Schizochytrium strain rich in n-3 fatty acids and methods for its selection and cultivation

Abstract

The present invention pertains to the field of biotechnology. It provides a Schizochytrium strain rich in n-3 fatty acids and methods for its selection and cultivation. Compared to original strains, the strain provided in the present invention has significantly increased yields of EPA and n-3 DPA, and exhibits good genetic stability, a rapid growth rate, and promising prospects for industrial application. The present invention also provides a means for mutagenesis of a Schizochytrium sp. and a means for the selection of a Schizochytrium sp., as well as a suitable mutagenesis method and a suitable selection method.

Description

A Schizochytrium algae strain rich in n-3 fatty acids and its screening and cultivation method Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Schizochytrium algae strain rich in n-3 fatty acids and a screening and culturing method thereof. Background Art

[0002] n-3 fatty acids are a class of polyunsaturated fatty acids, primarily including eicosapentaenoic acid (C20:5, cis-5,8,11,14,17, EPA), docosapentaenoic acid (C22:5, cis-7,10,13,16,19, n-3DPA), and docosahexaenoic acid (C22:6, cis-4,7,10,13,16,19, DHA). They are found in deep-sea marine animals and marine microorganisms. n-3 fatty acids are essential nutrients for humans, playing a vital role in neurodevelopment and the prevention of chronic diseases. Traditionally, n-3 fatty acids have been obtained from fish oil. However, due to increasing depletion of marine resources and frequent marine pollution, the supply of high-quality fish oil has declined significantly. Marine microalgae, as the primary producer of n-3 fatty acids, offer a safe, controllable fermentation process that does not require land resources, making them a green and sustainable alternative to fish oil.

[0003] Schizochytrium is one of the most important oil-producing microalgae in commercial production, primarily producing DHA, with yields reaching 40 g / L. Schizochytrium can also simultaneously produce unsaturated fatty acids such as EPA and n-3DPA. However, the wild-type strain's ability to synthesize both EPA and n-3DPA is limited, failing to meet the requirements of industrial production. Therefore, exploring how Schizochytrium can simultaneously produce high levels of n-3 fatty acids has been a hot topic for researchers. Numerous studies have shown that the fatty acid synthase pathway for Schizochytrium to produce fatty acids is primarily divided into the fatty acid synthase pathway and the ketone polymerase pathway. The fatty acid synthase pathway, common to most microorganisms, involves a series of dehydration, condensation, and elongation steps involving fatty acid precursors. Fatty acid synthesis begins with the reaction of acetyl-CoA and malonyl-CoA to produce palmitic acid (C16:00). Palmitic acid is then converted to linoleic acid (C18:2n-6) catalyzed by elongases and desaturases. Linoleic acid then enters the n-6 and n-3 pathways catalyzed by Δ15 and Δ6 desaturases, respectively. The n-3 pathway ultimately synthesizes unsaturated fatty acids such as EPA, n-3DPA, and DHA. The ketone polymerase pathway primarily involves the dehydration and isomerization of fatty acid acyl intermediates. Studies have shown that the ketone polymerase pathway can directly synthesize DPA and EPA in addition to DHA, but the specific synthesis pathway and mechanism remain controversial.

[0004] Therefore, there is a great need to find ideal methods such as strain mutagenesis, strain domestication and genetic engineering to enhance the n-3 pathway of fatty acid synthase in Schizochytrium to increase the content of EPA and n-3DPA. Summary of the Invention

[0005] SUMMARY OF THE INVENTION

[0006] To solve the above technical problems, the present invention provides a culture medium for inducing the mutation of Schizochytrium and a culture medium for screening Schizochytrium. Through suitable mutagenesis and screening methods, a Schizochytrium strain was obtained. Compared with the original strain, the EPA and DPA in this strain were greatly improved. This strain has good genetic stability and a fast growth rate, and has good industrialization prospects.

[0007] In a first aspect, the present invention provides a strain of Schizochytrium sp., designated Schizochytrium sp. HS08; deposited with the China General Microbiology Center (CGMCC) under the accession number CGMCC No. 40902. The fermentation broth obtained from Schizochytrium sp. HS08 exhibits significantly higher levels of EPA and n-3DPA in the total fatty acids of the fermentation broth, demonstrating unexpected technical benefits. Furthermore, the strain provided by the present invention can be cultured multiple times to consistently produce all n-3 fatty acids, exhibiting advantages such as good genetic stability and rapid growth, and thus has promising industrial prospects.

[0008] In a second aspect, the present invention provides a fermentation broth.

[0009] In a third aspect, the present invention provides a bacterial suspension.

[0010] In a fourth aspect, the present invention provides a microbial agent.

[0011] In a fifth aspect, the present invention provides a method for preparing the fermentation broth described in the second aspect.

[0012] In a sixth aspect, the present invention provides a method for preparing n-3 fatty acids.

[0013] In a seventh aspect, the present invention provides an n-3 fatty acid.

[0014] In an eighth aspect, the present invention provides a method for preparing microbial oil containing n-3 fatty acids.

