High-protein microalgal biomass with excellent pepsin digestibility, cultivation method and use thereof

Abstract

The present invention relates to a high-protein microalgal biomass with excellent pepsin digestibility, a cultivation method, and uses thereof. The composition presented in the present invention not only has a high protein content, but also contains omega-3 fatty acids, which are useful fatty acids like fish meal, and can directly replace fish meal when blending in feed.

Description

[Technical field]

[0001] Cross-reference to related application(s) This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0047721 dated April 18, 2022, and all contents disclosed in the documents of that Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a high-protein microalgal biomass having excellent pepsin digestibility, a culture method, and uses thereof. [Background technology]

[0003] Fishmeal is a protein source that is widely used not only in fish feed but also in feed for various livestock species. However, recent issues regarding fishing quota restrictions and environmental pollution such as heavy metals and microplastics have raised demand for the development of new protein sources that can replace fishmeal.

[0004] In addition, there is a recent trend to reduce the FIFO (Fish in / Fish out) ratio in the fish feed sector. At the same time, as fish catches vary depending on government policies and climate, there is an increasing demand for improved protein sources from a sustainability perspective.

[0005] To date, various new protein sources have been proposed as substitutes for fish meal, but they have the problem that they cannot replace the effective substances contained in fish meal, such as omega-3 fatty acids. In addition, the green algae-based whole cell type proteins proposed to date have a problem that the digestibility is significantly reduced, making it practically difficult to include them at a high ratio in feed. In this regard, Korean Patent No. 1564829 discloses a compound feed composition for fish farming containing microalgae by-products.

[0006] In order to solve these problems, the present inventors have conducted research and developed a new protein source containing omega-3 fatty acids, which are useful fatty acids produced through the fermentation of Thraustochytrid microalgae. Furthermore, they have found a composition with excellent pepsin digestibility, providing a new sustainable and environmentally friendly protein source. Summary of the Invention [Problem to be solved by the invention]

[0007] One example of the present application provides biomass derived from microalgae of the genus Schizochytrium, which contains 46% or more of total amino acids based on the dry weight of the biomass and has excellent pepsin digestibility.

[0008] Another embodiment of the present application provides a feed composition comprising the biomass derived from the microalgae of the genus Schizochytrium.

[0009] Another embodiment of the present application provides a food composition comprising the biomass derived from the microalgae of the genus Schizochytrium.

[0010] Another example of the present application provides a method for culturing microalgae of the genus Schizochytrium, comprising: culturing the microalgae under conditions in which a residual carbon source concentration in the culture solution is maintained at 6% or less relative to the entire culture solution. [Means for solving the problem]

[0011] Each description and embodiment disclosed in this application may be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application belong to the scope of this application. In addition, the specific descriptions described below should not be considered as limiting the scope of this application. In addition, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific aspects of this application described in this application. In addition, such equivalents are intended to be included in this application.

[0012] One example of the present application provides biomass derived from microalgae of the genus Schizochytrium, which contains 46% by weight or more of total amino acids based on the dry weight of the biomass and has excellent pepsin digestibility.

[0013] The term "Thraustochytrid" as used herein means microalgae of the order Thraustochytriales. The term "Schizochytrium sp." as used herein means one of the genus names of the Thraustochytriaceae family of the order Thraustochytriales, and may be used interchangeably with the term "genus Schizochytrium." The term "microalgae" means a microscopic organism that is not visible to the naked eye but can be seen only through a microscope and lives floating freely in water. There are various types of microalgae, including strains that cannot photosynthesize and grow only heterotrophically.

[0014] The microalgae of the genus Schizochytrium may be, but is not limited to, the Schizochytrium strain CD01-5004 deposited under the accession number KCTC14345BP.

[0015] The term "biomass" as used herein means an organism such as a plant, animal, or microorganism that can be used for chemical energy, i.e., an energy source of bioenergy, and may also mean the weight or amount of energy of a specific organism present in a unit time and space ecologically. The biomass may include, but is not limited to, compounds secreted by cells, and may contain not only extracellular substances but also cells and / or intracellular contents. In the present application, the biomass may be, but is not limited to, Schizochytrium microalgae itself, a culture thereof, a dried product thereof, a crushed product thereof, or a product produced by culturing or fermenting the microalgae, or a concentrate or dried product of the biomass.

