Oil that does not become cloudy at low temperatures, and method for producing the same

Abstract

To provide an oil which does not become cloudy when stored at a low temperature while containing a specific amount of palmitic acid.SOLUTION: An oil containing a specific amount of palmitic acid has at least one characteristic selected from the group consisting of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (iv) a predetermined content of C18 fatty acids, and (v) a predetermined DHA / EPA ratio. The oil does not become cloudy during low-temperature storage.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to oils that do not develop haze when stored at low temperatures and methods for making same. [Background technology]

[0002] Oils contain various fatty acid compositions depending on their origin. Animal oils such as beef tallow and lard contain approximately 40% or more saturated fatty acids and are typically solid at room temperature. Vegetable oils, on the other hand, typically contain several percent to over 20% saturated fatty acids, though this varies greatly depending on the source material. While fish oil contains approximately 30% saturated fatty acids, it also contains significant amounts of monounsaturated and polyunsaturated fatty acids and is typically liquid at room temperature. Liquid edible oils are widely used for cooking. If liquid edible oils become cloudy at low temperatures, such as winter temperatures, their appearance and other commercial value are impaired. Therefore, a cooling test for salad oils is specified in the Japanese Agricultural Standards. Heat-resistant liquid cooking oils that meet the cooling test criteria (i.e., remaining clear in ice water at 0°C for at least five and a half hours) have been developed (Patent Document 1: JP 61-249344 A). Furthermore, a technique has been developed for concentrating desired unsaturated fatty acids by wintering at low temperatures (Patent Document 2: Japanese Patent Application Laid-Open No. 10-176181, Patent Document 3: Japanese Patent Application Laid-Open No. 2-268133).

[0003] Edible oils contain saturated fatty acids such as palmitic acid (16 carbon atoms), myristic acid (14 carbon atoms), and stearic acid (18 carbon atoms). Oils containing a high concentration of saturated fatty acids have a higher melting point than oils containing unsaturated fatty acids, resulting in solidification at low temperatures and cloudiness. Palmitic acid, the most abundant saturated fatty acid in living organisms, is produced by chain elongation of acetyl-CoA and malonyl-CoA via the action of fatty acid synthase, a reaction common to a wide variety of organisms. As a result, palmitic acid is typically found in large amounts in biologically derived oils, including fish oils, vegetable oils, animal oils, and microbial oils. Palmitic acid is also the most abundant fatty acid in the human body.

[0004] It has been reported that high intakes of saturated fatty acids and trans fatty acids increase the risk of cardiovascular disease, while polyunsaturated fatty acids have been reported to improve blood lipid levels. Therefore, the need to pay attention to the fatty acid composition of ingested oils has been publicized, and the intake of oils rich in polyunsaturated fatty acids is recommended. Because omega-3 unsaturated fatty acids such as DHA and EPA have excellent physiological effects, attempts have been made to produce oils containing high concentrations of omega-3 unsaturated fatty acids. One such production method is a method for producing microbial oil using a strain that highly produces omega-3 unsaturated fatty acids (Patent Document 4: JP 2020-72676 A, Patent Document 5: JP 2022-126707 A, Patent Document 6: JP 2023-403200 A), and a method for concentrating unsaturated fatty acids has been developed (Patent Document 7: WO 2012 / 118173 A). A method for producing ω-3 unsaturated fatty acids as a solid fat composition has also been disclosed (Patent Document 8: JP-A-2009-500022). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 61-249344 [Patent Document 2] Japanese Patent Application Publication No. 10-176181 [Patent Document 3] Japanese Patent Application Publication No. 2-268133 [Patent Document 4] Japanese Patent Publication No. 2020-72676 [Patent Document 5] Japanese Patent Publication No. 2022-126707 [Patent Document 6] Special Publication No. 2023-403200 [Patent Document 7] International Publication No. 2012 / 118173 [Patent Document 8] Special Publication No. 2009-500022 Summary of the Invention [Problem to be solved by the invention]

[0006] Oils containing a certain amount of palmitic acid become cloudy when exposed to low temperatures and have poor low-temperature stability. Furthermore, oils containing a certain amount of palmitic acid may produce precipitates when exposed to low temperatures, making it difficult to maintain a homogeneous state and making them difficult to handle. Therefore, the objective of the present disclosure is to provide an oil that contains a certain amount of palmitic acid and does not become cloudy when exposed to low temperatures, as well as to provide a method for producing such an oil. [Means for solving the problem]

[0007] The present disclosure is based on the discovery that, when the oil contains a predetermined content of palmitic acid and has a predetermined composition of other fatty acids in the oil, the oil can be stored at low temperatures, for example, cooled to 0°C for 3 hours without becoming cloudy.

[0008] The specified palmitic acid content refers to palmitic acid present in an amount of 7% to 20%, 8% to 19%, or 9% to 18% by weight of the total weight of fatty acids in the oil. Palmitic acid is a fatty acid found in various oils and exhibits antioxidant properties. However, because palmitic acid is a saturated fatty acid, increasing its content reduces the low-temperature stability of the oil. For example, the palmitic acid content can be adjusted by wintering.

[0009] The oil disclosed herein contains a predetermined amount of palmitic acid (C16:0) and further has at least one characteristic selected from the group consisting of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (iv) a predetermined C18 fatty acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content, and does not become cloudy when cooled at 0°C for 3 hours. These characteristics may be used alone or in combination. For example, among characteristics (i) to (vi), the oil may be a combination of any two, any three, any four, any five, or all six of the characteristics.

[0010] When two characteristics are combined, any combination may be used. Examples include a combination of (i) a predetermined triglyceride content and (ii) a predetermined PUFA content, a combination of (i) a predetermined triglyceride content and (iii) a predetermined stearic acid content, a combination of (i) a predetermined triglyceride content and (iv) a predetermined 18 carbon fatty acid content, a combination of (i) a predetermined triglyceride content and (v) a predetermined DHA / EPA ratio, a combination of (i) a predetermined triglyceride content and (vi) a predetermined DHA content, a combination of (ii) a predetermined PUFA content and (iii) a predetermined stearic acid content, a combination of (ii) a predetermined PUFA content and (iv) a predetermined 18 carbon fatty acid content, ( Examples of such a combination include ii) a combination of a predetermined PUFA content and (v) a predetermined DHA / EPA ratio, a combination of (ii) a predetermined PUFA content and (vi) a predetermined DHA content, a combination of (iii) a predetermined stearic acid content and (iv) a predetermined C18 fatty acid content, a combination of (iii) a predetermined stearic acid content and (v) a predetermined DHA / EPA ratio, a combination of (iii) a predetermined stearic acid content and (vi) a predetermined DHA content, a combination of (iv) a predetermined C18 fatty acid content and (v) a predetermined DHA / EPA ratio, a combination of (iv) a predetermined C18 fatty acid content and (vi) a predetermined DHA content, and a combination of (v) a predetermined DHA / EPA ratio and (vi) a predetermined DHA content.

[0011] When the three characteristics are combined, any combination may be used. Examples include a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, and (iii) a predetermined stearic acid content, a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, and (iv) a predetermined content of a fatty acid having 18 carbon atoms, a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, and (v) a predetermined DHA / EPA ratio, and a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, and (vi) a predetermined combinations of (i) a predetermined triglyceride content with (iii) a predetermined stearic acid content with (iv) a predetermined 18 carbon fatty acid content; combinations of (i) a predetermined triglyceride content with (iii) a predetermined stearic acid content with (v) a predetermined DHA / EPA ratio; combinations of (i) a predetermined triglyceride content with (iii) a predetermined stearic acid content with (vi) a predetermined DHA content; combinations of (i) a predetermined triglyceride content with (iv) a predetermined 18 carbon fatty acid content with (v) a predetermined DHA / EPA ratio; (i) A combination of a predetermined triglyceride content and (iv) a predetermined 18-carbon fatty acid content and (vi) a predetermined DHA content; a combination of (i) a predetermined triglyceride content and (v) a predetermined DHA / EPA ratio and (vi) a predetermined DHA content; a combination of (ii) a predetermined PUFA content and (iii) a predetermined stearic acid content and (iv) a predetermined 18-carbon fatty acid content; a combination of (ii) a predetermined PUFA content and (iii) a predetermined stearic acid content and (v) a predetermined DHA / EPA ratio; a combination of (ii) a predetermined PUFA content and (iii) a predetermined stearic acid content and (v) a predetermined DHA / EPA ratio; a combination of (ii) a predetermined PUFA content with (iv) a predetermined 18 carbon fatty acid content and (v) a predetermined DHA / EPA ratio; a combination of (ii) a predetermined PUFA content with (iv) a predetermined 18 carbon fatty acid content and (vi) a predetermined DHA content; a combination of (ii) a predetermined PUFA content with (v) a predetermined DHA / EPA ratio and (vi) a predetermined DHA content; a combination of (iii) a predetermined stearic acid content with (iv) a predetermined 18 carbon fatty acid content and (v) a predetermined DHA / EPA ratio;Examples include a combination of (iii) a predetermined stearic acid content, (iv) a predetermined C18 fatty acid content, and (vi) a predetermined DHA content, a combination of (iii) a predetermined stearic acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content, and a combination of (iv) a predetermined C18 fatty acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content.

