Composition for freeze-preserving microalgae belonging to family thraustochytriaceae and method for freeze-preserving of microalgae belonging to thraustochytriaceae using same

Abstract

The present application relates to a composition for freeze-preserving microalgae belonging to the family Thraustochytriaceae and a method for freeze-preserving microalgae belonging to the family Thraustochytriaceae using same. By the composition for freeze preserving microalgae belonging to the family Thraustochytriaceae according to an aspect and the method for freeze-preserving microalgae belonging to the family Thraustochytriaceae using same, the microalgae may be stored stably for a long period of time, and the costs for preservation of the microalgae may be reduced by shortening a process. In addition, according to a method for preparing freeze-dried biomass of microalgae belonging to the family Thraustochytriaceae using the composition, even during long-term storage at room temperature, freeze-dried biomass in the form of powder that can maintain bacterial activity and is easy for storage and transportation may be manufactured through a simple process.

Description

Composition for cryopreservation of microalgae of the family Thraustochytridae and method for cryopreservation of microalgae of the family Thraustochytridae using the same

[0001] The present application relates to a composition for cryopreservation of microalgae in the family Thraustochytridae and a method for cryopreservation of microalgae in the family Thraustochytridae using the same.

[0002] Microalgae, the lowest level of the marine food chain, are called phytoplankton. Among these, microalgae belonging to the Thraustochytriaceae family differ from typical microalgae in that they are heterotrophic, rather than photosynthetic autotrophic. They produce and contain high concentrations of omega-3 polyunsaturated fatty acids, including docosahexaenoic acid and eicosapentaenoic acid, playing a crucial role in supplying these fatty acids to the marine ecosystem.

[0003] While general microorganisms can be stored long-term through cryopreservation or freeze-drying, thraustochytrid microalgae are difficult to effectively store long-term using these conventional methods. Therefore, to date, their preservation is primarily accomplished through subculture. However, due to their heterotrophic nature, thraustochytrid microalgae require more frequent subcultures than general microorganisms, depending on the storage environment. This makes them vulnerable to contamination and incurs significant storage costs. Therefore, the development of new methods for long-term storage of thraustochytrid microalgae is urgently needed.

[0004] [Prior Art Literature]

[0005] [Patent Document]

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

[0007] The purpose of the present application is to provide a composition for cryopreservation of microalgae of the Thraustochytriaceae family, comprising skim milk; sucrose; and sodium chloride.

[0008] Another object of the present application is to provide a method for freezing microalgae of the Thraustochytrid family using the composition for freezing microalgae of the Thraustochytrid family or a method for producing freeze-dried biomass of microalgae of the Thraustochytrid family.

[0009] Another object of the present application is to provide a freeze-dried biomass of microalgae of the Thraustochytrid family produced by a method for producing a freeze-dried biomass of the microalgae of the Thraustochytrid family.

[0010] Another object of the present application is to provide a use of a composition for cryopreservation of microalgae in the family Thraustochytrid, comprising the above-described skim milk; sucrose; and sodium chloride, for cryopreservation of microalgae in the family Thraustochytrid.

[0011] Another object of the present application is to provide a use of the composition for cryopreservation of microalgae in the family Thraustochytrid, comprising the skim milk; sucrose; and sodium chloride, for producing freeze-dried biomass of microalgae in the family Thraustochytrid.

[0012] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions set forth below. Furthermore, those skilled in the art will recognize or be able to ascertain, through routine experimentation alone, numerous equivalents to the specific embodiments of this application described in this application. Furthermore, such equivalents are intended to be encompassed by this application.

[0013]

[0014] One aspect provides a composition for cryopreservation of microalgae of the Thraustochytriaceae family, comprising skim milk; sucrose; and sodium chloride.

[0015] As used herein, the term "skim milk" refers to milk from which fat has been removed and may be used interchangeably with the terms "skim milk" or "skim milk".

