Polyunsaturated fatty acid oil microcapsule and oil for improving microcapsule flavor

Abstract

The present invention provides a polyunsaturated fatty acid oil microcapsule and an oil for improving the flavor of the microcapsule. The polyunsaturated fatty acid oil microcapsule includes polyunsaturated fatty acid oil; the content of polyunsaturated fatty acids in the polyunsaturated fatty acid oil is ≥50%, and the content of long-chain saturated fatty acids is ≥30%; the content of DHA in the polyunsaturated fatty acid oil is ≥35%, and the content of C14:0 in the polyunsaturated fatty acid oil is ≥12%. The specific (polyunsaturated fatty acid content ≥50%, DHA ≥35%; long-chain saturated fatty acid content ≥30%, C14:0 ≥12%) polyunsaturated fatty acid oil provided by the present invention can effectively improve the flavor of microcapsule powder prepared using the polyunsaturated fatty acid oil.

Description

Technical Field

[0001] The invention relates to the field of microbial oils and fats, and more specifically, to a polyunsaturated fatty acid oil microcapsule and an oil for improving the flavor of the microcapsule. Background Art

[0002] Most of the flavor compounds in oils and fats are generated by the reaction of unsaturated fatty acids in triglycerides or polar lipids with oxygen. The unpleasant flavor substances produced by oxidation reactions will greatly affect consumers' acceptance of oils and fats. The relative oxidation rate of fatty acid esters is closely related to their molecular unsaturation, such as: at 36.5°C, the relative oxidation rate of oleate is 1.0, linoleate is 8.0, linolenate is 21.7, and (EPA+DHA) ester is 39.1. Polyunsaturated fatty acid oils and fats, such as algae oil, have a high oxidation rate of polyunsaturated fatty acids EPA / DHA and the instability of their hydroperoxides, which leads to rapid secondary oxidation reactions of their hydroperoxides. The alkoxy radicals generated by the homolysis of various peroxide isomers are β-cleaved to obtain a series of products. The generated volatile aldehydes and other compounds then cause algae oil to produce unpleasant flavors such as fishy smell.

[0003] Microcapsule embedding technology can improve the instability, easy oxidation and fishy smell of such oils. Existing technologies such as CN 114098081 B and CN 109846853 B improve microcapsules by improving the components of microcapsule wall materials. However, the components are greatly restricted by processes, equipment, and regulations, and are often difficult to adapt to multiple factors. On the contrary, few people have studied the core embedded component oil. Summary of the invention

[0004] The first object of the present invention is to provide a polyunsaturated fatty acid oil microcapsule, wherein the microcapsule comprises polyunsaturated fatty acid oil, wherein the polyunsaturated fatty acid (PUFA) in the polyunsaturated fatty acid oil is ≥50%, wherein DHA accounts for ≥35% of the polyunsaturated fatty acid oil; and the long-chain saturated fatty acid is ≥30%, wherein C14:0 accounts for ≥12% of the polyunsaturated fatty acid oil. The present invention finds that the use of the specific type of polyunsaturated fatty acid oil to prepare microcapsules can effectively improve the flavor of the microcapsules.

[0005] The long-chain saturated fatty acids provided in the present invention refer to fatty acids containing more than 12 carbon atoms and having no unsaturated bonds, mainly including but not limited to myristic acid, palmitic acid, stearic acid and the like.

[0006] The polyunsaturated fatty acids provided in the present invention include, but are not limited to, one or more of γ-linoleic acid, DHA (docosahexaenoic acid), DPA (docosapentaenoic acid), EPA (eicosapentaenoic acid), and ARA (arachidonic acid, also known as AA).

[0007] In the art, polyunsaturated fatty acid oils with DHA as the main component have a special smell, which can be effectively masked after being made into microcapsules. However, after being stored for a long time or at high temperatures, the special smell will reappear. The present invention finds that this problem can be improved after microcapsules are made of polyunsaturated fatty acid oils with long-chain saturated fatty acids ≥30%, especially C14:0 ≥12%.

