TW202432824A - Novel strain of schizochytrium sp. strain and a method for producing oil containing arachidonic acid using the same - Google Patents

Abstract

The present application relates to a novel Schizochytrium sp. strain and a method for producing oil containing arachidonic acid using the same, and bio-oil produced by the novel Schizochytrium sp. strain according to one aspect has advantages of high flowability due to high contents of docosahexaenoic acid and eicosapentaenoic acid and a low content of palmitic acid, and comprises arachidonic acid which is an essential nutrient that plays an important role in development of a nervous system, so the microalgae, biomass or bio-oil produced therefrom can be usefully used as feed or food raw material, or the like.

Description

Novel strain of Schizophora strain and method for producing oil containing arachidonic acid using the same

The present invention relates to a novel strain of Schizophora species and a method for producing oil containing arachidonic acid using the strain. Cross-reference to related applications

This application is based on the benefit of priority claimed in Korean Patent Application No. 10-2023-0005633 filed on January 13, 2023, and the entire contents disclosed in the documents of the corresponding Korean Patent Application are incorporated as a part of this specification.

Thraustochytrids live and are distributed in a variety of environments in nature. They attach to organisms and live in symbiosis, or float in marine environments or freshwater and brackish water environments, or survive by being distributed in various sedimentary terrains. In this way, thraustochytrids belong to the lowest level of the marine ecological food chain and are also classified as heterotrophic protists as phytoplankton.

The omega-3s contained in thalassemia, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA), function as a source of supply for the marine ecosystem. In addition, docosahexaenoic acid and eicosapentaenoic acid are known to be essential fatty acids in the brain, eye tissues, and nervous system, and even play an important role in preventing cardiovascular disease. Arachidonic acid, which is present in similar amounts to docosahexaenoic acid in the brain, is known to be involved in the growth process of neurons and the development of the nervous system. Due to this role, supplementation with arachidonic acid in the early stages of Alzheimer's disease is known to reduce symptoms and slow the progression of the disease.

Most higher organisms cannot synthesize docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) by themselves, so they are essential nutrients and should be ingested from the outside. The main external source of the supply of docosahexaenoic acid and eicosapentaenoic acid is fish oil extracted from the oil of blueback fish (such as mackerel, saury, tuna, horse mackerel, sardines, anchovies, etc.), and this is very useful as a fish farming feed, such as a starter feed for marine fish. In the case of arachidonic acid, it is obtained from animal food when linoleic acid cannot be converted into arachidonic acid.

Polyunsaturated fatty acids extracted from fish oil are used in high proportions in the industry, but have many disadvantages: the quality of fish oil is affected by the fish species, the season and location of fishing, and the supply may be continuous and difficult depending on the amount of fish caught, and it may contain heavy metals, etc.

To address these issues, research is underway into producing unsaturated fatty acids including docosahexaenoic acid and eicosapentaenoic acid by culturing microorganisms rather than by providing omega-3 through fish catching.

Because fatty acids are synthesized using microalgae extracts through cultivation, they can be produced in a shorter time than unsaturated fatty acids extracted by catching fish, and because the quality is not affected by season or location, they can be prepared in a relatively constant composition. In addition, unsaturated fatty acids extracted using microalgae are composed of a simpler fatty acid composition than fish oil, so there is an advantage that the target product can be easily prepared.

Based on such advantages, the research and industrialization of polyunsaturated fatty acids including ω-3 unsaturated fatty acids such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), docosapentaenoic acid (DPA) and α-linolenic acid using microalgae have recently progressed very rapidly, and mainly, polyunsaturated fatty acids are produced using Thraustochytrium species and Schizophora species. For example, methods for preparing ω-3 polyunsaturated fatty acids using Schizobacterium sp. ATCC20888 and Schizobacterium sp. PTA10208, which are microorganisms of the genus Schizobacterium, have been disclosed (U.S. Patent No. 5,130,242), and further, methods for preparing docosahexaenoic acid and eicosapentaenoic acid using Thraustocystis sp. ATCC10212 (Thaustocystis sp. PTA10212), which is a thaumatocyst-based microorganism of the genus Thraustocystis, have been disclosed. Patent Documents

(Patent Document 1) U.S. Patent Publication No. US 5130242 A

Technical issues

One embodiment of the present application provides a novel Schizophora species microalgae. In a particular embodiment, the novel Schizophora species microalgae may be Schizophora species CD02-8025 strain, which is a microalgae deposited under the deposit number KCTC 15246BP and has the ability to produce ω-3 and ω-6 unsaturated fatty acids.

Another embodiment of the present application provides a biomass derived from a Schizozobacterium species strain, comprising the Schizozobacterium species strain, a culture of the strain, a dry substance of the culture, or a lysate of the dry substance.

Other embodiments of the present application provide a feed composition or a food composition comprising the Schizophrenia species strain, a culture of the strain, a dry substance of the culture, or a lysate of the dry substance.

Other embodiments of the present application provide a method for preparing biomass or bio-oil derived from the Schizochlum species strain.

Other embodiments of the present application provide a microbial agent for preparing docosahexaenoic acid, eicosapentaenoic acid, palmitic acid or arachidonic acid, which comprises the strain of the genus Schizophora or its culture solution as an active ingredient. Technical solution

Each description and implementation disclosed in this application is also applicable to each other description and implementation. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application should not be considered to be limited by the specific description described below. In addition, a person with ordinary knowledge in the art to which the invention belongs can identify or confirm many equivalents of the specific implementation of this application disclosed in this application using only ordinary experiments. In addition, it is intended to include such equivalents in this application.

One embodiment of the present application provides a novel Schizochytes species of microalgae.

