WO2019218839A1 - Dha微生物油脂中脂肪酸组合物成分调整的方法 - Google Patents

Dha微生物油脂中脂肪酸组合物成分调整的方法 Download PDF

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WO2019218839A1
WO2019218839A1 PCT/CN2019/083394 CN2019083394W WO2019218839A1 WO 2019218839 A1 WO2019218839 A1 WO 2019218839A1 CN 2019083394 W CN2019083394 W CN 2019083394W WO 2019218839 A1 WO2019218839 A1 WO 2019218839A1
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content
dha
fatty acid
hours
composition
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PCT/CN2019/083394
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English (en)
French (fr)
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瞿瀚鹏
曹晟
王身健
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梁云
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Priority to AU2019271764A priority Critical patent/AU2019271764B2/en
Priority to EP19803477.9A priority patent/EP3795690A4/en
Priority to KR1020207032137A priority patent/KR102565648B1/ko
Publication of WO2019218839A1 publication Critical patent/WO2019218839A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/02—Other edible oils or fats, e.g. shortenings, cooking oils characterised by the production or working-up
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/02—Other edible oils or fats, e.g. shortenings, cooking oils characterised by the production or working-up
    • A23D9/04—Working-up
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • A23L29/035—Organic compounds containing oxygen as heteroatom
    • A23L29/04—Fatty acids or derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/008—Refining fats or fatty oils by filtration, e.g. including ultra filtration, dialysis
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/16—Refining fats or fatty oils by mechanical means
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B7/00—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils
    • C11B7/0075—Separation of mixtures of fats or fatty oils into their constituents, e.g. saturated oils from unsaturated oils by differences of melting or solidifying points
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor

Definitions

  • the invention relates to the technical field of microbial fermentation, in particular to a method for adjusting the composition of a fatty acid composition in a DHA microbial oil.
  • Microbial oils and fats also known as single-cell fats and oils, are a large number of fats and oils produced in the bacteria by using fat-producing microorganisms such as yeast, mold and microalgae under certain conditions using carbon sources, nitrogen sources and trace elements.
  • Microbial oils have high polyunsaturated fatty acids, and docosahexaenoic acid (DHA) and arachidonic acid (ARA) are essential fatty acids and have important physiological functions.
  • DHA docosahexaenoic acid
  • ARA arachidonic acid
  • the products such as DHA and ARA sold in the market are mainly extracted from deep-sea fish oil. Due to the limitation of raw materials, large-scale production cannot be carried out. At the same time, the content of unsaturated fatty acids is unstable, and the yield is low and the cost is high. Therefore, microbial oils and fats have become important for obtaining high value-added fatty acids such as linolenic acid (GLA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). raw material.
  • GLA linolenic acid
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • the microbial oil produced by microorganisms such as Mycobacterium, Schizochytrium, Thraustochytrid, Cryptophyceae, and yeast is high in DHA content, and is the main strain used in the production of DHA oil.
  • the DHA content is not high enough.
  • the oil also contains more EPA and some harmful fatty acids, such as myristic acid, lauric acid, erucic acid, etc., which affect the use of microbial oils.
  • the object of the present invention is to overcome the problem that the DHA microbial oil produced in the prior art has high harmful fatty acid content, high DHA content and high EPA content, and provides a method for adjusting the composition of the fatty acid composition in the DHA microbial oil.
  • the method utilizes microbial fermentation of microorganisms such as Mycobacterium, Schizochytrium, Thraustochytrid, Cryptophyllum, and yeast to produce DHA microbial oil, which can regulate the content of fatty acid composition in microbial oil and fat, and produce high DHA.
  • the content of microbial oil and fat, and the content of harmful fatty acids is very low.
  • the present invention provides a method for adjusting a composition of a fatty acid composition in a DHA microbial oil, comprising controlling the carbon to nitrogen ratio of the medium to 3 to 0 to 80 hours after the fermentation of the strain. 20:1, the culture temperature is controlled between 29 and 32 ° C; after the 81st hour of fermentation, the carbon-to-nitrogen ratio of the medium is controlled between 1 and 15:1, and the culture temperature is controlled between 19 and 28 ° C. .
  • the carbon to nitrogen ratio is controlled by controlling the amount of glucose and sodium glutamate in the medium to maintain the desired carbon to nitrogen ratio.
  • the process of controlling the content of glucose and sodium glutamate in the culture medium is: in the 0 to 16 hours of fermentation, the glucose content is 2-8 g/100 ml, and the content of sodium glutamate is 1 to 5 g/100 ml.
  • the glucose content is 2-6g/100ml, the content of sodium glutamate is 1-3g/100ml; the 56th to 80th hour, the glucose content is 1-5g/100ml, the content of sodium glutamate It is 1 ⁇ 2g/100ml; the 81st to 95th hour, the glucose content is 0.5 ⁇ 1.0g/100ml, the content of sodium glutamate is 0.75 ⁇ 1.0g/100ml; after the 95th hour, the glucose content is ⁇ 0.5g/100ml The content of sodium glutamate is 0.5 to 0.75 g/100 ml.
  • the method for adjusting the composition of the fatty acid composition in the DHA microbial oil provided by the present invention further comprises: in the refining process of the microbial oil, first heating the oil to 70-90 ° C for dehydration, and then gradually cooling to 20 to 30 ° C at room temperature in winter. After 16 to 24 hours, after filtration and dehydration, the temperature is gradually lowered to -10 to 1 °C according to the set procedure, and the temperature is 48 to 90 hours.
  • the present invention provides a DHA microbial fat, which is obtained by the method for adjusting the composition of a fatty acid composition in any of the DHA microbial oils according to the first aspect of the present invention, and based on the total amount of the microbial oil and fat.
  • the following components not less than 40% by weight of DHA, not more than 3% by weight of EPA; 0 to 5% by weight of C12:0 fatty acid; 0 to 8% by weight of C14:0, C14:1 fatty acid; 5 to 50% by weight of C16: 0 and C16:1 fatty acids; 0.5 to 10% by weight of C18:0, C18:1 and C18:2 fatty acids; 0 to 5% by weight of C22:1n9 fatty acids.
  • the ratio of DHA to EPA is from 13.3 to 600:1.
  • the DHA microbial fat of the present invention has a content ratio of unsaturated fatty acid to saturated fatty acid of not less than 0.6.
