Method for producing biomass containing protein and omega-3 fatty acids from a single microalgae and biomass produced thereby

Abstract

This application relates to a method for producing biomass containing protein and omega-3 fatty acids from a single microalga, and the biomass produced thereby. According to one aspect of the biomass production method, a single microalgae-derived biomass containing high protein and omega-3 fatty acids can be produced by continuously supplying a nitrogen source during the cultivation process, and the biomass produced thereby can be useful as a single microbial source of protein and omega-3 fatty acids.

Description

Technical Field

[0001] The present application relates to a method for producing biomass containing proteins and omega-3 fatty acids from a single microalgae and the biomass produced thereby.

Background Art

[0002] Unsaturated fatty acids are fatty acids having one or more double bonds within the fatty acid chain, and when containing two or more double bonds, they are called polyunsaturated fatty acids (PUFAs). Among these, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are typical omega-3 fatty acids and are essential fatty acids for the brain, eye tissues, and nervous system. Also, they are known to play important functions in nervous system development such as infant vision and motor nerve ability and in the prevention of cardiovascular diseases, and are the most abundant components in the structural lipids of the brain.

[0003] The main source for industrially supplying polyunsaturated fatty acids such as omega-3 fatty acids is fish oil extracted from the oil of bluefish, and fish meal is also widely used as a protein source for food or feed. However, due to the difficulties in continuously supplying fish oil and fish meal such as catch limits, there is a need to develop alternative sources of omega-3 fatty acids and proteins that can replace fish oil and fish meal.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Recently, research has been conducted on the production of polyunsaturated fatty acids by culturing microalgae. However, this research has focused on oil production such as fatty acids, and there is little research on methods that can produce high levels of protein from microalgae. Therefore, the inventors of the present application have derived a method for producing biomass containing omega-3 fatty acids and high levels of protein from a single microalgae and completed the present application.

[0006] An object of the present application is to provide a method for producing Thraustochytrid-derived biomass, which includes culturing a single strain of Thraustochytrid microalgae in a medium; and continuously supplying a nitrogen source during the culturing step, and Thraustochytrid-derived biomass of a single microalgae produced thereby.

Means for Solving the Problems

[0007] Each description and embodiment disclosed in the present application is applicable to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. Also, it cannot be said that the scope of the present application is limited by the specific descriptions described below. Also, those with ordinary knowledge in the technical field can recognize or confirm a number of equivalents to the specific aspects of the present application described in the present application using only ordinary experiments. Also, such equivalents are intended to be included in the present application.

[0008] One aspect provides a method for producing Thraustochytrid-derived biomass, which includes culturing a single strain of Thraustochytrid microalgae in a medium; and continuously supplying a nitrogen source during the culturing step.

[0009] As used herein, the term "Thraustochytrid" means microalgae of the order Thraustochytriales. The Thraustochytrid microalgae may be microalgae of the genus Thraustochytrium sp., Schizochytrium sp., Aurantiochytrium sp. or Thraustochytriidae sp.

[0010] As used herein, the terms "Thraustochytrium sp.", "Schizochytrium sp.", "Aurantiochytrium sp." and "Thraustochytriidae sp." are each one of the genus names belonging to the family Thraustochytriaceae of the order Thraustochytriales, and can be used interchangeably with the terms "genus Thraustochytrium", "genus Schizochytrium", "genus Aurantiochytrium" and "genus Thraustochytriidae", respectively.

[0011] Also, the term "microalgae" means organisms that are plants that perform photosynthesis with chlorophyll, are invisible to the naked eye and can only be seen through a microscope, and live floating freely in water, and are also called Phytoplankton.

[0012] In one specific example, the Thraustochytrium - based microalgae may be, for example, the microalgae CD01 - 6003 of the genus Thraustochytrium deposited under the accession number KCTC14346BP, or the microalgae CD01 - 5004 of the genus Schizochytrium deposited under the accession number KCTC14345BP, but is not limited thereto.

[0013] As used herein, the term "biomass" means organisms such as plants, animals, and microorganisms that can be used as chemical energy, i.e., the energy source of bioenergy, and may also mean the weight or energy amount of a specific organism present ecologically within a unit time and space. Further, the biomass includes compounds secreted by cells, but is not limited thereto, and may contain not only extracellular substances but also cells and / or intracellular contents. In the present application, the biomass may be the Thraustochytrium - based microalgae itself, its culture, its dried product, its crushed product, or a product produced by culturing or fermenting the microalgae, or may be a concentrate or dried product of the biomass, but is not limited thereto.