As used in this description, the term "Schizogonium species" is one of the genus names belonging to the Thraustochytriaceae family of the Thraustochytriales order and is used interchangeably with the term "Schizogonium genus". In addition, the term "microalgae" means an organism that cannot be seen with the naked eye and can only be seen with a microscope, lives freely floating in the water, belongs to plants that photosynthesize with chlorophyll, and is also called phytoplankton.

As used in this description, the term "Schizogonium sp. strain" is used interchangeably with "Schizogonium sp. microalgae".

The novel Schizozombia sp. strain may be a mutant strain obtained by mutating a wild-type Schizozombia sp. strain into a parent strain.

As used in this description, the term "parent strain" means a strain before induction and can be used interchangeably with "wild-type strain".

The parent strain may be a wild-type Schizozombia species strain, and in particular may be a wild-type Schizozombia species CD01-5000 strain, but is not limited thereto.

As used in the present description, the term "productivity" means the ability to produce bio-oil or unsaturated fatty acids, and can be used interchangeably with "production capacity", and can be confirmed by measuring the content of bio-oil or unsaturated fatty acids contained in a culture solution or biomass derived from the strain after culturing the strain producing bio-oil or unsaturated fatty acids.

The mutation method can be carried out by various methods known in the art to which the invention belongs, and one of the physical or chemical induction methods can be used. For example, as a physical mutation method applicable to the present invention, a method of irradiating gamma rays or ultraviolet rays can be used, but it is not limited thereto. In addition, as a chemical mutation method, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), dibutylene oxide, ethyl methanesulfonate, mustard compounds, hydrazine and nitrous acid can be used, but it is not limited thereto.

The mutation used to prepare the mutant strain may be induced by irradiation.

The radiation used to induce the mutation may be gamma radiation or ultraviolet radiation, and preferably gamma radiation.

The radiation may be in a dose of 4.0 to 7.0 kGy, 4.5 to 7.0 kGy, 5.0 to 7.0 kGy, 4.0 to 6.5 kGy, 4.5 to 6.5 kGy, 5.0 to 6.5 kGy, 4.0 to 6.0 kGy, 4.5 to 6.0 kGy or 5.0 to 6.0 kGy, particularly 5.0 kGy, 5.5 kGy or 6.0 kGy per hour.

In the present application, as an implementation method, mutations are generated by irradiating the wild-type Schizophrenia sp. CD01-5000 strain with gamma rays, and at the same time, strains with high docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) contents and low palmitic acid (PA) content are selected from the mutant strains, and this is named Schizophrenia sp. CD02-8025, and was deposited with the international depository Korea Research Institute of Bioscience and Biotechnology Korea Collection for Type Culture (KCTC) on December 15, 2022 in accordance with the Budapest Treaty, and was given the deposit number KCTC15246BP.

Therefore, in the present description, the novel Schizochacella species microalgae may be Schizochacella species CD02-8025 strain, which is a microalgae deposited under the accession number KCTC15246BP.

The Schizobacterium sp. CD02-8025 strain may produce and/or contain high levels of ω-3 and ω-6 unsaturated fatty acids.

The ω-3 unsaturated fatty acid may be docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA), but is not limited thereto.

The ω-6 unsaturated fatty acid may be arachidonic acid (ARA), but is not limited thereto.

The Schizobacterium sp. strain CD02-8025 can produce less saturated fatty acids than known Schizobacterium sp. strains.

The saturated fatty acid may be palmitic acid (PA), but is not limited thereto.

The known Schizozobacterium sp. strain may be a wild-type Schizozobacterium sp. strain, and in one embodiment, it may be the Schizozobacterium sp. CD01-5000 strain.

As used in this description, the term "docosahexaenoic acid (DHA)" is one of the polyunsaturated fatty acids, which has a chemical formula of C ₂₂ H ₃₂ O ₂ and corresponds to ω-3 fatty acids together with α-linolenic acid (ALA) and eicosapentaenoic acid (EPA), and its common name is cervonic acid, and it can be represented by the abbreviation 22:6 n-3.

As used in the present description, the term "eicosapentaenoic acid (EPA)" is one of the polyunsaturated fatty acids, which has a chemical formula of C ₂₀ H ₃₀ O ₂ and corresponds to ω-3 fatty acids together with ALA and DHA, and which can be represented by the abbreviation C20:5 n-3.

As used in this description, the term "palmitic acid (PA)" is one of the _{polyunsaturated fatty acids, which has a chemical formula of C16H32O2 and can} be represented by the abbreviation C16:0.

As used in this description, the term "arachidonic acid (ARA)" is one of the polyunsaturated fatty acids, which has a chemical formula of C ₂₀ H ₃₂ O ₂ , and which can be represented by the abbreviation C20:4n6 or C20:4. .

The novel Schizozomula species strain can produce bio-oil containing arachidonic acid, and contains docosahexaenoic acid and eicosapentaenoic acid at high concentrations.

The Schizozobacterium species strain may produce and/or comprise 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, or 57% or more docosahexaenoic acid (DHA) by weight, based on the total weight of the fatty acids. For example, the Schizozobacterium species strain may produce and/or comprise 35 to 70%, 35 to 65%, 35 to 60%, 35 to 59%, 35 to 58%, 40 to 70%, 40 to 65%, 40 to 60%, 40 to 59%, 40 to 58%, 45 to 70%, 45 to 65%, 45 to 60%, 45 to 59%, 45 to 58 %, 50 to 70%, 50 to 65%, 50 to 60%, 50 to 59%, 50 to 58%, 53 to 70%, 53 to 65%, 53 to 60%, 53 to 59%, 53 to 58%, 55 to 70%, 55 to 65%, 55 to 60%, 55 to 59%, 55 to 58% or 56 to 58% by weight of docosahexaenoic acid (DHA).

