used for the manufacturing container.

[0040] In addition, the addition of the above mixture to an aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 can be performed by transferring the mixture using a syringe equipped with a needle and then using a syringe pump, wherein the tip of the syringe needle is set perpendicular to the surface of the aqueous solution and the mixture is added by dropping it onto the aqueous solution.

[0041] In addition, the process of adding the above mixture to the above aqueous solution can be carried out while stirring the aqueous solution, and when manufacturing calcium alginate beads, the stirring speed of the aqueous solution can be performed at 500 to 700 rpm, and preferably at 600 rpm.

[0042] In the above conditions for manufacturing calcium alginate beads, if the stirring speed is less than 500 rpm, there is a risk that the beads will clump together, and if it exceeds 700 rpm, there is a risk that non-spherical beads will be manufactured. Therefore, in the present invention, when manufacturing calcium alginate beads, it is preferable to perform the stirring speed of the solution at 500 to 700 rpm.

[0043] In addition, the calcium alginate beads produced in the present invention have a spherical shape, and the shape of the beads during the manufacturing process may also be affected by the inner diameter of the needle, the angle of the needle tip, the distance between the needle and the solution, and the flow rate during the process of adding the mixture to an aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 using a syringe equipped with a needle.

[0044] After the calcium alginate beads supported with microalgae are formed in the first step, the calcium alginate beads supported with microalgae are then further stirred in an aqueous solution containing CaCl2 and washed with distilled water to obtain the calcium alginate beads supported with Porphyridium cruentum of the present invention.

[0045] Spherical calcium alginate beads loaded with porphyridium cruentum can be produced by the method described above.

[0046] Calcium alginate beads loaded with Porphyridium cruentum, manufactured by the method of the present invention, are characterized by having a uniform spherical shape. Since they are manufactured to have a spherical shape, when a blue light source is irradiated as a culture condition to increase the production of useful bioactive substances, the light source can be uniformly irradiated onto the beads. If the manufactured beads have a non-spherical shape, there is a problem that the light source cannot be uniformly irradiated onto the beads.

[0047] In addition, calcium alginate beads loaded with porphyridium cruentum produced by the method of the present invention have a diameter of 2 to 5 mm.

[0048] If the diameter of the bead is less than 2 mm, there is a risk that uniformity will be compromised during bead manufacturing, whereas if it exceeds 5 mm, there is a risk that wear resistance will be compromised. Preferably, the diameter of the calcium alginate bead supported with porphyridium cruentum of the present invention may be 2.5 mm.

[0049] In one embodiment of the present invention, calcium alginate beads having different diameter sizes (2.5 mm, 5 mm) were manufactured and an abrasion resistance evaluation was performed for each bead. It was found that the beads with a diameter of 2.5 mm had significantly superior abrasion resistance compared to the beads with a diameter of 5 mm. Therefore, the calcium alginate beads with a diameter of 2.5 mm can maintain their intact shape without abrasion even during long culture times and stirring.

[0050] In addition, the calcium alginate beads loaded with porphyridium cruentum of the present invention allow bioactive substances produced by porphyridium cruentum (sulfated polysaccharides, phycobili proteins, extracellular macromolecules, metabolites, etc.) loaded inside the beads to pass through the beads (pass through the calcium alginate wall) and be secreted outside the beads.

[0051] Therefore, due to these characteristics, using the calcium alginate beads of the present invention allows for obtaining bioactive substances without the process of crushing cultured microalgae or used beads, and also offers economic advantages as it eliminates the need for additional fixation such as centrifugation, coagulation, or filtration to separate the microalgae from the produced bioactive substances.

[0052] In addition, the present invention provides a method for fixing culture of Porphyridium cruentum using spherical calcium alginate beads loaded with Porphyridium cruentum prepared by the method of the present invention.

[0053] When using the calcium alginate beads of the present invention, Porphyridium cruentum can be encapsulated inside the beads and cultured in fixation, and since the alginate used to manufacture the beads of the present invention is non-toxic and transparent, the microalgae immobilized inside the beads can grow through photosynthesis.