[0016] The "culture" of the Schizochytrium microalgae refers to a product produced by culturing the microalgae, and may be specifically a culture solution containing the microalgae or a culture filtrate obtained by removing the microalgae from the culture solution, but is not limited thereto. The "dried product" of the Schizochytrium microalgae culture is a product obtained by removing water from the microalgae culture, and may be, for example, in the form of dried cells of the microalgae, but is not limited thereto. In addition, the "crushed product" of the dried product is a general term for a product obtained by crushing a dried product obtained by removing water from the microalgae culture, and may be, for example, a dried cell powder, but is not limited thereto. The Schizochytrium microalgae culture may be produced by inoculating the microalgae into a microalgae culture medium and culturing the microalgae according to a culturing method known in the art, and the dried product of the culture and the crushed product thereof may be produced by a processing or drying method of the microalgae or culture solution known in the art.

[0017] The biomass derived from a microalga of the genus Schizochytrium may contain 46% by weight or more, 48% by weight or more, 49% by weight or more, 50% by weight or more, 52% by weight or more, 46 to 60% by weight, 46 to 55% by weight, 46 to 53% by weight, 48 to 53% by weight, 50 to 55% by weight, 52 to 55% by weight, 52 to 53% by weight, 52.5 to 55% by weight, or 52.5 to 53% by weight of total amino acids based on the dry weight of the biomass, and the upper limit may be, but is not limited to, 80% by weight or less, 75% by weight or less, 70% by weight or less, or 65% by weight or less.

[0018] The biomass derived from a microalga of the genus Schizochytrium may contain crude protein in an amount of 60% by weight or more, 64% by weight or more, 67% by weight or more, or 70% by weight or more, 60 to 85% by weight, 60 to 80% by weight, 64 to 80% by weight, 64 to 78% by weight, 67 to 80% by weight, 67 to 78% by weight, 67 to 75% by weight, 67 to 70% by weight, 69 to 80% by weight, 69 to 78% by weight, 65 to 70% by weight, or 69 to 70% by weight, based on the dry weight of the biomass, and the upper limit may be 85% by weight or less, 80% by weight or less, or 78% by weight or less, but is not limited thereto.

[0019] The biomass derived from microalgae of the genus Schizochytrium may contain crude fat in an amount of 5% by weight or more, 7% by weight or more, 9% by weight or more, 10% by weight or more, 14% by weight or more, 20% by weight or more, 1 to 20% by weight, 4 to 23% by weight, 9 to 25% by weight, 9 to 23% by weight, 5 to 18% by weight, 10 to 18% by weight, 10 to 15% by weight, 12 to 18% by weight, 12 to 15% by weight, or 13 to 15% by weight, based on the dry weight of the biomass.

[0020] As used herein, the term "crude protein" may be used interchangeably with "crude protein" and refers to the protein determined by measuring total nitrogen in the general analysis of food and feed and multiplying this by 6.25 (the reciprocal of the average nitrogen content of protein, 1 / 0.16).

[0021] As used herein, the term "crude fat" refers to the component extracted with ether using a Soxhlet extractor in the general analysis of food and feed, and "crude fat content" may be interchangeably used with "crude fat amount" or "total lipids" or "total fatty acids."

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

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

[0024] The biomass derived from Schizochytrium microalgae may contain 39 wt% or more, 40 wt% or more, 41 wt% or more, 45 wt% or more, 49 wt% or more, 50 wt% or more, 39 to 60 wt%, 39 to 55 wt%, 39 to 50 wt%, 42 to 55 wt%, 42 to 50 wt%, 43 to 55 wt%, 43 to 50 wt%, 45 to 50 wt%, 47 to 50 wt%, or 49 to 50 wt% docosahexaenoic acid (DHA) based on the total crude fat content.

[0025] The biomass derived from a microalga of the genus Schizochytrium may contain, based on the total crude fat content, 0.5 wt% or more, 1.0 wt% or more, 1.5 wt% or more, 2.0 wt% or more, 2.1 wt% or more, 3.5 wt% or more, 0.5 wt% to 15.0 wt%, 0.5 wt% to 10.0 wt%, 0.5 wt% to 5.0 wt%, 0.5 wt% to 3.0 wt%, 1.0 wt% to 3.0 wt%, 1.9 wt% to 3.0 wt%, 1.9 wt% to 2.5 wt%, or 1.9 wt% to 2.2 wt% of eicosapentaenoic acid (EPA).