The Schizozobacterium species strain may produce and/or comprise 0.8 wt % or more, 1 wt % or more, 1.5 wt % or more, 2 wt % or more, 2.5 wt % or more, 2.6 wt % or more, 2.7 wt % or more or 2.8 wt % or more eicosapentaenoic acid (EPA), based on the total weight of fatty acids. For example, the Schizozomula species strain may produce and/or comprise 0.5-10%, 0.5-9%, 0.5-8%, 0.5-7%, 0.5-6%, 0.5-5%, 1-10%, 1-9%, 1-8%, 1-7%, 1-6%, 1-5%, 1.5-10%, 1.5-9%, 1.5-8%, 1.5-7%, 1.5-6%, 1.5-5%, 2-10%, 2-9%, 2-8%, 2-7%, 2-6%, 2-5%, 2.5-10%, 2.5-9%, 2.5-8%, 2.5-7%, 2.5-6% or 2.5-5% eicosapentaenoic acid (EPA) based on the total weight of fatty acids.

The Schizozobacterium species strain may produce and/or comprise 30% or less, 29% or less, 25% or less, 20% or less, 19% or less, 18% or less, 17% or less, or 16% or less palmitic acid (PA) by weight, based on the total weight of the fatty acids. For example, the Schizozobacterium species strain may produce and/or comprise 0.1 to 30%, 0.1 to 29%, 0.1 to 25%, 0.1 to 20%, 0.1 to 19%, 0.1 to 18%, 0.1 to 17%, 0.1 to 16%, 1 to 30%, 1 to 29%, 1 to 25%, 1 to 20%, 1 to 19%, 1 to 18%, 1 %, 10 to 29 wt %, 5 to 25 wt %, 5 to 20 wt %, 5 to 19 wt %, 5 to 18 wt %, 5 to 17 wt %, 5 to 16 wt %, 10 to 30 wt %, 10 to 29 wt %, 10 to 25 wt %, 10 to 20 wt %, 10 to 19 wt %, 10 to 18 wt %, 10 to 17 wt %, or 10 to 16 wt % of palmitic acid (PA).

The Schizozobacterium species strain may produce and/or comprise 0.001 wt % or more, 0.01 wt % or more, 0.05 wt % or more, 0.1 wt % or more, 0.5 wt % or more, 0.6 wt % or more, 0.7 wt % or more, 0.8 wt % or more, 0.9 wt % or more, 1 wt % or more or 1.1 wt % or more arachidonic acid (ARA), based on the total weight of fatty acids. For example, the Schizozomula species strain may produce and/or comprise 0.001 to 10 wt%, 0.001 to 5 wt%, 0.001 to 4 wt%, 0.001 to 3 wt%, 0.001 to 2 wt%, 0.001 to 1.5 wt%, 0.01 to 10 wt%, 0.01 to 5 wt%, 0.01 to 4 wt%, 0.01 to 3 wt%, 0.01 to 2 wt%, 0.01 to 1.5 wt%, 0.1 to 10 wt%, 0.1 to 5 wt%, 0.1 to 4 wt%, 0.1 to 3 wt%, 0 .1 to 2 wt%, 0.1 to 1.5 wt%, 0.5 to 10 wt%, 0.5 to 5 wt%, 0.5 to 4 wt%, 0.5 to 3 wt%, 0.5 to 2 wt%, 0.5 to 1.5 wt%, 0.7 to 10 wt%, 0.7 to 5 wt%, 0.7 to 4 wt%, 0.7 to 3 wt%, 0.7 to 2 wt%, 0.7 to 1.5 wt%, 0.7 to 1.3 wt%, 1 to 10 wt%, 1 to 5 wt%, 1 to 4 wt%, 1 to 3 wt%, 1 to 2 wt% or 1 to 1.5 wt% arachidonic acid (ARA).

Another aspect of the present application provides biomass or bio-oil derived from a Schizozombia species strain, comprising the Schizozombia species strain, a culture of the strain, a dry substance of the culture, or a lysate of the dry substance.

The matters used for the Schizochlum species strain are the same as those described above.

As used in this description, the term "biomass" means an organism such as a plant, animal, microorganism or the like, which can be used as a source of chemical energy, i.e., bioenergy, and ecologically also means the weight or amount of energy of a specific organism present in a unit of time and space. In addition, the biomass includes compounds secreted by cells, but is not limited thereto, and may contain cells and/or intracellular contents as well as extracellular substances. In this application, the biomass may be the product itself produced by culturing or fermenting the Schizophrenia species strain (microorganism), its culture, its dry matter, its lysate or the strain, or may be a concentrate or dry matter of the biomass, but is not limited thereto.

The "culture" of the Schizozobacterium species microalgae refers to a product produced by culturing the strain, and specifically, it may be a culture solution containing the strain or a culture filtrate in which the strain is removed from the culture solution, but is not limited thereto.

The "dry matter" of the culture of the Schizozobacterium species strain may be one in which water is removed from the culture of the strain, and for example, it may be in the form of dried microbial cells of the strain, but is not limited thereto. In addition, "lysate" refers in general to the result of lysing the dry matter in which water is removed from the culture of the strain, and for example, it may be dried microbial cell powder, but is not limited thereto. The culture of the Schizozobacterium species strain may be produced by inoculating the strain in the culture medium of the strain according to a culturing method known in the art to which the invention belongs, and the dry matter of the culture and its lysate may also be prepared according to a treatment or drying method of a strain (microalgae) or a culture solution known in the art to which the invention belongs.

As used in this description, the term "bio-oil" means oil obtained from biomass by biological, thermochemical, and physical and chemical extraction procedures.

In the present description, the bio-oil may include an extract of biomass.

