Schizochytrium sp. mutant strain and application of oil produced by the mutant strain in microcapsules

Abstract

The application provides a schizochytrium mutant and application thereof. The schizochytrium mutant is schizochytrium mutant CABIO-A-2-VII, and the preservation number is CCTCC M 20242741, and the schizochytrium mutant is preserved in the China Center for Type Culture Collection. The schizochytrium mutant CABIO-A-2-VII provided in the application has a great increase in the content of EPA in the polyunsaturated fatty acid oil obtained by fermentation, and the content of C14:0 is high, the content of DHA in the oil obtained by using the strain under a conventional fermentation process is greater than or equal to 40%, the content of EPA is greater than 2%, and the content of C14:0 is greater than or equal to 12% (the content of C14:0 can reach 19% under a preferred fermentation process).

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically, to the application of a Schizochytrium mutant strain and its produced oil in microcapsules. Background Technology

[0002] Schizochytrium sp., also known as the fissochrytic algae, belongs to the family Schizochytriaceae. It is a single-celled, spherical marine fungus rich in docosahexaenoic acid (DHA). It exhibits strong environmental adaptability, rapid growth and reproduction, and can be produced on a large scale through heterotrophic fermentation. Currently, high-density fermentation of Schizochytrium sp. has been used industrially to produce DHA and other unsaturated fatty acids.

[0003] EPA (eicosapentaenoic acid) is an omega-3 polyunsaturated fatty acid with numerous health benefits, including improved blood circulation, reduced blood viscosity, and prevention of cardiovascular disease. Currently, most EPA on the market is derived from deep-sea fish oil, and its yield and quality fluctuate greatly due to factors such as environment, climate, and extraction methods. Marine algae and other microorganisms are primary producers of omega-3 long-chain polyunsaturated fatty acids and possess highly efficient EPA synthesis mechanisms. With the increasing demand for EPA, the fermentation production of EPA using modified microbial strains is gradually becoming the mainstream method.

[0004] The unsaturated fatty acids synthesized by *Schizochytridica* are mainly DHA, with a certain proportion of EPA also present. Studies show that during the fermentation process of *Schizochytridica*, changes in biomass and fatty acid content are affected by factors such as initial glucose content, C / N ratio, liquid volume, and citric acid addition. *Schizochytridica* exhibits two fatty acid synthesis pathways during fermentation: the aerobic desaturase / elongase (FAS) pathway and the polyketide synthase (PKS) pathway. In the FAS pathway, the substrate of the desaturase typically binds to phosphatidylcholine (PC), while the fatty acid substrate utilized by the elongase binds to coenzyme A. During the synthesis of long-chain unsaturated fatty acids, intermediates need to be repeatedly transported between PC and coenzyme A, which is a metabolic bottleneck in the biosynthesis of EPA and DHA, significantly limiting the content of EPA and DHA in heterologous biosynthesis. It has been reported that overexpression of the *Phaeodactylum tricornutum* lysophosphatidylcholine acyltransferase (LPCAT) gene can increase the relative EPA content, and the LPCAT gene copy number is positively correlated with EPA content. However, the specific pathways of EPA anabolic metabolism are still unclear, which limits the application of genetic engineering technology in the industrialization of EPA metabolic regulation. Summary of the Invention

[0005] The first objective of this invention is to provide a Schizochytrium mutant strain, namely Schizochytrium mutant strain CABIO-A-2-VII, with the following accession information: accession number CCTCC M 20242741, classified as Schizochytrium, deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, and deposited on December 6, 2024.

[0006] Ion beam microbial mutagenesis breeding technology (refer to CN111235035A for ion beam mutagenesis methods) is a relatively mature strain selection technology. Combined with high-throughput screening methods designed for the unique physicochemical properties of various products, it can achieve high-throughput selection of high-yield industrial strains. In the process of studying strains with high EPA content, this invention discovered that a strain produced polyunsaturated fatty acid oils with significantly increased EPA content and high C14:0 content. Using this strain, the oil obtained under conventional fermentation processes had DHA content ≥40%, EPA content >2%, and C14:0 ≥12% (under optimized fermentation processes, C14:0 can reach 19%, and EPA content >6%). Furthermore, the oil produced by this strain showed unexpected results in the preparation of microcapsules.

[0007] Another objective of this invention is to provide a method for preparing a polyunsaturated fatty acid oil, which includes the following steps: fermentation culture using the *Schizochytrium* mutant strain CABIO-A-2-VII provided by this invention. Since this fatty acid oil has the highest DHA content, the polyunsaturated fatty acid oil obtained by this invention can also be simply referred to as DHA oil, and will not be described in detail below.

[0008] In a specific embodiment of the present invention, the fermentation culture can use the conventional steps of fermenting strains in the art to obtain oils.

[0009] In a preferred embodiment of the present invention, the concentration of chloride ions in the fermentation medium during the fermentation step is 2.0% to 2.8%, the concentration of magnesium ions is 0.4% to 0.8%, and the concentration of calcium ions is 0.01% to 0.05%. In a preferred embodiment of the present invention, the specific substances added to the fermentation medium are preferably such that the concentrations of chloride ions, magnesium ions, and calcium ions are within the above-mentioned ranges, within which the fermentation effect is better.