[0012] When the four features are combined, they may be any combination, but as an example: a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, and (iv) a predetermined content of a fatty acid having 18 carbon atoms; a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, and (v) a predetermined DHA / EPA ratio; a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, and (vi) a predetermined DHA content. a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iv) a predetermined 18-carbon fatty acid content, and (v) a predetermined DHA / EPA ratio; a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iv) a predetermined 18-carbon fatty acid content, and (vi) a predetermined DHA content; a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content; (i) a combination of a predetermined triglyceride content, (iii) a predetermined stearic acid content, (iv) a predetermined 18-carbon fatty acid content, and (v) a predetermined DHA / EPA ratio; A combination of (i) a predetermined triglyceride content, (iii) a predetermined stearic acid content, (iv) a predetermined 18-carbon fatty acid content, and (vi) a predetermined DHA content; a combination of (i) a predetermined triglyceride content, (iii) a predetermined stearic acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content; a combination of (i) a predetermined triglyceride content, (iv) a predetermined 18-carbon fatty acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content; a combination of (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (iv) a predetermined 18-carbon fatty acid content, and (v) a predetermined DHA / EPA ratio. combinations of (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (iv) a predetermined 18-carbon fatty acid content, and (vi) a predetermined DHA content; combinations of (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content; combinations of (ii) a predetermined PUFA content, (iv) a predetermined 18-carbon fatty acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content; combinations of (iii) a predetermined stearic acid content, (iv) a predetermined 18-carbon fatty acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content.

[0013] When the five features are combined, any combination may be used. For example, A combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (iv) a predetermined 18-carbon fatty acid content, and (v) a predetermined DHA / EPA ratio; a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (iv) a predetermined 18-carbon fatty acid content, and (vi) a predetermined DHA content; a combination of (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content; Examples of such a combination include a combination of a predetermined triglyceride content with (ii) a predetermined PUFA content with (iv) a predetermined 18-carbon fatty acid content with (v) a predetermined DHA / EPA ratio and (vi) a predetermined DHA content, a combination of (i) a predetermined triglyceride content with (iii) a predetermined stearic acid content with (iv) a predetermined 18-carbon fatty acid content with (v) a predetermined DHA / EPA ratio and (vi) a predetermined DHA content, and a combination of (ii) a predetermined PUFA content with (iii) a predetermined stearic acid content with (iv) a predetermined 18-carbon fatty acid content with (v) a predetermined DHA / EPA ratio and (vi) a predetermined DHA content.

[0014] It may have a combination of six characteristics, including (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (iv) a predetermined 18-carbon fatty acid content, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content.

[0015] (i) A predetermined triglyceride content refers to a ratio of triglyceride weight to the total weight of glycerides contained in the oil of 83% or more, 85% or more, 88% or more, or 90% or more. Without intending to be limited by theory, it is believed that low monoglyceride and diglyceride content contributes to high low-temperature stability. The triglyceride content can be adjusted by adjusting the lipase treatment conditions. Furthermore, in the case of microbial oils, the triglyceride content can be increased by culturing the oil under carbon source depletion conditions at a predetermined low temperature. Carbon source depletion conditions refer to, for example, when glucose is used as a carbon source, a glucose concentration of less than 0.5 g / L. The glucose concentration can be measured using a glucose sensor.

[0016] (ii) A predetermined PUFA (polyunsaturated fatty acid) content means that the total weight of polyunsaturated fatty acids relative to the total weight of fatty acids in the oil is 70% or more, 75% or more, or 80% or more. Polyunsaturated fatty acids refer to fatty acids with 18 or more carbon atoms and multiple double bonds, including α-linolenic acid (C18:3, n-3), stearidonic acid (C18:4, n-3), linoleic acid (C18:2, n-6), γ-linolenic acid (C18:3, n-6), eicosatetraenoic acid (C20:4, n-3), eicosapentaenoic acid (C20:5, n-3), docosapentaenoic acid (C22:5, n-3), docosahexaenoic acid (C22:6, n-3), dihomo-γ-linolenic acid (C20:3, n-6), arachidonic acid (C20:4, n-6), and docosapentaenoic acid (C22:5, n-6). Without intending to be limited by theory, it is believed that a higher polyunsaturated fatty acid content results in higher low-temperature stability, even when the oil contains a given palmitic acid content. This can be achieved by using fish oil or microbial oil with a high polyunsaturated fatty acid content. Furthermore, in the case of microbial oil, the polyunsaturated fatty acid content can be increased by culturing under carbon source depletion conditions. Furthermore, the polyunsaturated fatty acid content can be adjusted by lipase treatment or wintering treatment.

[0017] (iii) The specified stearic acid content means that the weight of stearic acid relative to the total weight of fatty acids in the oil is 3% or less than 1%. Stearic acid is a C18 saturated fatty acid, and the melting point increases as the saturated fatty acid content relative to the total weight of fatty acids in the oil increases. Therefore, achieving a specified stearic acid content reduces the melting point, thereby improving the low-temperature stability of the oil. The specified stearic acid content can be achieved by using raw materials with a low stearic acid content. The stearic acid content can also be adjusted by lipase treatment.

[0018] (iv) A predetermined C18 fatty acid content refers to a ratio of the weight of C18 fatty acids to the total weight of fatty acids in the oil being less than 50%, less than 25%, less than 10%, less than 5%, or less than 2%. Examples of C18 fatty acids include stearic acid, oleic acid, linoleic acid, and linolenic acid. Reducing the C18 fatty acid content improves low-temperature stability. The C18 fatty acid content can be set low to exclude vegetable oils that are rich in oleic acid or linoleic acid and do not cause turbidity. A predetermined C18 fatty acid content can be achieved by using raw materials rich in unsaturated fatty acids such as DHA and EPA. The C18 fatty acid content can be adjusted by lipase treatment.

[0019] (v) The specified DHA / EPA ratio means that the ratio of DHA to EPA in the oil is 11 to 20. A low DHA content relative to the EPA content increases low-temperature stability. Within the specified DHA / EPA ratio range, oil without turbidity is obtained. The specified DHA / EPA ratio depends on the raw material used, but in the case of microbial oil, the DHA / EPA ratio increases when cultured after carbon source depletion, so it must be adjusted to fall within the range of 11 to 20. The DHA and EPA contents can also be adjusted by lipase treatment or wintering treatment.

[0020] (vi) A predetermined DHA content refers to a ratio of DHA weight to the total weight of fatty acids in the oil of 60% or more, 65% or more, 68% or more, or 70% or more. Without intending to be limited by theory, it is believed that a high content of DHA, a polyunsaturated fatty acid, increases low-temperature stability even when a predetermined amount of palmitic acid is contained. For example, this can be achieved by using fish oil or microbial oil with a high DHA content. The DHA content can also be adjusted by lipase treatment or wintering treatment.

[0021] The oil according to the present disclosure can be derived from any source. For example, the oil according to the present disclosure can be a microbial oil derived from a specific microorganism. The present disclosure can also relate to a method for processing the microbial oil, a method for producing the microbial oil, and the specific microorganism. More specifically, the present disclosure relates to: [1] An oil containing palmitic acid (C16:0) at a concentration of 7% to 20% by weight of the total weight of fatty acids in the oil, which does not become cloudy when cooled at 0°C for 3 hours, and which has the following characteristics: (i) triglycerides are 83% or more, 85% or more, 88% or more, or 90% or more; (ii) the PUFA content is 70% or more, 75% or more, or 80% or more; (iii) a stearic acid content of less than 3% or 1%; (iv) less than 50%, less than 25%, less than 10%, less than 5%, or less than 2% of fatty acids have 18 carbon atoms; and (v) DHA / EPA ratio is 11 to 20 An oil having at least one characteristic selected from the group consisting of: [2] Furthermore, (vi) the oil according to Item 1, wherein the DHA content is 60% or more, 65% or more, 68% or more, or 70% or more. [3] The oil is selected from the group consisting of: (ii) the PUFA content is 70% or more, 75% or more, or 80% or more; and (v) DHA / EPA ratio is 11 to 20 Item 1. The oil according to item 1, having the characteristics represented by: [4] The oil is selected from the group consisting of: (iii) a stearic acid content of less than 3% or 1%; and (v) DHA / EPA ratio is 11 to 20 Item 1. The oil according to item 1, having the characteristics represented by: [5] The oil is selected from the group consisting of: (iv) less than 50%, less than 25%, less than 10%, less than 5%, or less than 2% of fatty acids have 18 carbon atoms; and (v) DHA / EPA ratio is 11 to 20 Item 1. The oil according to item 1, having the characteristics represented by: [6] The oil according to any one of items 3 to 5, further characterized in that (i) the triglyceride content is 83% or more, 85% or more, 88% or more, or 90% or more. [7] The oil according to any one of items 2, 4, and 5, further characterized in that (ii) the PUFA content is 70% or more, 75% or more, or 80% or more. [8] The oil according to any one of items 2, 3, and 5, further comprising (iii) a stearic acid content of less than 3% or 1%. [9] The oil according to any one of items 2 to 4, further characterized in that (iv) the content of fatty acids having 18 carbon atoms is less than 50%, less than 25%, less than 10%, less than 5%, or less than 2%.

[10] The oil according to item 2 or 3, further comprising (v) a DHA / EPA ratio of 11 to 20.

[11] The oil according to any one of items 1 to 10, which is a microbial oil.

[12] The oil according to item 11, which is a refined oil.

[13] The oil according to Item 12, which is a refined oil obtained by degumming and deacidifying a crude oil obtained by hexane extraction.