  • the DHA microbial fat of the present invention has a content ratio of unsaturated fatty acid to saturated fatty acid of not less than 2.3.
  • the present invention provides a composition comprising the DHA microbial oil of the second aspect of the present invention, which is suitable for use in the preparation of infant formula (infant formula), health food, health food, and Ordinary food, etc.
  • the fermentation strains of the DHA microbial oil and fat are all used in Schizochytrium, and the strains and various culture materials used are commercially available, and the reagents and detection methods involved are all obtained. Implemented according to national standards.
  • Production strain The original strain is connected to a sterilized and cooled medium, and cultured in an shaking shake flask.
  • the shake flask medium formula is: glucose 4.5%, sodium glutamate 3.2%, yeast extract 0.62%, sodium chloride 1.8%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.55%, calcium chloride 0.03%, trace elements (Sodium sulfate, copper sulfate, sodium molybdate, manganese chloride, cobalt chloride, zinc sulfate, ferrous sulfate, etc.) 0.1%, vitamins (B2, B6, B12, etc.) 0.1%.
  • the culture conditions were as follows: shaking at 200 rpm, shaking at 28 ⁇ 1°C for 36-44 hours, taking off the hyphae, and then adding the bottle to the first-stage seed tank.
  • the strain is connected to a sterilized and cooled medium and cultured in a primary seed tank.
  • the formulation of the medium is: glucose 3%, yeast powder 0.83%, sodium glutamate 0.62%, yeast extract 0.62%, sodium chloride 0.62%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.52%, calcium chloride 0.02 %, sodium bicarbonate 0.02%, sodium sulfate 0.93%, ammonium sulfate 0.10%, potassium chloride 0.08%, trace elements (same as above) 0.10%, vitamins (same as above) 0.10%, epoxy silicone ether 0.03%.
  • the culture conditions were as follows: temperature 30 ⁇ 2 ° C, rotation speed of 90 rpm, aeration amount of 0.50 vvm, and culture for 18 to 24 hours.
  • Secondary seed 4% of the primary strain is connected to a sterilized and cooled medium and cultured in a secondary seed tank.
  • the formulation of the medium is: glucose 5%, sodium glutamate 2.0%, yeast extract 1.0%, sodium chloride 0.14%, potassium dihydrogen phosphate 0.16%, magnesium sulfate 0.50%, calcium chloride 0.02%, sodium bicarbonate 0.02%, sodium sulfate 0.91%, ammonium sulfate 0.10%, potassium chloride 0.08%, trace elements (same as above) 0.09%, vitamins (same as above) 0.09%, epoxy silicone ether 0.36%.
  • the culture conditions were as follows: temperature 30 ⁇ 2 ° C, rotation speed 90 rpm, and culture for 14 to 16 hours.
  • the medium formula is: glucose 5%, sodium glutamate 2.5%, yeast extract 1.0%, sodium chloride 0.29%, potassium dihydrogen phosphate 0.28%, magnesium sulfate 0.66%, calcium chloride 0.03%, sodium bicarbonate 0.02 %, sodium sulfate 0.58%, ammonium sulfate 0.13%, potassium chloride 0.11%, trace elements (same as above) 0.13%, vitamins (same as above) 0.13%, epoxy silicone ether 0.03%.
  • the culture conditions were: rotation speed of 90 rpm, temperature control of 30 ° C for 0 to 80 hours, temperature control of 22 ° C after 80 hours; ventilation of 0.5 VVm; addition of glucose to sugar cans containing 50% glucose, concentration of 60
  • the sodium glutamate solution adjusts the content of glucose and sodium glutamate in the medium to control the carbon to nitrogen ratio.
  • the contents of glucose and sodium glutamate in the medium and the control of pH are shown in Table 1, and cultured for 5 days.
  • Extracting the oil After the fermentation is completed, the mixture is dehydrated by a three-phase centrifuge, the mycelium is separated from the water, dehydrated with 95% ethanol, and then extracted with hexane to obtain a hair oil.
  • Acid refining The oil was heated to 75 ° C, citric acid having an oil weight of 4 Torr was added, and stirred at 80 rpm for 40 minutes. Further, hot water of 85 ° C with an oil weight of 10% was added, and the mixture was stirred for 20 minutes, and allowed to stand for 3 hours, and then water was separated.
  • Normal temperature winterization The oil after dehydration naturally cools down, and the oil temperature drops to 25 °C to maintain the crystal. Normal temperature winter time is 20 hours.
  • Filtration The grease after frequent warming is filtered using a plate and frame filter press.
  • the filter medium is an industrial filter cloth, and the filtration pressure is 0.3 MPa.
  • Low temperature winterization The dehydrated oil is cooled according to the setting procedure: firstly, the grease is rapidly cooled at a rate of 10 ° C every half hour, and when it reaches 45 ° C, the temperature is lowered by 3 ° C per hour, and gradually reduced to 1 ° C per hour. . When the oil temperature drops to 14 °C, the temperature is raised back to the temperature, the temperature is 1.5 °C, the temperature is 4.5 hours, and the temperature is lowered to -5 °C at a rate of 1.5 °C per hour after warming.
  • the low temperature winterization time is 70 hours.
  • Re-filtration The grease after frequent warming is filtered using a plate and frame filter press.
  • the filter medium is an industrial filter cloth, and the filtration pressure is 0.2 MPa.
  • Deodorization The deodorization temperature starts at 175 ⁇ 2°C, the deodorization temperature is maintained at 175 ⁇ 2°C, the deodorization time is 4 hours, the steam pressure is maintained at 0.2MPa ⁇ 0.3MPa, the vacuum degree is maintained at 50Pa, and the steam consumption is controlled.
  • the oil weighs about 5%. Cooling and breaking the vacuum to obtain finished fats and oils.
  • Production strain The original strain is connected to a sterilized and cooled medium, and cultured in an shaking shake flask.
  • the shake flask medium formula is: glucose 4.5%, sodium glutamate 3.2%, yeast extract 0.62%, sodium chloride 1.8%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.55%, calcium chloride 0.03%, trace elements (Sodium sulfate, copper sulfate, sodium molybdate, manganese chloride, cobalt chloride, zinc sulfate, ferrous sulfate, etc.) 0.1%, vitamins (B2, B6, B12, etc.) 0.1%.