[0010] In an optional embodiment of the present invention, the concentration of sodium chloride in the fermentation medium during the fermentation step is 2% to 2.5%. 、The concentrations of magnesium sulfate, calcium chloride, sodium sulfate, potassium chloride, and sodium bicarbonate were 0.4%–0.8%, 0.01%–0.05%, 0.2%–0.6%, 0.04%–0.08%, and 0.005%–0.02%, respectively. To further increase both DHA and EPA content while simultaneously increasing C14:0 content, the fermentation medium in the fermentation step contained 2.3%–2.4% sodium chloride, 0.55%–0.6% magnesium sulfate, 0.011%–0.012% calcium chloride, 0.45%–0.48% sodium sulfate, 0.07%–0.072% potassium chloride, and 0.018%–0.019% sodium bicarbonate.

[0011] In an optional embodiment of the present invention, an artificial seawater formulation can be used in the fermentation medium to prepare DHA lipids. When using an artificial seawater formulation, the concentration of chloride ions must be at least 2.0%–2.8%, the concentration of magnesium ions must be 0.4%–0.8%, and the concentration of calcium ions must be 0.01%–0.05%. Typically, the main components of artificial seawater include sodium chloride, magnesium sulfate, sodium sulfate, potassium chloride, calcium chloride, and sodium bicarbonate.

[0012] In specific embodiments of the present invention, the fermentation medium for the fermentation step further includes a carbon source commonly used in the art. The carbon source may include, but is not limited to, one or more of glucose and yeast extract, preferably including glucose and yeast extract. In a preferred embodiment, the concentration of glucose in the fermentation medium is 7%–12%, and the concentration of yeast extract is 0.75%–1%.

[0013] In a specific embodiment of the present invention, the fermentation culture medium may further include monosodium glutamate, and the concentration of monosodium glutamate may be 2% to 4%.

[0014] In an optional embodiment of the present invention, the fermentation medium may further include potassium dihydrogen phosphate, a trace element stock solution (i.e., solution M), and / or a vitamin mixture (i.e., solution V). The concentration of potassium dihydrogen phosphate may be a concentration commonly used in the art, for example, 0.1%. Commonly used concentrations of trace element stock solution and vitamin mixture may be added as needed.

[0015] In this invention, the trace element mother liquor (i.e., M liquor) may include elements such as nickel, molybdenum, manganese, cobalt, copper, zinc, and iron. For example, the trace element mother liquor may include nickel sulfate hexahydrate, sodium molybdate dihydrate, manganese chloride tetrahydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, zinc sulfate heptahydrate, and ferrous sulfate heptahydrate to provide the aforementioned elements. The vitamin mixture (i.e., V liquor) may include thiamine (VB1), calcium pantothenate (VB5), biotin (VBH), etc. The concentration of each substance can be selected from conventional concentrations in the art, or commercially available trace element mother liquor can be used directly.

[0016] In a preferred embodiment of the present invention, the fermentation temperature of the fermentation step is 24℃~33℃, preferably 24℃~28℃.

[0017] In specific embodiments of the present invention, other parameters in the fermentation step can be commonly used fermentation parameters in the art. In a preferred embodiment of the present invention, the rotation speed in the fermentation step can be 100-800 rpm, and the aeration rate can be 0.5-2 vvm. In a preferred embodiment, citric acid can be used to reduce the pH of the fermentation system to less than 7.8. In a preferred embodiment of the present invention, the glucose content and sugar consumption rate are monitored during fermentation to ensure that the glucose content in the culture medium is not completely depleted, and fermentation is terminated after 144 hours to obtain the Schizochytrium fermentation broth.

[0018] In one specific embodiment of the present invention, the process typically includes, before the fermentation step, activating and expanding the strain to OD. 600 It ranges from 10 to 20.

[0019] In one specific embodiment of the present invention, the strain activation culture includes the following steps: inoculating the Schizochytrium mutant strain provided by the present invention into a primary activation medium to obtain a seed activation culture solution. The primary activation medium preferably comprises: 4%–8% glucose, 2%–3% monosodium glutamate, 0.5%–0.75% yeast extract, 2%–3% sodium chloride, 0.5%–0.6% potassium dihydrogen phosphate, 0.5%–0.8% magnesium sulfate, 0.01%–0.03% calcium chloride, 0.05%–0.1% V solution, and 0.1%–0.2% M solution (trace element stock solution). The culture temperature can be 25–30°C, the culture rotation speed can be 200–250 r / min, and the culture time can be 30–40 h.

[0020] In this field, it is common practice to inoculate the bacterial strain into the bacterial expansion medium at an inoculation rate of 5-20% (volume ratio) when the bacterial concentration in the seed activation culture medium reaches 3-6% (volume ratio) for bacterial expansion culture.

