NOVEL SCHIZOCHYTRIUM STRAIN AND METHOD FOR PRODUCING POLYUNSATURATED FATTY ACIDS USING THE SAME

Abstract

The present invention relates to a novel Schizochytrium sp. strain and a method for producing polyunsaturated fatty acids using the same. In one embodiment, the novel Schizochytrium microalgae has a high fat content in the biomass, particularly a high content of polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The microalgae, biomass, or bio-oil produced therefrom can be usefully utilized as feed ingredients, etc.

Description

[Technical field]

[0001] The present invention relates to a novel Schizochytrium sp. strain and a method for producing polyunsaturated fatty acids using the same. [Background technology]

[0002] Unsaturated fatty acids are fatty acids that have one or more double bonds in the fatty acid chain, and when they contain two or more double bonds, they are called polyunsaturated fatty acids (PUFAs). Among them, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are representative omega-3 fatty acids and are essential fatty acids for the brain, eye tissue, and nervous system. They are also known to play important roles in nervous system development, such as infant vision and motor skills, and in preventing cardiovascular disease, and are the most abundant components of structural lipids in the brain.

[0003] Most higher organisms, including humans, cannot synthesize polyunsaturated fatty acids on their own, so they must ingest them as essential nutrients, and polyunsaturated fatty acids are mainly supplied from deep-sea fish such as tuna and salmon, which are dominant in the marine ecological environment. The main industrial source of polyunsaturated fatty acids identified to date is fish oil extracted from blue-backed fish such as mackerel, saury, tuna, horse mackerel, sardine, and herring, which is also very useful for fish farming feed such as the initial feed for marine fish. However, the quality of fish oil varies depending on the fish species, season, and fishing location, and there are difficulties in continuously supplying fish oil. In addition to the problems of contamination of fish oil with heavy metals and organic chemicals, the fishy odor peculiar to fish oil, the oxidation of double bonds during the processing process, and the limited production volume, there is an increasing need to find alternative sources of polyunsaturated fatty acids.

[0004] In order to solve such problems, research has been conducted recently on a method for producing polyunsaturated fatty acids including docosahexaenoic acid by microbial culture. In particular, microalgae can produce lipids, are easy to culture, and enable the production of biomass with a relatively constant biochemical composition. In addition, lipids produced by microalgae do not have an unpleasant odor like fish oil, have a simpler fatty acid composition than fish oil, and are convenient for separating specific fatty acids. Therefore, new microalgae that can stably supply specific fatty acids at high contents are of great industrial value, and the development of such microalgae is necessary. Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present application is to provide a microalga of the genus Schizochytrium, which is deposited under accession number KCTC14344BP or accession number KCTC14345BP and has the ability to produce docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and palmitic acid (PA). Another object of the present application is to provide a biomass derived from the Schizochytrium microalgae, and a feed composition containing the same. Yet another object of the present application is to provide a method for producing biomass or bio-oil derived from the Schizochytrium microalgae. [Means for solving the problem]

[0006] Each description and embodiment disclosed in this application also applies to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application belong to the scope of this application. In addition, the specific descriptions described below cannot be used to limit the scope of this application. In addition, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific aspects of this application described herein. In addition, such equivalents are intended to be included in this application.

[0007] One embodiment provides a microalga of the genus Schizochytrium, deposited under accession number KCTC14344BP or accession number KCTC14345BP, and capable of producing docosahexaenoic acid, eicosapentaenoic acid, and palmitic acid.

[0008] The term "Thraustochytrid" as used herein means microalgae of the order Thraustochytriales. The term "Schizochytrium sp." as used herein means one of the genus names belonging to the family Thraustochytriaceae of the order Thraustochytriales, and is also used interchangeably with the term "genus Schizochytrium." The term "microalgae" refers to plants that perform photosynthesis using chlorophyll, cannot be seen with the naked eye, can only be seen through a microscope, and live freely floating in water, and is also called phytoplankton.

[0009] As used herein, the term "docosahexaenoic acid (DHA)" refers to 22 H 32It is one of the polyunsaturated fatty acids with the chemical formula O2, and along with α-linolenic acid (ALA) and eicosapentaenoic acid (EPA), it belongs to the omega-3 fatty acids. Its common name is cervonic acid, and it is also abbreviated as 22:6n-3.

[0010] As used herein, the term "eicosapentaenoic acid (EPA)" refers to C 20 H 30 It is one of the polyunsaturated fatty acids with the chemical formula O2, and along with ALA and DHA, it belongs to the omega-3 fatty acids and can also be abbreviated as 20:5n-3. As used herein, the term "palmitic acid (PA)" refers to C 16 H 32 It refers to one of the saturated fatty acids that have the chemical formula O2.

[0011] The Schizochytrium microalgae is a novel wild-type Schizochytrium microalgae CD01-5000 deposited under the accession number KCTC14344BP or a mutant thereof, and is also Schizochytrium microalgae CD01-5004 deposited under the accession number KCTC14345BP.