  • the culture conditions were as follows: shaking at 200 rpm, shaking at 28 ⁇ 1°C for 36-44 hours, taking off the hyphae, and then adding the bottle to the first-stage seed tank.
  • the strain is connected to a sterilized and cooled medium and cultured in a primary seed tank.
  • the formulation of the medium is: glucose 3%, yeast powder 0.83%, sodium glutamate 0.62%, yeast extract 0.62%, sodium chloride 0.62%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.52%, calcium chloride 0.02 %, sodium bicarbonate 0.02%, sodium sulfate 0.93%, ammonium sulfate 0.10%, potassium chloride 0.08%, trace elements (same as above) 0.10%, vitamins (same as above) 0.10%, epoxy silicone ether 0.03%.
  • the culture conditions were as follows: temperature 30 ⁇ 2 ° C, rotation speed of 90 rpm, aeration amount of 0.5 vvm, and culture for 18 to 22 hours.
  • Secondary seed 4% of the primary strain is connected to a sterilized and cooled medium and cultured in a secondary seed tank.
  • the formulation of the medium is: glucose 5%, sodium glutamate 2.0%, yeast extract 1.0%, sodium chloride 0.14%, potassium dihydrogen phosphate 0.16%, magnesium sulfate 0.50%, calcium chloride 0.02%, sodium bicarbonate 0.02%, sodium sulfate 0.91%, ammonium sulfate 0.10%, potassium chloride 0.08%, trace elements (same as above) 0.09%, vitamins (same as above) 0.09%, epoxy silicone ether 0.36%.
  • the culture conditions were as follows: temperature 30 ⁇ 2 ° C, rotation speed of 90 rpm, and culture for 14 to 16 hours.
  • the medium formula is: glucose 5%, sodium glutamate 2.5%, yeast extract 1.0%, sodium chloride 0.29%, potassium dihydrogen phosphate 0.28%, magnesium sulfate 0.66%, calcium chloride 0.03%, sodium bicarbonate 0.02 %, sodium sulfate 0.58%, ammonium sulfate 0.13%, potassium chloride 0.11%, trace elements (same as above) 0.13%, vitamins (same as above) 0.13%, epoxy silicone ether 0.03%.
  • the culture conditions were: rotation speed of 90 rpm, temperature control of 29 ° C for 0 to 80 hours, temperature control of 19 ° C after 80 hours; ventilation of 0.45 VVm; addition of glucose with sugar cans containing 50% glucose, addition of 60%
  • the sodium glutamate solution adjusts the content of glucose and sodium glutamate in the medium to control the carbon to nitrogen ratio.
  • the contents of glucose and sodium glutamate in the medium and the control of pH are shown in Table 2, and cultured for 5 days.
  • Extracting the oil After the fermentation is completed, the mixture is dehydrated by a three-phase centrifuge, the mycelium is separated from the water, dehydrated with 95% ethanol, and then extracted with hexane to obtain a hair oil.
  • Acid refining The oil was heated to 75 ° C, citric acid having an oil weight of 4 Torr was added, and stirred at 80 rpm for 40 minutes. Further, hot water of 85 ° C with an oil weight of 10% was added, and the mixture was stirred for 20 minutes, and allowed to stand for 3 hours, and then water was separated.
  • Normal temperature winterization The oil after dehydration naturally cools down, and the oil temperature drops to 20 °C to maintain the crystal. Normal temperature winter time is 16 hours.
  • Filtration The grease after frequent warming is filtered using a plate and frame filter press.
  • the filter medium is an industrial filter cloth, and the filtration pressure is 0.3 MPa.
  • Low temperature winterization The dehydrated oil is cooled according to the setting procedure: firstly, the grease is rapidly cooled at a rate of 10 ° C every half hour, and when it reaches 45 ° C, the temperature is lowered by 3 ° C per hour, and gradually reduced to 1 ° C per hour. . When the oil temperature drops to 15 °C, the temperature is raised back to the temperature, the temperature is 1 °C, and the temperature is 4 hours. After the temperature is returned, the temperature is lowered to 1 °C at a rate of 2 °C per hour, and the temperature is lowered for 48 hours.
  • Re-filtration The grease after frequent warming is filtered using a plate and frame filter press.
  • the filter medium is an industrial filter cloth, and the filtration pressure is 0.2 MPa.
  • Deodorization The deodorization temperature starts at 175 ⁇ 2°C, the deodorization temperature is maintained at 175 ⁇ 2°C, the deodorization time is 4 hours, the steam pressure is maintained at 0.2MPa ⁇ 0.3MPa, the vacuum degree is maintained at 50Pa, and the steam consumption is controlled.
  • the oil weighs about 5%. Cooling and breaking the vacuum to obtain finished fats and oils.
  • Production strain The original strain is connected to a sterilized and cooled medium, and cultured in an shaking shake flask.
  • the shake flask medium formula is: glucose 4.5%, sodium glutamate 3.2%, yeast extract 0.62%, sodium chloride 1.8%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.55%, calcium chloride 0.03%, trace elements (Sodium sulfate, copper sulfate, sodium molybdate, manganese chloride, cobalt chloride, zinc sulfate, ferrous sulfate, etc.) 0.1%, vitamins (B2, B6, B12, etc.) 0.1%.
  • the culture conditions were as follows: shaking at 200 rpm, shaking at 28 ⁇ 1°C for 36-44 hours, taking off the hyphae, and then adding the bottle to the first-stage seed tank.
  • the strain is connected to a sterilized and cooled medium and cultured in a primary seed tank.
  • the formulation of the medium is: glucose 3%, yeast powder 0.83%, sodium glutamate 0.62%, yeast extract 0.62%, sodium chloride 0.62%, potassium dihydrogen phosphate 0.7%, magnesium sulfate 0.52%, calcium chloride 0.02 %, sodium bicarbonate 0.02%, sodium sulfate 0.93%, ammonium sulfate 0.10%, potassium chloride 0.08%, trace elements (same as above) 0.10%, vitamins (same as above) 0.10%, epoxy silicone ether 0.03%.
  • the culture conditions were as follows: temperature 30 ⁇ 2° C., rotation speed of 90 rpm, aeration amount of 0.50 vvm, and culture for 18 to 22 hours.
  • Secondary seed 4% of the primary strain is connected to a sterilized and cooled medium and cultured in a secondary seed tank.