[0008] In a specific embodiment of the present invention, plant extraction, fish extraction, microbial fermentation, and oil transesterification can be used to prepare the specific type of polyunsaturated fatty acid oil of the present invention, thereby preparing the corresponding microcapsules. The present invention can use one of the methods to prepare the specific type of polyunsaturated fatty acid oil of the present invention, or two or more of the methods can be used together to prepare the specific type of polyunsaturated fatty acid oil of the present invention.

[0009] In one embodiment of the present invention, the specific type of polyunsaturated fatty acid oil provided by the present invention can be obtained by microbial fermentation, for example, it can be prepared by fermentation of one or more microorganisms selected from Cryptodinium cohnii, Schizochytrium, Biphasic dinoflagellate, Microsphaeria, and Thraustochytrium. In another embodiment of the present invention, the inventors have found that the specific polyunsaturated fatty acid oil of the present invention can be prepared by fermentation of Schizochytrium.

[0010] In a preferred embodiment of the present invention, the inventor unexpectedly discovered that the specific polyunsaturated fatty acid oil of the present invention can be prepared by fermentation of the Schizochytrium mutant strain CABIO-A-2-VII. In a preferred embodiment of the present invention, the deposit information of the Schizochytrium mutant strain CABIO-A-2-VII is: the deposit number is CCTCC M 20242741, the classification name is Schizochytrium sp. CABIO-A-2-VII, the deposit unit is China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, and the deposit date is December 6, 2024.

[0011] In some specific embodiments of the present invention, the fermentation method for preparing the specific polyunsaturated fatty acid oil of the present invention by Schizochytrium fermentation can be a fermentation method commonly used in the art, and specifically can include: strain activation culture, strain expansion culture and fermentation culture.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In the scheme of the present invention, when the OD600 in the culture medium is 10-20, 5-10% (volume ratio) of the inoculation amount is inoculated into the fermentation medium for fermentation culture.

[0016] 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.

[0017] 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.

[0018] 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%.

[0019] 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.

[0020] 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%.

[0021] 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%.

[0022] 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.

[0023] 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.

[0024] 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 26°C.

[0025] 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 control the pH in the fermentation system to be 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 in the culture medium is not completely consumed, and the fermentation is terminated after 144 hours to obtain the Schizochytrium fermentation broth.

[0026] In the present invention, the extraction method of oil is not particularly limited. On the premise of obtaining oil with a low degree of oxidation, both solvent-based and solvent-free extraction methods are acceptable. In a specific embodiment of the present invention, the fermentation method for preparing the specific polyunsaturated fatty acid oil of the present invention by Schizochytrium fermentation also includes the following steps: breaking the cell wall of the fermentation broth obtained by fermentation culture with alkaline protease, adding n-hexane for extraction, taking the mixed oil, desolventizing, and obtaining DHA oil, that is, the microbial oil of the present invention.

[0027] In a specific embodiment of the present invention, DHA oil can also be obtained by the solvent-free extraction method in CN202010421634.X.

[0028] In an optional embodiment of the present invention, the polyunsaturated fatty acid oil provided by the present invention is obtained by transesterifying a polyunsaturated fatty acid oil raw material with one or two of stearic acid and myristic acid under lipase, wherein the polyunsaturated fatty acid oil raw material is derived from plant extraction, fish extraction, and algae fermentation.