[0012] The Schizochytrium microalgae may have an 18S rRNA base sequence of SEQ ID NO: 1, but is not limited thereto. For example, the Schizochytrium microalgae may have an 18S rRNA having a base sequence that shows 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more sequence identity to the base sequence of SEQ ID NO: 1, but is not limited thereto.

[0013] The Schizochytrium microalgae may produce 35 to 60% by weight of DHA based on the total weight of fatty acids. For example, the Schizochytrium microalgae may produce 40 to 60% by weight, 45 to 60% by weight, 50 to 60% by weight, 35 to 58% by weight, 40 to 58% by weight, 45 to 58% by weight, or 50 to 58% by weight of DHA based on the total weight of fatty acids. The Schizochytrium microalgae may also contain 35 to 60% by weight of DHA based on the total weight of fatty acids.

[0014] The microalgae of the genus Schizochytrium may produce 0.5 to 10% by weight of EPA based on the total weight of fatty acids. For example, the microalgae of the genus Schizochytrium may produce 0.5 to 8% by weight, 0.5 to 5% by weight, 0.5 to 3% by weight, 0.8 to 10% by weight, 0.8 to 8% by weight, 0.8 to 5% by weight, 0.8 to 3% by weight, 1 to 10% by weight, 1 to 8% by weight, 1 to 5% by weight, or 1 to 3% by weight of EPA based on the total weight of fatty acids. The microalgae of the genus Schizochytrium may also contain 0.5 to 10% by weight of EPA based on the total weight of fatty acids.

[0015] The Schizochytrium microalgae may produce 10 to 30% by weight of PA based on the total weight of fatty acids. For example, the Schizochytrium microalgae may produce 15 to 30% by weight, or 20 to 30% by weight of PA based on the total weight of fatty acids. The Schizochytrium microalgae may also contain 10 to 30% by weight of PA based on the total weight of fatty acids.

[0016] The Schizochytrium microalgae also have a carotenoid producing ability. For example, the Schizochytrium microalgae produce one or more selected from the group consisting of β-carotene, lutein, astaxanthin, capsanthin, annatto, canthaxanthin, lycopene, β-apo-8-carotenal, zeaxanthin, and β-apo-8-carotenal-ester.

[0017] The term "carotenoid" as used herein means a terpenoid pigment synthesized from phytoene, which is composed of 40 carbon elements, and is produced in microalgae, bacteria, fungi, and higher plants. Carotenoids are often used as feed and food additives due to their unique color, and because they have a long double bond chain and a ketone group (C=O) or a hydroxyl group (-OH), they have a strong antioxidant effect and are also being studied as medicines and health foods. Carotenoids include β-carotene, lutein, astaxanthin, capsanthin, annatto, canthaxanthin, lycopene, β-apo-8-carotenal, zeaxanthin, and β-apo-8-carotenal-ester.

[0018] Another aspect provides a biomass derived from Schizochytrium microalgae, including Schizochytrium microalgae deposited under Accession No. KCTC14344BP or Accession No. KCTC14345BP and capable of producing docosahexaenoic acid, eicosapentaenoic acid, and palmitic acid, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product. The Schizochytrium microalgae are as described above.

[0019] The term "biomass" as used herein means an organism such as a plant, animal, or microorganism that can be used as chemical energy, i.e., an energy source of bioenergy, and also means the weight or amount of energy of a specific organism present in a unit time and space ecologically. The biomass includes, but is not limited to, compounds secreted by cells, and includes not only extracellular substances but also cells and / or intracellular contents. In the present application, the biomass may be Schizochytrium microalgae itself, a culture thereof, a dried product thereof, a crushed product thereof, or a product produced by culturing or fermenting the microalgae, or a concentrate or dried product of the biomass, but is not limited thereto.

[0020] The "culture" of the Schizochytrium microalgae refers to a product produced by culturing the microalgae, specifically, a culture solution containing the microalgae, or a culture filtrate obtained by removing the microalgae from the culture solution, but is not limited thereto. The "dried product" of the Schizochytrium microalgae culture refers to a product obtained by removing water from the microalgae culture, for example, a dried cell form of the microalgae, but is not limited thereto. In addition, the "crushed product" of the dried product refers to a product obtained by crushing a dried product obtained by removing water from the microalgae culture, for example, a dried cell powder, but is not limited thereto. The Schizochytrium microalgae culture is produced by inoculating the microalgae into a microalgae culture medium and culturing the microalgae according to a culture method known in the art, and the dried product and crushed product of the culture may also be produced by a treatment method or drying method of the microalgae or culture solution known in the art.

[0021] The biomass derived from the Schizochytrium microalgae may contain 35 to 60% by weight of DHA based on the total weight of fatty acids, 0.5 to 10% by weight of EPA based on the total weight of fatty acids, or 10 to 30% by weight of PA based on the total weight of fatty acids.

[0022] The biomass derived from Schizochytrium microalgae also contains a carotenoid, for example, one or more selected from the group consisting of β-carotene, lutein, astaxanthin, capsanthin, annatto, canthaxanthin, lycopene, β-apo-8-carotenal, zeaxanthin, and β-apo-8-carotenal-ester. The biomass is also produced by a method for producing biomass derived from Schizochytrium microalgae according to one embodiment.

