Schizochytrium and Its Applications, and Methods and Products for Producing EPA, DHA and Heptadecanoic Acid

Abstract

The present invention relates to the technical field of microbial preparation, specifically to Schizochytrium sp. and its applications, as well as methods and products for producing EPA, DHA, and heptadecanoic acid. Schizochytrium sp. SK-3 was deposited at the China Center for Type Culture Collection on October 23, 2024, with the deposit number CCTCC NO: M 20242315. The application of Schizochytrium sp. SK-3 in the production of EPA, DHA, and high-yield heptadecanoic acid. This strain is cultured in a fermentation medium containing a carbon source, a nitrogen source, and inorganic salts at 20-30 °C for 72-96 hours to obtain oils containing long-chain fatty acids such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and heptadecanoic acid, which can be used to prepare food products, cosmetics, or pharmaceutical compositions for animals or humans.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial preparation, specifically to Schizochytrium and its applications, as well as methods and products for producing EPA, DHA, and heptadecanoic acid. Background Art

[0002] The development and application of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have attracted much attention in the health care products and pharmaceutical fields. However, at present, the main commercial sources of EPA and DHA are limited to deep-sea fish oil, which not only has high production costs but also limited yields, making it difficult to meet the growing market demand. Given the broad application prospects of EPA and DHA and the increasing market demand, it is particularly urgent and important to find and develop new sources of EPA and DHA raw materials. This need has driven researchers to explore alternative sources of EPA and DHA in order to achieve more economical, efficient, and sustainable production of EPA and DHA. Polyunsaturated fatty acids (PUFAs) are synthesized by microorganisms such as microalgae and fungi. The polyunsaturated fatty acids extracted from microalgae, commonly known as "algal oil", contain varying amounts of EPA and DHA. In addition to certain specific algae, especially microalgal cells, such as those of the order Thraustochytriales, have become important sources for providing high-quality PUFA lipids.

[0003] The oils produced by microorganisms usually have a simpler polyunsaturated fatty acid profile than the corresponding fish, making it possible to enrich high concentrations of EPA and DHA. Schizochytrium is currently the most commercially successful strain for producing EPA and DHA, but the current content of EPA and DHA in its body is still low. At present, people in the market are more willing to accept high-content EPA or / and DHA triglyceride-type oils. Therefore, it is necessary to develop a green and low-cost technology for enriching EPA or / and DHA triglyceride-type oils.

[0004] Schizochytrium can also be used to produce odd-chain fatty acids. In EP0823475A, the odd-chain fatty acids of Schizochytrium sp. SR21 strain contain 10.1% pentadecanoic acid (C15:0) and 1.8% heptadecanoic acid (C17:0). In recent years, studies have found that heptadecanoic acid has many important physiological functions, including maintaining cell membrane integrity, regulating blood lipid and blood glucose levels, and promoting nervous system function. Therefore, heptadecanoic acid is of great significance to human health. However, at present, no products of odd-chain fatty acids have been introduced to the market. The main reason is that the sources of odd-chain fatty acids are very few and their contents in animal and vegetable oils are very low. Microorganisms are excellent materials for producing odd-chain fatty acids. Summary of the Invention

[0005] The object of the present invention is: aiming at the deficiencies of the current existing technologies, to provide a Schizochytrium sp. for producing EPA, DHA and high-yield heptadecanoic acid, and a method for preparing algal oil with high contents of EPA, DHA and triglyceride heptadecanoate by using the same. This method has the advantages of being green, having mild conditions, high reaction selectivity, few by-products, low cost, etc.

[0006] In order to achieve the above invention objects, the technical solution of the present invention is as follows:

[0007] One of the invention objects of the present application is to protect a Schizochytrium sp. SK-3, which was deposited at the China Center for Type Culture Collection on October 23, 2024, with the deposit number CCTCC NO: M 20242315, and the address of the depositary institution is Wuhan University, Wuhan, China.

[0008] Another invention object of the present application is to protect a Schizochytrium sp. microorganism isolated from the strain with the deposit number CCTCC NO: M 20242315 or its derived strain.

[0009] Further, the strain derived from the above-mentioned Schizochytrium sp. microorganism is a mutant strain.

[0010] Another invention object of the present invention is to protect a Schizochytrium sp. biomass, which comprises the aforementioned Schizochytrium sp. SK-3, the isolated Schizochytrium sp. microorganism or their mixture.

