WO2019218841A1 - 被孢霉属微生物油脂中脂肪酸组合物成分调整的方法 - Google Patents

被孢霉属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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WO2019218841A1
WO2019218841A1 PCT/CN2019/083419 CN2019083419W WO2019218841A1 WO 2019218841 A1 WO2019218841 A1 WO 2019218841A1 CN 2019083419 W CN2019083419 W CN 2019083419W WO 2019218841 A1 WO2019218841 A1 WO 2019218841A1
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hours
carbon
fatty acid
temperature
composition
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PCT/CN2019/083419
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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 AU2019271766A priority Critical patent/AU2019271766B2/en
Priority to KR1020207032138A priority patent/KR102565649B1/ko
Priority to EP19804372.1A priority patent/EP3795691A4/en
Publication of WO2019218841A1 publication Critical patent/WO2019218841A1/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
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • 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
    • 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
    • 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
    • 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

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 microbial oil of the genus Mortierella.
  • 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 raw materials for obtaining high value-added fatty acids such as linolenic acid (GLA), ARA, eicosapentaenoic acid (EPA), DHA and the like.
  • GLA linolenic acid
  • ARA eicosapentaenoic acid
  • DHA eicosapentaenoic acid
  • the microbial oil produced by the microorganism of the genus Mortierella is high in ARA content.
  • the fats and oils obtained by the preparation method of the existing microbial oil of the genus Mortierella are not high enough, and the fats and oils also contain some harmful fatty acids, such as myristic acid, lauric acid, erucic acid, etc., which affect the use of microbial oils and fats. .
  • the object of the present invention is to overcome the problem of high content of harmful fatty acids, high content of saturated fatty acids, and insufficient content of ARA in the microbial oil of the genus Mortierella produced by the prior art, and to provide a microorganism oil of the genus Mortierella.
  • a method for adjusting the composition of a fatty acid composition which can regulate the content of a fatty acid composition in a microbial oil and fat, and produce a microbial oil having a high ARA content, wherein the harmful fatty acid content is low.
  • This high ARA content, low harmful fatty acid content can be used to make infant foods and health foods.
  • the present invention provides a method for adjusting a composition of a fatty acid composition in a microbial oil of the genus Mortierella, which comprises controlling the carbon-nitrogen ratio of the culture from the initial period of 2.5 to 5% during the main fermentation. 20:1 keeps falling until it is 0.
  • the culture temperature is controlled between 29 and 32 °C during the main fermentation for 0 to 120 hours. After 120 hours of the main fermentation, the culture temperature is controlled between 15 and 28 °C.
  • the "high temperature, high carbon to nitrogen ratio process” in the early stage of fermentation enables microbial cells to grow and reproduce rapidly, so that the biomass can be rapidly increased, and the microbial oil can be rapidly converted and enriched.
  • the "low temperature, low carbon to nitrogen ratio process” in the late fermentation stage promotes The fatty acid is further transformed to control the levels of non-essential fatty acids and harmful fatty acids.
  • the process of controlling the carbon to nitrogen ratio is: in the 0 to 40 hours of fermentation, the carbon to nitrogen ratio is 2.5 to 25:1; 41 to 65 hours, and the carbon to nitrogen ratio is 2 to 10:1; 66 to 89.
  • carbon to nitrogen ratio is 2 to 8:1; 90 to 113 hours, carbon to nitrogen ratio is 1 to 5:1; 114 to 122 hours, carbon to nitrogen ratio is 1 to 2:1; 123 to 130 hours, carbon to nitrogen ratio It is 1 to 0.5:1; after 131 hours, the carbon to nitrogen ratio is 0.
  • the carbon to nitrogen ratio is controlled by not replenishing the nitrogen source after the initial input of the nitrogen source, and periodically supplementing the carbon source as needed to maintain the desired carbon to nitrogen ratio.