[0023] Another embodiment provides a composition comprising a microalga of the genus Schizochytrium deposited under Accession No. KCTC14344BP or Accession No. KCTC14345BP and capable of producing docosahexaenoic acid, eicosapentaenoic acid, and palmitic acid, a culture of the microalga, a dried product of the culture, or a crushed product of the dried product. The composition also comprises a biomass or bio-oil derived from the microalga of the genus Schizochytrium.

[0024] Another aspect provides a feed composition comprising a biomass derived from Schizochytrium microalgae deposited under accession number KCTC14344BP or accession number KCTC14345BP and capable of producing docosahexaenoic acid, eicosapentaenoic acid, and palmitic acid, or a concentrate or dried product of the biomass. The Schizochytrium microalgae, the biomass, the culture of the microalgae, the dried product of the culture, and the crushed product of the dried product are as described above. The biomass concentrate or dry matter may also be produced by any method known in the art for treating, concentrating or drying microbial biomass.

[0025] As used herein, the term "bio-oil" refers to oil obtained from biomass by biological, thermochemical and physicochemical extraction processes, and the bio-oil produced in this application may contain polyunsaturated fatty acids, specifically, but is not limited to, DHA and EPA.

[0026] The composition may be in the form of, but is not limited to, a solution, a powder or a suspension, for example a food composition, a feed composition or a feed additive composition.

[0027] The term "feed composition" as used herein refers to a food fed to an animal. The feed composition refers to a substance that provides organic or inorganic nutrients necessary for an animal to sustain life or produce meat, milk, etc. The feed composition may also contain additional nutritional components necessary for an animal to sustain life or produce meat, milk, etc. The feed composition may be prepared in various forms of feed known in the art, and specifically may include concentrated feed, roughage, and / or special feed.

[0028] The term "feed additive" as used herein includes substances added to feed for various purposes such as supplementing nutrients and preventing weight loss, increasing digestibility of fiber in feed, improving milk quality, preventing reproductive disorders and improving conception rate, and preventing high temperature stress in summer. The feed additive of the present application corresponds to supplementary feed under the Feed Management Act, and may additionally include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, mineral preparations that are trace minerals such as zinc, copper, cobalt, and selenium, vitamin preparations such as keratin, vitamin E, vitamins A, D, and E, nicotinic acid, and vitamin B complex, protected amino acid preparations such as methionine and lysine, protected fatty acid preparations such as fatty acid calcium salts, live bacteria preparations (lactic acid bacteria preparations), live bacteria such as yeast cultures and mold fermentation products, and yeast preparations.

[0029] The term "food composition" as used herein includes all forms such as functional foods, nutritional supplements, health foods, and food additives, and food compositions of the above types can be prepared in various forms by general methods known in the art.

[0030] The composition of the present application may further comprise grains, such as crushed or crushed wheat, oats, wheat, corn and rice; vegetable protein feeds, such as feeds based on beans and sunflower; animal protein feeds, such as blood meal, meat meal, bone meal and fish meal; sugars and dairy products, such as various milk powders and whey powders, as well as other dry ingredients, such as nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

[0031] The compositions of the present application can be administered to animals alone or in an edible carrier in combination with other feed additives. The compositions can also be easily administered to animals as a top dressing, or by mixing them directly with the feed or in a separate oral dosage form from the feed. When the compositions are administered separately from the feed, they can be combined with a pharma- ceutically acceptable edible carrier and made into immediate or sustained release dosage forms, as is well known in the art. Such edible carriers can be solid or liquid, such as corn starch, lactose, sucrose, pea flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the composition can be in the form of a tablet, capsule, powder, lozenge, or lozenge, or a top dressing in microdispersible form. When a liquid carrier is used, the composition can be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution.

[0032] The composition of the present application may also include, for example, a preservative, a stabilizer, a wetting or emulsifying agent, a cryoprotectant, or an excipient, etc. The cryoprotectant may be one or more selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder, and starch.

[0033] The preservative, stabilizer or excipient is contained in the composition in an effective amount sufficient to reduce deterioration of the Schizochytrium microalgae contained in the composition, and the cryoprotectant is contained in the composition in an effective amount sufficient to reduce deterioration of the Schizochytrium microalgae contained in the composition when the composition is in a dried state. The compositions may be applied by needle injection (drip), spraying or mixing into the animal feed.

[0034] The composition of the present application can be applied to many animal diets, including, but not limited to, mammals, birds, fish, crustaceans, cephalopods, reptiles, and amphibians. For example, the mammals can include pigs, cows, sheep, goats, laboratory rodents, or pets, and the birds can include domestic animals, which can include, but are not limited to, chickens, turkeys, ducks, geese, pheasants, or quails. The fish can include commercially farmed fish and their juveniles, ornamental fish, and the crustaceans can include, but are not limited to, shrimp, barnacles, and the like. The composition can also be applied to the diet of rotifers, which are zooplankton.