[0021] In one specific embodiment of the present invention, the strain expansion culture includes the following steps: inoculating the seed activation culture solution into a secondary expansion medium to obtain a seed expansion culture solution. The secondary expansion medium preferably comprises: 4%–8% glucose, 2%–3% monosodium glutamate, 0.5%–1% yeast extract, 2%–3% sodium chloride, 0.4%–0.6% sodium sulfate, 0.018%–0.019% sodium bicarbonate, 0.4%–0.5% potassium dihydrogen phosphate, 0.3%–0.5% magnesium sulfate, 0.01%–0.03% calcium chloride, 0.1%–0.12% potassium chloride, 0.1%–0.15% ammonium sulfate, 0.05%–0.1% solution V, and 0.1%–0.2% solution M. The culture temperature can be 25–30°C, the culture rotation speed can be 200–250 r / min, and the culture time is 10–20 h.

[0022] In the scheme of the present invention, when OD in the culture medium 600 When the concentration is 10-20, inoculate it into the fermentation medium at an inoculation rate of 5-10% (volume ratio) for fermentation culture.

[0023] In this invention, the extraction method of the oil is not specifically limited. As long as the oil has a low degree of oxidation, either solvent-based or solvent-free extraction methods are acceptable. In one specific embodiment of this invention, the method further includes the following steps: after the fermentation broth obtained from fermentation culture is subjected to alkaline protease to disrupt the cell wall, hexane is added for extraction, the mixed oil is collected, and the solvent is removed to obtain DHA oil.

[0024] In one specific embodiment of the present invention, the solvent-free extraction method in CN202010421634.X can also be used to obtain DHA oil.

[0025] Another object of the present invention is to provide a polyunsaturated fatty acid oil obtained by the above preparation method.

[0026] In a preferred embodiment of the present invention, the polyunsaturated fatty acid oil contains C14:0 ≥ 12%, EPA content > 2%, and DHA content ≥ 40%.

[0027] An unexpected discovery of this invention is that the microcapsules obtained by fermenting the oil using this strain have low surface oil content and peroxide value, and are odorless.

[0028] Another object of the present invention is to provide a polyunsaturated fatty acid microcapsule comprising a polyunsaturated fatty acid oil, which is obtained by fermentation of the Schizochytrium mutant strain CABIO-A-2-VII provided by the present invention.

[0029] The preferred embodiment of the method for preparing polyunsaturated fatty acid oils provided by the present invention is described above and will not be detailed here.

[0030] In a preferred embodiment of the present invention, the content of polyunsaturated fatty acid oil in the polyunsaturated fatty acid microcapsule is 10% to 45%, wherein the content of DHA (accounting for 5% to 20% of the weight of the polyunsaturated fatty acid microcapsule) is 5%.

[0031] In this invention, the applicant discovered that microcapsules prepared using the polyunsaturated fatty acid oil (also known as DHA oil) provided by this invention and different microcapsule raw materials using commonly used microcapsule preparation methods in the art have lower surface oil content and peroxide value compared to microcapsules prepared from DHA oil obtained by fermentation of other strains in the prior art. Therefore, in this invention, the raw materials and preparation methods for microcapsules can use commonly used raw materials and preparation methods in the art.

[0032] In a specific embodiment of the present invention, the raw materials for the polyunsaturated fatty acid microcapsules may include an emulsifier, which is one or more selected from plant gums, modified starch, and protein, and the mass percentage of the emulsifier may be 4%-20%. The plant gums include, but are not limited to, gellan gum, gum arabic, xanthan gum, vegan gum, gelatin, dextran, pullulan, agar, pectin, carrageenan, alginate, and gelatin, or any combination of at least two of these. Modified starch includes, but is not limited to, sodium octenyl succinate starch. The protein may be a water-soluble protein, which may be plant protein or animal protein. Preferably, the emulsifier is a water-soluble protein. The water-soluble protein may be plant protein or animal protein, wherein the animal protein is preferably one or more selected from sodium caseinate, whey protein, and gelatin, and the plant protein is preferably one or more selected from pea protein, soy protein isolate, hemp seed protein, perilla seed protein, flaxseed protein, rice protein, and chickpea protein. It should be clarified that although the emulsifying properties of various proteins differ, those skilled in the art should possess the ability to adjust the compounding to achieve suitable emulsifying properties for preparing stable microcapsule emulsions from water-soluble proteins. In some embodiments of the present invention, the emulsifier is sodium caseinate, or a mixture of sodium caseinate and whey protein in a mass ratio of (0.5-3.5):1, or a mixture of soy protein and sodium caseinate in a mass ratio of (0.5-3.5):1. In other embodiments of the present invention, the emulsifier is modified starch.

[0033] The raw materials for the microcapsules provided by the present invention may also include one or more selected from water-soluble filler wall materials, pH adjusters, and antioxidants.

[0034] The water-soluble wall filler may include one or more selected from lactose, starch, cellulose, syrup, and dextrin. The water-soluble wall filler preferably comprises no less than 30% by mass.

[0035] The pH adjuster may include one or more selected from citric acid, sodium citrate, potassium hydroxide, sodium hydroxide, sodium bicarbonate, potassium bicarbonate, and sodium carbonate. The main function of the pH adjuster is to maintain the solution at a neutral or slightly acidic level, and it can be added selectively depending on the raw materials.

