Method for producing omega-3 polyunsaturated fatty acids

Abstract

The present invention relates to the field of microbial fermentation technology and discloses a method for producing ω-3 polyunsaturated fatty acids by culturing Schizochytrium seed liquid. The resulting Schizochytrium seed liquid is inoculated into a fermentation medium at an inoculum concentration of 30 to 40%, and fermented to produce ω-3 polyunsaturated fatty acids. The Schizochytrium fermentation method of the present invention can significantly improve the production yield of various ω-3 polyunsaturated fatty acids.

Description

Technical Field

[0001] (Cross-reference to Related Applications) This application claims the priority and benefits of Chinese Patent Application No. 202310489514.7, filed with the China National Intellectual Property Administration on May 4, 2023, the entire content of which is incorporated herein by reference.

[0002] (Technical Field) The present invention relates to the field of microbial fermentation technology, and particularly to a method for producing ω-3 polyunsaturated fatty acids.

Background Art

[0003] EPA, also known as eicosapentaenoic acid, is a long-chain ω-3 series polyunsaturated fatty acid and has various functional effects in regulating the physiological health of the human body. For example, it regulates blood pressure, blood lipids and blood sugar, and has anti-inflammatory and antioxidant activities, etc., and also has potential beneficial effects on atherosclerotic plaque factors and diet. Since the human body cannot synthesize EPA by itself, it has to be ingested from food.

[0004] DPA, also known as docosapentaenoic acid, has two types of structures. One is ω-3 DPA, which has functions such as regulating blood lipids, softening blood vessels, reducing blood viscosity, improving eyesight, promoting growth and development, and enhancing the immune function of the human body. Its function of regulating blood lipids is many times stronger than that of EPA, which is called a blood vessel cleaner, and is more suitable for middle-aged and elderly people with high blood lipids. At the same time, it is an important immune factor in human breast milk and is an essential fatty acid especially in the growth process of infants. The other is ω-6 DPA. According to research, ω-6 DPA can be converted into arachidonic acid in the animal body, and the metabolites derived from ω-6 DPA can regulate macrophage function, relieve inflammatory diseases such as experimental colitis, and at the same time can effectively inhibit platelet activation and thrombosis after vascular injury, playing a role in preventing cardiovascular diseases. It has been proven that when combined with DHA, it can enhance the anti-inflammatory and neuroprotective effects of DHA. Both ω-3 DPA and ω-6 DPA are beneficial to improving the lipoprotein distribution of high-cholesterol diet hamsters, and especially the former has a better effect on aortic function.

[0005] DHA, also known as docosahexaenoic acid, is an essential omega-3 series polyunsaturated fatty acid for the human body. Commonly called "brain gold," it has unique physiological functions, including promoting cerebral nerve development, improving vision, alleviating brain diseases, anti-inflammatory and anti-cancer properties, boosting immunity, enhancing lipid metabolism, and maintaining intestinal and cardiovascular health. It is especially crucial for the intellectual and visual development of fetuses and babies.

[0006] Currently, deep-sea fish oil is a major commercial source of omega-3 fatty acids, but marine microalgae are primary producers of polyunsaturated fatty acids and have the ability to synthesize omega-3s (e.g., DHA, EPA). Moreover, algal oil is odorless and low in cholesterol. Microorganisms capable of producing DHA / EPA mainly include Schizochytrium sp., Mortierella alpina, Pythium irregulare, Chlorella, and Phaeodactylum tricornutum. Among these, Schizochytrium has a large biomass, a high fatty acid content, and is an industrial production strain of DHA (docosahexaenoic acid), and is also one of the potential strains that can realize industrial production of EPA and DPA.

[0007] While there are existing studies on using schizochtrium to ferment and produce DHA, EPA, or DPA, conventional schizochtrium fermentation methods cannot effectively increase the content of DHA, EPA, and DPA simultaneously. For example, patent CN202111291970.8 discloses a method for producing schizochtrium oil containing EPA and DHA, which can increase the DHA content but cannot effectively increase the EPA content in the schizochtrium extract.

[0008] Therefore, there is still an urgent need for production methods that can simultaneously increase the production of various omega-3 polyunsaturated fatty acids. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] CN202111291970.8 [Overview of the project] [Problems that the invention aims to solve]

[0010] This invention provides a method for producing ω-3 polyunsaturated fatty acids, thereby solving the problem of reduced yield of EPA content in schizochytrium extracts. [Means for solving the problem]

[0011] The present invention relates to a method for producing ω-3 polyunsaturated fatty acids, which includes the following: Schizochytrium is taken, cultured, and a schizochytrium seed solution is obtained. The obtained schizochytrium seed solution is inoculated into a fermentation medium and fermented, with the fermentation pH and temperature adjusted in stages, and a batch-based method of replenishing the material is employed to maintain the residual sugar concentration in the fermentation medium at 20-40 g / L during fermentation, controlling the dissolved oxygen level to 50% or higher during the fermentation process, and at this stage approximately 20-48 hours after the start of fermentation, 3-10 g / L of amino acids is added by fed-batch culture, with a total fermentation time of approximately 144-168 hours. Selectable fermentation conditions include fermentation at room temperature and a fermentation pH of 5.0-6.0. The above method is advantageous for the growth of schizochytrium, and is advantageous for improving the weight of the microbial cells after drying, as well as the content and yield of ω-3 polyunsaturated fatty acids.