[0011] Further, for the above-mentioned Schizochytrium sp. biomass, at least about 50% of the dry cells of the biomass are fatty acids by weight, and at least about 45% of the fatty acids are ω-3 fatty acids.

[0012] Another invention object of the present application is to protect the application of the above-mentioned Schizochytrium sp. in the production of EPA and DHA.

[0013] As a preferred embodiment in the present application, Schizochytrium sp. SK-3 is cultured in a fermentation medium containing a carbon source, a nitrogen source and inorganic salts at 20 - 30 °C for 72 - 96 hours to obtain an oil containing long-chain polyunsaturated fatty acids, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and heptadecanoic acid, which can be applied to food products, cosmetics or pharmaceutical compositions for animals or humans.

[0014] As a preferred embodiment of the present application, after culturing Schizochytrium sp. SK-3, the weight of the oil in the cells is 43-55% of the dry weight of the cell mass. Among them, the triglyceride of docosahexaenoic acid (DHA) accounts for 30-45% of the weight of the oil, the triglyceride of eicosapentaenoic acid (EPA) accounts for 2-6% of the weight of the oil, and the triglyceride of heptadecanoic acid accounts for 4-8% of the weight of the oil.

[0015] The third object of the invention of the present application is to protect a method for preparing algal oil with high contents of EPA, DHA and triglyceride of heptadecanoic acid, and the aforementioned Schizochytrium sp. is used in this method;

[0016] The specific preparation method includes the following steps:

[0017] 1) Activation and culture: First, transfer Schizochytrium sp. into an activated plate medium for culture; then pick a single colony on the activated plate medium and inoculate it into an activated liquid medium for culture to obtain a primary seed solution; then take the primary seed solution and inoculate it again into the activated liquid medium for culture to obtain a secondary seed solution;

[0018] 2) Fermentation production: Transfer the secondary seed solution obtained in step 1) into a fermentation medium, introduce sterile air, and supplement glucose for fermentation culture;

[0019] 3) Cell disruption and oil collection: Centrifuge to collect all the cells, add an appropriate amount of pure water and cell wall-breaking enzyme for enzymatic hydrolysis, and then use a high-pressure homogenizer for disruption; after the disruption is completed, centrifuge to collect the upper-layer oil to obtain algal oil;

[0020] 4) Enrichment of EPA, DHA and heptadecanoic acid in the algal oil;

[0021] a. Add a certain amount of the algal oil, pure water and enzyme obtained in step 3) into a reaction kettle, fill it with nitrogen for protection, stir and react at a certain temperature. After the reaction is completed, centrifuge and separate the obtained oil-water mixture. The upper layer is the oil phase and the lower layer is the water phase;

[0022] b. Subject the oil phase obtained in step a) to molecular distillation, and collect the residue in the molecular distillation, which is the enriched EPA and DHA, and the distillate is the enriched heptadecanoic acid;

[0023] c. Enzymatically hydrolyze the residue obtained in step b) under certain conditions, and centrifuge to collect the upper-layer oil phase to obtain free fatty acids rich in EPA and DHA;

[0024] d. Add the free fatty acids rich in EPA and DHA, glycerol, and lipase obtained in step c to a reaction kettle for an esterification reaction, and fill it with nitrogen for protection. React under the conditions of stirring and vacuum pumping. After the reaction is completed, filter, and then remove the unreacted free fatty acids by molecular distillation. The obtained oil is the triglyceride-type algal oil enriched with EPA and DHA.

[0025] e. Add the heptadecanoic acid, glycerol, and lipase obtained in step b to a reaction kettle for an esterification reaction, and fill it with nitrogen for protection. React under the conditions of stirring and vacuum pumping. After the reaction is completed, filter, and then remove the unreacted free fatty acids by molecular distillation. The obtained oil is the triglyceride-type algal oil enriched with heptadecanoic acid.

[0026] As a preferred embodiment of the present application, in step 1) of the method for preparing triglyceride algal oil with high contents of EPA, DHA, and heptadecanoic acid, the culture temperature in the plate medium is 25°C - 30°C, and the culture time is 2 - 3 days; the culture temperature in the activated liquid medium is 25°C - 30°C, and the culture time is 1 - 2 days; the inoculation amount when the primary seed liquid is re-introduced into the activated liquid medium is 10%, the culture temperature is 25°C - 30°C, and the culture time is 1 - 2 days.

