A novel Schizochytrium strain with high intracellular oil content and a method for producing oil containing omega-3 using the same

Abstract

The present application relates to a novel Schizochytrium sp. strain having a high intracellular oil content and a method for producing an oil containing omega-3 using the same, and the novel Schizochytrium sp. microalgae according to the present invention has a high fat content in the biomass, in particular a high content of unsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid, so that it is very easy to extract the biomass itself or the biomass produced by culture and fermentation, and the fat components containing unsaturated fatty acids from the biomass. Therefore, the microalgae, the dried biomass and the bio-oil produced therefrom can be usefully used in feed compositions or food compositions.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0152561 dated November 8, 2021, and all contents disclosed in the documents of that Korean patent application are incorporated herein by reference.

[0002] The present application relates to a novel Schizochytrium sp. strain having a high intracellular oil content and a method for producing an oil containing omega-3 using the same. [Background technology]

[0003] Thraustochytrids live and distribute in a variety of environments in nature. They attach to organisms and live symbiotically, or they float in marine, freshwater, and brackish environments, distributing across a variety of sedimentary layers. These Thraustochytrids belong to the lowest level of the marine ecological food chain and are sometimes classified as phytoplankton, or organoheterotrophic protists (microalgae). In the natural environment, Thraustochytrids function to circulate and purify natural circulating elements such as sulfur, nitrogen, phosphorus, and potassium. They also contain high concentrations of polyunsaturated fatty acids (PUFAs), including omega-3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), serving as a resource for the marine ecosystem.

[0004] Most higher organisms, including humans, cannot synthesize polyunsaturated fatty acids, including docosahexaenoic acid and eicosapentaenoic acid, on their own and must ingest them as essential nutrients. Among polyunsaturated fatty acids, docosahexaenoic acid and eicosapentaenoic acid are essential fatty acids for the brain, eye tissue, and nervous system, and are known to play important roles in nervous system development, particularly in infants' vision and motor skills, and in preventing cardiovascular disease. They are also the most abundant components of structural lipids in the brain.

[0005] Until now, the primary source of polyunsaturated fatty acids has been fish oil extracted from blue-backed fish such as mackerel, saury, tuna, horse mackerel, sardines, and herring. This oil is highly useful as aquaculture feed, such as a starter feed for marine fish. While the extraction and consumption of polyunsaturated fatty acids from fish oil has become widespread, there are drawbacks. The quality of fish oil varies depending on the fish species, season, and fishing location. Since fish oil is generated through fishing, it is difficult to ensure a sustainable supply. Furthermore, there are limitations on the manufacturing process and production volume due to issues with contamination from heavy metals and organic chemicals contained in fish oil, its distinctive fishy odor, and the oxidation of double bonds during processing.

[0006] To address these issues, research has recently been conducted into methods for producing polyunsaturated fatty acids, including docosahexaenoic acid and eicosapentaenoic acid, through microbial culture. In particular, microalgae offer various advantages over fish oil, including the ability to naturally synthesize fatty acids de novo. They can be stably supplied through industrial-scale culture, enabling the production of biomass with a relatively consistent biochemical composition. Unlike fish oil, lipids produced by microalgae do not have any unpleasant odor. Furthermore, they have a simpler fatty acid composition than fish oil, which facilitates the process of separating the major fatty acids.

[0007] Based on these advantages, research and industrialization into the production of polyunsaturated fatty acids, including omega-3 unsaturated fatty acids (ω-unsaturated fatty acids) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), docosapentaenoic acid (DPA), and α-linolenic acid, using microalgae has been progressing rapidly recently. Polyunsaturated fatty acid production is primarily being carried out using marine microalgae from the genera Thraustochytrium and Schizochytrium. As examples, a method for producing omega-3 polyunsaturated fatty acids using Schizochytrium sp. ATCC20888 and Schizochytrium sp. PTA10208, which are microorganisms of the genus Schizochytrium, has been disclosed (U.S. Patent No. 5,130,242), and a method for producing docosahexaenoic acid and eicosapentaenoic acid using Thraustochytrid microorganisms of the genus Thraustochytrium, Thraustochytrium sp. ATCC10212, which is also a Thraustochytrid microorganism of the genus Thraustochytrium, has also been disclosed. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 5,130,242 Summary of the Invention [Problem to be solved by the invention]

[0009] One example of the present application provides a novel Schizochytrium microalgae. In one specific example, the novel Schizochytrium microalgae may be Schizochytrium microalgae CD01-1821 (Accession Number: KCTC14660BP).