As a method for preparing the bio-oil extract, depending on the method for lysing or solubilizing cell membrane or cell wall components, a method for using enzymes such as protease, cellulase, pectinase or chitinase or the like, a method for physically lysing cell membrane or cell wall components using a homogenizer, an ultrasonic oscillator or bead treatment or the like, a method for extracting by directly adding a solvent and penetrating the cell interior, a solvent-free extraction procedure for separation by a centrifugation procedure after various lysis procedures or the like can be used, but are not limited thereto.

The biomass or bio-oil prepared in the present invention may contain ω-3 and ω-6 unsaturated fatty acids, and in particular, the ω-3 unsaturated fatty acids may be any one or more selected from the group consisting of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and palmitic acid (PA), and the ω-6 unsaturated fatty acids may be arachidonic acid (ARA), but are not limited thereto.

The biomass or bio-oil derived from a Schizozombia species strain may comprise 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, or 57% or more docosahexaenoic acid (DHA) by weight, based on the total weight of the fatty acids. For example, the biomass derived from a Schizozombia species strain may comprise 35 to 70%, 35 to 65%, 35 to 60%, 35 to 59%, 35 to 58%, 40 to 70%, 40 to 65%, 40 to 60%, 40 to 59%, 40 to 58%, 45 to 70%, 45 to 65%, 45 to 60%, 45 to 59%, 45 to 58% by weight, based on the total weight of the fatty acids. %, 50 to 70%, 50 to 65%, 50 to 60%, 50 to 59%, 50 to 58%, 53 to 70%, 53 to 65%, 53 to 60%, 53 to 59%, 53 to 58%, 55 to 70%, 55 to 65%, 55 to 60%, 55 to 59%, 55 to 58% or 56 to 58% by weight of docosahexaenoic acid (DHA).

The biomass or bio-oil derived from a Schizozomula species strain may comprise 0.8 wt % or more, 1 wt % or more, 1.5 wt % or more, 2 wt % or more, 2.5 wt % or more, 2.6 wt % or more, 2.7 wt % or more, or 2.8 wt % or more eicosapentaenoic acid (EPA), based on the total weight of fatty acids. For example, the biomass or bio-oil derived from a Schizozomula sp. strain may comprise 0.5-10%, 0.5-9%, 0.5-8%, 0.5-7%, 0.5-6%, 0.5-5%, 1-10%, 1-9%, 1-8%, 1-7%, 1-6%, 1-5%, 1.5-10%, 1.5-9%, 1.5-8%, 1.5-7%, 1.5-6%, 1.5-5%, 2-10%, 2-9%, 2-8%, 2-7%, 2-6%, 2-5%, 2.5-10%, 2.5-9%, 2.5-8%, 2.5-7%, 2.5-6% or 2.5-5% eicosapentaenoic acid (EPA) based on the total weight of fatty acids.

The biomass or bio-oil derived from the Schizozombia species strain may contain 30 wt% or less, 29 wt% or less, 25 wt% or less, 20 wt% or less, 19 wt% or less, 18 wt% or less, 17 wt% or less, or 16 wt% or less of palmitic acid (PA) based on the total weight of the fatty acids. For example, the biomass or bio-oil derived from the Schizozombia species strain may contain 0.1 to 30 wt%, 0.1 to 29 wt%, 0.1 to 25 wt%, 0.1 to 20 wt%, 0.1 to 19 wt%, 0.1 to 18 wt%, 0.1 to 17 wt%, 0.1 to 16 wt%, 1 to 30 wt%, 1 to 29 wt%, 1 to 25 wt%, 1 to 20 wt%, 1 to 19 wt%, 1 to 18 wt% %, 1 to 17 wt %, 1 to 16 wt %, 5 to 30 wt %, 5 to 29 wt %, 5 to 25 wt %, 5 to 20 wt %, 5 to 19 wt %, 5 to 18 wt %, 5 to 17 wt %, 5 to 16 wt %, 10 to 30 wt %, 10 to 29 wt %, 10 to 25 wt %, 10 to 20 wt %, 10 to 19 wt %, 10 to 18 wt %, 10 to 17 wt % or 10 to 16 wt % of palmitic acid (PA).

The biomass or bio-oil derived from a Schizozomyces sp. strain may comprise 0.001 wt % or more, 0.01 wt % or more, 0.05 wt % or more, 0.1 wt % or more, 0.5 wt % or more, 0.6 wt % or more, 0.7 wt % or more, 0.8 wt % or more, 0.9 wt % or more, 1 wt % or more, or 1.1 wt % or more arachidonic acid (ARA), based on the total weight of fatty acids. For example, the biomass or bio-oil derived from a Schizozomula sp. strain may comprise 0.001 wt % or more, 0.01 wt % or more, 0.05 wt % or more, 0.1 wt % or more, 0.5 wt % or more, 0.6 wt % or more, 0.7 wt % or more, 0.8 wt % or more, 0.9 wt % or more, 1 wt % or more, or 1.1 wt % or more arachidonic acid (ARA), based on the total weight of fatty acids. For example, the biomass or bio-oil derived from a Schizozomula species strain may comprise 0.001 to 10 wt%, 0.001 to 5 wt%, 0.001 to 4 wt%, 0.001 to 3 wt%, 0.001 to 2 wt%, 0.001 to 1.5 wt%, 0.01 to 10 wt%, 0.01 to 5 wt%, 0.01 to 4 wt%, 0.01 to 3 wt%, 0.01 to 2 wt%, 0.01 to 1.5 wt%, 0.1 to 10 wt%, 0.1 to 5 wt%, 0.1 to 4 wt%, 0.1 to 3 wt% based on the total weight of the fatty acids. %, 0.1 to 2 wt%, 0.1 to 1.5 wt%, 0.5 to 10 wt%, 0.5 to 5 wt%, 0.5 to 4 wt%, 0.5 to 3 wt%, 0.5 to 2 wt%, 0.5 to 1.5 wt%, 0.7 to 10 wt%, 0.7 to 5 wt%, 0.7 to 4 wt%, 0.7 to 3 wt%, 0.7 to 2 wt%, 0.7 to 1.5 wt%, 0.7 to 1.3 wt%, 1 to 10 wt%, 1 to 5 wt%, 1 to 4 wt%, 1 to 3 wt%, 1 to 2 wt% or 1 to 1.5 wt% arachidonic acid (ARA).