[0054] In addition, the present invention may provide a method for mass-producing sulfated polysaccharides or phycobili proteins from Porphyridium cruentum, comprising the step of culturing using spherical calcium alginate beads loaded with Porphyridium cruentum.

[0055] In one embodiment of the present invention, an experiment was conducted to establish culture conditions that can enhance the production of sulfated polysaccharides or phycobiliri proteins, which are useful physiologically active substances from Porphyridium cruentum, using the calcium alginate beads of the present invention. As a result of analyzing cell productivity and sulfated polysaccharide and phycobiliri protein productivity for a group cultured without calcium alginate beads (control group), a group using calcium alginate beads, and a group cultured by irradiating calcium alginate beads and a blue light source, it was found that the group using calcium alginate beads showed increased cell productivity and sulfated polysaccharide and phycobiliri protein productivity compared to the group not using calcium alginate beads, and the group that simultaneously applied calcium alginate beads and a blue light source showed the highest cell productivity and sulfated polysaccharide and phycobiliri protein productivity.

[0056] Through this, the inventors found that when Porphyridium cruentum is cultured using calcium alginate beads and irradiated with a blue light source, sulfated polysaccharides or phycobili proteins can be mass-produced from Porphyridium cruentum, and the preferred culture conditions are 60 μmol m⁻² at 25°C with stirring at 120 rpm. -2 s -1 It can be performed by irradiating a blue light source with an intensity of .

[0057] In another embodiment of the present invention, to determine whether the production of sulfated polysaccharides or phycobili proteins can be enhanced under different conditions of the culture medium, calcium alginate beads loaded with Porphyridium cruentum were cultured under nutrient-deficient conditions, and then cell productivity, sulfated polysaccharide productivity, and phycobili protein productivity were analyzed according to culture time.

[0058] As a result, cell productivity was found to be the best when cultured under conditions where vitamins were bound. In addition, in the analysis of productivity of sulfated polysaccharides and phycobili proteins, vitamin-deficient conditions showed the highest efficiency during the initial culture up to day 15, and the group cultured under nitrogen-deficient conditions showed the highest efficiency during the subsequent culture up to day 30.

[0059] Therefore, through these results, the inventors found that when culturing Porphyridium cruentum using the spherical calcium alginate beads loaded with Porphyridium cruentum of the present invention, the production of sulfated polysaccharides and phycobili proteins from Porphyridium cruentum can be maximized by irradiating with a blue light source, culturing in a nutrient-deficient medium, or performing these simultaneously.

[0060] In addition, the isolation and acquisition of the sulfated polysaccharide or phycobili protein produced from the above-mentioned Porphyridium cruentum can be performed from a culture medium of spherical calcium alginate beads supported with Porphyridium cruentum, wherein the sulfated polysaccharide or phycobili protein is produced and secreted from Porphyridium cruentum and passes through the beads to exist in the culture medium.

[0061] Therefore, after collecting only the culture medium, these substances can be easily obtained in large quantities through a process of separating them from the culture medium.

[0062] In addition, the spherical calcium alginate beads loaded with Porphyridium cruentum of the present invention have the characteristic of being continuously usable through the process of replacing only the culture medium.

[0063] That is, there is a characteristic that allows for the continuous induction of the production of sulfated polysaccharides or phycobili proteins through a process in which useful physiologically active substances are isolated from a culture medium in which spherical calcium alginate beads loaded with porphyridium cruentum are cultured, and then the spherical calcium alginate beads loaded with porphyridium cruentum are transferred to a fresh culture medium and cultured again.

[0064] Therefore, spherical calcium alginate beads loaded with Porphyridium cruentum can produce sulfated polysaccharides or phycobili proteins semi-continuously through a simple process of replacing only the medium.

[0065] Furthermore, it was confirmed that the sulfated polysaccharides or phycobili proteins produced from Porphyridium cruentum using the calcium alginate beads of the present invention have the same properties and activity as the sulfated polysaccharides or phycobili proteins produced by conventional methods.