[14] The oil according to Item 13, which is a refined oil obtained by further subjecting the refined oil to lipase treatment and deacidification treatment.

[15] The oil according to item 14, wherein the microbial oil is obtained from a microorganism belonging to the order S tramenopiles.

[16] The oil according to item 15, wherein the microbial oil is obtained from a microorganism belonging to the class Labyrinthulea.

[17] The oil according to item 16, wherein the microbial oil is obtained from a microorganism belonging to the Thraustochytrid fungus family.

[18] The oil according to item 17, wherein the microbial oil is obtained from the microorganism Thraustochytrium aggregatum.

[19] The oil according to item 18, wherein the microbial oil is obtained from the microorganism Thraustochytrium aggregatum having the deposit number FERM BP-22486.

[20] A method for screening a strain capable of low-temperature growth, comprising: A step of inducing mutations in microorganisms belonging to the genus Stramenopiles (S tramenopiles) by chemical or physical treatment; Culturing the microorganism in which the mutation has been induced; a step of obtaining a strain with high low-temperature growth ability from the cultured microorganism; wherein the low temperature is 5°C to 10°C lower than the optimum growth temperature of the microorganism.

[21] The method according to Item 20, wherein the step of culturing the microorganism in which the mutation has been induced is carried out using a solid medium.

[22] The method according to Item 21, wherein in the step of obtaining a strain having high low-temperature growth capability from the cultured microorganism, the strain having high low-temperature growth capability is obtained by isolating colonies formed on the solid medium.

[23] The method according to Item 20, wherein the chemical treatment is a mutagenic agent treatment, or the physical treatment is an ultraviolet ray, an X-ray, a gamma ray, or an ion beam treatment.

[24] The method according to Item 21, wherein the solid medium is selected from the group consisting of an agar medium, a gelatin medium, and a gellan gum medium.

[25] The method according to any one of Items 20 to 24, wherein the low temperature is a culture temperature of 13 to 18°C.

[26] A method for producing a microbial oil, comprising: inoculating a carbon source-containing medium with the strain screened by the method according to any one of Items 20 to 25; Cultivating the inoculated carbon source-containing medium at a temperature below 20°C, below 18°C, or below 16°C; recovering the bacterial cells; A step of subjecting the recovered bacterial cells to solvent extraction to obtain crude oil; and subjecting the solvent extracted crude oil to degumming and deacidification; wherein in the culturing step, culturing is carried out for 12 hours or more, 1 day or more, 2 days or more, 3 days or more, or 4 days or more after carbon source depletion. Method for producing microbial oil.

[27] The production method according to Item 26, wherein the strain screened by the method according to any one of Items 22 to 25 is a microorganism of Thraustochytrium aggregatum having deposit number FERM BP-22486.

[28] A method for producing a microbial oil, comprising: A step of inoculating a carbon source-containing medium with microorganisms belonging to the genus Stramenopiles; culturing the inoculated microorganism in the carbon source-containing medium; recovering the cultured microorganism cells; extracting oils and fats from the collected microbial cells; subjecting the extracted oil or fat to lipase treatment; and Deoxidation process A method for producing a microbial oil, comprising:

[29] The method for producing a microbial oil according to Item 28, wherein the step of extracting oils and fats from the collected microbial cells is performed by solvent extraction.

[30] The method according to Item 28, wherein the lipase treatment is carried out for 3 hours or less.

[31] The method according to Item 28, further comprising a step of carrying out a degumming treatment before or after the lipase treatment.

[32] The method according to any one of Items 28 to 31, further comprising a step of performing a deacidification treatment before the lipase treatment.

[33] Microorganism of Thraustochytrium aggregatum with accession number FERM BP-22486. [Effects of the Invention]

[0022] According to the present disclosure, an oil containing a predetermined amount of palmitic acid (C16:0) and having high low-temperature stability is provided. Because palmitic acid has physiological functions such as antioxidant activity, such an oil has high functionality and high low-temperature stability. As a result, the oil of the present disclosure has good storage stability at low temperatures. [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 is a graph showing the changes in the glucose concentration (g / L) and fatty acid content (%) in the medium when the TaM411 mutant strain was cultured at a normal culture temperature (23°C) and a low temperature (15°C). At the normal culture temperature, glucose in the medium was depleted on day 9, and at the low temperature, glucose in the medium was depleted on day 10. After glucose depletion, the fatty acid content decreased. [Figure 2] Figure 2 is a graph showing the changes in the fatty acid composition of the oil obtained when the TaM411 mutant strain was cultured at a normal culture temperature (23°C). After the 9th day, glucose in the medium became depleted. [Figure 3] 3 is a graph showing the change in fatty acid composition of the oil obtained when the TaM411 mutant strain was cultured at a low temperature (15°C). After the 10th day, glucose in the medium became depleted. [Figure 4] Figure 4 is a graph showing the glyceride ratio of the microbial oil obtained after culturing the TaM411 mutant strain at a normal culture temperature (23°C) and a low temperature (15°C) and continuing the culture even after the glucose in the medium was depleted (14 days). DETAILED DESCRIPTION OF THE INVENTION

[0024] (1) Oil The term "oil" is generally defined as a liquid, water-immiscible, flammable substance that undergoes phase separation with water. Furthermore, "fat" is defined as a solid, water-immiscible, flammable substance that undergoes phase separation with water, and these two are collectively referred to as "oil and fat." The term "lipid" is generally defined as a substance of biological origin that is insoluble in water. Conventionally, solid animal fats and oils are referred to as "fats," and liquid vegetable or fish fats and oils are referred to as "oil." However, whether a substance is solid or liquid depends on its composition, and even vegetable and fish oils can be solid. Therefore, in this disclosure, the term "oil" does not necessarily mean liquid, and for convenience, it may be used interchangeably with "oils and fats" or "lipids."

[0025] The terms "oil" and "fats" refer in the narrow sense to triglycerides, but in the present disclosure are defined to include other lipid components such as diglycerides, monoglycerides, phospholipids, glycolipids, cholesterol, free fatty acids, etc. In the present disclosure, oil is used as a term that refers to both crude oil and refined oil without distinction.

[0026] "Crude oil" refers to oil that has been pressed or extracted from an oilseed feedstock. Refined oil is obtained from crude oil by removing impurities such as phospholipids and cholesterol. Since crude oil is oil before refining, its fatty acid composition depends on the fatty acid composition of the oilseed feedstock.

[0027] "Refined oil" refers to oil obtained after a refining process, including degumming, deacidification, bleaching, deodorization, and any combination thereof, to remove undesired substances such as phospholipids and sterols. Refining typically results in an increased proportion of triglycerides.

[0028] The oil may be derived from any source, such as a vegetable oil derived from a plant, an animal oil derived from an animal, a fish oil derived from a fish, or a microbial oil derived from a microorganism. The oil may be produced by a pressing method in which the raw material is pressed to extract the oil, or by an extraction method in which the raw material is extracted with a solvent. When a microorganism is used as the raw material, the microbial oil can be obtained by culturing the microorganism in an appropriate medium and collecting the microbial cells by a method such as solvent extraction. Whether these oils are crude oils or refined oils, they are only refined to the extent that some components characteristic of the raw material remain, and can be distinguished from concentrates of specific fats and oils.

[0029] Oils have fatty acid compositions influenced by the composition of the raw material. The fatty acid composition of oils can be determined by conventional fatty acid analysis. For example, the oil to be measured is esterified using a lower alcohol and a catalyst to obtain fatty acid lower alcohol esters. The resulting fatty acid lower alcohol esters are then analyzed by gas chromatography. Peaks corresponding to each fatty acid are identified in the resulting gas chromatography, and the peak area of ​​each fatty acid can be determined, for example, using the Agilent ChemStation integration algorithm (revision C.01.03

[37] , Agilent Technologies). In this disclosure, the term "area %" used in the fatty acid composition refers to the ratio (percentage) of the peak area of ​​each component to the total peak area in the chromatogram obtained by fatty acid analysis. For example, it refers to the content of the component of a peak determined by gas chromatography or thin-layer chromatography / flame ionization detector (TLC / FID) analysis of an oil containing various fatty acids as constituent components. The fatty acid composition was determined by gas chromatography, for example, according to the method described in the Examples. The fatty acid composition can also be expressed as the weight (wt%) of a particular fatty acid relative to the total weight of fatty acids in the oil, with or without taking into account the molecular weight of the fatty acid in the "area %" determined from the analytical chart. In this disclosure, "%" refers to "area %" unless otherwise specified.

[0030] For convenience, the term "fatty acid" may refer not only to free saturated or unsaturated fatty acids themselves, but also to fatty acids as structural units contained in alkyl esters, triglycerides, diglycerides, monoglycerides, phospholipids, steryl esters, etc., and may also be referred to as "constituent fatty acids." In the present disclosure, unless otherwise specified, the form of a compound containing a fatty acid may be omitted. Examples of the form of a compound containing a fatty acid include free fatty acid form, fatty acid alkyl ester form, glyceryl ester form, phospholipid form, steryl ester form, etc. Compounds containing the same fatty acid may be contained in an oil in a single form or as a mixture of two or more forms.