[0016] As used herein, the term "Thraustochytriaceae" refers to microalgae belonging to the order Thraustochytriales, for example, the microalgae include the genera Thraustochytrium sp., Schizochytrium sp., Aurantiochytrium sp., Thraustochytriidaes p., Japonochytrium sp., Monorhizochytrium sp., Sicyoidochytrium sp., Ulkenia sp., Parietichytrium sp., Botryochytrium sp., Hondaeasp., and It may be at least one selected from the group consisting of, but is not limited to, Labyrinthulochytrium sp.

[0017] The composition may comprise skim milk in an amount of 0.5 to 20 wt% based on the total weight of the composition. For example, the composition may comprise skim milk in an amount of 0.5 to 15 wt%, 0.5 to 10 wt%, 0.5 to 8 wt%, 1 to 20 wt%, 1 to 15 wt%, 1 to 10 wt%, 1 to 8 wt%, 2 to 20 wt%, 2 to 15 wt%, 2 to 10 wt%, 2 to 8 wt%, 3 to 20 wt%, 3 to 15 wt%, 3 to 10 wt%, or 3 to 8 wt% based on the total weight of the composition.

[0018] The composition may comprise 1 to 20 wt% of sucrose based on the total weight of the composition. For example, the composition may comprise 1 to 15 wt%, 1 to 12 wt%, 2 to 20 wt%, 2 to 15 wt%, 2 to 12 wt%, 4 to 20 wt%, 4 to 15 wt%, 4 to 12 wt%, 6 to 20 wt%, 6 to 15 wt%, 6 to 12 wt%, or 6 to 9 wt% of sucrose based on the total weight of the composition.

[0019] The composition may contain 0.1 to 10 wt% of sodium chloride based on the total weight of the composition. For example, the composition may contain 0.1 to 9 wt%, 0.1 to 8.5 wt%, 0.5 to 10 wt%, 0.5 to 9 wt%, 0.5 to 8.5 wt%, 1 to 10 wt%, 1 to 9 wt%, 1 to 8.5 wt%, 2 to 10 wt%, 2 to 9 wt%, 2 to 8.5 wt%, 3 to 10 wt%, 3 to 9 wt%, 3 to 8.5 wt%, 5 to 8.5 wt%, or 6 to 8.5 wt% of sodium chloride based on the total weight of the composition.

[0020] The composition may comprise skim milk and sucrose in a weight ratio of 1:0.5 to 1:10. For example, the composition may 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.

[0021] The composition may comprise skim milk and sodium chloride in a weight ratio of 1:0.5 to 1:10. For example, the composition may 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.

[0022] As used herein, the term "cryopreservation" refers to preserving a liquid substance by freezing it into a solid state, and may include "freeze-dried preservation." For example, the composition may be a composition for freeze-dried preservation of microalgae in the family Thraustochytridae. The term "freeze-drying" refers to a drying method in which a liquid sample is frozen and left under reduced pressure to remove moisture in the sample by sublimation. It can be used for long-term preservation of microorganisms, but an appropriate cryoprotectant must be used to protect cells from damage during the freeze-drying process.

[0023] The composition for cryopreservation of microalgae of the family Thraustochytridae, including the above-mentioned skim milk; sucrose; and sodium chloride, may be used in combination with other cryoprotectants. For example, sodium chloride, dimethylsulfoxide (DMSO), dextran, sucrose, glycerol, mannitol, sorbitol, fructose, raffinose, serum albumin, and the like may be used in combination according to the purpose, but is not limited thereto.

[0024]

[0025] Another aspect provides a method for cryopreservation of microalgae in the family Thraustochytrid, comprising the steps of: 1) culturing microalgae in the family Thraustochytrid in a medium containing a composition for cryopreservation of microalgae in the family Thraustochytrid, comprising skim milk; sucrose; and sodium chloride; 2) recovering the culture in step 1); and 3) freeze-drying the culture to produce biomass.

[0026] The composition for cryopreservation of the above-mentioned Thraustochytrid microalgae is as described above.

[0027] As used herein, the term "cultivation" refers to growing the microalgae under appropriately controlled environmental conditions. The cultivation process of the present application can be performed using appropriate media and cultivation conditions known in the art. This cultivation process can be easily adjusted and used by those skilled in the art depending on the selected microalgae.

