Compositions for cryopreservation of thraustochytrid microalgae and methods of cryopreservation of thraustochytrid microalgae using the same

Abstract

The present application relates to a composition for cryopreservation of microalgae belonging to the Thraustochytridaceae family, and a method for cryopreservation of microalgae belonging to the Thraustochytridaceae family using the same. According to one aspect, by the composition for cryopreservation of microalgae belonging to the Thraustochytridaceae family and the method for cryopreservation of microalgae belonging to the Thraustochytridaceae family using the same, the microalgae can be stably stored for a long time, and the cost of preserving the microalgae can be reduced by shortening the process. Furthermore, according to the method of preparing a freeze-dried biomass of microalgae belonging to the Thraustochytridaceae family using the same, even during long-term storage at room temperature, a freeze-dried biomass in the form of a powder capable of maintaining bacterial activity and easy to store and transport can be manufactured through a simple process.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition for cryopreservation of Thraustochytriaceae microalgae and a method for cryopreservation of Thraustochytriaceae microalgae. BACKGROUND

[0002] Microalgae are phytoplankton, which occupy the lowest level in the food chain of marine ecosystems. Among them, microalgae belonging to the Thraustochytriaceae family are different from general microalgae in that they are heterotrophs rather than autotrophs derived from photosynthesis, and play an important role in supplying omega-3 polyunsaturated fatty acids (including docosahexaenoic acid and eicosapentaenoic acid) to marine ecosystems by producing and containing high concentrations of omega-3 polyunsaturated fatty acids (including docosahexaenoic acid and eicosapentaenoic acid).

[0003] General microorganisms can be stored for a long time through a cryopreservation or freeze-drying preservation method. However, the general storage method of these microorganisms is not suitable for long-term effective storage of Thraustochytriaceae microalgae. Therefore, to date, such microalgae have been preserved in the form of subculture. However, since the microalgae of the Thraustochytriaceae family are characterized as heterotrophs, they need more frequent subculturing than general microorganisms depending on the storage environment, thus making them vulnerable to contamination and resulting in high storage costs. Therefore, there is a need to develop a new method for long-term storage of Thraustochytriaceae microalgae.

[0004] PRIOR ART DOCUMENTS

[0005] PATENT DOCUMENTS

[0006] (Patent Document 1) U.S. Patent Publication No. US 2013 / 0089901 A1 SUMMARY

[0007] TECHNICAL PROBLEM

[0008] The present application provides a composition for cryopreservation of Thraustochytriaceae microalgae, the composition comprising skimmed milk, sucrose, and sodium chloride.

[0009] The present application provides a method for cryopreservation of Thraustochytriaceae microalgae using a composition for cryopreservation of Thraustochytriaceae microalgae, or a method for preparing freeze-dried biomass of Thraustochytriaceae microalgae.

[0010] The present application provides freeze-dried biomass of Thraustochytriaceae microalgae prepared by using a method for preparing freeze-dried biomass of Thraustochytriaceae microalgae.

[0011] The present application provides a composition for cryopreservation of Thraustochytriaceae microalgae for use in cryopreservation of Thraustochytriaceae microalgae, the composition comprising skimmed milk, sucrose, and sodium chloride.

[0012] The present application provides use of a composition for cryopreservation of Thraustochytriaceae microalgae comprising skimmed milk, sucrose and sodium chloride in the manufacture of a freeze-dried biomass of Thraustochytriaceae microalgae.

[0013] Technical Solution

[0014] The various descriptions and implementations described herein can also apply to other descriptions and implementations. That is, all combinations of claimed elements in the descriptions and implementations described herein are within the scope of the present application. In addition, the described aspects of the application are not limited to the specific descriptions and implementations described herein. Rather, features and characteristics of the aspects described herein can be combined with each other and / or combined with other features and characteristics not expressly described but falling within the scope of the application. Moreover, the described aspects of the application are not limited to the specific examples described herein. Rather, features and characteristics of the aspects described herein can be combined with each other and / or combined with other features and characteristics not expressly described but falling within the scope of the application.

[0015] In one aspect, a composition for cryopreservation of Thraustochytriaceae microalgae is provided, the composition comprising skimmed milk, sucrose and sodium chloride.

[0016] The term "skimmed milk" refers to milk from which fat has been removed.

