Application of schizochytrium limacinum mutant strain and grease produced by schizochytrium limacinum mutant strain in microcapsules

Abstract

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

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and more specifically to a schizochytrium mutant and application of oil produced thereby in microcapsules. Background Art

[0002] Schizochytrium sp., also known as Schizochytrium sp., belongs to the Thraustochytriaceae family. It is a unicellular, spherical marine fungus rich in docosahexaenoic acid (DHA). It has strong environmental adaptability, rapid growth and reproduction, and can be fermented and produced on a large scale through heterotrophic growth. Currently, the industry has achieved the production of unsaturated fatty acids such as DHA through high-density fermentation of Schizochytrium sp.

[0003] EPA (Eicosapentaenoic Acid) is a polyunsaturated fatty acid of the ω-3 series, which has many benefits for human health, including improving blood circulation, reducing blood viscosity, and preventing cardiovascular diseases. Currently, EPA on the market mainly comes from deep-sea fish oil. Its production and quality fluctuate greatly due to factors such as the environment, climate, and extraction method. Microorganisms such as marine algae are primary producers of ω-3 ultra-long-chain polyunsaturated fatty acids and have an efficient EPA synthesis mechanism. With the increasing demand for EPA, the method of producing EPA by fermentation using modified microbial strains has gradually become the mainstream.

[0004] The unsaturated fatty acids synthesized by Schizochytrium are mainly DHA, and there is also a certain proportion of EPA. Studies have shown that during the fermentation process of Schizochytrium, the changes in biomass and fatty acid content are affected by factors such as the initial glucose content, carbon-nitrogen ratio, liquid volume, and citric acid addition. Schizochytrium has two fatty acid synthesis pathways during the fermentation process: the aerobic desaturase / elongase (FAS) pathway and the polyketide synthase (PKS) pathway. In the FAS pathway, the substrate of the desaturase is usually bound to phosphatidylcholine (PC), while the fatty acid substrate used by the elongase is bound to coenzyme A. During the synthesis of long-chain unsaturated fatty acids, the intermediates need to be repeatedly transported between PC and coenzyme A, which is a metabolic bottleneck in the biosynthesis of EPA and DHA, greatly limiting the content of EPA and DHA in heterologous biosynthesis. It has been reported that overexpression of the triangular lysophosphatidylcholine acyltransferase (LPCAT) gene of Phaeodactylum triangularis can increase the relative content of EPA, and the copy number of the LPCAT gene is positively correlated with the EPA content. However, the specific pathway of EPA anabolism is still unclear, which limits the application of genetic engineering technology in the industrialization of EPA metabolic regulation. Summary of the invention

[0005] The first object of the present invention is to provide a Schizochytrium mutant strain, which is the Schizochytrium mutant strain CABIO-A-2-VII, and the preservation information is: the preservation number is CCTCC M 20242741, the classification name is Schizochytrium, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan University, and the preservation date is December 6, 2024.

[0006] Ion beam microbial mutagenesis breeding technology (reference for ion beam mutagenesis method: CN111235035A) is a relatively mature strain selection technology. Combined with a high-throughput screening method designed for the unique physical and chemical properties of various products, it can achieve high-throughput selection of industrial high-yield strains. In the process of studying strains with high EPA content, the present invention found that in addition to a significant increase in EPA content, the C14:0 content of the polyunsaturated fatty acid oil produced by a strain was relatively high. The DHA content of the oil obtained by using the strain under conventional fermentation technology was ≥40%, the EPA content was >2%, and the C14:0 was ≥12% (C14:0 could reach 19% and EPA content >6% under the preferred fermentation technology), and it was found that the oil produced by the strain had an unexpected effect in the application of preparing microcapsules.

[0007] Another object of the present invention is to provide a method for preparing a polyunsaturated fatty acid oil, the preparation method comprising the following steps: fermenting and culturing the mutant strain CABIO-A-2-VII of Schizochytrium provided by the present invention. Since the content of DHA in the fatty acid oil is the highest, the polyunsaturated fatty acid oil obtained by the present invention may also be referred to as DHA oil, which will not be described in detail below.

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

[0009] In a preferred embodiment of the present invention, the concentration of chloride ions in the fermentation medium in 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 ranges, and within the above ranges, the fermentation effect is better.