Another aspect of the present application provides a composition comprising the Schizochacella sp. CD02-8025 strain, a culture of the strain, a dry substance of the culture, or a lysate of the dry substance.

The composition may comprise the biomass or bio-oil derived from the Schizozombus species strain or a combination thereof.

The materials used for the Schizozobacterium species strain, biomass, bio-oil, culture of the strain, dry matter of the culture and lysate of the dry matter are as described above.

The composition may be in the form of a solution, powder or suspension, but is not limited thereto. The composition may be, for example, a food composition, a feed composition or a feed supplement composition.

As used in the present description, the term "feed composition" refers to feed fed to animals. The feed composition refers to a substance that supplies organic or inorganic nutrients required for maintaining the life of an animal or producing meat, milk, etc. The feed composition may additionally contain nutritional components required for maintaining the life of an animal or producing meat, milk, etc. The feed composition may be prepared in the form of various feeds known in the art, and in particular, it may include concentrated feeds, roughages and/or special feeds.

As used in this description, the term "feed additive" includes substances to be added to feed for various purposes, such as supplementing nutrients and preventing weight loss, enhancing the digestibility of fiber in feed, improving milk quality, preventing reproductive failure and increasing fertility, preventing summer heat stress, etc. The feed additive in this application corresponds to the Control of Livestock and Fish Feed Act. The invention relates to a supplementary feed for feeding young animals under the Food and Drug Administration (FDA) and may further include mineral preparations (such as sodium bicarbonate, bentonite, magnesium oxide, complex minerals, etc.), mineral preparations in the form of trace amounts of trace substances (such as zinc, copper, cobalt, selenium, etc.), vitamin preparations (such as carotene, vitamin E, vitamins A, D, E, niacin, vitamin B complex, etc.), protected amino acid preparations (such as methionine, lysine, etc.), protected fatty acid preparations (such as fatty acid calcium salts, etc.), live cells and yeast preparations (such as probiotics (lactic acid bacteria preparations), yeast cultures, fungal fermentation materials, etc.).

As used in this description, the term "food composition" includes all forms, such as functional foods, nutritional supplements, health foods and food additives, etc., and the food compositions in the above forms can be produced in various forms according to common methods known in the art to which the invention belongs.

The composition of the present application may further include grains, such as powdered or crushed wheat, oats, barley, corn and rice; plant protein feed, such as feed containing soybean and sunflower as main components; animal protein feed, such as blood meal, meat meal, bone meal and fish meal; sugar and dairy products, such as dry components composed of various types of milk powder and whey powder, etc., and in addition to the above, it may further include dietary supplements, digestion and absorption enhancers, growth promoters, etc.

The composition of the present application can be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the composition can be easily administered to animals as a topping or mixed directly in the feed or as an oral formulation separated from the feed. When the composition is administered separately from the feed, it can be produced in the form of an immediate release or sustained release formulation in combination with a pharmaceutically acceptable edible carrier, as is well known in the art to which the invention belongs. This edible carrier can be a solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the composition can be a tablet, capsule, buccal tablet or lozenge or a topping in a microdispersible form. When a liquid carrier is used, the composition may be a formulation in a gelatin soft capsule or a syrup or a suspension, an emulsion or a solution.

The composition of the present application may contain, for example, a preservative, a stabilizer, a wetting agent or an emulsifier, a cryoprotectant or a shaping agent, etc. The cryoprotectant may be at least one selected from the group consisting of glycerol, honey, maltodextrin, skimmed milk powder and starch.

The preservative, stabilizer or excipient may be included in the composition in an amount effective enough to reduce the deterioration of the Schizozoon species microalgae contained in the composition. In addition, the cryoprotectant may be included in an amount effective enough to reduce the deterioration of the Schizozoon species microalgae contained in the composition when the composition is in a dry condition.

The composition can be applied by adding to the animal's feed by immersion, spraying or mixing.

The composition of the present application can be applied to the diet of some animals, including mammals, birds, fish, crustaceans, cephalopods, reptiles and amphibians, but not limited thereto. For example, the mammals can include pigs, cattle, sheep, goats, laboratory rodents or pets, and the birds can include poultry, and the poultry can include chickens, turkeys, ducks, geese, pheasants or quails, etc., but not limited thereto. In addition, the fish can include commercially farmed fish and their fry, ornamental fish, etc., and the crustaceans can include shrimps, rattan pots, etc., but not limited thereto. In addition, the composition can also be applied to the diet of rotifers (which are zooplankton).

Other aspects of the present application provide a method for preparing biomass derived from a Schizophrenia sp. strain, comprising culturing the Schizophrenia sp. CD02-8025 strain; and recovering biomass from the strain, a culture of the strain, a dry material of the culture, or a lysate of the dry material.

The Schizozobacterium species strain, biomass, culture of the strain, dry matter of the culture and lysate of the dry matter are the same as those described above.

As used in this description, the term "culture" means growing the strain under suitable controlled environmental conditions. The culture procedure of this application can be carried out according to suitable culture media and culture conditions known in the art to which the invention belongs. Such a culture procedure can be easily adjusted and used by a person of ordinary skill in the art to which the invention belongs based on the strain to be selected.

Specifically, the culture of the Schizophrenia species strain of the present application can be carried out under heterotrophic conditions, but is not limited thereto.

As used in this description, the term "heterotrophic" is a nutritional method that relies on organic substances obtained from outside the body for energy sources or nutrient sources, and is a term corresponding to autotrophic organisms, and it can be used interchangeably with the term "dark culture".

