Schizochytrium limacinum culture medium and method for fermentation production of n-3 fatty acid by using same

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a schizochytrium limacinum culture medium and a method for producing n-3 fatty acid through fermentation of the schizochytrium limacinum culture medium. Specifically, in the culture medium disclosed by the invention, glycerol and glucose in a certain proportion are used as carbon sources, and the dry weight of thalli and the yield of EPA, DPA and DHA are greatly improved in cooperation with the culture method disclosed by the invention.

Description

Technical Field

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

[0002] According to the number of carbon chains of fatty acids, fatty acids can be divided into short-chain fatty acids, medium-chain fatty acids and long-chain fatty acids; according to the presence or absence of double bonds between carbon atoms, fatty acids can be divided into saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids; according to the position of the first double bond at the methyl end of the fatty acid carbon chain, polyunsaturated fatty acids can be divided into n-3 fatty acids (the first double bond appears at the third carbon of the carbon chain methyl end), n-6 fatty acids (the first double bond appears at the sixth carbon of the carbon chain methyl end), and n-9 fatty acids (the first double bond appears at the ninth carbon of the carbon chain methyl end). n-3 fatty acids and n-6 ​​fatty acids have important physiological functions in the human body, and docosahexaenoic acid (22:6), docosapentaenoic acid (22:5), and eicosapentaenoic acid (20:5) are common n-3 fatty acids, which play an important role in enhancing immunity, regulating lipid metabolism, preventing cardiovascular diseases, preventing tumors, and promoting human 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, tuna, etc. The quality of polyunsaturated fatty acids is affected by many factors, such as species, fishing season, and location. Due to the depletion of global marine resources and marine pollution, the global supply of high-quality fish oil is insufficient.

[0004] Schizochytrium is a heterotrophic marine fungus that reproduces mostly by budding and spores. It is commonly found in the ocean and mangroves. It has the advantages of fast growth, strong adaptability, simple culture medium, short fermentation cycle, and simple fatty acid composition. It is widely used in the industrial production of DHA. In recent years, researchers have found that Schizochytrium also has the potential to synthesize EPA. Therefore, as the original producer of n-3 fatty acids, marine microalgae are well-deserved sustainable alternative resources.

[0005] However, the yield of EPA from industrialized microalgae fermentation is very low, and it is still a challenge to use Schizochytrium to mass produce EPA. Therefore, finding suitable fermentation conditions and culture medium conditions to increase the yield of EPA 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 medium, which can produce at least 40% DHA and at least 5% EPA by culturing Schizochytrium using the 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 may be at least 0.05 ug / L; the sodium molybdate may be at least 0.05 ug / L; and the nickel sulfate may be 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 yeast extract 10-15 g / L, anhydrous sodium sulfate 10-15 g / L, anhydrous calcium chloride 0.1-0.2 g / L, potassium chloride 0.3-0.6 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, ammonium sulfate 2.0-4.0, zinc sulfate 1.0-4.0 g / L, magnesium sulfate 2.0-6.0 g / L, and potassium sulfate 0.5-1.5 g / L.

[0016] In addition, the present invention also provides a Schizochytrium fermentation method, wherein glucose and glycerol are added in a repeated batch feeding manner during the initial fermentation process, and 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, 5:1; in the middle stage of fermentation, the glucose / glycerol concentration ratio is at least 4:1, 3:1, 2:1; in the late stage of fermentation, the glucose / glycerol concentration ratio is at least 1:0, 10:1, 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 is between 2:1-5:1 70 hours before 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 5:1-1:0, 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 may be at least 0.05 ug / L; the sodium molybdate may be at least 0.05 ug / L; and the nickel sulfate may be 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, 0.8 mg / L cobalamin, 0.09 mg / L sodium molybdate, and 1.2 mg / L nickel sulfate mixture is added during 0-5 h; and 1 mg / L cobalamin, 0.1 mg / L sodium molybdate, and 2.0 mg / L nickel sulfate mixture 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 0-70 hours of fermentation; and at 4.5-5.5 after 70 hours 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 r / min for 48 hours to obtain a seed solution;

[0033] S2: Fermentation culture; the seed liquid is transferred to a fermentation tank at an inoculation rate 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 primary seed culture and secondary seed culture. That is, the seed culture is first performed, and then the secondary seed culture is performed by inoculation; wherein the culture medium for the primary seed culture can be the same as or different from the culture medium for the secondary culture. The conditions for the primary seed culture can be the same as or different from the conditions for the secondary culture.

[0035] Terminology

[0036] Certain embodiments of the present invention are now described in detail. The present invention is intended to cover all substitutions, modifications and equivalent technical solutions, which are all included in the scope of the present invention as defined in the claims. It should be appreciated by those skilled in the art that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is by no means limited to the methods and materials described herein. In the event that one or more of the combined documents, patents and similar materials are different from or contradictory to the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0037] It should be further appreciated that certain features of the invention, which for clarity are described in the context of multiple 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 sub-combination.