[0029] In an optional embodiment of the present invention, DHA oil raw material fermented by algae is exchanged with myristic acid under lipase. DHA oil can be obtained by fermentation with schizochytrium in the prior art, such as CCTCCM2019990, and the C14:0 content in the obtained DHA oil is not higher than 6%, the C16:0 content is not higher than 15.00%, the C18:0 content is not higher than 1%, and the C22:6 content is not higher than 60%. The specific transesterification method preferably includes the following steps: mixing the above-mentioned DHA oil with myristic acid in a mass ratio of 1:2 to 1:6, adding solvent and lipase, and reacting at 40 to 60°C for 8 to 24 hours. In a preferred embodiment of the present invention, the lipase can be Lipozyme RM lipase. The amount of lipase can be 5 to 8% of the total substrate mass. In a specific embodiment of the present invention, the solvent can be an organic solvent commonly used in the art, such as n-hexane. After the transesterification reaction is completed, the lipase is filtered to remove the lipase, the solvent is removed by desolventizing, and the free fatty acids are removed by molecular distillation. In a preferred embodiment of the present invention, the method for preparing microbial oil may include the following steps: mixing DHA oil and myristic acid in a mass ratio of 1:3, adding a solvent and lipase, and reacting at 50°C for 18 hours.

[0030] The above-mentioned preparation method can also be used to obtain the specific polyunsaturated fatty acid oil of the present invention, that is, the content of polyunsaturated fatty acids in the polyunsaturated fatty acid oil is ≥50%, and the content of long-chain saturated fatty acids is ≥30%; among the polyunsaturated fatty acids, the content of DHA in the polyunsaturated fatty acid oil is ≥35%; among the long-chain saturated fatty acids, the content of C14:0 in the polyunsaturated fatty acid oil is ≥12%.

[0031] The present invention finds that the microcapsules obtained by using the specific polyunsaturated fatty acid oil have low surface oil content and peroxide value and no fishy smell.

[0032] In a preferred embodiment of the present invention, the polyunsaturated fatty acid oil content in the polyunsaturated fatty acid microcapsules is 10-45%. Preferably, the DHA content in the polyunsaturated fatty acid microcapsules is 5-20% (accounting for the weight of the polyunsaturated fatty acid microcapsules).

[0033] In the present invention, the applicant has found that the microcapsules prepared by using the polyunsaturated fatty acid 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, and can maintain good sensory flavor in the stability experiment. 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.

[0034] 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 sodium octenyl succinate.

[0035] 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.

[0036] 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%.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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:

[0042] (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;

[0043] (2) homogenizing the mixture obtained in step (1) to obtain an emulsion;

[0044] (3) drying the emulsion obtained in step (2);

[0045] (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.

[0046] 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.

[0047] Another object of the present invention is to provide a polyunsaturated fatty acid oil, that is, a polyunsaturated fatty acid oil for improving the flavor of microcapsules, wherein the content of polyunsaturated fatty acids in the polyunsaturated fatty acid oil is ≥50%, and the content of long-chain saturated fatty acids is ≥30%; among the polyunsaturated fatty acids, DHA accounts for ≥35% of the polyunsaturated fatty acid oil; and among the long-chain saturated fatty acids, C14:0 accounts for ≥12% of the polyunsaturated fatty acid oil.

[0048] For other preferred solutions, please refer to the above contents and will not be described in detail here.

[0049] The specific (polyunsaturated fatty acid content ≥50%, DHA ≥35%, long-chain saturated fatty acid ≥30%, C14:0 ≥12%) polyunsaturated fatty acid oil provided by the present invention can effectively improve the flavor of microcapsule algae powder prepared by using the polyunsaturated fatty acid oil. DETAILED DESCRIPTION

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0051] 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.

[0052] 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);

[0053] 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.

[0054] 1. Breeding of Schizochytrium mutants

[0055] (1) Starting strain: Schizochytrium sp., with a deposit number of CCTCC NO: M 2015716 (disclosed in CN202311743382.2).

[0056] (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:

[0057] ① Activation culture of the starting strain: culture temperature 28°C, shaking speed of 250r / min, culture for 48h until the logarithmic growth phase.

[0058] 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.

[0059] 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 .