  • the formulation of the medium is: glucose 5%, sodium glutamate 2.0%, yeast extract 1.0%, sodium chloride 0.14%, potassium dihydrogen phosphate 0.16%, magnesium sulfate 0.50%, calcium chloride 0.02%, sodium bicarbonate 0.02%, sodium sulfate 0.91%, ammonium sulfate 0.10%, potassium chloride 0.08%, trace elements (same as above) 0.09%, vitamins (same as above) 0.09%, epoxy silicone ether 0.36%.
  • the culture conditions were as follows: temperature 30 ⁇ 2 ° C, rotation speed of 90 rpm, and culture for 14 to 16 hours.
  • the medium formula is: glucose 5%, sodium glutamate 2.5%, yeast extract 1.0%, sodium chloride 0.29%, potassium dihydrogen phosphate 0.28%, magnesium sulfate 0.66%, calcium chloride 0.03%, sodium bicarbonate 0.02 %, sodium sulfate 0.58%, ammonium sulfate 0.13%, potassium chloride 0.11%, trace elements (same as above) 0.13%, vitamins (same as above) 0.13%, epoxy silicone ether 0.03%.
  • the culture conditions were: rotation speed of 90 rpm, temperature control of 32 ° C for 0 to 80 hours, temperature control of 28 ° C after 80 hours, ventilation of 0.5 VVm, addition of glucose with sugar cans containing 50% glucose, and addition of 60%
  • the sodium glutamate solution adjusts the content of glucose and sodium glutamate in the medium to control the carbon to nitrogen ratio.
  • the contents of glucose and sodium glutamate in the medium and the control of pH are shown in Table 3, and cultured for 5 days.
  • Extracting the oil After the fermentation is completed, the mixture is dehydrated by a three-phase centrifuge, the mycelium is separated from the water, dehydrated with 95% ethanol, and then extracted with hexane to obtain a hair oil.
  • Acid refining The oil was heated to 75 ° C, citric acid having an oil weight of 4 Torr was added, and stirred at 80 rpm for 40 minutes. Further, hot water of 85 ° C with an oil weight of 10% was added, and the mixture was stirred for 20 minutes, and allowed to stand for 3 hours, and then water was separated.
  • Normal temperature winterization The oil after dehydration naturally cools down, and the oil temperature drops to 30 °C to maintain the crystal. Normal temperature winter time is 24 hours.
  • Filtration The grease after frequent warming is filtered using a plate and frame filter press.
  • the filter medium is an industrial filter cloth, and the filtration pressure is 0.3 MPa.
  • Low temperature winterization The dehydrated oil is cooled according to the setting procedure: firstly, the grease is rapidly cooled at a rate of 10 ° C every half hour, and when it reaches 45 ° C, the temperature is lowered by 3 ° C per hour, and gradually reduced to 1 ° C per hour. . When the oil temperature drops to 13 °C, the temperature is raised back to the temperature, the temperature is 2 °C, and the temperature is 5 hours. After the temperature is returned, the temperature is lowered to -10 °C at a rate of 1 °C per hour to ensure that the crystal growth time is not less than 16 hour. The low temperature winterization time is 90 hours.
  • Re-filtration The grease after frequent warming is filtered using a plate and frame filter press.
  • the filter medium is an industrial filter cloth, and the filtration pressure is 0.2 MPa.
  • Deodorization The deodorization temperature starts at 175 ⁇ 2°C, the deodorization temperature is maintained at 175 ⁇ 2°C, the deodorization time is 4 hours, the steam pressure is maintained at 0.2MPa ⁇ 0.3MPa, the vacuum degree is maintained at 50Pa, and the steam consumption is controlled.
  • the oil weighs about 5%. Cooling and breaking the vacuum to obtain finished fats and oils.
  • the strain and the fermentation were cultured according to the method of Example 1, except that the main fermentation medium contained 4.5% glucose, 2% sodium glutamate, 0.9% yeast extract, (carbon to nitrogen ratio 10.7:1), and 28
  • the temperature was cultured at a constant temperature of 5 °C for 5 days; at the low temperature during winterization, the temperature was rapidly lowered to 0 ° C for 48 hours.
  • Example 1 The finished fats and oils obtained in Example 1, Example 2, Example 3 and the comparative examples were respectively subjected to gas chromatography to detect the fatty acid components therein, and the proportion of each fatty acid in the finished fats and oils is shown in Table 4.
  • the method of adjusting the composition of the fatty acid composition in the DHA microbial oil of the present invention has a higher DHA content and a lower harmful fatty acid content. It also has better low temperature setting properties.
  • the microbial oil provided by the invention has high DHA content, low EPA content, low content of harmful fatty acids, good low temperature solidification performance, and can be used for preparing infant formula foods, especially infant formula milk powder; and can also be made into health care products, according to the present invention.
  • the relationship between DHA and many diseases known to those skilled in the field is provided for the treatment and health care needs of people with relevant needs; it can also be made into healthy foods and common foods to provide the required nutrients for the human body, supplementing daily intake. insufficient.

