A culture medium for Schizochytrium and a method for producing n-3 fatty acids by fermentation thereof

Abstract

The present invention relates to the field of microbial fermentation technology, specifically to a Schizochytrium algae culture medium and a method for producing n-3 fatty acids through fermentation. Specifically, the culture medium of the present invention uses a certain ratio of glycerol and glucose as carbon sources, and in combination with the culture method of the present invention, significantly increases the dry weight of the microorganisms and the yields of EPA, DPA, and DHA.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a culture medium of Schizochytrium and a method for producing n-3 fatty acids by fermentation. Background Art

[0002] Fatty acids can be classified as short-chain, medium-chain, and long-chain fatty acids based on the number of carbon chains. Based on the presence or absence of double bonds between carbon atoms, fatty acids can be classified as saturated, monounsaturated, and polyunsaturated fatty acids. Based on the position of the first double bond at the methyl end of the fatty acid chain, polyunsaturated fatty acids can be further classified as n-3 fatty acids (the first double bond occurs at the third carbon from the methyl end of the carbon chain), n-6 fatty acids (the first double bond occurs at the sixth carbon from the methyl end of the carbon chain), and n-9 fatty acids (the first double bond occurs at the ninth carbon from the methyl end of the carbon chain). n-3 and n-6 ​​fatty acids play important physiological roles in the human body. Docosahexanoic acid (22:6), docosapentaenoic acid (22:5), and eicosapentaenoic acid (20:5) are common n-3 fatty acids that play a crucial role in enhancing immunity, regulating lipid metabolism, preventing cardiovascular disease and tumors, and promoting brain development. Studies have shown that docosahexaenoic acid (DHA) exists in cell membranes, especially in retinal cells and brain gray matter, with a content between 10% and 40%. Eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) have the function of treating coronary atherosclerosis and lowering blood lipids. Since the human body cannot synthesize n-3 long-chain fatty acids, it is particularly important to supplement these nutrients through diet.

[0003] The main dietary source of polyunsaturated fatty acids is deep-sea fish, such as sardines, salmon, and tuna. The quality of polyunsaturated fatty acids is affected by many factors, including species, fishing season, and location. Due to global marine resource depletion and ocean pollution, there is a global shortage of high-quality fish oil.

[0004] Schizochytrium is a heterotrophic marine fungus that primarily reproduces by budding and spores. It is commonly found in oceans and mangroves. Its rapid growth, strong adaptability, simple culture medium, short fermentation cycle, and simple fatty acid profile make it widely used in industrial DHA production. In recent years, researchers have discovered that Schizochytrium also has the potential to synthesize EPA. Therefore, as the primary producer of n-3 fatty acids, marine microalgae are a well-deserved sustainable alternative resource.

[0005] However, the yield of EPA from industrial microalgae fermentation is very low, and large-scale production of EPA using Schizochytrium algae remains a challenge. Therefore, finding suitable fermentation and culture medium conditions to increase EPA production will help solve the problem of insufficient EPA supply. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a Schizochytrium fermentation culture medium, which can produce at least 40% DHA and at least 5% EPA by culturing Schizochytrium using the culture medium.

[0007] Specifically, the present invention provides a Schizochytrium fermentation medium, wherein the carbon source of the fermentation medium contains glycerol and glucose in a ratio of (1-5):(0-1); the glucose concentration is controlled at 10-40 g / L, and the glycerol concentration is controlled at 0-8 g / L.

[0008] In some embodiments, the carbon source of the fermentation medium may further include other carbon sources. Other carbon sources may be one or more of formic acid, acetic acid, succinic acid, valeric acid, methanol, ethanol, butanol, butanediol, propanol, propylene glycol, and mannose.

[0009] The carbon source concentration is at least greater than 40 g / L, at least greater than 30 g / L, at least greater than 20 g / L, at least greater than 10 g / L.

[0010] In some embodiments, the fermentation medium further comprises a limiting nutrient factor, and the limiting nutrient factor is selected from one or more of cobalamin, sodium molybdate, cobalt nitrate, nickel sulfate, copper sulfate, inositol, cobalt chloride, and biotin.

[0011] In some embodiments, the limiting nutritional factors include at least cobalamin, sodium molybdate and nickel sulfate; wherein the cobalamin is at least 0.05 ug / L; the sodium molybdate is at least 0.05 ug / L; and the nickel sulfate is at least 0.05 ug / L.