[0026] The biomass derived from Schizochytrium microalgae may contain omega-3 fatty acids in an amount of 41% by weight or more, 42% by weight or more, 50% by weight or more, 42 to 55% by weight, 42 to 52% by weight, 43 to 55% by weight, 43 to 52% by weight, 49 to 55% by weight, or 49 to 52% by weight, based on the total crude fat content.

[0027] The biomass derived from microalgae of the genus Schizochytrium may have a pepsin digestibility of 68.7% or more, 69% or more, 70% or more, 73% or more, 75% or more, 77% or more, 80% or more, 83% or more, 89% or more, 69% to 95%, 69% to 90%, 70% to 95%, 70% to 90%, 73% to 95%, or 73% to 90%, 75% to 95%, 75% to 90%, 85% to 95%, 85% to 90%, 87% to 95%, 87% to 90%, 89% to 95%, or 89% to 90%.

[0028] The term "pepsin digestibility" as used herein is used in the quality analysis of animal protein raw materials and refers to an index that allows comparison of the extent to which digestion is carried out in a certain environment using pepsin extracted from an animal's stomach.

[0029] The biomass may be cultured by a method for culturing microalgae of the genus Schizochytrium according to one embodiment.

[0030] Another aspect of the present application provides a composition comprising biomass derived from microalgae of the genus Schizochytrium, which contains 46% or more of total amino acids by weight based on the dry weight of the biomass and has excellent pepsin digestibility.

[0031] The composition may be characterized by good pepsin digestibility and may be used in food and / or feed production.

[0032] Another aspect of the present application provides a feed composition comprising biomass derived from microalgae of the genus Schizochytrium, which contains 46% by weight or more of total amino acids based on the dry weight of the biomass and has excellent pepsin digestibility.

[0033] The biomass derived from microalgae of the genus Schizochytrium is as described above.

[0034] The term "feed composition" as used herein refers to a feed given to an animal. The feed composition refers to a substance that provides organic or inorganic nutrients necessary for the survival of an animal or the production of meat, milk, etc. The feed composition may further include nutritional components necessary for the survival of an animal or the production of meat, milk, etc. The feed composition may be prepared in various forms of feed known in the art, and may specifically include concentrated feed, roughage, and / or special feed.

[0035] The term "feed additive" as used herein includes substances added to feed for various purposes such as supplementing nutrients and preventing weight loss, increasing digestibility of fiber in feed, improving milk quality, preventing reproductive disorders and improving conception rates, preventing high temperature stress in summer, etc. The feed additive of the present application corresponds to a supplementary feed under the Feed Management Act, and may additionally include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, complex minerals, etc., mineral preparations which are trace minerals such as zinc, copper, cobalt, selenium, etc., vitamin preparations such as carotene, vitamin E, vitamin A, D, E, nicotinic acid, vitamin B complex, etc., protected amino acid preparations such as methionine, lysine, etc., protected fatty acid preparations such as fatty acid calcium salts, live bacteria preparations (lactic acid bacteria preparations), live bacteria such as yeast cultures, mold fermentation products, yeast preparations, etc.

[0036] Another aspect of the present application provides a food composition comprising biomass derived from microalgae of the genus Schizochytrium, which contains 46% by weight or more of total amino acids based on the dry weight of the biomass and has excellent pepsin digestibility.

[0037] The biomass derived from microalgae of the genus Schizochytrium is as described above.

[0038] The term "food composition" as used herein includes all forms of functional foods, nutritional supplements, health foods, and food additives, and food compositions of the above types can be prepared in various forms by conventional methods known in the art.

[0039] The compositions of the present application may further comprise grains, such as crushed or crushed wheat, oats, barley, corn and rice; vegetable protein feeds, such as feeds based on beans and sunflower; animal protein feeds, such as blood meal, meat meal, bone meal and fish meal; dry ingredients consisting of sugars and dairy products, such as various milk powders and whey powders, and may further comprise nutritional supplements, digestion and absorption enhancers, growth promoters, etc.