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Abstract

一种DHA微生物油脂中脂肪酸组合物成分调整的方法，包括：在菌种发酵的第0～80小时，将培养基的碳氮比控制在3～20:1，培养温度控制在29～32℃之间；在菌种发酵的第81小时以后，将培养基的碳氮比控制在1～15:1，培养温度控制在19～28℃之间，以提高DHA的转化率，降低其它脂肪酸的含量。还涉及由上述方法制得的DHA微生物油脂以及包含该油脂的组合物。

Description

DHA微生物油脂中脂肪酸组合物成分调整的方法 技术领域
本发明涉及微生物发酵技术领域，具体涉及一种DHA微生物油脂中脂肪酸组合物成分调整的方法。
背景技术
微生物油脂，又称为单细胞油脂，是由酵母、霉菌和微藻等产脂微生物在一定条件下利用碳源、氮源、微量元素，在菌体内产生的大量油脂。
产油微生物资源丰富，能在多种培养条件下生长，进行工业规模生产和开发有着巨大的潜力。微生物油脂的多不饱和脂肪酸含量较高，其中的二十二碳六烯酸(DHA)、花生四烯酸(ARA)等为人体必须脂肪酸，具有重要的生理功能。
婴儿缺乏ARA和DHA，可造成永久性智力低下和视力障碍，6～12岁儿童的皮肤瘙痒、眼角干燥、上课注意力不集中；对高血压、高血脂、糖尿病、病毒感染等疾病的预防和治疗也有显著的效果。
市场销售的DHA、ARA等产品，主要从深海鱼油中提取，由于原料的限制，无法进行规模化生产；同时不饱和脂肪酸含量不稳定，而且产量低、成本高。因此，微生物油脂已成为获取高附加值脂肪酸，如亚麻酸(GLA)、花生四烯酸(ARA)、二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)等的重要原料。
但现有的研究表明，过量的EPA对胎儿和婴幼儿的生长、代谢存在不良的影响，因此对孕妇和婴幼儿食用的微生物油脂，应严格控制其中的EPA含量。
由于吾肯氏菌属、裂殖壶菌属、破囊壶菌目、隐甲藻属、酵母等微生物 发酵生产的微生物油脂DHA含量较高，是生产DHA油脂所主要使用的菌种。而现有的利用吾肯氏菌属、裂殖壶菌属、破囊壶菌目、隐甲藻属、酵母等微生物发酵生产微生物油脂的方法所获取的油脂中，DHA的含量还不够高，油脂中还含有较多的EPA和一些有害脂肪酸，如豆蔻酸、月桂酸、芥酸等，影响了微生物油脂的使用。
发明内容
本发明的目的是为了克服现有技术生产的DHA微生物油脂中有害脂肪酸含量偏高、以及DHA的含量不够高、EPA含量较高的问题，提供一种DHA微生物油脂中脂肪酸组合物成份调整的方法，该方法利用吾肯氏菌属、裂殖壶菌属、破囊壶菌目、隐甲藻属、酵母等微生物发酵生产DHA微生物油脂,可以调控微生物油脂中脂肪酸组合物的含量，生产高DHA含量的微生物油脂，且其中的有害脂肪酸含量很低。
为了实现上述目的，第一方面，本发明提供了一种DHA微生物油脂中脂肪酸组合物成分调整的方法，包括在菌种发酵的第0～80小时，将培养基的碳氮比控制在3～20:1，培养温度控制在29～32℃之间；在菌种发酵的第81小时以后，将培养基的碳氮比控制在1～15:1，培养温度控制在19～28℃之间。
优选地，所述控制碳氮比的方式为，控制培养基中葡萄糖和谷氨酸钠的含量，以维持所需的碳氮比。
优选地，所述控制培养基中葡萄糖和谷氨酸钠含量的过程为，在发酵的第0～16小时，葡萄糖含量为2～8g/100ml，谷氨酸钠的含量为1～5g/100ml；第17～55小时，葡萄糖含量为2～6g/100ml，谷氨酸钠的含量为1～3g/100ml；第56～80小时，葡萄糖含量为1～5g/100ml，谷氨酸钠的含量为1～2g/100ml；第81～95小时，葡萄糖含量为0.5～1.0g/100ml，谷氨酸钠的含量为0.75～ 1.0g/100ml；第95小时以后，葡萄糖含量为<0.5g/100ml，谷氨酸钠的含量为0.5～0.75g/100ml。
优选地，本发明提供的DHA微生物油脂中脂肪酸组合物成分调整的方法，还包括在微生物油脂的精制过程中，先将油脂升温至70～90℃脱水，然后逐渐冷却至20～30℃常温冬化16～24小时，过滤、脱水后，再按设定的程序逐渐降温到-10～1℃保温，低温冬化48～90小时。
第二方面，本发明提供了一种DHA微生物油脂，根据本发明第一方面所述的任一种DHA微生物油脂中脂肪酸组合物成分调整的方法制得，以微生物油脂的总量为基准，含有以下组分：不低于40wt％的DHA，不超过3wt％的EPA；0～5wt％的C12:0脂肪酸；0～8wt％的C14:0、C14:1脂肪酸；5～50wt％的C16:0和C16:1脂肪酸；0.5～10wt％的C18:0、C18:1和C18:2脂肪酸；0～5wt％的C22:1n9脂肪酸。
优选地，本发明的DHA微生物油脂中，DHA与EPA的比例为13.3～600:1。
优选地，本发明的DHA微生物油脂中不饱和脂肪酸与饱和脂肪酸的含量比不低于0.6。
进一步优选地，本发明的DHA微生物油脂中不饱和脂肪酸与饱和脂肪酸的含量比不低于2.3。
第三方面，本发明提供了一种组合物，包含本发明第二方面所述的DHA微生物油脂，所述组合物适用于制作婴幼儿配方食品(婴幼儿配方奶粉)、保健食品、健康食品以及普通食品等。
通过上述技术方案，不仅微生物油脂的产量较高，微生物油脂中DHA的含量高、EPA的含量低，有害脂肪酸豆蔻酸、月桂酸和芥酸的含量很低，可在-10～5℃条件下可保障澄清透明状态。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
在本发明的实施例和对照例中，DHA微生物油脂的发酵菌种均使用裂壶藻(Schizochytrium)，所用菌种和各种培养用品均由市售而得，所涉及的试剂和检测方法均按国家标准执行。
实施例1
1)生产菌种：将原始菌种接入灭菌冷却好的培养基中，在振荡摇瓶中培养。