[0031] When describing fatty acids, numerical notation is sometimes used to abbreviate the number of carbon atoms, the number of double bonds, and the location of the double bonds using numbers and letters, respectively. For example, a saturated fatty acid with 20 carbon atoms is represented as "C20:0," and a triunsaturated fatty acid with 20 carbon atoms and three double bonds in the carbon chain is represented as "C20:3." For example, behenic acid may be represented as "C22:0," arachidonic acid as "C20:4n-6," etc. "n-" indicates the position of the first double bond counting from the methyl end of the fatty acid; for example, "n-6" indicates the position of the double bond at the sixth position counting from the methyl end of the fatty acid, and "n-3" indicates the position of the double bond at the third position counting from the methyl end of the fatty acid. This method is well known to those skilled in the art, and those skilled in the art can easily identify fatty acids represented according to this method.

[0032] The oil of the present disclosure contains a predetermined content of palmitic acid, and with respect to other fatty acids in the oil, has at least one characteristic selected from the group consisting of: (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (iv) a predetermined content of fatty acids with 18 carbon atoms, (v) a predetermined DHA / EPA ratio, and (vi) a predetermined DHA content. Such oils do not become cloudy even when cooled at 0°C for 3 hours. The degree of clarity can be confirmed visually or determined using a device such as a spectrophotometer. More specifically, cloudiness can be determined by the STEARIN test described in the SPECIFIC TESTS for USP-NF Fish Oil Containing Omega-3 Acids, an official method in the United States. In the STEARIN test, oils that remain transparent even when cooled at 0°C for 3 hours are considered to have excellent low-temperature stability. Transparent oils as defined in the STEARIN test correspond to the "oils that do not become cloudy" of the present disclosure. The oils of the present disclosure can contain a predetermined amount of palmitic acid and have the above-mentioned characteristics by simply performing degumming, deacidification, and optionally lipase treatment without undergoing wintering, which increases the content of specific fatty acids. On the other hand, the oils of the present disclosure may either exclude or include oils that have been subjected to wintering, which increases the content of specific fatty acids.

[0033] In the present invention, DHA refers to docosahexaenoic acid (C22:6n-3) and EPA refers to eicosapentaenoic acid (C20:5n-3).

[0034] The oils according to the present disclosure can be incorporated into pharmaceutical compositions, cosmetic compositions, foods, supplements, or animal feeds. Palmitic acid contained in the oils according to the present disclosure can have antioxidant and moisturizing effects.

[0035] (2) Microbial oil In this disclosure, unless otherwise specified, microbial oil broadly refers to all lipids obtained from microorganisms, and is used as a term to refer to both crude oil and refined oil without distinction in this disclosure. Microbial oil is obtained by culturing microorganisms in an appropriate medium and collecting the microbial cells by a method such as solvent extraction. Even if the microbial oil is a refined oil, it is only refined to the extent that some components characteristic of the microorganism remain, and can be distinguished from a concentrate of a specific oil or fat.

[0036] The microbial oil of the present disclosure refers to oil obtained from any microorganism. Depending on the type of microorganism, it can also be called yeast oil, fungal oil, or algae oil. In addition, oil can also be obtained using cells instead of microorganisms, and can also be called cell oil.

[0037] (3) Microorganisms In the present disclosure, "microorganisms" include eubacteria, archaea, eukaryotes, etc. Eukaryotes include algae, protists, fungi, and slime molds. Ascomycetes such as yeast, filamentous fungi such as molds, heterokonts, etc. can be used to produce oil. In particular, microorganisms belonging to the Stramenopiles can be used to produce oils characterized by (i) a predetermined triglyceride content, (ii) a predetermined PUFA content, (iii) a predetermined stearic acid content, (iv) a predetermined 18-carbon fatty acid content, (v) a predetermined DHA / EPA ratio, and / or (vi) a predetermined DHA content. Stramenopiles include Bicosoecida, Labyrinthulea, Blastocystis, Actinophyida, Opalinata, Placidida, Oomycetes, Hyphochytriomycetes, Developayella, Chrysophyceae, Eustigmatophyceae, Phaeothamniophyceae, Pinguiophyceae, and Raphidophyceae. Examples of suitable microorganisms include the Raphidophyceae, Synurophyceae, Xantexyophyceae, Phaeophyceae, Schizocladiophyceae, Chrysomerophyceae, Dictyochophyceae, Bolidophyceae, Pelagophyceae, and Bacillariophyceae. However, among microorganisms belonging to the Stramenopiles, microorganisms belonging to the Labyrinthulea are particularly useful. Among microorganisms belonging to the Labyrinthulea, microorganisms belonging to the Thraustochytrids are particularly preferred.Examples of microorganisms belonging to the Thraustochytrium fungi include those of the genera Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizocytrium, Thraustochytrium, Ulkenia, Aurantiochytrium, Oblongichytrium, Botryochytrium, Parietichytrium, and Sicyoidochytrium, and among these, those of the genus Thraustochytrium are particularly preferred. Microorganisms belonging to the genus Thraustochytrium, particularly Thraustochytrium aggregatum, are used.

[0038] In the present disclosure, among microorganisms, microorganisms belonging to the genus Stramenopiles that grow at low temperatures can be used. A low-temperature growing microorganism refers to a microorganism that has a predetermined growth rate at temperatures 5 to 10°C, 6 to 9°C, or 7 to 8°C lower than the optimal growth temperature. The predetermined growth rate refers to a growth rate that is 98% or more, 95% or more, or 90% or more of the growth rate at the optimal growth temperature. The optimal growth temperature refers to the temperature at which the growth rate is maximized depending on the strain, and can be determined by culturing the strain under different temperature conditions and measuring the number of bacteria. For Stramenopile microorganisms, the optimal growth temperature is often 20 to 30°C, and 20°C, 23°C, or 25°C can be used as the optimal growth temperature, for example. Such low-temperature growing microorganisms can be obtained by screening low-temperature growing strains from mutant-derived strains. When used in oil production, a low-temperature growing strain may have the characteristic that the fatty acid content is less likely to decrease during the culture period after carbon source depletion. In yet another embodiment, a microorganism of the strain of Thraustochytrium aggregatum (strain Tam411) having deposit number FERM BP-22486 can be used.

[0039] The present disclosure also relates to a method for screening for a cold-growing strain. More specifically, A step of inducing mutations in microorganisms belonging to the genus Stramenopiles (S tramenopiles) by chemical or physical treatment; Culturing the microorganism in which the mutation has been induced; a step of obtaining a strain with high low-temperature growth ability from the cultured microorganism; Includes. More specifically, the mutagenesis step involves applying a suspension of cultured cells of microorganisms belonging to the genus S tramenopiles to a solid medium, and inducing mutations by chemical or physical treatment. The step of obtaining a strain with high low-temperature growth ability is carried out by culturing the solid medium inoculated with the mutated microorganisms at a predetermined low temperature, and isolating the formed colonies to obtain a strain with high low-temperature growth ability. Any solid medium capable of growing microorganisms belonging to the genus S tramenopiles can be used as the solid medium. Examples include agar medium, gelatin medium, gellan gum medium, or a mixture thereof. Mutation induction in microorganisms belonging to the genus S tramenopiles can be performed by any method known in the art. For example, chemical treatments include treatment with mutagenic agents such as nitrosoguanidine, ethyl methanesulfonate, and diazinium compounds. The concentration of the agent treatment can be selected appropriately based on the mutation rate. Physical treatments include treatment with radiation capable of inducing mutations, such as ultraviolet light, X-rays, gamma rays, or ion beam treatment. The intensity, duration, and frequency of the physical treatment can be selected appropriately based on the mutation rate. Ultraviolet light irradiation is preferred due to its ease of operation. The wavelength and intensity of the ultraviolet light, as well as the irradiation time, can be set appropriately. For example, UV treatment is performed by irradiating the microorganisms with 254 nm UV light at 8 W for 10 to 20 seconds, once or multiple times, particularly twice. At this UV intensity, treatment times longer than 10 to 20 seconds result in the death of the stramenopiles, while treatment times shorter than 10 to 20 seconds fail to achieve the desired mutagenesis efficiency. Microorganisms belonging to the Stramenopiles can be selected as appropriate, and the NBRC 110819 strain can be used as an example. This strain has an optimal growth temperature of 23°C. After UV irradiation, the microorganisms are cultured at a temperature (referred to as a predetermined low temperature) that is 5 to 10°C, 6 to 9°C, or 7 to 8°C lower than the optimal growth temperature. The predetermined low temperature is selected as a temperature at which the microorganisms before mutagenesis exhibit low or no growth. Strains capable of low-temperature growth can be obtained by isolating the strain from colonies formed during culture at the predetermined low temperature. Strains capable of low-temperature growth can be used for the production of microbial oils. A strain capable of growing at low temperatures has the characteristic that, when used for oil production, the fatty acid content is less likely to decrease during the culture period after carbon source depletion.

[0040] (4) Oil manufacturing method The microbial oil according to the present disclosure can be produced according to the production method described below. Specifically, the following: a step of inoculating a cold-growing strain belonging to the genus S tramenopiles into a carbon source-containing liquid medium; Cultivating the inoculated carbon source-containing liquid medium at a temperature below 20°C, below 18°C, or below 16°C; recovering the bacterial cells; A step of subjecting the recovered bacterial cells to solvent extraction to obtain crude oil; and subjecting the solvent extracted crude oil to degumming and deacidification; wherein in the culturing step, culturing is carried out for 12 hours or more, 1 day or more, 2 days or more, 3 days or more, or 4 days or more after carbon source depletion. During the culture period after carbon source depletion, the fatty acid composition changes, for example, the DHA content and / or EPA content increases, while the saturated fatty acid content decreases. By using a strain capable of low-temperature growth, the fatty acid content is less likely to decrease during the culture period after carbon source depletion, and therefore, oils with high DHA and / or EPA contents can be obtained with high productivity by using a strain capable of low-temperature growth. This method allows the production of oil containing a predetermined amount of palmitic acid without generating turbidity when cooled for 3 hours at 0° C. Microorganisms that can be used in this production method include the strain of Thraustochytrium aggregatum (Tam411 strain) with the accession number FERM BP-22486.