[0027] As a preferred embodiment of the present application, in step 1) of the method for preparing triglyceride algal oil with high contents of EPA, DHA, and heptadecanoic acid,

[0028] The components of the plate medium are: 30 g / L of glucose, 4 g / L of yeast powder, 18 g / L of sodium glutamate, 2.1 g / L of magnesium chloride hexahydrate, 4.5 g / L of sodium sulfate, 0.4 g / L of potassium chloride, 0.4 g / L of sodium chloride, 0.5 g / L of magnesium sulfate, 1.5 g / L of potassium dihydrogen phosphate, and 1.5 g / L of agar;

[0029] The components of the liquid medium in step (2) are: 30 g / L of glucose, 4 g / L of yeast powder, 18 g / L of sodium glutamate, 2.1 g / L of magnesium chloride hexahydrate, 4.5 g / L of sodium sulfate, 0.4 g / L of potassium chloride, 0.4 g / L of sodium chloride, 0.5 g / L of magnesium sulfate, and 1.5 g / L of potassium dihydrogen phosphate.

[0030] As a preferred embodiment of the present application, in step 2) of the method for preparing triglyceride algal oil with high contents of EPA, DHA, and heptadecanoic acid, the addition amount of glucose is adjusted to maintain its concentration at 5 - 20 g / L; during fermentation production, the dissolved oxygen in the culture medium is maintained at 10% - 50% by regulating the ventilation volume and stirring speed; ferment for 72 - 96 h; the inoculation amount of the seed liquid is 5 - 10%, the rotation speed is 100 - 500 rpm, the ventilation volume is 1 - 5 L / min, and the fermentation temperature is 25 - 30°C.

[0031] As a preferred embodiment of the present application, in step 2) of the method for preparing algal oil with high contents of EPA, DHA and heptadecanoin, the components of the fermentation medium are: glucose 50 g / L, peptone 2 g / L, yeast powder 4 g / L, sodium glutamate 18 g / L, magnesium chloride hexahydrate 4.2 g / L, sodium sulfate 18 g / L, potassium chloride 0.8 g / L, sodium chloride 1 g / L, magnesium sulfate 1.5 g / L, and potassium dihydrogen phosphate 3 g / L.

[0032] As a preferred embodiment of the present application, in step 3) of the method for preparing algal oil with high contents of EPA, DHA and heptadecanoin, the addition amount of the cell wall-breaking enzyme is 1-10% of the cell mass; the enzymatic hydrolysis conditions are: 30-40 °C, 100-200 rpm, enzymatic hydrolysis for 12-24 h, and the enzyme is alkaline protease 20FL from Xiasheng; after crushing with a high-pressure homogenizer for 2-5 times, the pressure is 700-1200 bar.

[0033] As a preferred embodiment of the present application, in step 4) a of the method for preparing algal oil with high contents of EPA, DHA and heptadecanoin, the oil-water mass ratio is 50-200%; the enzyme is porcine pancreatic enzyme; the addition amount of the enzyme is 0.02-2% of the algal oil mass; the temperature of the stirring reaction is 40-60 °C, the reaction time is 0.5-3 h, and the stirring speed is 150-300 r / min.

[0034] As a preferred embodiment of the present application, in step 4) b of the method for preparing algal oil with high contents of EPA, DHA and heptadecanoin, the specific steps of molecular distillation are: adding the upper oil phase obtained in step a into the feed tank of the secondary molecular distillation device; through the first-stage molecular distillation, dehydrating and degassing the oil phase at 90 °C; setting the vacuum degree of the second-stage molecular distillation to 0.1 Pa, the scraping film speed to 300 rpm, and the feed flow rate to 1.0 mL / min, separating the oil phase at 150-200 °C, and collecting the obtained residue.

[0035] As a preferred embodiment of the present application, in step 4) c of the method for preparing algal oil with high contents of EPA, DHA and heptadecanoin, the enzymatic hydrolysis conditions are: the oil-water mass ratio is 50-200%, the addition amount of the enzyme is 1-5% of the algal oil weight; the enzyme is Novozym 435 immobilized lipase; the enzymatic hydrolysis temperature is 45-60 °C, the time is 3-6 h, and the stirring speed is 200-300 rpm.

[0036] As a preferred embodiment of the present application, in step 4) d of the method for preparing algal oil with high contents of EPA, DHA and heptadecanoic acid triglyceride, the esterification reaction is as follows: the reaction temperature is 40 - 60 °C, the reaction time is 4 - 12 h; the dosage of glycerol is 4 - 8% of the weight of fatty acids, and the dosage of enzyme is 2 - 8% of the mass of algal oil; the enzyme is Novozym 435 immobilized lipase.