[0028] Specifically, the cultivation of the Thraustochytrid microalgae of the present application may be performed under heterotrophic conditions, but is not limited thereto.

[0029] The term "heterotrophy" as used herein refers to a nutritional method that relies on organic matter obtained from outside the body as an energy source or nutrient source, and is a term corresponding to autotrophy, and may be used interchangeably with the term "dark culture."

[0030] The step of culturing the above-mentioned Thraustochytrid microalgae is not particularly limited thereto, but may be performed using a known batch culture method, continuous culture method, fed-batch culture method, etc. The medium used for culturing the microalgae of the present application may be any culture medium that allows the Thraustochytrid microalgae to grow without limitation. Specifically, the microalgae of the present application may be used in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0031] The carbon source included in the medium used in the step of culturing the above-mentioned microalgae of the Thraustochytrid family may be at least one selected from the group consisting of glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose, and glycerol, but is not limited thereto as long as it is a carbon source used in culturing the above-mentioned microalgae.

[0032] The nitrogen source included in the medium used in the step of culturing the above-described microalgae of the Thraustochytrid family may 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 MSG (Monosodium glutamate), but is not limited thereto as long as it is a nitrogen source used in culturing the above-described microalgae.

[0033] The medium used in the step of culturing the above-mentioned thraustochytrid microalgae may individually or mixedly contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts as phosphorus sources, but is not limited thereto.

[0034] The cultivation conditions of the step of culturing the above-mentioned Thraustochytrid microalgae can be applied without limitation as long as they are conditions that can grow the Thraustochytrid microalgae. For example, the cultivation may be performed under aerobic conditions while controlling temperature, pH, etc.

[0035] Specifically, the appropriate pH of the culture (e.g., pH 5 to 9, specifically pH 6 to 8) can be adjusted using, but is not limited to, a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid).

[0036] In addition, in order to maintain an aerobic state of the culture, oxygen or an oxygen-containing gas may be injected into the culture, or in order to maintain an anaerobic or microaerobic state, no gas may be injected or nitrogen, hydrogen or carbon dioxide gas may be injected, but this is not limited thereto.

[0037] Additionally, the culture temperature may be maintained at 20 to 45°C, or 25 to 40°C, and the culture may be performed for about 10 to 160 hours, about 10 to 120 hours, about 10 to 80 hours, about 10 to 50 hours, or about 10 to 40 hours, but is not limited thereto. Additionally, during the culture, a defoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation, but is not limited thereto.

[0038] The step of recovering the culture may involve collecting the desired culture using a suitable method known in the art. Examples of methods that may be used include, but are not limited to, centrifugation, filtration, and anion exchange chromatography.

[0039] The term "biomass" as used herein refers to living organisms such as plants, animals, and microorganisms that can be used as chemical energy, i.e., the energy source of bioenergy, and ecologically, it also refers to the weight or energy amount of a specific organism existing within a unit of time and space. In addition, the biomass includes, but is not limited to, compounds secreted by cells, and may contain cells and / or intracellular contents as well as extracellular substances. In the present application, the biomass may be, but is not limited to, microalgae of the family Thraustochytridae themselves, a culture thereof, a product produced by culturing or fermenting the microalgae, or a concentrate of the biomass.

[0040] The "culture" of the above-mentioned microalgae of the Thraustochytrid family refers to a product produced by culturing the microalgae, and may specifically be a culture solution containing the microalgae or a culture filtrate from which the microalgae have been removed, but is not limited thereto. The culture of the above-mentioned microalgae of the Thraustochytrid family can be produced by inoculating the microalgae into a microalgae culture medium and using a culture method known in the art.

[0041] The step of producing biomass by freeze-drying the above-mentioned culture may be performed according to a freeze-drying method known in the art. For example, the microalgae culture may be recovered, placed in a freeze-drying vial (FD vial), connected to a freeze-drying device while maintaining a vacuum, and then moisture may be removed while maintaining constant temperature and pressure conditions. The constant temperature conditions may be, for example, a temperature of -50°C or lower, and the constant pressure conditions may be, but are not limited to, a pressure of 0.133 mbar or lower.