[0017] The term "Thraustochytriaceae" as used herein refers to microalgae belonging to the order Thraustochytriales, which can comprise at least one selected from the group consisting of Thraustochytrium sp., Schizochytrium sp., Aurantiochytrium sp., Thraustochytriidae sp., Japonochytrium sp., Monorhizochytrium sp., Sicyoidochytrium sp., Ulkenia sp., Parietichytrium sp., Botryochytrium sp., Hondaea sp., and Labyrinthulochytrium sp. However, embodiments of the present disclosure are not limited thereto.

[0018] The composition comprises skim milk in an amount of 0.5% to 20% by weight, based on the total weight of the composition. For example, the amount of skim milk included in the composition can be 0.5% to 15%, 0.5% to 10%, 0.5% to 8%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 8%, 2% to 20%, 2% to 15%, 2% to 10%, 2% to 8%, 3% to 20%, 3% to 15%, 3% to 10%, or 3% to 8%, by weight, based on the total weight of the composition.

[0019] The composition comprises sucrose in an amount of 1% to 20% by weight, based on the total weight of the composition. For example, the amount of sucrose included in the composition can be 1% to 15%, 1% to 12%, 2% to 20%, 2% to 15%, 2% to 12%, 4% to 20%, 4% to 15%, 4% to 12%, 6% to 20%, 6% to 15%, 6% to 12%, or 6% to 9%, by weight, based on the total weight of the composition.

[0020] The composition can comprise sodium chloride in an amount of 0.1% to 10% by weight, based on the total weight of the composition. For example, the amount of sodium chloride included in the composition can be 0.1% to 9%, 0.1% to 8.5%, 0.5% to 10%, 0.5% to 9%, 0.5% to 8.5%, 1% to 10%, 1% to 9%, 1% to 8.5%, 2% to 10%, 2% to 9%, 2% to 8.5%, 3% to 10%, 3% to 9%, 3% to 8.5%, 5% to 8.5%, or 6% to 8.5%, by weight, based on the total weight of the composition.

[0021] The composition comprises skim milk and sucrose in a weight ratio of 1:0.5 to 1:10. For example, the composition can comprise skim milk and sucrose in a weight ratio of 1:0.5 to 1:8, 1:0.5 to 1:5, 1:0.5 to 1:3, 1:1 to 1:10, 1:1 to 1:8, 1:1 to 1:5, 1:1 to 1:3, 1:1.2 to 1:10, 1:1.2 to 1:8, 1:1.2 to 1:5, or 1:1.2 to 1:3.

[0022] The composition comprises skim milk and sodium chloride in a weight ratio of 1:0.5 to 1:10. For example, the composition can comprise skim milk and sodium chloride in a weight ratio of 1:0.5 to 1:8, 1:0.5 to 1:5, 1:0.5 to 1:3, 1:1 to 1:10, 1:1 to 1:8, 1:1 to 1:5, 1:1 to 1:3, 1:1.2 to 1:10, 1:1.2 to 1:8, 1:1.2 to 1:5, or 1:1.2 to 1:3.

[0023] The term "cryopreservation" as used herein refers to preservation of a material in a solid state after being frozen in a liquid state, and can include "lyophilization preservation". For example, the composition can be a composition for lyophilization preservation of a breviportid microalga. The term "lyophilization" as used herein refers to a drying method in which a sample in a liquid state is frozen and placed under reduced pressure to sublimate and remove water from the sample. Lyophilization can be used for long-term preservation of microorganisms. However, a suitable cryoprotective agent should be used to prevent damage to cells during lyophilization treatment.

[0024] The composition for cryopreservation of breviportid microalgae comprising skim milk, sucrose, and sodium chloride can be used together with other cryoprotective agents. For example, sodium chloride, dimethyl sulfoxide (DMSO), dextran, sucrose, glycerol, mannitol, sorbitol, fructose, raffinose, serum albumin, etc. can be used in combination according to the purpose, but embodiments of the present disclosure are not limited thereto.

[0025] In another aspect, there is provided a method for cryopreservation of breviportid microalgae, the method comprising: 1) culturing breviportid microalgae in a medium comprising a composition for cryopreservation of breviportid microalgae comprising skim milk, sucrose, and sodium chloride; 2) recovering the culture product of 1); and 3) lyophilizing the culture product to produce a biomass.

[0026] The composition for cryopreservation of breviportid microalgae is the same as described above.

[0027] The term "culture" as used herein refers to growth of microalgae under appropriate controlled environmental conditions. The culture process of the present application can be performed according to suitable culture media and culture conditions known in the art. The culture process can be easily adjusted and used by a person skilled in the art according to the selected microalgae.