[0037] As a preferred embodiment of the present application, in step 4) e of the method for preparing algal oil with high contents of EPA, DHA and heptadecanoic acid triglyceride, the esterification reaction is as follows: the reaction temperature is 40 - 60 °C, the reaction time is 4 - 12 h; the dosage of glycerol is 4 - 8% of the weight of fatty acids, and the dosage of enzyme is 2 - 8% of the mass of algal oil; the enzyme is Novozym 435 immobilized lipase.

[0038] The present application also protects algal oil with high contents of EPA, DHA and heptadecanoic acid triglyceride obtainable by using the above method steps or any combination of the method steps.

[0039] The present application also protects an isolated Schizochytrium culture, which comprises the above-mentioned Schizochytrium microorganism or a mixture thereof.

[0040] Furthermore, for the above-mentioned microbial oil, the oil is crude oil extracted from the biomass of microorganisms without further processing or refined oil obtained by treating the crude microbial oil with further processing steps selected from refining, bleaching and / or deodorization.

[0041] Microbial oil from Schizochytrium sp. comprises at least 80% triglyceride fraction by weight, wherein the DHA content in the triglyceride fraction is at least 40% by weight, the EPA content is at least 6% by weight, and the heptadecanoic acid is at least 7% by weight.

[0042] Food, cosmetic or pharmaceutical compositions for animals or humans, which comprise the above-mentioned Schizochytrium microorganism, or the above-mentioned Schizochytrium biomass, or a mixture thereof and any one of the microbial oils.

[0043] Compared with the existing technologies, the beneficial effects of the present invention are as follows:

[0044] (1) A strain of Schizochytrium sp. SK-3 is provided. This strain has a fast growth rate and a short fermentation time, and can be used for preparing algal oil with high contents of EPA and DHA triglyceride.

[0045] (2) During the production process, the selectivity of pancreatic lipase for long-chain fatty acids is utilized, and by strictly controlling the reaction degree, the purpose of enriching EPA, DHA and heptadecanoic acid is achieved.

[0046] (3) In the present invention, both the hydrolysis and esterification reactions use enzymes as catalysts. Compared with traditional chemical reaction methods, they have the advantages of being green, having mild reaction conditions, high reaction selectivity, few by-products, and low costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is the cell morphology diagram of Schizochytrium sp. SK-3 in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0048] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any manner.

[0049] Any feature disclosed in this specification (including the claims and abstract), unless specifically stated, can be replaced by other equivalent or similar-purpose alternative features. That is, unless specifically stated, each feature is only an example in a series of equivalent or similar features.

[0050] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0051] Schizochytrium sp. SK-3 in the following embodiments was deposited with the China Center for Type Culture Collection on October 23, 2024, and the deposit number is CCTCC NO: M 20242315.

[0052] %, unless otherwise specified in this application, all represent mass percentages. In the following embodiments, the alkaline protease used was produced by Xiasheng Company.

[0053] Example 1:

[0054] Screening of Schizochytrium sp. SK-3:

[0055] Decayed leaves and seawater were collected from the mangrove forest by the seaside in Xiamen, Fujian, and isolated by the pine pollen fishing method, and stored at -80 °C for future use. After identification by morphological and molecular biological means, the strain was identified as the genus Schizochytrium, so it was named Schizochytrium sp. SK-3. Under an optical microscope, its cells are spherical, with a diameter of 3 - 15 μm, and there are granular oils in the cells (see Figure 1 ). After gas phase analysis, it was found that its polyunsaturated fatty acid oils are mainly composed of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The gene sequence of this strain is shown in SEQ ID NO.1:

[0056] SEQ ID NO.1:

[0057] AATTTCGGAGGATACAATGTTTTGCCTGTGGTTTTTTTTTTTTGTTTGCTATTTCTTTTTAATTAAATACCAAAAATCAGACATGACTTTCAGCGATGGGTGTCTAGGCTCTCGTAACGATGAAGAACGCAGCGAAATGCGATACGTAGTGTGAATTGCAAAATTAGCGAGCATCAAATCTTTGAACGCACATTGCGCTTCCGTGTGATGCGAGAGCATTCCTGCTTGAGTGTTTGTACGTTTGTCTCATTGTCGCTTGGCGATGATGCTCTGTGGATGGGTCGATGTAATTTGATTGATCAGATTGGCCTGAAAAAGCGCGCTATAAGAGCAAAGGGTTGCTTGATTGCAAATGATCCAAGCATCCGATAGAATCAAACCTCAAGTTGGAAAGAAGACCTGCTGAATTTAATCATATCAATAAGCGGAGGACAG.