[0010] Another example of the present application provides a biomass or bio-oil derived from the microalgae of the genus Schizochytrium.

[0011] Another example of the present application provides a feed composition comprising biomass, bio-oil, or a combination thereof derived from the microalgae of the genus Schizochytrium.

[0012] Another example of the present application provides a food composition comprising biomass, bio-oil, or a combination thereof derived from the microalgae of the genus Schizochytrium.

[0013] Another example of the present application provides a method for producing biomass or bio-oil derived from the microalgae of the genus Schizochytrium.

[0014] Another example of the present application provides a use of the microalgae of the genus Schizochytrium for producing biomass or bio-oil.

[0015] Yet another example of the present application provides a use of the Schizochytrium microalgae for producing a feed composition or a food composition. [Means for solving the problem]

[0016] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions set forth below are not intended to limit the scope of this application. Furthermore, those skilled in the art will recognize or be able to ascertain, through no more than routine experimentation, numerous equivalents to the specific aspects of this application described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0017] One example of the present application provides a novel Schizochytrium sp. microalgae.

[0018] As used herein, the term "Thraustochytrid" refers to microalgae of the order Thraustochytriales. The term "Schizochytrium sp." as used herein refers to a genus belonging to the family Thraustochytriaceae of the order Thraustochytriales and may be used interchangeably with the term "genus Schizochytrium." The term "microalgae" refers to a plant that performs photosynthesis using chlorophyll, is invisible to the naked eye, can only be seen through a microscope, and lives as a free-floating organism in water. There are many different types of microalgae, including strains that are unable to photosynthesize and grow solely heterotrophically.

[0019] As an example, in this application, environmental samples were collected from coastal areas to obtain pure isolated colonies of Thraustochytrids. Then, as a result of crude fat and fatty acid analysis, two strains, CD01-1821 and CD01-1822, with high intracellular DHA content were obtained. As a result of culture evaluation, it was confirmed that CD01-1821 strain has high total biomass production and total oil content under the same fermentation conditions, making it easier to scale up.

[0020] Therefore, in this specification, the novel Schizochytrium sp. microalgae may be Schizochytrium sp. microalgae CD01-1821 (accession number: KCTC14660BP).

[0021] Furthermore, the (wild-type) Schizochytrium strain may have, but is not limited to, the 18s rRNA base sequence of SEQ ID NO: 1. For example, the Schizochytrium microalga may have, but is not limited to, an 18S rRNA composed of 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.

[0022] As used herein, the term "docosahexaenoic acid (DHA)" refers to C 22 H 32 It is a polyunsaturated fatty acid with the chemical formula O2, and is one of the omega-3 fatty acids along with alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA). Its common name is cervonic acid, and it can also be abbreviated as 22:6n-3.

[0023] As used herein, the term "eicosapentaenoic acid (EPA)" refers to C 20 H 30It is a polyunsaturated fatty acid with the chemical formula O2, and along with ALA and DHA, it is classified as an omega-3 fatty acid and can also be abbreviated as 20:5n-3.

[0024] The microalgae of the genus Schizochytrium may produce and / or contain 35 to 60% by weight of DHA based on the total weight of fatty acids. For example, the microalgae of the genus Schizochytrium may produce and / or contain 40 to 65%, 45 to 65%, 50 to 65%, 40 to 60%, 45 to 60%, 50 to 60%, (35 to 58%, 40 to 58%, 45 to 58%, or 48 to 52% by weight) or 55 to 60% by weight of DHA based on the total weight of fatty acids.

[0025] The microalgae of the genus Schizochytrium may produce and / or contain 0.1 to 2 wt% of EPA based on the total weight of fatty acids. For example, the microalgae of the genus Schizochytrium may produce and / or contain 0.2 to 2 wt%, 0.2 to 1.5 wt%, 0.2 to 1 wt%, 0.3 to 2 wt%, 0.3 to 1.5 wt%, 0.3 to 1 wt%, 0.5 to 2 wt%, 0.5 to 1.5 wt%, 0.5 to 1 wt%, 0.5 to 0.9 wt%, or 0.6 to 0.8 wt% of EPA based on the total weight of fatty acids.

