Fermentation broth rich in hexadecenoic acid and astaxanthin, and fermentation method and application thereof

Abstract

The present application belongs to the field of microorganisms and microbial fermentation technology, and particularly relates to a fermentation broth rich in hexadecenoic acid and astaxanthin as well as a fermentation method and application thereof. The three-in-one combined regulation strategy of'stepwise temperature variation + salt concentration stress + key precursor (various salt combinations / amino acids) feeding' can simultaneously increase the yield of hexadecenoic acid and astaxanthin in the fermentation process by gradually increasing the fermentation temperature, synchronously adding a second salt combination, a third salt combination or a fourth salt combination different from the fermentation medium, and through osmotic pressure stress, salt concentration stress and medium optimization (such as adding amino acids, organic acids and other adjustment factors).

Description

Technical Field

[0001] This invention relates to the field of microbial and microbial fermentation technology, specifically to a fermentation broth rich in hexadecenoic acid and astaxanthin, its fermentation method, and its application. Background Technology

[0002] Palmitoleic acid (C16:1, ω-7, POA, hexadecenoic acid) is a monounsaturated fatty acid with important physiological activities. In 2008, it was first revealed that POA can act as a lipid hormone to stimulate insulin response in muscles and inhibit hepatic steatosis. Subsequently, numerous studies have further confirmed the positive effects of POA in improving skin condition, enhancing insulin sensitivity, reducing hepatic lipid accumulation and inflammation, and alleviating coronary heart disease and hypertension. Therefore, POA is considered a highly promising nutritional pharmaceutical and functional food ingredient. Currently, POA is commonly found in vegetable and animal oils, with major sources including marine fish (anchovies) and macadamia nuts (POA content 15%–22%), and sea buckthorn fruit oil (POA content 30%–35%).

[0003] Marine resources are affected by seasonal limitations, nuclear pollution, heavy metal pollution, and resource depletion, while plant-based sources face challenges such as low yields and high prices. For example, commercially available products made from anchovy oil have a POA purity of approximately 50%, with a minimum production cost as high as $3,000–5,000 per kilogram. Given the limitations of natural sources, the synthesis of POA using microbial fermentation has become a research hotspot. Researchers have heterologously expressed relevant genes in non-lipid microorganisms such as *E. coli* and *Saccharomyces cerevisiae*, achieving a POA content of 45%–55% in total fatty acids, but the overall yield is extremely low. For example, the yield in *E. coli* is only 37.7 mg / L, and the yield in *Saccharomyces cerevisiae* after 7 days of shake-flask fermentation is only about 3 g / L, far from meeting industrial needs. Although some lipid microalgae (such as *Eustigmatos*) can accumulate a higher proportion of POA, the cultivation period is long and the biomass is only 4–8 g / L, resulting in low overall productivity. The recently reported oily yeast *Scheffersomyces segobiensis* DSM 27193 possesses a naturally high POA accumulation capacity (POA > 10%), exhibits rapid cell growth and high density, and its genome sequence and lipid metabolism pathways have been elucidated, demonstrating its potential as a chassis cell for POA production. However, regardless of the microorganism used, the fermentation of single POA products still faces bottlenecks.

[0004] Astaxanthin is a natural antioxidant, chemically named 3,3'-dihydroxy-β,β'-carotene-4,4'-dione. It possesses many important biological functions, including strong antioxidant, anti-aging, antiviral, and anti-staining properties, and has broad application prospects in the pharmaceutical, chemical, food, and aquaculture industries. *Schizochytrium* is a mature strain for industrial DHA production, characterized by high lipid content, large fermentation biomass, and the presence of an astaxanthin synthesis pathway.

[0005] Therefore, it is of great significance to develop a fermentation broth rich in hexadecenoic acid and astaxanthin, as well as its fermentation method and application. Summary of the Invention

[0006] To achieve the above-mentioned technical objectives, this invention provides a fermentation broth rich in hexadecenoic acid and astaxanthin, along with its fermentation method and applications. The fermentation method provided by this invention, on the one hand, can utilize the strong antioxidant properties of carotenoids to protect oils and improve their oxidative stability; on the other hand, it can significantly reduce production costs and improve the comprehensive utilization rate of raw materials, providing a feasible foundation for the industrial production of poly(acetic acid) (POA).

[0007] The *Schizochytrium* used in this invention is named *Schizochytrium HS08*; its preservation information is: deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 40902. It has been disclosed in patent CN117701403A.

[0008] In a first aspect, the present invention provides a fermentation broth obtained by fermenting Schizochytrium.

[0009] In some embodiments, the *Schizochytridica* is *Schizochytridica HS08*.

[0010] In some embodiments, the fermentation broth includes one or more of the following: Schizochytrium, microbial algal oil, sugar, protein, amino acids, and inorganic salts.

[0011] In some embodiments, the mass percentage of microbial algal oil in the fermentation broth dry matter is 20%-45%, and can be any one of the following ranges: 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, and any two of the above values.

[0012] In some embodiments, the microbial algal oil comprises one or more of total fatty acids, astaxanthin, phospholipids, fat-soluble substances, and sterols; the total fatty acids include hexadecenoic acid.

[0013] In some embodiments, the content of hexadecenoic acid in the fermentation broth accounts for more than 2% by mass of the microbial algal oil. In some embodiments, the content of hexadecenoic acid in the fermentation broth accounts for 2%-11% by mass of the microbial algal oil. In some embodiments, the content of hexadecenoic acid in the fermentation broth accounts for 2%, 2.1%, 2.2%, 3%, 4%, 5%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.2%, 6.3%, 6.4%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, or 11%, or any two of the above values ​​forming a range.