[0012] The present invention further relates to a method for producing ω-3 polyunsaturated fatty acids, which includes the following: (1) Take the schizochytrium, cultivate it, and obtain a schizochytrium seed solution. (2) The schizochytrium seed solution obtained in step (1) is inoculated into the fermentation medium at a dose of 30-40% and fermented under the following conditions: The fermentation temperature is 25-28°C, the fermentation pH is 5.0-6.0, and the fermentation pH and temperature are adjusted in stages, and a batch replenishment method is used to maintain the residual sugar concentration in the fermentation medium at 20-40 g / L during fermentation, and the dissolved oxygen during the fermentation process is controlled to 50% or more, and the total fermentation is 144-168 hours, yielding ω-3 polyunsaturated fatty acids, and 3-10 g / L of amino acids is added by fed-batch culture at a stage of 20-48 hours after the start of fermentation.

[0013] The method described above is advantageous for the growth of schizochytrium, and is beneficial for increasing the weight of the microbial cells after drying following fermentation, as well as the content and yield of ω-3 polyunsaturated fatty acids.

[0014] (Detailed description of the invention) The present invention relates to a method for producing ω-3 polyunsaturated fatty acids, and includes the following: Schizochytrium is taken, cultured, and a schizochytrium seed solution is obtained. The obtained schizochytrium seed solution is inoculated into a fermentation medium and fermented, the fermentation pH and temperature are adjusted in stages, a batch replenishment method is adopted to maintain the residual sugar concentration in the fermentation medium at 20-40 g / L during fermentation, the dissolved oxygen during the fermentation process is controlled to 20% or more, amino acids are added by fed-batch culture about 15 hours after the start of fermentation, for example, a mixture of glutamic acid, lysine and / or tyrosine is added by fed-batch culture, or amino acids are added by fed-batch culture starting about 20 hours after the start of fermentation, and the amino acids are selected from a mixture of lysine, threonine and / or arginine, or amino acids are added by fed-batch culture starting about 15 hours after the start of fermentation, and the amino acids are selected from a mixture of lysine, glycine and / or phenylalanine. In some examples, approximately 3 to 10 g / L of amino acids are added by fed-batch culture at the 20 to 48 hour stage, or approximately 10 g / L of amino acids are added by fed-batch culture at the 25 to 40 hour stage, or approximately 5 g / L of amino acids are added by fed-batch culture at the 26 to 35 hour stage. In some examples, the total fermentation time is approximately 120 hours or more, or approximately 125 hours or more, or approximately 130 hours or more, or approximately 135 hours or more, or approximately 140 hours or more, or approximately 142 hours or more, or approximately 144 hours or more, and preferably the total fermentation time is approximately 144 to 168 hours.

[0015] In some examples, schizochytrium seed extract was inoculated into the fermentation medium at inoculation levels of approximately 25%, 28%, 29%, 30%, 31%, 33%, 35%, 37%, 39%, 40%, 41%, 43%, 45%, 48%, and 50%, and fermentation was carried out. In some examples, schizochytrium seed extract was inoculated into the fermentation medium at inoculation levels of approximately 30-40%, and fermentation was carried out.

[0016] In some examples, a batch replenishment method is used to maintain the residual sugar concentration in the fermentation medium during fermentation at approximately 15 g / L, 17 g / L, 19 g / L, 21 g / L, 23 g / L, 25 g / L, 27 g / L, 29 g / L, 31 g / L, 33 g / L, 35 g / L, 37 g / L, 39 g / L, 41 g / L, 43 g / L, 45 g / L, and 47 g / L. In some examples, a batch replenishment method is used to maintain the residual sugar concentration in the fermentation medium during fermentation at approximately 20 g / L to 40 g / L.

[0017] In several examples, the dissolved oxygen during the fermentation process is controlled to be approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 42% or more, approximately 45% or more, approximately 47% or more, approximately 49% or more, or approximately 50% or more, or approximately 55% or more.

[0018] In some examples, selectable fermentation conditions include fermentation at room temperature, such as approximately 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C.

[0019] In some examples, the process of adjusting the fermentation pH stepwise can be selected from the 0 to 48-hour stage of the fermentation process, controlling the pH to approximately 5.5 to 6.0. After 48 hours of fermentation, the pH is controlled to approximately 5.0 to 6.0.

[0020] In some examples, the method for producing ω-3 polyunsaturated fatty acids includes the following: (1) Take the schizochytrium, cultivate it, and obtain a schizochytrium seed solution. (2) Inoculate the schizochytrium seed liquid obtained in step (1) into the fermentation medium at an inoculation amount of 30-40% and ferment. The fermentation conditions are as follows. The fermentation temperature is 25-28 °C, the fermentation pH is 5.0-6.0, and the fermentation pH and temperature are adjusted step by step. The batch supplementation method is adopted to maintain the residual sugar concentration in the fermentation medium during fermentation at 20-40 g / L. The dissolved oxygen during the fermentation process is controlled at 50% or more, and the total fermentation time is 144-168 hours to obtain ω-3 polyunsaturated fatty acids. At the stage of 20-48 hours after the start of fermentation, 3-10 g / L of amino acids are added by fed-batch culture.

[0021] In some embodiments, the amino acid is at least one of glutamic acid, lysine, tyrosine, threonine, arginine, glycine, or phenylalanine. In some embodiments, the amino acid is at least two or more of glutamic acid, lysine, tyrosine, threonine, arginine, glycine, or phenylalanine. In some embodiments, the amino acids are glutamic acid, lysine, and tyrosine, and the mass ratio of glutamic acid:lysine:tyrosine is about 1:1:1.

