High-protein microalgae 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, filed April 18, 2022, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

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

[0003] Fishmeal is a protein source widely used in feed for a variety of livestock, including fish farming. However, recent concerns about catch limits 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 in the fish feed sector to reduce the FIFO (fish in / fish out) ratio, and as catches fluctuate due to government policies and climate, there is an increasing demand for improved protein sources from a sustainability perspective.

[0005] To date, various novel protein sources have been proposed as substitutes for fish meal, but they have had the problem of being unable to replace the beneficial substances contained in fish meal, such as omega-3 fatty acids. Furthermore, the green algae-based whole cell proteins proposed to date have a significantly low digestibility, making it practically difficult to include them at high ratios in feed. In relation to this, Korean Patent No. 1564829 discloses a compound feed composition for fish farming containing microalgae by-products.

[0006] 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 discovered a composition with excellent pepsin digestibility, providing a sustainable and environmentally friendly new 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 total amino acids based on the dry weight of the biomass and has excellent pepsin digestibility.

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

[0009] Another example 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, including culturing the microalgae under conditions in which the concentration of a residual carbon source in the culture solution is maintained at 6% or less relative to the total culture solution. [Means for solving the problem]

[0011] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application belong to the scope of this application. Furthermore, the specific descriptions set forth below should not be construed as limiting the scope of this application. Furthermore, 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 herein. Furthermore, such equivalents are intended to be encompassed by 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] As used herein, the term "Thraustochytrid" refers to microalgae of the order Thraustochytriales. The term "Schizochytrium sp." as used herein refers to a genus belonging to the family Thraustochytriaceae of the order Thraustochytriales, and may be used interchangeably with the term "Schizochytrium." The term "microalgae" refers to microscopic organisms that are not visible to the naked eye but only through a microscope, and that live freely floating in water. There are many different types of microalgae, including strains that are unable to photosynthesize and grow solely through heterotrophy.

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

[0015] As used herein, the term "biomass" refers to organisms, such as plants, animals, and microorganisms, that can be used for chemical energy, i.e., a source of bioenergy. Ecologically, it also refers to the weight or energy amount of a specific organism present within a unit of time and space. Furthermore, biomass includes, but is not limited to, compounds secreted by cells, and may contain not only extracellular substances but also cells and / or intracellular contents. In the present application, the biomass may be Schizochytrium microalgae itself, a culture thereof, a dried product thereof, or a crushed product thereof, or a product produced by culturing or fermenting the microalgae, or a concentrate or dried product of the biomass, but is not limited thereto.

[0016] The "culture" of the Schizochytrium microalgae refers to a product produced by culturing the microalgae, and may specifically be, but is not limited to, a culture solution containing the microalgae or a culture filtrate obtained by removing the microalgae from the culture solution. The "dried product" of the Schizochytrium microalgae culture refers to the microalgae culture from which water has been removed, and may be, for example, in the form of dried cells of the microalgae, but is not limited to this. Furthermore, the "crushed product" of the dried product collectively refers to the product obtained by crushing the microalgae culture from which water has been removed, and may be, for example, a dried cell powder, but is not limited to this. The Schizochytrium microalgae culture may be produced by inoculating the microalgae into a microalgae culture medium and culturing it using a method known in the art. The dried product of the culture and its crushed product may also be produced by treating or drying the microalgae or culture solution using a method known in the art.

[0017] The biomass derived from microalgae 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 microalgae 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, 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, but is not limited to, 85% by weight or less, 80% by weight or less, or 78% by weight or less.

[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] The term "crude fat" as used herein 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 used interchangeably with "crude fat amount," "total lipids," or "total fatty acids."

[0022] As used herein, the term "docosahexaenoic acid (DHA)" refers to C 22 H 32 It is a polyunsaturated fatty acid with the chemical formula O2, and is classified as an omega-3 fatty acid along with α-linolenic acid (ALA) and eicosapentaenoic acid (EPA). 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 a polyunsaturated fatty acid with the chemical formula O2, and along with ALA and DHA, it is classified as an omega-3 fatty acid, and is also abbreviated as 20:5n-3.

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

[0025] The biomass derived from microalgae 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 % eicosapentaenoic acid (EPA).

[0026] The biomass derived from microalgae of the genus Schizochytrium 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 can be used to compare the degree of digestion that occurs in a certain environment using pepsin extracted from the stomach of an animal.

[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% by weight or more of total amino acids 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 fed to an animal. The feed composition refers to a substance that provides organic or inorganic nutrients necessary for sustaining the life of an animal or for producing meat, milk, etc. The feed composition may additionally contain nutritional components necessary for sustaining the life of an animal or for producing meat, milk, etc. The feed composition may be prepared in various forms known in the art, and may specifically include concentrated feed, roughage, and / or special feed.