[0014] In some embodiments, the astaxanthin content in the fermentation broth is at least 50 mg / kg microbial algal oil, at least 100 mg / kg microbial algal oil, at least 200 mg / kg microbial algal oil, or at least 300 mg / kg microbial algal oil. In some embodiments, the astaxanthin content in the fermentation broth is 50-400 mg / kg of microbial algal oil, preferably 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg, 150 mg / kg, 160 mg / kg, 170 mg / kg, 180 mg / kg, 190 mg / kg, 200 mg / kg, 210 mg / kg, 220 mg / kg, 230 mg / kg, 240 mg / kg, 250 mg / kg, 260 mg / kg, 270 mg / kg, 280 mg / kg, 290 mg / kg, 300 mg / kg, 310 mg / kg, 320 mg / kg, 330 mg / kg, 340 mg / kg, 350 mg / kg, 360 mg / kg, etc. mg / kg, 370 mg / kg, 380 mg / kg, 390 mg / kg, 400 mg / kg, and any two of the above values ​​constitute any one of the ranges.

[0015] In a second aspect, the present invention provides a microbial inoculant, the microbial inoculant comprising the fermentation broth described in the first aspect.

[0016] Thirdly, the present invention provides a method for preparing the fermentation broth described in the first aspect, comprising: seed culture and fermentation culture of *Schizochytrium*.

[0017] In some embodiments, the seed culture includes seed culture of *Schizochytrium HS08* in an expansion culture medium to obtain a seed culture solution.

[0018] In some embodiments, the fermentation culture includes transferring the obtained seed culture medium to a first fermenter containing fermentation medium for cultivation, and then transferring it to a second fermenter containing fermentation medium for fermentation to obtain a fermentation broth.

[0019] In some embodiments, the fermentation culture includes transferring the obtained seed culture medium to a first fermenter containing fermentation medium for 15-36 hours of culture, and then transferring it to a second fermenter containing fermentation medium for 96-120 hours of fermentation to obtain a fermentation broth.

[0020] In some embodiments, a pH adjuster is used to adjust the pH during fermentation.

[0021] In some embodiments, the pH adjuster is ammonia and citric acid.

[0022] In some embodiments, the pH adjuster is ammonia and citric acid with a mass concentration of 50%.

[0023] In some embodiments, the expansion culture medium comprises the following components: solute composition: carbon source 60-100 g / L, yeast extract 10-25 g / L, sodium chloride 20-30 g / L, sodium sulfate 5-20 g / L, potassium sulfate 1-3 g / L, ammonium sulfate 1-2 g / L, magnesium sulfate 0.1-0.5 g / L, potassium dihydrogen phosphate 1-10 g / L, and dipotassium hydrogen phosphate 1-5 g / L.

[0024] In some embodiments, the expansion culture medium comprises the following components: glucose 60-100 g / L, yeast extract 15-25 g / L, sodium chloride 20-30 g / L, sodium sulfate 10-20 g / L, potassium sulfate 1-3 g / L, ammonium sulfate 1-2 g / L, magnesium sulfate 0.1-0.5 g / L, potassium dihydrogen phosphate 5-10 g / L, and dipotassium hydrogen phosphate 1-2 g / L.

[0025] In some embodiments, the expansion culture medium comprises the following components: 80-85 g / L glucose, 20 g / L yeast extract, 25 g / L sodium chloride, 15 g / L sodium sulfate, 2 g / L potassium sulfate, 1.5 g / L ammonium sulfate, 0.25 g / L magnesium sulfate, 8 g / L potassium dihydrogen phosphate and 1.5 g / L dipotassium hydrogen phosphate, with water as the solvent and a pH of 5.0-6.0.

[0026] In some embodiments, the fermentation medium comprises the following components: 5-30 g / L nitrogen source, 60-150 g / L glucose, and 10-60 g / L first salt combination.

[0027] In some embodiments, the fermentation medium comprises the following components: nitrogen source 5-30 g / L, glucose 60-150 g / L, first salt combination 10-60 g / L and second salt combination 0.5-7 g / L.

[0028] Preferably, the nitrogen source can be one or more of the following: soybean protein, whey protein, corn steep liquor, peptone, yeast extract, yeast extract, yeast flakes, betaine, creatine, amino acids (high arginine, arginine, branched-chain amino acids, cysteine, threonine, glycine, valine, ornithine, glutamic acid, histidine), and ammonium sulfate. In some embodiments, the nitrogen source is added to the base material all at once; in some embodiments, the nitrogen source is added in a continuous stream to the base material. Preferably, in the fermentation medium, the nitrogen source is one or more of the following: yeast extract 1-10 g / L, betaine 1-2 g / L, arginine 0.5-1 g / L, histidine 0.1-0.5 g / L, and ammonium sulfate 1-5.0 g / L. Preferably, in the fermentation medium, the nitrogen source is one or more of the following: yeast extract 5-7 g / L, betaine 1-2 g / L, arginine 0.5 g / L, histidine 0.1 g / L, and ammonium sulfate 4.0 g / L.

[0029] Preferably, the first salt combination can be one or more of sodium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, ammonium chloride, magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium pyrophosphate, calcium phosphate, and calcium sulfate. More preferably, the first salt combination can be one or more of sodium sulfate, ammonium sulfate, ammonium chloride, magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, calcium carbonate, and calcium phosphate. In some embodiments, the first salt combination is added to the base material in a single step. In some embodiments, the first salt combination is added as a supplementary addition to the base material.

[0030] Preferably, in the fermentation medium, the first salt combination can be at least one of the following: anhydrous sodium sulfate 5-20 g / L, magnesium sulfate 3-5 g / L, potassium dihydrogen phosphate 5-15 g / L, dipotassium hydrogen phosphate 1-5 g / L, ammonium sulfate 2-5 g / L, calcium chloride 0.1-0.5 g / L, and calcium phosphate 0.01-0.05 g / L; preferably, the first salt combination can be at least one of the following: anhydrous sodium sulfate 10-12 g / L, magnesium sulfate 4 g / L, potassium dihydrogen phosphate 5-10 g / L, dipotassium hydrogen phosphate 1 g / L, ammonium sulfate 4.0 g / L, calcium chloride 0.1 g / L, and calcium phosphate 0.01 g / L.

[0031] Preferably, the second salt combination can be one or more of ferric citrate, ferric ammonium citrate, sodium molybdate, cobalt chloride, cobalt sulfate, manganese chloride, zinc sulfate, ferric sodium EDTA, ferrous sulfate, and ferrous chloride. In some embodiments, the second salt combination is added to the base material in a single step. In some embodiments, the second salt combination is added in a continuous feed after being added to the base material.