[0022] In some embodiments, the process of adjusting the fermentation pH step by step is as follows. At the stage of 0-48 hours in the fermentation process, the pH is controlled at about 5.5-6.0. After 48 hours of fermentation, the pH is controlled at about 5.0-about 6.0.

[0023] In some embodiments, the fermentation process uses an acid and / or an alkali to adjust the pH. The alkali is a mixture of one or more of ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, and calcium carbonate. The acid is a mixture of one or more of citric acid, malic acid, acetic acid, propionic acid, succinic acid, oxalic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and phytic acid.

[0024] In some embodiments, the process of adjusting the fermentation temperature step by step is as follows. At the stage of 0-48 hours in the fermentation process, the fermentation temperature is controlled at about 28 °C. After 48 hours of fermentation, the fermentation temperature is controlled at about 25 °C.

[0025] In some examples, the fermentation medium contains 40-60 g / L of initial glucose, 5-15 g / L of yeast extract powder, 2-5 g / L of yeast powder, 2-10 g / L of corn steep liquor powder, 10-30 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.4-0.8 g / L of magnesium sulfate, 0.5-1.5 g / L of potassium dihydrogen phosphate, 0.5-1.5 g / L of ammonium sulfate, 15-40 mg / L of vitamin B12, 5-15 mg / L of vitamin B1, and 5-15 mg / L of biotin.

[0026] In some examples, in step (1), schizochytrium is cultured in a shake flask, a primary seed culture, and a secondary seed stepwise culture to obtain the schizochytrium seed solution, of which the shake flask culture includes the following: Schizochytrium is inoculated into a shake flask seed medium and cultured to obtain the shake flask seed solution, and the culture conditions in the shake flask culture are to set the culture temperature to 28-30°C and to culture for 24-30 hours while shaking the shake flask at 180-220 rpm.

[0027] Primary seed culture is performed by inoculating a shake flask seed solution into primary seed medium at an inoculation volume of 2-4% and culturing to obtain a primary seed solution. The culture conditions for primary seed culture are a culture temperature of 28-30°C and adjusting the fermentation pH to approximately 5.0-6.0 with alkali and / or acid.

[0028] Secondary seed culture involves culturing the primary seed medium until the residual sugar content is 20 g / L or less, then inoculating the primary seed solution into the secondary seed medium at a dose of 10-20% and culturing to obtain schizochytrium seed solution. The cultivation conditions for secondary seed culture are a culture temperature of 28-30°C, adjusting the fermentation pH to 5.0-6.0 with alkali and / or acid, and culturing the secondary seeds until the residual sugar content in the secondary seed medium is 20 g / L or less.

[0029] In some examples, the shake flask seed medium contains 40-80 g / L of glucose, 10-15 g / L of yeast extract powder, 10-15 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.5-1.5 g / L of potassium dihydrogen phosphate, 2.0-4.0 g / L of ammonium sulfate, 1.0-4.0 g / L of zinc sulfate, 2.0-6.0 g / L of magnesium sulfate, and 0.5-1.5 g / L of potassium sulfate.

[0030] The primary seed medium contains 40-80 g / L of glucose, 5-15 g / L of yeast extract powder, 10-15 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.5-1.5 g / L of potassium dihydrogen phosphate, 2.0-4.0 g / L of ammonium sulfate, 1.0-4.0 g / L of zinc sulfate, 2.0-6.0 g / L of magnesium sulfate, and 0.5-1.5 g / L of potassium sulfate.

[0031] The secondary seed medium contains 40-60 g / L of glucose, 5-15 g / L of yeast extract powder, 5-10 g / L of yeast powder, 10-20 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.5-1.5 g / L of potassium dihydrogen phosphate, 2.0-4.0 g / L of ammonium sulfate, 1.0-4.0 g / L of zinc sulfate, 2.0-6.0 g / L of magnesium sulfate, and 0.5-1.5 g / L of potassium sulfate.

[0032] In some embodiments, the batch supplementation is glucose supplementation.

[0033] After fermentation for a certain period of time, the weight of the fermented liquid after drying the microbial cells was measured, and at the same time, the percentage of crude fat content was measured, as well as the content of related ω-3 polyunsaturated fatty acids in the crude fat. In several examples, the weight of the fermented liquid after drying the microbial cells was approximately 135 g / L, 140 g / L, 145 g / L, 150 g / L, 155 g / L, 160 g / L, 165 g / L, 170 g / L, 175 g / L, 180 g / L, 185 g / L, 190 g / L, 195 g / L, 200 g / L, 205 g / L, and 209 g / L.

[0034] The above-described production method of the present invention also helps to increase the fatty acid biomass content in schizochytrium, the biomass containing at least one of DHA, EPA, and / or DPA, and in particular, increases the EPA content in the fermented liquid cells. The mass percentage of EPA in the weight of the cells after drying is greater than or equal to 8.0%, or the mass percentage of unsaturated fatty acids in the cell oil of EPA is greater than or equal to 15%, or about 8.0% to 15%, or about 9.0% to 12%. In some examples, the EPA content reaches about 8% or more, or about 8.5% or more, or about 9.0% or more, or about 9.5% or more, or about 10.0% or more, or about 12.0% or more, or about 15.0% or more.