The culturing of the Schizophrenia species strain can be performed by known batch culture methods, continuous culture methods, fed batch culture methods, etc., but is not particularly limited thereto. For the culture medium and other culture conditions used to culture the strain of the present application, any one can be used without particular limitation as long as it is a common culture medium for culturing microalgae. Specifically, the strain of the present application can be cultured in a common culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and/or vitamin, etc., by adjusting the temperature, pH, etc.

Specifically, a suitable pH (e.g., pH 5 to 9, specifically pH 6 to 8, most specifically pH 6.8) can be adjusted using an alkaline compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid), but not limited thereto.

Furthermore, in order to maintain aeration conditions for culture, oxygen or oxygen-containing gas may be injected into the culture, or for anaerobic and non-aeration conditions, no gas may be injected or nitrogen, hydrogen or carbon dioxide gas may be injected into the culture, but is not limited thereto.

In addition, 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. In addition, during the culture period, an antifoaming agent (such as fatty acid polyglycol ester, but not limited thereto) may be used to suppress the generation of bubbles.

The carbon source contained in the culture medium used for culturing the Schizozombia species strain 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 for culturing microalgae.

The nitrogen source contained in the culture medium used for culturing the Schizozombia species strain may be i) at least one organic nitrogen source selected from the group consisting of yeast extract, beef extract, peptone and pancreas, or ii) at least one inorganic nitrogen source selected from the group consisting of ammonium acetate, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, urea and MSG (monosodium glutamate), but is not limited thereto, as long as it is a nitrogen source for culturing microalgae.

The culture medium used for culturing the Schizozombia species strain may contain potassium dihydrogen phosphate, potassium hydrogen phosphate, sodium salts corresponding thereto, etc. as a phosphorus source, individually or in combination, but is not limited thereto.

The biomass recovery from the strain, the culture of the strain, the dry matter of the culture or the lysate of the dry matter can be collected using a suitable method known in the art to which the invention belongs. For example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC etc. can be used, and it can further include a purification procedure.

Other aspects of the present application provide a method for preparing a bio-oil derived from a Schizophrenia sp. strain, comprising culturing the Schizophrenia sp. CD02-8025 strain; and recovering lipids from the strain, a culture of the strain, a dry substance of the culture, or a lysate of the dry substance.

The culture of the Schizozombus species strain, the biological oil, the culture of the strain, the dry matter of the culture, the lysate of the dry matter, and the culture of the strain are the same as those described above.

The lipids recovered from the strain, the culture of the strain, the dried material of the culture or the lysate of the dried material can be collected using a suitable method known in the art to which the invention belongs. For example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC etc. can be used, and it can further include a purification procedure.

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. Such lipids and lipid derivatives can also be extracted using methods such as liquefaction, oil liquefaction, and supercritical _CO2 extraction. In addition, known methods for recovering microalgae lipids include, for example, the following methods: i) collecting cells by centrifugation and washing with distilled water, and then drying by freeze drying and ii) pulverizing the obtained cell powder and then extracting lipids with n-hexane (Miao, X and Wu, Q, Biosource Technology (2006) 97: 841-846).

Other aspects of the present application provide a microbial agent for preparing docosahexaenoic acid, eicosapentaenoic acid, palmitic acid or arachidonic acid, which comprises the strain CD02-8025 of the genus Schizophora or its culture solution as an active ingredient. Beneficial effects

Because the bio-oil produced by the novel Schizophora species strain having the ability to produce ω-3 and ω-6 unsaturated fatty acids of the present invention has the advantage of high fluidity due to the high content of unsaturated fatty acids of docosahexaenoic acid and eicosapentaenoic acid and low content of palmitic acid (which is a saturated fatty acid), it is easy to store and use, and therefore the strain itself, the biomass produced by culturing and fermenting the strain, its concentrate and extract can be usefully used as a feed component or food component or the like. In addition, because the bio-oil produced by the strain contains arachidonic acid, which is an essential nutrient that plays an important role in the development of the nervous system, it has the advantage of being a feed component or food component.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to be used to illustratively describe at least one specific embodiment, and the scope of the present invention is not limited to these examples. Example 1. Development of novel artificial mutant strains of Schizophora microalgae

The artificial mutant strain irradiated with gamma rays was developed from the purified wild-type Schizochlum sp. CD01-5000 strain (Deposit No.: KCTC 14344BP) (Korean Patent Publication No. 10-2022-0080594) by the following method.

Specifically, the isolated Schizochlum sp. CD01-5000 strain (Deposit No.: KCTC 14344BP) was cultured in a modified GYEP medium (glucose 10 g/L, yeast extract 1 g/L, peptone 1 g/L, MgSO ₄ ∙7H ₂ O 2 g/L, H ₃ BO ₃ 5.0 mg/L, MnCl ₂ 3.0 mg/L, CuSO ₄ 0.2 mg/L, NaMo ₄ ∙2H ₂ O 0.05 mg/L, CoSO ₄ 0.05 mg/L, ZnSO ₄ ∙7H ₂ O 0.7 mg/L) culture medium for about 24 hours or more to allow the early exponential growth phase to be reached, and then the culture solution sample is centrifuged to harvest the microbial cells. The harvested microbial cells are suspended in a 0.1 M phosphate buffer solution containing NaCl 1.0% so that the cell number becomes about 10 ⁹ cells/mL, and used for gamma irradiation.