[0014] The "culture" of the Thraustochytrium - based microalgae refers to a product produced by culturing the microalgae, and specifically may be a culture solution containing the microalgae or a culture solution from which the microalgae have been removed, but is not limited thereto. The "dried product" of the Thraustochytrium - based microalgae culture is a product from which moisture has been removed from the microalgae culture, and may be, for example, the dried cell form of the microalgae, but is not limited thereto. Further, the "crushed product" of the dried product is a general term for the product obtained by crushing the dried product from which moisture has been removed from the microalgae culture, and may be, for example, dried cell powder, but is not limited thereto.

[0015] The culture of the Slothkitridophyceae microalgae can be produced by a culturing method including the steps of inoculating the microalgae into a microalgae culture medium and culturing them, and continuously supplying a nitrogen source during the culturing step. The dried product and the crushed product of the culture can be produced by a method for treating or drying microalgae or a culture solution known in the art.

[0016] In the biomass production method, the nitrogen source may be supplied from immediately after inoculating the microalgae into the medium until the end of the culture.

[0017] Alternatively, the nitrogen source may be continuously supplied so that the total nitrogen concentration in the culture solution becomes 300 ppm or more. For example, the nitrogen source may be continuously supplied so that the total nitrogen concentration in the culture solution is maintained within the range of 300 to 10,000 ppm, 300 to 8,000 ppm, 300 to 5,000 ppm, 300 to 3,000 ppm, 300 to 2,000 ppm, 300 to 1,500 ppm, 350 to 10,000 ppm, 350 to 8,000 ppm, 350 to 5,000 ppm, 350 to 3,000 ppm, 350 to 2,000 ppm, or 350 to 1,500 ppm.

[0018] In the biomass production method, the medium may contain a carbon source and a nitrogen source.

[0019] In the biomass production method, the nitrogen source in the step of continuously supplying the nitrogen source and the nitrogen source contained in the medium may be any one or more organic nitrogen sources selected from the group consisting of i) yeast extract, beef extract, peptone, and tryptone, or ii) any one or more inorganic nitrogen sources selected from the group consisting of ammonium acetate, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, urea, MSG (Monosodium glutamate), and ammonia, but it is not limited to this as long as it is a nitrogen source used for culturing Slawskitrid-based microalgae.

[0020] The step of continuously supplying the nitrogen source can be carried out, for example, by continuously supplying a medium containing the nitrogen source to the culture solution, or by continuously supplying ammonia gas to the fermenter for culturing the microalgae, but it is not limited to this. Continuously supplying a medium containing the nitrogen source to the culture solution can be carried out, for example, by a fed-batch culture method of intermittently supplying the medium or a continuous culture method of continuously supplying the medium.

[0021] The method can further include a step of continuously supplying a carbon source during the culturing step. The step of continuously supplying the carbon source can be carried out, for example, by continuously supplying a medium containing the carbon source to the culture solution, but it is not limited to this. Continuously supplying a medium containing the carbon source to the culture solution can be carried out, for example, by a fed-batch culture method or a continuous culture method. The step of continuously supplying the carbon source can be carried out while maintaining the carbon source concentration in the culture solution at 5% (w / v) or less.

[0022] The carbon source in the stage of continuously supplying the carbon source and the carbon source contained in the medium may be any one or more selected from the group consisting of glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose, and glycerol, but is not limited thereto as long as it is a carbon source used for culturing Slawskitrid-based microalgae.

[0023] The medium may further contain a suitable phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc. used for culturing Slawskitrid-based microalgae. For example, the phosphorus source may individually contain or contain a mixture of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts, etc., but is not limited thereto.

[0024] The culturing stage can be carried out within 95 hours or less. For example, the culturing stage can be carried out for 30 - 95 hours, 35 - 95 hours, 40 - 95 hours, 45 - 95 hours, 30 - 90 hours, 35 - 90 hours, 40 - 90 hours, or 45 - 90 hours.