[0035] Any of the above technical solutions has the following beneficial effects: In this invention, complex amino acids are added by fed-batch culture within a fermentation period of 20 to 48 hours, which helps in the growth of Schizochytrium. The dry weight of the bacterial cells reaches 150 g / L or more, improving the content of DHA and EPA. The EPA content in the dry weight of the bacterial cells can reach 8% or more. Simultaneously, during the fermentation process, the fermentation pH is adjusted from 5.5 to 6.0 to 5.0 to 6.0, and the fermentation temperature is adjusted from 28°C to 25°C. These three culture conditions work synergistically, allowing the EPA content in the fermented liquid bacterial cells to reach 15% or more.

[0036] Explanation of terms In this specification, relational terms such as "1st" and "2nd" are used solely to distinguish one entity or action from another, and do not necessarily require or imply that there is an actual relationship or order between these entities or actions. The terms "includes," "contains," or any other form are intended to represent a non-exclusive inclusion relationship, including, but not limited to, the explicitly stated elements, other elements not explicitly mentioned, and elements inherent to the process, method, or article itself, when a process, method, or article is described as including a set of elements. Unless otherwise specified, elements limited by expressions such as "includes" or "contains" do not preclude the existence of other elements in the process, method, or article encompassing those elements. In this specification, "greater than," "less than," "greater than," etc., are understood to be non-numerical. "Greater than or equal to," "less than or equal to," "less than or equal to," etc., are understood to be non-numerical.

[0037] In this invention, "room temperature" and "normal temperature" refer to ambient temperatures ranging from approximately 10°C to approximately 40°C. In some embodiments, "room temperature" or "normal temperature" refers to temperatures ranging from approximately 20°C to approximately 30°C. In other embodiments, "room temperature" or "normal temperature" refers to temperatures ranging from approximately 25°C to approximately 30°C. In yet another embodiment, "room temperature" or "normal temperature" refers to temperatures such as 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc.

[0038] In this specification, any reference to the terms “one example,” “several examples,” “exhibit,” “specific example,” or “several examples” means that the specific features, structures, materials, or properties described in relation to that example or example are included in at least one example or example of the present invention. In this specification, exemplary expressions of the above terms do not necessarily refer to the same example or example. The specific features, structures, materials, or properties described may be combined in appropriate ways in any or more examples or examples. A person skilled in the art may combine different examples or examples and features of different embodiments or examples without contradiction.

[0039] "Inoculation volume" refers to the ratio of the volume of the introduced seed juice to the volume of the culture medium after inoculation.

[0040] "g / L" is a relative value to the volume of liquid in the culture medium or fermentation tank. For example, 40-80 g / L of glucose in the culture medium means that there are 40-80 g of glucose per liter of culture medium.

[0041] All numerical values ​​disclosed in this invention are considered approximations, and these values ​​may differ by 1%, 2%, 5%, and in some cases by 10-20%, whether or not the words "approximately" or "approximate" are explicitly attached. Whenever a numerical range having a lower limit RL and an upper limit RU is disclosed, any numerical value within that range is considered to be specifically disclosed. Specifically, the following numerical values ​​within the range are specifically disclosed: R = RL + k * (RU - RL), where k is a variable, the range of variation is from 1% to 100%, and the increment is 1%, i.e., k is 1%, 2%, 3%, 4%, 5%, ..., 50%, 51%, 52%, ..., 95%, 96%, 97%, 98%, 99%, or 100%. Furthermore, any numerical range defined by the two R numerical values ​​defined above is also specifically disclosed.

[0042] Schizochytrium sp. is a species of marine fungus belonging to the group of chytrid fungi. The genus Schizochytrium includes, but is not limited to, Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium species (S31) (ATCC 20888), Schizochytrium sp. LX0809, Schizochytrium genus (S8) (ATCC 20889), Schizochytrium genus (LCRM) (ATCC 18915), Schizochytrium genus (SR 21), preserved strain ATCC 28209, preserved Schizochytrium limacinum strain IFO 32693, recombinant Schizochytrium, the Schizochytrium-related fungus Thraustochytrium, and mixtures thereof.

[0043] In the term "adding amino acids by fed-batch culture," "fed-batch culture" refers to the slow and continuous addition of one or more amino acid complexes, which can be continued for several hours or more, depending on the amount of amino acids. [Modes for carrying out the invention]

[0044] The following examples are provided for illustrative purposes only and are not limiting. The following examples illustrate, and are not limiting, the method of the present invention and the ω-3 polyunsaturated fatty acids produced by the method of the present invention. Other appropriate modifications and adjustments to the various conditions and parameters commonly encountered when inoculating schizochytrium seed extract into fermentation media for fermentation will be apparent to those skilled in the art and are within the spirit and scope of the present invention.

[0045] Abbreviation: DHA (Docosahexaenoic Acid) DPA (Docosapentaenoic Acid) EPA (eicosapentaenoic acid)

[0046] This invention verifies the method for producing ω-3 polyunsaturated fatty acids using the Schizochytrium strain disclosed in Chinese application number 201711102734.0 as an example, but the method of this invention is not limited to inoculation into a fermentation medium for fermentation.

[0047] The preservation information for the aforementioned strain is as follows: The species name is Schizochytrium, the Latin name is Schizochytrium, the strain number is HS01, the preservation institution is the "Chinese Microbial Species Preservation and Management Committee Ordinary Microbial Center", the abbreviation of the preservation institution is CGMCC, the address is "No. 3, Courthouse 1, Beichen West Road, Chaoyang District, Beijing", the preservation date is March 10, 2017, and the preservation center registration number is CGMCC No. 13746.