[0060] 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 comprises: 40-60 g / L glucose, 4-9 g / L yeast extract, 20-40 g / L sodium glutamate, 1-5 g / L sodium chloride, 1-5 g / L potassium dihydrogen phosphate, 5-10 g / L magnesium sulfate, 0.5-1 g / L calcium chloride, 0.5-1 g / L sodium bicarbonate, 5-10 g / L sodium sulfate, 5-20 g / L ammonium sulfate, 0.5-1 g / L potassium chloride, and natural pH.

[0061] (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.

[0062] Strain fermentation

[0063] 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 a starting strain (CCTCC NO: M 2015716) and a production strain (CCTCCM2019990, disclosed in CN201911399018.2). The specific method is as follows:

[0064] (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.

[0065] (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.

[0066] (3) Fermentation tank culture: 200 mL of the secondary expansion strain (OD600 is 15) was inoculated into the fermentation medium, the inoculation amount was 5%, and the culture temperature was 28°C.

[0067] The fermentation medium formula of Example 1 is: 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.

[0068] The culture medium formula of Example 2 is: 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, and 0.1% potassium dihydrogen phosphate.

[0069] (4) Schizochytrium fermentation process

[0070] The fermentation temperature of Example 1 was 28°C.

[0071] The fermentation temperature of Example 2 was 33°C.

[0072] 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.

[0073] (5) Post-treatment: After the fermentation broth obtained by fermentation culture is broken by alkaline protease, the extractant n-hexane is added for extraction, and the residue obtained after extraction is transferred into an extraction container for repeated extraction, and the extraction process is terminated when there is no oil in the extract. The extract is filtered and separated to obtain a mixed oil, and the microbial crude oil is obtained by desolventizing. The microbial crude oil is obtained by conventional refining in the field to obtain microbial oil (i.e., polyunsaturated fatty acid oil).

[0074] The oils and fats of Example 1 and Example 2 were obtained by fermentation, and the main fatty acid compositions were shown in Table 1.

[0075] Example 3

[0076] The selected DHA finished oil is produced by fermentation of Schizochytrium CCTCCM2019990, and its main fatty acid composition is: C14:0 content is 5.27%, C16:0 content is 15.00%, C18:0 content is 0.41%, and C22:6 content is 52.37%.

[0077] The microbial oil and myristic acid were transesterified under the catalysis of lipase, and the microbial oil and myristic acid were added to the reactor as substrates in a mass ratio of 1:3, and n-hexane was added to mix the substrates evenly, n-hexane: substrate = 1:1 (V / M), and Lipozyme RM lipase accounting for 5% of the total substrate mass was added, and the reaction was stirred at 50°C for 18h. After the reaction was completed, the lipase was removed by filtration, the solvent was removed by desolventization, and the free fatty acids were removed by molecular distillation to obtain the final product (i.e., polyunsaturated fatty acid oil) with the main fatty acid composition as shown in Table 1.

[0078] Comparative Example 1

[0079] The raw material oil was produced by fermentation of Schizochytrium CCTCCM2019990, and the fermentation method was the same as that of Example 3 of CN111235035A. The main fatty acid composition is shown in Table 1.

[0080] Comparative Example 2:

[0081] Our company (JiaBiYo) also has a strain of Schizochytrium that produces DHA. The main fatty acid composition of DHA oil prepared by conventional fermentation is shown in Table 1 below.

[0082] Table 1 Main fatty acid composition of oils and fats

[0083] Sample name Example 1# Example 2# Example 3# Comparative Example 1# Comparative Example 2# C14:0 12.86 16.4 18.31 5.2 2.0 C16:0 21.13 24.26 12.53 15.5 39.1 C18:0 0.53 0.71 0.17 0.51 1.9 C18:1 0.33 0.4 1.45 0.61 1.00 C18:2 / / 2.16 1.31 2.1 C18:3 0.33 0.23 / / / C20:4 0.62 0.5 2.61 3.3 0.8 C20:5 2.50 4.36 1.49 2.5 1 C22:5 11.89 9.85 13.75 17.9 9.2 C22:6 43.59 41.99 44.52 50.4 40.5

[0084] Among them, fatty acids such as C15:0, C16:1, and C17:0 are not listed in this table due to their extremely low content.