[0015] In a ninth aspect, the present invention provides a microbial oil containing n-3 fatty acids.

[0016] In a tenth aspect, the present invention provides the aforementioned Schizochytrium fungus, fermentation broth, bacterial suspension, microbial agent, fermentation broth prepared by the aforementioned preparation method, n-3 fatty acids prepared by the aforementioned preparation method, n-3 fatty acids, microbial oil containing n-3 fatty acids prepared by the aforementioned preparation method, and applications of microbial oil containing n-3 fatty acids.

[0017] In an eleventh aspect, the present invention provides a mutagenesis culture medium for mutagenizing Schizochytrium sp.

[0018] In a twelfth aspect, the present invention provides a screening culture medium for screening Schizochytrium sp.

[0019] In a thirteenth aspect, the present invention provides a method for screening Schizochytrium sp.

[0020] Detailed Description of the Invention

[0021] To solve the above technical problems, the present invention provides a culture medium for inducing the mutation of Schizochytrium and a culture medium for screening Schizochytrium. Through suitable mutagenesis and screening methods, a Schizochytrium strain was obtained. Compared with the original strain, the EPA and DPA in this strain were greatly improved. This strain has good genetic stability and a fast growth rate, and has good industrialization prospects.

[0022] Specifically, in the first aspect, the present invention provides a strain of Schizochytrium sp., named Schizochytrium sp. HS08; preservation information: deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC), with a preservation number of CGMCC No. 40902.

[0023] In a second aspect, the present invention provides a fermentation broth.

[0024] A fermentation broth is prepared by fermenting the Schizochytrium sp. described in the first aspect.

[0025] In some embodiments, the content of EPA in the fermentation broth is greater than 9% of the total fatty acids. In some embodiments, the content of EPA in the fermentation broth is between 9% and 10% of the total fatty acids. In some embodiments, the content of EPA in the fermentation broth is 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9% or 10% of the total fatty acids.

[0026] In some embodiments, the content of n-3DPA in the fermentation broth accounts for more than 5% of the total fatty acids. In some embodiments, the content of n-3DPA in the fermentation broth accounts for 5%, 5.5%, 6%, 6.5% or 7% of the total fatty acids.

[0027] In some embodiments, the content of DHA in the total fatty acids in the fermentation broth is greater than 35%. In some embodiments, the content of DHA in the total fatty acids in the fermentation broth is 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% or 45%.

[0028] In some embodiments, the fermentation broth is a mixture of one or more of Schizochytrium sp., sugars, proteins, amino acids, and inorganic salts; and the dry weight of Schizochytrium sp. biomass cells in the fermentation broth accounts for 5% or more.

[0029] In some embodiments, the oil content of the Schizochytrium sp. in the fermentation broth accounts for at least 1%, 2%, 3%, 4%, 5%, or 6% of the mixture.

[0030] In some embodiments, the oil contains one or more of total fatty acids, phospholipids, fat-soluble substances, and sterols; the total fatty acids contain at least one of saturated fatty acids, unsaturated fatty acids, and n-3 fatty acids; in some embodiments, the mass ratio of the total fatty acids in the oil can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%.

[0031] In some embodiments, the oil content in the biomass cell dry weight is at least 50% or more.

[0032] In some embodiments, the n-3 fatty acids contained in the oil account for at least 42% of the cell dry matter.

[0033] In some embodiments, the n-3 fatty acids in the oil account for at least 50% or more of the total fatty acids. In some embodiments, the n-3 fatty acids in the oil account for at least 55% or more of the total fatty acids.

[0034] In some embodiments, the oil content of the Schizochytrium sp. in the mixture is at least 1%-10%, the oil content in the biomass cell dry weight is at least 50% or more, and the n-3 fatty acids contained in the oil account for at least 45% or more of the cell dry matter.

[0035] In some embodiments, the oil comprises at least 9% or less EPA.

[0036] In some embodiments, the oil contains at least 10% or less EPA; and at least 35% or more DHA.

[0037] In some embodiments, the oil contains at least 10% or less EPA; and at least 5% or more n-3DPA.

[0038] In some embodiments, the oil contains at least 10% or less EPA, more than 40% DHA, and more than 5% n-3DPA.

[0039] In some embodiments, the ratio of DHA to EPA in the fatty acids is no less than 4:1.

[0040] In some embodiments, the ratio of DHA to n-3DPA in the fatty acids is not less than 8:1.

[0041] In some embodiments, the ratio of DHA to EPA in the fatty acids is not less than 4:1; and the ratio of DHA to n-3DPA in the fatty acids is not less than 6:1.

[0042] In a third aspect, the present invention provides a bacterial suspension.

[0043] A bacterial suspension comprising the Schizochytrium sp. described in the first aspect.

[0044] In a fourth aspect, the present invention provides a microbial agent.

[0045] A microbial agent, characterized in that the microbial agent comprises at least one of the Schizochytrium described in the first aspect, the fermentation broth described in the second aspect, and the bacterial suspension described in the third aspect.

[0046] In a fifth aspect, the present invention provides a method for preparing the fermentation broth described in the second aspect.