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

[0011] In an optional embodiment of the present invention, an artificial seawater formula can be used in the fermentation medium to prepare DHA oil, and when using the artificial seawater formula, at least the concentration of chloride ions needs to be determined to be 2.0% to 2.8%, the concentration of magnesium ions needs to be 0.4% to 0.8%, and the concentration of calcium ions needs to be 0.01% to 0.05%. Generally, the main components of artificial seawater include sodium chloride, magnesium sulfate, sodium sulfate, potassium chloride, calcium chloride, and sodium bicarbonate.

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

[0013] In a specific embodiment of the present invention, the fermentation medium may further include sodium glutamate, and the concentration of sodium 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 mother solution (i.e., M solution), and / or a vitamin mixture solution (i.e., V solution). The concentration of potassium dihydrogen phosphate may be a concentration commonly used in the art, for example, 0.1%. The trace element mother solution and the vitamin mixture solution of commonly used concentrations may be added according to actual needs.

[0015] In the present invention, the trace element mother solution (i.e., M solution) may include elements such as nickel, molybdenum, manganese, cobalt, copper, zinc, and iron. For example, the trace element mother solution 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 above elements. The vitamin mixture (i.e., V solution) may include thiamine VB1, calcium pantothenate (VB5), biotin (VBH), etc. The concentration of each substance may be selected from the conventional concentrations in the art, or commercially available trace element mother solutions may be directly used.

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

[0017] In a specific embodiment of the present invention, other parameters in the fermentation step may be fermentation parameters commonly used in the art. In a preferred embodiment of the present invention, the rotation speed in the fermentation step may be 100 to 800 rpm, and the ventilation volume may be 0.5-2 vvm. In a preferred embodiment, citric acid may be used to reduce the pH in 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 the fermentation process to ensure that the glucose content in the culture medium is not completely consumed, and the fermentation is terminated after 144 hours to obtain the Schizochytrium fermentation broth.

[0018] In one embodiment of the present invention, before the fermentation step, the method further includes activating the strain and expanding the strain to an OD of 600 It is 10 to 20.

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

[0020] In the art, when the bacterial concentration in the seed activation culture solution reaches 3-6% (volume ratio), the seed is usually inoculated into the bacterial expansion medium at an inoculation rate of 5-20% (volume ratio) for strain expansion culture.

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

[0022] In the scheme of the present invention, when the OD 600 When the concentration is 10 to 20, the inoculation amount is 5 to 10% (volume ratio) inoculated into the fermentation medium for fermentation culture.

[0023] In the present invention, the extraction method of oil is not particularly limited, and the extraction method can be either solvent-containing or solvent-free, provided that low-oxidation oil is obtained. In a specific embodiment of the present invention, the following steps are also included: the fermentation broth obtained by fermentation culture is broken by alkaline protease, and then n-hexane is added for extraction, and the mixed oil is taken and desolventized to obtain DHA oil.

[0024] In a specific embodiment of the present invention, DHA oil can also be obtained by the solvent-free extraction method in CN202010421634.X.

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

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

[0027] The present invention unexpectedly found that the microcapsules obtained by using the oil fermented by the strain have low surface oil content and peroxide value and no fishy smell.

[0028] Another object of the present invention is to provide a polyunsaturated fatty acid microcapsule, wherein the polyunsaturated fatty acid microcapsule comprises polyunsaturated fatty acid oil, and the polyunsaturated fatty acid oil is produced by fermenting the Schizochytrium mutant CABIO-A-2-VII provided by the present invention.

[0029] Among them, the preferred embodiments of the method for preparing the polyunsaturated fatty acid oil provided by the present invention are referred to the above content and will not be described in detail here.

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

[0031] In the present invention, the applicant has found that the microcapsules prepared by using the polyunsaturated fatty acid oil (also called DHA oil) provided by the present invention and different microcapsule raw materials using the microcapsule preparation method commonly used in the art have lower surface oil content and peroxide value than the microcapsules prepared by DHA oil fermented by other strains in the prior art. Therefore, in the present invention, the raw materials and preparation methods of the microcapsules can use the raw materials and preparation methods commonly used in the art.

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

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

[0034] The water-soluble filling wall material may include one or more selected from lactose, starch, cellulose, syrup, and dextrin. The mass percentage of the water-soluble filling wall material is preferably not less than 30%.

[0035] The pH regulator may include one or more selected from citric acid, sodium citrate, potassium hydroxide, sodium hydroxide, sodium bicarbonate, potassium bicarbonate, and sodium carbonate. The pH regulator is mainly used to maintain the feed liquid at a neutral or weakly acidic level, and can be selectively added according to the raw material conditions.