[0032] Preferably, the second salt combination is selected from at least one of the following: ferric citrate 0.01-0.05 g / L, ferric ammonium citrate 0.002-0.008 g / L, sodium molybdate 0.021-0.035 g / L, cobalt chloride 0.0014-0.005 g, zinc sulfate 0.0001-0.0002 g / L, ferric sodium ethylenediaminetetraacetate 0.45-5 g / L, ferrous sulfate 0.04-1.42 g / L, and ferrous chloride 0.10-0.13 g / L. Preferably, the second salt combination is selected from at least one of the following: ferric citrate 0.023 g / L, ferric ammonium citrate 0.004 g / L, sodium molybdate 0.025 g / L, cobalt chloride 0.0014 g / L, zinc sulfate 0.0001 g / L, ferric sodium ethylenediaminetetraacetate 0.45 g / L, ferrous sulfate 0.04 g / L, and ferrous chloride 0.1~0.11 g / L.

[0033] In some embodiments, the expansion culture medium uses water as a solvent.

[0034] In some embodiments, the fermentation medium uses water as a solvent.

[0035] In some embodiments, the pH of the expansion medium is not adjusted additionally (i.e., the pH is natural).

[0036] In some embodiments, the pH of the fermentation medium is not adjusted additionally (i.e., the pH is natural).

[0037] In some embodiments, the seed culture time is 24h-144h. In some embodiments, the seed culture time is any one of the following ranges: 12h, 15h, 20h, 24h, 25h, 30h, 35h, 40h, 45h, 46h, 47h, 48h, 49h, 50h, 55h, 60h, 65h, 70h, 75h, 80h, 85h, 90h, 95h, 100h, 105h, 110h, 115h, 120h, 125h, 130h, 135h, 140h, or 144h. In some embodiments, the seed culture time is 48h.

[0038] In some embodiments, the seed culture temperature is 20℃-30℃. In some embodiments, the seed culture temperature is 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, or 30℃.

[0039] In some embodiments, the rotation speed of the seed culture is 100 rpm to 250 rpm. In some embodiments, the rotation speed of the seed culture is any one of the following: 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, or any two of the above values ​​forming a range.

[0040] In some embodiments, the seed culture includes primary culture, secondary culture and tertiary culture.

[0041] In some embodiments, the primary culture involves inoculating the activation solution into a primary shake flask at an inoculum of 10-20% and culturing it at 23°C and 200 r / min for 24-48 h to obtain the primary seed culture.

[0042] In some embodiments, the secondary culture involves inoculating the primary seed culture at 10-20% into a secondary shake flask and culturing it at 23°C and 200 r / min for 24-48 h to obtain the secondary seed culture.

[0043] In some embodiments, the tertiary culture involves inoculating the secondary seed culture at 4-5% into a tertiary seed tank and culturing it at 22-30°C and 150 r / min for 24-48 h to obtain the seed culture solution.

[0044] In some embodiments, the fermentation culture time is 90-150 h. In some embodiments, the fermentation culture time is 90 h, 95 h, 96 h, 100 h, 110 h, 120 h, 130 h, 140 h, or 150 h, or any two of the above values ​​forming a range.

[0045] In some embodiments, the fermentation culture temperature is 22-30℃. In some embodiments, the fermentation culture temperature is 22℃, 23℃, 24℃, 25℃, 25.5℃, 26℃, 26.5℃, 27℃, 27.5℃, 28℃, 28.5℃, 29℃, 29.5℃, or 30℃.

[0046] In some embodiments, the fermentation rotation speed is 50 rpm - 200 rpm. In some embodiments, the fermentation rotation speed is 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, or 200 rpm, or any two of the above values ​​forming a range.

[0047] In some embodiments, the pH of the fermentation culture can be 3.0-6.0, preferably any one of the ranges of 3, 3.5, 4, 4.5, 5, 5.5, 6, or any two of the above values. In some embodiments, the pH is adjusted using ammonia and citric acid during the fermentation culture. In some embodiments, the pH is adjusted using ammonia and 50% (w / w) citric acid during the fermentation culture.

[0048] In some embodiments, the dissolved oxygen in the fermentation culture can be 0-100%, preferably any one of the ranges of 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and any two of the above values.

[0049] In some embodiments, during the fermentation culture, the inoculation amount of the seed culture medium is 4-40%, preferably 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or any two of the above values ​​forming a range.

[0050] In some embodiments, the fermentation culture involves transferring the seed culture medium to a fermenter at an inoculum of 4-40%, with a fermentation temperature of 22-28°C and a fermentation pH of 3.0-6.0. During fermentation, a pH adjuster is used to adjust the pH, and the fermentation time is 96-120 h to obtain the fermentation product.

[0051] In some embodiments, the fermentation culture involves transferring the seed culture solution at an inoculum of 4-40% to a first fermenter containing fermentation medium for cultivation, followed by inoculation into a second fermenter containing fermentation medium for fermentation.

[0052] In some embodiments, the fermentation culture involves transferring the seed culture solution at an inoculum of 4-40% to a first fermenter containing fermentation medium and culturing it for 15-36 hours, followed by inoculation into a second fermenter containing fermentation medium for fermentation.

[0053] In some embodiments, the fermentation culture involves transferring the seed culture medium at an inoculum of 4-40% to a first fermenter containing fermentation medium for cultivation, followed by inoculation into a second fermenter containing fermentation medium for fermentation for 96-120 h.

[0054] In some embodiments, the fermentation culture involves transferring the seed culture solution at an inoculum of 4-40% to a first fermenter containing fermentation medium and culturing it for 15-36 hours, followed by inoculation into a second fermenter containing fermentation medium and fermenting for 96-120 hours.

[0055] In some embodiments, during the fermentation culture, the sugar concentration is controlled at 70-100 g / L, preferably 70 g / L, 80 g / L, 90 g / L, or 100 g / L.

[0056] In some embodiments, during the fermentation culture, at least one of a second salt combination, a third salt combination, or a fourth salt combination is added to the second fermenter.