[0047] A method for preparing the fermentation broth according to the second aspect is characterized by comprising: subjecting the Schizochytrium sp. according to the first aspect to seed culture and fermentation culture.

[0048] In some embodiments, the seed culture comprises culturing the Schizochytrium HS08 in a seed culture medium to obtain a seed culture solution.

[0049] In some embodiments, the fermentation culture comprises fermenting the seed culture solution obtained by seed culture of the Schizochytrium HS08 in a fermentation medium.

[0050] In some embodiments, the seed culture medium comprises the following components: 50 g / L glucose, 10 g / L yeast extract, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, and 0.17 g / L anhydrous calcium chloride.

[0051] In some embodiments, the fermentation medium comprises the following components: 50 g / L glucose, 12 g / L yeast extract, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, 0.17 g / L anhydrous calcium chloride, and 10 g / L agar powder.

[0052] In some embodiments, the seed culture medium uses water as a solvent.

[0053] In some embodiments, the fermentation medium uses water as a solvent.

[0054] In some embodiments, the pH of the seed culture medium is not additionally adjusted (ie, the pH is natural).

[0055] In some embodiments, the pH of the fermentation medium is not additionally adjusted (ie, the pH is natural).

[0056] In some embodiments, the seed culture is cultured for 12 hours to 60 hours. In some embodiments, the seed culture is cultured for 12 hours, 15 hours, 20 hours, 24 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 55 hours, or 60 hours. In some embodiments, the seed culture is cultured for 48 hours.

[0057] In some embodiments, the culturing temperature of the seed culture is 20° C.-30° C. In some embodiments, the culturing temperature of the seed culture is 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° C.

[0058] In some embodiments, the rotation speed of the seed culture is 100 rpm-250 rpm. In some embodiments, the rotation speed of the seed culture is 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm or 250 rpm.

[0059] In some embodiments, the fermentation culture is cultured for 100 h to 300 h. In some embodiments, the fermentation culture is cultured for 100 h, 110 h, 120 h, 130 h, 140 h, 150 h, 200 h, 250 h, or 300 h.

[0060] In some embodiments, the fermentation culture is cultured at a temperature of 25-30° C. In some embodiments, the seed culture is cultured at a temperature of 25° C., 25.5° C., 26° C., 26.5° C., 27° C., 27.5° C., 28° C., 28.5° C., 29° C., 29.5° C., or 30° C.

[0061] In some embodiments, the fermentation culture is operated at a rotation speed of 150 rpm to 200 rpm. In some embodiments, the seed culture is operated at a rotation speed of 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, or 200 rpm.

[0062] In a sixth aspect, the present invention provides a method for preparing n-3 fatty acids.

[0063] A method for preparing n-3 fatty acids, comprising: extracting n-3 fatty acids from the fermentation broth described in the second aspect or the fermentation broth prepared by the preparation method described in the fifth aspect.

[0064] In some embodiments, the n-3 fatty acid comprises at least one of EPA, n-3 DPA, and DHA.

[0065] In a seventh aspect, the present invention provides an n-3 fatty acid.

[0066] An n-3 fatty acid prepared according to the preparation method of the sixth aspect.

[0067] In an eighth aspect, the present invention provides a method for preparing microbial oil containing n-3 fatty acids.

[0068] A method for preparing microbial oil containing n-3 fatty acids comprises: extracting microbial oil containing n-3 fatty acids from the fermentation broth described in the second aspect or the fermentation broth prepared by the preparation method described in the fifth aspect.

[0069] In some embodiments, the n-3 fatty acid-containing microbial oil comprises at least one of EPA, n-3 DPA, and DHA.

[0070] In a ninth aspect, the present invention provides a microbial oil containing n-3 fatty acids.

[0071] A microbial oil containing n-3 fatty acids prepared according to the preparation method of the eighth aspect.

[0072] In a tenth aspect, the present invention provides the aforementioned Schizochytrium fungus, fermentation broth, bacterial suspension, microbial agent, fermentation broth prepared by the aforementioned preparation method, n-3 fatty acids prepared by the aforementioned preparation method, n-3 fatty acids, microbial oil containing n-3 fatty acids prepared by the aforementioned preparation method, and applications of microbial oil containing n-3 fatty acids.

[0073] A use of the Schizochytrium fungus described in the first aspect, the fermentation broth described in the second aspect, the bacterial suspension described in the third aspect, the microbial agent described in the fourth aspect, the fermentation broth prepared by the preparation method described in the fifth aspect, the n-3 fatty acid prepared by the preparation method described in the sixth aspect, the n-3 fatty acid described in the seventh aspect, the microbial oil containing n-3 fatty acids prepared by the preparation method described in the eighth aspect, or the microbial oil containing n-3 fatty acids described in the ninth aspect in the preparation of food, cosmetics, health products, feed and / or medicine.

[0074] In the eleventh aspect, the present invention provides a mutagenesis culture medium for mutagenizing Schizochytrium sp., wherein the mutagenesis culture medium contains a mutagen, and the mutagen is selected from one or more of nitrosoguanidine, ethyl methanesulfonate, and diethyl sulfate; the concentration of the mutagen is 0.1%-5.0%.