[0026] Another aspect of the present application provides biomass or bio-oil derived from Schizochytrium microalgae, including the Schizochytrium CD01-1821 microalgae, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product.

[0027] The microalgae of the genus Schizochytrium are as described above.

[0028] As used herein, the term "biomass" refers to organisms, such as plants, animals, and microorganisms, that can be used as chemical energy, i.e., a source of bioenergy. Ecologically, it can also refer to the weight or energy amount of a specific organism present within a unit of time and space. Furthermore, biomass includes, but is not limited to, compounds secreted by cells, and may contain not only extracellular substances but also cellular and / or intracellular contents. In the present application, the biomass may be Schizochytrium microalgae itself, a culture thereof, a dried product thereof, or 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.

[0029] The "culture" of the Schizochytrium microalgae refers to a product produced by culturing the microalgae, and may specifically be, but is not limited to, a culture solution containing the microalgae or a culture filtrate obtained by removing the microalgae from the culture solution. The "dried product" of the Schizochytrium microalgae culture refers to the microalgae culture from which water has been removed, and may be, for example, in the form of dried cells of the microalgae, but is not limited to this. Furthermore, the "crushed product" of the dried product collectively refers to the product obtained by crushing the dried product from which water has been removed from the microalgae culture, and may be, for example, but is not limited to, a dried cell powder. The Schizochytrium microalgae culture may be produced by inoculating the microalgae into a microalgae culture medium and using a culture method known in the art. The dried product of the culture and its crushed product may also be produced by treating or drying microalgae or culture solution known in the art.

[0030] The biomass derived from the Schizochytrium genus CD01-1821 microalgae may contain 40 to 85 wt%, 45 to 80 wt%, 50 to 75 wt%, 50 to 70 wt%, or 54 to 66 wt% crude fat based on the total weight of the biomass.

[0031] The biomass derived from the microalgae of the genus Schizochytrium may contain 5 to 25 wt %, 5 to 20 wt %, 10 to 20 wt %, or 15 to 20 wt % crude protein based on the total weight of the biomass.

[0032] The biomass derived from the Schizochytrium microalgae may contain 35 wt % or more, or 35 to 60 wt % DHA based on the total weight of fatty acids, 0.1 wt % or more, or 0.1 to 2 wt % EPA based on the total weight of fatty acids, and 30 to 40 wt % or more palmitic acid based on the total weight of fatty acids.

[0033] The microalgae of the genus Schizochytrium have a high oil content, among which the high omega-3 content, which is advantageous for the scale-up process by shortening the culture time in complex sugars.

[0034] In this specification, the term "oil content" may be used interchangeably with "crude fat content."

[0035] The biomass may be produced by a method for producing biomass derived from microalgae of the genus Schizochytrium according to one embodiment.

[0036] Another aspect of the present application provides a composition comprising microalgae of the genus Schizochytrium CD01-1821, a culture of the microalgae, a dried product of the culture, and a crushed product of the dried product.

[0037] The composition may comprise biomass, bio-oil, or a combination thereof derived from the microalgae of the genus Schizochytrium.

[0038] Another aspect of the present application provides a feed composition comprising biomass derived from Schizochytrium sp. CD01-1821 microalgae, or a concentrate or dry matter of said biomass.

[0039] The Schizochytrium microalgae, biomass, culture of the microalgae, dried product of the culture, and crushed product of the dried product are as described above.

[0040] The biomass concentrate or dry product may be produced by any method known in the art for processing, concentrating or drying microbial biomass.

[0041] 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.

[0042] As used herein, the bio-oil may comprise an extract of biomass.

[0043] The method for producing the bio-oil extract may include, but is not limited to, a method using enzymes such as protease, cellulase, pectinase, or chitinase to disrupt or dissolve cell membranes or cell wall components; a method using a homogenizer, an ultrasonic crusher, or bead treatment to physically disrupt cell membranes or cell wall components; a method of extracting by directly adding a solvent and allowing it to penetrate into the cells; and a solvent-free extraction process in which separation is performed through a centrifugation process after various disruption processes.

[0044] The composition may be in the form of, but is not limited to, a solution, a powder, or a suspension, and may be, for example, a food composition, a feed composition, or a feed additive composition.