[0028] In particular, the culture of breviportid microalgae of the present application can be performed under heterotrophic conditions, but is not limited thereto.

[0029] The term "heterotrophic" is a nutritional method that relies on organic matter obtained from an energy source or a nutrient source outside the body, is a term that distinguishes from autotrophic, and can be used interchangeably with the term "dark culture".

[0030] The method of culturing the Thraustochytriales microalgae is not particularly limited, and batch culture, continuous culture, fed-batch culture, and the like, which are well known in the art, can be performed. Any medium for culturing the microalgae of the present application can be used without limitation, as long as the medium is one in which the Thraustochytriales microalgae grow. Specifically, a conventional medium containing a carbon source, a nitrogen source, a phosphorus source, an inorganic compound, an amino acid, and / or a vitamin suitable for the microalgae of the present application can be used.

[0031] The carbon source contained in the medium for culturing the Thraustochytriales microalgae can be at least one selected from the group consisting of glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose, and glycerol. However, any carbon source can be used as long as it is used for culturing the microalgae.

[0032] The nitrogen source contained in the medium for culturing the Thraustochytriales microalgae can be i) at least one organic nitrogen source selected from the group consisting of yeast extract, beef extract, peptone, and tryptone, or ii) at least one inorganic nitrogen source selected from the group consisting of ammonium acetate, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, urea, and monosodium glutamate (MSG). However, the embodiments of the present disclosure are not limited thereto, and any nitrogen source used for culturing the microalgae can be used herein.

[0033] The medium for culturing the Thraustochytriales microalgae can include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt thereof, or a combination thereof as a phosphorus source. However, the phosphorus source is not limited thereto.

[0034] The culture conditions for culturing the Thraustochytriales microalgae can be any culture conditions in which the Thraustochytriales microalgae grow. For example, culturing under aerobic conditions can be performed while controlling the temperature, pH, and the like.

[0035] Specifically, an alkaline compound such as sodium hydroxide, potassium hydroxide, or ammonia, or an acidic compound such as phosphoric acid or sulfuric acid is used to adjust the pH to an appropriate level for culturing (e.g., pH 5 to pH 9, specifically, pH 6 to pH 8). However, the embodiments of the present disclosure are not limited thereto.

[0036] In addition, in order to maintain an aerobic state of the culture, oxygen or an oxygen-containing gas can be injected into the culture; or in order to maintain an anaerobic and microaerobic condition, no gas or nitrogen gas, hydrogen gas, or carbon dioxide gas can be injected. However, the embodiments of the present disclosure are not limited thereto.

[0037] Further, the culture temperature can be maintained at about 20°C to about 45°C or about 25°C to about 40°C for about 10 hours to about 160 hours, about 10 hours to about 120 hours, about 10 hours to about 80 hours, about 10 hours to about 50 hours, or about 10 hours to about 40 hours. However, the culture conditions are not limited thereto. In addition, during the culture process, a defoaming agent such as a fatty acid polyethylene glycol ester can be used to inhibit the generation of bubbles, but the embodiments of the present application are not limited thereto.

[0038] The recovered culture can be collected using a suitable method known in the art. For example, centrifugation, filtration, anion exchange chromatography, etc. can be used, but are not limited thereto.

[0039] The term "biomass" used herein refers to organisms such as plants, animals, and microorganisms that can be used as a source of chemical energy, i.e., a source of energy of bioenergy, and can refer to the weight or energy of organisms present in a unit of time and space from an ecological point of view. Further, the biomass includes, but is not limited to, a compound secreted by a cell, and can include extracellular material as well as cells and / or contents within the cells. In the present application, the biomass can be the thraustochytrid microalgae itself, a culture thereof, or a product produced by culturing or fermenting the microalgae, or can be a concentrate of the biomass. However, the biomass is not limited thereto.

[0040] The "culture product" of the thraustochytrid microalgae refers to a product produced by culturing the microalgae. Specifically, the culture product can be a culture medium containing the microalgae or a culture filtrate in which the microalgae are removed from the culture medium. However, the culture product is not limited thereto. The culture product of the thraustochytrid microalgae can be prepared by inoculating the microalgae into a microalgae culture medium and then culturing the same according to a culturing method known in the art.