  • the method for adjusting the composition of the fatty acid composition in the microbe oil of the genus Mortierella according to the present invention further comprises adopting a secondary winterization process in the refining process of the oil and fat: first heating the oil to 70-90 ° C for dehydration, and then gradually Cool to 20 ⁇ 30 ° C at room temperature for 16 to 24 hours, filter, dehydrate, and then gradually reduce the temperature to -10 ⁇ 1 ° C according to the set procedure, low temperature winterization for 48 to 90 hours.
  • the secondary winterization process of normal temperature winterization and low temperature winterization can further reduce the content of harmful fatty acids with higher melting point in the oil and improve the low temperature flow performance of the oil.
  • the present invention provides a method for modulating the composition of a fatty acid composition in any of the microorganisms of the genus Mortierella according to the first aspect of the present invention, wherein the microbial oil is obtained by the method of adjusting the composition of the fatty acid composition of the microorganism of the genus Mortierella according to the first aspect of the present invention.
  • the following components are contained: not less than 40% by weight of arachidonic acid; 0 to 5% by weight of C12:0 fatty acid; 0 to 5% by weight of C14:0 and C14:1 fatty acid; and 1% to 25% by weight C16:0 and C16:1 fatty acids; 2wt% to 35wt% C18:0, C18:1 and C18:2 fatty acids; 0-5wt% C22:1n9 fatty acids.
  • the weight ratio of the unsaturated fatty acid to the saturated fatty acid in the microorganism of the genus Mortierella is not less than 0.6.
  • the weight ratio of the unsaturated fatty acid to the saturated fatty acid in the microorganism of the Mortierella microorganism is not less than 2.3.
  • the present invention provides a composition comprising the microorganism of the genus Mortierella according to the second aspect of the present invention, which is suitable for the preparation of an infant formula (infant formula), a health food, Healthy food and general food.
  • Mortierella alpina is used by the microorganism of the genus Mortierella, and the strains and various culture materials used are commercially available, and the reagents and detection methods involved are National standards are implemented.
  • 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: 3% glucose, 1.5% yeast powder, and the pH is controlled to 8.0-8.5 with sodium hydroxide solution.
  • the culture condition is: 200 rpm on a shaker, and cultured at 28 ⁇ 1 ° C for 44 to 50 hours. After the hyphae are long, remove the bottle, and then access the first-stage seed tank.
  • the strain is connected to a sterilized and cooled medium and cultured in a primary seed tank.
  • the medium formula was: 3% glucose, 1.5% yeast powder, 0.06% antifoaming agent (epoxy silicone ether), and 0.09% sodium hydroxide.
  • the culture conditions were: 200 rpm, the aeration amount was 0.55 VVm, and the culture was carried out at 29 ⁇ 1 ° C for 48 hours.
  • Secondary seed The strain is connected to a sterilized and cooled medium and cultured in a secondary seed tank.
  • the medium formula was: 4% glucose, 1.5% yeast powder, 0.095% antifoaming agent (epoxy silicone ether), and 0.11% sodium hydroxide.
  • the culture conditions were: 200 rpm, the aeration amount was 0.55 VVm, and the culture was carried out at 29 ⁇ 1 ° C for 48 hours.
  • Main fermentation The seed liquid was inoculated in a fermentation medium of a fermenter at a ratio of 15% for fermentation.
  • the medium formulation was: 8% glucose, 2% yeast powder, and 0.04% antifoam (epoxy silicone ether).
  • the culture conditions were as follows: the rotation speed was 160 rpm, the temperature was set to 30 ° C for 0 to 80 hours, the temperature was set to 22 ° C after 80 hours, the ventilation was 0.8 VVm for 0 to 80 hours, and 0.67 VVm for 80 hours; Glucose is added to the sterile sugar solution of glucose to control the carbon to nitrogen ratio.
  • the carbon to nitrogen ratio and pH control are shown in Table 1, and cultured for 7 days.
  • Drying drying with “boiling drying tower”, the hot air temperature is 180 ° C, and the moisture content of the bacteria after drying is ⁇ 5%.