[0042] The above method for freezing microalgae of the Thraustochytrid family may not further include a step of freezing the microalgae before or after step 3). For example, a pre-freezing process may not be included before step 3). The method can preserve microalgae in a simpler and more cost-effective manner by producing freeze-dried biomass without a pre-freezing process.

[0043]

[0044] Another aspect provides a method for producing a freeze-dried biomass of a Thraustochytrid microalgae, comprising the steps of: 1) culturing a Thraustochytrid microalgae in a medium containing a composition for cryopreservation of a Thraustochytrid microalgae, comprising skim milk; sucrose; and sodium chloride; 2) recovering the culture of step 1); and 3) producing a biomass by freeze-drying the culture.

[0045] The composition for freezing and preserving the above-mentioned Thraustochytrid microalgae, the step of culturing the Thraustochytrid microalgae, the step of recovering the culture, and the step of producing freeze-dried biomass are as described above.

[0046] The method for producing freeze-dried biomass of the above-described Thraustochytrid microalgae may not further include a step of freezing the microalgae before and after step 3). For example, the method may not include a pre-freezing process before step 3). By producing freeze-dried biomass without including a pre-freezing process, the method can produce freeze-dried biomass in a simpler manner at reduced costs.

[0047]

[0048] Another aspect provides a) a composition for cryopreservation of microalgae of the family Thraustochytriaceae, comprising skim milk; sucrose; and sodium chloride; and b) a freeze-dried biomass of microalgae of the family Thraustochytriaceae, comprising microalgae of the family Thraustochytriaceae, and produced by a method for producing a freeze-dried biomass of the microalgae of the family Thraustochytriaceae.

[0049] The composition for freezing and preserving the above-mentioned Thraustochytrid microalgae and the method for producing freeze-dried biomass of the above-mentioned Thraustochytrid microalgae are as described above.

[0050] The freeze-dried biomass may be stored at 15 to 25°C for 12 weeks or more. For example, the freeze-dried biomass may 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, or 3 months to 6 months.

[0051] The term "room temperature" as used herein may mean a temperature of about 15 to 25°C, and may be used interchangeably with the term "room temperature".

[0052] As used herein, the term "storable" may mean that the lyophilized biomass is capable of growing when cultured in a medium again after storage, or may mean that the lyophilized biomass maintains 60% or more, 70% or more, 80% or more, or 90% or more of its activity compared to live, non-lyophilized bacteria.

[0053] The above freeze-dried biomass was stored at 15 to 25°C for more than 12 weeks and then 1.0 X 10 per 1 mL of biomass 7 Dog or 1.0 X 10 12 It may contain live bacteria. For example, the freeze-dried biomass may contain 1.0 X 10 per mL of biomass after storage at 15 to 25°C for 12 weeks or more. 7 Dog or 1.0 X 10 11 Dog, 1.0 X 10 7 Dog or 1.0 X 10 10 Dog, 1.0 X 10 8 Dog or 1.0 X 10 12 Dog, 1.0 X 10 8 Dog or 1.0 X 10 11 Dog, 1.0 X 10 8 Dog or 1.0 X 10 10 Dog, 1.0 X 10 9 Dog or 1.0 X 10 12 Dog, 1.0 X 10 9 Dog or 1.0 X 10 11 dog, or 1.0 X 10 9 Dog or 1.0 X 10 10 It may contain live bacteria.

[0054] The above freeze-dried biomass was stored at 15 to 25°C for more than 12 weeks and then 1.0 X 10 7 1.0 X 10 12 It may contain viable cells of CFU (colony forming unit) / mL. For example, the freeze-dried biomass may contain viable cells of 1.0 X 10 after storage at 15 to 25°C for more than 12 weeks.7 1.0 X 10 11 , 1.0 X 10 7 1.0 X 10 10 , 1.0 X 10 8 1.0 X 10 12 , 1.0 X 10 8 1.0 X 10 11 , 1.0 X 10 8 1.0 X 10 10 , 1.0 X 10 9 1.0 X 10 12 , 1.0 X 10 9 1.0 X 10 11 , or 1.0 X 10 9 1.0 X 10 10 It may contain live bacteria of CFU / mL.