[0045] As used herein, the term "feed composition" refers to a feed fed to an animal. The feed composition refers to a substance that provides organic or inorganic nutrients necessary for sustaining the life of an animal or producing meat, milk, etc. The feed composition may additionally contain nutritional components necessary for sustaining the life of an animal or producing meat, milk, etc. The feed composition may be prepared in various forms known in the art, and may specifically include concentrated feed, roughage, and / or special feed.

[0046] The term "feed additive" as used herein includes substances added to feed for various purposes, such as supplementing nutrients and preventing weight loss, increasing the digestibility of fiber in feed, improving milk quality, preventing reproductive disorders and improving conception rates, and preventing heat stress in summer. The feed additives of the present application fall under the category of 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 of 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 acids such as methionine and lysine, protected fatty acids such as fatty acid calcium salts, probiotics (lactic acid bacteria preparations), live bacteria such as yeast cultures and mold fermentation products, yeast preparations, etc.

[0047] The term "food composition" as used herein includes all forms of functional foods, nutritional supplements, health foods, food additives, etc., and food compositions of the above types can be prepared in various forms by conventional methods known in the art.

[0048] The compositions of the present application may further comprise grains, such as crushed or crushed wheat, oats, barley, corn and rice; vegetable protein feeds, such as feeds based on soybeans and sunflowers; animal protein feeds, such as blood meal, meat meal, bone meal and fish meal; dry ingredients consisting of sugars and dairy products, such as various milk powders and whey powders, and may further comprise nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

[0049] The compositions of the present application may be administered to animals alone or in combination with other feed additives in an edible carrier. Alternatively, the compositions may be administered to animals as a top dressing, or by directly mixing them into the feed or as an oral formulation separate from the feed. When the compositions are administered separately from the feed, they can be combined with a pharmaceutically acceptable edible carrier to prepare immediate-release or sustained-release formulations, as is well known in the art. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the composition may 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 may be in the form of a soft gelatin capsule, or a syrup, suspension, emulsion, or solution.

[0050] The composition of the present application may contain, for example, a preservative, a stabilizer, a humectant or emulsifier, 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.

[0051] The preservative, stabilizer, or excipient may be contained in the composition in an amount effective enough to reduce deterioration of the Schizochytrium microalgae contained in the composition, and the cryoprotectant may be contained in the composition in an amount effective enough to reduce deterioration of the Schizochytrium microalgae contained in the composition when the composition is in a dried state.

[0052] The composition may be applied by dipping, spraying or mixing into animal feed.

[0053] The composition of the present application can be used in the feed of many animals, 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 pet animals. The birds can include poultry, including, but not limited to, chickens, turkeys, ducks, geese, pheasants, and quails. The fish can include commercially farmed fish and their fry, as well as ornamental fish. The crustaceans can include, but are not limited to, shrimp and barnacles. The composition can also be used as a feed for zooplankton rotifers.

[0054] Another aspect of the present application provides a method for producing biomass derived from microalgae of the genus Schizochytrium, the method including the steps of culturing microalgae of the genus Schizochytrium CD01-1821 and recovering biomass from the microalgae, a culture of the microalgae, a dried product of the culture, or crushed products of the dried product.

[0055] The Schizochytrium microalgae, biomass, culture of the microalgae, dried product of the culture, and crushed product of the dried product are as described above.

[0056] The term "cultivation" as used herein means growing the microalgae under appropriately controlled environmental conditions. The culturing process of the present application may be carried out using appropriate media and culture conditions known in the art. Such a culturing process may be easily adjusted by those skilled in the art depending on the selected microalgae.

[0057] Specifically, the cultivation of the microalgae of the genus Schizochytrium of the present application may be carried out under heterotrophic conditions, but is not limited thereto.

[0058] 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."

[0059] 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 commonly used for culturing microalgae, without any particular limitations. Specifically, the microalgae of the present application may be cultured in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, under aerobic conditions while controlling the temperature, pH, etc.

[0060] Specifically, a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid) may be used to adjust the pH to an appropriate value (e.g., pH 5 to 9, specifically, pH 6 to 8, most specifically, pH 6.8), but this is not limitative.