The gamma irradiation experiment was conducted at the Korean Atomic Energy Research Institute Advanced Radiation Technology Institute, and irradiated with a dose of 5.5 kGY of gamma rays. The microalgae culture solution sample irradiated with gamma rays was cultured in a GYEP medium containing 20 g/L of agar and octyl gallate to inhibit the synthesis of fatty acids, and then passed through an O/N recovery process in a dark room. The microalgae colonies that grew during the culture period of about 2 weeks were selected and subcultured under the same culture medium and culture environment conditions. Strains that can grow continuously between subcultures and have excellent colony growth were preferentially selected. In addition, colonies that are red in morphology and grow rapidly were selected. The selected colonies were isolated and cultured as a single cell line, and the corresponding strain was named CD02-8025 strain, and deposited to the Korea Research Institute of Bioscience and Biotechnology Korea Collection for Type Culture (KCTC) on December 15, 2022, and was given the deposit number KCTC15246BP. Example 2. Confirmation of the culture characteristics of the mutant Schizophrenia species CD02-8025 strain Example 2-1. Analysis of crude fat and fatty acid content of the initial culture solution of CD02-8025 strain

In order to confirm the culture characteristics of the mutant Schizophrenia sp. CD02-8025 strain selected in Example 1, fermentation culture evaluation was performed.

Specifically, before the main culture on a 30 L scale, in order to confirm the crude fat and fatty acid content of the Schizophrenia sp. CD02-8025 strain compared to the wild-type strain, it was inoculated in a GYEP medium containing a working volume of 50 ml and 30 g/L of glucose in a 250 ml flask, and cultured in a shaking incubator at 30°C and 180 rpm for about 48 hours. The culture solution in which the preliminary culture was completed was dried so that the moisture content became 5 to 8%, and then the crude fat and fatty acid content was analyzed and is shown in the following Table 1. Table 1 Results of analysis of crude fat and fatty acids in the flask-level culture solution of the novel CD02-8025 mutant strain Strain name Sample Crude fat content (%) Fatty acid content in crude fat (%) C16:0 C20:3n3 EPA DPA DHA Omega-3 CD02-8025 1 16.75 12.85 0.45 4.61 14.32 52.13 56.74 2 18.67 12.64 0.50 5.61 12.54 42.91 48.52 3 17.42 13.96 0.52 5.04 13.30 44.53 49.57 4 15.33 15.47 0.24 3.54 12.08 38.92 42.45 5 14.86 15.26 0.22 3.33 11.70 37.81 41.14 6 15.16 18.92 0.19 2.55 11.73 42.87 45.41 7 15.75 18.07 0.23 3.39 11.68 42.31 45.70 8 10.88 16.97 0.23 3.88 13.37 42.06 45.94 9 13.86 16.39 0.21 3.71 12.62 40.99 44.70

As a result, as shown in Table 1, it was confirmed that the flask-level culture solution of the CD02-8025 strain of the genus Schizophrenia had a high ω-3 content, and in particular, it had a high content of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Example 2-2. Analysis of crude fat and fatty acid content of culture solution samples of CD02-8025 mutant strain and wild-type CD01-5000 strain

The strain of the genus CD02-8025 of the genus Schizophora, which was confirmed to have a high ω-3 content in Example 2-1, was inoculated in a 30 L fermenter containing a culture medium under the same conditions and fermented and cultured with a total working volume of 20 L. Glucose corresponding to 20% of the working volume was continuously added and used for cell culture under the conditions of 30°C, 500 rpm, 0.5-1 vvm, pH 5-7, and injected so that the glucose concentration was maintained at a level of 20 g/L at this time. When the glucose (the supplied carbon source) was completely consumed, the culture was terminated. The fermented solution in which the culture was completed was dried using a freeze dryer so that the moisture content became about 5 to 8%, and the obtained dry microbial cells were used to analyze the crude fat and fatty acid content.

In order to analyze the lipid and polyunsaturated fatty acid contents in cells of wild-type Schizobacter sp. CD01-5000 strain and mutant Schizobacter sp. CD02-8025 strain, the following method was used for the experiment.

Specifically, the following procedure: 8.3 M hydrochloric acid solution was applied to each of 2 g of the obtained dried microalgae microbial cells at 80°C to hydrolyze the cell wall of the microalgae microbial cells, and then 30 mL of ether and 20 mL of petroleum ether were added and mixed for 30 seconds, and then centrifugation was repeated 3 times or more. The separated solvent layer was recovered and moved to a flask whose weight had been measured previously, and then nitrogen was injected to remove the solvent, and cooled and weighed in a desiccator. The total oil content was calculated by measuring the weight of the dried oil. The DHA content contained in the oil was measured by gas chromatography with pre-treated methanol 0.5 N NaOH and 14% trifluoroborane methanol (BF3). The measured results are shown in Table 2 below. ■ Total oil content (%, *g oil/g dry microbial cell weight x 100) *g oil: flask weight after acid hydrolysis and solvent removal – empty flask weight Table 2 Evaluation of cultures of novel CD02-8025 strain and wild-type CD01-5000 strain and crude fat analysis results Schizochlum species CD01-5000 Schizochlum sp.CD02-8025 Cultivation time ( hr ) 36 53.6 OD ( 680nm ) 158.4 163 DCW ( g/L ) 115.1 104.1 Crude fat content in biomass ( % ) 46.2 34.6 Fatty acids in crude fat ( % ) - C16:0 30.3 16 - C20:5 n-3 1 4.3 - C22:6 n-3 50.3 57.1

As a result, as shown in Table 2, the wild-type CD01-5000 strain and the mutant CD02-8025 strain produced biomass at a level of about 100 – 120 g/L. In particular, it was confirmed that the biomass produced by the CD02-8025 strain contained a content that was reduced by about 47.2% compared to the wild-type strain, as the content of palmitic acid (C16:0) in crude fat was shown to be 16%. In addition, it was confirmed that it contained a higher content of ω-3 components, as the EPA (C20:5 n-3) content was 430% or higher and the DHA (C22:6 n-3) content was 13.5% or higher compared to the biomass derived from the wild-type strain. Example 3 Analysis of crude fat and fatty acid content of oil extracts of CD02-8025 mutant strain and wild-type CD01-5000 strain

After preparing the oil extracts of the wild-type CD01-5000 strain and the mutant CD02-8025 strain of the present invention, the crude fat and fatty acid contents were analyzed by the following method.