[0048] Measurement method: For the detection methods of cell weight after drying and total lipids, refer to the detection methods in "Research on Fermentation Technology with High DHA Production by Schizochytrium and Analysis of Metabolic Mechanisms." For the detection methods of DHA content, DPA content, and EPA content, refer to the measurement methods in GB 5009.168-2016 and calculate using the fatty acid area normalization method.

[0049] The dry weight and fatty acid detection methods used in Table 1 are as follows: 1. Method for measuring the weight of the bacterial cells after drying: Place 4 mL of fermentation solution into a 10 mL centrifuge tube and centrifuge at 4300 r / min for 3 minutes. Remove the supernatant, add another 4 mL of distilled water, repeat the centrifugation process, remove the supernatant, place the precipitate on a weighed plate (M), dry in an oven at 105°C, and weigh after cooling (M). 1) .

number

[0050] Gas chromatography conditions: Column SP2560 (100m x 0.25mm ID, 0.20μm film) Injection conditions: injection volume 1μL, split flow ratio 1:10 Carrier gas: Nitrogen: 24 mL / min Detector temperature: 280℃ Column temperature conditions: Maintain an initial temperature of 100°C for 13 minutes, then increase to 180°C at a rate of 10°C / min and maintain for 6 minutes. Further increase to 200°C at a rate of 1°C / min and maintain for 20 minutes. Further increase to 230°C at a rate of 4°C / min and maintain for 10.5 minutes.

[0051] The formula for calculating the EPA content (Y1) is as follows:

number

[0052] Method for measuring residual sugar (glucose) 1. Reagents Sodium potassium tartrate, dinitrosalicylic acid, sodium hydroxide, phenol, sodium sulfite, glucose standard 2. Experimental equipment 10 mL centrifuge tube, volumetric bottle, spectrophotometer 3. Glucose measurement by the colorimetric method using 3,3,5-dinitrosalicylic acid (DNS).

[0053] 3.1 Preparation of DNS solution Weigh out exactly 185g of potassium sodium tartrate, add water to make a total volume of 500mL, heat and prepare as needed. Then weigh out 6.3g of dinitrosalicylic acid, weigh out 262mL of 2mol / L NaOH solution and add it to the hot potassium sodium tartrate solution, stirring thoroughly until uniformly mixed. After that, add 5g of phenol and 5g of sodium sulfite, stir thoroughly until dissolved, and then put the solution into a brown bottle and prepare as needed.

[0054] 3.2 Construction of a glucose standard curve Weigh out 0.01g, 0.02g, 0.03g, 0.04g, and 0.05g of glucose standard, add water to make a total volume of 50mL, add 1mL of glucose dilution and 2mL of DNS solution to a colorimetric tube with a stopper, heat in hot water for 2 minutes, and after cooling, add 9mL of distilled water and shake thoroughly to ensure uniformity. Measure the absorbance at a wavelength of 540nm. Use the sample without glucose standard as the control group, and draw a standard curve with glucose concentration as the x-coordinate and absorbance as the y-coordinate.

[0055] The present invention provides a method for producing ω-3 polyunsaturated fatty acids, which includes the following: (1) Take the schizochytrium, cultivate it, and obtain a schizochytrium seed solution. (2) The schizochytrium seed solution obtained in step (1) is inoculated into the fermentation medium at a dose of 30-40% and fermented under the following conditions: The fermentation temperature is 25-28°C, the fermentation pH is 5.0-6.0, and the fermentation pH and temperature are adjusted in stages. A batch replenishment method is used to maintain the residual sugar concentration in the fermentation medium at 20-40 g / L during fermentation. Dissolved oxygen during the fermentation process is controlled to 50% or more, and the total fermentation is 144-168 hours, yielding ω-3 polyunsaturated fatty acids. At 20-48 hours after the start of fermentation, 3-10 g / L of amino acids are added by fed-batch culture.

[0056] Example 1: Preparation of schizochytrium seed solution The cultivation process for schizochytrium seed solution is as follows: 1. Seed culture in a shake flask Take 10 ml of Schizochytrium fungal strain from a glycerin tube stored in a cryogenic refrigerator, inoculate it into a 250 ml shake flask containing 50 ml of culture medium, and culture in a 25°C shake flask at a rotation speed of 220 rpm for 24 hours. Then, inoculate a 10% inoculation into a 1000 ml shake flask containing 400 ml of culture medium, and culture in a 25°C shake flask at a rotation speed of 220 rpm for 24 hours to obtain shake flask seed solution. The shake flask seed medium (g / L) contains 40-80 g / L of glucose, 10-15 g / L of yeast extract powder, 10-15 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.5-1.5 g / L of potassium dihydrogen phosphate, 2.0-4.0 g / L of ammonium sulfate, 1.0-4.0 g / L of zinc sulfate, 2.0-6.0 g / L of magnesium sulfate, and 0.5-1.5 g / L of potassium sulfate. Culture conditions for shake flask seed culture: Shake flask culture at 28-30°C at 180-220 rpm for 24-30 hours.