[0012] The cobalamin in the limiting nutritional factor can be at least 0.05 ug / L, at least 0.5 mg / L, or at least 1 g / L.

[0013] The sodium molybdate may be at least 0.05 ug / L, at least 0.5 mg / L, or at least 1 g / L.

[0014] The nickel sulfate may be at least 0.05 ug / L, at least 0.5 mg / L, or at least 50 mg / L.

[0015] In some embodiments, the fermentation medium further contains 10-15 g / L yeast extract, 10-15 g / L anhydrous sodium sulfate, 0.1-0.2 g / L anhydrous calcium chloride, 0.3-0.6 g / L potassium chloride, 0.5-1.5 g / L potassium dihydrogen phosphate, 2.0-4.0 g / L ammonium sulfate, 1.0-4.0 g / L zinc sulfate, 2.0-6.0 g / L magnesium sulfate, and 0.5-1.5 g / L potassium sulfate.

[0016] In addition, the present invention also provides a Schizochytrium fermentation method, in which glucose and glycerol are added in a repeated batch feeding manner during the initial fermentation process. During the fermentation process, the glucose / glycerol concentration ratio is (1-5):(0-1); the glucose concentration is controlled at 10-40 g / L, and the glycerol concentration is controlled at 0-8 g / L.

[0017] In some embodiments, in the early stage of fermentation, the glucose / glycerol concentration ratio is at least 1:0, 10:1, or 5:1; in the middle stage of fermentation, the glucose / glycerol concentration ratio is at least 4:1, 3:1, or 2:1; and in the late stage of fermentation, the glucose / glycerol concentration ratio is at least 1:0, 10:1, or 100:1.

[0018] In some embodiments, 70 hours before fermentation, the glucose / glycerol concentration ratio is between 5:1-1:0, the glucose concentration is controlled at 20-40 g / L, and the glycerol concentration is controlled at 0-6.7 g / L; after 70 hours of fermentation, the glucose / glycerol concentration ratio is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3 g / L, and the glycerol concentration is controlled at 3.7-6.7 g / L.

[0019] In some embodiments, the glucose / glycerol concentration ratio 70 hours before fermentation is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3 g / L, and the glycerol concentration is controlled at 3.7-6.7 g / L; after 70 hours of fermentation, the glucose / glycerol concentration ratio is between 5:1-1:0, the glucose concentration is controlled at 20-40 g / L, and the glycerol concentration is controlled at 0-6.7 g / L.

[0020] In some embodiments, during the fermentation process, a limiting nutrient factor is fed, and the limiting nutrient factor is selected from one or more of cobalamin, sodium molybdate, cobalt nitrate, nickel sulfate, copper sulfate, inositol, cobalt chloride, and biotin.

[0021] In some embodiments, the limiting nutritional factors include at least cobalamin, sodium molybdate and nickel sulfate; wherein the cobalamin is at least 0.05 ug / L; the sodium molybdate is at least 0.05 ug / L; and the nickel sulfate is at least 0.05 ug / L.

[0022] The cobalamin in the limiting nutritional factor can be at least 0.05 ug / L, at least 0.5 mg / L, or at least 1 g / L.

[0023] The sodium molybdate may be at least 0.05 ug / L, at least 0.5 mg / L, or at least 1 g / L.

[0024] The nickel sulfate may be at least 0.05 ug / L, at least 0.5 mg / L, or at least 50 mg / L.

[0025] In some embodiments, a mixture of 1.8 mg / L cobalamin, 0.19 mg / L sodium molybdate, and 2.2 mg / L nickel sulfate is fed during 0-5 h during the fermentation process.

[0026] In some embodiments, during the fermentation process, a mixture of 0.8 mg / L cobalamin, 0.09 mg / L sodium molybdate, and 1.2 mg / L nickel sulfate is added during 0-5 h; and a mixture of 1 mg / L cobalamin, 0.1 mg / L sodium molybdate, and 2.0 mg / L nickel sulfate is added during 70-75 h.

[0027] In some embodiments, the temperature during fermentation is controlled at 25-30° C., and the fermentation pH is controlled at 4.5-6.5.

[0028] In some embodiments, the temperature during the fermentation process is controlled at 25-30° C., and the pH is controlled at 5.5-6.5 during fermentation 0-70 h; and at 4.5-5.5 after 70 h of fermentation.