[0057] In some embodiments, during the fermentation culture, after 45 hours of fermentation, at least one of a second salt combination, a third salt combination, or a fourth salt combination is added to the second fermenter.

[0058] In some embodiments, during the fermentation culture, after 72 hours of fermentation, at least one of a second salt combination, a third salt combination, or a fourth salt combination is added to the second fermenter.

[0059] In some embodiments, during the fermentation culture, at least one of a second salt combination, a third salt combination, or a fourth salt combination is added to the second fermenter at 45 h and 72 h of fermentation, respectively.

[0060] Preferably, the third salt combination can be one or more of sodium chloride, sodium sulfate, potassium sulfate, ammonium sulfate, ammonium chloride, magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, sodium pyrophosphate, calcium phosphate, and calcium sulfate. More preferably, the third salt combination can be one or more of sodium sulfate, ammonium sulfate, ammonium chloride, magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, calcium carbonate, and calcium phosphate. In some embodiments, the third salt combination is added to the base material in a single step. In some embodiments, the third salt combination is added as a supplementary addition to the base material.

[0061] Preferably, the third salt combination can be at least one of the following: magnesium sulfate 1-5 g / L, potassium dihydrogen phosphate 10-15 g / L, dipotassium hydrogen phosphate 1-3 g / L, ammonium sulfate 1-4 g / L, calcium chloride 0.1-0.5 g / L, and calcium phosphate 0.01-0.05 g / L; preferably, the third salt combination can be at least one of the following: magnesium sulfate 4-5 g / L, potassium dihydrogen phosphate 10 g / L, dipotassium hydrogen phosphate 1 g / L, ammonium sulfate 4.0 g / L, calcium chloride 0.1 g / L, and calcium phosphate 0.01 g / L.

[0062] Preferably, the fourth salt combination can be one or more of sodium acetate, sodium phenylpropionate, ammonium acetate, sodium pyruvate, sodium citrate, sodium acetate, sodium succinate, sodium malate, sodium lactate, sodium oxalate, and potassium sodium tartrate. In some embodiments, the fourth salt combination is added to the base material in a single step. In some embodiments, the fourth salt combination is added as a supplementary addition to the base material.

[0063] Preferably, the fourth salt combination can be at least one of the following: sodium pyruvate 0.1-5 g / L, sodium citrate 1-10 g / L, sodium acetate 1-1.5 g / L, sodium succinate 1-1.5 g / L, sodium malate 1-6.5 g / L, sodium lactate 0.1-0.9 g / L, sodium oxalate 0.1-0.5 g / L, and potassium sodium tartrate 0.1-0.6 g / L.

[0064] More preferably, in the fermentation medium, the fourth salt combination is selected from at least one of: sodium pyruvate 0.5-1 g / L, sodium citrate 2.0-4.0 g / L, and sodium malate 2-4 g / L.

[0065] In some embodiments, during the fermentation culture, the second salt combination is supplemented with at least one selected from the following: ferric citrate 0.01-0.05 g / L, ferric ammonium citrate 0.002-0.008 g / L, sodium molybdate 0.021-0.035 g / L, zinc sulfate 0.0001-0.0002 g / L, ferric sodium ethylenediaminetetraacetate 0.45-5 g / L, ferrous sulfate 0.04-1.42 g / L, and ferrous chloride 0.1-0.13 g / L.

[0066] In some embodiments, during the fermentation culture, the second salt combination is supplemented with at least one selected from ferric citrate 0.023 g / L, ferric ammonium citrate 0.004 g / L, sodium molybdate 0.025 g / L, zinc sulfate 0.0001 g / L, ferric sodium EDTA 0.45 g / L, ferrous sulfate 0.04 g / L, and ferrous chloride 0.1 g / L.

[0067] In some embodiments, during the fermentation culture, the initial temperature is set to 22-23°C, preferably 22°C or 23°C.

[0068] In some embodiments, during the fermentation culture, the temperature is increased to 24°C-25°C, preferably 24°C or 25°C, after 45 hours of fermentation.

[0069] In some embodiments, during the fermentation culture, the temperature is increased to 26°C-28°C, preferably 26°C or 27°C, after 72 hours of fermentation.

[0070] Fourthly, the present invention provides a method for preparing microbial algal oil, comprising: extracting microbial algal oil from the fermentation broth described in the first aspect or the fermentation broth prepared by the preparation method described in the second aspect.

[0071] In some embodiments, the microbial algal oil includes at least one of hexadecenoic acid and astaxanthin.

[0072] Fifthly, the present invention provides a microbial algal oil prepared according to the preparation method described in the fourth aspect.

[0073] Sixthly, the present invention provides an application of the aforementioned fermentation broth, microbial agent, and microbial algal oil.

[0074] In some embodiments, the application is for the preparation of food, cosmetics, health products, or feed.

[0075] Beneficial effects Compared with the prior art, the present invention has the following beneficial technical effects: This invention provides a fermentation regulation strategy mediated by temperature and salt ions, specifically a three-pronged joint regulation strategy of "stepwise temperature variation + salt concentration stress + key precursor (multiple salt combinations / amino acids) feeding". During fermentation, the fermentation temperature is gradually increased, and a second, third, or fourth salt combination different from the fermentation medium is added simultaneously. Through osmotic pressure stress, salt concentration stress, and medium optimization (such as adding regulatory factors such as amino acids and organic acids), the yields of POA and astaxanthin can be increased simultaneously.

[0076] The fermentation method provided by this invention results in a significantly higher content of hexadecenoic acid and astaxanthin in the microbial algal oil of the fermentation broth, which has unexpected technical effects.

[0077] This invention has good prospects for industrialization.

[0078] Terminology Explanation In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0079] In the following content, all numbers disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. 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 numbers with values ​​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 "+ / -" indicates addition or subtraction. Detailed Implementation

[0080] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0081] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0082] The microalgae fermentation method of the present invention uses an oil-producing microorganism, namely, *Schizochytrium HS08*, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40902.

[0083] Test method: The biomass detection method was as follows: accurately pipette 4 mL of fermentation broth into a centrifuge and centrifuge at 6000 r / min for 4 min. Discard the supernatant, wash repeatedly with distilled water and centrifuge twice, then place in a freeze dryer to dry. After drying, weigh the refrigerated algal powder. Biomass = mass of freeze-dried algal powder / 4 * 1000.