[0058] Example 2:

[0059] A method for preparing algal oil with high contents of EPA, DHA and tricosanoin, which adopts high-density fermentation of Schizochytrium sp. SK-3:

[0060] Transfer Schizochytrium sp. SK-3 preserved at -80°C in Example 1 into an activated plate medium, and culture it at 28°C for 48 h; pick a single colony on the activated plate medium, inoculate it into 500 mL of activated liquid medium, and culture it at 200 rpm and 28°C for 48 h to obtain a primary seed solution; then transfer the primary seed solution into 5 L of activated liquid medium, culture it at 200 rpm and 28°C with an inoculation amount of 10% for 1.5 days to obtain a secondary seed solution.

[0061] Among them, the components of the plate medium are 30 g / L of glucose, 4 g / L of yeast powder, 18 g / L of sodium glutamate, 2.1 g / L of magnesium chloride hexahydrate, 4.5 g / L of sodium sulfate, 0.4 g / L of potassium chloride, 0.4 g / L of sodium chloride, 0.5 g / L of magnesium sulfate, 1.5 g / L of potassium dihydrogen phosphate, and 1.5 g / L of agar.

[0062] The components in the activated liquid medium are 30 g / L of glucose, 4 g / L of yeast powder, 18 g / L of sodium glutamate, 2.1 g / L of magnesium chloride hexahydrate, 4.5 g / L of sodium sulfate, 0.4 g / L of potassium chloride, 0.4 g / L of sodium chloride, 0.5 g / L of magnesium sulfate, and 1.5 g / L of potassium dihydrogen phosphate.

[0063] Then, inoculate the secondary seed liquid into a 50 L fermenter at an inoculation amount of 10%, with a fermentation temperature of 28 °C, a stirring speed of 350 rpm, and an aeration rate of 3 L / min. Control the dissolved oxygen saturation in the fermenter at about 30% by controlling the aeration rate and stirring speed. After 24 h of fermentation, measure the glucose content in the fermentation broth every 1 h, and supplement the carbon and nitrogen source concentrations in the fermentation broth to remain unchanged. Stop fermentation after 96 hours.

[0064] Centrifuge to collect the thalli, wash them once with pure water with a volume one-third of the fermentation volume, centrifuge to remove the washing liquid, collect the thalli (wet-weight thalli), weigh the cell weight, and calculate that the dry weight of Schizochytrium sp. SK-3 is 74.52 g / L.

[0065] Example 3

[0066] On the basis of Example 2, Schizochytrium sp. SK-3 was disrupted:

[0067] Weigh the dry-weight thalli obtained in Example 2, add pure water to prepare a bacterial liquid with a concentration of 300 g / L, and perform high-pressure homogenization and cell wall breaking 3 times at a temperature of 10 °C and a pressure of 1300 bar to obtain a disrupted bacterial liquid.

[0068] Add alkaline protease (a commercially available product, here alkaline protease 20FL from Xiasheng Company) to the disrupted bacterial liquid, with an addition amount of 1 g of protease powder per 100 g of thalli, and perform enzymatic hydrolysis at 45 °C and 200 rpm for 6 h to obtain an enzymatically hydrolyzed and disrupted bacterial liquid.

[0069] Perform high-pressure homogenization and cell wall breaking on the enzymatically hydrolyzed and disrupted bacterial liquid 3 times at a temperature of 10 °C and a pressure of 1300 bar; then add n-hexane with a volume one-third of the disrupted liquid, shake well, let it stand for stratification, take the upper layer liquid, and repeat 2 - 4 times until the upper layer liquid is colorless. Combine the upper layer liquids to obtain an n-hexane extraction liquid.

[0070] Perform rotary evaporation on the n-hexane extraction liquid, with the working temperature and pressure being 50 °C and -0.1 MPa respectively. Recover n-hexane by rotary evaporation and obtain crude oil at the same time. After calculation, the weight of the oil is 53.14% of the cell dry weight. Then, degum the crude oil with phosphoric acid and wash it with water to obtain degummed crude oil (i.e., degummed algal oil).