[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 description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

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

[0041] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of 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] The components of the primary culture medium are: 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 is 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] The 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) Take out a Schizochytrium glycerol tube (Schizochytrium HS08; deposit information: deposited in the General Microbiological Center of China Microbiological Culture Collection (CGMCC), deposit number is CGMCC No.40902) from a -80°C refrigerator, put it into the primary seed culture medium, and culture it at 28°C and 180r / min for 48h to obtain the primary seed solution;

[0050] 2) Inoculate the primary seed solution into the secondary seed culture medium at a 10% inoculation rate, and culture at 28° C. and 180 r / min for 24 h to obtain the secondary seed solution.

[0051] 2. Fermentation

[0052] Example 1

[0053] The secondary seed solution obtained in the first part of this article "Cultivating Seed Solution" was transferred to the fermentation tank at an inoculum of 35%. The temperature was controlled at 28°C and the fermentation pH was controlled at 5.0-6.0. Ammonia and 50% malic acid were used to adjust the pH during the fermentation.

[0054] Among them, the fermentation carbon sources are glycerol and glucose. Glucose and glycerol are added in repeated batch feeding 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 part of this article "Cultivating Seed Solution" was transferred to the fermentation tank at an inoculum of 35%. The temperature was controlled at 28°C and the fermentation pH was controlled at 5.0-6.0 during the fermentation. Ammonia water and 50% malic acid were used to adjust the pH during the fermentation.

[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 part of this article "Cultivating Seed Solution" was transferred to the fermentation tank at an inoculum of 35%. The temperature was controlled at 28°C and the fermentation pH was controlled at 5.0-6.0 during the fermentation. Ammonia water and 50% malic acid were used to adjust the pH during the fermentation.

[0062] The fermentation carbon sources are glycerol and glucose. Glucose and glycerol are added in a repeated batch feeding mode 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 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 5:1-1:0, 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 part of this article "Cultivating Seed Solution" was transferred to the fermentation tank at an inoculum of 35%. The temperature was controlled at 28°C and the fermentation pH was controlled at 5.0-6.0 during the fermentation. Ammonia water and 50% malic acid were used to adjust the pH during the fermentation.

[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 fed during the fermentation process 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 part of this article "Cultivating Seed Solution" was transferred to the fermentation tank at an inoculum of 35%. The temperature was controlled at 28°C and the fermentation pH was controlled at 5.0-6.0 during the fermentation. Ammonia water and 50% malic acid were used to adjust the pH during the fermentation.

[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, 0.8 mg / L cobalamin, 0.09 mg / L sodium molybdate, and 1.2 mg / L nickel sulfate mixture was added during 0-5 h; and 1 mg / L cobalamin, 0.1 mg / L sodium molybdate, and 2.0 mg / L nickel sulfate mixture 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 part of this article "Cultivation of seed solution" was transferred to the fermentation tank at an inoculum of 35%. During the fermentation process, the temperature was controlled at 28°C, the pH was controlled at 5.5-6.5 for 0-70h of fermentation, and at 4.5-5.5 after 70h of fermentation. Ammonia water and 10% acetic acid + 50% citric acid were used to adjust the pH during the 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, 0.8 mg / L cobalamin, 0.09 mg / L sodium molybdate, and 1.2 mg / L nickel sulfate mixture was added during 0-5 h; and 1 mg / L cobalamin, 0.1 mg / L sodium molybdate, and 2.0 mg / L nickel sulfate mixture 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 part of this article "Cultivating Seed Solution" was transferred to the fermentation tank at an inoculum of 30%. The temperature during the fermentation was controlled at 28°C, the pH during the fermentation was controlled at 5.0-6.0, and ammonia and 50% malic acid were used to adjust the pH during the fermentation.

[0081] 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, 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 part of this article "Cultivating Seed Solution" was transferred to the fermentation tank at an inoculum of 30%. The temperature during the fermentation was controlled at 28°C, the pH during the fermentation was controlled at 5.0-6.0, and ammonia and 50% malic acid were used to adjust the pH during the fermentation.

[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 part of this article "Cultivating Seed Solution" was transferred to the fermentation tank at an inoculum of 35%. The temperature was controlled at 28°C and the fermentation pH was controlled at 5.0-6.0 during the fermentation. Ammonia water and 50% malic acid were used to adjust the pH during the fermentation.

[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 solution obtained in the first part of this article "Cultivation of seed solution" was transferred to the fermentation tank at an inoculation rate 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 the glycerol concentration is controlled 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 Comparative Examples are shown in Table 1.