[0067] Therefore, when using spherical calcium alginate beads loaded with Porphyridium cruentum, Porphyridium cruentum can be stably immobilized inside the beads, and the production of bioactive substances such as sulfated polysaccharides and phycobili proteins can be enhanced through the control of culture conditions, and the bioactive substances can be easily separated from the cells and collected, as well as bioactive substances can be continuously produced from the spherical calcium alginate beads loaded with Porphyridium cruentum through the replacement of the culture medium.

[0069] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0071] <Example 1>

[0072] Manufacturing of calcium alginate beads

[0073] Calcium alginate beads were prepared by the following method. 5 mL of cell culture (culture containing Porphyridium cruentum) was mixed with 10 mL of an aqueous solution containing 1.92% (w / v) sodium alginate and 1% (w / v) Tween 80. Subsequently, the mixture was transferred to a 5 mL syringe equipped with a needle (inner diameter 0.97 mm), and using a syringe pump, 0.4 mL min was added to a beaker (inner diameter 85 mm) containing 150 mL of an aqueous solution containing 45% (w / v) PEG 6000 and 3% (w / v) CaCl2. -1 Pumping was performed at a flow rate. At this time, the tip of the needle was set vertically 3 cm above the surface of the solution, and the beaker was stirred at 600 rpm using a stirrer. After pumping was finished, the beaker was stirred for an additional 15 minutes, and the prepared calcium alginate beads were washed three times with sterile distilled water. The washed calcium alginate beads were transferred to 100 mL of an aqueous solution containing 1% (w / v) CaCl2, stirred for an additional 30 minutes, and then washed three times with sterile distilled water to prepare calcium alginate beads encapsulated with Porphyridium cruentum according to the present invention.

[0075] <Example 2>

[0076] Evaluation of wear resistance of calcium alginate beads

[0077] A wear resistance evaluation was conducted on calcium alginate beads. For the evaluation, cell-free calcium alginate beads without Porphyridium cruentum were prepared and used. The beads were prepared with diameters of 2.5 mm and 5 mm, respectively. Calcium alginate beads without added microalgae cells were placed in flasks containing 150 mL of culture medium and shaken at 150 rpm and 25°C. After 15 and 30 days, the beads were removed, dried at 60°C for 24 hours, and their dry weight was measured. Subsequently, the wear rate was calculated according to the following formula.

[0078] Wear rate (%) = (1 - DW / DW0) × 100

[0079] In the above formula, DW represents the dry weight on the day of the experiment, and DW0 represents the dry weight on day 0.

[0081] The inventors established bead conditions suitable for the culture process of microalgae through wear resistance analysis and confirmed the relationship between the diameter of the bead and wear resistance. As a result of the analysis, as shown in Figure 2, the weight of a bead with a diameter of 5 mm decreased by 45.2 ± 2.9% and 60.0 ± 9.3% on days 15 and 30 of the experiment, respectively, under culture conditions. On the other hand, the weight of a bead with a diameter of 2.5 mm decreased by 18.5 ± 6.7% and 20.4 ± 3.7% on days 15 and 30 of the experiment, respectively.

[0082] Through these results, the inventors found that using beads with a diameter of 2.5 mm is more suitable for fixed culture conditions because it has better wear resistance than using beads with a diameter of 5 mm.

[0084] <Example 3>

[0085] Establishment of optimal culture conditions for Porphyridium cruentum using the calcium alginate beads of the present invention

[0086] Porphyridium cruentum was cultured using an improved f / 2 medium; specifically, the composition of the medium was as follows per 950 mL of filtered seawater: NaNO3 1.5 g, Na2SiO3·9H2O 30 mg, NaH2PO4·H2O 5 mg, Na2EDTA·2H2O 4.36 mg, FeCl3·6H2O 3.15 mg, MnCl2·4H2O 180 μg, thiamine·HCl 100 μg, ZnSO4·7H2O 22 μg, CoCl2·6H2O 10 μg, CuSO4·5H2O 9.8 μg, Na2MoO4·2H2O 6.3 μg, vitamin B 12 A medium containing 0.5 μg of biotin and 0.5 μg of sodium nitrate was used, and prior to culture, it was sterilized using an autoclave at 121°C for 20 minutes.