[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 the antioxidant is the conventional amount in the art.

[0037] The raw materials of the microcapsules provided by the present invention may further include an anticaking agent, which 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 filling wall material, 0-0.1 parts of pH regulator, 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 comprises the following steps: preparing the raw materials into an emulsion, and then drying the emulsion. In some specific embodiments, the preparation method may comprise the following steps:

[0040] (1) mixing an emulsifier, a water-soluble wall material, an antioxidant, a pH regulator and water, and then adding a polyunsaturated fatty acid oil to mix to obtain a mixture, wherein the mixing includes stirring and shearing;

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

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

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

[0044] In some specific embodiments of the present invention, the shear rate is 8000-12000r / min, and the shear time is 10-30min. The homogenization pressure can be 600-1000bar. The number of homogenizations can be 2-3 times. The drying method includes but is not limited to boiling drying, spray drying, freeze drying, fluidized drying, etc. in the presence of a base material, with the purpose of removing water from the liquid to form solid particles, which is not limited here. Optionally, if spray drying is selected, the process conditions of spray drying may include: an inlet air temperature of 160-180°C, an outlet air temperature of 60-80°C, and a pressure of 100-200bar. If boiling drying is selected, the temperature of boiling drying can be 30°C-90°C. Those skilled in the art can determine the mesh size of the sieving according to actual needs, such as 20 mesh, 25 mesh, 30 mesh, etc.

[0045] The invention provides a novel Schizochytrium mutant strain CABIO-A-2-VII. In polyunsaturated fatty acid oils obtained by fermentation using the Schizochytrium mutant strain CABIO-A-2-VII, the EPA content is greatly improved, and the C14:0 content is relatively high. In the oils obtained by using the strain under a conventional fermentation process, the DHA content is ≥40%, the EPA content is >2%, and the C14:0 content is ≥12% (under a preferred fermentation process, the C14:0 content can reach 19%, and the EPA content can be greater than 6%). DETAILED DESCRIPTION

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0047] In the present invention, unless otherwise specified, "%" means mass percentage. In the present invention, parts by weight may be μg, mg, g, kg, or other weight units known in the art, 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), 0.06 g / L biotin (VBH);

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

[0050] Example 1

[0051] 1. Breeding of Schizochytrium mutants

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

[0053] (2) Strain mutagenesis: In order to further increase the EPA content in Schizochytrium, ultraviolet mutagenesis combined with ion beam mutagenesis technology was used to screen Schizochytrium mutant strains through 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°C, shaking speed of 250r / min, culture for 48h until the logarithmic growth phase.

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

[0056] ③ Ion beam mutagenesis: The bacterial solution after UV mutagenesis was air-dried in a sterile environment, and then transferred into a high-energy particle beam implanter (sterile) for mutagenesis by high-energy N+ ion beam implantation with an energy of 20 keV. The N+ ion beam implantation dose was 100*10 17 ions / cm 2 .

[0057] ④ Mutation strain culture: The bacterial film after the above mutagenesis treatment was washed with sterile water, diluted, and spread on the activation medium plate for culture. The solid activation medium is: glucose 10g / L, sodium glutamate 25g / L, yeast extract 10g / L, sodium chloride 20g / L, magnesium sulfate 0.5g / L, agar powder 20g / L, pH natural. Collect the single colonies after mutagenesis, and use toothpicks to pick them into a porous plate filled with fermentation medium for culture. The culture temperature is 28°C, the shaking speed of the shaker is 200r / min, and the culture is 96h; the fermentation liquid in the porous plate is transferred to a glass test tube, and the sedimentation is allowed to stand to observe the stratification; the fermentation liquid of the single colony with slow cell sedimentation is selected for detection and analysis; the selected cells are vacuum dried, and 20mg samples are taken for fatty acid composition analysis. The single colony with a DHA content higher than 35wt% and an EPA content higher than 1.5wt% in the fatty acid composition is used as the starting strain for the next mutagenesis. The fermentation medium is: 5% glucose, 3% sodium 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 sorting: In the process of strain selection and domestication, the present invention uses a deep-well plate 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 myristic acid C14:0 content.