[0036] The antioxidant may include one or more selected from sodium ascorbate, ascorbic acid, ascorbyl palmitate, vitamin E, phospholipids, and tea polyphenols. The amount of antioxidant used is the conventional amount used in the art.

[0037] The raw materials of the microcapsules provided by this invention may also include an anti-caking agent. The anti-caking agent may include one or more selected from tricalcium phosphate, silicon dioxide, microcrystalline cellulose, and magnesium stearate.

[0038] In some specific embodiments of the present invention, the raw materials of the microcapsules may include the following components in parts by weight: 20-50 parts of polyunsaturated fatty acid oil, 4-20 parts of emulsifier, 30-60 parts of water-soluble filler wall material, 0-0.1 parts of pH adjuster, 1-10 parts of antioxidant, and 0-2 parts of anti-caking agent.

[0039] In some specific embodiments of the present invention, the preparation method of the polyunsaturated fatty acid oil microcapsules includes the following steps: preparing the raw materials into an emulsion, and then drying the emulsion. In some specific embodiments, the preparation method may include the following steps:

[0040] (1) Mix emulsifier, water-soluble wall material, antioxidant, pH adjuster and water, and then add polyunsaturated fatty acid oil to mix, and the mixing includes stirring and shearing;

[0041] (2) Homogenize the mixture obtained in step (1) to obtain an emulsion;

[0042] (3) Dry the emulsion obtained in step (2);

[0043] (4) Optionally, the dried material obtained in step (3) is mixed with an anti-caking agent and then sieved; or the dried material obtained in step (3) is sieved and then mixed with an anti-caking agent.

[0044] In some specific embodiments of the present invention, the shearing rate is 8000-12000 r / min, and the shearing time is 10-30 min. The homogenization pressure can be 600-1000 bar. The homogenization can be performed 2-3 times. The drying methods include, but are not limited to, fluidized bed drying, spray drying, freeze drying, and fluidized bed drying with a base material, all aimed at removing moisture from the liquid material to form solid particles, and are not limited herein. Optionally, if spray drying is used, the process conditions may include: inlet air temperature of 160-180℃, outlet air temperature of 60-80℃, and pressure of 100-200 bar. If fluidized bed drying is used, the temperature may be 30℃-90℃. Those skilled in the art can determine the mesh size according to actual needs, such as 20 mesh, 25 mesh, 30 mesh, etc.

[0045] This invention provides a novel Schizochytrium mutant strain CABIO-A-2-VII. The polyunsaturated fatty acid oil obtained by fermentation using this Schizochytrium mutant strain CABIO-A-2-VII has a significantly increased EPA content and a high C14:0 content. The oil obtained by using this strain under conventional fermentation process has a DHA content ≥40%, an EPA content >2%, and a C14:0 content ≥12% (under the preferred fermentation process, C14:0 can reach 19%, and EPA can be greater than 6%). Detailed Implementation

[0046] The specific embodiments of the present invention will be described in further detail below with reference to the examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0047] In this invention, unless otherwise specified, "%" refers to mass percentage. In this invention, parts by weight can be weight units known in the art such as μg, mg, g, kg, or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0048] In a specific embodiment of the present invention, the vitamin mixture (V) is: 9.5 g / L thiamine (VB1), 3.2 g / L calcium pantothenate (VB5), and 0.06 g / L biotin (VBH);

[0049] The trace element stock solution (M) consists of: 2 g / L nickel sulfate hexahydrate, 0.04 g / L sodium molybdate dihydrate, 3 g / L manganese chloride tetrahydrate, 0.04 g / L cobalt chloride hexahydrate, 2 g / L copper sulfate pentahydrate, 3 g / L zinc sulfate heptahydrate, and 10 g / L ferrous sulfate heptahydrate.

[0050] Example 1

[0051] 1. Breeding of Schizochytrium mutant strains

[0052] (1) Starting strain: Schizochytrium sp., accession number CCTCC NO:M 2015716 (disclosed in CN202311743382.2).

[0053] (2) Strain Mutagenesis: To further increase the EPA content in Schizochytrium, ultraviolet mutagenesis combined with ion beam mutagenesis was used. Mutant strains of Schizochytrium were screened by utilizing high-throughput culture and the sedimentation characteristics of Schizochytrium. The specific methods are as follows:

[0054] ① Activation culture of the starting strain: culture temperature 28℃, shaking speed 250r / min, culture for 48h to the logarithmic growth phase.

[0055] ② UV mutagenesis of the starting strain: Take 1 ml of the activated seed culture solution from step ① above and spread it evenly on a sterile petri dish. Irradiate with a UV lamp for 60 seconds. The UV lamp power is 30 watts and the irradiation distance is 30 cm.

[0056] ③ Ion beam mutagenesis: The bacterial culture after UV mutagenesis was air-dried under sterile conditions and then transferred to a high-energy particle beam injector (sterile). It underwent high-energy N+ ion beam implantation mutagenesis at an energy of 20 keV, with an N+ ion beam implantation dose of 100*10⁻⁶. 17 ions / cm 2 .