[0041] The preparation of the biomass by freeze-drying the culture product can be performed according to a freeze-drying method known in the art. For example, the microalgae culture product is recovered, put into a freeze-drying vial (FD vial), and connected to a freeze-drying device while maintaining a vacuum, and then moisture is removed under conditions of maintaining a constant temperature and pressure. The constant temperature condition can be, for example, a temperature of -50°C or less, and the constant pressure condition can be a pressure of 0.133 mbar or less, but the temperature and pressure conditions are not limited thereto.

[0042] The method of cryopreserving the thraustochytrid microalgae can further include freezing the microalgae neither before nor after process 3). For example, the pre-freezing process can not be performed before process 3). According to the method, by manufacturing the freeze-dried biomass without including the pre-freezing process, the microalgae can be preserved at a low cost through a simple process.

[0043] In another aspect, there is provided a method of preparing a freeze-dried biomass of a Thraustochytrid microalga, the method comprising: 1) culturing a Thraustochytrid microalga in a medium comprising a composition for cryopreservation of a Thraustochytrid microalga comprising skim milk, sucrose, and sodium chloride; 2) recovering the culture product of 1); and 3) freeze-drying the culture product to produce a biomass.

[0044] The composition for cryopreservation of a Thraustochytrid microalga and the method of preparing a freeze-dried biomass of a Thraustochytrid microalga are as described above.

[0045] The method of preparing a freeze-dried biomass of a Thraustochytrid microalga can further comprise not freezing the microalga either before process 3) or after process 3). For example, the pre-freezing process can not be performed before process 3). According to the method, the freeze-dried biomass can be prepared at a low cost by a simple process of manufacturing a freeze-dried biomass without including a pre-freezing process.

[0046] In another aspect, there is provided a freeze-dried biomass of a Thraustochytrid microalga, comprising: a) a composition for cryopreservation of a Thraustochytrid microalga, the composition comprising skim milk, sucrose, and sodium chloride; and b) a Thraustochytrid microalga, wherein the freeze-dried biomass is prepared by a method of preparing a freeze-dried biomass of a Thraustochytrid microalga.

[0047] The composition for cryopreservation of a Thraustochytrid microalga and the method of preparing a freeze-dried biomass of a Thraustochytrid microalga are as described above.

[0048] The freeze-dried biomass can be stored at 15°C to 25°C for 12 weeks or more. For example, the freeze-dried biomass can be stored at room temperature for 3 months to 5 years, 3 months to 3 years, 3 months to 2 years, 3 months to 1 year, 3 months to 10 months, 3 months to 8 months, 3 months to 6 months.

[0049] As used herein, the term "room temperature" can refer to a temperature of about 15°C to 25°C, and can be used interchangeably with the term "ambient temperature".

[0050] As used herein, the term "storable" can refer to a feature that when the freeze-dried biomass is stored, then cultured in a medium, cells can grow, or cell viability is maintained at 60% or more, 70% or more, 80% or more, or 90% or more, as compared to live cells that are not freeze-dried.

[0051] The freeze-dried biomass can comprise 1.0 x 10 7 to 1.0 x 10 12viable cells per 1 mL of biomass. For example, the freeze-dried biomass can comprise 1.0 x 10 7 to 1.0 x 10 11 viable cells per 1 mL of biomass. For example, the freeze-dried biomass can comprise 1.0 x 10 7 to 1.0 x 10 10 viable cells per 1 mL of biomass. For example, the freeze-dried biomass can comprise 1.0 x 10 8 to 1.0 x 10 12 viable cells per 1 mL of biomass. For example, the freeze-dried biomass can comprise 1.0 x 10 8 to 1.0 x 10 11 viable cells per 1 mL of biomass. For example, the freeze-dried biomass can comprise 1.0 x 10 8 to 1.0 x 10 10 viable cells per 1 mL of biomass. For example, the freeze-dried biomass can comprise 1.0 x 10 9 to 1.0 x 10 12 viable cells per 1 mL of biomass. For example, the freeze-dried biomass can comprise 1.0 x 10 9 to 1.0 x 10 11 viable cells per 1 mL of biomass. For example, the freeze-dried biomass can comprise 1.0 x 10 9 to 1.0 x 10 10 viable cells per 1 mL of biomass.