[0029] In some embodiments, one or more of sodium hydroxide, ammonia, ammonium sulfate, sodium acetate, formic acid, acetic acid, succinic acid, fumaric acid, malic acid, and citric acid are used to adjust the pH during fermentation.

[0030] In some embodiments, aqueous ammonia and 50% malic acid are used to adjust the pH during fermentation; preferably, aqueous ammonia and 10% acetic acid + 50% citric acid are used to adjust the pH during fermentation.

[0031] In some embodiments, the fermentation method comprises the steps of:

[0032] S1: Seed culture: The seed culture medium includes 40-80 g / L glucose, 10-15 g / L yeast extract, 10-15 g / L anhydrous sodium sulfate, 0.1-0.2 g / L anhydrous calcium chloride, 0.3-0.6 g / L potassium chloride, 0.5-1.5 g / L potassium dihydrogen phosphate, 2.0-4.0 g / L ammonium sulfate, 1.0-4.0 g / L zinc sulfate, 2.0-6.0 g / L magnesium sulfate, and 0.5-1.5 g / L potassium sulfate; the seeds are cultured at 25-30°C and 150-250 rpm for 48 hours to obtain a seed solution;

[0033] S2: Fermentation culture; the seed liquid is transferred to a fermentation tank at an inoculum size of 20-50% for fermentation culture, the fermentation temperature is 20-37°C, the fermentation pH is 2.0-8.0, and the fermentation culture time is 156-190h; wherein the carbon source concentration is controlled to be 10-50g / L.

[0034] In some embodiments, the seed culture includes a primary seed culture and a secondary seed culture. That is, the primary seed culture is performed first, followed by inoculation for the secondary seed culture. The culture medium for the primary seed culture and the secondary culture may be the same or different. The conditions for the primary seed culture and the secondary culture may be the same or different.

[0035] Terminology

[0036] Certain embodiments of the present invention are now described in detail. The present invention is intended to encompass all alternatives, modifications, and equivalent technical solutions, which are all included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the combined documents, patents, and similar materials differ from or contradict the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0037] It will be further appreciated that certain features of the invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0038] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0039] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0040] In the following disclosure, all numerical values ​​disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The numerical value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number having a value of N is disclosed, any number having a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% is expressly disclosed, where "+ / -" means plus or minus. DETAILED DESCRIPTION

[0041] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0042] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0043] 1. Cultivating seed solution

[0044] Culture medium:

[0045] Components of the primary culture medium: glucose 40-80, yeast extract powder 10-15, anhydrous sodium sulfate 10-15, anhydrous calcium chloride 0.1-0.2, potassium chloride 0.3-0.6, potassium dihydrogen phosphate 0.5-1.5, ammonium sulfate 2.0-4.0, zinc sulfate 1.0-4.0, magnesium sulfate 2.0-6.0, potassium sulfate 0.5-1.5; unit: g / L.

[0046] Secondary culture medium composition: glucose 40-80, yeast extract powder 10-15, anhydrous sodium sulfate 10-15, anhydrous calcium chloride 0.1-0.2, potassium chloride 0.3-0.6, potassium dihydrogen phosphate 0.5-1.5, ammonium sulfate 2.0-4.0, zinc sulfate 1.0-4.0, magnesium sulfate 2.0-6.0, potassium sulfate 0.5-1.5; unit g / L.

[0047] Fermentation tank culture medium composition: yeast extract powder 2.0-6.0, anhydrous sodium sulfate 20-30, anhydrous calcium chloride 0.1-0.2, potassium chloride 0.3-0.6, potassium dihydrogen phosphate 0.5-1.5, ammonium sulfate 2.0-4.0, zinc sulfate 1.0-4.0, magnesium sulfate 2.0-6.0; unit g / L.

[0048] Seed liquid fermentation steps:

[0049] 1) Remove a glycerol tube of Schizochytrium sp. (Schizochytrium sp. HS08; deposited at the China General Microbiology Center (CGMCC), with the accession number CGMCC No. 40902) from a -80°C freezer and place it in a primary seed culture medium. Incubate at 28°C and 180 rpm for 48 h to obtain a primary seed solution.

[0050] 2) The primary seed solution was inoculated into the secondary seed culture medium at an inoculum rate of 10%, and cultured at 28° C. and 180 rpm for 24 h to obtain the secondary seed solution.

[0051] 2. Fermentation Culture

[0052] Example 1

[0053] The secondary seed solution obtained in the first section of this article, "Cultivating Seed Solution," was transferred to a fermenter at a 35% inoculum level. The fermentation temperature was maintained at 28°C and the pH was maintained between 5.0 and 6.0. Ammonia and 50% malic acid were used to adjust the pH.