[0041] The microbial oil according to the present disclosure can also be produced according to the production method described below. A step of inoculating a carbon source-containing medium with microorganisms belonging to the genus Stramenopiles; culturing the inoculated microorganism in the carbon source-containing medium; recovering the cultured microorganism cells; Extracting oils and fats from the collected microbial cells; and A step of subjecting the extracted oil and fat to lipase treatment. The present invention also relates to a method for producing a microbial oil, including the steps of: (1) performing lipase treatment; (2) increasing the DHA content in the oil; (3) producing a microbial oil containing a predetermined amount of palmitic acid while not becoming cloudy when cooled at 0°C for 3 hours; and (4) performing a degumming step before or after lipase treatment. The degumming step can remove gums, such as phospholipids, contained in the crude oil. Because phospholipids cause oil cloudiness, removing them through degumming can adjust the cloudiness. The method may further include a deacidification step before lipase treatment. Because lipase treatment produces free fatty acids as a product, removing the free fatty acids before lipase treatment can facilitate the progress of the lipase treatment reaction. Both degumming and deacidification steps may be performed before lipase treatment, in which case the order of the steps may be arbitrary. For example, a degumming step may be performed before lipase treatment, followed by a deacidification step.

[0042] Microbial culture may be performed using a method commonly used in the art. Examples include aeration culture, shaking culture, or static culture, and can be selected appropriately depending on the cells to be cultured. There are no particular limitations on the incubator used for the culture, and any incubator typically used for microbial culture can be used. The incubator can be selected appropriately depending on the scale of the culture. For example, when using a microorganism of the genus Thraustochytrium, aeration culture is typically performed for 1 to 3 weeks. From the perspective of continuing the culture after carbon source depletion, the culture may be performed for 7 days or more, 10 days or more, or 14 days or more. From the perspective of productivity, the culture is typically performed for 35 days or less, 28 days or less, or 21 days or less. The aeration rate in aeration culture may be the same as that typically used. Any medium known in the art can be used for the culture depending on the microorganism used.

[0043] The temperature at which Thraustochytrium microorganisms are cultured is typically 20 to 25°C, for example, 22°C, 23°C, or 24°C. After carbon source depletion, the fatty acid content significantly decreases at normal culture temperatures. Therefore, to prevent this decrease in fatty acid content, culture can be performed at a lower temperature than normal, for example, below 20°C, below 18°C, or below 16°C. Culture after carbon source depletion changes the fatty acid composition. To change the fatty acid composition, culture is performed at 10°C or higher, 12°C or higher, or 15°C or higher. Culture can be performed at a temperature lower than normal culture temperature by 7°C or higher, 10°C or higher, or 12°C or higher. The change in fatty acid composition allows the production of oil with a characteristic composition that does not become cloudy even after cooling at 0°C for 3 hours, making it easy to handle at low temperatures, simply by degumming and deacidifying. The temperature can also be changed during culture. For example, culture can be performed at a normal culture temperature before carbon source depletion, and then at a lower temperature after carbon source depletion. This allows a high growth rate in the presence of a carbon source to be achieved, and the DHA content to be increased while suppressing a decrease in the fatty acid content after carbon source depletion.

[0044] A carbon source-containing medium refers to a medium capable of culturing the microorganisms of the present disclosure and containing a carbon source. Any commonly used carbon source can be used as the carbon source, including, but not limited to, glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol, etc. Glucose is typically used as the carbon source. The carbon source concentration can be selected appropriately depending on the strain and culture conditions, and, for example, 10 g / L to 80 g / L can be used. To ensure sufficient growth, a concentration of 10 g / L or more, 20 g / L or more, 30 g / L or more, or 40 g / L or more can be selected. To prevent carbon source depletion during the culture process, a concentration of 80 g / L or less, 70 g / L or less, 60 g / L or less, or 50 g / L or less can be selected. In addition to a carbon source, the medium for culturing the microorganisms disclosed herein may contain natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acids, and corn steep liquor, organic nitrogen sources such as urea, and inorganic nitrogen sources such as sodium nitrate, ammonium nitrate, and ammonium sulfate, as well as trace nutrient sources such as inorganic salts and vitamins, as needed. The aqueous medium that can be used as the base material for the liquid medium is basically water, and distilled water or purified water can be used. Liquid media are commonly used, but solid media such as agar media may also be used.

[0045] After culturing, the cells are collected from the culture. The culture contains the cells, secretions secreted from the cells, and the medium. If oil is secreted, the method may further include a step of collecting the secretions. A washing step may also be included. The cells are collected by centrifugation and dried to dryness. The drying may be any drying method used in the art, including freeze-drying, air-drying, and heat-drying. When the cells are cultured in a solid medium, the solid medium and the cells can be disrupted using a homogenizer or the like without separating them from the medium, and the resulting disrupted material can be directly subjected to the oil collection step.

[0046] Solvent extraction is carried out by drying the fungal cells and using an organic solvent. The organic solvent used may be any solvent used in this technical field, such as ether, hexane, methanol, ethanol, chloroform, dichloromethane, or petroleum ether. Hexane is typically used to extract oil. Crude oil is obtained by distilling off the organic solvent from the extract under reduced pressure. Alternatively, extraction can be carried out using wet fungal cells. A water-compatible solvent, such as methanol or ethanol, or a water-compatible mixed solvent containing this solvent and water and / or other solvents is used. Other procedures are the same as those described above.

[0047] The crude oil obtained by solvent extraction can be subjected to at least one refining step selected from the group consisting of a deacidification step, a degumming step, and a lipase treatment step. The order of the degumming and deacidification steps is not particularly limited. The deacidification step is carried out by adding sodium hydroxide in an amount corresponding to the amount of free fatty acids contained in the crude oil, removing the aqueous phase, and then performing solvent extraction. The deacidified oil can be recovered by removing the solvent under reduced pressure. The degumming step is a step in which phospholipids are hydrated by adding warm water and then separated from the oil as a gum using a centrifuge. Typical refining steps for oils and fats include degumming and deacidification, as well as bleaching, deodorization, and dewaxing (wintering). These treatments may be carried out by methods well known in the art. Examples of bleaching treatments include treatment with activated clay, activated carbon, silica gel, etc. Examples of deodorization methods include steam distillation.

[0048] Lipase treatment produces free fatty acids due to the enzymatic activity of lipase, but transesterification can also occur. Degumming and / or deacidification treatment can be performed before or after lipase treatment, or both. Lipase treatment for a specified period of time yields an oil with a characteristic composition, which does not become cloudy even after cooling at 0°C for 3 hours and has excellent low-temperature handling properties. While not intending to be limited by theory, lipase catalytic activity does not act equally on all fatty acids; its activity varies depending on the bond position in the glyceride, the carbon chain length of the fatty acid, and the double bond. Lipases are highly reactive with saturated fatty acids such as palmitic acid and stearic acid, but are less reactive with long-chain unsaturated fatty acids. As a result, lipase treatment converts saturated fatty acids into free fatty acids, which is thought to change the composition of the oil's constituent fatty acids. Lipase treatment using a lipase with particularly low reactivity with DHA increases the DHA content in the oil. The time and reaction temperature for lipase treatment may be varied depending on the type and activity of lipase, but for example, when lipase OF 200 units / g oil is used, lipase treatment may be carried out at 18°C ​​for 3 hours or less, or 2 hours or less. To ensure sufficient treatment time, lipase treatment may be carried out for 30 minutes or more, or 1 hour or more.

[0049] Any lipase can be used for the lipase treatment, but it is preferable that the lipase has the property of being less reactive to highly unsaturated fatty acids and concentrating highly unsaturated fatty acids in the undecomposed glyceride fraction in the hydrolysis reaction or alcoholysis reaction. Examples of such lipases that can be used include lipases obtained from microorganisms belonging to Alcaligenes sp. (Lipase QLM, Lipase QLC, Lipase PL, all manufactured by Meito Sangyo Co., Ltd.), lipases obtained from microorganisms belonging to Burkholderia cepacia (Lipase PS, manufactured by Amano Enzyme Co., Ltd.), lipases obtained from microorganisms belonging to Pseudomonas fluorescens (Lipase AK, manufactured by Amano Enzyme Co., Ltd.), lipases obtained from microorganisms belonging to Thermomyces lanuginosa (Lipozyme TLIM, manufactured by Novozymes), and lipases produced by the genus Candida, particularly Candida rugosa and Candida cylindracea.

[0050] The production method described in the present disclosure can produce oil that does not become cloudy when cooled at 0°C for 3 hours using microorganisms of the genus Thraustochytrium, particularly Thraustochytrium aggregatum. Even if the strain of the microorganism is not particularly limited, by performing lipase treatment, oil that does not become cloudy when cooled at 0°C for 3 hours and has a characteristic composition that is easy to handle at low temperatures can be obtained. On the other hand, even if lipase treatment is not performed, by using Thraustochytrium aggregatum, a microorganism of the genus Thraustochytrium, with accession number FERM BP-22486, it is possible to produce oil that does not become cloudy when cooled at 0°C for 3 hours simply by performing deacidification and degumming treatment.