[0084] The method for detecting oil content refers to Method I of GB 5009.6-2016.

[0085] The method for detecting hexadecenoic acid content is the normalization method in GB5009.168-2016.

[0086] The astaxanthin detection method refers to QB / T5948-2023.

[0087] Preliminary Example Preparation steps and methods of seed culture medium: 1. Inoculate one tube of *Schizochytrium* glycerol into the expansion culture medium and incubate at 23°C and 200 r / min for 36 h.

[0088] 2. Inoculate the activation solution at a rate of 10% into a primary shake flask containing expansion culture medium, and incubate at 23℃ and 200 r / min for 48 h to obtain the primary seed culture.

[0089] 3. Inoculate the primary seed culture at 10% into a secondary shake flask containing expansion culture medium, and incubate at 23℃ and 200 r / min for 24 h to obtain the secondary seed culture.

[0090] 4. Inoculate the secondary seed culture at 4-5% into the tertiary seed tank containing the expansion culture medium, and culture at 24℃ and 150 r / min for 36 h to obtain the seed culture solution.

[0091] The culture medium consisted of: 80-85 g / L glucose, 20 g / L yeast extract, 25 g / L sodium chloride, 15 g / L sodium sulfate, 2 g / L potassium sulfate, 1.5 g / L ammonium sulfate, 0.25 g / L magnesium sulfate, 8 g / L potassium dihydrogen phosphate and 1.5 g / L dipotassium hydrogen phosphate, with water as the solvent and a pH of 5.0-6.0.

[0092] Comparative Example 1 The *Schizochytrium* seed culture obtained in the preliminary example was inoculated at a 4% inoculum into a 1-ton fermenter containing fermentation medium and cultured for 15 hours before being transferred to an 8-ton fermenter to begin fermentation. The sugar concentration was controlled at 45-50 g / L, and the pH was adjusted during fermentation using ammonia and 50% citric acid to maintain a range of 5.5-6.0. The fermentation temperature was 25℃. The fermentation medium contained 5 g / L yeast extract as the basic nitrogen source, and the inorganic salt combination was: 15 g / L anhydrous sodium sulfate, 4 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, and 4.0 g / L ammonium sulfate. After 96 hours of fermentation, the fermentation broth was obtained, and the biomass, oil content, hexadecenoic acid content, and astaxanthin content in the microbial algal oil were measured.

[0093] Example 1: The *Schizochytrium* seed culture obtained in the preliminary example was inoculated at a 4% inoculum into a 1-ton fermenter containing fermentation medium and cultured for 24 hours before being transferred to an 8-ton fermenter to begin fermentation. The sugar concentration was controlled at 70-95 g / L, and the pH was adjusted during fermentation using ammonia and 50% citric acid to maintain a range of 4.5-5.0. The fermentation temperature was 25℃. The fermentation medium contained 90 g / L glucose; the nitrogen source consisted of 5 g / L yeast extract, 2 g / L betaine, 0.5 g / L arginine, 0.1 g / L histidine, and 4.0 g / L ammonium sulfate; the first salt combination consisted of 10 g / L anhydrous sodium sulfate, 4 g / L magnesium sulfate, 5 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 4.0 g / L ammonium sulfate, 0.1 g / L calcium chloride, and 0.01 g / L calcium phosphate.

[0094] After 96 hours of fermentation, the fermentation broth was obtained, and the biomass, the content of microbial algal oil in the fermentation broth, the content of hexadecenoic acid in the microbial algal oil, and the content of astaxanthin were detected.

[0095] Example 2 The *Schizochytrium* seed culture obtained in the preliminary example was inoculated into a 1-ton fermenter containing fermentation medium at a 4% inoculation rate and cultured for 20 h before being transferred to an 8-ton fermenter to start fermentation. The sugar concentration was controlled at 80-100 g / L, and the pH was adjusted during fermentation with ammonia and 50% citric acid to be controlled at 4.0-4.5. The fermentation temperature was 25℃. The fermentation medium contained 90 g / L glucose; the nitrogen sources were yeast extract (5.5 g / L), betaine (2 g / L), arginine (0.5 g / L), histidine (0.1 g / L), and ammonium sulfate (4.0 g / L); the first salt combination consisted of anhydrous sodium sulfate (10 g / L), magnesium sulfate (4 g / L), potassium dihydrogen phosphate (10 g / L), dipotassium hydrogen phosphate (1 g / L), ammonium sulfate (4.0 g / L), calcium chloride (0.1 g / L), and calcium phosphate (0.01 g / L); the second salt combination consisted of ferric citrate (0.023 g / L), ferric ammonium citrate (0.004 g / L), sodium molybdate (0.025 g / L), cobalt chloride (0.0014 g / L), zinc sulfate (0.0001 g / L), ferric sodium EDTA (0.45 g / L), ferrous sulfate (0.04 g / L), and ferrous chloride (0.1 g / L).

[0096] After 45 hours of fermentation, a third salt combination was added to the fermenter: magnesium sulfate 4 g / L, potassium dihydrogen phosphate 10 g / L, dipotassium hydrogen phosphate 1 g / L, ammonium sulfate 4.0 g / L, calcium chloride 0.1 g / L, and calcium phosphate 0.01 g / L.

[0097] After 96 hours of fermentation, the fermentation broth was obtained, and the biomass, the content of microbial algal oil in the fermentation broth, the content of hexadecenoic acid in the microbial algal oil, and the content of astaxanthin were detected.