[0061] In addition, to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas may be injected into the culture, or to maintain the anaerobic and microaerobic state, no gas may be injected or nitrogen, hydrogen or carbon dioxide gas may be injected, but this is not limitative.

[0062] 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, but is not limited thereto. During the culture, foam formation may be suppressed using an antifoaming agent such as a fatty acid polyglycol ester, but is not limited thereto.

[0063] The carbon source contained in the medium used in the step of culturing the Schizochytrium microalgae may be any one or more 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.

[0064] The nitrogen source contained in the medium used in the step of culturing the Schizochytrium microalgae may be 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 MSG (Monosodium glutamate), but is not limited thereto as long as it is a nitrogen source used in culturing microalgae.

[0065] The culture medium used in the step of culturing the Schizochytrium microalgae may contain, as a phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, etc., individually or in combination, but is not limited thereto.

[0066] 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 any suitable method known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, and may further include a purification step.

[0067] Another aspect of the present application provides a method for producing bio-oil derived from microalgae of the genus Schizochytrium, the method including the steps of culturing microalgae of the genus Schizochytrium CD01-1821 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.

[0068] The Schizochytrium microalgae, bio-oil, culture of the microalgae, dried product of the culture, crushed product of the dried product, and the step of culturing the microalgae are as described above.

[0069] 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.

[0070] 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) collecting cells by centrifugation, washing with distilled water, and then drying by lyophilization; (ii) grinding the resulting cell powder and then extracting lipids with n-hexane (Miao, X and Wu, Q, Biosource Technology (2006) 97:841-846).

[0071] Another aspect of the present application provides use of Schizochytrium sp. CD01-1821 microalgae, a culture of the microalgae, a dried product of the culture, or a crushed product of the dried product for producing biomass or bio-oil.

[0072] The Schizochytrium microalgae, biomass, culture of the microalgae, dried product of the culture, and crushed product of the dried product are as described above.

[0073] Another example of the present application provides use of Schizochytrium sp. CD01-1821 microalgae, a culture of the microalgae, a dried product of the culture, or crushed product of the dried product for producing a feed composition or a food composition.

[0074] The Schizochytrium microalgae, biomass, culture of the microalgae, dried product of the culture, and crushed product of the dried product are as described above. [Effects of the Invention]

[0075] The novel Thraustochytrid microalgae of the present invention have a high fat content in their biomass, particularly a high content of unsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid, making it very easy to extract fat components, including unsaturated fatty acids, from the biomass itself or the biomass produced by culture and fermentation. Therefore, the microalgae, and the dried biomass and bio-oil produced therefrom can be usefully used in feed compositions or food compositions. [Brief explanation of the drawings]

[0076] [Figure 1] FIG. 1 is a schematic diagram showing the process of isolating a Thraustochytrid microalgae strain. [Figure 2] Figure 2 shows the results of analyzing the total lipid content and the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) contents of 29 isolated species of Thraustochytrid microalgae. [Figure 3] FIG. 3 is a photograph of the wild-type Schizochytrium sp. strain CD01-1821 observed under an optical microscope. DETAILED DESCRIPTION OF THE INVENTION

[0077] The present invention will be described in more detail with reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0078] Example 1. Isolation of Thraustochytrid microalgae To isolate Thraustochytrid microalgae, environmental samples in the form of seawater, leaves, and sediment were collected from over 40 locations along the coasts of Seochon, Gunsan, Buan, and Yeonggwang-gun, South Korea's west coast. Sampling was conducted in specific areas where organic sediments were observed to have accumulated. The collected environmental samples were then transported to a laboratory within seven days to remove any other contaminants, such as bacteria, microorganisms, fungi, and protozoa, excluding the Thraustochytrid microalgae to be isolated. Through continuous microscopic examination, Thraustochytrid microalgae cells were isolated from samples that showed the characteristic morphology of Thraustochytrid microalgae and formed observable zoospores during their life cycle or contained an ectoplasmic network (Figure 1). The isolation and culture medium used in the isolation process was modified YEP medium (yeast extract 0.1 g / L, peptone 0.5 g / L, MgSO4·7H2O 2 g / L, sea salt 50 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, ZnSO4·7H2O 0.7 mg / L, agar 15 g / L). Through several isolation and subculture processes, pure isolated colonies were obtained from which contaminants had been removed. The isolated colonies were then placed on solid medium containing an antibiotic cocktail solution (streptomycin sulfate 0-50mg / L, ampicillin 0-30mg / L, penicillin G 0-30mg / L, kanamycin sulfate 0-30mg / L) to once again control and remove contaminants, yielding pure, isolable colonies.