[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 the digestibility of fiber in feed, improving milk quality, preventing reproductive disorders and improving conception rates, and preventing heat stress in summer. The feed additives of the present application fall under the category of supplementary feed under the Feed Management Act, and may additionally include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals; mineral preparations such as trace minerals like zinc, copper, cobalt, and selenium; vitamin preparations such as carotene, vitamin E, vitamins A, D, and E, nicotinic acid, and vitamin B complex; protected amino acids such as methionine and lysine; protected fatty acid preparations such as fatty acid calcium salts; probiotics (lactic acid bacteria preparations), probiotics such as yeast cultures and mold fermentation products; and yeast preparations.

[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 composition 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 such as 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 can be administered to animals alone or in combination with other feed additives in an edible carrier. Alternatively, the compositions can be easily administered to animals as a top dressing, directly mixed with feed, or in an oral dosage form separate from feed. When administered separately from feed, the compositions can be combined with a pharmaceutically acceptable edible carrier to produce immediate-release or sustained-release formulations, as is well known in the art. Such edible carriers can 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 can be in the form of a tablet, capsule, powder, lozenge, or lozenge, or a slightly acidic top dressing. When a liquid carrier is used, the composition can 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 agent or emulsifier, a cryoprotectant, or an excipient, etc. The cryoprotectant may be one or more selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder, and starch.

[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 used by dipping, spraying or mixing it into animal feed.

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

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

[0046] The microalgae of the genus Schizochytrium may be, but is not limited to, the Schizochytrium strain CD01-5004 deposited under 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 can 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 of the total culture medium.

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

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

[0053] The step of culturing the microalgae of the genus Schizochytrium may be carried out by known methods such as batch culture, continuous culture, and fed-batch culture, without being particularly limited thereto. The medium and other culture conditions used to culture the microalgae of the present application may be any medium commonly used for culturing microalgae, without any particular limitations. Specifically, the microalgae of the present application may be cultured under aerobic conditions in a conventional medium containing an appropriate 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 microalgae of the genus Schizochytrium 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 at least one selected from the group consisting of glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose, and glycerol, and specifically may be at least one 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 microalgae of the genus Schizochytrium may include at least one selected from the group consisting of urea, aqueous ammonia, 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, as a phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, etc., individually or in combination, but is not limited thereto.

[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 carried out for about 10 to 70 hours, but is not limited thereto.

[0061] In the method for culturing microalgae of the genus Schizochytrium, the residual carbon source concentration in the culture solution is maintained within a range of up to 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 and increasing the pepsin digestibility. [Effects 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, a useful fatty acid similar to fish meal, making it a direct substitute for fish meal when formulated into feed. Furthermore, the high-protein microalgae biomass proposed in the present invention exhibits a high pepsin digestibility of over 70% when analyzed using a pepsin digestibility analysis method. Therefore, even when the biomass is incorporated into feed at a high ratio, it does not negatively affect overall digestibility. Furthermore, because it is a protein source produced based on microalgae, it has the advantage of being free from environmental pollution issues and minimizing the impact of the external environment, allowing for stable production. [Brief explanation of the drawings]

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

[0064] The present invention will be described in more detail below with reference to examples. These examples are intended to more specifically explain the present invention, and it will be obvious to those skilled in the art 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> The Schizochytrium sp. CD01-5004 strain, a Thraustochytrid microalgae species deposited under 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, and vitamin mixture 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 aliquoted 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°C, 300 rpm, 0.6 vvm, and pH 6.8. The residual carbon source was supplied to maintain a concentration of 7-9% (w / v) of the total culture medium throughout the 45-hour culture. Nitrogen was supplied from the initiation of the culture until its completion. To maintain an appropriate nitrogen source concentration, the nitrogen source was supplied to the culture medium by repeatedly turning it on and off. The carbon source was glucose, and the nitrogen source was ammonium sulfate. After the culture was completed, the bacterial culture medium was freeze-dried or drum-dried to obtain dry biomass for the control group (Comparative Example 1).

[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 of Comparative Example 1 (control condition, control group), the composition of the MJW01 medium was maintained, and carbon sources were supplied so that the residual carbon source concentration in the culture medium was maintained within the range of 0-2% (w / v ratio) of the total culture medium during the culture period (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 the 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 2-5% (w / v ratio) of the total culture medium during the culture period (45 hours). The remaining culture conditions were the same as in Comparative Example 1, and the 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 the 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 within the range of 4 to 7% (w / v ratio) of 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 dried biomass obtained in Comparative Example 1 and Examples 1 to 3, the following experiment was carried out.