[0025] The culturing stage can be carried out at 20 - 35°C. For example, the culturing stage can be carried out at 25 - 35°C, 20 - 30°C, or 25 - 30°C, but is not necessarily limited thereto.

[0026] In the culturing stage, in order to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas can be injected into the culture, or without gas injection to maintain the anaerobic and anoxic states, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but is not limited thereto.

[0027] The culturing step can be carried out while adjusting the pH. For example, it can be carried out while adjusting to maintain a pH of 3.5 - 9.0, pH 4.0 - 9.0, pH 4.5 - 9.0, pH 5.0 - 9.0, pH 3.5 - 8.0, pH 4.0 - 8.0, pH 4.5 - 8.0 or pH 5.0 - 8.0 using a basic compound (e.g., sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid), but is not limited thereto.

[0028] The biomass production method may further include a step of recovering biomass from the strain, the culture of the strain, the dried product of the culture or the crushed product of the dried product.

[0029] In the step of recovering the biomass, the target biomass can be collected using a suitable method known in the art. For example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC etc. can be used, and a purification step can further be included.

[0030] The biomass produced by the production method may contain 50 wt% or more of protein and 37 wt% or less of fat based on the total weight of the biomass. The biomass may contain, for example, 50 - 80 wt%, 50 - 75 wt%, 50 - 70 wt%, 55 - 80 wt%, 55 - 75 wt% or 55 - 70 wt% of protein; and 5 - 37 wt%, 5 - 35 wt%, 5 - 30 wt%, 5 - 25 wt%, 10 - 37 wt%, 10 - 35 wt%, 10 - 30 wt%, 10 - 25 wt%, 15 - 37 wt%, 15 - 35 wt%, 15 - 30 wt% or 15 - 25 wt% of fat.

[0031] The biomass produced by the manufacturing method may contain 50% by weight or more of protein and omega-3 fatty acids based on the total weight of the biomass. The omega-3 fatty acids may contain any one or more of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

[0032] The term "docosahexaenoic acid (DHA)" used in this specification is one of the polyunsaturated fatty acids having the chemical formula of C 22 H 32 O2, which corresponds to omega-3 fatty acids together with alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA), and its common name is cervonic acid, and it can also be denoted by the abbreviation 22:6 n-3.

[0033] The term "eicosapentaenoic acid (EPA)" used in this specification is one of the polyunsaturated fatty acids having the chemical formula of C 20 H 30 O2, which corresponds to omega-3 fatty acids together with ALA and DHA, and it can also be denoted by the abbreviation 20:5 n-3.

[0034] In the biomass containing 50% by weight or more of protein and omega-3 fatty acids, the biomass may contain 3 to 30% by weight, 3 to 25% by weight, 3 to 20% by weight, or 3 to 15% by weight of omega-3 fatty acids based on the total weight of the biomass. Also, the biomass may contain 15 to 60% by weight, 15 to 55% by weight, or 15 to 50% by weight of omega-3 fatty acids based on the total weight of total fatty acids.

[0035] Another aspect provides a biomass derived from a single strain of Thraustochytrium microalgae, which is produced by a biomass production method including culturing a single strain of Thraustochytrium microalgae in a medium; and continuously supplying a nitrogen source during the culturing step.

[0036] The biomass production method is as described above.

[0037] The biomass may contain 50% by weight or more of protein and 37% by weight or less of fat based on the total weight of the biomass. The biomass may contain, for example, 50 - 80% by weight, 50 - 75% by weight, 50 - 70% by weight, 55 - 80% by weight, 55 - 75% by weight or 55 - 70% by weight of protein; and 5 - 37% by weight, 5 - 35% by weight, 5 - 30% by weight, 5 - 25% by weight, 10 - 37% by weight, 10 - 35% by weight, 10 - 30% by weight, 10 - 25% by weight, 15 - 37% by weight, 15 - 35% by weight, 15 - 30% by weight or 15 - 25% by weight of fat.

[0038] The biomass may contain 50% by weight or more of protein and omega - 3 fatty acids based on the total weight of the biomass, and the omega - 3 fatty acids may contain any one or more of docosahexaenoic acid and eicosapentaenoic acid.