[0057] 2. Primary seed culture The shaken flask seed solution is inoculated into primary seed medium at a concentration of 2-4% and cultured until the residual sugar in the primary seed medium is less than 20 g / L to obtain a primary seed solution. The primary seed medium (g / L) contains 40-80 g / L of glucose, 5-15 g / L of yeast extract powder, 10-15 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.5-1.5 g / L of potassium dihydrogen phosphate, 2.0-4.0 g / L of ammonium sulfate, 1.0-4.0 g / L of zinc sulfate, 2.0-6.0 g / L of magnesium sulfate, and 0.5-1.5 g / L of potassium sulfate. The culture conditions for primary seed cultivation are as follows: a culture temperature of 28-30°C, adjusting the fermentation pH to approximately 5.0-6.0 with alkali and / or acid, culturing for 24-30 hours, and, for example, adopting a pH condition of a constant concentration of ammonia water.

[0058] 3, Secondary seed culture The primary seed solution is inoculated into the secondary seed medium at a concentration of 10-20% and cultured until the residual sugar in the secondary seed medium is 20 g / L or less, thereby obtaining the schizochytrium seed solution. The secondary seed medium contains 40-60 g / L of glucose, 5-15 g / L of yeast extract powder, 5-10 g / L of yeast powder, 10-20 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.5-1.5 g / L of potassium dihydrogen phosphate, 2.0-4.0 g / L of ammonium sulfate, 1.0-4.0 g / L of zinc sulfate, 2.0-6.0 g / L of magnesium sulfate, and 0.5-1.5 g / L of potassium sulfate, and the above components are dissolved in purified water. The culture conditions for secondary seed culture are as follows: culture temperature of 28-30°C, fermentation pH adjusted to 5.0-6.0 with alkali and / or acid, and cultured for 24-30 hours. The following example experiments will be conducted using the schizochytrium seed solution obtained from the secondary seed culture described above.

[0059] Example 2 The schizochytrium seed solution obtained in Example 1 was inoculated into a fermentation medium at a dose of 37.5% and fermented. The fermentation conditions were as follows: The fermentation temperature was 28°C, the fermentation pH was 5.5-6.0, the residual sugar concentration was maintained at 20-40 g / L by replenishing materials in batches, the dissolved oxygen during the fermentation process was controlled to over 50%, and the total fermentation took 144 hours to obtain the fermented liquid. At the stage of fermentation for 20-48 hours, 5 g / L of complex amino acids (lysine:glycine = 1:1) was added by fed-batch culture. The fermentation medium consists of 50 g / L glucose, 12 g / L yeast extract powder, 3 g / L yeast powder, 5 g / L corn steep liquor powder, 15 g / L anhydrous sodium sulfate, 0.17 g / L anhydrous calcium chloride, 0.5 g / L potassium chloride, 0.65 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, 1 g / L ammonium sulfate, 20 mg / L vitamin B12, 10 mg / L vitamin B1, and 10 mg / L biotin. After 144 hours of fermentation, the dried weight of the microbial cells in the fermentation liquid is measured, and at the same time, the percentage of crude fat content is measured, as well as the content of related ω-3 polyunsaturated fatty acids in the crude fat.

[0060] Example 3 The schizochytrium seed solution obtained in Example 1 was inoculated into a fermentation tank at 30%, and the differences compared to Example 2 are as follows. The total fermentation time is 144 hours. During this stage, from 20 to 48 hours of fermentation, 3 g / L of a complex amino acid (glutamic acid:lysine:tyrosine = 1:1:1) is added using a fed-batch culture. The fermentation medium consists of 40 g / L glucose, 15 g / L yeast extract powder, 2 g / L yeast powder, 5 g / L corn steep liquor powder, 15 g / L anhydrous sodium sulfate, 0.1 g / L anhydrous calcium chloride, 0.3 g / L potassium chloride, 0.65 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L ammonium sulfate, 40 mg / L vitamin B12, 10 mg / L vitamin B1, and 10 mg / L biotin.

[0061] Example 4 The schizochytrium seed solution obtained in Example 1 was inoculated into a fermentation tank at 35%, and the differences compared to Example 2 are as follows. The total fermentation time is 144 hours. During this stage, from 20 to 48 hours into fermentation, 10 g / L of a complex amino acid (lysine:threonine:arginine in a mass ratio of 1:0.5:0.5) is added using a fed-batch culture. The fermentation medium consists of 60 g / L glucose, 5 g / L yeast extract powder, 5 g / L yeast powder, 2 g / L corn steep liquor powder, 10 g / L anhydrous sodium sulfate, 0.2 g / L anhydrous calcium chloride, 0.3 g / L potassium chloride, 0.4 g / L magnesium sulfate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L ammonium sulfate, 15 mg / L vitamin B12, 5 mg / L vitamin B1, and 5 mg / L biotin.

[0062] Example 5 The schizochytrium seed solution obtained in Example 1 was inoculated into a fermentation tank at 40%, and the differences compared to Example 2 are as follows. The total fermentation time is 144 hours. At this stage, 20-48 hours into fermentation, 10 g / L of a complex amino acid (lysine:glycine:phenylalanine in a mass ratio of 1:1:1) is added using a fed-batch culture. The fermentation medium consists of 50 g / L glucose, 15 g / L yeast extract powder, 5 g / L yeast powder, 10 g / L corn steep liquor powder, 30 g / L anhydrous sodium sulfate, 0.2 g / L anhydrous calcium chloride, 0.6 g / L potassium chloride, 0.8 g / L magnesium sulfate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L ammonium sulfate, 40 mg / L vitamin B12, 15 mg / L vitamin B1, and 15 mg / L biotin.

[0063] Comparative Example 1 The schizochytrium seed solution obtained in Example 1 was inoculated into a fermentation tank at 37.5%, and the differences compared to Example 2 are as follows. The total fermentation time was 144 hours, and no amino acids were added via fed-batch culture throughout the entire fermentation process. The other steps are the same as in Example 2.