[0051] In this disclosure, the term "process" refers not only to an independent process, but also to processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. In this disclosure, numerical ranges indicated using "to" indicate ranges that include the numerical values ​​before and after the range as the minimum and maximum values, respectively. In this disclosure, when multiple substances corresponding to each component are present in the mixture, the content of each component in the mixture means the total content of the multiple substances present in the mixture, unless otherwise specified. In this disclosure, the terms "equal to or less than" or "less than" in relation to percentages include 0%, i.e., "not contained," unless a lower limit is specifically specified, or include a range that includes values ​​that are undetectable by current means.

[0052] All documents mentioned herein are incorporated by reference in their entirety.

[0053] The examples of the present disclosure described below are for illustrative purposes only and do not limit the technical scope of the present disclosure. The technical scope of the present disclosure is limited only by the claims. The present disclosure may be modified, for example, by adding, deleting, or substituting components of the present disclosure, provided that the modifications do not depart from the spirit of the present disclosure. [Example]

[0054] Example 1: Obtaining and cryopreserving NBRC 110819 strain The NBRC 110819 strain, a species closely related to Thraustochytrium aff. aggregatum, was obtained from the screening strains provided by the Biotechnology Center of the National Institute of Technology and Evaluation. 4 ml of liquid medium (1) was placed in a 16.5 mm diameter x 165 mm rimmed glass test tube, stoppered with silicone, and sterilized with moist heat. After cooling, 0.2 ml of cultured bacterial fluid obtained from NBRC was inoculated into the test tube and cultured with shaking at 26°C and 180 rpm for 2 to 5 days. The shaker was placed inside a low-temperature constant temperature chamber. In this way, cultured bacterial suspension (1) was prepared.

[0055] 100 mL of liquid medium (1) was placed in a 500 mL fluted Erlenmeyer flask, stoppered with silicone, and sterilized by moist heat. After cooling, 1 mL of cultured bacterial cell suspension (1) was inoculated into the Erlenmeyer flask and cultured with shaking at 26°C and 120 rpm for 2 to 5 days. In this way, cultured bacterial cell suspension (2) was prepared.

[0056] 20 mL of the resulting cultured bacterial cell suspension (2) was placed in a centrifuge tube and centrifuged at 5,000 x g for 3 minutes at 25°C to recover wet bacterial cells. The recovered wet bacterial cells were suspended in 20 mL of artificial seawater and centrifuged at 5,000 x g for 3 minutes at 25°C to wash the wet bacterial cells. This washing step was repeated twice. The recovered wet bacterial cells were then resuspended in 20 mL of artificial seawater containing a final concentration of 10 wt% glycerol and 5 wt% trehalose. This was used as a frozen cultured bacterial solution of the NBRC 110819 strain and was stored frozen at -80°C until use in each experiment.

[0057] The liquid medium (1) and artificial seawater used to culture and cryopreserve the NBRC 110819 strain are shown below. The compositions of the vitamin solution (*) and metal salt solution (**) in the liquid medium (1) are also shown below. [Table 1] [Table 2] [Table 3] [Table 4]

[0058] Example 2: Obtaining TaM411 mutant strain 100 mL of liquid medium (1) was placed in a 500 mL fluted Erlenmeyer flask, stoppered with silicone, and sterilized by moist heat. After the medium temperature had dropped to approximately 20°C, 0.1 mL of a frozen culture of the NBRC 110819 strain was inoculated into the Erlenmeyer flask and cultured for 2 days at 26°C with shaking at 120 rpm. Thus, a cultured bacterial suspension (3) was obtained.

[0059] The agar medium composition shown in Table 6 was placed in a glass beaker, covered with an aluminum foil, and sterilized with moist heat. The mixture was aseptically dispensed into plastic petri dishes with an outer diameter of approximately 90 mm in a clean bench and allowed to stand to prepare agar medium. 0.1 mL of the cultured bacterial suspension (3) was applied to this agar medium (1), which was then irradiated with 254 nm, 8W x 2 UV light for 10 to 20 seconds. A HandyUVLanp SUV-16 (AS ONE Corporation) was used for UV irradiation. The agar medium was then cultured at 20°C for 2 to 5 days until colony formation of the NBRC 110819 strain was confirmed. [Table 5] [Table 6]

[0060] A single colony formed on the agar medium was picked in a clean bench, streaked onto a new agar medium, and cultured. This procedure was repeated twice to isolate mutant strains. Frozen culture broth of each mutant strain was obtained from the isolated colonies according to the method described in Example 1. Furthermore, 100 ml of liquid medium (1) was placed in a 500 ml fluted Erlenmeyer flask, stoppered with silicone, and sterilized by moist heat. After cooling, 1 ml of frozen cultured bacterial cell fluid of each mutant strain was inoculated into the Erlenmeyer flask and cultured with shaking at 26°C and 120 rpm for 2 to 5 days. In this way, each cultured bacterial cell suspension (3) was prepared.

[0061] 1 to 10 mL of the resulting cultured bacterial cell suspension (3) was placed in a sterilized disposable centrifuge tube and centrifuged at 5,000 x g for 3 minutes at 25°C to recover the wet bacterial cells. The recovered wet bacterial cells were suspended in 20 mL of artificial seawater and centrifuged at 5,000 x g for 3 minutes at 25°C to wash the wet bacterial cells. This washing step was repeated twice. The resulting suspension was then freeze-dried at -80°C to obtain freeze-dried mutant bacterial cells.

[0062] Total lipids were extracted from the obtained freeze-dried cells with chloroform:methanol (2:1, v / v) according to the method of Folch et al. (J. Biological and Chemistry. 226: 497-509 (1957)). The obtained total lipids were methyl-esterified to obtain fatty acid methyl esters (FAMEs). The obtained FAMEs were analyzed by gas chromatography. The gas chromatograph conditions were set as follows: Column: DB-WAX 0.530 mm x 30 m, film thickness 1.00 μm (Agilent Technologies, Inc.) Carrier gas conditions: Helium 1.0 ml / min, separation ratio 100:1 Column temperature conditions: 140°C for 5 minutes, increase temperature to 240°C at 4°C / min, and then hold at 240°C for 10 minutes Detection: FID Detector temperature: 260℃ ·Inlet temperature: 250℃ ·Injection volume: 1μL

[0063] FAME analysis failed to identify any strains with a 5% or greater increase in EPA and DHA concentrations in total fatty acids. All mutant strains shared the ability to grow at temperatures below 20°C, more than 5°C lower than the optimal growth temperature of 26°C. One of the mutants with a rapid growth rate at low temperatures was designated TaM411 and deposited on October 17, 2023, with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number FERM BP-22486.

[0064] Example 3: Production of low-temperature stable oil by enzymatic reaction 100 mL of liquid medium (1) was placed in a 500 mL fluted Erlenmeyer flask, stoppered with silicone, and sterilized with moist heat. After cooling, 0.1 mL of frozen cultured bacterial cell liquid of the NBRC 110819 strain was inoculated into the Erlenmeyer flask and cultured with shaking at 23°C and 120 rpm for 2 days. The cultured bacterial cell suspension thus obtained was used as a preculture liquid for culture in a fermentation culture tank.

[0065] A 5-L fermentation / culture tank was charged with 3 L of liquid medium (1) and 0.2 g of antifoaming agent KM-72F (Shin-Etsu Chemical Co., Ltd.), and then subjected to moist heat sterilization. After the medium temperature had dropped to approximately 20°C, approximately 30 mL of preculture solution was inoculated. Culture was carried out for 12 days under the following conditions: culture temperature 23°C, agitation speed 250 rpm, aeration rate 1.2 L / min, internal gauge pressure 0 MPa, and pH 6.8 ± 0.2.

[0066] The cultured bacterial cell suspension was then centrifuged at 5,000 x g for 3 minutes at 25°C to obtain wet bacterial cells. The collected wet bacterial cells were suspended in 200 mL of artificial seawater and centrifuged at 5,000 x g for 3 minutes at 25°C to wash the wet bacterial cells. This washing process was repeated three times, and then the cells were dried in an oven at 100°C to obtain dry bacterial cells.

[0067] 120 g of dried cells were weighed into an Erlenmeyer flask, 1.2 L of hexane was added, and the mixture was stirred with a stirrer for 1 hour. The mixture was filtered under reduced pressure and collected in a recovery flask, and the solvent was removed by vacuum distillation at 40°C. The above extraction was repeated three times for the same cells, and the resulting oils were combined and the solvent was removed using a vacuum pump, yielding 36.4 g of extracted oil.

[0068] The acid value of the extracted oil was measured as follows: 0.1 g of oil was placed in an Erlenmeyer flask, 10 mL of a 1:1 ethanol:diethyl ether solution and one drop of phenolphthalein solution were added, and 0.1 mol / L potassium hydroxide solution was added while stirring until the red color persisted for 30 seconds. The acid value was calculated using the following formula:

number

[0069] (deoxidation treatment) The extracted oil was subjected to solvent deacidification treatment as follows: 31.8 g of oil was transferred to a separatory funnel along with 2.5 volumes (v / w) of hexane. An equal volume of acetone to the hexane and an aqueous solution containing 1.3 equivalents of free fatty acids (calculated from the acid value) of sodium hydroxide (2.5 volumes (v / w) of the oil) were added, vigorously shaken, and then allowed to stand. After collecting the upper layer, the lower layer was returned to the separatory funnel and extracted twice with 2.5 volumes (v / w) of hexane. The collected upper layer (hexane layer) was returned to the analytical funnel and repeatedly washed with 2.5 volumes (v / w) of water until the pH reached 7. The solvent from the hexane layer was removed by vacuum distillation, and 26.7 g of deacidified oil was recovered.