[0072] Specifically, 2 g of dried microalgae cells from Comparative Example 1, Examples 1, and 3 was added to an 8.3 M hydrochloric acid solution and hydrolyzed at 80°C. Then, 30 mL of ethyl ether and 20 mL of petroleum ether were added, and the mixture was mixed for 30 seconds and centrifuged. This process was repeated at least three times. The separated solvent layer was transferred to a pre-weighed round flask and then purged with nitrogen to remove the solvent and residual moisture. The total oil content was calculated by measuring the weight of the remaining oil after drying, and the omega-3 (DHA and EPA) content in the oil was measured by gas chromatography after pre-treatment with 0.5 N methanolic 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 an analysis tube for a Kjeldahl apparatus (Kjeltec2100), and sulfuric acid and a catalyst were added and digested in a digester. The digestion tube was then attached to an autosampler. The ammonia gas generated by heating and distilling with caustic soda and steam in the Kjeldahl apparatus was cooled and collected, and then titrated with hydrochloric acid to automatically calculate the nitrogen content. This method measured 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 dried biomass obtained in Comparative Example 1 and Examples 1 to 3, the following experiment was carried out.

[0076] Specifically, 0.5 to 1 g of dried microalgae cells from each of Comparative Example 1 and Examples 1 to 3 was subjected to acid hydrolysis, followed by total amino acid analysis using liquid chromatography. The analysis results for individual amino acids were standardized by the amount of dried microalgae cells used to calculate the content ratio of each 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 dried microalgae 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 Example 1 was able to increase the total amino acid content in the microalgae biomass by maintaining the residual carbon source concentration in the culture solution in a low range of 0 to 2% during the culture process, but that the crude fat content increased.

[0080] In Example 2, the concentration of the carbon source remaining in the culture medium during cultivation was adjusted to a range of 2-5%. Comparing the component contents between 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, the total amino acid content in the biomass was 52.78%, which was the highest value among the culture conditions.

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

[0082] Experimental Example 2. Analysis of pepsin digestibility of dried microalgae biomass under different culture conditions Since protein digestibility is an important indicator for using microalgal biomass as feed and food, analysis was performed using a pepsin digestibility analysis method. 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 using the method described in Official Method of Analysis 971.09, Association of Official Analytical Chemists, 1990, and the pepsin digestibility was calculated using the following calculation formula 1.

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

[0084] The Kjeldahl nitrogen content in Calculation 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 shown to be relatively low at 68.6%. However, by changing the culture conditions and altering 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%, showing high pepsin digestibility.

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

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

[0090] As a result, as shown in Figures 1 to 3, among the components of biomass, the crude fat content, crude protein, and omega-3 fatty acid content relative to the weight of crude fat showed no 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 biomass and pepsin digestibility (R 2 =0.9527). Generally, total amino acids are included in crude protein content, and therefore crude protein content and total amino acids are known to fluctuate 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 has been confirmed that the pepsin digestibility increases as the total amino acid content in biomass increases.

[0091] [Accession number] Depository institution: Korea Center for Biological Resources (KCTC) Accession number: KCTC14345BP Date of acceptance: 20201026 TIFF0007793802000003.tif236169

Claims

1. A method for producing biomass derived from microalgae of the genus Schizochytrium, which comprises culturing microalgae of the genus Schizochytrium deposited under accession number KCTC14345BP under conditions in which the concentration of residual carbon sources in the culture solution is maintained at 7% or less of the total culture solution.

2. A method for producing biomass derived from Schizochytrium microalgae as described in claim 1, wherein the carbon source is one or more selected from the group consisting of glucose, fructose and sucrose.

3. The method for producing biomass derived from Schizochytrium microalgae according to claim 1, wherein the culture is carried out in a medium containing one or more nitrogen sources selected from the group consisting of urea, aqueous ammonia, ammonium sulfate, yeast extract, peptone, and sodium nitrate.

4. The culture is carried out in the presence of 0.01 to 5 g / L of yeast extract, KH ２ P.O. ４ 1.0-3.0g / L, K ２ HPO ４ The method for producing biomass derived from microalgae of the genus Schizochytrium according to claim 1, wherein the method is carried out in a medium containing 5 to 20 g / L of the schizochytrium.

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

6. A method for producing biomass derived from microalgae of the genus Schizochytrium as described in claim 1, wherein the pepsin digestibility of the biomass derived from microalgae of the genus Schizochytrium is 70% or more.

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