[0098] Example 3 The *Schizochytrium* seed culture obtained in the preliminary example was inoculated at a 20% inoculum into a 1-ton fermenter containing fermentation medium and cultured for 15 hours before being transferred to an 8-ton fermenter to begin fermentation. The sugar concentration was controlled at 80-100 g / L, and the pH was adjusted during fermentation using ammonia and 50% malic acid to maintain a range of 4.5-5.0. The fermentation temperature was 22℃. The fermentation medium contained 90 g / L glucose. The initial basal nitrogen source for fermentation consisted of 6 g / L yeast extract, 2 g / L betaine, 0.5 g / L arginine, 0.1 g / L histidine, and 4.0 g / L ammonium sulfate. The first salt combination consisted of 12 g / L anhydrous sodium sulfate, 4 g / L magnesium sulfate, 10 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 4.0 g / L ammonium sulfate, 0.1 g / L calcium chloride, and 0.01 g / L calcium phosphate. The second salt combination consists of: ferric citrate 0.023 g / L, ferric ammonium citrate 0.004 g / L, sodium molybdate 0.025 g / L, cobalt chloride 0.0014 g / L, zinc sulfate 0.0001 g / L, ferric sodium ethylenediaminetetraacetate 0.45 g / L, ferrous sulfate 0.04 g / L, and ferrous chloride 0.11 g / L.

[0099] A third salt combination was added after 45 h of fermentation: magnesium sulfate 4 g / L, potassium dihydrogen phosphate 10 g / L, and dipotassium hydrogen phosphate 1 g / L. A fourth salt combination was added after 72 h of fermentation: sodium pyruvate 0.67 g / L, sodium citrate 3 g / L, and sodium malate 3 g / L.

[0100] After 96 hours of fermentation, the fermentation broth was obtained, and the biomass, the content of microbial algal oil in the fermentation broth, the content of hexadecenoic acid in the microbial algal oil, and the content of astaxanthin were detected.

[0101] Example 4 The *Schizochytrium* seed culture obtained in the preliminary example was inoculated into a 1-ton fermenter containing fermentation medium at a 20% inoculation rate and cultured for 15 hours before being transferred to an 8-ton fermenter to begin fermentation. The sugar concentration was controlled at 80-100 g / L. During fermentation, the pH was adjusted using ammonia and 50% citric acid, maintained at 5.0-6.0. The initial fermentation temperature was set at 23℃, increased to 25℃ after 45 hours. The fermentation medium contained 90 g / L glucose. The initial basal nitrogen source was yeast extract (5 g / L), betaine (2 g / L), arginine (0.5 g / L), histidine (0.1 g / L), and ammonium sulfate (4.0 g / L). The first salt combination consisted of anhydrous sodium sulfate (12 g / L), magnesium sulfate (4 g / L), potassium dihydrogen phosphate (10 g / L), dipotassium hydrogen phosphate (1 g / L), ammonium sulfate (4.0 g / L), calcium chloride (0.1 g / L), and calcium phosphate (0.01 g / L). The second salt combination consisted of ferric citrate (0.023 g / L), ferric ammonium citrate (0.004 g / L), sodium molybdate (0.025 g / L), cobalt chloride (0.0014 g / L), and zinc sulfate (0.0001 g / L). 0.45 g / L ferric sodium ethylenediaminetetraacetate, 0.04 g / L ferrous sulfate, and 0.1 g / L ferrous chloride.

[0102] At 45 h and 72 h of fermentation, a third salt combination was added simultaneously: magnesium sulfate 4 g / L, potassium dihydrogen phosphate 10 g / L, and dipotassium hydrogen phosphate 1 g / L, and a fourth salt combination: sodium pyruvate 0.67 g / L, sodium citrate 2.5 g / L, and sodium malate 3 g / L.

[0103] After 96 hours of fermentation, the fermentation broth was obtained, and the biomass, the content of microbial algal oil in the fermentation broth, the content of hexadecenoic acid in the microbial algal oil, and the content of astaxanthin were detected.

[0104] Example 5 The *Schizochytrium* seed culture obtained in the preliminary example was inoculated at a 20% inoculum into a 1-ton fermenter containing fermentation medium and cultured for 15 hours before being transferred to an 8-ton fermenter to begin fermentation. The sugar concentration was controlled at 80-100 g / L, and the pH was adjusted during fermentation using ammonia and 50% citric acid to maintain a range of 4.5-5.0. The initial fermentation temperature was set at 22℃, increased to 24℃ at 45 hours, and further increased to 26℃ at 72 hours. The fermentation medium contained 90 g / L glucose. The initial basal nitrogen source consisted of 6 g / L yeast extract, 2 g / L betaine, 0.5 g / L arginine, 0.1 g / L histidine, and 4.0 g / L ammonium sulfate. The first salt combination consisted of 10 g / L anhydrous sodium sulfate, 4 g / L magnesium sulfate, 10 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 4.0 g / L ammonium sulfate, 0.1 g / L calcium chloride, and 0.01 g / L calcium phosphate.

[0105] At 45 h and 72 h of fermentation, the following salt combinations were added: a third salt combination: 4 g / L magnesium sulfate, 10 g / L potassium dihydrogen phosphate, and 1 g / L dipotassium hydrogen phosphate; a second salt combination: 0.023 g / L ferric citrate, 0.004 g / L ferric ammonium citrate, 0.025 g / L sodium molybdate, 0.0001 g / L zinc sulfate, 0.45 g / L ferric sodium EDTA, 0.04 g / L ferrous sulfate, and 0.1 g / L ferrous chloride; and a fourth salt combination: 0.67 g / L sodium pyruvate, 3 g / L sodium citrate, and 3 g / L sodium malate.

[0106] After 96 hours of fermentation, the fermentation broth was obtained, and the biomass, the content of microbial algal oil in the fermentation broth, the content of hexadecenoic acid in the microbial algal oil, and the content of astaxanthin were detected.

[0107] Example 6 The *Schizochytrium* seed culture obtained in the preliminary example was inoculated into a 1-ton fermenter containing fermentation medium at a 20% inoculation rate and cultured for 15 hours before being transferred to an 8-ton fermenter to begin fermentation. The sugar concentration was controlled at 80-100 g / L. During fermentation, the pH was adjusted using ammonia and 50% citric acid to maintain a range of 4.5-5.0. The initial fermentation temperature was set at 22℃, increased to 24℃ after 45 h, and further increased to 26℃ after 72 h. The fermentation medium contained 90 g / L glucose. The initial basal nitrogen source consisted of 6 g / L yeast extract, 2 g / L betaine, 0.5 g / L arginine, 0.1 g / L histidine, and 4.0 g / L ammonium sulfate. The first salt combination consisted of 10 g / L anhydrous sodium sulfate, 4 g / L magnesium sulfate, 10 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 4.0 g / L ammonium sulfate, 0.1 g / L calcium chloride, and 0.01 g / L calcium phosphate. The second salt combination consisted of 0.023 g / L ferric citrate, 0.004 g / L ferric ammonium citrate, 0.025 g / L sodium molybdate, and 0.0014 g / L cobalt chloride. 0.0001 g / L zinc sulfate, 0.45 g / L ferric sodium EDTA, 0.04 g / L ferrous sulfate, and 0.1 g / L ferrous chloride.