[0075] In some embodiments, the mutagen is 0.5-2% ethyl methanesulfonate or 0.5-2% diethyl sulfate; preferably, the mutagen is 1% ethyl methanesulfonate or 1% diethyl sulfate.

[0076] In some embodiments, the mutagenesis medium further contains 40-60 g / L glucose, 9-15 g / L yeast extract, 15-20 g / L anhydrous sodium sulfate, 0.5-1 g / L potassium chloride, 3-5 g / L magnesium sulfate heptahydrate, 0.25-0.9 g / L potassium sulfate, 0.5-3.5 g / L potassium dihydrogen phosphate, 0.5-3.5 g / L ammonium sulfate and 0.1-2 g / L anhydrous calcium chloride, and the pH value is natural.

[0077] In some embodiments, the mutagenesis medium contains 50 g / L glucose, 10 g / L yeast extract powder, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, and 0.17 g / L anhydrous calcium chloride.

[0078] In the twelfth aspect, the present invention provides a screening culture medium for screening Schizochytrium sp., the screening culture medium contains a screening agent, and the screening agent is selected from one or more of quizalofop-p-ethyl, cerulenin, VB12, and diphenylamine; the concentration of cerulenin is 0.001~1mM, the concentration of quizalofop-p-ethyl is 0.001~1mM, the concentration of diphenylamine is 0.001~1mM, and the concentration of VB12 is 1~100ug / L.

[0079] In some embodiments, the concentration of quizalofop-ethyl is 0.01-0.08 mM; the concentration of cerulenin is 0.01-0.02 mM; and the concentration of VB12 is 0.01 ug / L.

[0080] In some embodiments, the screening culture medium further contains 40-60 g / L of glucose, 9-15 g / L of yeast extract powder, 15-20 g / L of anhydrous sodium sulfate, 0.5-1 g / L of potassium chloride, 3-5 g / L of magnesium sulfate heptahydrate, 0.25-0.9 g / L of potassium sulfate, 0.5-3.5 g / L of potassium dihydrogen phosphate, 0.5-3.5 g / L of ammonium sulfate, 0.1-2 g / L of anhydrous calcium chloride, and 5-20 g / L of agar powder, and the pH value is natural.

[0081] In some embodiments, the screening medium contains 50 g / L glucose, 12 g / L yeast extract powder, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, 0.17 g / L anhydrous calcium chloride, and 10 g / L agar powder.

[0082] In a thirteenth aspect, the present invention provides a method for screening Schizochytrium sp., comprising the steps of:

[0083] (1) inoculating Schizochytrium spp. into a seed culture medium and culturing for 24 h to obtain a Schizochytrium spp. seed solution;

[0084] (2) aspirating the seed liquid, centrifuging and discarding the supernatant, washing with a phosphate buffer solution of pH = 8.0 and resuspending to obtain a seed suspension; inoculating the seed suspension into the mutagenesis culture medium of the present invention and culturing to obtain a mutant Schizochytrium liquid;

[0085] (3) aspirating the mutant Schizochytrium liquid, diluting it with a phosphate buffer solution of pH=8.0 to a certain gradient, and then aspirating the diluted mutant Schizochytrium liquid to apply it on the screening medium of the present invention for cultivation;

[0086] (4) selecting a single colony with a fast growth rate and regular edges and smearing it on the screening medium of the present invention for cultivation;

[0087] (5) The colonies with fast growth rate and moderate size were selected and transferred to seed culture medium. After culturing for 24 hours, the seeds were preserved to obtain the high-yield Schizochytrium strain to be purified.

[0088] In (2), washing with a phosphate buffer at pH = 8.0 and resuspension were washing with a phosphate buffer at pH = 8.0 and resuspension with a phosphate buffer at pH = 8.0.

[0089] In some embodiments, the Schizochytrium sp. species is Schizochytrium sp. species HS01, which is deposited in the China General Microbiology Center (CGMCC) with a deposit number of CGMCC No. 13746.

[0090] In some embodiments, the seed culture medium includes 40-60 g / L glucose, 9-15 g / L yeast extract, 15-20 g / L anhydrous sodium sulfate, 0.5-1 g / L potassium chloride, 3-5 g / L magnesium sulfate heptahydrate, 0.25-0.9 g / L potassium sulfate, 0.5-3.5 g / L potassium dihydrogen phosphate, 0.5-3.5 g / L ammonium sulfate and 0.1-2 g / L anhydrous calcium chloride, and the pH value is natural.

[0091] In some embodiments, the seed culture medium contains 50 g / L glucose, 12 g / L yeast extract, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, and 0.17 g / L anhydrous calcium chloride.

[0092] In some embodiments, step (2) is culturing at 28° C. and 180 rpm for 1-6 hours.

[0093] In some embodiments, step (3) is to culture the cells upside down in a 37°C incubator for 48 hours.

[0094] In some embodiments, step (4) is to culture the cells upside down in a 37°C incubator for 48 hours.