[0055]

[0056] Another aspect provides a use of the composition for cryopreservation of microalgae in the family Thraustochytrid, comprising skim milk; sucrose; and sodium chloride.

[0057] Another aspect provides a use of the composition for cryopreservation of microalgae in the family Thraustochytrid, comprising the above skim milk; sucrose; and sodium chloride, for producing freeze-dried biomass of microalgae in the family Thraustochytrid.

[0058] The composition for freezing and preserving the above-mentioned Thraustochytrid microalgae, the method for freezing and preserving the above-mentioned Thraustochytrid microalgae, and the method for producing freeze-dried biomass of the above-mentioned Thraustochytrid microalgae are as described above.

[0059] According to a composition for cryopreservation of microalgae in the family Thraustochytrid and a cryopreservation method using the same, the microalgae can be stably stored for a long period of time, and the process can be shortened, thereby reducing the cost of preserving the microalgae. In addition, according to a method for producing freeze-dried biomass of microalgae in the family Thraustochytrid using the composition, the cell activity is maintained even when stored at room temperature for a long period of time, and a freeze-dried biomass in powder form that is easy to store and transport can be produced through a simple process.

[0060] FIG. 1 shows freeze-dried vials (A to D) manufactured according to one aspect, and photographs (E to H) showing whether colonies were formed after the contents of the vials were inoculated onto an agar plate (AH: A is a photograph showing a freeze-dried vial manufactured according to condition 1-1, E is a photograph showing whether colonies were formed after the contents of vial A were inoculated onto an agar plate, B is a photograph showing a freeze-dried vial manufactured according to condition 1-2, F is a photograph showing whether colonies were formed after the contents of vial B were inoculated onto an agar plate, C is a photograph showing a freeze-dried vial manufactured according to condition 1-3, G is a photograph showing whether colonies were formed after the contents of vial C were inoculated onto an agar plate, D is a photograph showing a freeze-dried vial manufactured according to condition 1-4, and H is a photograph showing whether colonies were formed after the contents of vial D were inoculated onto an agar plate.).

[0061] Figure 2 is a photograph showing whether colonies were formed after inoculating freeze-dried biomass according to a daily pattern onto an agar plate (A-E: A is a photograph showing whether colonies were formed after inoculating freeze-dried biomass according to condition 3-1 onto an agar plate, B is a photograph showing whether colonies were formed after inoculating freeze-dried biomass according to condition 3-2 onto an agar plate, C is a photograph showing whether colonies were formed after inoculating freeze-dried biomass according to condition 3-3 onto an agar plate, D is a photograph showing whether colonies were formed after inoculating freeze-dried biomass according to condition 3-4 onto an agar plate, and E is a photograph showing whether colonies were formed after inoculating freeze-dried biomass according to condition 3-5 onto an agar plate.).

[0062] Figure 3 is a growth curve graph showing the absorbance measured according to the culture time after inoculating freeze-dried biomass according to the daily aspect into a culture flask.

[0063] Figure 4 is a growth curve graph showing the absorbance measured at each culture time after freeze-dried biomass was stored at room temperature for 7 days according to the daily pattern and then inoculated into a culture flask.

[0064] Figure 5 is a photograph showing the formation of colonies by inoculating freeze-dried biomass on an agar plate after storing it at room temperature for 12 weeks according to the daily pattern.

[0065] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify one or more specific examples, and the scope of the present invention is not limited to these examples.

[0066]

[0067] Example 1. Confirmation of cell viability according to freeze-drying conditions of microalgae.

[0068] Example 1-1. Production of freeze-dried preservative

[0069] A total of 4 mL of lyophilized preservative was prepared by mixing 0.8 mL of 4% skim milk solution, 1.6 mL of 20% sucrose solution, and 1.6 mL of 20% sodium chloride solution, each dissolved in distilled water.