  • 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.
  • 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. .
  • the oil temperature drops to 15 °C, the temperature is raised back to the temperature, the temperature is 1.5 °C, the temperature is back to 5 hours, and after the temperature is returned, the temperature is lowered to -5 °C at a rate of 2 °C per hour to ensure that the crystal growth time is not less than 16 hour.
  • Low temperature winterization time 80 hours.
  • Re-filtration The oil 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.
  • the deodorization temperature starts at 175 ⁇ 2°C, the deodorization temperature is maintained at 175 ⁇ 2°C, the deodorization time is 4 hours, the vapor pressure is maintained at 0.2MPa ⁇ 0.3MPa, the vacuum degree is maintained at 50Pa, and the steam consumption is controlled. About 5% of oil
  • 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: 3% glucose, 1.5% yeast powder, and the pH is controlled to 8.0-8.5 with sodium hydroxide solution.
  • the culture condition is: 200 rpm on a shaker, and cultured at 28 ⁇ 1 ° C for about 44 to 50 hours. After the hyphae are long, the bottle is removed, and then the first stage seed tank is connected.
  • the strain is connected to a sterilized and cooled medium and cultured in a primary seed tank.
  • the medium formula was: 3% glucose, 1.5% yeast powder, 0.06% antifoaming agent (epoxy silicone ether), and 0.09% sodium hydroxide.
  • the culture conditions were: 200 rpm, the aeration amount was 0.55 VVm, and the culture was carried out at 29 ⁇ 1 ° C for 48 hours.
  • Secondary seed The strain is connected to a sterilized and cooled medium and cultured in a secondary seed tank.
  • the medium formula was: 4% glucose, 1.5% yeast powder, 0.095% antifoaming agent (epoxy silicone ether), and 0.11% sodium hydroxide.
  • the culture conditions were: 200 rpm, the aeration amount was 0.55 VVm, and the culture was carried out at 29 ⁇ 1 ° C for 48 hours.
  • Main fermentation The seed liquid was inoculated in a fermentation medium of a fermenter at a ratio of 10% for fermentation.
  • the medium formula was: 8% glucose, 2% yeast powder, and 0.03% antifoaming agent (epoxy silicone ether).
  • the culture conditions are: rotation speed 120 rpm, temperature setting from 0 to 80 hours is 29 ° C, temperature is set to 15 ° C after 80 hours; ventilation is 0 to 80 hours is 0.75 VVm, after 80 hours is 0.65 VVm; use is 25% Glucose is added to the sterile sugar solution to control the carbon to nitrogen ratio.
  • the carbon to nitrogen ratio and pH control are shown in Table 2, and cultured for 7 days.
  • Drying drying with “boiling drying tower”, the hot air temperature is 180 ° C, and the moisture content of the bacteria after drying is ⁇ 5%.
  • 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.
  • 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 1 °C, and the temperature is 4 hours. After the temperature is returned, the temperature is lowered to 1 °C at a rate of 1 °C per hour to ensure that the crystal growth time is not less than 16. hour. Low temperature winterization time of 48 hours.
  • Re-filtration The oil 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.
  • the deodorization temperature starts at 175 ⁇ 2°C, the deodorization temperature is maintained at 175 ⁇ 2°C, the deodorization time is 4 hours, the vapor pressure is maintained at 0.2MPa ⁇ 0.3MPa, the vacuum degree is maintained at 50Pa, and the steam consumption is controlled. About 5% of oil
  • 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: 3% glucose, 1.5% yeast powder, and the pH is controlled to 8.0-8.5 with sodium hydroxide solution.
  • the culture condition is: 200 rpm on a shaker, and cultured at 28 ⁇ 1 ° C for about 44 to 50 hours. After the hyphae are long, the bottle is removed, and then the first stage seed tank is connected.
  • the strain is connected to a sterilized and cooled medium and cultured in a primary seed tank.