[0054] Among them, the fermentation carbon sources are glycerol and glucose. Glucose and glycerol are added in a repeated batch feeding manner during the initial fermentation process. The sugar concentration and glycerol concentration are detected every 2-4 hours during the fermentation process. The glucose / glycerol concentration ratio is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3 g / L, and the glycerol concentration is controlled at 3.7-6.7 g / L.

[0055] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0056] Example 2

[0057] The secondary seed solution obtained in the first section of this article, "Cultivating Seed Solution," was transferred to a fermenter at a 35% inoculum. The fermentation temperature was controlled at 28°C and the pH was controlled at 5.0-6.0. Ammonia and 50% malic acid were used to adjust the pH.

[0058] Among them, the fermentation carbon sources are glycerol and glucose. Glucose and glycerol are added in a repeated batch feeding manner during the initial fermentation process. The sugar concentration and glycerol concentration are detected every 2-4 hours during the fermentation process. The glucose / glycerol concentration ratio is between 5:1-1:0 in the first 70 hours of fermentation, the glucose concentration is controlled at 20-40 g / L, and the glycerol concentration is controlled at 0-6.7 g / L. After 70 hours of fermentation, the glucose / glycerol concentration ratio is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3 g / L, and the glycerol concentration is controlled at 3.7-6.7 g / L.

[0059] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0060] Example 3

[0061] The secondary seed solution obtained in the first section of this article, "Cultivating Seed Solution," was transferred to a fermenter at a 35% inoculum. The fermentation temperature was controlled at 28°C and the pH was controlled at 5.0-6.0. Ammonia and 50% malic acid were used to adjust the pH.

[0062] The fermentation carbon sources are glycerol and glucose. During the initial fermentation process, glucose and glycerol are added in a repeated batch feeding manner. The sugar concentration and glycerol concentration are detected every 2-4 hours during the fermentation process. The glucose / glycerol concentration ratio is between 2:1 and 5:1 in the first 70 hours of fermentation, the glucose concentration is controlled at 13.3-33.3 g / L, and the glycerol concentration is controlled at 3.7-6.7 g / L. After 70 hours of fermentation, the glucose / glycerol concentration ratio is between 5:1 and 1:0, the glucose concentration is controlled at 20-40 g / L, and the glycerol concentration is controlled at 0-6.7 g / L.

[0063] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0064] Example 4

[0065] The secondary seed solution obtained in the first section of this article, "Cultivating Seed Solution," was transferred to a fermenter at a 35% inoculum. The fermentation temperature was controlled at 28°C and the pH was controlled at 5.0-6.0. Ammonia and 50% malic acid were used to adjust the pH.

[0066] Among them, the fermentation carbon sources are glycerol and glucose. Glucose and glycerol are added in a repeated batch feeding manner during the initial fermentation process. The sugar concentration and glycerol concentration are detected every 2-4 hours during the fermentation process. 70 hours before fermentation, the glucose / glycerol concentration ratio is between 5:1-1:0, the glucose concentration is controlled at 20-40 g / L, and the glycerol concentration is controlled at 0-6.7 g / L. After 70 hours of fermentation, the glucose / glycerol concentration ratio is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3 g / L, and the glycerol concentration is controlled at 3.7-6.7 g / L.

[0067] In addition, a mixture of 1.8 mg / L cobalamin, 0.19 mg / L sodium molybdate, and 2.2 mg / L nickel sulfate was added during the fermentation period from 0 to 5 h.

[0068] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0069] Example 5

[0070] The secondary seed solution obtained in the first section of this article, "Cultivating Seed Solution," was transferred to a fermenter at a 35% inoculum. The fermentation temperature was controlled at 28°C and the pH was controlled at 5.0-6.0. Ammonia and 50% malic acid were used to adjust the pH.

[0071] Among them, the fermentation carbon sources are glycerol and glucose. Glucose and glycerol are added in a repeated batch feeding manner during the initial fermentation process. The sugar concentration and glycerol concentration are detected every 2-4 hours during the fermentation process. The glucose / glycerol concentration ratio is between 5:1-1:0 in the first 70 hours of fermentation, the glucose concentration is controlled at 20-40 g / L, and the glycerol concentration is controlled at 0-6.7 g / L. After 70 hours of fermentation, the glucose / glycerol concentration ratio is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3 g / L, and the glycerol concentration is controlled at 3.7-6.7 g / L.