[0039] In the biomass containing 50% by weight or more of protein and omega - 3 fatty acids, the biomass may contain 3 - 30% by weight, 3 - 25% by weight, 3 - 20% by weight or 3 - 15% by weight of omega - 3 fatty acids based on the total weight of the biomass. Also, the biomass may contain 15 - 60% by weight, 15 - 55% by weight or 15 - 50% by weight of omega - 3 fatty acids based on the total weight of fatty acids.

[0040] Another aspect provides a composition comprising biomass produced by a biomass production method including culturing a single strain of Thraustochytrium microalgae in a medium and continuously supplying a nitrogen source during the culturing step, or a concentrate or dried product of the biomass.

[0041] The biomass production method and the Thraustochytrium single microalgae-derived biomass are as described above.

[0042] The concentrate or dried product of the biomass can be produced by methods for treating, concentrating, or drying microbial biomass known in the art.

[0043] 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 additive composition.

[0044] As used herein, the term "feed composition" refers to feed given to animals. The feed composition refers to a substance that supplies organic or inorganic nutrients necessary to maintain the life of animals or produce meat, milk, etc. The feed composition can additionally contain nutritional components necessary to maintain the life of animals or produce meat, milk, etc. The feed composition can be manufactured as various forms of feed known in the art, specifically including concentrated feed, roughage, and / or special feed.

[0045] As used herein, the term "feed additive" includes substances added to feed for various purposes such as nutrient supplementation and weight loss prevention, enhancing the digestibility and utilization of fiber in feed, improving milk quality, preventing reproductive disorders and increasing conception rates, and preventing summer heat stress. The feed additives of this application fall under the category of supplementary feeds under the Feed Control Law and can additionally include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals; mineral preparations that are trace minerals such as zinc, copper, cobalt, and selenium; vitamin preparations such as carotene, vitamin E, vitamin A, D, E, nicotinic acid, and vitamin B complex; protected amino acid preparations such as methionine and lysine; protected fatty acid preparations such as calcium salts of fatty acids; live bacteria preparations (lactic acid bacteria preparations), live bacteria such as yeast cultures and mold fermentations, and yeast preparations, etc.

[0046] As used herein, the term "food composition" includes all forms such as functional foods, nutritional supplements, health foods, and food additives. Food compositions of the above types can be manufactured in various forms by ordinary methods known in the art.

[0047] The compositions of this application can further include grains such as ground or crushed wheat, oats, barley, corn, and rice; vegetable protein feeds such as feeds mainly composed of beans and sunflowers; animal protein feeds such as blood meal, meat meal, bone meal, and fish meal; sugars and dairy products such as dry components composed of various powdered milks and milk plasma powders, etc., and can also further include nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

[0048] The composition of the present application can be administered to animals alone or in combination with other feed additives in an edible carrier. Further, the composition can be easily administered to animals as a top dressing or by directly mixing them into the feed or as a separate oral dosage form from the feed. When the composition is administered separately from the feed, as is well known in the art, it can be manufactured in an immediate release or sustained release dosage form in combination with a pharmaceutically acceptable edible carrier. Such edible carriers can be solid or liquid, such as corn starch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the composition may be in the form of tablets, capsules, powders, troches or sugar-coated tablets or a top dressing in an undispersed form. When a liquid carrier is used, the composition may be in the form of soft gelatin capsules, or in the form of syrups, suspensions, emulsions or solutions.

[0049] The composition of the present application can contain, for example, preservatives, stabilizers, wetting agents or emulsifiers, cryoprotectants or excipients. The cryoprotectant may be one or more selected from the group consisting of glycerol, trehalose, maltodextrin, non-fat dry milk and starch.

[0050] The preservative, stabilizer or excipient may be contained in the composition in an effective amount sufficient to reduce the deterioration of the Thraustochytrium-based microalgae contained in the composition. Further, the cryoprotectant may be contained in the composition in an effective amount sufficient to reduce the deterioration of the Thraustochytrium-based microalgae contained in the composition when the composition is in a dried state.

[0051] The composition can be used by adding it to animal feed by liquid immersion, spraying or mixing.

[0052] The compositions of the present application can be applied to, but are not limited to, a number of animal diets including mammals, birds, fish, crustaceans, cephalopods, reptiles and amphibians. For example, the mammals can include pigs, cows, sheep, goats, laboratory rodents or pets, etc.; the birds can include poultry, and the poultry can include, but are not limited to, chickens, turkeys, ducks, geese, pheasants or quails, etc. Also, the fish can include commercially farmed fish and their fry, ornamental fish, etc.; the crustaceans can include, but are not limited to, shrimps, barnacles, etc. Further, the compositions can also be applied to the diet of rotifers which are zooplankton.