  • the medium formula was: 3% glucose, 1.5% yeast powder, 0.06% antifoaming agent (epoxy silicone ether), and 0.09% sodium hydroxide.
  • the culture conditions were: 200 rpm, the aeration amount was 0.55 VVm, and the culture was carried out at 29 ⁇ 1 ° C for 48 hours.
  • Secondary seed The strain is connected to a sterilized and cooled medium and cultured in a secondary seed tank.
  • the medium formula was: 4% glucose, 1.5% yeast powder, 0.095% antifoaming agent (epoxy silicone ether), and 0.11% sodium hydroxide.
  • the culture conditions were: 200 rpm, the aeration amount was 0.55 VVm, and the culture was carried out at 29 ⁇ 1 ° C for 48 hours.
  • Main fermentation The seed liquid was inoculated in a fermentation medium of a fermenter at a ratio of 20% for fermentation.
  • the medium formulation was: 8% glucose, 2% yeast powder, and 0.04% antifoam (epoxy silicone ether).
  • the culture conditions are: rotation speed 200 rpm, temperature control at 32 ° C for 0 to 80 hours, temperature control at 28 ° C after 80 hours, 0.9 VVm for 0 to 80 hours, 0.7 VVm after 80 hours, and 25% for use Glucose is added to the sterile sugar solution to control the carbon to nitrogen ratio.
  • the carbon to nitrogen ratio and pH control are shown in Table 3, and cultured for 7 days.
  • Drying drying with “boiling drying tower”, the hot air temperature is 185 ° C, and the moisture content of the bacteria after drying is ⁇ 5%.
  • Acid refining The oil was heated to 75 ° C, citric acid having an oil weight of 4 Torr was added, and stirred at a speed of 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.
  • 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 12 °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 2 °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 oil 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.
  • the deodorization temperature starts at 175 ⁇ 2°C, the deodorization temperature is maintained at 175 ⁇ 2°C, the deodorization time is 4 hours, the vapor pressure is maintained at 0.2MPa ⁇ 0.3MPa, the vacuum degree is maintained at 50Pa, and the steam consumption is controlled. About 5% of oil
  • the strain and the fermentation were cultured in the same manner as in Example 1, except that the main fermentation medium contained 5% glucose and 2% yeast powder (carbon to nitrogen ratio 11.8:1), and cultured at 28 ° C for 7 days. In the low temperature winterization process, the temperature is rapidly lowered to -5 ° C, and the crystals are crystallized for 48 hours.
  • Example 1 The finished fats and oils obtained in Example 1, Example 2, Example 3, and Comparative Example 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.
  • Example 1 Example 2
  • Example 3 Control case Lauric acid (C12:0) 0.02 0.028 0.03 1.12 Myristic acid (C14:0) 0.386 0.412 0.397 1.35 Myristic acid C14:1 0.025 0.031 0.028 1.28 Palmitic acid (C16:0) 6.25 6.35 6.34 27.47 Palmitoleic acid C16:1 0.144 0.213 0.196 0.113 Stearic acid (C18:0) 5.776 5.634 4.236 17.36 Oleic acid (C18:1, n9) 7.192 6.21 5.78 0.19 Linoleic acid (C18: 2, n6) 7.45 6.599 6.707 0.16 ⁇ -linolenic acid (C18:3n6) 3.949 3.672 3.11 0.13 ARA (C20: 4, n6) 51.7 54.76 56.9 35.87 Arachiic acid (C22:0) 3.411 3.42 3.395 4.12 EPA (C20: 5, n3) 0.099 0.087
  • Unsaturated fatty acids saturated fatty acids 2.41 2.54 2.66 0.59
  • the microorganism oil obtained by the embodiment of the method for adjusting the composition of the fatty acid composition in the microorganism microorganism oil of the present invention has a higher ARA content and a lower harmful fatty acid content. It also has better low temperature setting properties.
  • microorganisms of the genus Mortierella provided by the present invention have higher ARA content.
  • a microorganism of the genus Mortierella having an ARA content of more than 50% can be produced, wherein the content of the harmful fatty acid component is also lower.