[0085] Experimental example

[0086] Preparation of polyunsaturated fatty acid microcapsules

[0087] Recipe 1:

[0088] The polyunsaturated fatty acid oil microcapsule comprises the following components (in percentage by mass): 8% sodium caseinate, 10% whey protein powder, polyunsaturated fatty acid oil whose DHA content accounts for 20% of the total raw materials, 2.5% sodium ascorbate, 0.6% tricalcium phosphate, 0.01% sodium citrate, and the rest is maltodextrin.

[0089] Recipe 2:

[0090] The polyunsaturated fatty acid oil microcapsules with starch formula include the following components (mass percentage): 20% sodium starch octenylsuccinate, polyunsaturated fatty acid oil with DHA content accounting for 20% of the total raw materials, 2.5% sodium ascorbate, 0.6% tricalcium phosphate, 0.01% sodium citrate, and the rest is maltodextrin.

[0091] The polyunsaturated fatty acid oils are provided by the polyunsaturated fatty acid oils of Examples 1 to 3 and Comparative Examples 1 to 2, respectively.

[0092] The method for preparing the polyunsaturated fatty acid oil microcapsules comprises the following steps:

[0093] (1) mixing and dissolving an emulsifier, sodium ascorbate, sodium citrate, and maltodextrin with water, and then adding microbial oil for shear emulsification, wherein the shearing is performed at a speed of 10,000 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] Embedding and accelerated test effects

[0098] The content of DHA in the microcapsules obtained in the embodiment and the comparative example is basically the same, and the embedding effect and the sensory evaluation results of the microcapsules at 60° C. for 30 days are shown in Table 2. The embedding effect of the microcapsules is good.

[0099] (1) Determination of surface oil content: Weigh 50.00 g of microcapsules into a 250 mL conical flask, add 100 mL of petroleum ether, shake for 1 min, let stand to separate layers, and filter the supernatant into a flat-bottom flask of constant weight. Then add 100 mL of petroleum ether to rinse the iodine volume flask and the microcapsules in the flask, and filter them into a flat-bottom flask. After desolventizing, put it in a vacuum oven at 60°C 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.

[0100] The surface oil content (X%) is calculated as follows:

[0101] Surface oil content (X%) = (m1-m0) / m

[0102] Where: m1—mass of flat-bottom flask plus surface oil, g;

[0103] m0—mass of the flat-bottom flask after constant weight, g;

[0104] m—the mass of the weighed powder, g.

[0105] (2) Peroxide value: The peroxide value shall be tested in accordance with GB 5009.227.

[0106] (3) Sensory evaluation: Open the bag and smell the sample every three days, and directly rate the fishy and pungent smell of the sample: 1 for slight smell, 2 for slight smell, 3 for moderate smell, and 4 for strong smell.

[0107] Table 2 Microcapsule encapsulation effect and 60℃ accelerated sensory results

[0108]

[0109] As can be seen from Table 2, the surface oil content of the embodiment and the comparative example is better than that of the comparative example. Although the initial peroxide value is at a lower level in each group, the peroxide value of the embodiment is better. In the accelerated sensory test at 60°C, the sensory results of the embodiment show that it can maintain a lighter fishy smell for a longer time. At the same time, the overall effect of the DHA oil obtained by direct fermentation is slightly stronger than that of the DHA oil obtained by ester exchange, which may be because the native fermented DHA oil is more suitable for preparing microcapsules in terms of fatty acid distribution and other fatty acid content.

[0110] Sensory evaluation:

[0111] The microcapsule powder prepared by using the microbial oils provided in the examples and comparative examples was placed at 37° C. for 30 days, and the fishy smell intensity and overall preference were evaluated. The sensory results are shown in Table 3.