[0095] In some embodiments, step (5) is to culture at 28°C and 180 rpm in a shaking incubator for 24 hours. Beneficial effects

[0096] Compared with the existing technology, the strain provided by the present invention has the following beneficial technical effects:

[0097] (1) The fermentation broth obtained by fermenting the strain provided by the present invention has significantly higher contents of EPA and n-3DPA in the total fatty acids of the fermentation broth, which has unexpected technical effects.

[0098] (2) The strain provided by the present invention can stably produce n-3 fatty acids after multiple subcultures, and has the advantages of good genetic stability and fast growth rate, and has good industrialization prospects.

[0099] Terminology

[0100] 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.

[0101] 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

[0102] 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.

[0103] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0104] The present invention uses the original polyunsaturated fatty acid-producing strain Schizochytrium HS01 as the starting strain (deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) with the deposit number CGMCC No. 13746). The DHA content of the total fatty acids in this strain can reach more than 40%, but the EPA content and n-3DPA content are only about 0.5%. The invention intends to obtain a new strain rich in EPA, n-3DPA and DHA through a mutagenesis screening method.

[0105] Culture medium:

[0106] Unless otherwise specified, the formula of the culture medium used in the Examples or Comparative Examples of the present invention is as follows:

[0107] Seed culture medium formula: glucose 50g / L, yeast extract powder 10g / L, anhydrous sodium sulfate 15g / L, potassium chloride 0.5g / L, magnesium sulfate heptahydrate 4.1g / L, potassium sulfate 0.65g / L, potassium dihydrogen phosphate 1.0g / L, ammonium sulfate 1.0g / L and anhydrous calcium chloride 0.17g / L, natural pH value, water as the solvent.

[0108] Fermentation medium formula: glucose 50g / L, yeast extract powder 12g / L, anhydrous sodium sulfate 15g / L, potassium chloride 0.5g / L, magnesium sulfate heptahydrate 4.1g / L, potassium sulfate 0.65g / L, potassium dihydrogen phosphate 1.0g / L, ammonium sulfate 1.0g / L and anhydrous calcium chloride 0.17g / L, natural pH value, water as the solvent.

[0109] Solid culture medium formula: glucose 50g / L, yeast extract powder 12g / L, anhydrous sodium sulfate 15g / L, potassium chloride 0.5g / L, magnesium sulfate heptahydrate 4.1g / L, potassium sulfate 0.65g / L, potassium dihydrogen phosphate 1.0g / L, ammonium sulfate 1.0g / L, anhydrous calcium chloride 0.17g / L and agar powder 10g / L, natural pH value, water as the solvent.

[0110] Test method:

[0111] The DHA content, DPA content, and EPA content were detected according to GB 5009.168-2016 and calculated using the fatty acid area normalization method.

[0112] Example 1: Schizochytrium screening

[0113] 1. Mutation of Schizochytrium

[0114] HS01 Schizochytrium (deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC), with the deposit number CGMCC No. 13746) seeds were inoculated into a seed culture medium at an inoculum size of 2% and cultured in a shaker at 28°C and 180 r / min for 24 h to obtain a seed solution; 10 mL of the seed solution was aspirated, centrifuged at 5000 r / min for 3 min, and the supernatant was discarded. The solution was then washed twice with a pH = 8.0 phosphate buffer and resuspended; the seed suspension was added to a liquid seed culture medium containing 1.0% ethyl methanesulfonate and cultured at 28°C and 180 r / min for 6 h.

[0115] 2. Screening of Schizochytrium

[0116] 4 mL of the mutant Schizochytrium solution was taken and diluted to 10% using phosphate buffer at pH 8.0. -2 , 10 -3 , 10 -4 , 10 -5Gradient, draw 10 -5 200uL of the gradient mutant Schizochytrium dilution solution was spread on solid culture media containing 0.02mM and 0.08mM quizalofop-ethyl, respectively, and cultured upside down in a 37°C incubator for 48h; single colonies of appropriate size and regular edges were picked and spread again on solid culture media containing 0.02mM and 0.08mM quizalofop-ethyl, and cultured in a 37°C incubator for 48h.

[0117] 3. Fermentation culture of Schizochytrium

[0118] Three strains with the fastest growth rates were selected and incubated in a seed culture medium at 28°C and 180 rpm for 48 hours to obtain a seed culture solution. This seed culture solution was then inoculated into a fermentation medium at a 4% inoculum and incubated for 120 hours at 28°C and 180 rpm to obtain a fermentation broth. The fermentation broth was then extracted and the content of EPA, n-3DPA, and DHA in the total fatty acids in the resulting broth was determined. The results are shown in Table 1.

[0119] Table 1: Contents of EPA, n-3DPA, and DHA in the total fatty acids of the fermentation broth

[0120] Example 2

[0121] 1. Mutation of Schizochytrium

[0122] 10 mL of the seed solution cultured in Example 1 was aspirated and centrifuged at 5000 rpm for 3 min. The supernatant was discarded and washed twice with pH 8.0 phosphate buffer and resuspended. The solution was then added to a fermentation medium containing 1.0% diethyl sulfate and cultured at 28°C and 180 rpm for 1 h.