[0040] The compositions of the present application may be administered to animals alone or in combination with other feed additives in an edible carrier. The compositions may also be readily administered to animals in oral dosage forms, either as a top dressing or by mixing them directly into the feed or separately from the feed. When the compositions are administered separately from the feed, they may be combined with a pharma- ceutically acceptable edible carrier to produce immediate or sustained release dosage forms, as is well known in the art. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, pea flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the composition may be a tablet, capsule, powder, lozenge, or lozenge or a top dressing in a slightly acidic form. When a liquid carrier is used, the composition may be in the form of a soft gelatin capsule, or a syrup, suspension, emulsion, or solution.

[0041] The composition of the present application may contain, for example, a preservative, a stabilizer, a wetting or emulsifying agent, a cryoprotectant, or an excipient, etc. The cryoprotectant may be one or more selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder, and starch.

[0042] The preservative, stabilizer, or excipient may be contained in the composition in an effective amount sufficient to reduce deterioration of the Schizochytrium microalgae contained in the composition, and the cryoprotectant may be contained in the composition in an effective amount sufficient to reduce deterioration of the Schizochytrium microalgae contained in the composition when the composition is in a dried state.

[0043] The composition can be added to animal feed by immersion, spraying or mixing.

[0044] The composition of the present application can be applied to many animal diets, including, but not limited to, mammals, birds, fish, crustaceans, cephalopods, reptiles, and amphibians. For example, the mammals can include pigs, cows, sheep, goats, laboratory rodents, or pets, and the birds can include poultry, which can include, but are not limited to, chickens, turkeys, ducks, geese, pheasants, or quails. In addition, the fish can include commercially farmed fish and their juveniles, ornamental fish, and the crustaceans can include, but are not limited to, shrimp, barnacles, and the like. In addition, the composition can be applied to the diet of rotifers, which are zooplankton.

[0045] Another aspect of the present application provides a method for culturing microalgae of the genus Schizochytrium, comprising: culturing the microalgae under conditions in which a residual carbon source concentration in the culture solution is maintained at 6% or less relative to the entire culture solution.

[0046] The microalgae of the genus Schizochytrium may be, but is not limited to, the Schizochytrium strain CD01-5004 deposited under the accession number KCTC14345BP.

[0047] The biomass derived from microalgae of the genus Schizochytrium is as described above.

[0048] The term "cultivation" as used herein means growing the microalgae under appropriately controlled environmental conditions. The culturing process of the present application may be carried out using appropriate media and culture conditions known in the art. Such a culturing process may be easily adjusted and used by those skilled in the art depending on the selected microalgae.

[0049] The term "culture medium" as used herein may refer to a medium for culturing microalgae, or may refer to a medium for culturing microalgae that further contains microalgae and / or products produced by the microalgae.

[0050] In the step of culturing the Schizochytrium microalgae, the culture conditions may be specifically such that the residual carbon source concentration in the culture medium is maintained at 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less relative to the total culture medium.

[0051] Specifically, the cultivation of the Schizochytrium microalgae of the present application may be carried out under heterotrophic conditions, but is not limited thereto.

[0052] The term "heterotrophy" as used herein refers to a nutritional method that depends on organic matter obtained from outside the body for energy or nutrients, and is a term corresponding to autotrophy, and may be used interchangeably with the term "dark culture."

[0053] The step of culturing the Schizochytrium microalgae is not particularly limited, and may be performed by known batch culture methods, continuous culture methods, fed-batch culture methods, etc. The medium and other culture conditions used for culturing the microalgae of the present application may be any medium used for culturing conventional microalgae without any particular limitations. Specifically, the microalgae of the present application may be cultured under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, while controlling the temperature, pH, etc.

[0054] Specifically, the appropriate pH (e.g., pH 5 to 9, specifically pH 5 to 8) can be adjusted 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.

[0055] The medium used in the step of culturing the Schizochytrium microalgae of the present application may be, but is not limited to, MJW01 medium.

[0056] The carbon source contained in the medium used in the step of culturing the Schizochytrium microalgae may be any one or more selected from the group consisting of glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose, and glycerol, and specifically may be one or more selected from the group consisting of glucose, fructose, and sucrose, but is not limited thereto.