[0087] For the production of sulfated polysaccharides, the experimental group was cultured by fixing Porphyridium cruentum on calcium alginate beads, and the control group was cultured for 30 days in the same environment without using calcium alginate beads.

[0088] In addition, the culture of Porphyridium cruentum for the production of sulfide polysaccharides was performed at 25℃, 120 rpm, and 60 μmol m⁻² -2 s -1 The experiment was conducted in a photobioreactor set to LED conditions, and blue LEDs were used for culture to verify the possibility of simultaneous application of the fixation culture of calcium alginate beads and the light source. In addition, the fixation culture of Porphyridium cruentum using calcium alginate beads was performed as a semi-continuous culture in which 70% of the medium was replaced with fresh medium after 15 days of culture, and the following analyses were performed.

[0090] <3-1> Cell Biomass Analysis of Porphyridium cruentum

[0091] The cell biomass of Porphyridium cruentum cultured under the culture conditions described above was analyzed. Changes in cell biomass during culture were assessed by collecting equal proportions of control and experimental groups, measuring their dry weights, and comparing them. For the experimental group, 10 mL of a 3% (w / v) tri-sodium citric acid solution was added, and the cells were sonicated for 15 minutes to remove calcium alginate. Subsequently, extracellular macromolecules were removed by washing three times with sterile water (pH = 4), and the cells were filtered using a 1.2 μm glass fiber filter. The cells were then dried at 60°C for 24 hours, and their weight was measured.

[0093] As a result, as shown in Figure 3a, the cell biomass of Porphyridium cruentum was found to be significantly higher in the experimental group in which cells were fixed and cultured with calcium alginate beads compared to the control group (microalgae culture group without using calcium alginate beads). In addition, among the groups in which cells were fixed and cultured with calcium alginate beads, the group irradiated with blue light showed a higher cell biomass compared to the group not irradiated with blue light.

[0094] Through these results, the inventors found that the use of the calcium alginate beads of the present invention and treatment with a blue light source is a method that can effectively promote cell growth of Porphyridium cruentum.

[0096] <3-2> Daily Sulfated Polysaccharide Productivity Analysis

[0097] The inventors analyzed the daily sulfated polysaccharide productivity for each experimental group. The productivity of sulfated polysaccharides was analyzed according to the modified phenol-sulfuric acid method. 2 mL of the supernatant was taken from each culture, 50 μL of 80% (v / v) phenol and 5 mL of 95.5% (v / v) H2SO4 were added, and the reaction mixture was left at 25°C for 10 minutes, then transferred to a water bath and left at 30°C for 20 minutes. Afterward, the mixture was stabilized for 4 hours under dark conditions at 25°C, and the optical density value at 480 nm was measured using a multi-detection microplate reader. The calibration curve was prepared and used based on glucose.

[0099] As a result, as shown in Fig. 3b, for the group cultured for 3 to 15 days, the daily productivity of sulfated polysaccharides was found to be similar for both the control group without calcium alginate beads and the group cultured with cells fixed using calcium alginate beads; however, the group using calcium alginate beads and simultaneously irradiated with blue light showed increased productivity of daily sulfated polysaccharides. In addition, for the group cultured for 18 to 30 days, it was confirmed that the productivity of sulfated polysaccharides in the group using the calcium alginate beads of the present invention increased significantly compared to the control group without calcium alginate beads.

[0101] <3-3> Analysis of Phycobiliary Protein Productivity

[0102] Next, phycobiliri protein productivity was analyzed under each culture condition. Phycobiliri protein productivity was assessed by extracting pigments via a freeze-thaw method and measuring absorbance using a spectrometer. For the experimental group, calcium alginate was removed using the same method as described above. Harvested cells were transferred to a 15 mL conical tube, 5 mL of 0.1 M phosphate buffer (pH=6) was added, and the cells were frozen at -20°C. Subsequently, the cells were thawed at 25°C, vigorously vortexed for 1 minute, and then frozen again at -20°C, a process repeated three times. After three cycles, the cells were centrifuged at 3500 rpm for 10 minutes to remove cell debris, and the supernatant was collected to measure absorbance at 565, 620, and 650 nm. The concentrations of phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (AC) were then calculated according to the following formula.