[0070] (Degumming and deoxidation treatment) The solvent-deacidified oil was subjected to degumming and deoxidation as follows. 20.0 g of the solvent-deacidified oil was placed in an eggplant-shaped flask, and 0.13% (w / w) 85% phosphoric acid and 2.5% (w / w) water were added. The flask was then filled with nitrogen gas and heated and stirred at 75°C for 15 minutes. After cooling, 1.3 equivalents (molar ratio) of 20% aqueous sodium hydroxide, sufficient to neutralize the phosphoric acid and the free fatty acids in the oil (calculated from the acid value), was added, and the mixture was heated and stirred at 75°C for 15 minutes. The contents were centrifuged at 1,910 x g for 15 minutes, and the upper layer was transferred to a new eggplant-shaped flask, 20% (w / w) water was added, and the mixture was heated and stirred at 75°C for 15 minutes, followed by a hot water wash. The contents were centrifuged as above, and the upper layer was vacuum dried at 120°C for 15 minutes. 12.4 g of degummed and deacidified oil was obtained.

[0071] (Lipase treatment) The resulting degummed and deacidified oil was then treated with an enzyme to concentrate DHA. The reaction conditions were as follows: ·Oil:Water=3:2(w / w) Reaction temperature: 18℃ Reaction time: 2, 4, 8, 24, 48 hours Enzyme: Lipase OF 200 units / g oil

[0072] The lipase OF used was a lipase OF derived from Candida rugosa, manufactured by Meito Sangyo Co., Ltd., with a capacity of 360,000 units / g. After the reaction, the reaction mixture was heated at 80°C for 10 minutes to inactivate the enzyme. After cooling, saturated saline was added in an amount equal to the amount of the reacted oil (w / w), and the mixture was heated at 80°C until the oil was dissolved. The mixture was then centrifuged to recover the upper layer of the enzyme-reacted oil.

[0073] The enzyme-reacted oil was subjected to solvent deacidification treatment in the same manner as above. The fatty acid composition and lipid composition of the degummed and deacidified oil before the enzyme reaction and five samples of the deacidified oil after the enzyme reaction were analyzed.

[0074] (Analysis of fatty acid composition) Regarding the fatty acid composition, the oil was converted into fatty acid methyl esters (FAMEs) as follows, and FAMEs were analyzed by gas chromatography (GC). 20 μL of oil was placed in a plastic tube and 1 mL of 1N sodium methylate solution was added. The inside of the tube was replaced with nitrogen, and after mixing, the tube was heated at 80°C for 2 minutes. After cooling, 1 mL of 1N hydrochloric acid was added and mixed. 1 mL of hexane and 5 mL of saturated saline were added and mixed. The tube was centrifuged at 1,910 x g for 3 minutes, and the supernatant was analyzed by GC. The GC conditions were as follows: ·GC:6890N Network GC System(Agilent Technologies) Column: DB-WAX 0.250 mm x 30 m, film thickness 0.25 μm (Agilent Technologies, Inc.) Carrier gas conditions: Helium 1.5 mL / min, separation ratio 30:1 Column temperature conditions: 180°C to 240°C at 3°C / min, then 15 min at 230°C Detection: FID Detector temperature: 250℃ ·Inlet temperature: 250℃ ·Injection volume: 1μL

[0075] (Analysis of lipid composition) Lipid composition was analyzed by TLC-FID. 20 μL of oil was dissolved in 1 mL of hexane, and 1 μL of the solution was injected into a Chroma Rod using a syringe. The primary development was performed using chloroform:methanol = 95:5 (v / v), and the secondary development was performed using hexane:diethyl ether:acetic acid = 70:30:1 (v / v / v) for 5 and 30 minutes, respectively, before analysis. An IATROSCAN MK-6 (Mitsubishi Chemical Iatron Corporation) was used for TLC-FID.

[0076] (Turbidity analysis) In the STEARIN test, a standard method in the United States, as specified in the USP-NF SPECIFIC TESTS for Fish Oil Containing Omega-3 Acids, the oil must remain transparent even after cooling to 0°C for three hours. When the deacidified oil was subjected to the STEARIN test after enzymatic reaction, only the sample after two hours of enzymatic reaction was transparent and passed the test; all samples after four hours became cloudy and failed. Regarding fatty acid composition, the proportion of docosahexaenoic acid (DHA) increased with increasing reaction time, while the proportion of palmitic acid (C16:0) decreased. Regarding lipid composition, the proportion of triacylglycerol (TAG) decreased with increasing reaction time, while the proportions of diacylglycerol (DAG) and monoacylglycerol (MAG) increased.

[0077] [Table 7] ND: Not Detected

[0078] Example 4: Method for obtaining microbial oil with a high TAG ratio The mutant strain capable of low-temperature cultivation created in Example 1 was cultured for a certain period at 23°C (normal cultivation temperature) and 15°C (low temperature), and sampling was carried out over time to confirm the changes in fatty acid composition.

[0079] A preculture solution for cultivation in a fermentation culture tank was prepared using the method described in Example 3. A 1-L fermentation culture tank was charged with 0.6 L of liquid medium (2) and 0.18 g of antifoaming agent KM-72F (Shin-Etsu Chemical Co., Ltd.), and sterilized at 121°C for 20 minutes. The same vitamin solution and metal salt solution as those used in liquid medium (1) were used. After the medium temperature had dropped to approximately 20°C, approximately 10 mL of preculture solution was inoculated. Cultivation was carried out for 14 days under the following conditions: a cultivation temperature of 23°C or 15°C, an agitation speed of 300 rpm, an aeration rate of 0.25 L / min, an internal gauge pressure of 0 MPa, and a pH of 6.8±0.2. [Table 8]

[0080] The cultured bacterial cell suspension was then centrifuged at 5,000 x g for 3 minutes at 25°C to obtain wet bacterial cells. The collected wet bacterial cells were suspended in 200 mL of artificial seawater and centrifuged again at 5,000 x g for 3 minutes at 25°C to wash the wet bacterial cells. This washing process was repeated three times, and then the suspension was freeze-dried at -80°C to obtain freeze-dried bacterial cells.

[0081] The culture medium was sampled daily throughout the culture period to measure the glucose concentration and dry cell weight (DCW) (g / L). Furthermore, freeze-dried cells were obtained using the method described in Example 2, and the fatty acid content (g / L, %) and fatty acid composition of the dried cells were measured. The analytical methods are described below.

[0082] The glucose concentration was measured using a biochemistry analyzer YSI 2900D (Xylem Japan Co., Ltd.) in the supernatant obtained by centrifuging the culture medium at 8,000 xg for 3 minutes at 25°C.

[0083] Dry cell weight (DCW) was determined by weighing the wet cells obtained by centrifuging 1 mL of the culture medium at 8,000 xg for 3 minutes at 25°C and then dry-drying them at 105°C for 3 hours. The fatty acid content and fatty acid composition of the dried cells were determined by weighing 20±3 mg of lyophilized cells, adding 2 mL of hydrogen chloride-methanol reagent (5-10%) as an esterifying agent and 1 mL of an approximately 0.5 mg / mL internal standard solution (C13:0-methyl ester / dichloromethane solution), mixing, and then heating at 50°C for 3 hours. During the 3-hour heating period, mixing was performed once per hour for approximately 20 seconds. After cooling, 4 mL of water and 1 mL of hexane were added and the mixture was vigorously mixed by inversion. The mixture was centrifuged at 1,000 x g for 3 minutes at 25°C to separate the lower aqueous layer and the upper organic solvent layer. GC analysis of the organic solvent layer was performed as in Example 2.

[0084] The fatty acid content in the dried cells was calculated from the results of DCW and fatty acid analysis. It was calculated from the ratio of the amount and area of ​​the internal standard C13:0-methyl ester to the total area of ​​other detected fatty acids, and the weight of the freeze-dried cells used in the analysis sample. Total lipids were extracted from a portion of the lyophilized cells obtained after 14 days of culture using the Bligh & Dyer method (EG Bligh & WJ Dyer: Can. J. Biochem. Physiol., 37, 911 (1959)). Lipid composition (glyceride ratio) was analyzed by TLC-FID. 2 g of lyophilized cells were weighed into a 50 mL glass centrifuge tube, and 8 mL of chloroform, 16 mL of methanol, and 6.4 mL of water were added. The cells were disrupted by sonication at 40 kHz for 40 minutes. An additional 8 mL of chloroform and 8 mL of water were added, and the mixture was mixed by inversion and sonicated for an additional 20 minutes. After centrifugation at 1,910 x g for 3 minutes, the lower organic layer was transferred to a new 50 mL glass centrifuge tube, and the lipids were collected. 16 mL of chloroform was added to the remaining upper layer, mixed, and centrifuged. The organic lower layer was transferred to the same 50 mL glass centrifuge tube as before to recover lipids. The solvent was removed from the resulting lower layer using vacuum distillation to recover oil. The recovered oil was subjected to TLC-FID analysis using the method described in Example 2. Figure 1 shows the relationship between the number of days of culture at each temperature and the glucose concentration and fatty acid content. Figures 2 and 3 also show the change in fatty acid composition depending on the number of days of culture.