[0035] Another aspect provides a method for producing biomass derived from Schizochytrium microalgae, the method comprising: culturing Schizochytrium microalgae deposited under Accession No. KCTC14344BP or Accession No. KCTC14345BP and capable of producing docosahexaenoic acid, eicosapentaenoic acid, and palmitic acid; and recovering biomass from the microalgae, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product. The Schizochytrium microalgae, the biomass, the culture of the microalgae, the dried product of the culture, and the crushed product of the dried product are as described above.

[0036] The term "cultivation" as used herein means growing the microalgae under appropriately controlled environmental conditions. The culture process of the present application may be performed using appropriate media and culture conditions known in the art. Such a culture process may be easily adjusted and used by a person skilled in the art depending on the microalgae selected. Specifically, the cultivation of Schizochytrium microalgae of the present application is carried out under heterotrophic conditions, but is not limited thereto.

[0037] The term "heterotrophy" as used herein refers to a nutritional method that relies on organic matter obtained from outside the body as a source of energy or nutrients, and is a term that corresponds to autotrophy, and may be used interchangeably with the term "dark culture."

[0038] The step of culturing the Schizochytrium microalgae is not particularly limited, and may be performed by known batch culture methods, continuous culture methods, fed-batch culture methods, etc. The medium and other culture conditions used to culture the microalgae of the present application may be any medium used for culturing conventional microalgae without any particular limitations. Specifically, the microalgae of the present application may be cultured in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, under aerobic conditions while controlling temperature, pH, etc.

[0039] Specifically, a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia hydroxide) or an acidic compound (e.g., phosphoric acid or sulfuric acid) can be used to adjust the pH to an appropriate level (e.g., pH 5 to 9, specifically, pH 6 to 8, most specifically, pH 6.8), but this is not intended to be limiting.

[0040] In addition, to maintain the culture in an aerobic state, oxygen or an oxygen-containing gas can be injected into the culture, or to maintain anaerobic and microaerobic states, no gas can be injected or nitrogen, hydrogen or carbon dioxide gas can be injected, but this is not intended to be limiting.

[0041] The culture temperature can be maintained at 20 to 45° C., or 25 to 40° C., and the culture can be performed for about 10 to 160 hours, but is not limited thereto. During the culture, an antifoaming agent such as a fatty acid polyglycol ester can be used to suppress the generation of bubbles, but is not limited thereto.

[0042] The carbon source contained in the medium used in the step of culturing the Schizochytrium microalgae 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 microalgae.

[0043] The nitrogen source contained in the medium used in the step of culturing the Schizochytrium microalgae may be any one or more organic nitrogen sources selected from the group consisting of i) yeast extract, beef extract, peptone, and tryptone, or ii) any one or more inorganic nitrogen sources selected from the group consisting of ammonium acetate, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, urea, and monosodium glutamate (MSG), but is not limited thereto as long as it is a nitrogen source used in culturing microalgae.

[0044] The medium used in the step of culturing the Schizochytrium microalgae may contain, as a phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a corresponding sodium-containing salt thereof, either individually or in combination, but is not limited thereto.

[0045] The step of recovering biomass from the microalgae, the culture of the microalgae, the dried product of the culture, or the crushed product of the dried product may involve collecting the desired biomass using a suitable method known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, and may further include a purification step.

[0046] Another aspect provides a method for producing a bio-oil derived from Schizochytrium microalgae, the method comprising: culturing Schizochytrium microalgae deposited under Accession No. KCTC14344BP or Accession No. KCTC14345BP and capable of producing docosahexaenoic acid, eicosapentaenoic acid, and palmitic acid; and recovering lipids from the microalgae, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product.

[0047] The Schizochytrium microalgae, the bio-oil, the culture of the microalgae, the dried product of the culture, the crushed product of the dried product, and the step of culturing the microalgae are as described above.

[0048] The step of recovering lipids from the microalgae, the culture of the microalgae, the dried product of the culture, or the crushed product of the dried product may involve collecting the lipids of interest using a suitable method known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, and may further include a purification step.

[0049] For example, lipids and lipid derivatives, such as fatty aldehydes, fatty alcohols, and hydrocarbons (e.g., alkanes), can be extracted with hydrophobic solvents, such as hexane. Lipids and lipid derivatives can also be extracted using methods such as liquefaction, oil liquefaction, and supercritical CO2 extraction. Known methods for recovering microalgae lipids include, for example, i) harvesting cells by centrifugation, washing with distilled water, and drying by freeze-drying, and ii) grinding the resulting cell powder and extracting lipids with n-hexane (Miao, X and Wu, Q, Biosource Technology (2006) 97: 841-846). Effect of the Invention

[0050] According to one embodiment, the novel Schizochytrium sp. microalgae has a high fat content in the biomass, including a high content of polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The microalgae, and the biomass or bio-oil produced therefrom can be usefully used as feed ingredients, etc. [Brief description of the drawings]