[0079] Example 2. Cultivation evaluation of isolated microalgae and selection of superior strains The colonies isolated in Example 1 were subjected to a culture evaluation, and superior strains were selected.

[0080] Specifically, the pure colonies isolated in Example 1 were cultured in a modified GGYEP medium (5 g / L glucose, 5 g / L glycerol, 0.1 g / L yeast extract, 0.5 g / L peptone, 2 g / L MgSO4·7H2O, 50 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 approximately two days. Based on the culture results, 29 species of microalgae were selected that could grow at temperatures above 30°C, had excellent growth rates, and were capable of maintaining a sufficient bacterial mass. The selected microalgae strains were cultured at a scale in a 500ml flask for two days at 30°C, 150 rpm, in modified GYEP medium containing 30g / L glucose as a carbon source. After confirming that the carbon source had been completely consumed during the two-day culture, the entire culture medium was collected and dried overnight in a dry oven at 60°C to obtain biomass.

[0081] The lipid and polyunsaturated fatty acid content of the cultured microalgae was analyzed using the following method. The fatty acid-containing oil derived from the microalgae using dried microalgae was measured as follows: 5 g of dried microalgae was added to 8.3 M hydrochloric acid (HCl) solution and heated at 80°C to hydrolyze the cell walls of the microalgae. 30 mL of ethyl ether and 20 mL of petroleum ether were then added, mixed for 30 seconds, and centrifuged. This process was repeated at least three times. The separated solvent layer was collected and transferred to a pre-weighed round flask. The solvent was removed using a nitrogen purge and cooled to a constant weight in a desiccator. After drying, the weight of the dried oil was calculated by subtracting the weight of the empty flask from the weight of the flask to calculate the total oil content. The docosahexaenoic acid (DHA) content in the oil was measured by gas chromatography after pretreatment with methanolic 0.5 N NaOH and 14% trifluoroborane methanol (BF3).

[0082] [Formula 1] Total oil content (%) = (* oil g / dry cell mass g) x 100 *Oil g: weight of flask after acid hydrolysis and solvent removal - weight of empty flask

[0083] "Biomass" in Table 1 below means the concentration of bacterial cells in the culture medium, and can be used interchangeably with DCW (dry cell weight) in Table 2 below.

[0084] [Table 1]

[0085] (In the above table, TFA means total fatty acids and can be used interchangeably with crude fat or total lipids.)

[0086] As a result, as shown in Table 1 and Figure 2, two strains, CD01-1821 and CD01-1822, showed very high intracellular DHA content of 50% or more.

[0087] Based on the fatty acid analysis results, two strains, CD01-1821 and CD01-1822, which had excellent intracellular DHA content, were cultured and evaluated in a 5L-scale fermentor. Seed cultures were prepared in sterilized 500mL flasks using MJW02 medium (glucose 30g / L, MgSO4·7H2O 3.0g / L, Na2SO4 15g / L, NaCl 0.8g / L, yeast extract 1.0g / L, MSG·1H2O 1.0g / L, NaNO3 1.0g / L, KH2PO4 0.8g / L, K2HPO4 1.5g / L, CaCl2 0.5g / L, and vitamin mixture solution 10ml / L) at 30°C and 150 rpm for approximately 24 hours. The seed culture flasks were then dispensed and inoculated into 5L fermentors. Glucose was supplied as a carbon source at 28% of the total culture medium, and the culture was carried out for approximately 72 hours under the conditions of 30°C, 500 rpm, 1.5 vvm, and pH 5-8 in sterilized MJW02 medium and culture environment.

[0088] [Table 2]

[0089] As a result, as shown in Table 2, the CD01-1821 strain exhibited higher total biomass production and crude fat content than the CD01-1822 strain under the same fermentation conditions, demonstrating its ease of scale-up. Therefore, the CD01-1821 strain was selected and used for identifying the strain sequence and developing additional strains. The morphology of the selected CD01-1821 strain was observed using an optical microscope and is shown in Figure 3.