[0123] 2. Screening of Schizochytrium

[0124] 4 mL of the mutant Schizochytrium solution was taken and diluted to 10% using phosphate buffer at pH 8.0. -2 , 10 -3 , 10 -4、 10 -5 Gradient, draw 10 -5 200uL of the gradient mutation Schizochytrium bacterial solution was spread on a solid culture medium containing 0.01mM cerulenin and cultured upside down in a 37°C incubator for 48h; a single colony of appropriate size and regular edges was picked and spread again on a solid culture medium containing 0.01mM cerulenin and cultured in a 37°C incubator for 48h.

[0125] 3. Fermentation culture of Schizochytrium

[0126] Three strains with relatively fast growth rates were selected and cultured according to the fermentation method described in "3. Fermentation and Culture of Schizochytrium" in Example 1 to obtain a fermentation broth. The fermentation broth was then used to extract oil and fat using the "Hydrolysis-Extraction Method" in accordance with GB 5009.168-2016. The content of EPA, n-3DPA, and DHA in the total fatty acids in the resulting fermentation broth was determined. The results are shown in Table 2.

[0127] Table 2: Contents of EPA, n-3DPA, and DHA in the total fatty acids of the fermentation broth

[0128] Example 3

[0129] The No. 3-1 seeds in Example 2 were inoculated into the seed culture medium at an inoculum rate of 2%, and cultured in a shaking incubator at 28° C. and 180 rpm for 24 hours to obtain a seed solution.

[0130] 1. Screening of Schizochytrium

[0131] Take 4 mL of the mutagenic bacterial solution and dilute it to 10% with phosphate buffer at pH 8.0. -2 , 10 -3 , 10 -4 , 10 -5 Gradient, draw 10 -5 200uL of the gradient mutation Schizochytrium liquid dilution was spread on a solid culture medium containing 0.01mM cerulenin and 0.08mM quizalofop-ethyl, and cultured upside down in a 37°C incubator for 48h. Single colonies of appropriate size and regular edges were picked and spread again on a solid culture medium containing 0.01mM cerulenin and 0.08mM quizalofop-ethyl, and cultured in a 37°C incubator for 48h.

[0132] 2. Fermentation culture of Schizochytrium

[0133] Three strains with relatively rapid growth were selected and cultured according to the fermentation method described in "3. Fermentation and Culture of Schizochytrium" in Example 1. The fermentation broth was then extracted for oil and fat according to the "Hydrolysis-Extraction Method" in GB 5009.168-2016. The content of EPA, n-3DPA, and DHA in the total fatty acids in the resulting broth was determined. The results are shown in Table 3.

[0134] Table 3: Contents of EPA, n-3DPA, and DHA in the total fatty acids of the fermentation broth

[0135] Example 4

[0136] The No. 3-1 seeds in Example 2 were inoculated into the seed culture medium at an inoculum rate of 2%, and cultured in a shaking incubator at 28° C. and 180 rpm for 24 hours to obtain a seed solution.

[0137] 1. Screening of Schizochytrium

[0138] Take 4 mL of the mutagenic bacterial solution and dilute it to 10% with phosphate buffer at pH 8.0. -2 , 10 -3 , 10 -4 , 10 -5 Gradient, draw 10 -5 200uL of the gradient mutation Schizochytrium liquid dilution was spread on a solid culture medium containing 0.01mM cerulenin and 0.01ug / L VB12 (vitamin B12), and cultured in a 37°C incubator for 48h. Single colonies of appropriate size and regular edges were picked and spread again on a solid culture medium containing 0.01mM cerulenin and 0.01ug / L VB12, and cultured in a 37°C incubator for 48h.

[0139] 2. Fermentation culture of Schizochytrium

[0140] Three strains with relatively fast growth rates were selected and cultured according to the fermentation method described in "3. Fermentation and Culture of Schizochytrium sp." in Example 1. (During the seed preservation step after seed culture, strain 5-1 was named Schizochytrium sp. HS08 and is currently deposited with the China General Microbiological Culture Collection Center (CGMCC) under the deposit number CGMCC No. 40902.) Fermentation broth was obtained. The fermentation broth was then extracted for oil and fat according to the "Hydrolysis-Extraction Method" in GB 5009.168-2016. The contents of EPA, n-3DPA, and DHA in the total fatty acids in the resulting fermentation broth were determined. The results are shown in Table 4.

[0141] Table 4: Contents of EPA, n-3DPA, and DHA in the total fatty acids of the fermentation broth

[0142] As shown in Tables 1 and 2, the strains obtained after mutagenesis screening according to the present invention successfully increased the EPA and n-3DPA content in the total fatty acids of Schizochytrium sp. In Example 4, the strains obtained from the mutagenesis screening were further subjected to cerulenin combined with VB12 targeted screening. The fermentation broth of strain 5-1 obtained by targeted screening increased the EPA content of the total fatty acids in the fermentation broth to 9.85%, and the n-3DPA content of the total fatty acids to 5.81% (see Table 4), demonstrating excellent and unexpected technical results. After multiple subcultures, strain 5-1 was found to be capable of stably producing each n-3 fatty acid. Strain 5-1 was designated Schizochytrium sp. HS08 and is currently deposited with the General Microbiology Center of the China General Culture Collection (CGMCC) under the deposit number CGMCC No. 40902. This strain exhibits excellent genetic stability, rapid growth, and promising industrialization prospects.