[0104] PE (mg L-1) = (A565 - 2.8 × PC - 1.34 × AP) / 12.7

[0105] PC (mg L-1) = (A620 - 0.7 × A650) / 7.38

[0106] AP (mg L-1) = (A650 - 0.19 × A620) / 5.65

[0107] Phycobiliprotein (mg L-1) = PE + PC + AP

[0108] From each calculated result, the pigment content per cell was analyzed by dividing the pigment content calculated from the above formula by the measured dry weight of the cell.

[0110] As a result of the analysis, on day 30 of culture, the phycobiliprotein content for the control group, the group cultured with cells fixed using the beads of the present invention, and the group cultured with cells fixed using the beads of the present invention and irradiated with blue light was 1.52 ± 0.01, 2.34 ± 0.12, and 5.48 ± 0.23 mg L, respectively. -1 It was found that (Fig. 3c). In particular, in the group using beads and irradiated with blue light, the content of all pigments PE (phycoerythrin), PC (phycocyanin), and AP (allophycocyanin) was significantly higher, and their content was found to be 372 ± 10%, 295 ± 10%, and 233 ± 27%, respectively, compared to the control group (Fig. 3c).

[0112] In addition, the analysis of intracellular phycobiliri protein content also showed that the group irradiated with blue light while performing cell fixation culture with the beads of the present invention had the highest intracellular phycobiliri protein content (Fig. 3d).

[0114] Based on the above results, the inventors found that when cells are fixed and cultured with calcium alginate beads while simultaneously irradiating them with blue light, cell growth can be induced most effectively, and the productivity of useful components such as sulfated polysaccharides and phycobili proteins can be improved.

[0116] <Example 4>

[0117] Analysis of the constituent elements of extracellular macromolecules produced from Porphyridium cruentum cultured by fixation with the calcium alginate beads of the present invention

[0118] The composition of extracellular polymeric substances produced from Porphyridium cruentum cultured by fixation with the calcium alginate beads of the present invention was analyzed using an elemental analyzer. For this purpose, extracellular polymeric substances were extracted from the supernatant collected from the cell culture using the alcohol precipitation method. For extraction, the collected supernatant was added to 95% (v / v) ethanol in a volume ratio of 1:3 and precipitated at 4°C for 24 hours. The resulting precipitate was then centrifuged and freeze-dried. After complete drying, 1.8 g of the precipitate was analyzed using an elemental analyzer at 1150°C with sulfanilic acid and benzoic acid as standards.

[0120] As a result, it was found that there was no significant difference in elemental composition between the group in which cells were fixed and cultured with calcium alginate beads and the group in which cells were cultured by dispensing them into the medium without using calcium alginate beads (control group). On the 15th day of culture, the sulfur content obtained from the control group, the calcium alginate bead culture group, and the calcium alginate bead and blue light irradiation culture group was found to be 16.0 ± 0.6 wt%, 16.4 ± 1.2 wt%, and 17.9 ± 1.0 wt%, respectively (Fig. 4a), and on the 30th day of culture, the sulfur content was found to be 17.0 ± 1.0 wt%, 15.7 ± 0.3 wt%, and 16.3 ± 0.3 wt%, respectively (Fig. 4b).

[0122] These results indicate that fixing and culturing cells with calcium alginate beads does not cause significant abnormalities in the cells themselves, nor does it pose a major problem for the production of physiologically active substances.

[0124] <Example 5>

[0125] Physiological activity analysis of extracellular macromolecules produced from Porphyridium cruentum cultured by fixation with calcium alginate beads of the present invention

[0126] The physiological activity of extracellular polymers produced from Porphyridium cruentum cultured and immobilized with the calcium alginate beads of the present invention was analyzed, and the antioxidant activity of the extracellular polymers produced from the cells was analyzed using the DPPH (2,2-diphenyl-1-picryhydrazyl) colorimetric assay. After dissolving the extracellular polymers produced from Porphyridium cruentum in 20 μL of distilled water, a mixture of DPPH and methanol (0.09 mg mL) -1 ) 180 μL was added to a microplate and reacted for 30 minutes under dark conditions at 25°C. Afterwards, the absorbance was measured at 517 nm using an equal amount of DPPH solution dissolved in distilled water as a blank, and the antioxidant activity was quantified.