[0064] Comparative Example 2 The schizochytrium seed solution obtained in Example 1 was inoculated into a fermentation tank at 37.5%, and the differences compared to Example 2 are as follows. The total fermentation time is 144 hours. During this stage, from 20 to 144 hours into fermentation, 5 g / L of a complex amino acid (lysine:glycine = 1:1) is added using a fed-batch culture. The other steps are the same as in Example 2.

[0065] The detection results of the fermentation liquids from Examples 2-5 and Comparative Examples 1-2 are shown in Table 1.

[0066] [Table 1]

[0067] Of these, palmitic acid (%), DHA (%), EPA (%), and DPA (%) represent the content of the corresponding unsaturated fatty acids in the bacterial lipids, respectively.

[0068] As can be seen by comparing Examples 2-5 with Comparative Example 1, in the present invention, by adding amino acids in the fermentation step using fed-batch culture, the dry weight of the microbial cells in the fermented liquid after fermentation can be effectively increased, which also helps to increase the DHA and EPA content produced by schizochytrium.

[0069] As can be seen by comparing Examples 2-5 with Comparative Example 2, the timing of adding amino acids by fed-batch culture in the present invention is at the 20-48 hour stage of fermentation. Compared to adding them during the long-term fed-batch culture in Comparative Example 2, the method of the present invention only requires adding amino acids by fed-batch culture within a short period, thereby reducing fermentation costs, and the final fermentation effect (dry weight of bacterial cells, unsaturated fatty acid content) was also superior to that of Comparative Example 2.

[0070] Example 6 The schizochytrium seed solution obtained in Example 1 is inoculated into a fermentation tank, and the temperature of the fermentation culture conditions is adjusted on the base of Example 2. The cultivation conditions in the fermentation tank involve controlling the temperature to 28°C 48 hours before fermentation, and then lowering the temperature to 25°C after 48 hours.

[0071] Example 7 The schizochytrium seed solution obtained in Example 1 was inoculated into a fermentation tank, and the pH of the fermentation culture environment was adjusted on the base from Example 2. The fermentation conditions involve controlling the pH to 5.5-6.0 48 hours before fermentation and adjusting it to 5.0-6.0 after 48 hours. The residual sugar concentration is maintained at 20-40 g / L using a batch replenishment method, and the dissolved oxygen level during the fermentation process is controlled to over 50%. During the fermentation process, the pH is adjusted using an acid or alkali, wherein the alkali is one or a combination of several of the following: ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, and calcium carbonate. The acid is one or a combination of several of the following: citric acid, malic acid, acetic acid, propionic acid, succinic acid, oxalic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and phytic acid.

[0072] Example 8 The schizochytrium seed solution obtained in Example 1 was inoculated into a fermentation medium, and the temperature and pH of the fermentation culture conditions were adjusted on the basis of Example 2. The fermentation culture conditions involve controlling the temperature to 28°C and the pH to 5.5-6.0 48 hours before fermentation. After 48 hours, the temperature is lowered to 25°C and the pH is adjusted to 5.0-6.0.

[0073] Comparative Example 3 The schizochytrium seed solution obtained in Example 1 was inoculated into a fermentation medium. The difference from Example 2 is that the temperature and pH of the fermentation culture conditions were adjusted during the fermentation process, and amino acids were not added by fed-batch culture throughout the entire fermentation process. The fermentation culture conditions involve controlling the temperature to 28°C and the pH to 5.5-6.0 48 hours before fermentation. After 48 hours, the temperature is lowered to 25°C and the pH is adjusted to 5.0-6.0.

[0074] The detection results of the fermentation liquids from Examples 6-8 and Comparative Example 3 are shown in Table 2 below.

[0075] [Table 2]

[0076] Of these, palmitic acid (%), DHA (%), EPA (%), and DPA (%) represent the content of the corresponding unsaturated fatty acid in the weight after drying of the bacterial cells, respectively.

[0077] Comparing Examples 6-8 with Comparative Example 3, the present invention adds complex amino acids in a fed-batch culture during the fermentation step, simultaneously adjusting the pH and temperature according to the fermentation process. The three culture conditions work synergistically with each other, thus improving the dry cell weight (maximum 209 g / L) and EPA content (which can reach 15.15%).

[0078] Although the embodiments described above have been explained, those skilled in the art, knowing the basic creativity concept, can make additional changes and modifications to these embodiments. Therefore, the above are merely embodiments of the present invention and do not thereby limit the scope of patent protection of the present invention. For the same reasons, equivalent structures or equivalent flow transformations, or direct or indirect applications in other related technical fields, made using the contents of the specification and accompanying drawings of the present invention are all included within the scope of patent protection of the present invention.

Claims

1. A method for producing omega-3 polyunsaturated fatty acids, Schizochytrium is taken and cultured to obtain schizochytrium seed solution. The obtained schizochytrium seed solution is inoculated into a fermentation medium and fermented. The fermentation pH and temperature are adjusted in stages, and a batch replenishment method is used to maintain the residual sugar concentration in the fermentation medium at 20-40 g / L during fermentation, while controlling the dissolved oxygen level to over 50% during the fermentation process. A method for producing ω-3 polyunsaturated fatty acids, characterized by continuously adding amino acids containing 3 to 10 g / L of lysine by fed-batch culture for 20 to 48 hours after the start of fermentation.