[0057] The nitrogen source contained in the medium used in the step of culturing the Schizochytrium microalgae may include at least one selected from the group consisting of urea, ammonia water, ammonium sulfate ((NH4)2SO4), yeast extract, peptone, and sodium nitrate (NaNO3), but is not limited thereto as long as it is a nitrogen source used in culturing microalgae.

[0058] The medium used in the step of culturing the Schizochytrium microalgae may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, etc., individually or in combination as a phosphorus source, but is not limited thereto.

[0059] The medium used in the step of culturing the Schizochytrium microalgae may contain, but is not limited to, 0.01 to 5 g / L of yeast extract, 1.0 to 3.0 g / L of KH2PO4, and 5 to 20 g / L of K2HPO4.

[0060] The culture temperature can be maintained at 20 to 45° C. or 25 to 40° C., and the culture can be performed for about 10 to 70 hours, but is not limited thereto.

[0061] The method for culturing Schizochytrium microalgae can increase the pepsin digestibility by supplying a residual carbon source in the culture solution so as to maintain the concentration of the residual carbon source in the culture solution at a maximum of 6% or less relative to the total culture solution, thereby increasing the total amino acid content relative to the dry weight of the microalgae biomass. Effect of the Invention

[0062] The present invention has developed an alternative protein source that can replace fish meal through a microalgae-based fermentation process. The composition proposed in the present invention not only has a high protein content, but also contains omega-3 fatty acids, which are useful fatty acids like fish meal, and can directly replace fish meal when mixed into feed. Furthermore, the high-protein microalgae biomass proposed in the present invention has a high pepsin digestibility of 70% or more as analyzed by a pepsin digestibility analysis method, so that even if the mixing ratio in the feed is high, it does not have a negative effect on the overall digestibility, and since it is a protein source produced based on microalgae, it has the advantage of being free from environmental pollution problems and being able to be produced stably while minimizing the influence of the external environment. [Brief description of the drawings]

[0063] [Figure 1] 1 is a graph showing the correlation coefficient between the pepsin digestibility of microalgae dried biomass under different culture conditions and the crude fat content in the biomass (average ± standard deviation, n=4). [Diagram 2] 1 is a graph showing the correlation coefficient between the pepsin digestibility of microalgae dried biomass under different culture conditions and the crude protein content in the biomass (average ± standard deviation, n=4). [Diagram 3] 1 is a graph showing the correlation coefficient between the pepsin digestibility of microalgae dried biomass under different culture conditions and the omega-3 fatty acid content relative to the crude fat weight in the biomass (average ± standard deviation, n=4). [Figure 4] 1 is a graph showing the correlation coefficient between the pepsin digestibility of microalgae dried biomass under different culture conditions and the total amino acid content in the biomass (average ± standard deviation, n=4). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] The present invention will be described in more detail below with reference to examples. These examples are provided to more specifically explain the present invention, and it will be obvious to those skilled in the art to which the present invention pertains that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

[0065] <Seed culture of microalgae strains> Schizochytrium sp. CD01-5004 strain, which belongs to Thraustochytrid microalgae and was deposited under the accession number KCTC14345BP, was used. The strain was cultured in a 250 mL flask containing 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, vitamin mixed solution 10 ml / L) at 26°C and 200 rpm for 30 hours.

[0066] Comparative Example 1. Production of biomass by culturing a strain at a carbon source concentration of 7-9% (w / v ratio) in the culture medium The seed cultured strain was dispensed into a 5L fermenter containing sterilized MJW01 medium (glucose 30g / L, MgSO4·7H2O 3.0g / L, Na2SO4 10g / L, NaCl 1.0g / L, yeast extract 0.5g / L, MSG·1H2O 1.0g / L, NaNO3 5.0g / L, KH2PO4 1.5g / L, K2HPO4 10g / L, CaCl2 0.5g / L, vitamin mixture solution 10ml / L) and cultured at 28℃, 300rpm, 0.6vvm, pH 6.8. Carbon sources were supplied so that the residual carbon source concentration in the culture solution was maintained within the range of 7-9% (w / v ratio) of the entire culture solution during the culture (45 hours). The culture was continued while supplying nitrogen source from immediately after the start of the culture to the end of the culture. In order to maintain the appropriate nitrogen source concentration, the nitrogen source was repeatedly supplied to the culture medium. Glucose was used as the carbon source, and ammonium sulfate was used as the nitrogen source. After the culture was completed, the bacterial culture medium was freeze-dried or drum-dried to obtain the dry biomass of Comparative Example 1, which is the control group.