[0052] The freeze-dried biomass can comprise 1.0 x 10 7 to 1.0 x 10 12 CFU (colony forming units) per mL of viable cells after storage at 15 °C to 25 °C for at least 12 weeks. For example, the freeze-dried biomass can comprise 1.0 x 10 7 to 1.0 x 10 11 CFU per mL of viable cells after storage at 15 °C to 25 °C for at least 12 weeks. For example, the freeze-dried biomass can comprise 1.0 x 10 7 to 1.0 x 10 10 CFU per mL of viable cells after storage at 15 °C to 25 °C for at least 12 weeks. For example, the freeze-dried biomass can comprise 1.0 x 10 8 to 1.0 x 10 12 CFU per mL of viable cells after storage at 15 °C to 25 °C for at least 12 weeks. For example, the freeze-dried biomass can comprise 1.0 x 10 8 to 1.0 x 10 11 CFU per mL of viable cells after storage at 15 °C to 25 °C for at least 12 weeks. For example, the freeze-dried biomass can comprise 1.0 x 10 8 to 1.0 x 10 10 CFU per mL of viable cells after storage at 15 °C to 25 °C for at least 12 weeks. For example, the freeze-dried biomass can comprise 1.0 x 10 9 to 1.0 x 10 12 CFU per mL of viable cells after storage at 15 °C to 25 °C for at least 12 weeks. For example, the freeze-dried biomass can comprise 1.0 x 10 9 to 1.0 x 10 11 CFU per mL of viable cells after storage at 15 °C to 25 °C for at least 12 weeks. For example, the freeze-dried biomass can comprise 1.0 x 10 9 to 1.0 x 10 10 CFU per mL of viable cells after storage at 15 °C to 25 °C for at least 12 weeks.

[0053] In another aspect, there is provided a composition for cryopreservation of a Break- sea family microalgae for use in cryopreservation of a Break-sea family microalgae, the composition comprising skimmed milk, sucrose and sodium chloride.

[0054] In another aspect, there is provided a use of a composition for cryopreservation of a thraustochytrid microalgae for the preparation of a freeze-dried biomass of a thraustochytrid microalgae, the composition comprising skimmed milk, sucrose, and sodium chloride.

[0055] The composition for cryopreservation of a thraustochytrid microalgae, the method for cryopreservation of a thraustochytrid microalgae, and the method for preparing a freeze-dried biomass of a thraustochytrid microalgae as described above.

[0056] Advantages

[0057] The composition for cryopreservation of a thraustochytrid microalgae and the cryopreservation method using the same according to the present application can allow the microalgae to be stably preserved for a long time and reduce the cost of preserving the microalgae by shortening the process. In addition, the method for preparing a freeze-dried biomass of a thraustochytrid microalgae using the composition can maintain the microbial activity even when stored for a long time at room temperature, and the freeze-dried biomass is in a powder form, which is easy to store and transport, and is prepared by a simple process. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Images of freeze-dried vials (A to D) prepared according to one aspect, and images of confirmation of colony formation after inoculating the contents of the vials onto agar plates (E to H) are shown (A-H: A shows an image of a freeze-dried vial prepared according to Condition 1-1, E shows an image of confirmation that colonies have formed after inoculating the contents of the A vial onto an agar plate, B shows an image of a freeze-dried vial prepared according to Condition 1-2, F shows an image of confirmation that colonies have formed after inoculating the contents of the B vial onto an agar plate, C shows an image of a freeze-dried vial prepared according to Condition 1-3, G shows an image of confirmation that colonies have formed after inoculating the contents of the C vial onto an agar plate, D shows an image of a freeze-dried vial prepared according to Condition 1-4, and H shows an image of confirmation that colonies have formed after inoculating the contents of the D vial onto an agar plate).

[0059] Figure 2 Confirmation of colonies after inoculating freeze-dried biomass according to one aspect onto agar plates is shown (A-E: A shows confirmation of colonies after inoculating freeze-dried biomass according to Condition 3-1 onto an agar plate, B shows confirmation of colonies after inoculating freeze-dried biomass according to Condition 3-2 onto an agar plate, C shows confirmation of colonies after inoculating freeze-dried biomass according to Condition 3-3 onto an agar plate, D shows confirmation of colonies after inoculating freeze-dried biomass according to Condition 3-4 onto an agar plate, and E shows confirmation of colonies after inoculating freeze-dried biomass according to Condition 3-5 onto an agar plate).

[0060] Figure 3 A graph showing a growth curve in which absorbance was measured for each cultivation time after inoculating a freeze-dried biomass according to an aspect in a culture flask is shown.

[0061] Figure 4 A graph showing a growth curve in which absorbance was measured for each cultivation time after a freeze-dried biomass according to an aspect was stored at room temperature for 7 days and then inoculated into a culture flask is shown.