[0085] From 2 g of freeze-dried cells on the 14th day of cultivation, 0.40 g of oil was obtained at 23°C and 0.24 g at 15°C. The glyceride ratios were higher at 23°C than at 15°C, with a higher proportion of TAG and a relatively lower proportion of MAG, especially DAG (Figure 4).

[0086] Example 5: Low-temperature scale-up cultivation of mutant strains Scale-up culture of Example 4 was carried out. A preculture solution for cultivation in a fermentation culture tank was prepared using the method described in Example 3. A 50-L fermentation culture tank was charged with 30 L of liquid medium (2) and 9 g of antifoaming agent KM-72F (Shin-Etsu Chemical Co., Ltd.), followed by moist heat sterilization. The same vitamin solution and metal salt solution as those used in liquid medium (1) were used. After the medium temperature was lowered to approximately 15°C, approximately 300 mL of preculture solution was inoculated. Cultivation was carried out for 14 days under the following conditions: a cultivation temperature of 15°C, an agitation speed of 200 rpm, an aeration rate of 15 L / min, an internal gauge pressure of 0.02 MPa, and a pH of 6.8±0.2. The entire cultured bacterial cell suspension was then centrifuged at 5,000 x g for 3 minutes at 25°C to obtain wet bacterial cells. The collected wet bacterial cells were suspended in 1 L of artificial seawater and centrifuged at 5,000 x g for 3 minutes at 25°C to wash the wet bacterial cells. This washing process was repeated twice, and the cells were then dried at 100°C. Furthermore, throughout the culture period, the culture medium was sampled every day, and the glucose concentration, dry cell weight (DCW), fatty acid content, fatty acid composition, and lipid composition in the medium were measured in the same manner as in Example 4. The trends in changes in glucose concentration and fatty acid content were the same as in Example 4. Furthermore, the trends in changes in fatty acid composition and the composition values ​​were not significantly different from those in Example 4. As for the change in lipid composition (glyceride ratio) over time, the ratio of TAG increased from immediately after glucose depletion toward the end of the culture, and by the 14th day of culture, it had reached a ratio similar to the glyceride ratio in Example 4.

[0087] Approximately 130 g of dry-heat dried cells obtained after 14 days of cultivation were subjected to hexane extraction in the same manner as in Example 2, yielding 3.7 g of oil. Furthermore, the acid value of the hexane-extracted oil was measured and subjected to degumming and deacidification. The acid value, fatty acid composition, and lipid composition of the hexane-extracted oil and the degummed and deacidified oil were measured. The acid value decreased to 0.1 meq / kg after degumming and deacidification. Regarding the fatty acid composition, there was a slight decrease in the proportion of palmitic acid (C16:0) and an increase in the DHA concentration before and after degumming and deacidification, but the differences were not significant (Table 9). Regarding the lipid composition, the proportion of phospholipids decreased with the increase in the proportion of TAG due to degumming and deacidification. Each oil was subjected to the STEARIN test in the same manner as in Example 3. The degummed and deacidified oil remained transparent even after cooling at 0°C for 3 hours. [Table 9] ND: Not Detected [Accession number]

[0088] FERM BP-22486

Claims

1. 1. An oil containing palmitic acid (C16:0) at a concentration of 7% to 20% by weight of the total weight of fatty acids in the oil, which does not produce turbidity when cooled at 0°C for 3 hours, and which has the following characteristics: (i) triglycerides are 83% or more, 85% or more, 88% or more, or 90% or more; (ii) the PUFA content is 70% or more, 75% or more, or 80% or more; (iii) a stearic acid content of less than 3% or 1%; (iv) less than 50%, less than 25%, less than 10%, less than 5%, or less than 2% of fatty acids having 18 carbon atoms; and (v) the DHA / EPA ratio is 11 to 20 An oil having at least one characteristic selected from the group consisting of:

2. 10. The oil of claim 1, further comprising: (vi) a DHA content of 60% or more, 65% or more, 68% or more, or 70% or more.

3. The oil may be one of the following: (ii) the content of PUFAs is 70% or more, 75% or more, or 80% or more; and (v) the DHA / EPA ratio is 11 to 20 2. The oil of claim 1, characterized by:

4. The oil may be one of the following: (iii) a stearic acid content of less than 3% or 1%; and (v) the DHA / EPA ratio is 11 to 20 2. The oil of claim 1, characterized by:

5. The oil may be one of the following: (iv) less than 50%, less than 25%, less than 10%, less than 5%, or less than 2% of fatty acids having 18 carbon atoms; and (v) the DHA / EPA ratio is 11 to 20 2. The oil of claim 1, characterized by:

6. The oil of any one of claims 3 to 5, further characterized in that (i) it has a triglyceride content of 83% or more, 85% or more, 88% or more, or 90% or more.

7. 6. The oil of any one of claims 2, 4, and 5, further characterized in that (ii) the PUFA content is 70% or more, 75% or more, or 80% or more.

8. 6. The oil of any one of claims 2, 3, and 5, further comprising: (iii) a stearic acid content of less than 3%, or less than 1%.

9. 5. The oil of claim 2, further characterized in that (iv) the oil contains less than 50%, less than 25%, less than 10%, less than 5%, or less than 2% of fatty acids having 18 carbon atoms.

10. The oil according to claim 2 or 3, further comprising (v) a DHA / EPA ratio of 11 to 20.

11. 11. The oil according to any one of claims 1 to 10, which is a microbial oil.

12. 12. The oil of claim 11, which is a refined oil.

13. The oil according to claim 12, which is a refined oil obtained by degumming and deacidifying a crude oil obtained by hexane extraction.

14. The oil according to claim 13, which is a refined oil obtained by further subjecting the refined oil to lipase treatment and deacidification treatment.

15. 15. The oil according to claim 14, wherein the microbial oil is obtained from a microorganism belonging to the Stramenopiles.

16. 16. The oil of claim 15, wherein the microbial oil is obtained from a microorganism belonging to the class Labyrinthulea.

17. 17. The oil of claim 16, wherein the microbial oil is obtained from a microorganism belonging to the Thraustochytrid fungus family.

18. 18. The oil of claim 17, wherein the microbial oil is obtained from the microorganism Thraustochytrium aggregatum.

19. 19. The oil of claim 18, wherein the microbial oil is obtained from the microorganism Thraustochytrium aggregatum having the deposit number FERM BP-22486.

20. A method for screening for a cold-growing strain, comprising the steps of: A step of inducing mutations in microorganisms belonging to Stramenopiles by chemical or physical treatment; Culturing the microorganism in which the mutation has been induced; a step of obtaining a strain with high low-temperature growth ability from the cultured microorganism; wherein the low temperature is 5°C to 10°C lower than the optimum growth temperature of the microorganism.

21. The method according to claim 20, wherein the step of culturing the microorganism in which the mutation has been induced is carried out using a solid medium.

22. The method according to claim 21, wherein in the step of obtaining a strain having high low-temperature growth ability from the cultured microorganism, the strain having high low-temperature growth ability is obtained by isolating colonies formed on the solid medium.

23. 21. The method of claim 20, wherein the chemical treatment is a mutagenic agent treatment, or the physical treatment is ultraviolet light, X-rays, gamma rays, or ion beam treatment.

24. 22. The method of claim 21, wherein the solid medium is selected from an agar medium, a gelatin medium, and a gellan gum medium.

25. The method according to any one of claims 20 to 24, wherein the low temperature is a culture temperature of 13 to 18°C.

26. 1. A method for producing a microbial oil, comprising: a step of inoculating a carbon source-containing medium with the strain screened by the method according to any one of claims 20 to 25; Cultivating the inoculated carbon source-containing medium at a temperature below 20°C, below 18°C, or below 16°C; recovering the bacterial cells; A step of subjecting the recovered bacterial cells to solvent extraction to obtain crude oil; and a step of subjecting the solvent extracted crude oil to degumming and deacidification; wherein in the culturing step, culturing is carried out for 12 hours or more, 1 day or more, 2 days or more, 3 days or more, or 4 days or more after carbon source depletion. Method for producing microbial oil.

27. The method according to claim 26, wherein the strain screened by the method according to any one of claims 22 to 25 is a microorganism of Thraustochytrium aggregatum having deposit number FERM BP-22486.

28. 1. A method for producing a microbial oil, comprising: a step of inoculating a carbon source-containing medium with a microorganism belonging to the Stramenopiles; culturing the inoculated microorganism in the carbon source-containing medium; a step of recovering the cultured microbial cells; extracting oils and fats from the collected microbial cells; subjecting the extracted oil and fat to lipase treatment; and Deoxidation process A method for producing a microbial oil, comprising:

29. The method for producing microbial oil according to claim 28, wherein solvent extraction is performed in the step of extracting oils and fats from the collected microbial cells.

30. The method according to claim 28, wherein the lipase treatment is carried out for 3 hours or less.

31. 29. The method of claim 28, further comprising the step of degumming before or after the lipase treatment.

32. The method according to any one of claims 28 to 31, further comprising a step of carrying out a deacidification treatment before the lipase treatment.

33. The microorganism Thraustochytrium aggregatum has accession number FERM BP-22486.

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