Advantages of the Invention

[0053] According to the biomass production method in a uniform phase, by continuously supplying a nitrogen source during the culture process, a single microalgae-derived biomass containing a high content of protein and omega-3 fatty acids can be produced, and the biomass thus produced can be usefully utilized as a single microbial source of protein and omega-3 fatty acids.

Modes for Carrying Out the Invention

[0054] Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustratively explaining one or more specific examples, and the scope of the present invention is not limited to these examples.

Examples

[0055] 〔Example 1〕 Confirmation of Fat and Protein Production by the Culture Method of Microalgae of the Genus Thraustochytrium 1-1. Preparation of Microalgae and Inoculum Culture The microalgae CD01-6003 of the genus Slaucistrochytrium (Accession No.: KCTC14346BP) was inoculated into sterilized MJW01 medium (glucose 30 g / L, MgSO4·7H2O 3.0 g / L, Na2SO4 10 g / L, NaCl 1.0 g / L, yeast extract 9.0 g / L, MSG·1H2O 1.0 g / L, NaNO3 1.0 g / L, KH2PO4 0.1 g / L, K2HPO4 0.5 g / L, CaCl2 0.5 g / L, and vitamin mixed solution 10 mL / L) and cultured in a 250 mL flask at 20 - 35 °C and 100 - 300 rpm for 10 - 30 hours.

[0056] 1 - 2. This culture 1 - 2 - 1. Comparative Example (1) The seed culture prepared in Example 1 - 1 above was dispensed into a 5 L fermenter containing sterilized MJW01 medium supplemented with 6 g / L of ammonium sulfate ((NH4)2·SO4) and 5 g / L of yeast extract as nitrogen sources, and cultured at 20 - 35 °C, 100 - 500 rpm, and 0.5 - 1.5 vvm until the volume of the culture solution in the fermenter reached 2.8 L, for a total of 101 hours of culture. Ammonia gas was continuously supplied to the fermenter from immediately after the start of the culture as an additional nitrogen source so that the total nitrogen concentration in the culture solution would be 300 ppm or more for 14 hours, and then the supply of ammonia gas was interrupted. During the entire culture process, a Feed containing a carbon source was continuously supplied so that the carbon source concentration in the culture solution would be maintained at 5% or less.

[0057] 1 - 2 - 2. Example (1) The culture was carried out in the same manner as described in 1 - 2 - 1 above, except that ammonia gas was continuously supplied to the fermenter so that the total nitrogen concentration in the culture solution would be 300 ppm or more during the entire culture process, for a total of 86 hours of culture.

[0058] 1 - 2 - 3. Example (2) During the entire culturing process, ammonia gas was continuously supplied to the fermenter so that the total nitrogen concentration in the culture solution became 300 ppm or more. Culturing was carried out in the same manner as described in 1-2-1 for a total of 75 hours, except that only 18 g / L of ammonium sulfate was added as a nitrogen source to the sterilized MJW01 medium.

[0059] 1-2-4. Example (3) During the entire culturing process, ammonia gas was continuously supplied to the fermenter so that the total nitrogen concentration in the culture solution became 300 ppm or more. Culturing was carried out in the same manner as described in 1-2-1 for a total of 55 hours, except that 10 g / L of ammonium sulfate was further added as a nitrogen source to the Feed containing a carbon source and supplied.

[0060] 1-2-5. Example (4) During the entire culturing process, ammonia gas was continuously supplied to the fermenter so that the total nitrogen concentration in the culture solution became 300 ppm or more. Culturing was carried out in the same manner as described in 1-2-1 for a total of 92 hours, except that only 6 g / L of ammonium sulfate was added as a nitrogen source to the sterilized MJW01 medium, and 12 g / L of ammonium sulfate was further added as a nitrogen source to the Feed containing a carbon source and supplied.