[0070]

[0071] Example 1-2. Freeze-drying of Thraustochytrid microalgae

[0072] The microalgae of the genus Schizochytrium, CD01-5004 (accession number: KCTC14345BP), was inoculated into GYEP medium (glucose 10 g / L, yeast extract 1 g / L, peptone 1 g / L, MgSO4·7H2O 2 g / L, sea salt 20 g / L, H3BO3 5.0 mg / L, MnCl2 3.0 mg / L, CuSO4 0.2 mg / L, NaMo4·2H2O 0.05 mg / L, CoSO4 0.05 mg / L, and ZnSO4·7H2O 0.7 mg / L), which is a medium in which microalgae of the family Thraustochytrid can grow, and cultured overnight in a 500 mL flask at 28°C and 180 rpm. The culture was recovered, centrifuged, and the supernatant was removed, and then suspended in the freeze-dried preservative prepared in Example 1-1 to prepare a microalgae biomass suspension. The prepared biomass suspension was placed in an ampoule-type freeze-drying vial (FD vial), and conditions 1-1 to 1-3 were pre-frozen under the conditions shown in Table 1 below, while condition 1-4 was prepared as a suspension that was not pre-frozen.

[0073] Experimental group Pre-freezing conditions Condition 1-1 General freezing using a freezing mixture of 99% ethanol and dry ice Pre-freezing of biomass suspension. Condition 1-2 Slow freezing Pre-freezing of biomass suspension by leaving it at 4℃ for 60 minutes, then at -20℃ for 60 minutes, and then at -80℃ for 60 minutes under gentle conditions. Condition 1-3 Slow freezing + drying at room temperature Pre-freezing under the same conditions as Condition 1-2 above, then drying at room temperature (25℃). Condition 1-4 No pre-freezing -

[0074]

[0075] The freeze-drying vials of each condition above were connected to a freeze-drying device and freeze-dried under the conditions of maintaining a temperature below -50℃ and a pressure below 0.133 mbar in a vacuum state. Since connecting a large number of ampoules to the freeze-drying device may increase the pressure of the device, which may affect the growth of freeze-dried cells, one ampoule was connected to the device, and then the next ampoule was connected again after the device pressure dropped below 0.133 mbar. After all ampoules were connected to the device, freeze-drying was performed for approximately 3 hours, and the freeze-dried ampoules were sealed with a gas torch so that a vacuum state could be maintained.

[0076]

[0077] Example 1-3. Confirmation of the viability of freeze-dried cells

[0078] In order to confirm the viability of the freeze-dried cells produced in Example 1-2, a powdered dried biomass sample was suspended in distilled water and spread on a GYEP agar plate, and colony growth was visually confirmed.

[0079] As a result, as shown in Fig. 1, it was confirmed that the freeze-dried biomass of conditions 1-1 to 1-3 that had undergone the pre-freezing process could not grow and form colonies on an agar plate, but only the freeze-dried biomass of condition 1-4 that had not undergone the pre-freezing process could grow and form colonies on an agar plate.

[0080]

[0081] Example 2. Confirmation of the viability of freeze-dried cells according to the components of the freeze-dried preservative.

[0082] In order to evaluate the freeze-drying preservation effect according to the components of the freeze-drying preservative, 4 mL of the freeze-drying preservative containing the corresponding components at the indicated final concentrations for each condition group was prepared as shown in Table 2 below.

[0083] Experimental groupSkim milk(%)Sucrose(%)Trehalose(%)Methanol(%)Peptone(%)Glycerol(%)Sodium chloride(%)Conditional group 2-11010-----Conditional group 2-210-10----Conditional group 2-310--10---Conditional group 2-4--10-5--Conditional group 2-51044----Conditional group 2-668----4Conditional group 2-710----10-

[0084]

[0085] Microalgae CD01-5004 (accession number: KCTC14345BP) of the genus Schizochytrium was inoculated into GYEP medium containing 30 g / L of glucose, and a biomass suspension was prepared in the same manner as described in Example 1-2, except that a freeze-dried preservative prepared with the composition of Table 2 was used, and the suspension was freeze-dried as is without a pre-freezing process.