[0067] Example 1. Production of biomass by culturing a strain at a carbon source concentration of 0 to 2% (w / v ratio) in the culture medium In the culture conditions (control conditions, control group) of Comparative Example 1, the composition of MJW01 medium was maintained, and the residual carbon source concentration in the culture solution was maintained within the range of 0 to 2% (w / v ratio) relative to the entire culture solution during the culture (45 hours). The remaining culture conditions were the same as in Comparative Example 1, and the dry biomass of Example 1 was obtained.

[0068] Example 2. Production of biomass by culturing a strain at a carbon source concentration of up to 2-5% (w / v ratio) in the culture medium Under the culture conditions of Comparative Example 1 (control conditions, control group), the composition of MJW01 medium was maintained, and the residual carbon source was supplied so that the concentration in the culture medium was maintained within the range of 2 to 5% (w / v ratio) relative to the total culture medium during the culture (45 hours). The remaining culture conditions were the same as in Comparative Example 1, and dry biomass of Example 3 was obtained.

[0069] Example 3. Production of biomass by culturing a strain at a carbon source concentration of up to 4-7% (w / v ratio) in the culture medium Under the culture conditions of Comparative Example 1 (control conditions, control group), the composition of MJW01 medium was maintained, and the residual carbon source was supplied so that the concentration of the residual carbon source in the culture medium was maintained in the range of 4 to 7% (w / v ratio) relative to the total culture medium during the culture period (45 hours).

[0070] Experimental Example 1. Component analysis of dried microalgae biomass under different culture conditions

[0071] 1-1. Analysis of crude fat and fatty acid contents of dried microalgae biomass In order to analyze the crude fat content and fatty acid content of each of the dried biomasses obtained in Comparative Example 1 and Examples 1 to 3, the following experiment was carried out.

[0072] Specifically, 2 g of dried microalgae cells of Comparative Example 1 and Examples 1 to 3 were added with 8.3 M hydrochloric acid solution and hydrolysis reaction was carried out at 80°C. Then, 30 mL of ethyl ether and 20 mL of petroleum ether were added, and the process of mixing for 30 seconds and centrifuging was repeated three times or more. The separated solvent layer was transferred to a round flask whose weight was measured in advance, and then placed in a container in which the solvent and residual moisture were removed through nitrogen purging and dried. The solvent was dried, and the weight of the remaining oil was measured to calculate the total oil content, and the omega-3 (DHA and EPA) content in the oil was measured by gas chromatography after pretreatment with methanolic 0.5N NaOH and 14% trifluoroborane methanol (BF3).

[0073] 1-2. Analysis of crude protein content of dried microalgae biomass In order to analyze the crude protein content of each of the dried biomasses obtained in Comparative Example 1 and Examples 1 to 3, the following experiment was carried out using an automatic Kjeldahl analyzer.

[0074] Specifically, each microalgae dry biomass sample obtained in Comparative Example 1 and Examples 1 to 3 was placed in a Kjeldahl system (Kjeltec2100) analysis tube, and sulfuric acid and a catalyst were added to decompose the sample in a decomposer, after which the decomposition tube was attached to an autosampler. The gaseous ammonia generated by heating and distilling with caustic soda and steam in the Kjeldahl system was cooled and collected, and then titrated with hydrochloric acid to automatically calculate the nitrogen content, thereby measuring the crude protein content of the microalgae dry biomass.

[0075] 1-3. Analysis of amino acid content in dried microalgae biomass In order to analyze the amino acid content of each of the dried biomasses obtained in Comparative Example 1 and Examples 1 to 3, the following experiment was carried out.