[0090] Example 3. Confirmation of the culture characteristics of the CD01-1821 strain under complex carbon source conditions Heterotrophic microbial fermentation primarily uses glucose as a carbon source. Glucose is a monosaccharide that is over 90% purified, which means it is more expensive to ferment on an industrial scale than other carbon source raw materials. To utilize inexpensive carbon source raw materials and thereby ensure price competitiveness, it is important to discover strains that can be used in fermentation without using purified glucose and that can be successfully cultivated using inexpensive carbon source materials.

[0091] Therefore, the CD01-1821 and CJM01 (registered patent 10-2100650) strains selected in Example 2 were subjected to fermentation culture evaluation using raw sugars containing glucose, fructose, or sucrose as the main components to confirm their culture characteristics. Cultures were conducted in 30 L fermentors using modified MJW02 medium as the base medium, with the main carbon source components being 450 g / L glucose, a mixture of 225 g / L glucose and 225 g / L fructose, and a crude sugar hydrolyzate containing 225 g / L glucose, 220 g / L fructose, and 1.51 g / L sulfate. Culture conditions were identical: 30°C, 500 rpm, 1.5 vvm, and pH 5-8. The carbon source was supplied at 35% of the total culture volume.

[0092] [Table 3]

[0093] As a result, as shown in Table 3, the CD01-1821 strain exhibited equal or higher levels of total biomass production and crude fat production when fermented in a medium containing a fructose mixture or crude sugar hydrolysate, rather than a single glucose component. In contrast, the CJM01 strain exhibited a diauxic growth pattern and a dual carbon source consumption pattern when sugar components other than glucose were added to the medium, resulting in a longer total cultivation time. These experimental results confirmed the potential of the CD01-1821 strain for scale-up fermentation under complex carbon source conditions.

[0094] Example 4. Identification of the novel Schizochytrium strain CD01-1821 For molecular biological identification of the microalgae strain CD01-1821 isolated and selected in Examples 1 and 2, the 18S rRNA gene sequence was analyzed.

[0095] Specifically, gDNA was extracted and separated from a colony of the purely isolated microalgae CD01-1821, and then PCR amplification was performed using the gene amplification primers 18s-Fwd and LABY-ARev for the 18s rRNA region listed in Table 4.

[0096] [Table 4]

[0097] The PCR reaction was carried out using a reaction solution containing Taq polymerase, with denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, and polymerization at 72°C for 2 minutes, 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 1000 bp in size had been amplified, and then subjected to base sequence sequencing analysis. Through an NCBI BLAST search, the sequence identified showed 95.11% identity with the 18S rRNA gene sequence of Schizochytrium limacinum strain OUC109, a microalga belonging to the Thraustochytrid family, and 95.0% identity with the 18S rRNA gene sequence of Schizochytrium sp. strain LY-2012. This confirmed that the isolated microalgae CD01-1821 is a new Schizochytrium strain, which was named Schizochytrium sp. CD01-1821 and deposited with the Korea Center for Biological Resources (KCTC) at the Korea Institute of Bioscience and Biotechnology (KCTC) on August 23, 2021, and assigned the accession number KCTC14660BP.

[0098] Example 5. Analysis of crude protein content of CD01-1821 strain culture samples The crude protein content of culture samples of Schizochytrium sp. CD01-1821 (and CD01-2147) was analyzed using the following method.

[0099] Specifically, each fermentation broth dry matter (sample) equivalent to approximately 20–30 mg of dried bacterial cells was precisely measured and placed in a digestion tube with two digestion accelerators. The digestion accelerator requires a sulfuric acid (H2SO4) to potassium sulfate (K2SO4) ratio of 1.4–2.0:1.0 for efficient digestion. Then, 12–15 mL of concentrated sulfuric acid (H2SO4) was added to the digestion tube and digested in a digestion apparatus at 420°C for 45–60 minutes until the digestion solution turned clear light blue (when using a copper catalyst) or clear yellow (when using a selenium catalyst). After cooling, 80 mL of distilled water was added to the digested test solution. 25 mL of the mixed indicator-mixed collection solution was placed in an Erlenmeyer flask and placed in a distillation apparatus. The Erlenmeyer flask stand was raised to allow the distillate to enter the collection solution during distillation. 50 mL of sodium hydroxide solution (NaOH) (equivalent to four times the amount of sulfuric acid used during digestion) was placed in the digestion tube and distilled for 3 to 4 minutes in a distillation apparatus. The collecting solution in the Erlenmeyer flask of the distillation apparatus was confirmed to turn green as it collected the ammonia (NH3) contained in the distillate. The distillate was titrated with hydrochloric acid solution (typically 0.1 N or 0.2 N) until the end point turned pale pink, and the amount of acid used in the titration was recorded. In the case of an automated apparatus, the distillation, titration, and calculation processes were all performed automatically. Using the experimental results, the nitrogen percentage was calculated using the following equation 2. The protein content was calculated by multiplying the nitrogen percentage calculated earlier by the average nitrogen coefficient of 6.25.