[0072] In addition, during the fermentation process, a mixture of 0.8 mg / L cobalamin, 0.09 mg / L sodium molybdate, and 1.2 mg / L nickel sulfate was added during 0-5 h; and a mixture of 1 mg / L cobalamin, 0.1 mg / L sodium molybdate, and 2.0 mg / L nickel sulfate was added during 70-75 h.

[0073] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0074] Example 6

[0075] The secondary seed solution obtained in the first section of this article, "Cultivating Seed Solution," was transferred to a fermenter at a 35% inoculum level. The fermentation temperature was maintained at 28°C, and the pH was maintained between 5.5 and 6.5 for 0-70 hours, and between 4.5 and 5.5 after 70 hours. Ammonia and 10% acetic acid + 50% citric acid were used to adjust the pH during fermentation.

[0076] Among them, the fermentation carbon sources are glycerol and glucose. Glucose and glycerol are added in a repeated batch feeding manner during the initial fermentation process. The sugar concentration and glycerol concentration are detected every 2-4 hours during the fermentation process. The glucose / glycerol concentration ratio is between 5:1-1:0 in the first 70 hours of fermentation, the glucose concentration is controlled at 20-40 g / L, and the glycerol concentration is controlled at 0-6.7 g / L. After 70 hours of fermentation, the glucose / glycerol concentration ratio is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3 g / L, and the glycerol concentration is controlled at 3.7-6.7 g / L.

[0077] In addition, during the fermentation process, a mixture of 0.8 mg / L cobalamin, 0.09 mg / L sodium molybdate, and 1.2 mg / L nickel sulfate was added during 0-5 h; and a mixture of 1 mg / L cobalamin, 0.1 mg / L sodium molybdate, and 2.0 mg / L nickel sulfate was added during 70-75 h.

[0078] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0079] Comparative Example 1-1

[0080] The secondary seed solution obtained in the first section of this article, "Cultivating Seed Solution," was transferred to a fermenter at a 30% inoculum size. The fermentation temperature was maintained at 28°C, and the pH was maintained between 5.0 and 6.0. Ammonia and 50% malic acid were used to adjust the pH.

[0081] The fermentation carbon source is glucose, which is added in a repeated batch feeding manner during the fermentation process. The sugar concentration is detected every 2-4 hours and the glucose concentration is controlled at 20-40 g / L.

[0082] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0083] Comparative Example 1-2

[0084] The secondary seed solution obtained in the first section of this article, "Cultivating Seed Solution," was transferred to a fermenter at a 30% inoculum size. The fermentation temperature was maintained at 28°C, and the pH was maintained between 5.0 and 6.0. Ammonia and 50% malic acid were used to adjust the pH.

[0085] Among them, the fermentation carbon source is glucose, and glucose is added in a repeated batch feeding manner during the fermentation process. The sugar concentration is detected every 2-4 hours. The glucose concentration is controlled at 20-40g / L before 70 hours of fermentation, and the glucose concentration is controlled at 0-10g / L after 70 hours of fermentation.

[0086] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0087] Comparative Example 2-1

[0088] The secondary seed solution obtained in the first section of this article, "Cultivating Seed Solution," was transferred to a fermenter at a 35% inoculum. The fermentation temperature was controlled at 28°C and the pH was controlled at 5.0-6.0. Ammonia and 50% malic acid were used to adjust the pH.

[0089] The fermentation carbon source is glycerol, which is added in a repeated batch feeding manner. During the fermentation process, the glycerol concentration is detected every 2-4 hours and controlled at 20-40 g / L.

[0090] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0091] Comparative Example 2-2

[0092] The secondary seed liquid obtained in the first part of this article "Cultivation of seed liquid" was transferred to a fermentation tank with an inoculum size of 35%. During the fermentation process, the temperature was controlled at 28°C and the fermentation pH was controlled at 5.0-6.0. Ammonia water and 50% malic acid were used to adjust the pH during the fermentation.

[0093] The fermentation carbon source is glycerol, which is added in a repeated batch feeding manner. The glycerol concentration is detected every 2-4 hours during the fermentation process. The glycerol concentration is controlled at 20-40 g / L before 70 hours of fermentation and at 0-10 g / L after 70 hours of fermentation.