2. (1) Take the schizochytrium and cultivate it to obtain a schizochytrium seed solution, (2) The schizochytrium seed solution obtained in step (1) is inoculated into a fermentation medium at a concentration of 30-40% and fermented under the following conditions: The fermentation temperature was 25-28°C, the fermentation pH was 5.0-6.0, and the fermentation pH and temperature were adjusted in stages. A batch replenishment method was employed to maintain the residual sugar concentration in the fermentation medium at 20-40 g / L during fermentation. Dissolved oxygen was controlled to over 50% during the fermentation process, and the total fermentation time was 144-168 hours. Omega-3 polyunsaturated fatty acids were obtained, and amino acids containing lysine at a concentration of 3-10 g / L were continuously added by fed-batch culture for 20-48 hours after the start of fermentation. The ω-3 polyunsaturated fatty acid according to claim 1, comprising the above steps Production method.

3. The method according to claim 1, characterized in that the amino acid includes at least one of glutamic acid, tyrosine, threonine, arginine, glycine, and phenylalanine in addition to lysine, and the batch supplementation is glucose supplementation.

4. The process of adjusting the fermentation pH in stages is as follows: The method according to claim 1, characterized in that the pH is controlled to 5.5 to 6.0 during the 0 to 48-hour stage of the fermentation process, and then controlled to 5.0 to 6.0 after 48 hours of fermentation.

5. The method according to claim 3, characterized in that the pH is adjusted using an acid and / or alkali during the fermentation process, wherein the alkali is one or more of ammonia water, sodium hydroxide, potassium hydroxide, sodium carbonate, and calcium carbonate, and the acid is one or more of a combination of citric acid, malic acid, acetic acid, propionic acid, succinic acid, oxalic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and phytic acid.

6. The method according to claim 1, characterized in that, in the process of adjusting the fermentation temperature step by step, the fermentation temperature is controlled to 28°C during the 0 to 48-hour stage of the fermentation process, and after 48 hours of fermentation, the fermentation temperature is controlled to 25°C.

7. The method according to claim 1, characterized in that the fermentation medium contains 40-60 g / L of initial glucose, 5-15 g / L of yeast extract powder, 2-5 g / L of yeast powder, 2-10 g / L of corn steep liquor powder, 10-30 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.4-0.8 g / L of magnesium sulfate, 0.5-1.5 g / L of potassium dihydrogen phosphate, 0.5-1.5 g / L of ammonium sulfate, 15-40 mg / L of vitamin B12, 5-15 mg / L of vitamin B1, and 5-15 mg / L of biotin.

8. In step (1), the schizochytrium is cultured in a shake flask, a primary seed culture, and a secondary seed stepwise culture to obtain the schizochytrium seed solution. Of these, the shake flask culture includes the following: Schizochytrium was inoculated into shake flask seed medium and cultured to obtain shake flask seed solution. The culture conditions for the shake flask culture were to set the culture temperature to 28-30°C and to shake the shake flask at 180-220 rpm for 24-30 hours. It is about nourishing, Primary seed culture involves inoculating a shake flask seed solution into primary seed medium at an inoculum concentration of 2-4% to obtain a first-grade seed solution. The culture conditions for primary seed culture are a culture temperature of 28-30°C and adjusting the fermentation pH to 5.0-6.0 with alkali and / or acid. The method according to claim 1, characterized in that the secondary seed culture is performed by culturing the primary seed medium until the residual sugar content is 20 g / L or less in the primary seed culture step, then inoculating the primary seed solution into the secondary seed medium at an inoculation rate of 10-20% to obtain a schizochytrium seed solution, and the culture conditions for the secondary seed culture are a culture temperature of 28-30°C, adjusting the fermentation pH to 5.0-6.0 with alkali and / or acid, and culturing the secondary seeds until the residual sugar content in the secondary seed medium is 20 g / L or less.

9. The aforementioned shake flask seed medium contains 40-80 g / L of glucose, 10-15 g / L of yeast extract powder, 10-15 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.5-1.5 g / L of potassium dihydrogen phosphate, 2.0-4.0 g / L of ammonium sulfate, 1.0-4.0 g / L of zinc sulfate, 2.0-6.0 g / L of magnesium sulfate, and 0.5-1.5 g / L of potassium sulfate. The primary seed medium contains 40-80 g / L of glucose, 5-15 g / L of yeast extract powder, 10-15 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.5-1.5 g / L of potassium dihydrogen phosphate, 2.0-4.0 g / L of ammonium sulfate, 1.0-4.0 g / L of zinc sulfate, 2.0-6.0 g / L of magnesium sulfate, and 0.5-1.5 g / L of potassium sulfate. The method according to 8, characterized in that the secondary seed medium contains 40-60 g / L of glucose, 5-15 g / L of yeast extract powder, 5-10 g / L of yeast powder, 10-20 g / L of anhydrous sodium sulfate, 0.1-0.2 g / L of anhydrous calcium chloride, 0.3-0.6 g / L of potassium chloride, 0.5-1.5 g / L of potassium dihydrogen phosphate, 2.0-4.0 g / L of ammonium sulfate, 1.0-4.0 g / L of zinc sulfate, 2.0-6.0 g / L of magnesium sulfate, and 0.5-1.5 g / L of potassium sulfate.

10. The method according to any one of claims 1 to 8, characterized in that the ω-3 polyunsaturated fatty acid comprises at least one of eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid.

11. The method according to 10, characterized in that the eicosapentaenoic acid is at least 8.0% by weight of the total lipid.

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