[0076] Specifically, 0.5 to 1 g of each of the dried microalgae cells of Comparative Example 1 and Examples 1 to 3 was subjected to acid hydrolysis, and then total amino acid analysis was performed using liquid chromatography. The analysis results of individual amino acids were standardized by the amount of dried microalgae cells used to calculate the content ratio of each individual amino acid in the dried cells, and the content ratios of all amino acids detected in the analysis results were summed to calculate the total amino acid content ratio in the cells.

[0077] 1-4. Component analysis results of microalgae dried biomass under different culture conditions The components of the dried biomass obtained in Comparative Example 1 and Examples 1 to 3 were analyzed using the methods of Experimental Examples 1-1 to 1-3. The results are shown in Table 1 below.

[0078] [Table 1]

[0079] As a result, as shown in Table 1, when comparing Comparative Example 1 and Example 1, it was confirmed that in Example 1, the total amino acid content in the microalgae biomass was increased by maintaining the residual carbon source concentration in the culture solution in a low range of 0 to 2% during the culture process, but the crude fat content increased.

[0080] In Example 2, the range of carbon source concentration remaining in the culture medium during cultivation was adjusted to 2-5%. Comparing the component contents of Comparative Example 1 and Example 2, it was confirmed that when the carbon source concentration was maintained in the range of 2-5% in Example 2, the crude protein content was maintained at a level of 70%, and the crude fat content was in the intermediate range between Comparative Example 1 and Example 1. In particular, it was shown that the total amino acid content in the biomass was 52.78%, which was the highest value among the cultivation conditions.

[0081] In Example 3, the carbon source concentration remaining in the culture medium during the culture was maintained in the range of 4 to 7%. When the carbon source concentration was maintained in the range of 4 to 7% in Example 3, it was confirmed that the crude protein content was 77.20%, the total amino acid content was 52.18%, and the crude fat content was 12.20%, which was the highest crude protein content among the culture conditions.

[0082] Experimental Example 2. Analysis of pepsin digestibility of microalgae dried biomass under different culture conditions Since protein digestibility is an important index for using microalgal biomass as feed and food, analysis was performed using a pepsin digestibility analysis method. In order to analyze the pepsin digestibility of the microalgal dried biomass under different culture conditions, the pepsin digestibility of each dried biomass obtained in Comparative Example 1 and Examples 1 to 3 was measured according to the method described in the Association of Official Analytical Chemists, 1990, Official Method of Analysis 971.09, and the pepsin digestibility was calculated using the following calculation formula 1.

[0083] Specifically, 150 mL of 0.2% pepsin hydrochloric acid solution preheated to 42-45°C was added to 1 g of each dried biomass sample, and the mixture was digested for 16 hours while shaking in a 45°C hot water bath, and then filtered through No. 2A filter paper. After washing the fluorinated product with hot water, the crude protein content of the fluorinated product and the filter paper was determined by Kjeldahl analysis, and the results are shown in Table 2 below.

[0084] The Kjeldahl nitrogen content in formula 1 was determined by the method described in the crude protein analysis using an automatic Kjeldahl analyzer in Experimental Example 1-3.

[0085] [Formula 1] Pepsin digestibility (%) = 100*(AB) / A *A:% Kjeldahl nitrogen content of the original sample *B: % content of Kjeldahl nitrogen insoluble in acid pepsin solution

[0086] [Table 2]

[0087] As a result, as shown in Table 2, the pepsin digestibility of the microalgae dried biomass of Comparative Example 1 was 68.6%, which was relatively low. However, by changing the culture conditions to alter the component ratio of the biomass, it was confirmed that the pepsin digestibility of the microalgae dried biomass of Example 1 was 73.40%, the pepsin digestibility of the microalgae dried biomass of Example 2 was 89.62%, and the pepsin digestibility of the microalgae dried biomass of Example 3 was 83.20%, which were all high.

[0088] Experimental Example 3: Confirmation of the correlation between the pepsin digestibility and total amino acid content of microalgae dried biomass under different culture conditions In the above Experimental Example 2, it was confirmed that the pepsin digestibility increased as a result of changing the ratio of components of the biomass by changing the culture conditions. Therefore, it was confirmed which indicator of the components of the biomass has a correlation with the pepsin digestibility.