  • the microbial oil provided by the invention has high ARA content, low content of harmful fatty acids, good low temperature solidification performance, and can be used for preparing infant formula, especially infant formula; and can also be made into health care products according to those skilled in the art.
  • the known relationship between ARA and many diseases is provided to people with relevant needs for treatment and health care needs; it can also be made into healthy foods and common foods to provide the body with the required nutrients, supplementing the daily intake.

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Abstract

一种被孢霉属微生物油脂中脂肪酸组合物成分调整的方法，在菌体主发酵的过程中，控制培养的碳氮比从培养初期的2.5～25：1不断下降，直至为0，随着氮源的消耗、再控制碳源的补充，营造一个营养缺陷型生长环境，有助于脂肪酸的转化。同时，在发酵的0～120h，将发酵温度控制在29～32℃；在发酵120h以后，将发酵温度控制在15～28℃的温度条件。精炼阶段采用变温与程序控温的冬化工艺，去除相对饱和度高的脂肪酸含量，提高多不饱和脂肪酸的相对含量，改善油脂的低温流动性能。该方法取得的组合物可用于制备婴幼儿配方食品、保健品、健康食品以及普通食品。

Description

被孢霉属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，具体涉及一种被孢霉属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)、ARA、二十碳五烯酸(EPA)、DHA等的重要原料。
由被孢霉属微生物发酵生产的微生物油脂ARA含量较高。而现有的被孢霉属微生物油脂的制备方法所获取的油脂，ARA的含量还不够高，油脂中也含有一些有害脂肪酸，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被孢霉属微生物油脂中有害脂肪酸含量偏高、饱和脂肪酸含量偏高、以及ARA的含量不够高的问题，提供一种被孢霉属微生物油脂中脂肪酸组合物成份调整的方法，该方法可以调控微生物油脂中脂肪酸组合物的含量，生产高ARA含量的微生物油脂，且其中的有害脂肪酸含量很低。而这种高ARA含量、低有害脂肪酸含量的特性，可用于制作婴幼儿食品和健康保健食品等。
为了实现上述目的，第一方面，本发明提供了一种被孢霉属微生物油脂中脂肪酸组合物成分调整的方法，包括在主发酵的过程中，控制培养的碳氮比从培养初期的2.5～20：1不断下降，直至为0；同时，在主发酵的0～120小时，培养温度控制在29～32℃之间，在主发酵的120小时以后，培养温度控制在15～28℃之间；在发酵前期的“高温、高碳氮比工艺”使微生物细胞快速生长繁殖，使生物量快速增值，同时也使微生物油脂快速转化富集；发酵后期的“低温、低碳氮比工艺”促使脂肪酸进一步转化，控制非必需脂肪酸、有害脂肪酸的含量水平。
优选地，所述控制碳氮比的过程为，在发酵的第0～40小时，碳氮比为2.5～25：1；41～65小时，碳氮比为2～10：1；66～89小时，碳氮比为2～8：1；90～113小时，碳氮比为1～5：1；114～122小时，碳氮比为1～2：1；123～130小时，碳氮比为1～0.5：1；131小时以后，碳氮比为0。
优选地，所述控制碳氮比的方式为，在初始投入氮源后不再补充氮源，而定期按需补充碳源，维持所需的碳氮比。
优选地，本发明提供的被孢霉属微生物油脂中脂肪酸组合物成分调整的方法，还包括在油脂的精炼过程中采用二次冬化工艺：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。 采用常温冬化、低温冬化的二次冬化工艺，可进一步降低油脂中熔点较高的有害脂肪酸的含量，改善油脂的低温流动性能。
第二方面，本发明提供了一种被孢霉属微生物油脂，根据本发明第一方面所述的任一被孢霉属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％的花生四烯酸；0～5wt％的C12:0脂肪酸；0～5wt％的C14:0和C14:1脂肪酸；1wt％～25wt％的C16:0和C16:1脂肪酸；2wt％～35wt％的C18:0、C18:1和C18:2脂肪酸；0～5wt％的C22:1n9脂肪酸。
优选地，所述被孢霉属微生物油脂中不饱和脂肪酸与饱和脂肪酸的重量比不低于0.6。