[0071] After inspection, the recovery rate of the degummed crude oil is 94.67%, and the contents of EPA, DHA, and heptadecanoic acid are 5.82%, 39.54%, and 7.12% respectively.

[0072] Example 4

[0073] Based on Example 3, an optimization experiment on the hydrolysis conditions of Schizochytrium sp. SK-3 algal oil was carried out:

[0074] All the enzymes used in this example are commercially available products. Here, trypsin produced by Xiasheng Company was selected.

[0075] The degummed algal oil, pure water and enzyme (commercially available product, here (porcine) trypsin produced by Xiasheng Company) obtained in Example 3 were added to the reaction kettle. The oil-water mass ratios were 1:2, 1:1 and 2:1 respectively. Nitrogen was filled for protection, and the reaction was stirred at 50 °C for 1 h. The enzyme addition amount was 0.02% of the weight of the algal oil. After the reaction, the obtained oil-water mixture was centrifuged for liquid separation, and the contents of EPA and DHA in the hydrolyzed algal oil and the content of heptadecanoic acid in the distillate were measured. The results are shown in Table 1.

[0076] Table 1 Hydrolysis results at different oil-water ratios

[0077]

[0078] A certain amount of the degummed algal oil, pure water and enzyme obtained in Example 3 were added to the reaction kettle. The oil-water ratio was 1:2. Nitrogen was filled for protection, and the reaction was stirred at 40 °C, 50 °C and 60 °C for 1 h respectively. The enzyme addition amount was 0.02% of the weight of the algal oil. After the reaction, the obtained oil-water mixture was centrifuged for liquid separation, and the obtained oil was separated by molecular distillation. The residue and distillate were collected, and the contents of EPA and DHA in the residue and the content of heptadecanoic acid in the distillate were measured. The results are shown in Table 2.

[0079] Table 2 Hydrolysis results at different temperatures

[0080]

[0081] A certain amount of the degummed algal oil, pure water and enzyme obtained in Example 3 were added to the reaction kettle. The oil-water ratio was 2:1. Nitrogen was filled for protection, and the reaction was stirred at 50 °C. Samples were taken at 0.5 h, 1 h, 2 h and 3 h, and then the sampled oil was separated by molecular distillation, and the contents of EPA, DHA and heptadecanoic acid were measured. The enzyme addition amount in the reaction was 0.02% of the weight of the algal oil. The results are shown in Table 3.

[0082] Table 3 Hydrolysis results at different reaction times

[0083]

[0084] Add a certain amount of degummed algal oil, pure water, and enzyme obtained in Example 3 to a reaction kettle. The oil-water ratio is 2:1. Charge nitrogen for protection and stir and react at 50 °C for 2 h. The enzyme addition amounts are 0.02%, 0.05%, 0.10%, and 0.20% of the weight of the algal oil respectively. The results obtained are shown in Table 4.

[0085] Table 4 Hydrolysis results with different enzyme amounts

[0086]

[0087] Through condition optimization, the optimal hydrolysis conditions are obtained as follows: the oil-water ratio is 2:1, the temperature is 50 °C, the time is 2 h, and the enzyme amount is 0.02%.

[0088] Example 5

[0089] Based on Example 4, prepare Schizochytrium sp. SK-3 EPA and DHA triglycerides:

[0090] Perform molecular distillation on the hydrolyzed oil prepared under the optimal hydrolysis conditions in Example 4, and collect the residue in the molecular distillation, which is the enriched algal oil containing EPA and DHA; then perform complete hydrolysis on the obtained residue with Novozym 435 enzyme, with the enzyme amount being 4% of the oil weight, and react until the acid value no longer increases, then separate the oil and water; add the free fatty acids obtained from the complete hydrolysis, 9% by weight of glycerol, and 6% by weight of Novozym 435 enzyme (Novozym 435 immobilized lipase) to the reaction kettle for esterification reaction, and charge nitrogen for protection, and react under stirring, at 60 °C and under vacuum for 6 h. After the reaction is completed, filter the enzyme, and then remove the excess free fatty acids through molecular distillation. The obtained oil is the algal oil of triglyceride type enriched with EPA and DHA. Add 8% by weight of bleaching earth to the obtained triglyceride to remove and 1% of activated carbon to remove impurities such as peroxides, pigments, and heavy metals, and then perform winterization at 0 °C to remove part of the saturated fatty acids.