[0051] [Figure 1] 1 is a photograph of Schizochytrium sp. strain CD01-5000 observed under an optical microscope. [Diagram 2] FIG. 1 is a growth curve graph of Schizochytrium sp. strain CD01-5000 and Schizochytrium sp. strains CD01-5000 and CD01-5004CD01-5004. [Diagram 3] 1 is a growth curve graph of Schizochytrium sp. strain CD01-5004. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The present invention will be described in more detail below through examples. However, these examples are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0053] [Example 1. Isolation of Thraustochytrid Microalgae] In order to isolate Thraustochytrid microalgae, environmental samples in the form of seawater, soil, and sediment were collected from a total of 50 locations along the coasts of Taean, South Chungcheong Province, and Gunsan, North Jeolla Province, South Korea. The collected environmental samples were brought into a laboratory environment, and contamination sources such as mold, other bacteria, and protozoa were removed within 7 days. Thraustochytrid microalgae were isolated using direct plating and pine pollen baiting. During the continuous pollution source removal process and microalgae isolation process, samples showing morphology similar to cells containing zoospores, which are characteristic of Thraustochytrid microalgae, were isolated through a microscope and smeared on modified YEP medium (yeast extract 1g / L, peptone 1g / L, MgSO4·7H2O 2g / L, sea salt 20g / L, H3BO3 5.0mg / L, MnCl2 3.0mg / L, CuSO4 0.2mg / L, NaMo4·2H2O 0.05mg / L, CoSO4 0.05mg / L, ZnSO4·7H2O 0.7mg / L, and agar 15g / L), a medium for isolating marine microalgae. The obtained colonies were isolated as pure species through several subcultures. Colonies that remained contaminated were exposed to an antibiotic cocktail solution (streptomycin sulfate 0-100 mg / L, ampicillin 0-100 mg / L, penicillin G 0-100 mg / L, and kanamycin sulfate 0-100 mg / L) to control and remove the source of contamination. Through this process, more than 50 types of colonies were isolated and obtained.

[0054] The isolated colonies were cultured in modified 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) in a 250 mL flask at 10-35°C and 100-200 rpm for about 7 days. From these, one microalgae that could grow at temperatures above 25°C, had a good growth rate, and was able to secure a sufficient amount of bacteria was finally selected and named CD01-5000. The morphology of the selected strain was observed using an optical microscope (Figure 1).

[0055] Example 2. Identification of a novel Schizochytrium strain, CD01-5000 For molecular biological identification of the microalgae strain CD01-5000 isolated and selected in Example 1, the 18S rRNA gene sequence was analyzed. Specifically, gDNA was extracted and separated from a colony of the purely isolated microalgae CD01-5000, and then a PCR reaction was performed using the primers in Table 1 below as gene amplification primers for the 18S rRNA region.

[0056] [Table 1]

[0057] The PCR reaction was carried out using a reaction solution containing Taq polymerase, which was denatured at 95°C for 5 minutes, followed by 38 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute and 30 seconds, followed by polymerization at 72°C for 5 minutes. The reaction solution amplified through the PCR process was electrophoresed on a 1% agarose gel to confirm that a DNA fragment of approximately 1,600 to 2,000 bp in size had been amplified, and then sequencing analysis was carried out. As a result of the analysis, a base sequence of approximately 1,800 bp in size (SEQ ID NO: 1) was obtained, and through an NCBI BLAST search, it was confirmed that the sequence showed 99.3% homology with the 18S rRNA gene base sequence of Schizochytrium limacinum strain OUC192 ​​(NCBI accession No.: HM042913.2), which belongs to the Thraustochytrid microalgae, and 99.3% homology with the 18S rRNA gene base sequence of Schizochytrium sp. strain SH104 (NCBI accession No.: KX379459.1). Through this, the isolated microalgae CD01-5000 was confirmed to be a novel Schizochytrium strain, named Schizochytrium sp. CD01-5000, and deposited with the Korean Collection for Type Cultures (KCTC), an international depository institution under the Budapest Treaty, on October 26, 2020, with the accession number KCTC14344BP.

[0058] Example 3. Development of mutant microalgae strains. Example 3-1. Measurement of mortality rate due to gamma ray irradiation In order to develop an artificial mutant strain from the novel microalgae CD01-5000 confirmed in Example 2, the mortality rate according to the gamma ray dose was measured and the gamma ray irradiation conditions were selected.

[0059] Specifically, the novel microalgae CD01-5000 was cultured in a modified GYEP medium containing 3% glucose for more than about 20 hours until it reached the exponential phase. The cultured cell culture solution sample was centrifuged at 4,000 rpm for 15 minutes to harvest the cells, and then the cells were separated into 100 ml of ... 9 A microalgae culture sample suspended in PBS to give cells / mL was placed in a 50 mL conical tube and used for gamma ray irradiation experiments. The gamma ray irradiation experiments were carried out at the Advanced Radiation Research Institute of the Korea Atomic Energy Research Institute, and the microalgae culture samples were irradiated with 2,000 Gy, 2,500 Gy, 3,000 Gy, 3,500 Gy, 4,000 Gy, 4,500 Gy, 5,000 Gy, 5,500 Gy, 6,000 Gy, 7,000 Gy, or 8,000 Gy of gamma rays. The irradiated microalgae culture samples were centrifuged, the supernatant was removed, and the microalgae culture samples were then resuspended in GCBS medium containing 1% glucose (glucose 10g / L, corn teep liquor 5g / L, beef extract 5g / L, MgSO4·7H2O 5g / L, sea salt 15g / L, citric acid 20.08mg / L, FeSO4·7H2O 5.3mg / L, ZnSO4·7H2O 0.5mg / L, MnCl2 1.0mg / L, CuSO4 0.1mg / L, NaMo4·2H2O 0.1mg / L, CoSO4 0.1mg / L, ZnSO4·7H2O 0.5mg / L, biotin 1.0mg / L, thiamine hydrochloride 1.0mg / L, CAPA 1.0 mg / L, and vitamin B 12 The bacteria were inoculated at 0.01 mg / L and cultured at 30° C. for approximately 48 hours. The culture solution was then inoculated and smeared on GYEP medium containing 2% (20 g / L) agar, and cultured at 30° C. for 48 hours. The number of grown colonies was then counted, and the mortality rate was calculated for each gamma radiation dose according to the following formula 1.