进一步优选地，所述被孢霉属微生物油脂中不饱和脂肪酸与饱和脂肪酸的重量比不低于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被孢霉属微生物油脂，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ARA的含量也很高，有害脂肪酸豆蔻酸、月桂酸和芥酸的含量很低，还可在-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，被孢霉属微生物使用高山被孢霉 (Mortierella alpina)，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3％，酵母粉1.5％，用氢氧化钠溶液将PH控制在8.0～8.5。
培养条件为：200转/分摇床上，28±1℃培养44～50小时，待菌丝长好后取下，并瓶，然后接入一级种子罐。
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。
培养基配方为：葡萄糖3％，酵母粉1.5％，消泡剂(环氧硅醚)0.06％，氢氧化钠0.09％。
培养条件为：200转/分，通气量0.55VVm，29±1℃培养48小时。
3)二级种子：将菌种接入灭菌冷却好的培养基中，在二级种子罐中培养。
培养基配方为：葡萄糖4％，酵母粉1.5％，消泡剂(环氧硅醚)0.095％，氢氧化钠0.11％。
培养条件为：200转/分，通气量0.55VVm，29±1℃培养48小时。
4)主发酵：将种子液按15％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8％，酵母粉2％，消泡剂(环氧硅醚)0.04％。
培养条件为：转速160转/分，0～80小时温度设置为30℃，80小时以后温度设置为22℃；通气量0～80小时为0.8VVm，80小时以后为0.67VVm；用含25％葡萄糖的无菌糖液添加葡萄糖的方式来控制碳氮比，碳氮比及PH的控制见表1，培养7天。
表1
时间 0～40 41～65 66～89 90～113 114～122 123～130 ≥131
碳氮比 10～15 6～8 4～6 2～4 1～2 0.5～1 0
PH 自然 6.2～6.5 6.5～6.7 6.7～7.0 7.0～7.4 7.0～7.4 7.4～8.0
5)过滤：采用“板框压滤机”将菌丝体与水分离，分离得到的菌丝体用“摇摆颗粒机”粉碎造粒。
6)干燥：采用“沸腾干燥塔”干燥，热风温度180℃，干燥后菌体水分含量≤5％。
7)萃取：“丁烷”萃取制得毛油。
8)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。
9)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。
10)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。
11)脱水：将油脂加热到85℃，在-0.1MPa负压下脱水35分钟。
12)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。
13)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。
14)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。
15)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1.5℃，回温时间5小时，回温后以每小时2℃的速度继续降温至-5℃，确保养晶时间不少于16小时。低温冬化时间80小时。
16)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。
17)脱色：添加1.5％活性炭、1.5％活性白土，脱色70分钟。
18)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
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3％，酵母粉1.5％，用氢氧化钠溶液将PH控制在8.0～8.5。
培养条件为：200转/分摇床上，28±1℃培养44～50小时左右，待菌丝长好后取下，并瓶，然后接入一级种子罐。
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。
培养基配方为：葡萄糖3％，酵母粉1.5％，消泡剂(环氧硅醚)0.06％，氢氧化钠0.09％。
培养条件为：200转/分，通气量0.55VVm，29±1℃培养48小时。
3)二级种子：将菌种接入灭菌冷却好的培养基中，在二级种子罐中培养。
培养基配方为：葡萄糖4％，酵母粉1.5％，消泡剂(环氧硅醚)0.095％，氢氧化钠0.11％。
培养条件为：200转/分，通气量0.55VVm，29±1℃培养48小时。
4)主发酵：将种子液按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8％，酵母粉2％，消泡剂(环氧硅醚)0.03％。
培养条件为：转速120转/分，0～80小时温度设置为29℃，80小时以后温度设置为15℃；通气量0～80小时为0.75VVm，80小时以后为0.65VVm；用含25％葡萄糖的无菌糖液添加葡萄糖的方式来控制碳氮比，碳氮比及PH的控制见表2，培养7天。
表2
时间 0～40 41～65 66～89 90～113 114～122 130～144 ≥144