[0128] In addition, the collagenase inhibitory activity of extracellular macromolecules produced from Porphyridium cruentum was analyzed using the Collagenase activity colorimetric assay kit MAK293 according to the manufacturer's instructions. First, the extracellular macromolecules produced from Porphyridium cruentum were dissolved in distilled water, and 2 μL of the solution and 10 μL of the enclosed collagenase were added to a 96-well plate. 10 μL of collagenase without added solution was used as a negative control, and the volume of the sample and negative control was adjusted to 100 μL using the enclosed collagenase assay buffer. Subsequently, the mixture was reacted at room temperature for 10 minutes, and the inhibition rate (%) was calculated using the following formula.

[0129] Inhibition Rate (%) = [(Activity 음성 대조군 - Activity 시료 ) / Activity 음성 대조군 ] × 100

[0131] In addition, the elastase inhibitory activity of extracellular macromolecules produced from Porphyridium cruentum was evaluated using the Neutrophil elastase activity assay kit MAK246. First, 200 μL of the enclosed assay buffer was added to the enclosed enzyme standard. Subsequently, 2 μL of the solution and 48 μL of the assay buffer were added to a 96-well plate. The inhibition control was diluted 1:25 using the assay buffer. Distilled water containing the dissolved extracellular macromolecule, the inhibition control, and 25 μL of the assay buffer were added to the 96-well plate to establish the sample, positive control, and negative control, respectively, and the mixture was incubated at 37°C for 5 minutes. Afterward, 23 μL of the assay buffer and 2 μL of the enclosed substrate were added, and the mixture was incubated at 37°C for 30 minutes. After the reaction, fluorescence was measured at 450 and 505 nm. Next, the fluorescence values ​​at T1 and T2 were measured and set as R1 and R2, respectively, and the inhibition rate (%) was calculated according to the following formula.

[0132] ΔRelative fluorescence units (ΔRFU) = R2- R1

[0133] Inhibition rate (%) = (ΔRFU 시료 / ΔRFU 대조군 ) × 100

[0135] In addition, the hyaluronidase inhibitory activity of extracellular macromolecules produced from Porphyridium cruentum was analyzed by spectrophotometry. An analytical medium was prepared consisting of 100 μL of hyaluronidase and 100 μL of sodium phosphate buffer (100 mM, pH 7, 37°C) containing 50 mM NaCl and 0.01% BSA (bovine serum albumin). Subsequently, 50 μL of distilled water containing dissolved extracellular macromolecules was mixed with the analytical medium and reacted at 37°C for 10 minutes. Then, 100 μL of a substrate in the form of a hyaluronic acid solution (0.03% in 300 mM sodium phosphate, pH 5.35) was added, and the mixture was reacted at 37°C for 45 minutes. Undigested hyaluronic acid was precipitated using 1 mL of acid albumin solution (0.1% BSA in 24 mM sodium acetate and 79 mM CH3COOH, pH 3.75). The mixture was reacted at 25°C for 10 minutes, and the absorbance was measured at 600 nm. The absorbance in the absence of the enzyme was used as the control with the maximum inhibition rate, and ascorbic acid was used as the positive control. The inhibition rate (%) was calculated according to the following formula.

[0136] Inhibition rate (%) = (A 시료 / A 대조군 ) × 100

[0138] As a result of the analysis, as shown in Figures 4c and 4d, the activity analysis of bioactive substances extracted from the group cultured with cells fixed with calcium alginate beads and the group cultured in suspension without using calcium alginate beads showed no significant difference between all experimental and control groups.

[0140] Therefore, through these results, the inventors confirmed that the method of fixing culture of Porphyridium cruentum using the calcium alginate beads of the present invention does not significantly affect the activity of the physiologically active components and does not affect the cells, and thus can be used without major problems. As a result, it was found that the present invention, which has the advantage of being able to obtain a large amount of physiologically active components, can replace conventional culture methods.