[0143] 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 strain of Schizochytrium sp., named Schizochytrium sp. HS08; deposit information: deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number of CGMCC No.40902.

2. A fermentation broth, characterized in that: The method is prepared by fermenting the Schizochytrium sp. according to claim 1.

3. The fermentation broth according to claim 2, characterized in that The content of EPA in the fermentation broth is 9% or more of the total fatty acids in the fermentation broth; and / or the content of n-3DPA in the fermentation broth is 5% or more of the total fatty acids in the fermentation broth, and / or the content of DHA in the fermentation broth is 35% or more of the total fatty acids in the fermentation broth.

4. The fermentation broth according to claim 2, characterized in that The content of EPA in the fermentation broth is 9-10% of the total fatty acids in the fermentation broth; and / or the content of n-3DPA in the fermentation broth is 5-7% of the total fatty acids in the fermentation broth, and / or the content of DHA in the fermentation broth is 35-45% of the total fatty acids in the fermentation broth.

5. A bacterial suspension, characterized in that: The bacterial suspension comprises the Schizochytrium sp. according to claim 1.

6. A microbial agent, characterized in that: The microbial agent comprises at least one of the Schizochytrium sp. described in claim 1, the fermentation broth described in any one of claims 2 to 4, and the bacterial suspension described in claim 5.

7. A method for preparing the fermentation broth according to any one of claims 2 to 4, characterized in that: include: The Schizochytrium sp. according to claim 1 is subjected to seed culture and fermentation culture.

8. The preparation method according to claim 7, wherein the seed culture comprises culturing the Schizochytrium HS08 in a seed culture medium to obtain a seed culture solution; and / or The fermentation culture comprises fermenting the seed culture liquid obtained after seed culture of the Schizochytrium HS08 in a fermentation medium; Optionally, the seed culture medium comprises the following components: 50 g / L glucose, 10 g / L yeast extract, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate and 0.17 g / L anhydrous calcium chloride; Optionally, the fermentation medium comprises the following components: 50 g / L glucose, 12 g / L yeast extract, 15 g / L anhydrous sodium sulfate, 0.5 g / L potassium chloride, 4.1 g / L magnesium sulfate heptahydrate, 0.65 g / L potassium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, 0.17 g / L anhydrous calcium chloride and 10 g / L agar powder; Optionally, the seed culture is carried out for a culture time of 12 h to 60 h or 48 h; Optionally, the fermentation culture has a culture time of 100 h to 300 h or 120 h.

9. A method for preparing n-3 fatty acids, characterized in that: include: Extracting n-3 fatty acids from the fermentation broth according to claims 2-4 or the fermentation broth prepared by the preparation method according to any one of claims 7-8; Optionally, the n-3 fatty acid comprises at least one of EPA, n-3 DPA and DHA.

10. An n-3 fatty acid prepared according to the preparation method of claim 9.

11. A method for preparing a microbial oil containing n-3 fatty acids, characterized in that: include: Extracting microbial oil containing n-3 fatty acids from the fermentation broth according to claims 2-4 or the fermentation broth prepared by the preparation method according to any one of claims 7-8; Optionally, the n-3 fatty acid-containing microbial oil comprises at least one of EPA, n-3DPA and DHA.

12. A microbial oil containing n-3 fatty acids prepared according to the preparation method of claim 11.

13. Use of the Schizochytrium fungus according to claim 1, the fermentation broth according to any one of claims 2 to 4, the bacterial suspension according to claim 5, the microbial agent according to claim 6, the fermentation broth prepared by the preparation method according to any one of claims 7 to 8, the n-3 fatty acids prepared by the preparation method according to claim 9, the n-3 fatty acids according to claim 10, the microbial oil containing n-3 fatty acids prepared by the preparation method according to claim 11, or the microbial oil containing n-3 fatty acids according to claim 12 in the preparation of food, cosmetics, health products, feed and / or medicine.

14. A mutagenesis medium for mutagenizing Schizochytrium sp., characterized in that: The mutagenesis culture medium contains a mutagen, which is selected from one or more of nitrosoguanidine, ethyl methanesulfonate, and diethyl sulfate; the concentration of the mutagen is 0.1%-5.0%.

15. The mutagenesis medium according to claim 14, characterized in that The mutagen is 0.5-2% ethyl methanesulfonate or 0.5-2% diethyl sulfate; preferably, the mutagen is 1% ethyl methanesulfonate or 1% diethyl sulfate.