[0086] Powdered dried biomass samples were suspended in distilled water and diluted to an optical density (OD) of 0.1 at 680 nm. 1 mL of each was spread on GYEP agar plates, and the number of colonies formed per plate was counted. A portion of the cell culture collected before preparing the biomass suspension was not suspended in a freeze-dried preservative, and 1 mL of the diluted solution to an OD of 0.1 at 680 nm was spread on GYEP agar plates, and the number of colonies formed was counted and compared with the control group. 1 x 10 per colony 6 Since there are 10 cells, the number of colonies formed for each experimental group is 10 6 The number of viable cells per 1 mL of culture was calculated by multiplying the number of viable cells per mL of culture, and the survival rate of each condition was calculated as a percentage of the control group.

[0087] Experimental group Formed colony aquatic bacteria survival rate Control group 1500 1.5 X 10 9 100% conditional 2-1202.0 X 10 7 1.33% conditional 2-2000% conditional 2-3000% conditional 2-4000% conditional 2-522.0 X 10 6 0.13% conditional 2-62502.5 X 10 8 16.67% conditional 2-7000%

[0088]

[0089] As a result, as shown in Table 3, it was confirmed that colonies were formed within 72 hours after plating on the plate in condition groups 2-1, 2-5, and 2-6. The freeze-dried preservative used in these condition groups commonly contained 6 to 10% skim milk and 4 to 10% sucrose, and it was confirmed that the survival rate was significantly high at 16.67% in condition group 2-6, which used a freeze-dried preservative containing skim milk, sucrose, and sodium chloride.

[0090]

[0091] Example 3. Confirmation of the growth degree of freeze-dried cells according to the ingredient ratio of the freeze-dried preservative.

[0092] Example 3-1. Confirmation of colony formation on agar plates

[0093] In order to evaluate the freeze-drying preservation effect according to the concentration ratio of skim milk, sucrose, and sodium chloride, which are the most effective preservative ingredients derived from Example 2, 4 mL of freeze-dried preservatives containing the corresponding ingredients at the indicated final concentrations for each condition group were prepared as shown in Table 4 below. Using the freeze-dried preservative for each condition group, microalgae in the family Thraustochytridae were freeze-dried in the same manner as described in Example 2, and then suspended in distilled water and plated on GYEP agar plates to count the number of colonies formed. In the conditions where colonies were formed, colony formation was confirmed visually from 24 hours after plating, and the presence or absence of cell viability and the degree of colony formation could be clearly distinguished from 40 hours.

[0094] Experimental groupSkim milk(%)Sucrose(%)Sodium chloride(%)Colony numberCondition group 3-168430Condition group 3-2-10532Condition group 3-348834Condition group 3-410--0Condition group 3-55-50

[0095]

[0096] As a result, as shown in Fig. 2 and Table 4, no colonies were observed in condition groups 3-4 using a preservative containing only 10% skim milk and condition groups 3-5 using a preservative containing only 5% skim milk and 5% sodium chloride, and it was confirmed that more than 30 colonies were formed in the remaining condition groups.

[0097]

[0098] Example 3-2. Confirmation of cell growth in culture flasks

[0099] The degree of cell growth in the culture flask was measured for the freeze-dried cells of conditions 3-1 to 3-3 in which colony formation was confirmed in Example 3-1.

[0100] Specifically, GYEP medium containing 30 g / L of 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 under conditions of 28°C and 180 rpm. The culture of each condition was taken at each culture time, and the OD value was measured at 680 nm using a spectrophotometer to confirm the degree of cell growth.

[0101] As a result, as shown in Fig. 3, the freeze-dried cells of condition groups 3-1 to 3-3 showed an increase in OD value through full-scale cell growth from about 12 hours after culture, and all condition groups showed similar growth curves.

[0102]

[0103] Example 4. Confirmation of storage stability of freeze-dried samples according to the ingredient ratio of freeze-dried preservative.

[0104] The freeze-dried biomass of conditions 3-1 to 3-3 prepared in Example 3-1 was stored in a freeze-drying vial at room temperature for 7 days, and then 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 according to the culture time.