[0108] At 45 h and 72 h of fermentation, the following salt combinations were added: a third salt combination: 4 g / L magnesium sulfate, 10 g / L potassium dihydrogen phosphate, and 1 g / L dipotassium hydrogen phosphate; a second salt combination: 0.023 g / L ferric citrate, 0.004 g / L ferric ammonium citrate, 0.025 g / L sodium molybdate, 0.0001 g / L zinc sulfate, 0.45 g / L ferric sodium EDTA, 0.04 g / L ferrous sulfate, and 0.1 g / L ferrous chloride; and a fourth salt combination: 0.75 g / L sodium pyruvate, 3 g / L sodium citrate, and 3 g / L sodium malate.

[0109] After 96 hours of fermentation, the fermentation broth was obtained, and the biomass, the content of microbial algal oil in the fermentation broth, the content of hexadecenoic acid in the microbial algal oil, and the content of astaxanthin were detected.

[0110] Table 1. Biomass, microbial algal oil, hexadecenoic acid, and astaxanthin content in Comparative Example 1 and Examples 1-6

[0111] Note: The content of microbial algal oil = microbial algal oil / fermentation broth dry matter × 100%; the content of hexadecenoic acid = hexadecenoic acid / microbial algal oil × 100%. The unit of astaxanthin is mg / kg microbial algal oil.

[0112] As shown in Table 1, this invention significantly improved the accumulation levels of hexadecenoic acid (POA) and astaxanthin in the fermentation broth of Schizochytrium through a systematic fermentation regulation strategy.

[0113] Compared to Comparative Example 1, the amino acid combination (betaine, arginine, histidine, etc.) added to the fermentation medium in Example 1 of this invention provides additional nitrogen source and methyl donor for the MVA pathway, further promoting the accumulation of astaxanthin and increasing the concentrations of biomass, microbial algal oil, hexadecenoic acid, and astaxanthin.

[0114] Compared to Example 1, Example 2 achieved high salt concentration stress in the fermentation medium and added salt during the fermentation process, which effectively activated the gene expression and enzyme activity of desaturase in the FAS pathway. In Example 2, the biomass, microbial algal oil, hexadecenoic acid, and astaxanthin content were all significantly increased.

[0115] Compared to Example 2, the addition of a fourth salt combination after 72 hours of fermentation in Example 3 further promoted the accumulation of astaxanthin, resulting in a significant increase in astaxanthin content.

[0116] Compared to Example 3, Example 4 added high salt at both 45h and 72h of fermentation, effectively activating gene expression and enzyme activity of desaturases in the FAS pathway. The exogenously added TCA cycle intermediates (sodium pyruvate, sodium citrate, and sodium malate) provided sufficient acetyl-CoA precursors and reducing power (NADPH) for fatty acid synthesis, thus overcoming the metabolic bottleneck of POA synthesis. In Example 4, the astaxanthin content reached 339.06 mg / kg, approximately seven times higher than the 43.36 mg / kg in Comparative Example 1, confirming the significant promoting effect of this regulatory strategy on astaxanthin synthesis.

[0117] Compared to Example 4, Example 5 used only the first salt combination in the initial fermentation medium, and the second salt combination was added after 45 and 72 hours of fermentation. The initial high salt concentration stress was slightly worse and insufficient for activating the FAS pathway. With low temperature induction, although the astaxanthin content and microbial algal oil were slightly lower, the hexadecenoic acid content was slightly higher.

[0118] Compared to Example 4, Example 6 effectively activated the gene expression and enzyme activity of desaturases in the FAS pathway through low-temperature induction (stepwise temperature change from 22 to 26°C) combined with high salt concentration stress. Simultaneously, the exogenously added TCA cycle intermediates (sodium pyruvate, sodium citrate, and sodium malate) provided sufficient acetyl-CoA precursors and reducing power (NADPH) for fatty acid synthesis, thus overcoming the metabolic bottleneck in POA synthesis. In Example 6, the POA content reached 10.64%, approximately 80 times higher than the 0.13% in Comparative Example 1, fully validating the effectiveness of this strategy.

[0119] At the metabolic mechanism level, fatty acid synthesis in the fermentation broth of *Schizochytrium* mainly relies on two pathways: PKS and FAS. The target product, hexadecenoic acid (POA), is primarily catalyzed by the key enzymes Δ9-desaturase and Δ12-desaturase in the FAS pathway. Regarding astaxanthin synthesis, astaxanthin is synthesized stepwise from acetyl-CoA via the MVA (mevaleric acid) pathway. The stepwise temperature stress (22℃→24℃→26℃) and dynamic changes in salt ion concentration employed in this invention, on the one hand, induce oxidative stress, prompting cells to divert metabolic flow towards carotenoid synthesis; on the other hand, the supplemented amino acid combination (betaine, arginine, histidine, etc.) provides additional nitrogen and methyl donors for the MVA pathway, further promoting astaxanthin accumulation.

[0120] In summary, the three-pronged joint regulation strategy of "stepwise temperature variation + salt concentration stress + key precursor (multiple salt combinations / amino acids) feeding" established in this invention not only synergistically enhances the carbon flux allocation and enzyme activity expression of the two metabolic pathways of FAS and MVA, but also achieves efficient co-production of POA and astaxanthin in the same fermentation process, providing reliable technical support and theoretical basis for the industrial production of high-value-added lipid products from Schizochytrium.