[0091] After the treatment is completed, through detection, the triglyceride content is 97.45%, and the EPA and DHA contents are 10.01% and 51.13% respectively.

[0092] Example 6

[0093] Based on Example 5, prepare Schizochytrium sp. heptadecanoic acid triglyceride:

[0094] Add the heptadecanoic acid obtained by molecular distillation concentration, 9% by weight of glycerol and 6% by weight of Novozym 435 enzyme to the reaction kettle for esterification reaction, and fill with nitrogen for protection. React for 6 h under the conditions of stirring, 60 °C and vacuum pumping. After the reaction is completed, filter the enzyme, and then remove the excess free fatty acids by molecular distillation. The obtained oil is the triglyceride-type algal oil enriched with heptadecanoic acid. The obtained triglyceride is added with 8% by weight of clay to remove and 1% of activated carbon to remove impurities such as peroxides, pigments and heavy metals, and then winterized at 0 °C to remove some saturated fatty acids.

[0095] Detection of the quality of Schizochytrium sp. SK-3 EPA, DHA and triglyceride of heptadecanoic acid:

[0096] Perform quality determination on the obtained triglyceride, and the results are shown in Table 5 and Table 6.

[0097] Table 5 Quality identification table of Schizochytrium sp. SK-3 EPA and DHA triglyceride.

[0098]

[0099] Table 6 Quality identification table of Schizochytrium sp. SK-3 triglyceride of heptadecanoic acid.

[0100]

[0101] As can be seen from Table 5 and Table 6, for the triglyceride obtained in this application, all indicators meet the quality requirements of algal oil on the market. Therefore, the produced algal oil can be used as an additive and applied to at least one of food, medicine or feed.

[0102] The above-described embodiments only express the specific implementation manners of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the protection scope of this application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of this application, several deformations and improvements can still be made, and these all belong to the protection scope of this application.

[0103] This background art section is provided to generally present the context of the present invention. The work of the currently named inventors, to the extent described in this background art section, and aspects of the work that are not prior art as of the time of filing this application are neither expressly nor implicitly admitted to be prior art to the present invention.

Claims

1. A Schizochytrium sp. SK-3 strain for producing EPA and DHA, characterized in that: It was deposited at the China Center for Type Culture Collection on October 23, 2024, with the deposit number CCTCC NO: M 20242315.

2. Application of Schizochytrium sp. SK-3 as claimed in claim 1 in the preparation of Schizochytrium biomass, the production of EPA, DHA and heptadecanoic acid.

3. The application according to claim 2, wherein: When preparing Schizochytrium biomass, at least 50% by weight of the stem cells of the biomass are fatty acids, and at least 45% of the fatty acids are ω-3 fatty acids.

4. A method for preparing algal oil containing high contents of EPA, DHA and heptadecanoin, characterized in that Schizochytrium sp. SK-3 as claimed in claim 1 is used, and the specific preparation method comprises the following steps: 1) Activation and cultivation: First, transfer Schizochytrium into an activated plate medium for cultivation; then pick a single colony on the activated plate medium and inoculate it into an activated liquid medium for cultivation to obtain a primary seed liquid; then take the primary seed liquid and inoculate it again into the activated liquid medium for cultivation to obtain a secondary seed liquid; 2) Fermentation production: Transfer the secondary seed liquid obtained in step 1) into a fermentation medium, introduce sterile air, and supplement glucose for fermentation cultivation; 3) Cell disruption and oil collection: Centrifuge to collect all cells, add an appropriate amount of pure water and cell wall-breaking enzyme for enzymatic hydrolysis, and then use a high-pressure homogenizer for disruption; after the disruption is completed, centrifuge to collect the upper-layer oil to obtain algal oil; 4) Enrichment of EPA, DHA and heptadecanoic acid in algal oil; a. Add the algal oil, pure water and enzyme obtained in step 3) into a reaction kettle, fill it with nitrogen for protection, and carry out stirring reaction. After the reaction is completed, centrifuge and separate the obtained oil-water mixture. The upper layer is the oil phase and the lower layer is the water phase; b. Carry out molecular distillation on the oil phase obtained in step a, and collect the residue and distillate in the molecular distillation. That is, the residue is the enriched EPA and DHA, and the distillate is the enriched heptadecanoic acid; c. Carry out enzymatic hydrolysis on the residue obtained in step b, centrifuge to collect the upper-layer oil phase, that is, obtain free fatty acids rich in EPA and DHA; d. Add the free fatty acids rich in EPA and DHA obtained in step c, glycerol and lipase into a reaction kettle for esterification reaction, fill it with nitrogen for protection, and react under the conditions of stirring and vacuum pumping. After the reaction is completed, filter, and then remove the unreacted free fatty acids by molecular distillation. The obtained oil is algal oil in the form of triglyceride enriched with EPA and DHA; e. Add the heptadecanoic acid obtained in step b, glycerol and lipase into a reaction kettle for esterification reaction, fill it with nitrogen for protection, and react under the conditions of stirring and vacuum pumping. After the reaction is completed, filter, and then remove the unreacted free fatty acids by molecular distillation. The obtained oil is algal oil in the form of triglyceride enriched with heptadecanoic acid.