16. The mutagenesis medium according to claim 14, characterized in that The mutagenesis medium also contains 40-60 g / L of glucose, 9-15 g / L of yeast extract, 15-20 g / L of anhydrous sodium sulfate, 0.5-1 g / L of potassium chloride, 3-5 g / L of magnesium sulfate heptahydrate, 0.25-0.9 g / L of potassium sulfate, 0.5-3.5 g / L of potassium dihydrogen phosphate, 0.5-3.5 g / L of ammonium sulfate and 0.1-2 g / L of anhydrous calcium chloride, and the pH value is natural; preferably, in the mutagenesis medium, glucose is 50 g / L, yeast extract is 10 g / L, anhydrous sodium sulfate is 15 g / L, potassium chloride is 0.5 g / L, magnesium sulfate heptahydrate is 4.1 g / L, potassium sulfate is 0.65 g / L, potassium dihydrogen phosphate is 1.0 g / L, ammonium sulfate is 1.0 g / L, and anhydrous calcium chloride is 0.17 g / L.

17. A screening medium for screening Schizochytrium sp., characterized in that: The screening culture medium contains a screening agent, which is selected from one or more of quizalofop-p-ethyl, cerulenin, VB12, and diphenylamine; the concentration of cerulenin is 0.001-1mM, the concentration of quizalofop-p-ethyl is 0.001-1mM, the concentration of diphenylamine is 0.001-1mM, and the concentration of VB12 is 1-100ug / L.

18. The screening medium according to claim 17, characterized in that The concentration of quizalofop-p-ethyl is 0.01-0.08 mM; the concentration of cerulenin is 0.01-0.02 mM; and the concentration of VB12 is 0.01 ug / L.

19. The screening medium according to claim 17 or 18, characterized in that The screening medium also contains 40-60 g / L of glucose, 9-15 g / L of yeast extract, 15-20 g / L of anhydrous sodium sulfate, 0.5-1 g / L of potassium chloride, 3-5 g / L of magnesium sulfate heptahydrate, 0.25-0.9 g / L of potassium sulfate, 0.5-3.5 g / L of potassium dihydrogen phosphate, 0.5-3.5 g / L of ammonium sulfate, 0.1-2 g / L of anhydrous calcium chloride, and 5-20 g / L of agar powder, and the pH value is natural; preferably, in the screening medium, glucose 50 g / L, yeast extract 12 g / L, anhydrous sodium sulfate 15 g / L, potassium chloride 0.5 g / L, magnesium sulfate heptahydrate 4.1 g / L, potassium sulfate 0.65 g / L, potassium dihydrogen phosphate 1.0 g / L, ammonium sulfate 1.0 g / L, anhydrous calcium chloride 0.17 g / L, and agar powder 10 g / L.

20. A method for screening Schizochytrium sp., characterized in that: Includes steps: (1) inoculating Schizochytrium spp. into a seed culture medium and culturing for 24 hours to obtain a Schizochytrium spp. seed solution; (2) aspirating the seed liquid, centrifuging and discarding the supernatant, washing with a phosphate buffer having a pH value of 8.0 and resuspending to obtain a seed suspension; inoculating the seed suspension into the mutagenesis medium according to any one of claims 14 to 16, and culturing to obtain a mutant Schizochytrium liquid; (3) taking the mutant Schizochytrium liquid, diluting it with a phosphate buffer of pH=8.0 to a certain gradient, and then taking the diluted mutant Schizochytrium liquid and applying it on the screening medium according to any one of claims 17 to 19 for culturing; (4) selecting a single colony with a fast growth rate and regular edges and applying it to the screening medium according to any one of claims 17 to 19 for cultivation; (5) Selecting a colony with a fast growth rate and a moderate size and transferring it to a seed culture medium, culturing it for 24 hours and preserving the seed, thereby obtaining a high-yield Schizochytrium strain to be purified; Optionally, the seed culture medium comprises 40-60 g / L of glucose, 9-15 g / L of yeast extract, 15-20 g / L of anhydrous sodium sulfate, 0.5-1 g / L of potassium chloride, 3-5 g / L of magnesium sulfate heptahydrate, 0.25-0.9 g / L of potassium sulfate, 0.5-3.5 g / L of potassium dihydrogen phosphate, 0.5-3.5 g / L of ammonium sulfate and 0.1-2 g / L of anhydrous calcium chloride, and the pH value is natural; preferably, the seed culture medium comprises 50 g / L of glucose, 12 g / L of yeast extract, 15 g / L of anhydrous sodium sulfate, 0.5 g / L of potassium chloride, 4.1 g / L of magnesium sulfate heptahydrate, 0.65 g / L of potassium sulfate, 1.0 g / L of potassium dihydrogen phosphate, 1.0 g / L of ammonium sulfate and 0.17 g / L of anhydrous calcium chloride; Optionally; step (2) is cultured at 28°C, 180r / min for 1-6h; and / or Step (3) is to incubate the mixture upside down in a 37°C incubator for 48 hours; and / or Step (4) is to incubate the mixture in an inverted position at 37°C for 48 hours; and / or Step (5) is to culture in a shaking incubator at 28°C and 180 rpm for 24 h.