[0057] ④ Culture of Mutagenic Strains: The mutagenic bacterial membrane was washed with sterile water, diluted, and spread onto activated medium plates for culture. The solid activated medium consisted of: 10 g / L glucose, 25 g / L sodium glutamate, 10 g / L yeast extract, 20 g / L sodium chloride, 0.5 g / L magnesium sulfate, 20 g / L agar powder, and natural pH. Single colonies after mutagenesis were collected and spotted onto multi-well plates containing fermentation medium using toothpicks for culture at 28°C and 200 r / min for 96 h. The fermentation broth was then transferred to glass test tubes and allowed to settle, and the stratification was observed. Single colonies with slow cell settling were selected for analysis. The selected cells were vacuum-dried, and 20 mg of the sample was taken for fatty acid composition analysis. Single colonies with DHA content higher than 35 wt% and EPA content higher than 1.5 wt% were selected as the starting strains for the next mutagenesis step. The fermentation medium consisted of: 5% glucose, 3% monosodium glutamate, 0.9% yeast extract, 0.26% sodium chloride, 0.53% sodium sulfate, 0.016% sodium bicarbonate, 0.25% potassium dihydrogen phosphate, 0.6% magnesium sulfate, 0.029% calcium chloride, 0.1% potassium chloride, 0.12% ammonium sulfate, 0.1% V solution, and 0.1% M solution.

[0058] (3) Strain selection: In the process of strain selection and domestication, the present invention uses deep well plates for high-throughput culture of Schizochytrium. After fermentation, near-infrared detection is used for screening to obtain a mutant strain CABIO-A-2-VII with a significantly increased content of myristic acid C14:0.

[0059] Example 2

[0060] Fermentation of strains

[0061] Using the *Schizochytrium* mutant strain CABIO-A-2-VII obtained in Example 1, docosahexaenoic acid (DHA) oil was produced using an artificial seawater formulation. Comparative experiments were conducted using the starting strain (CCTCC NO: M 2015716) and strain (CCTCCM2019990, disclosed in CN201911399018.2). The specific methods are as follows:

[0062] (1) Activation culture of the strain: The Schizochytrium strain was inoculated into the primary activation medium (500 μL of glycerol bacteria was inoculated into 150 mL of activation medium) and cultured at 28℃ with a shaking speed of 250 r / min for 40 h. The primary medium formula was: 4% glucose, 3% monosodium glutamate, 0.6% yeast extract, 2% sodium chloride, 0.6% potassium dihydrogen phosphate, 0.8% magnesium sulfate, 0.03% calcium chloride, 0.1% V solution, and 0.1% M solution.

[0063] (2) Strain expansion culture: 10 mL of primary activation solution was inoculated into 200 mL of secondary expansion culture medium, with an inoculation amount of 5%. The culture temperature was 28℃, the rotation speed was 250 r / min, and the culture time was 24 h. The secondary culture medium formula was: 5% glucose, 3% monosodium glutamate, 0.9% yeast extract, 0.22% sodium chloride, 0.44% sodium sulfate, 0.013% sodium bicarbonate, 0.21% potassium dihydrogen phosphate, 0.5% magnesium sulfate, 0.024% calcium chloride, 0.08% potassium chloride, 0.1% ammonium sulfate, 0.1% V solution, and 0.1% M solution.

[0064] (3) Fermentation tank culture: 200 mL of secondary expansion strain (OD600 is 12) is inoculated into the fermentation medium at an inoculation amount of 5% and the culture temperature is 28℃.

[0065] The fermentation medium formula for Fermentation Example 1 is as follows: 10% glucose, 2% monosodium glutamate, 1.75% yeast extract, 2% sodium chloride, 0.5% magnesium sulfate, 0.01% calcium chloride, 0.4% sodium sulfate, 0.06% potassium chloride, and 0.016% sodium bicarbonate.

[0066] The concentrations of other substances in the fermentation medium of Fermentation Example 2 were the same as those in Fermentation Example 1, but the content of inorganic salts (magnesium sulfate, calcium chloride, sodium sulfate, potassium chloride, sodium bicarbonate) was 1.2 times that of Fermentation Example 1.

[0067] (4) Schizochytrium fermentation process

[0068] Fermentation temperature was 28℃; culture speed was 220 rpm; aeration rate was 1.5 vvm; pH was controlled to be less than 7.8 with 30% citric acid; 10% glucose was added before inoculation; glucose content and sugar consumption rate were monitored during fermentation to ensure that the glucose content in the culture medium was not completely consumed; fermentation was terminated after 144 hours to obtain the Schizochytrium fermentation broth.

[0069] (5) Post-processing: After the fermentation broth obtained from fermentation culture is broken by alkaline protease, hexane is added as an extractant for extraction. The solid phase obtained after extraction is transferred to an extraction vessel for repeated extraction. This process is repeated until there is no oil in the extract. 200 ml of hexane is added for the first extraction, and 150 ml of hexane is added each time thereafter. The mixed oil obtained after each extraction is filtered and desolventized to obtain microbial oil.

[0070] (6) Gas chromatography analysis: The microbial oil obtained in step (5) was analyzed by gas chromatography.