[0060]

number

[0061] [Table 2]

[0062] As a result, when gamma rays were irradiated at a dose of 6,000 Gy or more, all of the microalgae died and no colonies could be obtained, so a gamma ray dose of 5,500 Gy, which showed a mortality rate of 99.99%, was selected.

[0063] Example 3-2. Isolation of mutant microalgae strains Using the same method as described in Example 3-1, the novel microalgae strain CD01-5000 was irradiated with gamma rays at a dose of 5,000 Gy, and the microalgae culture solution sample was inoculated and smeared on GYEP solid medium and GYEP solid medium supplemented with butanol and isoniazid, respectively, and cultured at 30°C. Microalgae colonies that grew on the solid plates for about 4 weeks were selected and subcultured in the same medium and culture conditions. Each subcultured colony was determined to be a different mutant strain, and colonies and strains that could grow sustainably were additionally selected.

[0064] Example 3-3. Selection of superior mutant microalgae strains The mutant strains selected in Example 3-2 were cultured and evaluated in a flask scale, and superior mutant strains were selected through the analysis of crude fat and fatty acids. Specifically, in order to culture the novel wild-type microalgae CD01-5000 confirmed in Example 2 and the mutant microalgae selected in Example 3-2 on a flask scale, the final volume (working volume) was adjusted to 50 mL in a 500 mL flask, and the microalgae were cultured in GYEP medium containing 3% (30 g / L) glucose at 30° C. and 180 rpm for 24 hours to obtain a certain amount of bacterial cells that can be analyzed. Then, the culture solution was centrifuged using a 50 mL conical tube, and the supernatant was removed to obtain bacterial cells, which were then washed three times with PBS at pH 7.5 and dried overnight in a dry oven at 60° C. to obtain each dried bacterial cell.

[0065] 8.3M hydrochloric acid solution was added to 2g of each dried cell, and the cell walls of the microalgae cells were hydrolyzed at 80℃. Then, 30mL of ethyl ether and 20mL of petroleum ether were added, and the mixture was mixed for 30 seconds and centrifuged. This process was repeated at least three times. The separated solvent layer was collected and transferred to a round flask whose weight had been measured in advance, and the solvent was removed by nitrogen purging and dried in a desiccator until it reached a constant weight. After drying, the weight of the dried oil was measured by subtracting the weight of the empty flask from the weight of the flask. The dried oil was pretreated with 0.5N methanolic NaOH and 14% boron trifluoride methanol (BF3), and the palmitic acid (PA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) contents in the oil were measured by gas chromatography, and the contents were calculated as a percentage of the total oil, i.e., crude fat weight.

[0066] [Table 3]

[0067] As a result, as shown in Table 3, the novel Schizochytrium strain CD01-5000 showed a very high DHA content of 50% or more, and also showed higher levels of palmitic acid and EPA than other mutant strains. In addition, among the mutant strains tested, the strain "Mutant 3" showed a higher DHA content than the wild-type strain, and also showed higher levels of palmitic acid and EPA than other mutant strains. It was selected as an excellent mutant strain and named Schizochytrium CD01-5004. It was deposited at the Korean Collection for Type Cultures (KCTC), an international depository institution under the Budapest Treaty, on October 26, 2020, and received the accession number KCTC14345BP.

[0068] [Example 4. Confirmation of the cultural characteristics of wild-type Schizochytrium strain CD01-5000 and mutant Schizochytrium strain CD01-5004] Further cultural characteristics of the novel wild-type Schizochytrium strain CD01-5000 and its mutant Schizochytrium strain CD01-5004 were confirmed.

[0069] Example 4-1. Analysis of biomass content and fatty acid content The CD01-5000 strain, the CD01-5004 strain, and the control Schizochytrium sp. ATCC20888 strain were cultured in a 5 L fermenter until the total volume reached 3 L. The culture was performed in a GYEP medium containing 5 g / L yeast extract and 10 g / L peptone as a nitrogen source under conditions of 20 to 35° C., 100 to 500 rpm, and 0.5 to 1.5 vvm. Dry cells were obtained from the culture broth and the crude fat content and fatty acid content in the cells were analyzed in the same manner as described in Example 3-3.