[0100] [Formula 2] Nitrogen (%) = {(HCl amount mL - blank test mL) × M × 14.01 / sample amount mg} × 100 *14.01: Atomic weight of nitrogen *M: Molar concentration of HCl *Decomposition accelerator: Kjeltabs or equivalent *Boric acid solution: 100 g (or 400 g) of H3BO3, 100 mL of 0.1% bromocresol green solution, and 100 mL of 0.1% methyl red solution, adjusted to a volume of 10 L to make a 1% (or 4%) boric acid solution.

[0101] For amino acid content analysis, approximately 1 g of dried fermentation broth (specimen) sample was taken from the CD01-1821 culture. After acid hydrolysis of intracellular proteins using a 6N HCl solution, the sample was diluted with distilled water, filtered, and subjected to liquid chromatography.

[0102] [Table 5]

[0103] As a result, as shown in Table 5, the crude protein content of the dried CD01-1821 cells was confirmed to be 17%. Furthermore, the amino acid content in the dried cells was highest for glutamic acid, followed by tyrosine, alanine, phenylalanine, glycine, arginine, serine, valine, lysine, aspartic acid, methionine, isoleucine, and leucine, in that order. This confirmed that the amino acids in the dried CD01-1821 cells consist of glutamic acid, tyrosine, alanine, phenylalanine, glycine, arginine, serine, valine, lysine, aspartic acid, methionine, isoleucine, and leucine.

[0104] From the above description, a person skilled in the art to which the present application pertains should understand that the present application can be embodied in other specific forms without changing its technical spirit or essential characteristics. 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 and variations derived from the meaning and scope of the claims below, rather than the above detailed description, and equivalent concepts.

[0105] [Accession number] Depository institution: Korea Center for Biological Resources (KCTC) Accession number: KCTC14660BP Date of acceptance: 20210823 JPEG0007827851000006.jpg245168

Claims

1. Schizochytrium sp. CD01-1821 microalgae deposited under accession number KCTC14660BP.

2. The Schizochytrium microalgae according to claim 1, wherein the Schizochytrium CD01-1821 microalgae has the ability to produce omega-3 unsaturated fatty acids.

3. 3. The Schizochytrium microalgae according to claim 2, wherein the omega-3 unsaturated fatty acids are docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

4. The microalgae of the genus Schizochytrium according to claim 3, wherein the microalgae produce 35 to 60% by weight of docosahexaenoic acid based on the total weight of fatty acids.

5. The microalgae of the genus Schizochytrium according to claim 3, wherein the microalgae produce 0.1 to 2% by weight of eicosapentaenoic acid based on the total weight of fatty acids.

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 composition comprising the biomass derived from the Schizochytrium microalgae according to claim 6, a concentrate or dried product of said biomass, or an extract of said biomass.

8. The composition of claim 7, wherein the composition is a feed or food composition.

9. A method for producing biomass derived from Schizochytrium microalgae, comprising: culturing the Schizochytrium CD01-1821 microalgae according to claim 1; and recovering docosahexaenoic acid-containing biomass from the microalgae, a dried product thereof, or a crushed product thereof.

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 biomass production method according to claim 9 , wherein the culture is carried out using a medium containing a carbon source and a nitrogen source.

12. The biomass production method according to claim 11, 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. 12. The 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 MSG (Monosodium glutamate).

14. A method for producing bio-oil derived from Schizochytrium microalgae, comprising: culturing the Schizochytrium CD01-1821 microalgae according to claim 1; and recovering docosahexaenoic acid-containing biomass from the microalgae, a dried product thereof, or a crushed product thereof.

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