[0071] (7) The DHA oils in Table 1 were derived from three different strains: Comparative Example 1 was the starting strain, with accession number CCTCC NO: M2015716, and Comparative Example 2 had accession number CCTCC M2019990; Fermentation Examples 1 and 2 were both mutant strains of the present invention, CABIO-A-2-VII. Apart from this, the other fermentation, post-treatment, and gas chromatography analysis methods were as described above.

[0072] Table 1. Fatty acid content (wt%) in DHA lipids from different bacterial strains

[0073]

[0074] Fatty acids such as C15:0, C16:1, and C17:0 are not listed in Table 1 due to their extremely low content. Compared with the other two strains, the proportion of C14:0 in the fatty acid composition of the fermented oil of strain CABIO-A-2-VII is significantly increased, reaching 12.65%, and preferably up to 14.01%; the EPA content is slightly increased (>2%); and the content of characteristic fatty acids is superior to that of the starting strain, provided that the DHA content is suitable for application.

[0075] Example 3

[0076] Fermentation of CABIO-A-2-VII strain under different fermentation processes

[0077] In Table 2, methods 1-4 all use the CABIO-A-2-VII strain for microbial fermentation, and methods 5-8 all use the starting strain for microbial fermentation. The specific fermentation process and formula are as follows: (The steps of strain activation culture and strain expansion culture are the same as those in Example 2).

[0078] Method 1: Inoculate 200 mL of secondary expansion culture medium into the fermentation medium (inoculum size 5%), and incubate at 28℃. Fermentation medium formula: 10% glucose, 2% monosodium glutamate, 0.75% yeast extract, 2.4% sodium chloride, 0.6% magnesium sulfate, 0.012% calcium chloride, 0.48% sodium sulfate, 0.072% potassium chloride, and 0.019% sodium bicarbonate.

[0079] Method 2: Inoculate 200 mL of secondary expansion culture medium into the fermentation medium (inoculum size 5%), and incubate at 28℃. Fermentation medium formula: 10% glucose, 2% monosodium glutamate, 0.75% yeast extract, 2% sodium chloride, 0.5% magnesium sulfate, 0.01% calcium chloride, 0.4% sodium sulfate, 0.06% potassium chloride, and 0.016% sodium bicarbonate.

[0080] Method 3: Inoculate 200 mL of secondary expansion culture medium into the fermentation medium (inoculum size 5%), and incubate at 33℃. Fermentation medium formula: 10% glucose, 2% monosodium glutamate, 0.75% yeast extract, 2% sodium chloride, 0.8% magnesium sulfate, 0.01% calcium chloride, 0.2% sodium sulfate, 0.06% potassium chloride, 0.005% sodium bicarbonate, and 0.1% potassium dihydrogen phosphate.

[0081] Method 4: Inoculate 200 mL of secondary expansion culture medium into the fermentation medium (inoculum size 5%), and incubate at 28℃. Fermentation medium formula: 10% glucose, 2% monosodium glutamate, 0.75% yeast extract, 2% sodium chloride, 0.4% magnesium sulfate, 0.01% calcium chloride, 0.4% sodium sulfate, 0.04% potassium chloride, 0.016% sodium bicarbonate, 0.2% M solution. Micronutrient stock solution (M): 2 g / L nickel sulfate hexahydrate, 0.04 g / L sodium molybdate dihydrate, 3 g / L manganese chloride tetrahydrate, 0.04 g / L cobalt chloride hexahydrate, 2 g / L copper sulfate pentahydrate, 3 g / L zinc sulfate heptahydrate, 10 g / L ferrous sulfate heptahydrate.

[0082] Method 5: Inoculate 200 mL of secondary expansion culture medium into the fermentation medium (inoculum size 5%), and incubate at 24℃. Fermentation medium formula: 10% glucose, 2% monosodium glutamate, 0.75% yeast extract, 2.4% sodium chloride, 0.6% magnesium sulfate, 0.012% calcium chloride, 0.48% sodium sulfate, 0.072% potassium chloride, and 0.019% sodium bicarbonate.

[0083] Method 6: Inoculate 200 mL of secondary expansion culture medium into the fermentation medium (inoculum size 5%), and incubate at 28℃. Fermentation medium formula: 10% glucose, 2% monosodium glutamate, 0.75% yeast extract, 2.5% sodium chloride, 0.5% magnesium sulfate, 0.05% calcium chloride, 0.6% sodium sulfate, 0.06% potassium chloride, and 0.016% sodium bicarbonate.

[0084] Method 7: Inoculate 200 mL of secondary expansion culture medium into the fermentation medium (inoculum size 5%), and incubate at 33℃. Fermentation medium formula: 10% glucose, 2% monosodium glutamate, 0.75% yeast extract, 2% sodium chloride, 0.5% magnesium sulfate, 0.01% calcium chloride, 0.4% sodium sulfate, 0.06% potassium chloride, 0.016% sodium bicarbonate, and 0.1% potassium dihydrogen phosphate.