[0070] [Table 4]

[0071] As a result, as shown in Table 4, it was confirmed that the CD01-5000 and CD01-5004 strains exhibited biomass growth similar to that of the Schizochytrium sp. ATCC20888 strain cultivated as the control, and were capable of producing and accumulating high amounts of EPA and DHA intracellularly.

[0072] Example 4-2. Analysis of protein and amino acid contents Additional cultivation experiments were carried out on the CD01-5004 strain, which showed the highest biomass and EPA content, and analysis of the intracellular protein and amino acid contents was carried out.

[0073] Specifically, the culture was performed for a total of 63 hours under the same culture conditions as those described in Example 4-1, except that GYEP medium containing 10 g / L yeast extract and 10 g / L ammonium chloride was used as the nitrogen source. After the culture was completed, dried cells were obtained from the culture liquid in the same manner as that described in Example 3-3.

[0074] For protein content analysis, 0.5 to 1.0 g of dried cells was used to quantitatively analyze the nitrogen content in the sample using an elemental analyzer. The nitrogen weight ratio (TN%) in each sample was multiplied by 6.25 to calculate the crude protein content in the sample.

[0075] For amino acid content analysis, 0.5 to 1 g of dried cells were hydrolyzed with acid and then subjected to liquid chromatography. The percentage of each amino acid was calculated based on the percentage of the total amino acid concentration in the sample.

[0076] [Table 5]

[0077] As a result, as shown in Table 5, the total cell production was 60.5 g / L, and the crude protein content in the dry cells was confirmed to be 74.38%. It was confirmed that the intracellular amino acids were mainly composed of glutamate (23.31%) and phenylalanine (20.51%), followed by histidine, alanine, valine, proline, methionine, aspartic acid, glycine, leucine, isoleucine, and serine.

[0078] [Example 5. Confirmation of growth rates of wild-type Schizochytrium strain CD01-5000 and mutant Schizochytrium strain CD01-5004] The growth rates of a novel wild-type Schizochytrium strain, CD01-5000, and its mutant Schizochytrium strain, CD01-5004, were measured and compared.

[0079] Specifically, the CD01-5000 strain and the CD01-5004 strain were inoculated into GYEP medium containing 3% glucose in a 500 mL flask to a final working volume of 50 mL, and cultured in a shaking incubator at 30°C and 180 rpm. During the culture period, the absorbance was measured at a wavelength of 680 nm using a UV / Visible Spectrophotometer as an OD (optical density) value to measure the degree of cell growth at each stage.

[0080] As a result, as shown in FIG. 2, the mutant CD01-5004 showed equal or greater sugar consumption rate and cell growth than the wild-type CD01-5000, and reached exponential phase approximately 5 hours earlier than the wild-type.

[0081] Example 6. Confirmation of growth characteristics of wild-type Schizochytrium strain CD01-5000 and mutant Schizochytrium strain CD01-5004 It was confirmed whether the novel wild-type Schizochytrium sp. strain CD01-5000 and its mutant Schizochytrium sp. strain CD01-5004 could grow in a wide pH range during cultivation.

[0082] Specifically, the CD01-5000 strain and the CD01-5004 strain were inoculated into GYEP medium having a pH of 2, 3, 3.5, 4, 6, 8, 8.5, 9, or 9.5 in a 500 mL flask, respectively, and cultured in the same manner as described in Example 4. After culture, the absorbance was measured at 0, 24, 40, and 48 hours, respectively, to evaluate the degree of cell growth.

[0083] [Table 6]

[0084] As a result, as shown in Table 6 and FIG. 3, it was confirmed that the CD01-5004 strain was capable of growing over a wide range of pH from 3.5 to 9, and it was confirmed that the CD01-5000 strain also showed similar growth characteristics.

[0085] In addition, since the culture medium of the CD01-5000 and CD01-5004 strains turned deep red, it was found that they were strains that produce carotenoid antioxidant pigments, such as β-carotene, lutein, astaxanthin, capsanthin, annatto, canthaxanthin, lycopene, β-apo-8-carotenal, zeaxanthin, and β-apo-8-carotenal-ester.