[0112] The evaluation method and results are as follows:

[0113] The sensory evaluation method of the present invention is: smell the microcapsule powder for odor evaluation, and score the fishy smell intensity and comprehensive preference. The microcapsule powder is diluted with 45°C warm water to a concentration of about 6.5%, and the fishy smell intensity and comprehensive preference of the solution are evaluated.

[0114] The intensity of 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 attributes, the overall preference of the sample was scored on a 9-point scale (extremely like - 9 points; very like - 8 points; relatively like - 7 points; somewhat like - 6 points; neither like nor dislike - 5 points; somewhat dislike - 4 points; relatively dislike - 3 points; very dislike - 2 points; extremely dislike - 1 point), and the results were averaged (professional sensory evaluators were randomly selected for scoring, with 15 people in each group).

[0115] Table 3 Sensory evaluation results

[0116]

[0117] Sensory scoring: For the same attribute, the same letters indicate no significant difference, and no same letters indicate difference. From the perspective of fishy smell intensity and overall liking, there is no significant difference between Example 1# / 2# / 3#, and there is a significant difference with Comparative Example 1# / 2#, and the Example is significantly better than the Comparative Example.

[0118] 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 polyunsaturated fatty acid oil microcapsule, characterized in that: Including polyunsaturated fatty acid oil; the content of polyunsaturated fatty acids in the polyunsaturated fatty acid oil is ≥50%, and the content of long-chain saturated fatty acids is ≥30%; among the polyunsaturated fatty acids, the content of DHA in the polyunsaturated fatty acid oil is ≥35%; among the long-chain saturated fatty acids, the content of C14:0 in the polyunsaturated fatty acid oil is ≥12%.

2. The polyunsaturated fatty acid oil microcapsule according to claim 1, characterized in that: The polyunsaturated fatty acid oil is prepared by one or more methods including plant extraction, fish extraction, microbial fermentation and oil transesterification.

3. The polyunsaturated fatty acid oil microcapsules according to claim 2, wherein the microbial fermentation preparation is prepared by fermentation of one or more microorganisms selected from the group consisting of Cryptocoryneformis, Schizochytrium, Biphasic dinoflagellate, Microchlorophyll, and Thraustochytrium.

4. The polyunsaturated fatty acid oil microcapsule according to claim 3, characterized in that: The microbial oil is prepared by fermenting Schizochytrium.

5. The polyunsaturated fatty acid oil microcapsule according to claim 4, characterized in that: The Schizochytrium is a Schizochytrium mutant strain CABIO-A-2-VII, with a deposit number of CCTCC M 20242741, and is deposited in the China Center for Type Culture Collection.

6. The polyunsaturated fatty acid oil microcapsule according to claim 2, characterized in that: The oil transesterification is prepared by exchanging a polyunsaturated fatty acid oil raw material with one or two of stearic acid and myristic acid under the action of lipase, and the polyunsaturated fatty acid oil raw material is derived from plant extraction, fish extraction, and algae fermentation.

7. The polyunsaturated fatty acid oil microcapsule according to any one of claims 1 to 6, characterized in that: The polyunsaturated fatty acid oil content is 10-45%.

8. The polyunsaturated fatty acid oil microcapsule according to claim 7, characterized in that: The content of DHA accounts for 5-20% of the weight of the polyunsaturated fatty acid oil microcapsule.

9. A polyunsaturated fatty acid oil for improving the flavor of microcapsules, characterized in that: The content of polyunsaturated fatty acids in the polyunsaturated fatty acid oil is ≥50%, and the content of long-chain saturated fatty acids is ≥30%; among the polyunsaturated fatty acids, DHA accounts for ≥35% of the polyunsaturated fatty acid oil; among the long-chain saturated fatty acids, C14:0 accounts for ≥12% of the polyunsaturated fatty acid oil.

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