[0085] Method 8: Inoculate 200 mL of secondary expansion culture medium into the fermentation medium (inoculum size 5%), and incubate at 28℃. Fermentation medium formula: 10% glucose, 2% monosodium glutamate, 0.75% yeast extract, 2% sodium chloride, 0.5% magnesium sulfate, 0.01% calcium chloride, 0.4% sodium sulfate, 0.06% potassium chloride, 0.016% sodium bicarbonate, 0.2% M solution. Micronutrient stock solution (M): 2 g / L nickel sulfate hexahydrate, 0.04 g / L sodium molybdate dihydrate, 3 g / L manganese chloride tetrahydrate, 0.04 g / L cobalt chloride hexahydrate, 2 g / L copper sulfate pentahydrate, 3 g / L zinc sulfate heptahydrate, 10 g / L ferrous sulfate heptahydrate.

[0086] Table 2. Fatty acid content (wt%) of CABIO-A-2-VII strain under different fermentation processes

[0087]

[0088]

[0089] As shown in Table 2, the C14:0 content of strain CABIO-A-2-VII fluctuated in different fermentation methods, with the content reaching 19.61% in method 1. This indicates that different fermentation processes affect the C14:0 content, but overall the C14:0 content can be maintained above 12%. At the same time, compared with the starting strain, under the same process, the C14:0 content of strain CABIO-A-2-VII was significantly higher than that of the starting strain, and the contents of EPA and DHA were also increased, with EPA reaching over 6%.

[0090] Example 3

[0091] Preparation of polyunsaturated fatty acid microcapsules

[0092] The protein-formulated polyunsaturated fatty acid oil microcapsules are composed of the following components (by weight percentage): sodium caseinate 8%, whey protein powder 10%, DHA oil 43%, sodium ascorbate 2.5%, tricalcium phosphate 0.6%, sodium citrate 0.01%, and the remainder being maltodextrin. The preparation method of these polyunsaturated fatty acid oil microcapsules includes the following steps:

[0093] (1) Sodium caseinate, whey protein powder, sodium ascorbate, sodium citrate, maltodextrin and water are mixed and dissolved, and then DHA oil is added for shear emulsification, wherein the shearing is performed at 10000r / min for 15min.

[0094] (2) Homogenize the liquid obtained in step (1) twice at 800 bar to obtain an emulsion;

[0095] (3) The emulsion obtained in step (2) is directly spray-dried. The spray-drying conditions are: inlet air temperature 170±5℃, outlet air temperature 70±5℃, and spray pressure 20 bar.

[0096] (4) After passing the dried material obtained in step (3) through a 20-mesh sieve, mix it with tricalcium phosphate to obtain polyunsaturated fatty acid oil microcapsule product.

[0097] Among them, the DHA oils used in Experiment 1-2 are the oils obtained from fermentation Example 2 of CABIO-A-2-VII and the oils obtained from fermentation using Method 4 in Table 2, respectively. The oils produced in Experiment 1 and 2 are those produced by CCTCCM2019990 and CCTCC NO:M 2015716, and the fatty acids correspond to the fatty acid composition in Table 1.

[0098] The starch-based polyunsaturated fatty acid oil microcapsules are composed of the following ingredients (by mass percentage): 20% sodium octenyl succinate starch, 43% DHA oil, 2.5% sodium ascorbate, 0.6% tricalcium phosphate, 0.01% sodium citrate, and the remainder being maltodextrin. The preparation method is the same as that for polyunsaturated fatty acid oil microcapsules, resulting in the microcapsules of Experimental Example 3. The DHA oil used was the oil obtained in Example 2 of CABIO-A-2-VII. Comparative Experimental Example 3 used oil obtained using CCTCM2019990.

[0099] Determination methods

[0100] 1. Method for determining surface oil content: Weigh 50.00 g of microcapsules into a 250 mL Erlenmeyer flask, add 100 mL of petroleum ether, shake for 1 min, allow to stand for separation, and filter the supernatant into a constant-weight flat-bottom flask. Then add another 100 mL of petroleum ether to rinse the iodine flask and the microcapsules inside, and combine the rinses and filter into the flat-bottom flask. Desolvate, then place in a vacuum oven at 60 °C for 4 hours. After that, remove and place in a desiccator to cool to room temperature, and weigh on a 0.01 g electronic balance.

[0101] The surface oil content (X%) is calculated using the following formula:

[0102] Surface oil content (X%) = (m1 - m0) / m

[0103] Where: m1—mass of surface oil added to the flat-bottomed flask, g;

[0104] m0—mass of the flat-bottomed flask after constant weight, in g;

[0105] m — the mass of the powder weighed, in grams.

[0106] 2. Peroxide value: The peroxide value shall be tested in accordance with GB 5009.227.

[0107] 3. Sensory evaluation: Open the bag and smell the sample every three days. Rate the fishy and rancid smell of the sample directly: 1 represents faint, 2 represents slight, 3 represents moderate, and 4 represents strong.

[0108] Indicator evaluation: The same letter indicates no significant difference, and no identical letter indicates a difference.

[0109] (1) Microcapsule encapsulation effect detection

[0110] The results are shown in Table 3.