碳氮比 2.5～5 2～2.5 2～2.5 1～2 1～1.5 0.5～1 0
PH 自然 6.2～6.5 6.5～6.7 6.7～7.0 7.0～7.4 7.0～7.4 7.4～8.0
5)过滤：采用“板框压滤机”将菌丝体与水分离，分离得到的菌丝体用“摇摆颗粒机”粉碎造粒。
6)干燥：采用“沸腾干燥塔”干燥，热风温度180℃，干燥后菌体水分含量≤5％。
7)萃取：“丁烷”萃取制得毛油。
8)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。
9)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。
10)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。
11)脱水：将经水洗脱胶、碱炼脱皂后的油脂加热到70℃，在-0.1MPa负压下脱水35分钟。
12)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。
13)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。
14)再脱水：将经过滤后的油脂加热到70℃，在-0.1MPa负压下脱水35分钟。
15)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1℃，回温时间4小时，回温后以每小时1℃的速度继续降温至1℃保持，确保养晶时间不少于16小时。低温冬化时间48小时。
16)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。
17)脱色：添加1.5％活性炭、1.5％活性白土，脱色70分钟。
18)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
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3％，酵母粉1.5％，用氢氧化钠溶液将PH控制在8.0～8.5。
培养条件为：200转/分摇床上，28±1℃培养44～50小时左右，待菌丝长好后取下，并瓶，然后接入一级种子罐。
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。
培养基配方为：葡萄糖3％，酵母粉1.5％，消泡剂(环氧硅醚)0.06％，氢氧化钠0.09％。
培养条件为：200转/分，通气量0.55VVm，29±1℃培养48小时。
3)二级种子：将菌种接入灭菌冷却好的培养基中，在二级种子罐中培养。
培养基配方为：葡萄糖4％，酵母粉1.5％，消泡剂(环氧硅醚)0.095％，氢氧化钠0.11％。
培养条件为：200转/分，通气量0.55VVm，29±1℃培养48小时。
4)主发酵：将种子液按20％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8％，酵母粉2％，消泡剂(环氧硅醚)0.04％。
培养条件为：转速200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0～80小时为0.9VVm，80小时以后为0.7VVm；用含25％葡萄糖的无菌糖液添加葡萄糖的方式来控制碳氮比，碳氮比及PH的控制见表3，培养7天。
表3
时间 0～40 41～65 66～89 90～113 114～122 123～130 ≥131
碳氮比 20～25 8～10 6～8 4～5 1.5～2 0.5～1 0
PH 自然 6.2～6.5 6.5～6.7 6.7～7.0 7.0～7.4 7.0～7.4 7.4～8.0
5)过滤：采用“板框压滤机”将菌丝体与水分离，分离得到的菌丝体用“摇摆颗粒机”粉碎造粒。
6)干燥：采用“沸腾干燥塔”干燥，热风温度185℃，干燥后菌体水分含量≤5％。
7)萃取：“丁烷”萃取制得毛油。
8)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。
9)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。
10)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。
11)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。
12)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。
13)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。
14)再脱水：将经过滤后的油脂加热到90℃，在-0.1MPa负压下脱水35分钟。
15)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12℃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2℃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。
16)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。