[0105] As a result, as shown in Fig. 4, it was confirmed that there was a difference in the degree of cell growth by condition group, and specifically, condition group 3-3 showed the fastest growth rate, and growth was observed in condition group 3-1, albeit somewhat slowly, whereas cell growth was not observed in condition group 3-2 for up to 40 hours after culture.

[0106]

[0107] Example 5. Long-term storage stability of freeze-dried biomass samples

[0108] In order to confirm the long-term storage stability of freeze-dried biomass samples, a freeze-dried preservative containing 4% skim milk, 8% sucrose, and 8% sodium chloride, which had the same composition as Condition 3-3 of Example 3-1, was prepared, and then microalgae from the Thraustochytrid family were freeze-dried using the same method 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 plated on GYEP agar plates to count the number of colonies formed.

[0109] As a result, as shown in Fig. 5, 10 -2 Approximately 47 colonies were formed in the diluted sample, from which the viable cell count per mL of culture was approximately 4.7 X 10 9 It was confirmed that the freeze-dried biomass produced using the freeze-dried preservative according to the present invention maintained a certain number of viable cells even when preserved for at least 12 weeks.

[0110]

[0111] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering the technical concept or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

[0112]

Claims

1. A composition for cryopreservation of microalgae of the Thraustochytriaceae family, comprising skim milk; sucrose; and sodium chloride.

2. A composition according to claim 1, wherein the composition comprises skim milk in an amount of 0.5 to 20 wt% based on the total weight of the composition.

3. A composition according to claim 1, wherein the composition contains sucrose in an amount of 1 to 20 wt% based on the total weight of the composition.

4. A composition according to claim 1, wherein the composition contains 0.1 to 10 wt% of sodium chloride based on the total weight of the composition.

5. A composition according to claim 1, wherein the composition comprises skim milk and sucrose in a weight ratio of 1:0.5 to 1:

10.

6. A composition according to claim 1, wherein the composition comprises skim milk and sodium chloride in a weight ratio of 1:0.5 to 1:

10.

7. In claim 1, the Thraustochytridaceae microalgae is at least one microalga 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., Hondaeasp., and Labyrinthulochytrium sp. Composition. 8.1) A step of culturing microalgae of the Thraustochytriaceae family in a medium containing a composition for cryopreservation of microalgae of the Thraustochytriaceae family of claim 1; 2) a step of recovering the culture of step 1); and 3) A method for cryopreserving microalgae of the Thraustochytrid family, comprising the step of freeze-drying the culture to produce biomass.

9. A method for freezing and preserving microalgae of the Thraustochytrid family, wherein the method according to claim 8 does not further include a step of freezing the microalgae before and after step 3). 10.1) A step of culturing microalgae of the Thraustochytriaceae family in a medium containing a composition for cryopreservation of microalgae of the Thraustochytriaceae family of claim 1; 2) a step of recovering the culture of step 1); and 3) A method for producing a freeze-dried biomass of microalgae of the Thraustochytrid family, comprising the step of producing a biomass by freeze-drying the culture.

11. A method for producing freeze-dried biomass of microalgae of the Thraustochytrid family, wherein the method according to claim 10 does not further include a step of freezing the microalgae before and after step 3). 12.a) A composition for cryopreservation of microalgae of the Thraustochytriaceae family, comprising skim milk; sucrose; and sodium chloride; and b) comprising microalgae of the Thraustochytriaceae family, and A freeze-dried biomass of microalgae of the Thraustochytrid family, produced by the method of producing a freeze-dried biomass of microalgae of the Thraustochytrid family according to claim 10.

13. In claim 12, the freeze-dried biomass of microalgae of the Thraustochytrid family can be stored at 15 to 25°C for 12 weeks or longer.

14. In claim 13, the freeze-dried biomass has a viscosity of 1.0 X 10 per mL of biomass after storage at 15 to 25°C for 12 weeks or longer. 7 Dog 1.0 X 10 12 Freeze-dried biomass of microalgae of the family Thraustochytridae, comprising live bacteria.