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

[0097]

[0098]

[0099] Comparison of Comparative Examples 1-1 and 1-2 with Comparative Examples 2-1 and 2-2 shows that controlling the carbon source concentration and carbon source type in the fermentation broth in stages can not only increase the content of n-3 fatty acids in fatty acids, but also adjust 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 greatly increased, but it is not conducive to the accumulation of dry weight of the bacteria. The present invention combines glucose as a carbon source, controls the concentration ratio in stages, cooperates with directional flow to add limiting nutrient factors, and regulates pH in stages during the fermentation process, thereby greatly increasing the dry weight of the bacteria and the yield of EPA, DPA, and DHA.

[0100] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A Schizochytrium fermentation medium, characterized in that: 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.

2. The fermentation medium according to claim 1, characterized in that The fermentation medium also includes a limiting nutrient factor, which is selected from one or more of cobalamin, sodium molybdate, cobalt nitrate, nickel sulfate, copper sulfate, inositol, cobalt chloride, and biotin; preferably, the limiting nutrient factor includes at least cobalamin, sodium molybdate and nickel sulfate; wherein the cobalamin can be at least 0.05ug / L; the sodium molybdate can be at least 0.05ug / L; and the nickel sulfate can be at least 0.05ug / L.

3. The fermentation medium according to claim 1 or 2, characterized in that The fermentation medium also contains Yeast extract powder 2.0-6.0g / L, anhydrous sodium sulfate 20-30g / L, anhydrous calcium chloride 0.1-0.2g / L, potassium chloride 0.3-0.6g / L, potassium dihydrogen phosphate 0.5-1.5g / L, ammonium sulfate 2.0-4.0g / L, zinc sulfate 1.0-4.0g / L, magnesium sulfate 2.0-6.0g / L.

4. A Schizochytrium fermentation method, characterized in that: The method comprises fermentation culture, wherein glucose and glycerol are added in a repeated batch feeding manner in the initial fermentation process, and 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.

5. The fermentation method according to claim 4, characterized in that 70h before fermentation, the glucose / glycerol concentration ratio is between 5:1-1:0, the glucose concentration is controlled at 20-40g / L, and the glycerol concentration is controlled at 0-6.7g / L; 70h after fermentation, the glucose / glycerol concentration ratio is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3g / L, and the glycerol concentration is controlled at 3.7-6.7g / L; or 70h before fermentation, the glucose / glycerol concentration ratio is between 2:1-5:1, the glucose concentration is controlled at 13.3-33.3g / L, and the glycerol concentration is controlled at 3.7-6.7g / L; 70h after fermentation, the glucose / glycerol concentration ratio is between 5:1-1:0, the glucose concentration is controlled at 20-40g / L, and the glycerol concentration is controlled at 0-6.7g / L.

6. The fermentation method according to claim 4, characterized in that During the fermentation process, a limiting nutrient factor is added, 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; preferably, the limiting nutrient factor includes at least cobalamin, sodium molybdate and nickel sulfate; wherein the cobalamin can be at least 0.05ug / L; the sodium molybdate can be at least 0.05ug / L; and the nickel sulfate can be at least 0.05ug / L.

7. The fermentation method according to claim 6, characterized in that During the fermentation process, 1.8 mg / L cobalamin, 0.19 mg / L sodium molybdate, and 2.2 mg / L nickel sulfate mixture is added during 0-5 h; preferably, during the fermentation process, 0.8 mg / L cobalamin, 0.09 mg / L sodium molybdate, and 1.2 mg / L nickel sulfate mixture is added during 0-5 h; and 1 mg / L cobalamin, 0.1 mg / L sodium molybdate, and 2.0 mg / L nickel sulfate mixture is added during 70-75 h.

8. The fermentation method according to claim 4, characterized in that During the fermentation process, the temperature is controlled at 25-30°C, and the fermentation pH is controlled at 4.5-6.5; preferably, during the fermentation process, the temperature is controlled at 25-30°C, and the pH is controlled at 5.5-6.5 during fermentation 0-70h; and after 70h of fermentation, the pH is controlled at 4.5-5.

5.

9. The fermentation method according to claim 8, characterized in that During fermentation, one or more of sodium hydroxide, ammonia water, ammonium sulfate, sodium acetate, formic acid, acetic acid, succinic acid, fumaric acid, malic acid, and citric acid are used to adjust the pH; preferably, during fermentation, ammonia water and 50% malic acid are used to adjust the pH; preferably, during fermentation, ammonia water and 10% acetic acid + 50% citric acid are used to adjust the pH.

10. The fermentation method according to any one of claims 4 to 9, characterized in that: Includes 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 seed culture is cultured at 25-30°C and 150-250 r / min for 48 hours to obtain a seed solution; preferably, the seed culture includes primary seed culture and secondary seed culture; S2: Fermentation culture; the seed liquid is transferred to a fermentation tank at an inoculation rate 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.

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