[0059] Example 2

[0060] Strain fermentation

[0061] The Schizochytrium mutant CABIO-A-2-VII obtained in Example 1 was used to produce docosahexaenoic acid (DHA) oil using an artificial seawater formula. A comparative experiment was conducted using the starting strain (CCTCC NO: M 2015716) and the strain (CCTCCM2019990, disclosed in CN201911399018.2). The specific method is as follows:

[0062] (1) Strain activation culture: The Schizochytrium strain was inoculated into a primary activation medium (500 μL of glycerol was inoculated into 150 mL of activation medium), the culture temperature was 28°C, the shaking speed of the shaker was 250 r / min, and the culture was carried out for 40 hours. The primary culture medium formula: 4% glucose, 3% sodium 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, 0.1% M solution.

[0063] (2) Strain expansion culture: 10 mL of the first-level activation solution was inoculated into 200 mL of the second-level expansion medium, with an inoculation volume of 5%, a culture temperature of 28°C, a rotation speed of 250 r / min, and a culture time of 24 h. Second-level culture medium formula: 5% glucose, 3% sodium 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 the secondary expansion strain (OD600 is 12) was inoculated into the fermentation medium, the inoculation amount was 5%, and the culture temperature was 28°C.

[0065] The fermentation medium formula of Fermentation Example 1 is as follows: 10% glucose, 2% sodium glutamate, 1.75% yeast extract powder, 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, and 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] The fermentation temperature is 28°C; the culture speed is 220 rpm, the ventilation volume is 1.5 vvm, and the pH value is controlled to be less than 7.8 with 30% citric acid; 10% glucose is added before inoculation, and the glucose content and sugar consumption rate are monitored during the fermentation process to ensure that the glucose content in the culture medium will not be completely consumed. The fermentation is terminated after 144 hours to obtain the Schizochytrium fermentation liquid.

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

[0070] (6) Gas chromatography analysis: The microbial oil obtained in step (5) is subjected to gas chromatography analysis.

[0071] (7) The DHA oils in Table 1 come from three strains: Comparative Example 1 is the starting strain, with a preservation number of CCTCC NO: M2015716, and Comparative Example 2 has a preservation number of CCTCCM2019990; Fermentation Examples 1 and 2 are both mutant strains CABIO-A-2-VII of the present invention. In addition, the rest of the fermentation, post-treatment and gas chromatography analysis methods are as described above.

[0072] Table 1 Fatty acid content in DHA oils of different strains (wt%)

[0073]

[0074] Among them, 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 CABIO-A-2-VII strain was greatly increased to 12.65%, preferably up to 14.01%; the EPA content was slightly increased (>2%); and the characteristic fatty acid content was better than that of the starting strain under the premise that the DHA content was suitable for application.

[0075] Example 3

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

[0077] Methods 1-4 in Table 2 all use CABIO-A-2-VII strain for microbial fermentation, and methods 5-8 all use starting strain for microbial fermentation. The specific fermentation process and formula are as follows: (wherein, 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 the secondary expansion strain into the fermentation medium, with an inoculation volume of 5% and a culture temperature of 28°C. Fermentation medium formula: 10% glucose, 2% sodium glutamate, 0.75% yeast extract powder, 2.4% sodium chloride, 0.6% magnesium sulfate, 0.012% calcium chloride, 0.48% sodium sulfate, 0.072% potassium chloride, 0.019% sodium bicarbonate.

[0079] Method 2: Inoculate 200 mL of the secondary expansion strain into the fermentation medium, with an inoculation volume of 5% and a culture temperature of 28°C. Fermentation medium formula: 10% glucose, 2% sodium glutamate, 0.75% yeast extract powder, 2% sodium chloride, 0.5% magnesium sulfate, 0.01% calcium chloride, 0.4% sodium sulfate, 0.06% potassium chloride, 0.016% sodium bicarbonate.

[0080] Method 3: Inoculate 200 mL of the secondary expansion strain into the fermentation medium, with an inoculation volume of 5% and a culture temperature of 33°C. Fermentation medium formula: 10% glucose, 2% sodium glutamate, 0.75% yeast extract powder, 2% sodium chloride, 0.8% magnesium sulfate, 0.01% calcium chloride, 0.2% sodium sulfate, 0.06% potassium chloride, 0.005% sodium bicarbonate, 0.1% potassium dihydrogen phosphate.