[0089] Specifically, the correlation coefficients between the crude fat content, crude protein content, omega-3 fatty acid / crude fat content, and total amino acid content of each of the dried biomasses obtained in Comparative Example 1 and Examples 1 to 3 and the pepsin digestibility were calculated, and the results are shown in Figures 1 to 4.

[0090] As a result, as shown in Figures 1 to 3, it was shown that among the components of biomass, the crude fat content, crude protein, and omega-3 fatty acid content relative to the weight of crude fat did not have a significant correlation with pepsin digestibility. On the other hand, as shown in Figure 4, it was revealed that there was a significant correlation between the total amino acid content in the biomass and the pepsin digestibility (R 2 =0.9527). Generally, the total amino acids are included in the crude protein content, and it is known that the crude protein content and the total amino acids are values ​​that vary proportionally. However, as described in the present invention, even if the crude protein content is similar, the total amino acid content can be changed depending on the fermentation conditions, and it can be confirmed that the pepsin digestibility increases as the total amino acid content in the biomass increases.

[0091] [Accession number] Name of depository institution: Korea Comprehensive Biological Resource Center (KCTC) Accession number: KCTC14345BP Date of acceptance: 20201026 TIFF2025505439000004.tif236169

Claims

1. A method for producing biomass derived from microalgae of the genus Schizochytrium, comprising: culturing microalgae of the genus Schizochytrium under conditions in which a residual carbon source concentration in the culture solution is maintained at 7% or less relative to the entire culture solution.

2. The method for producing biomass derived from microalgae of the genus Schizochytrium according to claim 1, wherein the microalgae of the genus Schizochytrium are Schizochytrium strain CD01-5004 deposited under the accession number KCTC14345BP.

3. The method for producing biomass derived from Schizochytrium microalgae according to claim 1 , wherein the culturing step is performed in MJW01 medium.

4. 2. The method for producing biomass derived from Schizochytrium microalgae according to claim 1, wherein the carbon source of the medium is one or more selected from the group consisting of glucose, fructose and sucrose.

5. The method for producing biomass derived from Schizochytrium microalgae according to claim 1, wherein the medium contains one or more nitrogen sources selected from the group consisting of urea, aqueous ammonia, ammonium sulfate, yeast extract, peptone, and sodium nitrate.

6. The medium contains yeast extract 0.01-5 g / L, KH ２ P.O. ４ 1.0-3.0g / L, K ２ H.P.O. ４ The method for producing biomass derived from Schizochytrium microalgae according to claim 1, wherein the biomass contains 5 to 20 g / L.

7. The method for producing biomass derived from Schizochytrium microalgae according to claim 1, wherein the culture is carried out for 10 to 70 hours.

8. The method for producing biomass derived from Schizochytrium microalgae according to claim 1 , wherein the method for culturing microalgae increases a total amino acid content relative to the dry weight of the microalgae to increase a pepsin digestibility.

9. The method for producing biomass derived from Schizochytrium microalgae according to claim 1, wherein the pepsin digestibility is 70% or more.

10. 2. A biomass derived from microalgae belonging to the genus Schizochytrium, which has excellent pepsin digestibility and is produced by the production method according to claim 1.

11. The biomass according to claim 10, wherein the Schizochytrium microalgae is Schizochytrium sp. strain CD01-5004 deposited under deposit number KCTC14345BP.

12. 11. The biomass of claim 10, wherein the biomass contains 60% or more by weight crude protein based on the dry weight of the biomass.

13. 11. The biomass of claim 10, wherein the biomass contains 4% to 23% crude fat by weight based on the dry weight of the biomass.

14. 11. The biomass of claim 10, wherein the biomass contains 40% or more by weight of docosahexaenoic acid based on the total crude fat weight.

15. 11. The biomass of claim 10, wherein the biomass contains 0.5% or more by weight of eicosapentaenoic acid based on the total crude fat weight.

16. The biomass of claim 10, wherein the biomass contains 41% or more by weight of omega-3 fatty acids based on the total crude fat weight.

17. The biomass of claim 10, wherein the biomass has a pepsin digestibility of 70% or more.

18. A feed composition comprising the biomass derived from the microalgae of the genus Schizochytrium according to claim 10.

19. A food composition comprising the biomass derived from the microalgae of the genus Schizochytrium according to claim 10.

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