[0142] <Example 6>

[0143] Analysis of Changes in Cell Productivity and Daily Polysaccharide Productivity via Calcium Alginate Beads and Nutrient Deficiency Induction

[0144] Furthermore, to identify culture conditions capable of maximizing cell productivity and the productivity of useful bioactive substances in conjunction with cell fixation culture using the calcium alginate beads of the present invention, the inventors investigated changes in cell biomass and sulfated polysaccharide production under nutrient-deficient culture conditions. Nutrient-deficient conditions were analyzed for groups using nitrogen-removed media, vitamin-removed media, and media from which both nitrogen and vitamins were removed. In this case, the media consisted of the previously used modified f / 2 medium with a nitrogen source (NaNO3) and / or vitamin (vitamin B12). 12 Nutrient-deficient media were prepared and used by removing biotin and other nutrients. Additionally, the culture of Porphyridium cruentum using fixed culture and nutrient deprivation was conducted at 25°C, 120 rpm, and 60 μmol m⁻². -2 s -1 Semi-continuous culture was performed in a photobioreactor set to blue LED conditions, and cell biomass, sulfated polysaccharides, and phycobili protein productivity were analyzed using the same method as previously described. The analysis results are shown in Table 1 and Figure 5 below.

[0145]

[0147] As a result, as shown in Table 1 and Figure 5 above, it was found that cell productivity and the production of sulfated polysaccharides under nutrient deficiency induction conditions were such that the largest amount of sulfated polysaccharides could be produced when the culture up to the 15th day was cultured in a medium with vitamins removed (-V), and the culture up to the 30th day was cultured in a medium with nitrogen removed (-N).

[0149] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

Claim 1 (1) a step of mixing a culture of the microalgae Porphyridium cruentum with a sodium alginate solution; (2) 0.2 to 0.6 mL min of the mixture mixed in step (1); -1 A method for manufacturing calcium alginate beads supported with porphyridium cruentum, comprising the steps of: (3) adding to an aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 at a flow rate of 500 to 700 rpm to form calcium alginate beads supported with microalgae; and (3) washing the calcium alginate beads supported with microalgae with distilled water after further stirring in an aqueous solution containing CaCl2, wherein the porphyridium cruentum culture of step (1) and the sodium alginate solution are mixed in a volume ratio of 1:2, and the aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 is an aqueous solution containing 45% (w / v) polyethylene glycol (PEG) 6000 and 3% (w / v) CaCl2. Claim 2 delete Claim 3 A method for manufacturing calcium alginate beads loaded with porphyridium cruentum according to claim 1, characterized in that adding the mixture to an aqueous solution containing polyethylene glycol (PEG) 6000 and CaCl2 is performed by transferring the mixture using a syringe equipped with a needle and then using a syringe pump. Claim 4 delete Claim 5 delete Claim 6 Spherical calcium alginate beads loaded with porphyridium cruentum, having a diameter of 2 to 5 mm, manufactured by the method of claim 1. Claim 7 delete Claim 8 delete Claim 9 The method includes a step of culturing using spherical calcium alginate beads loaded with Porphyridium cruentum of claim 6, wherein the cultivation is performed at 25°C with stirring at 120 rpm at 60 μmol m⁻¹ -2 s -1 A method for mass-producing sulfated polysaccharides from Porphyridium cruentum, characterized by culturing while irradiating with a blue light source at a certain intensity, culturing in a vitamin-deficient medium until the 15th day of the initial culture, and then replacing it with a nitrogen-deficient medium and performing additional culture until the 30th day. Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 A method for mass-producing sulfide polysaccharides from Porphyridium cruentum, characterized in that, in claim 9, the sulfide polysaccharides are separated from a culture medium of spherical calcium alginate beads supported with Porphyridium cruentum. Claim 14 A method for mass-producing sulfide polysaccharides from Porphyridium cruentum according to claim 13, characterized in that the sulfide polysaccharides are separated from the culture medium, and then the spherical calcium alginate beads loaded with Porphyridium cruentum are transferred to a fresh culture medium and cultured again to continuously induce the production of sulfide polysaccharides.

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