[0081] Method 4: Inoculate 200 mL of the secondary expanded strain into the fermentation medium, with an inoculation volume of 5% and a culture temperature of 28°C. Formula of the fermentation medium: 10% glucose, 2% sodium 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. Trace element mother solution (M): 2g / L nickel sulfate hexahydrate, 0.04g / L sodium molybdate dihydrate, 3g / L manganese chloride tetrahydrate, 0.04g / L cobalt chloride hexahydrate, 2g / L copper sulfate pentahydrate, 3g / L zinc sulfate heptahydrate, 10g / L ferrous sulfate heptahydrate.

[0082] Method 5: Inoculate 200 mL of the secondary expansion strain into the fermentation medium, with an inoculation volume of 5% and a culture temperature of 24°C. Fermentation medium formula: 10% glucose, 2% sodium glutamate, 0.75% yeast extract powder, 2.4% sodium chloride, 0.6% magnesium sulfate, 0.012% calcium chloride, 0.48% sodium sulfate, 0.072% potassium chloride, 0.019% sodium bicarbonate.

[0083] Method 6: Inoculate 200 mL of the secondary expansion strain into the fermentation medium, with an inoculation volume of 5% and a culture temperature of 28°C. Fermentation medium formula: 10% glucose, 2% sodium glutamate, 0.75% yeast extract powder, 2.5% sodium chloride, 0.5% magnesium sulfate, 0.05% calcium chloride, 0.6% sodium sulfate, 0.06% potassium chloride, 0.016% sodium bicarbonate.

[0084] Method 7: Inoculate 200 mL of the secondary expansion strain into the fermentation medium, the inoculation amount is 5%, and the culture temperature is 33°C. Fermentation medium formula: 10% glucose, 2% sodium glutamate, 0.75% yeast extract powder, 2% sodium chloride, 0.5% magnesium sulfate, 0.01% calcium chloride, 0.4% sodium sulfate, 0.06% potassium chloride, 0.016% sodium bicarbonate, 0.1% potassium dihydrogen phosphate.

[0085] Method 8: Inoculate 200 mL of the secondary expanded strain into the fermentation medium, the inoculation volume is 5%, and the culture temperature is 28°C. Fermentation medium formula: 10% glucose, 2% sodium glutamate, 0.75% yeast extract powder, 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. Trace element mother solution (M): 2g / L nickel sulfate hexahydrate, 0.04g / L sodium molybdate dihydrate, 3g / L manganese chloride tetrahydrate, 0.04g / L cobalt chloride hexahydrate, 2g / L copper sulfate pentahydrate, 3g / L zinc sulfate heptahydrate, 10g / L ferrous sulfate heptahydrate.

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

[0087]

[0088]

[0089] It can be seen from Table 2 that the C14:0 content of the CABIO-A-2-VII strain fluctuates in different fermentation methods, and the content of method 1 reaches 19.61%, indicating that different fermentation processes will 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, it was found that under the same process, the C14:0 content of the CABIO-A-2-VII strain was significantly higher than that of the starting strain, and the EPA and DHA contents were also increased, with EPA reaching more than 6%.

[0090] Example 3

[0091] Preparation of polyunsaturated fatty acid microcapsules

[0092] The polyunsaturated fatty acid oil microcapsules of protein formula include the following components (by mass percentage): 8% sodium caseinate, 10% whey protein powder, 43% DHA oil, 2.5% sodium ascorbate, 0.6% tricalcium phosphate, 0.01% sodium citrate, and the rest is maltodextrin. The preparation method of the 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 a speed of 10000 r / min for 15 min;

[0094] (2) homogenizing the liquid obtained in step (1) at 800 bar for two times to obtain an emulsion;

[0095] (3) directly spray drying the emulsion obtained in step (2), wherein the spray drying conditions are an inlet air temperature of 170±5°C, an outlet air temperature of 70±5°C, and a spray pressure of 20 bar;

[0096] (4) The dried product obtained in step (3) is passed through a 20-mesh sieve and mixed with tricalcium phosphate to obtain a polyunsaturated fatty acid oil microcapsule product.

[0097] Among them, the DHA oils used in Experimental Examples 1-2 are the oil obtained by CABIO-A-2-VII fermentation example 2 and the oil obtained by fermentation using method 4 in Table 2, respectively. Comparative Experimental Examples 1 and 2 are the oils produced by CCTCCM2019990 and CCTCC NO:M 2015716, and the fatty acids correspond to the fatty acid composition in Table 1.