摇瓶培养基配方为：葡萄糖4.5％，谷氨酸钠3.2％，酵母浸膏0.62％，氯化钠1.8％，磷酸二氢钾0.7％，硫酸镁0.55％，氯化钙0.03％，微量元素(硫酸镍、硫酸铜、钼酸钠、氯化锰、氯化钴、硫酸锌、硫酸亚铁等)0.1％，维生素(B2、B6、B12等)0.1％。
培养条件为：200转/分摇床上，28±1℃培养36～44小时，待菌丝长好后取下，并瓶后接入一级种子罐。
2)一级种子：将菌种接入灭菌冷却好的培养基中，在一级种子罐中培养。
培养基的配方为：葡萄糖3％，酵母粉0.83％，谷氨酸钠0.62％，酵母浸膏0.62％，氯化钠0.62％，磷酸二氢钾0.7％，硫酸镁0.52％，氯化钙0.02％，碳酸氢钠0.02％，硫酸钠0.93％，硫酸氨0.10％，氯化钾0.08％，微量元素(成分同上)0.10％，维生素(成分同上)0.10％，环氧硅醚0.03％。
培养条件为：温度30±2℃，转速90rpm，通气量为0.50vvm，培养18～ 24小时。
3)二级种子：将4％的一级菌种接入灭菌冷却好的培养基中，在二级种子罐中培养。
培养基的配方为：葡萄糖5％，谷氨酸钠2.0％，酵母浸膏1.0％，氯化钠0.14％，磷酸二氢钾0.16％，硫酸镁0.50％，氯化钙0.02％，碳酸氢钠0.02％，硫酸钠0.91％，硫酸氨0.10％，氯化钾0.08％，微量元素(成分同上)0.09％，维生素(成分同上)0.09％，环氧硅醚0.36％。
培养条件为：温度30±2℃、转速90rpm，培养14～16小时。
4)主发酵：将二级种子液按10％的比例接种于发酵罐的发酵培养基中进行发酵。
培养基配方为：葡萄糖5％，谷氨酸钠2.5％，酵母浸膏1.0％，氯化钠0.29％，磷酸二氢钾0.28％，硫酸镁0.66％，氯化钙0.03％，碳酸氢钠0.02％，硫酸钠0.58％，硫酸氨0.13％，氯化钾0.11％，微量元素(成分同上)0.13％，维生素(成分同上)0.13％，环氧硅醚0.03％。
培养条件为：转速90转/分，0～80小时温度控制在30℃，80小时以后温度控制在22℃；通气量为0.5VVm；用含50％葡萄糖的糖罐添加葡萄糖、添加浓度为60％的谷氨酸钠溶液调整培养基中葡萄糖和谷氨酸钠的含量来控制碳氮比，培养基中葡萄糖和谷氨酸钠的含量及PH的控制见表1，培养5天。
表1
Figure PCTCN2019083394-appb-000001
5)提取毛油：发酵完成后，用三相离心机脱水，将菌丝体与水分离、用95％的乙醇脱水后再加入己烷萃取得到毛油。
6)水洗脱胶：加入油重10％的纯净水，加热到85℃，以80rpm的转速搅拌20分钟，静置2小时后分水。
7)酸炼：将油加热到75℃，加入占油重4‰的柠檬酸，以80rpm的转速搅拌40分钟。再加入油重10％的85℃热水，搅拌20分钟，静置3小时后分水。
8)碱炼：将油加热到45℃，根据油脂酸价计算加碱量(加碱量＝7.13×10 -4×酸价×油重)，加入40％的氢氧化钠溶液，以80rpm的转速搅拌50分钟；将油脂温度升温到80℃，再加入油重5％的85℃纯净水，以80rpm的速度搅拌15分钟，用二相离心机离心去除皂角。
9)脱水：碱炼后的油脂加热到80℃，在-0.1MPa负压下脱水35分钟。
10)常温冬化：脱水后的油脂自然降温，油温降至25℃保持养晶。常温冬化时间20小时。
11)过滤：经常温冬化后的油脂，使用板框压滤机过滤，过滤介质为工业滤布，过滤压力在0.3MPa。
12)再脱水：将经过滤后的油脂加热到80℃，在-0.1MPa负压下脱水35分钟。
13)低温冬化：将脱水后的油脂按设定程序降温：先将油脂以每半小时10℃的速度快速降温，达到45℃时以每小时降温3℃，逐渐减少为每小时降温1℃。油温降至14℃时开始回温养晶，回温温度1.5℃，回温时间4.5小时，回温后以每小时1.5℃的速度继续降温至-5℃保温，低温冬化时间70小时。
14)再过滤：经常温冬化后的油脂，使用板框压滤机过滤，过滤介质为工业滤布，过滤压力在0.2MPa。
15)脱色：添加1.5％活性炭、1.5％活性白土，脱色70分钟。
16)脱臭：脱臭温度在175±2℃时开始计时，脱臭温度维持在175±2℃， 脱臭时间为4小时，蒸汽压力维持在0.2MPa～0.3MPa，真空度维持在50Pa，蒸汽耗量控制在油重5％左右。降温、破真空，得到成品油脂。
实施例2
1)生产菌种：将原始菌种接入灭菌冷却好的培养基中，在振荡摇瓶中培养。
摇瓶培养基配方为：葡萄糖4.5％，谷氨酸钠3.2％，酵母浸膏0.62％，氯化钠1.8％，磷酸二氢钾0.7％，硫酸镁0.55％，氯化钙0.03％，微量元素(硫酸镍、硫酸铜、钼酸钠、氯化锰、氯化钴、硫酸锌、硫酸亚铁等)0.1％，维生素(B2、B6、B12等)0.1％。
培养条件为：200转/分摇床上，28±1℃培养36～44小时，待菌丝长好后取下，并瓶后接入一级种子罐。
2)一级种子：将菌种接入灭菌冷却好的培养基中，在一级种子罐中培养。
培养基的配方为：葡萄糖3％，酵母粉0.83％，谷氨酸钠0.62％，酵母浸膏0.62％，氯化钠0.62％，磷酸二氢钾0.7％，硫酸镁0.52％，氯化钙0.02％，碳酸氢钠0.02％，硫酸钠0.93％，硫酸氨0.10％，氯化钾0.08％，微量元素(成分同上)0.10％，维生素(成分同上)0.10％，环氧硅醚0.03％。
培养条件为：温度30±2℃，转速90转/分，通气量为0.5vvm，培养18～22小时。
3)二级种子：将4％的一级菌种接入灭菌冷却好的培养基中，在二级种子罐中培养。
培养基的配方为：葡萄糖5％，谷氨酸钠2.0％，酵母浸膏1.0％，氯化钠0.14％，磷酸二氢钾0.16％，硫酸镁0.50％，氯化钙0.02％，碳酸氢钠0.02％，硫酸钠0.91％，硫酸氨0.10％，氯化钾0.08％，微量元素(成分同上)0.09％，维生素(成分同上)0.09％，环氧硅醚0.36％。
培养条件为：温度30±2℃、转速90转/分，培养14～16小时。