Specifically, for the mutant strain CD02-8025 strain and the wild-type CD01-5000 strain, a 5 L scale fermentation culture was performed. The main culture of the 5 L scale was carried out under the conditions of 30°C, 800 rpm, 1 vvm, and glucose as a carbon source was supplied by a fed batch method. The microbial cells after culture were harvested and the two strains were analyzed for 37 FAME fatty acids by oil extraction, and the results are shown in the following Table 3. Table 3 Comparison of fatty acid content of oil extracts of mutant CD02-8025 strain and wild-type CD01-5000 strain Fatty acid type Schizochlum species CD01-5000 Schizochlum sp.CD02-8025 Fatty acid content ( % ) Fatty acid content ( % ) C14:0 1.61 ND C16:0 29.29 14.64 C18:0 1.04 0.86 C20:0 0.31 0.49 C20:3n6 0.32 ND C20:3n3 1.25 0.60 C20:4 ND 1.10 C22:2 0.54 0.76 C20:5 0.79 2.90 C22:6 50.13 57.08

As a result of comparing the fatty acid composition of the oil extract of each strain, as shown in Table 3, it was confirmed that it showed a tendency similar to the result of the analysis of the crude fat of the dry biomass. In other words, in the case of the oil extract, the content of palmitic acid (C16:0) of the oil extract of the Schizochlum sp. CD02-8025 strain was also lower than that of the wild-type strain oil extract, and also showed that the content of EPA (C20:5 n-3) and DHA (C22:6 n-3) was higher than that of the wild-type strain oil extract. It is known that when the palmitic acid content is high, it solidifies at room temperature, and when the palmitic acid content is low, it maintains the form of oil, so the lower the palmitic acid content, the higher the fluidity of the oil during oil extraction, and therefore it is easy to store and use.

Therefore, through the above results, it can be seen that the novel Schizozomula species CD02-8025 strain of the present invention can produce bio-oil with high fluidity, and the produced bio-oil has the advantages of being easy to store and use due to its low palmitic acid content.

In addition, as a result of comparing the arachidonic acid content in the oil extracts of the mutant Schizozobacterium species CD02-8025 strain and the wild-type Schizozobacterium species CD01-5000 strain, as shown in Table 3, it was confirmed that arachidonic acid (C20:4) that was not contained in the oil extract of the wild-type CD01-5000 strain was contained in the oil extract of the mutant CD02-8025 strain at 1.1%.

It is known that arachidonic acid is an essential nutrient that plays an important role in the development of the nervous system, and therefore, it can be seen that the novel Schizochlum sp. CD02-8025 strain of the present invention has the advantage of being a food or feed composition by containing arachidonic acid, which is an essential nutrient required for the development of the nervous system.

Based on the above description, a person with ordinary knowledge in the technical field to which this application belongs will be able to understand that this application can be implemented in other special forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as being illustrative and not restrictive in all aspects. The scope of this application should be interpreted as all changes or modifications of the meaning and scope of the patent scope described below, in addition to the above detailed description, and their equivalents are also included in the scope of this application.

without

without

Depository name: Korea Culture Center (KCTC), Korea Institute of Bioscience and Biotechnology Deposit number: KCTC15246BP Deposit date: 20221215

Claims (15)

A Schizochlum sp. CD02-8025 strain is a microalgae deposited with the deposit number KCTC15246BP and has the ability to produce ω-3 and ω-6 unsaturated fatty acids. The Schizozobacterium species strain of claim 1, wherein the ω-3 unsaturated fatty acids are docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The Schizozobacterium species strain of claim 1, wherein the ω-6 unsaturated fatty acid is arachidonic acid (ARA). The Schizozobacterium sp. strain of claim 1, wherein the strain produces 35 to 70 wt % of docosahexaenoic acid based on the total weight of fatty acids. The Schizozobacterium sp. strain of claim 1, wherein the strain produces 0.5 to 10 wt % of eicosapentaenoic acid based on the total weight of fatty acids. The Schizozobacterium species strain of claim 1, wherein the strain produces 10 to 20 wt % palmitic acid based on the total weight of fatty acids. The Schizozobacterium sp. strain of claim 1, wherein the strain produces 0.1 to 5 wt % arachidonic acid based on the total weight of fatty acids. A biomass derived from a strain of the genus Schizophrenia, comprising the strain of any one of claims 1 to 7, a culture solution of the strain, a dry substance of the culture solution, or a lysate of the dry substance. The biomass of claim 8, wherein the biomass comprises arachidonic acid. The biomass of claim 9, wherein the biomass comprises 0.1 to 5 wt % arachidonic acid based on the total weight of fatty acids. The biomass of claim 8, wherein the biomass comprises 0.1 to 20 wt % of palmitic acid based on the total weight of fatty acids. A feed composition comprising the strain of claim 1, a culture of the strain, a dry matter of the culture, or a lysate of the dry matter. A food composition comprising the strain of claim 1, a culture of the strain, a dry substance of the culture, or a lysate of the dry substance. A method for preparing biomass derived from a strain of the genus Schizophrenia, comprising: Cultivating a strain as claimed in any one of claims 1 to 7; and Recovering biomass from the strain, a culture thereof, a dry substance of the culture, or a lysate of the dry substance. A method for preparing biomass derived from a strain of the genus Schizophrenia, comprising: Cultivating a strain as claimed in any one of claims 1 to 7; and Recovering lipids from the strain, a culture thereof, a dry substance of the culture, or a lysate of the dry substance.