[0062] Figure 5 An image showing confirmation of colony formation after a freeze-dried biomass according to an aspect was stored at room temperature for 12 weeks and then inoculated into an agar plate is shown. DETAILED DESCRIPTION

[0063] Hereinafter, the present disclosure will be described in greater detail by examples. However, these examples are provided for the purpose of illustration in order to describe one or more embodiments, and the scope of the present disclosure is not limited to these examples.

[0064] Example 1. Confirmation of cell viability according to freeze-drying conditions of microalgae

[0065] Example 1-1. Production of freeze-drying protectant

[0066] A freeze-drying protectant having a total amount of 4 mL was prepared by mixing 0.8 mL of a 4% skim milk solution, 1.6 mL of a 20% sucrose solution, and 1.6 mL of a 20% sodium chloride solution, each dissolved in distilled water.

[0067] Example 1-2. Freeze-drying of a breviacinus microalgae

[0068] Microalgae of the genus *Schizochytrium*, CD01-5004 (accession number: KCTC14345BP), were inoculated on GYEP medium (10 g / L glucose, 1 g / L yeast extract, 1 g / L peptone, 2 g / L MgSO4·7H2O, 20 g / L sea salt, 5.0 mg / L H3BO3, 3.0 mg / L MnCl2, 0.2 mg / L CuSO4, 0.05 mg / L NaMo4·2H2O, 0.05 mg / L CoSO4, and 0.7 mg / L ZnSO4·7H2O), a medium suitable for the growth of *Schizochytrium* species, and cultured overnight in 500 mL flasks at 28 °C and 180 rpm. The culture was recovered and centrifuged, and the supernatant was removed. The remaining product was suspended in the freeze-drying protectant prepared in Example 1-1 to prepare a microalgae biomass suspension. After the prepared biomass suspension was placed in an ampoule-type freeze-drying vial (FD vial), it was pre-frozen under conditions 1-1 to 1-3 as shown in Table 1, and the suspension without pre-freezing was prepared according to condition 1-4.

[0069] Table 1

[0070]

[0071] Freeze-dried vials for each condition were connected to a freeze-drying apparatus and freeze-dried under vacuum conditions, maintaining a temperature below -50°C and a pressure below 0.133 mbar. When a large number of ampoules were connected to the freeze-drying apparatus, the pressure increased, affecting the growth of the freeze-dried cells. Therefore, one ampoule was connected, and then the next ampoule was connected only after the pressure in the apparatus had decreased to below 0.133 mbar. After all ampoules were connected to the apparatus, freeze-drying was performed for approximately 3 hours, and the freeze-dried ampoules were sealed with a gas torch to maintain a vacuum.

[0072] Examples 1-3. Confirmation of the viability of freeze-dried cells

[0073] To confirm the survival of the freeze-dried cells prepared in Examples 1-2, the powdered dried biomass sample was suspended in distilled water and spread on GYEP agar plates, and then the growth of colonies was visually observed.

[0074] As a result, Figure 1 As shown, in the case of freeze-dried biomass under pre-freezing conditions 1-1 to 1-3, no colonies grew or formed on agar plates, and only in the case of conditions 1-4 without pre-freezing was it confirmed that freeze-dried biomass could grow and form colonies on agar plates.

[0075] Example 2. Confirmation of the viability of freeze-dried cells according to the composition of freeze-drying protectants

[0076] To evaluate the freeze-drying preservation effect according to the composition of freeze-drying protectants, as shown in Table 2, 4 mL of freeze-drying protectants containing the indicated final concentration of the respective components were prepared for each condition.

[0077] Table 2

[0078]

[0079] Biomass suspensions were prepared using the same method as used in Examples 1-2, followed by freeze-drying without pre-freezing treatment, except that Schizochytrium sp. microalgae CD01-5004 (Accession No.: KCTC 14345BP) was inoculated into GYEP medium containing 30 g / L glucose, and freeze-drying protectants prepared in the compositions of Table 2 were used.

[0080] The dried biomass samples in powder form were suspended in distilled water and diluted so that the absorbance (optical density: OD) at 680 nm was 0.1. The resulting solution was spread on GYEP agar plates in an amount of 1 mL, and the number of colonies formed in each plate was counted. As a control, a portion of the recovered cell culture was not suspended in the freeze-drying protectant before preparing the biomass suspension, 1 mL of a solution diluted to an OD value of 0.1 at 680 nm was spread on GYEP agar plates, and the number of colonies formed was counted. Since there are 1 x 10 6 cells per colony, the number of viable cells per 1 mL of culture was calculated by multiplying the number of colonies formed in each experimental group by 10 6 , and the viability under each condition was calculated as a percentage of the control group.