[0111] Table 3. Microcapsule encapsulation effect

[0112] Serial Number Surface oil Peroxide value meq / kg Experimental Example 1 0.36％ 0.45 Experimental Example 2 0.33％ 0.46 Experimental Example 3 0.41％ 0.52 Comparative Experiment Example 1 0.45％ 0.50 Comparative Experiment Example 2 0.43％ 0.50 Comparative Experiment Example 3 0.48％ 0.55

[0113] Microcapsule encapsulation effect: As shown in Table 3, in terms of surface oil content, the experimental case showed better overall surface oil content compared to the control case with the same formulation. Although all groups were at a low level in the initial peroxide value determination, the experimental case showed better peroxide value performance.

[0114] (2) Simple sensory evaluation: accelerated treatment at 60℃. The results are shown in Table 4.

[0115] Table 4. Simple sensory evaluation after acceleration at 60℃

[0116] Serial Number 60℃ accelerates sensory perception Experimental Example 1# <![CDATA[30 days a , 1 fishy a > Experimental Example 2# <![CDATA[30 days a , 1 fishy a > Experimental Example 3# <![CDATA[30 days a , 2 fishy a > Comparative Experiment Example 1# <![CDATA[21 days b , 3 fishy b ; 30 days a , 2 ha b > Comparative Experiment Example 2# <![CDATA[21 days b , 4 fishy b ; 30 days a , 3 ha b > Comparative Experiment Example 3# <![CDATA[21 days b , 3 fishy b ; 30 days a , 3 halal b > It should be noted that the content of "3腥" and "3哈" seems rather unclear and may need further clarification in the original context to ensure a more accurate translation.

[0117] (3) Complex sensory evaluation:

[0118] The prepared microcapsules were treated at 37℃ for 30 days, followed by sensory evaluation. The results are shown in Table 5. The external odor was evaluated by scoring the intensity of the fishy smell on a 7-point scale (none – 0; possibly present – ​​1; slightly – 2; moderate – 3–4; fairly strong – 5–6; very strong – 7). Considering all attributes, the overall preference of the samples was scored.

[0119] The liking level was rated on a 9-point scale (extremely like - 9 points; very like - 8 points; somewhat like - 7 points; slightly like - 6 points; neither like nor dislike - 5 points; slightly dislike - 4 points; somewhat dislike - 3 points; very dislike - 2 points; extremely dislike - 1 point). The average of the results was taken (15 people were randomly selected from professional sensory evaluators for each group).

[0120] Table 5 Complex Sensory Evaluation

[0121]

[0122] Sensory evaluation: As shown in Tables 4 and 5, there was no significant difference between Experimental Examples 1# and 2# in terms of fishy smell intensity and overall preference, but there was a significant difference compared with Comparative Experimental Examples 1# and 2#. The sensory evaluation of Experimental Example 3 was better than that of Comparative Experimental Example 3, and the Example was significantly better than the Comparative Example.

[0123] Finally, the method of this invention is merely a preferred embodiment and is not intended to limit the scope of protection of this invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A Schizochytrium mutant strain, characterized in that, The Schizochytrium mutant strain is CABIO-A-2-VII, with accession number CCTCC M 20242741, and is deposited at the China Center for Type Culture Collection.

2. A method for preparing a polyunsaturated fatty acid oil, characterized in that, The method includes the following steps: obtaining the strain by fermentation culture using the Schizochytrium mutant strain described in claim 1.

3. The preparation method according to claim 2, characterized in that, The concentration of chloride ions in the fermentation medium of the fermentation step is 2.0%~2.8%, the concentration of magnesium ions is 0.4%~0.8%, and the concentration of calcium ions is 0.01%~0.05%.

4. The preparation method according to claim 3, characterized in that, The fermentation medium in the fermentation step has the following concentrations: sodium chloride 2%~2.5%, magnesium sulfate 0.4%~0.8%, calcium chloride 0.01%~0.05%, sodium sulfate 0.2%~0.6%, potassium chloride 0.04%~0.08%, and sodium bicarbonate 0.005%~0.02%.

5. The preparation method according to claim 4, characterized in that, The fermentation temperature for the fermentation step is 24℃~33℃.

6. The preparation method according to claim 5, characterized in that, The fermentation temperature for the fermentation step is 24℃~28℃.

7. The polyunsaturated fatty acid oil obtained by the preparation method according to any one of claims 2 to 6.

8. The polyunsaturated fatty acid oil according to claim 7, characterized in that, The polyunsaturated fatty acid oil contains C14:0 ≥ 12%, EPA content > 2%, and DHA content ≥ 40%.

9. A polyunsaturated fatty acid microcapsule, characterized in that, It includes polyunsaturated fatty acid oils, which are obtained by fermentation of the Schizochytrium mutant strain described in claim 1.

10. The polyunsaturated fatty acid microcapsules according to claim 9, characterized in that, The concentrations of chloride ions, magnesium ions, and calcium ions in the fermentation medium were 2.0%–2.8%, 0.4%–0.8%, and 0.01%–0.05%, respectively.

11. The polyunsaturated fatty acid microcapsules according to claim 9 or 10, characterized in that, The DHA content in oil ranges from 10% to 45%.

Images