[0061] 1-3. Fat content analysis The cells obtained after collecting each microalgae culture cultivated in the above 1-2-1 to 1-2-5, centrifuging, and then washing the cells three times with PBS were dried at 60°C for 16 hours. 8.3 M hydrochloric acid solution was added to 2 g of the dried cells, and a hydrolysis reaction was carried out at 80°C. Then, 30 mL of ethyl ether and 20 mL of petroleum ether were added to the reaction product, and the process of mixing for 30 seconds and centrifuging was repeated three or more times. The separated solvent layer was transferred to a round flask whose weight had been measured in advance, and then placed in a container where the solvent and residual moisture were removed through N2 purging and dried. The weight of the oil remaining after drying the solvent was measured to calculate the total oil content, and the content of omega-3 fatty acids (DHA and EPA) contained in the oil was measured by gas chromatography after pretreatment with 0.5 N NaOH in methanol and 14% boron trifluoride methanol solution (BF3-MeOH).

[0062]

Table 1

[0063] As a result, as shown in Table 1, the total fatty acid (TFA) content in the biomass decreased more in Examples (1) to (4) cultured with continuous supply of nitrogen source compared to Comparative Example (1), but the EPA, DHA, and omega-3 contents in the crude fat showed equal or higher contents in Examples (1) to (4) compared to Comparative Example 1.

[0064] 1-4. Protein content analysis Quantitative analysis of the nitrogen content present in the sample was performed using an elemental analyzer on 0.5 - 1 g of each dried cell obtained by the same method as described in 1-3 above. The nitrogen weight ratio (TN%) present in each sample was multiplied by 6.25 and calculated and shown as the crude protein content in the sample.

[0065]

Table 2

[0066] As a result, as shown in Table 2, in the cases of Examples (2) to (4), all showed a crude protein content of 55% or more. In the case of Example (1), considering that the crude fat content measured in 1-3 was 28.9%, the normal carbohydrate content of the microalgae biomass was about 2-3%, and the ash content was about 8-10%, it can be estimated that the crude protein content would reach a maximum of about 58.10%. Also, in the case of Comparative Example (1), it can be estimated that the maximum crude protein content would only be about 43.58% through the fact that the crude fat content measured in 1-3 was 43.42%. Through this, it was confirmed that when culturing microalgae under the condition of continuously supplying a nitrogen source, protein can be produced at a high content of 55% or more.

[0067] 〔Example 2〕 Confirmation of the production amounts of fat and protein by the method for culturing microalgae of the genus Schizochytrium 2-1. Preparation of microalgae and inoculum culture The microalgae of the genus Schizochytrium CD01-5004 (accession number: KCTC14345BP) was inoculum-cultured in the same manner as described in Example 1-1 above.

[0068] 2-2. Main culture 2-2-1. Comparative Example (2) The inoculum culture prepared in Example 2-1 was dispensed into a 5 L fermenter containing the sterilized MJW01 medium and cultured at 20-35 °C, 100-500 rpm, and 0.5-1.5 vvm until the volume of the culture solution in the fermenter reached 2.8 L, for a total of 105 hours of culture. As an additional nitrogen source, ammonia gas was supplied to the fermenter from immediately after the start of the culture for 10 hours so that the total nitrogen concentration in the culture solution would be 300 ppm or more, and then the supply of ammonia gas was interrupted. During the entire culture process, a Feed containing a carbon source was continuously supplied so that the carbon source concentration in the culture solution would be maintained at 5% or less.

[0069] 2-2-2. Example (5) During the entire cultivation process, ammonia gas was continuously supplied to the fermenter so that the total nitrogen concentration in the culture solution became 300 ppm or more. Cultivation was carried out in the same manner as described in 2-2-1 except that 6 g / L of ammonium sulfate and 10 g / L of yeast extract were added as nitrogen sources to the sterilized MJW01 medium, and the cultivation was carried out for a total of 84 hours.

[0070] 2-2-3. Example (6) Cultivation was carried out in the same manner as described in 2-2-2 except that the cultivation was carried out for a total of 53 hours.

[0071] 2-2-4. Examples (7) and (8) Cultivation was carried out in the same manner as described in 2-2-2 except that the cultivation was carried out for a total of 50 hours.

[0072] 2-2-5. Example (9) Cultivation was carried out in the same manner as described in 2-2-2 except that the cultivation was carried out for a total of 47 hours.

[0073] 2-2-6. Example (10) Cultivation was carried out in the same manner as described in 2-2-2 except that 5 g / L of MSG was further added as a nitrogen source to the sterilized MJW01 medium, and the cultivation was carried out for a total of 47 hours.