[0098] The starch-formulated polyunsaturated fatty acid oil microcapsules, whose constituent raw materials include the following components (mass percentage): 20% sodium starch octenylsuccinate, 43% DHA oil, 2.5% sodium ascorbate, 0.6% tricalcium phosphate, 0.01% sodium citrate, and the rest is maltodextrin. The preparation method is the same as the preparation method of the polyunsaturated fatty acid oil microcapsules to obtain the microcapsules of Experimental Example 3, and the DHA oil used is the oil obtained in Example 2 of CABIO-A-2-VII. Comparative Experimental Example 3 is the oil obtained using CCTCCM2019990.

[0099] Determination method

[0100] 1. Determination of surface oil content: weigh 50.00g of microcapsules into a 250mL conical flask, add 100mL of petroleum ether, shake for 1min, let stand for stratification, and filter the supernatant into a flat-bottom flask of constant weight. Then add 100mL of petroleum ether to rinse the iodine volume bottle and the microcapsules in the bottle, and filter them into the flat-bottom flask. Desolventize, then put it in a vacuum oven, keep it at 60℃ for 4 hours, take it out and put it in a desiccator, cool it to room temperature, and weigh it on a 1 / 10,000 electronic balance.

[0101] The surface oil content (X%) is calculated as follows:

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

[0103] Where: m1—mass of flat-bottom flask plus surface oil, g;

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

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

[0106] 2. Peroxide value: Test the peroxide value in accordance with GB 5009.227.

[0107] 3. Sensory evaluation: Open the bag and smell the samples every three days, and directly score the samples for fishy and pungent smells: 1 for slight smell, 2 for slight smell, 3 for medium smell, and 4 for strong smell.

[0108] Index evaluation: The same letters above indicate no significant difference, while the absence of the same letters indicates a difference.

[0109] (1) Microcapsule encapsulation effect detection

[0110] The results are shown in Table 3.

[0111] Table 3 Microcapsule embedding 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 embedding effect: As shown in Table 3, in terms of surface oil content between the experimental example and the comparative experimental example with the same formula, the surface oil of the experimental example is better overall. Although the initial peroxide value is measured at a low level in each group, the peroxide value of the experimental example is better.

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

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

[0116] Serial number 60℃ accelerated sensory 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 hala 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 hala b >

[0117] (3) Complex sensory evaluation:

[0118] The prepared microcapsules were treated at 37°C for 30 days and then subjected to sensory evaluation. The results are shown in Table 5. The external taste was evaluated and the intensity of the fishy smell was scored on a 7-point scale (absent - 0; somewhat present - 1; slight - 2; moderate - 3-4; relatively strong - 5-6; very strong - 7). After comprehensively considering all the attributes, the overall liking of the samples was scored.

[0119] The desirability was scored on a 9-point scale (like very much - 9 points; like very much - 8 points; like relatively - 7 points; like a little - 6 points; neither dislike nor hate - 5 points; hate a little - 4 points; hate relatively - 3 points; hate very - 2 points; hate extremely - 1 point) and the results were averaged (professional sensory evaluators were randomly selected to score, with 15 people in each group).

[0120] Table 5 Complex sensory evaluation

[0121]

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

[0123] Finally, the method of the present invention is only a preferred embodiment and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

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

2. A method for preparing polyunsaturated fatty acid oil, characterized in that: The method comprises the following steps: fermenting and culturing the mutant strain of Schizochytrium according to claim 1.

3. The preparation method according to claim 2, characterized in that: In the fermentation medium of the fermentation step, the concentration of chloride ions 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%.

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

5. The preparation method according to any one of claims 2 to 4, characterized in that: The fermentation temperature of the fermentation step is 24°C to 33°C, preferably 24°C to 28°C.

6. The polyunsaturated fatty acid oil obtained by the preparation method according to any one of claims 2 to 5.

7. The polyunsaturated fatty acid oil according to claim 6, characterized in that: In the polyunsaturated fatty acid oil, C14:0≥12%, EPA content>2%, and DHA content≥40%.

8. A polyunsaturated fatty acid microcapsule, characterized in that: The invention comprises polyunsaturated fatty acid oil, wherein the polyunsaturated fatty acid oil is obtained by fermenting the Schizochytrium mutant strain according to claim 1.

9. The polyunsaturated fatty acid microcapsule according to claim 8, characterized in that: The concentration of chloride ions in the fermentation medium 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%.

10. The polyunsaturated fatty acid microcapsule according to claim 8 or 9, characterized in that: The content of the DHA oil is 10% to 45%.

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