[0094] After 160 h of fermentation, the content of relevant n-3 polyunsaturated fatty acids in the fermentation broth was determined. The results are shown in Table 1.

[0095] The fermentation results of Examples 1-6 and the comparative example are shown in Table 1.

[0096] Table 1 Comparative example of Schizochytrium fermentation, fermentation results of implementations 1-5

[0097]

[0098]

[0099] Comparison of Comparative Examples 1-1 and 1-2 with Comparative Examples 2-1 and 2-2 shows that staged control of the carbon source concentration and type in the fermentation broth not only increases the content of n-3 fatty acids in the fatty acids, but also adjusts the content and ratio of palmitic acid (%), DHA (%), EPA (%), and DPA (%). When glycerol is used alone as a carbon source, the content of n-3 fatty acids, such as DHA and DPA, can be significantly increased, but this is not conducive to the accumulation of dry cell weight. The present invention combines glucose as a carbon source, controls the concentration ratio in stages, combines directional flow addition of limiting nutrient factors, and regulates the pH in stages during the fermentation process, thereby significantly increasing the dry cell weight and the yield of EPA, DPA, and DHA.

[0100] The methods of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art will be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and spirit of the present invention to implement and apply the technology of the present invention. Those skilled in the art may refer to the disclosure herein and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention.

Claims

1. A Schizochytrium fermentation method, characterized in that: The invention comprises a fermentation culture, wherein glucose and glycerol are added in a repeated batch feeding manner during the initial fermentation process, wherein the glucose / glycerol concentration ratio is (1-5): (0-1) during the fermentation process; the glucose concentration is controlled at 10-40 g / L, and the glycerol concentration is controlled at 0-8 g / L; the repeated batch feeding without glucose and glycerol addition is as follows: 70 hours before fermentation, the glucose / glycerol concentration ratio is between 5:1-1:0, the glucose concentration is controlled at 20-40 g / L, and the glycerol concentration is controlled at 0-6.7 g / L; after 70 hours of fermentation, the glucose / glycerol concentration ratio is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3 g / L, and the glycerol concentration is controlled at 3.7-6.7 g / L; and during the fermentation process of 0-5 hours and 70-75 hours, limiting nutrient factors are fed, wherein the limiting nutrient factors include cobalamin, sodium molybdate, and nickel sulfate; wherein 0.8 mg / L cobalamin, 0.09 The method comprises the following steps: adding a mixture of 1 mg / L sodium molybdate and 1.2 mg / L nickel sulfate; adding a mixture of 1 mg / L cobalamin, 0.1 mg / L sodium molybdate and 2.0 mg / L nickel sulfate during 70-75 hours; adjusting the pH value with aqueous ammonia and 10% acetic acid + 50% citric acid during the fermentation; controlling the temperature at 25-30°C during the fermentation process; controlling the pH value at 5.5-6.5 during the fermentation period from 0 to 70 hours; and controlling the pH value at 4.5-5.5 after 70 hours of fermentation. The fermentation medium comprises 2.0-6.0 g / L yeast extract, 20-30 g / L anhydrous sodium sulfate, 0.1-0.2 g / L anhydrous calcium chloride, 0.3-0.6 g / L potassium chloride, 0.5-1.5 g / L potassium dihydrogen phosphate, 2.0-4.0 g / L ammonium sulfate, 1.0-4.0 g / L zinc sulfate, and 2.0-6.0 g / L magnesium sulfate; and the fermentation culture time is 156-190 hours.

2. The fermentation method according to claim 1, characterized in that Including steps: S1: Seed culture: The seed culture medium includes 40-80 g / L glucose, 10-15 g / L yeast extract, 10-15 g / L anhydrous sodium sulfate, 0.1-0.2 g / L anhydrous calcium chloride, 0.3-0.6 g / L potassium chloride, 0.5-1.5 g / L potassium dihydrogen phosphate, 2.0-4.0 g / L ammonium sulfate, 1.0-4.0 g / L zinc sulfate, 2.0-6.0 g / L magnesium sulfate, and 0.5-1.5 g / L potassium sulfate; the seeds are cultured at 25-30°C and 150-250 r / min for 48 hours to obtain a seed solution; S2: Fermentation culture; transferring the seed liquid to a fermentation tank at an inoculum amount of 30-35% and fermenting and culturing in the manner described in claim 1.

3. The fermentation method according to claim 2, characterized in that The method comprises the following steps, wherein the seed culture comprises primary seed culture and secondary seed culture.

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