[0086] From the above description, a person skilled in the art to which the present application belongs can understand that the present application can be implemented in other specific forms without changing the technical idea or essential features. In this regard, it should be understood that the above described embodiments are illustrative in all respects and are not limiting. The scope of the present application should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts rather than the above detailed description. The present application provides the following: 1. A microalga of the genus Schizochytrium, deposited under accession number KCTC14344BP or accession number KCTC14345BP and capable of producing docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and palmitic acid (PA). 2. The Schizochytrium microalgae according to 1 above, which produces 35 to 60% by weight of docosahexaenoic acid based on the total weight of fatty acids. 3. The Schizochytrium microalgae according to 1 above, which produces 0.5 to 10% by weight of eicosapentaenoic acid based on the total weight of fatty acids. 4. The microalgae of the genus Schizochytrium described in 1 above, which produces 10 to 30% by weight of palmitic acid based on the total weight of fatty acids. 5. The microalgae of the genus Schizochytrium described in 1 above, which has carotenoid producing ability. 6. The Schizochytrium microalgae according to 5 above, wherein the carotenoid is one or more selected from the group consisting of β-carotene, lutein, astaxanthin, capsanthin, annatto, canthaxanthin, lycopene, β-apo-8-carotenal, zeaxanthin, and β-apo-8-carotenal-ester. 7. A biomass derived from Schizochytrium microalgae, comprising the Schizochytrium microalgae described in 1 above, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product. 8. A feed composition comprising a biomass derived from the microalgae of the genus Schizochytrium described in 7 above, or a concentrate or dried product of said biomass. 9. A step of culturing a microalga of the genus Schizochytrium, which is deposited under Accession No. KCTC14344BP or Accession No. KCTC14345BP and has an ability to produce docosahexaenoic acid, eicosapentaenoic acid, and palmitic acid; Recovering biomass from the microalgae, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product; The method for producing biomass derived from Schizochytrium microalgae comprises: 10. The method for producing biomass derived from Schizochytrium microalgae according to claim 9, wherein the culture is carried out under heterotrophic conditions. 11. The method for producing biomass derived from Schizochytrium microalgae according to claim 9, wherein the culture is carried out using a medium containing a carbon source and a nitrogen source. 12. A method for producing biomass derived from Schizochytrium microalgae described in 11 above, wherein the carbon source is one or more selected from the group consisting of glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose and glycerol. 13. A method for producing biomass derived from Schizochytrium microalgae according to claim 11, wherein the nitrogen source is i) any one or more organic nitrogen sources selected from the group consisting of yeast extract, beef extract, peptone and tryptone, or ii) any one or more inorganic nitrogen sources selected from the group consisting of ammonium acetate, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, urea and monosodium glutamate (MSG). 14. A step of culturing a microalga of the genus Schizochytrium, which is deposited under Accession No. KCTC14344BP or Accession No. KCTC14345BP and has an ability to produce docosahexaenoic acid, eicosapentaenoic acid, and palmitic acid; Recovering lipids from the microalgae, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product; The method for producing bio-oil derived from Schizochytrium microalgae comprises: JPEG0007621489000008.jpg163161JPEG0007621489000009.jpg155160

Claims

1. A microalga of the genus Schizochytrium, deposited under accession number KCTC14344BP or accession number KCTC14345BP, having the ability to produce docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) in an amount ranging from 1 to 10% by weight based on the total weight of fatty acids, and palmitic acid (PA).

2. 2. The Schizochytrium microalgae according to claim 1, wherein the microalgae produce 35 to 60% by weight of docosahexaenoic acid based on the total weight of fatty acids.

3. The microalgae of claim 1, which produces 10 to 30% by weight of palmitic acid based on the total weight of fatty acids.

4. The microalgae of the genus Schizochytrium according to claim 1 , wherein the microalgae have a carotenoid producing ability.

5. The Schizochytrium microalgae according to claim 4, wherein the carotenoid is at least one selected from the group consisting of β-carotene, lutein, astaxanthin, capsanthin, annatto, canthaxanthin, lycopene, β-apo-8-carotenal, zeaxanthin, and β-apo-8-carotenal ester.

6. A biomass derived from Schizochytrium microalgae comprising the Schizochytrium microalgae according to claim 1, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product.

7. A feed composition comprising the biomass derived from the Schizochytrium microalgae according to claim 6, or a concentrate or dry product of said biomass.

8. Cultivating a microalga of the genus Schizochytrium, which is deposited under Accession No. KCTC14344BP or Accession No. KCTC14345BP and has an ability to produce docosahexaenoic acid, eicosapentaenoic acid in an amount ranging from 1 to 10% by weight based on the total weight of fatty acids, and palmitic acid; Recovering biomass from the microalgae, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product; The method for producing biomass derived from Schizochytrium microalgae comprises:

9. The method for producing biomass derived from Schizochytrium microalgae according to claim 8, wherein the culture is carried out under heterotrophic conditions.

10. The method for producing biomass derived from Schizochytrium microalgae according to claim 8, wherein the culture is carried out using a medium containing a carbon source and a nitrogen source.

11. The method for producing biomass derived from Schizochytrium microalgae according to claim 10, wherein the carbon source is at least one selected from the group consisting of glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose and glycerol.

12. The method for producing biomass derived from Schizochytrium microalgae according to claim 10, wherein the nitrogen source is i) any one or more organic nitrogen sources selected from the group consisting of yeast extract, beef extract, peptone and tryptone, or ii) any one or more inorganic nitrogen sources selected from the group consisting of ammonium acetate, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, urea and monosodium glutamate (MSG).

13. Cultivating a microalga of the genus Schizochytrium, which is deposited under Accession No. KCTC14344BP or Accession No. KCTC14345BP and has an ability to produce docosahexaenoic acid, eicosapentaenoic acid in an amount ranging from 1 to 10% by weight based on the total weight of fatty acids, and palmitic acid; Recovering lipids from the microalgae, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product; The method for producing bio-oil derived from Schizochytrium microalgae comprises:

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