[0121] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A fermentation broth, characterized in that, The fermentation broth was obtained by fermenting *Schizochytrium*. In the fermentation broth, the mass percentage of microbial algal oil in the dry matter of the fermentation broth is 20%-45%; The content of hexadecenoic acid in the fermentation broth accounts for 2%-11% of the mass of the microbial algal oil; The astaxanthin content in the fermentation broth is 50-400 mg / kg of microbial algal oil.

2. The fermentation broth according to claim 1, characterized in that, The *Schizochytridica* species is *Schizochytridica HS08*.

3. A microbial inoculant, characterized in that, The microbial agent includes the fermentation broth according to claim 1.

4. A method for preparing the fermentation broth according to claim 1, characterized in that, include: The seed culture medium was used to culture *Schizochytrium* to obtain a seed culture solution. The obtained seed culture medium was transferred to a first fermenter containing fermentation medium and cultured for 15-36 hours, and then transferred to a second fermenter containing fermentation medium and fermented for 96-120 hours to obtain the fermentation broth. The fermentation medium comprises: nitrogen source 5-30 g / L, glucose 60-150 g / L and first salt combination 10-60 g / L; The nitrogen source is yeast extract, betaine, arginine, histidine, and ammonium sulfate; The first salt combination consists of anhydrous sodium sulfate, magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium chloride, and calcium phosphate. During the fermentation culture in the second fermenter, after 45 h of fermentation, at least one of the second salt combination, the third salt combination, or the fourth salt combination is added to the second fermenter. The second salt combination is ferric citrate, ferric ammonium citrate, sodium molybdate, cobalt chloride, zinc sulfate, ferric sodium ethylenediaminetetraacetate, ferrous sulfate, and ferrous chloride; or, the second salt combination is ferric citrate, ferric ammonium citrate, sodium molybdate, zinc sulfate, ferric sodium ethylenediaminetetraacetate, ferrous sulfate, and ferrous chloride. The third salt combination is selected from at least one of magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium chloride, and calcium phosphate. The fourth salt combination is selected from at least one of sodium pyruvate, sodium citrate, and sodium malate.

5. The preparation method according to claim 4, characterized in that, The culture medium is composed of: glucose 60-100 g / L, yeast extract 15-25 g / L, sodium chloride 20-30 g / L, sodium sulfate 10-20 g / L, potassium sulfate 1-3 g / L, ammonium sulfate 1-2 g / L, magnesium sulfate 0.1-0.5 g / L, potassium dihydrogen phosphate 5-10 g / L, and dipotassium hydrogen phosphate 1-2 g / L. In the fermentation medium, the nitrogen source is yeast extract 1-10 g / L, betaine 1-2 g / L, arginine 0.5-1 g / L, histidine 0.1-0.5 g / L and ammonium sulfate 1-5.0 g / L; The first salt combination consists of anhydrous sodium sulfate 5-20 g / L, magnesium sulfate 3-5 g / L, potassium dihydrogen phosphate 5-15 g / L, dipotassium hydrogen phosphate 1-5 g / L, ammonium sulfate 2-5 g / L, calcium chloride 0.1-0.5 g / L, and calcium phosphate 0.01-0.05 g / L.

6. The preparation method according to claim 4, characterized in that, The fermentation culture is as follows: the obtained seed culture is transferred to a first fermenter containing fermentation medium at an inoculation rate of 4-40% and cultured for 15-36 hours, and then transferred to a second fermenter containing fermentation medium for fermentation for 96-120 hours; the fermentation temperature is 22-28℃, the fermentation pH is 3.0-6.0, and the pH is adjusted using a pH adjuster during fermentation; Alternatively, the fermentation medium may further include a second salt combination of 0.5-7 g / L.

7. The preparation method according to claim 5 or 6, characterized in that, The pH adjuster is ammonia and citric acid; The second salt combination consists of 0.01-0.05 g / L ferric citrate, 0.002-0.008 g / L ferric ammonium citrate, 0.021-0.035 g / L sodium molybdate, 0.0014-0.005 g / L cobalt chloride, 0.0001-0.0002 g / L zinc sulfate, 0.45-5 g / L ferric sodium ethylenediaminetetraacetate, 0.04-1.42 g / L ferrous sulfate, and 0.10-0.13 g / L ferrous chloride; or the second salt combination consists of 0.01-0.05 g / L ferric citrate, 0.002-0.008 g / L ferric ammonium citrate, 0.021-0.035 g / L sodium molybdate, 0.0001-0.0002 g / L zinc sulfate, and 0.45-5 g / L ferric sodium ethylenediaminetetraacetate. 0.04-1.42 g / L of ferrous sulfate and 0.10-0.13 g / L of ferrous chloride; The third salt combination is 1-5 g / L magnesium sulfate, 10-15 g / L potassium dihydrogen phosphate, and 1-3 g / L dipotassium hydrogen phosphate, or the third salt combination is 1-5 g / L magnesium sulfate, 10-15 g / L potassium dihydrogen phosphate, 1-3 g / L dipotassium hydrogen phosphate, 1-4 g / L ammonium sulfate, 0.1-0.5 g / L calcium chloride, and 0.01-0.05 g / L calcium phosphate; The fourth salt combination consists of sodium pyruvate 0.5-1 g / L, sodium citrate 1-5 g / L, and sodium malate 1-5 g / L.

8. The preparation method according to claim 5 or 6, characterized in that, During the fermentation process, the initial temperature was set to 22-23℃, increased to 24℃-25℃ after 45 hours of fermentation, and increased to 26℃-28℃ after 72 hours of fermentation. Alternatively, during the fermentation culture, after 72 hours of fermentation, at least one of the second salt combination, the third salt combination, or the fourth salt combination is added to the second fermenter.

9. A method for preparing microbial algal oil, comprising: Microbial algal oil is extracted from the fermentation broth according to any one of claims 1-2 or the fermentation broth prepared by the preparation method according to any one of claims 4-8.

10. The application of the fermentation broth according to any one of claims 1-2, the microbial agent according to claim 3, the fermentation broth prepared by the preparation method according to any one of claims 4-8, or the microbial algal oil prepared by the preparation method according to claim 9, characterized in that, The applications are for preparing food, cosmetics, health products, or animal feed.

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