4)主发酵：将二级种子液按10％的比例接种于发酵罐的发酵培养基中进行发酵。
培养基配方为：葡萄糖5％，谷氨酸钠2.5％，酵母浸膏1.0％，氯化钠0.29％，磷酸二氢钾0.28％，硫酸镁0.66％，氯化钙0.03％，碳酸氢钠0.02％，硫酸钠0.58％，硫酸氨0.13％，氯化钾0.11％，微量元素(成分同上)0.13％，维生素(成分同上)0.13％，环氧硅醚0.03％。
培养条件为：转速90转/分，0～80小时温度控制为29℃，80小时以后温度控制在19℃；通气量为0.45VVm；用含50％葡萄糖的糖罐添加葡萄糖、添加60％的谷氨酸钠溶液调整培养基中葡萄糖和谷氨酸钠的含量来控制碳氮比，培养基中葡萄糖和谷氨酸钠的含量及PH的控制见表2，培养5天。
表2
Figure PCTCN2019083394-appb-000002
5)提取毛油：发酵完成后，用三相离心机脱水，将菌丝体与水分离、用95％的乙醇脱水后再加入己烷萃取得到毛油。
6)水洗脱胶：加入油重10％的纯净水，加热到85℃，以80rpm的转速搅拌20分钟，静置2小时后分水。
7)酸炼：将油加热到75℃，加入占油重4‰的柠檬酸，以80rpm的转速搅拌40分钟。再加入油重10％的85℃热水，搅拌20分钟，静置3小时后分水。
8)碱炼：将油加热到45℃，根据油脂酸价计算加碱量(加碱量＝7.13×10 -4×酸价×油重)，加入40％的氢氧化钠溶液，以80rpm的转速搅拌50 分钟；将油脂温度升温到80℃，再加入油重5％的85℃纯净水，以80rpm的速度搅拌15分钟，用二相离心机离心去除皂角。
9)脱水：碱炼后的油脂加热到70℃，在-0.1MPa负压下脱水35分钟。
10)常温冬化：脱水后的油脂自然降温，油温降至20℃保持养晶。常温冬化时间16小时。
11)过滤：经常温冬化后的油脂，使用板框压滤机过滤，过滤介质为工业滤布，过滤压力在0.3MPa。
12)再脱水：将经过滤后的油脂加热到80℃，在-0.1MPa负压下脱水35分钟。
13)低温冬化：将脱水后的油脂按设定程序降温：先将油脂以每半小时10℃的速度快速降温，达到45℃时以每小时降温3℃，逐渐减少为每小时降温1℃。油温降至15℃时开始回温养晶，回温温度1℃，回温时间4小时，回温后以每小时2℃的速度继续降温至1℃保温，低温冬化时间48小时。
14)再过滤：经常温冬化后的油脂，使用板框压滤机过滤，过滤介质为工业滤布，过滤压力在0.2MPa。
15)脱色：添加1.5％活性炭、1.5％活性白土，脱色70分钟。
16)脱臭：脱臭温度在175±2℃时开始计时，脱臭温度维持在175±2℃，脱臭时间为4小时，蒸汽压力维持在0.2MPa～0.3MPa，真空度维持在50Pa，蒸汽耗量控制在油重5％左右。降温、破真空，得到成品油脂。
实施例3
1)生产菌种：将原始菌种接入灭菌冷却好的培养基中，在振荡摇瓶中培养。
摇瓶培养基配方为：葡萄糖4.5％，谷氨酸钠3.2％，酵母浸膏0.62％，氯化钠1.8％，磷酸二氢钾0.7％，硫酸镁0.55％，氯化钙0.03％，微量元素(硫酸镍、硫酸铜、钼酸钠、氯化锰、氯化钴、硫酸锌、硫酸亚铁等)0.1％，维 生素(B2、B6、B12等)0.1％。
培养条件为：200转/分摇床上，28±1℃培养36～44小时，待菌丝长好后取下，并瓶后接入一级种子罐。
2)一级种子：将菌种接入灭菌冷却好的培养基中，在一级种子罐中培养。
培养基的配方为：葡萄糖3％，酵母粉0.83％，谷氨酸钠0.62％，酵母浸膏0.62％，氯化钠0.62％，磷酸二氢钾0.7％，硫酸镁0.52％，氯化钙0.02％，碳酸氢钠0.02％，硫酸钠0.93％，硫酸氨0.10％，氯化钾0.08％，微量元素(成分同上)0.10％，维生素(成分同上)0.10％，环氧硅醚0.03％。
培养条件为：温度30±2℃，转速90转/分，通气量为0.50vvm，培养18～22小时。
3)二级种子：将4％的一级菌种接入灭菌冷却好的培养基中，在二级种子罐中培养。
培养基的配方为：葡萄糖5％，谷氨酸钠2.0％，酵母浸膏1.0％，氯化钠0.14％，磷酸二氢钾0.16％，硫酸镁0.50％，氯化钙0.02％，碳酸氢钠0.02％，硫酸钠0.91％，硫酸氨0.10％，氯化钾0.08％，微量元素(成分同上)0.09％，维生素(成分同上)0.09％，环氧硅醚0.36％。
培养条件为：温度30±2℃、转速90转/分，培养14～16小时。
4)主发酵：将二级种子液按10％的比例接种于发酵罐的发酵培养基中进行发酵。
培养基配方为：葡萄糖5％，谷氨酸钠2.5％，酵母浸膏1.0％，氯化钠0.29％，磷酸二氢钾0.28％，硫酸镁0.66％，氯化钙0.03％，碳酸氢钠0.02％，硫酸钠0.58％，硫酸氨0.13％，氯化钾0.11％，微量元素(成分同上)0.13％，维生素(成分同上)0.13％，环氧硅醚0.03％。
培养条件为：转速90转/分，0～80小时温度控制在32℃，80小时以后 温度控制在28℃；通气量为0.5VVm；用含50％葡萄糖的糖罐添加葡萄糖、添加60％的谷氨酸钠溶液调整培养基中葡萄糖和谷氨酸钠的含量来控制碳氮比，培养基中葡萄糖和谷氨酸钠的含量及PH的控制见表3，培养5天。
表3
Figure PCTCN2019083394-appb-000003
5)提取毛油：发酵完成后，用三相离心机脱水，将菌丝体与水分离、用95％的乙醇脱水后再加入己烷萃取得到毛油。
6)水洗脱胶：加入油重10％的纯净水，加热到85℃，以80rpm的转速搅拌20分钟，静置2小时后分水。
7)酸炼：将油加热到75℃，加入占油重4‰的柠檬酸，以80rpm的转速搅拌40分钟。再加入油重10％的85℃热水，搅拌20分钟，静置3小时后分水。
8)碱炼：将油加热到45℃，根据油脂酸价计算加碱量(加碱量＝7.13×10 -4×酸价×油重)，加入40％的氢氧化钠溶液，以80rpm的转速搅拌50分钟；将油脂温度升温到80℃，再加入油重5％的85℃纯净水，以80rpm的速度搅拌15分钟，用二相离心机离心去除皂角。