5. A method for preparing algal oil containing high contents of EPA, DHA and tristearin according to claim 4, characterized in that: In step 1), the cultivation temperature in the plate medium is 25°C - 30°C, and the cultivation time is 2 - 3 days; the cultivation temperature in the activated liquid medium is 25°C - 30°C, and the cultivation time is 1 - 2 days; the inoculation amount of the primary seed liquid inoculated again into the activated liquid medium is 1 - 10%, the cultivation temperature is 25°C - 30°C, and the cultivation time is 1 - 2 days.

6. A method for preparing algal oil with high contents of EPA, DHA and tricosanoin according to claim 4, characterized in that, In step 2), the addition amount of glucose is based on maintaining its concentration at 5 - 20 g / L; during fermentation production, the dissolved oxygen content in the culture medium is maintained at 10 - 50% by regulating the aeration rate and stirring speed; fermentation is carried out for 72 - 96 h; the inoculation amount of the seed liquid is 5 - 10%, the rotation speed is 100 - 500 rpm, the aeration rate is 1 - 5 L / min, and the fermentation temperature is 25 - 30 °C.

7. A method for preparing algal oil with high contents of EPA, DHA and tricosanoin according to claim 4, characterized in that, In step 3), the addition amount of the cell wall-breaking enzyme is 1 - 10% of the cell mass; the enzyme hydrolysis conditions are: 30 - 40 °C, 100 - 200 rpm, and enzyme hydrolysis for 12 - 24 h; then use a high-pressure homogenizer to break 2 - 5 times, and the pressure is 700 - 1200 bar.

8. A method for preparing algal oil with high contents of EPA, DHA and tricosanoin according to claim 4, characterized in that, In step 4) a, the oil-water mass ratio is 50 - 200%; the enzyme is trypsin; the addition amount of trypsin is 0.02 - 2% of the algal oil mass; the temperature of the stirring reaction is 40 - 60 °C, the reaction time is 0.5 - 3 h, and the stirring speed is 150 - 300 r / min; In step 4) b, the specific steps of molecular distillation are: add the upper oil phase obtained in step a to the feed tank of the secondary molecular distillation device; through the first-stage molecular distillation, dehydrate and degas the oil phase at 90 °C; set the vacuum degree of the second-stage molecular distillation to 0.1 Pa, the scraping film rotation speed to 300 rpm, the feed flow rate to 1.0 mL / min, separate the oil phase at 150 - 200 °C, and collect the obtained residue; In step 4) c, the enzyme hydrolysis conditions are: the oil-water mass ratio is 50 - 200%, and the addition amount of the enzyme is 1 - 5% of the algal oil weight; the enzyme is Novozym 435 immobilized lipase; the enzyme hydrolysis temperature is 45 - 60 °C, the time is 3 - 6 h, and the stirring speed is 200 - 300 rpm; In step 4) d, the esterification reaction is: the reaction temperature is 40 - 60 °C, the reaction time is 4 - 12 h; the dosage of glycerol is 4 - 8% of the fatty acid weight, and the addition amount of the enzyme is 2 - 8% of the algal oil mass; the enzyme is Novozym 435 immobilized lipase.

9. A product of algal oil with high contents of EPA, DHA and heptadecanoin, characterized in that: The product contains the Schizochytrium sp. SK-3 described in claim 1, or Schizochytrium biomass prepared from Schizochytrium sp. SK-3.

10. The product according to claim 9, wherein: By weight, it contains at least 80% of the triglyceride fraction, wherein the DHA content of the triglyceride fraction is at least 40% by weight, the EPA content is at least 6% by weight, and the heptadecanoic acid is at least 7% by weight; the product includes food, cosmetics or pharmaceutical compositions for animals or humans.

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