[0081] Table 3

[0082] Experimental group Number of colonies formed Number of viable cells Viability Control 1500 1.5 X 10 9 ]]> 100％ Condition 2-1 20 2.0 X 10 7 ]] 1.33％ Condition 2-2 0 0 0％ Condition 2-3 0 0 0％ Condition 2-4 0 0 0％ Condition 2-5 2 2.0 X 10 6 ]]> 0.13％ Condition 2-6 250 2.5 X 10 8 ]]> 16.67％ Condition 2-7 0 0 0％

[0083] As a result, as shown in Table 3, it was confirmed that colonies were formed within 72 hours after spreading on plates under conditions 2-1, 2-5, and 2-6. The freeze-drying protectants used in these conditions generally contained 6% to 10% skim milk and 4% to 10% sucrose, and in particular, in condition 2-6, a freeze-drying protectant containing skim milk, sucrose, and sodium chloride was used, and the viability was significantly high up to 16.67%.

[0084] Example 3. Confirmation of the degree of growth of freeze-dried cells according to the ratio of the components of freeze-drying protectants

[0085] Example 3-1. Confirmation of colony formation on agar plate

[0086] To evaluate the preservation effect of freeze-drying according to the concentration ratio of skim milk, sucrose, and sodium chloride, which are the most effective protective agent ingredients from Example 2, 4 mL of a freeze-drying protective agent containing the respective ingredients at the final concentrations shown in Table 4 was prepared for each condition. After freeze-drying the Schizochytrium sp. microalgae using the freeze-drying protective agent for each condition in the same manner as described in Example 2, the number of colonies formed was counted by suspending in distilled water and spreading on a GYEP agar plate. In the colony-forming condition group, colony formation was visually confirmed from 24 hours after spreading, and from 40 hours after spreading, it was possible to clearly distinguish the presence or absence of cell survival and the degree of colony formation.

[0087] Table 4

[0088] Experimental group Skim milk (%) Sucrose (%) Sodium chloride (%) Number of colonies Condition 3-1 6 8 4 30 Condition 3-2 - 10 5 32 Condition 3-3 4 8 8 34 Condition 3-4 10 - - 0 Condition 3-5 5 - 5 0

[0089] As a result, as shown in Table 4, no colonies were formed in condition 3-4 using a protective agent containing only 10% skim milk, and condition 3-5 using a protective agent containing 5% skim milk and 5% sodium chloride, and colony formation was confirmed in the other conditions. Figure 2

[0090] Example 3-2. Confirmation of cell growth in a culture flask

[0091] Regarding the freeze-dried cells of conditions 3-1 to 3-3 in which colony formation was confirmed in Example 3-1, the degree of cell growth in a culture flask was measured.

[0092] Specifically, GYEP medium containing 30 g / L glucose was placed in a 500 mL flask, and the freeze-dried biomass of conditions 3-1 to 3-3 was inoculated therein and cultured at 28°C and 180 rpm. The culture of each condition was sampled for each culture time, and the degree of cell growth was confirmed by measuring the OD value at 680 nm using a spectrophotometer.

[0093] As a result, as shown in Table 4, the freeze-dried cells of conditions 3-1 to 3-3 showed an increase in OD value through overall cell growth from about 12 hours after culture, and showed similar growth in all condition groups. Figure 3

[0094] Example 4. Confirmation of storage stability of freeze-dried samples according to the ingredient ratio of freeze-drying protective agent

[0095] ​​After the freeze-dried biomass prepared in Example 3-1 under conditions 3-1 to 3-3 was stored in a freeze-dried vial at room temperature for 7 days, the biomass was cultured in a flask using the same method as described in Example 3-2, and the degree of growth of the cells was confirmed by measuring the absorbance at each culture time.

[0096] As a result, as shown in Figure 4 FIG. 4, it was confirmed that there was a difference in the degree of cell growth in each condition. Specifically, condition 3-3 showed the fastest growth rate, condition 3-1 showed slightly slower growth, and condition 3-2 showed cell growth only until 40 hours after culture.

[0097] Example 5. Long-term storage stability of freeze-dried biomass samples

[0098] To confirm the long-term storage stability of freeze-dried biomass samples, a freeze-drying protectant containing 4% skim milk, 8% sucrose, and 8% sodium chloride was prepared, which had the same freeze-drying protective composition as condition 3-3 of Example 3-1, and then a thraustochytrid microalga was freeze-dried in the same manner as described in Example 2. The prepared freeze-dried biomass was stored at room temperature for 12 weeks (84 days), and then suspended in distilled water and spread on a GYEP agar plate to count the number of colonies formed.