[0074] 2-3. Fat content analysis Each microalgae culture cultivated in 2-2-1 to 2-2-6 was collected, and the fatty acid content was analyzed in the same manner as described in Examples 1-3.

[0075]

Table 3

[0076] As a result, as shown in Table 3, the total fatty acid (TFA) content in the biomass decreased more in Examples (5) to (10) cultured with continuous supply of nitrogen source compared to Comparative Example 2. However, the EPA, DHA, and omega-3 contents in the crude fat showed equal or higher contents in Examples (5) to (10) compared to Comparative Example (2).

[0077] 2-4. Protein content analysis Each microalgae culture cultured in the above 2-2-1 to 2-2-6 was collected, and the protein content was analyzed in the same manner as described in Examples 1-4 above.

[0078]

Table 4

[0079] As a result, as shown in Table 4, in the case of Examples (5) to (10), all showed a crude protein content of 58% or more. In the case of Comparative Example 2, considering that the crude fat content measured in 2-3 above was 41.91%, the normal carbohydrate content of the microalgae biomass was about 2-3%, and the ash content was about 8-10%, it can be estimated that the crude protein content is at most about 45.1%. Through this, it was confirmed that when culturing microalgae under the condition of continuously supplying a nitrogen source, protein can be produced at a high content of 58% or more.

[0080] 2-5. Amino acid content and composition analysis The microalgae culture of Example (10) cultured in 2-2-6 above was collected, and after obtaining dried cells in the same manner as described in 1-3 above, 0.5-1 g of the dried cells was acid-hydrolyzed. Then, liquid chromatography was performed on this to analyze the total amino acid content and each individual amino acid content. The concentration of each individual amino acid in the sample was standardized by the amount of dried cells used, and the content (%) of each individual amino acid relative to the dry cell weight was calculated. The contents of all detected amino acids were summed up to calculate the total amino acid content (%) relative to the dry cell weight.

[0081]

Table 5

[0082]

Table 6

[0083] As a result, as shown in Tables 5 and 6, in the case of Example (10) in which microalgae were cultured under the condition of continuously supplying a nitrogen source, the total amino acid content in the biomass was 43.09%, and the content of glutamate among the individual amino acids was the highest, and the content ratio of glutamate to arginine was 2.04.

[0084] From the above description, those having ordinary knowledge in the technical field to which the present application pertains should be able to understand that the present application can be implemented in other specific forms without changing its technical idea and essential features. In this regard, it must be understood that the above-described embodiments are exemplary in all aspects and not restrictive. The scope of the present application should be interpreted to include the meaning and scope of the following claims and all changes or modified forms derived from the equivalent concepts thereof, rather than the above detailed description. [Deposit Number]

[0085] Name of depository institution: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) Deposit Number: KCTC14345BP Date of deposit: October 26, 2020 TIFF0007717157000007.tif199141

[0086] Name of depository institution: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) Deposit Number: KCTC14346BP Date of deposit: October 26, 2020 TIFF0007717157000008.tif199141

Claims

1. Culturing a single strain of microalgae belonging to the genus Thraustochytriidae sp. or Schizochytrium sp. in a medium; and During the culturing step, continuously supplying ammonia gas or ammonium sulfate as an additional nitrogen source from immediately after inoculating the microalgae into the medium until the end of culturing so that the total nitrogen concentration in the culture solution becomes 300 ppm or more, wherein the carbon source in the medium is glucose, A method for producing biomass derived from microalgae of the genus Thraustochytriidae sp. or Schizochytrium sp.

2. The method for producing biomass according to Claim 1, wherein the additional nitrogen source is ammonia gas.

3. The method for producing biomass according to Claim 1, further comprising the step of recovering biomass from the strain, a culture of the strain, a dried product of the culture, or a crushed product of the dried product.

4. The method for producing biomass according to Claim 1, wherein the biomass contains 50% by weight or more of protein and 37% by weight or less of fat based on the total weight of the biomass.

5. The method for producing biomass according to Claim 1, wherein the biomass contains 50% by weight or more of protein and omega-3 fatty acids based on the total weight of the biomass.

6. The method for producing biomass according to Claim 5, wherein the omega-3 fatty acids contain any one or more of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

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