17)脱色：添加1.5％活性炭、1.5％活性白土，脱色70分钟。
18)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
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5％，酵母粉2％(碳氮比为11.8：1)，采用28℃的恒温培养7天。在低温冬化工艺中，快速降温至-5℃，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，使用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
脂肪酸名称 实施例1 实施例2 实施例3 对照例
月桂酸(C12:0) 0.02 0.028 0.03 1.12
豆蔻酸(C14:0) 0.386 0.412 0.397 1.35
肉豆蔻酸C14:1 0.025 0.031 0.028 1.28
棕榈酸(C16:0) 6.25 6.35 6.34 27.47
棕榈油酸C16:1 0.144 0.213 0.196 0.113
硬脂酸(C18:0) 5.776 5.634 4.236 17.36
油酸(C18:1，n9) 7.192 6.21 5.78 0.19
亚油酸(C18:2,n6) 7.45 6.599 6.707 0.16
γ-亚麻酸(C18:3n6) 3.949 3.672 3.11 0.13
ARA(C20:4,n6) 51.7 54.76 56.9 35.87
花生酸(C22:0) 3.411 3.42 3.395 4.12
EPA(C20:5,n3) 0.099 0.087 0.095 0.12
DPA(C22:5,n6) 0.02 0.03 0.023 0.20
DHA(C22:6,n3) 0.022 0.031 0.025 0.032
二十四烷酸(C24:0) 9.342 8.16 8.45 15.12
芥酸(C22:1n9) 0.114 0.153 0.123 0.353
其他脂肪酸 4.1 4.21 4.165 4.71
不饱和脂肪酸:饱和脂肪酸 2.41 2.54 2.66 0.59
通过表4的结果可以看出，采用本发明的被孢霉属微生物油脂中脂肪酸组合物成分调整的方法的实施例得到的微生物油脂中ARA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。
本发明提供的被孢霉属微生物油脂中ARA含量更高。使用本发明提供的优选方法，可以生产出ARA含量超过50％的被孢霉属微生物油脂，其中有害脂肪酸成分的含量也更低。
本发明提供的微生物油脂中ARA含量高，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ARA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 (8)

  1. 一种被孢霉属微生物油脂中脂肪酸组合物成分调整的方法，其特征在于，包括在菌体主发酵的过程中，控制培养的碳氮比从培养初期的2.5～25：1不断下降，直至为0；同时，在菌体主发酵的0～120小时，培养温度控制在29～32℃之间，120小时以后，培养温度控制在15～28℃之间，以提高二十碳四烯酸的转化率，降低其它脂肪酸的含量。
  2. 根据权利要求1所述的被孢霉属微生物油脂中脂肪酸组合物成分调整的方法，其特征在于，所述控制培养的碳氮比的过程为，在发酵的第0～40小时，碳氮比为2.5～25：1；41～65小时，碳氮比为2～10：1；66～89小时，碳氮比为2～8：1；90～113小时，碳氮比为1～5：1；114～122小时，碳氮比为1～2：1；123～130小时，碳氮比为1～0.5：1，131小时以后，碳氮比为0。
  3. 根据权利要求1或2所述的被孢霉属微生物油脂中脂肪酸组合物成分调整的方法，其特征在于，所述控制培养的碳氮比的方式为，在初始投入氮源后不再补充氮源，而定期按需补充碳源，维持所需的碳氮比。
  4. 根据权利要求1所述的被孢霉属微生物油脂中脂肪酸组合物成分调整的方法，其特征在于，还包括在油脂的精炼过程中采用二次冬化工艺：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. 一种被孢霉属微生物油脂，其特征在于，根据权利要求1至4中任一项权利要求所述的被孢霉属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％的花生四烯酸；
    0～5wt％的C12:0脂肪酸；
    0～5wt％的C14:0和C14:1脂肪酸；
    1wt％～25wt％的C16:0和C16:1脂肪酸；
    2wt％～35wt％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所述的被孢霉属微生物油脂，其特征在于，其中不饱和脂肪酸与饱和脂肪酸的重量比不低于0.6。
  7. 根据权利要求6所述的被饱霉属微生物油脂，其特征在于，其中不饱和脂肪酸与饱和脂肪酸的重量比不低于2.3。
  8. 一种组合物，其特征在于，包含权利要求5-7任一项所述的被孢霉微生物油脂，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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