9)脱水：将经水洗脱胶、碱炼脱皂后的油脂加热到90℃，在-0.1MPa负压下脱水35分钟。
10)常温冬化：脱水后的油脂自然降温，油温降至30℃保持养晶。常温冬化时间24小时。
11)过滤：经常温冬化后的油脂，使用板框压滤机过滤，过滤介质为工 业滤布，过滤压力在0.3MPa。
12)再脱水：将经过滤后的油脂加热到85℃，在-0.1MPa负压下脱水35分钟。
13)低温冬化：将脱水后的油脂按设定程序降温：先将油脂以每半小时10℃的速度快速降温，达到45℃时以每小时降温3℃，逐渐减少为每小时降温1℃。油温降至13℃时开始回温养晶，回温温度2℃，回温时间5小时，回温后以每小时1℃的速度继续降温至-10℃，确保养晶时间不少于16小时。低温冬化时间90小时。
14)再过滤：经常温冬化后的油脂，使用板框压滤机过滤，过滤介质为工业滤布，过滤压力在0.2MPa。
15)脱色：添加1.5％活性炭、1.5％活性白土，脱色70分钟。
16)脱臭：脱臭温度在175±2℃时开始计时，脱臭温度维持在175±2℃，脱臭时间为4小时，蒸汽压力维持在0.2MPa～0.3MPa，真空度维持在50Pa，蒸汽耗量控制在油重5％左右。降温、破真空，得到成品油脂。
对照例
参照实施例1的方法培养菌种和发酵，不同的是主发酵培养基中含葡萄糖4.5％，谷氨酸钠2％，酵母浸膏0.9％，(碳氮比为10.7：1)，采用28℃的恒温培养5天；低温冬化时快速降温至0℃结晶养晶48小时。
实验结果
分别将实施例1、实施例2、实施例3和对照例得到的成品油脂，气相色谱检测其中的脂肪酸成分，各脂肪酸在成品油脂中所占比例见表4。
表4
Figure PCTCN2019083394-appb-000004
通过表4的结果可以看出，采用本发明的DHA微生物油脂中脂肪酸组 合物成分调整的方法实施例DHA含量更高，有害脂肪酸含量更低。另外还具有更好的低温凝固性能。
本发明提供的微生物油脂中DHA含量高、EPA含量低，有害脂肪酸的含量低，低温凝固性能好，可用于制作婴幼儿配方食品，尤其是婴幼儿配方奶粉；还可制作成保健品，根据本领域的技术人员已知的DHA与许多疾病的关系提供给有相关需求的人群作治疗、保健要求用；还可制作成健康食品与普通食品等为人体提供所需的营养，补充日常摄入的不足。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (9)

  1. 一种DHA微生物油脂中脂肪酸组合物成分调整的方法，其特征在于，包括在菌种发酵的第0～80小时，将培养基的碳氮比控制在3～20:1，培养温度控制在29～32℃之间；在菌种发酵的第81小时以后，将培养基的碳氮比控制在1～15:1，培养温度控制在19～28℃之间，以提高DHA的转化率，降低其它脂肪酸的含量。
  2. 根据权利要求1所述的DHA微生物油脂中脂肪酸组合物成分调整的方法，其特征在于，所述控制碳氮比的方式为，控制培养基中葡萄糖和谷氨酸钠的含量，以维持所需的碳氮比。
  3. 根据权利要求2所述的DHA微生物油脂中脂肪酸组合物成分调整的方法，其特征在于，所述控制培养基中葡萄糖和谷氨酸钠含量的过程为，在发酵的第0～16小时，葡萄糖含量为2～8g/100ml，谷氨酸钠的含量为1～5g/100ml；第17～55小时，葡萄糖含量为2～6g/100ml，谷氨酸钠的含量为1～3g/100ml；第56～80小时，葡萄糖含量为1～5g/100ml，谷氨酸钠的含量为1～2g/100ml；第81～95小时，葡萄糖含量0.5～1.0g/100ml，谷氨酸钠的含量为0.75～1.0g/100ml；第96小时以后，葡萄糖含量<0.5g/100ml，谷氨酸钠的含量为0.5～0.75g/100ml。
  4. 根据权利要求1所述的DHA微生物油脂中脂肪酸组合物成分调整的方法，其特征在于，还包括在微生物油脂的精制过程中，先将油脂升温至70～90℃脱水，然后逐渐冷却至20～30℃常温冬化16～24小时，过滤、脱水后，再按设定的程序逐渐降温到-10～1℃保温，低温冬化48～90小时。
  5. 一种DHA微生物油脂，其特征在于，根据权利要求1至4中任一项 所述的方法制得，以微生物油脂的总量为基准，含有以下组分：
    不低于40wt％的DHA，不超过3wt％的EPA；
    0～5wt％的C12:0脂肪酸；
    0～8wt％的C14:0、C14:1脂肪酸；
    5～50wt％的C16:0和C16:1脂肪酸；
    0.5～10wt％的C18:0、C18:1和C18:2脂肪酸；
    0～5wt％的C22:1n9脂肪酸。
  6. 根据权利要求5所述的DHA微生物油脂，其特征在于，所述DHA微生物油脂中，DHA与EPA的比例为(13.3～600):1。
  7. 根据权利要求5或6所述的DHA微生物油脂，其特征在于，所述DHA微生物油脂中不饱和脂肪酸与饱和脂肪酸的含量比不低于0.60。
  8. 根据权利要求7所述的DHA微生物油脂，其特征在于，所述DHA微生物油脂中不饱和脂肪酸与饱和脂肪酸的含量比不低于2.3。
  9. 一种组合物，其特征在于，包含权利要求5-8中任一项所述的DHA微生物油脂，所述组合物适用于制作婴幼儿配方食品、保健食品、健康食品以及普通食品。
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CN107746746A (zh) * 2017-10-17 2018-03-02 山东西王食品有限公司 一种连续结晶、养晶、脱蜡精炼提高植物油品质的方法及设备
CN109371071A (zh) * 2018-05-17 2019-02-22 梁云 Dha微生物油脂中脂肪酸组合物成分调整的方法

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