[0099] As a result, as shown in Figure 5 FIG. 5, about 47 colonies were formed in a 10 -2 fold diluted sample, and it was found that the number of viable cells per milliliter of culture was about 4.7 x 10 9 . Thus, it was confirmed that the freeze-dried biomass produced using the freeze-drying protectant according to the present disclosure could ensure a certain number or more of viable cell numbers even if stored for at least 12 weeks.

[0100] From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application can be implemented in other specific forms without changing the technical spirit or essential characteristics of the present application. In this regard, it should be understood that the above-described embodiments are provided only for the purpose of illustration in all aspects and do not limit the scope of the present application. The scope of the present application should be interpreted as including all modifications or variations derived from the meaning and scope of the appended claims and their equivalents, not just the above detailed description.

[0101]

Claims

1. The use of a composition for cryopreservation of microalgae of the genus *Schizochytrium* sp., said composition comprising the following: Skim milk with a final concentration of 0.5% w / w to 20% w / w, sucrose with a final concentration of 1% w / w to 20% w / w, sodium chloride with a final concentration of 0.1% w / w to 10% w / w, and water as the remaining component. In the composition, the weight ratio of skim milk to sucrose is 1:0.5 to 1:10, and The weight ratio of skim milk to sodium chloride in the composition is 1:0.5 to 1:

10.

2. A method for cryopreservation of microalgae of the genus *Schizochytrium* sp., the method comprising: 1) Microalgae of the genus Schizochytrium sp. are cultured in a culture medium containing the composition, said composition comprising: skim milk at a final concentration of 0.5% w / w to 20% w / w, sucrose at a final concentration of 1% w / w to 20% w / w, sodium chloride at a final concentration of 0.1% w / w to 10% w / w, and water as the remainder, wherein the weight ratio of skim milk to sucrose in said composition is 1:0.5 to 1:10, and wherein the weight ratio of skim milk to sodium chloride in said composition is 1:0.5 to 1:10; 2) Recover the culture product from step 1); and 3) Freeze-dry the recovered culture products to prepare biomass.

3. The method according to claim 2, wherein, The freezing of the microalgae is neither performed before nor after step 3).

4. A method for preparing freeze-dried biomass of microalgae of the genus *Schizochytrium* sp., the method comprising: 1) Microalgae of the genus Schizochytrium sp. are cultured in a culture medium containing the composition, said composition comprising: skim milk at a final concentration of 0.5% w / w to 20% w / w, sucrose at a final concentration of 1% w / w to 20% w / w, sodium chloride at a final concentration of 0.1% w / w to 10% w / w, and water as the remainder, wherein the weight ratio of skim milk to sucrose in said composition is 1:0.5 to 1:10, and wherein the weight ratio of skim milk to sodium chloride in said composition is 1:0.5 to 1:10; 2) Recover the culture product from step 1); and 3) Freeze-dry the recovered culture products to prepare biomass.

5. The method according to claim 4, wherein, The freezing of the microalgae is neither performed before nor after step 3).

6. A freeze-dried biomass of a microalgae of the genus *Schizochytrium* sp., wherein the freeze-dried biomass comprises: a) A composition comprising the following: The composition comprises skim milk with a final concentration of 0.5% w / w to 20% w / w, sucrose with a final concentration of 1% w / w to 20% w / w, sodium chloride with a final concentration of 0.1% w / w to 10% w / w, and water as the remainder, wherein the weight ratio of skim milk to sucrose in the composition is 1:0.5 to 1:10, and wherein the weight ratio of skim milk to sodium chloride in the composition is 1:0.5 to 1:10; and b) Microalgae of the genus *Schizochytrium* sp. The freeze-dried biomass is produced by the method described in claim 4 for preparing freeze-dried biomass of Schizochytrium sp. microalgae.

7. The freeze-dried biomass according to claim 6, wherein, The freeze-dried biomass can be stored at 15°C to 25°C for at least 12 weeks.

8. The freeze-dried biomass according to claim 7, wherein, After being stored at 15°C to 25°C for at least 12 weeks, the freeze-dried biomass contains 1.0 × 10⁻⁶ ppm. 7 Up to 1.0×10 12 One live cell per 1 mL of freeze-dried biomass.

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