WO2015135501A1 - 一种微生物油及其制备方法、一种微胶囊 - Google Patents

一种微生物油及其制备方法、一种微胶囊 Download PDF

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WO2015135501A1
WO2015135501A1 PCT/CN2015/074205 CN2015074205W WO2015135501A1 WO 2015135501 A1 WO2015135501 A1 WO 2015135501A1 CN 2015074205 W CN2015074205 W CN 2015074205W WO 2015135501 A1 WO2015135501 A1 WO 2015135501A1
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oil
content
microbial
microbial oil
weight
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PCT/CN2015/074205
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English (en)
French (fr)
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汪志明
周强
陆姝欢
肖敏
田勇
李翔宇
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嘉必优生物工程（武汉）有限公司
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Publication of WO2015135501A1 publication Critical patent/WO2015135501A1/zh

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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1528—Fatty acids; Mono- or diglycerides; Petroleum jelly; Paraffine; Phospholipids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
    • 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
    • A23D9/013—Other fatty acid esters, e.g. phosphatides
    • 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
    • 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
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • 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
    • C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
    • 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
    • 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
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, 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
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a microbial oil and a process for the preparation thereof, and to a microcapsule.
  • the present application is based on a Chinese patent application filed on March 14, 2014, the application number of which is incorporated herein by reference.
  • Polyunsaturated fatty acid Acid refers to a fatty acid containing two or more double bonds, typically having from 18 to 22 carbon atoms.
  • Industrialized PUFAs are mostly produced by single-cell microorganisms such as fungi and algae, and are therefore also called microbial oils.
  • the omega-3 series includes octadecatrienoic acid (commonly known as alpha-linolenic acid, ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).
  • the omega-6 series includes octadecadienoic acid (commonly known as linoleic acid, LA), octadecatrienoic acid (commonly known as ⁇ -linolenic acid, GLA), and eicosatetraenoic acid (commonly known as arachidonic acid).
  • Polyunsaturated fatty acids are the main components of human cell membrane phospholipids and have a decisive influence on cell membrane function. Some specific polyunsaturated fatty acids such as arachidonic acid and DHA are two major polyunsaturated fatty acids in the brain and retina, especially for fetuses and infants. Insufficient intake may lead to brain function and optic nerve developmental disorders.
  • the microbial oil obtained by industrial production is mainly in the form of glycerides.
  • Glyceryl ester is a compound obtained by esterifying glycerin with a fatty acid, and is classified into monoglyceride (monoglyceride, MG), diglyceride (diglyceride, DG), and triglyceride according to the degree of reaction.
  • Ester, TG triglyceride
  • triglyceride (TG) is esterified from 3 molecules of fatty acid and 1 molecule of glycerol, which is the main source of energy in the body and the main form of oil storage in various organisms in nature.
  • Diglyceride is a product obtained by esterification of two molecules of fatty acid with one molecule of glycerol. It is a natural component of oils and fats and an intermediate product of oil metabolism in humans. At the same time, diglycerides are also intermediates in the intracellular inositol phospholipid signaling pathway.
  • microcapsule embedding treatment mainly involves mixing, shearing, homogenizing and emulsifying the microbial oil core material with appropriate materials and water, and then embedding the wall material (for example, maltodextrin, etc.) while being spray-dried, so that the fat is tightly closed. Wrapped in the wall material.
  • Such a microcapsule product can prevent the oil from being oxidized and improve the flavor and mouthfeel of the product. Under normal circumstances, the stronger the emulsification performance of the oil, the better the embedding effect, and the better the flavor and stability of the produced microcapsules.
  • the patent application published as CN1662642A discloses a microbial oil containing at least 40% polyunsaturated fatty acids having a triglyceride content of greater than 90%.
  • the microbial oil has the following drawbacks: since the triglyceride has no hydrophilic group and no emulsifying property, the microbial oil has poor emulsifying properties. In the process of subsequent microcapsule production, the microbial oil can not form a good embedding, and the resulting microcapsule product has a high surface oil content, which is not conducive to subsequent further production and application.
  • One of the technical problems to be solved by the present invention is to provide a microbial oil which has good emulsifying properties and is advantageous for embedding.
  • a second technical problem to be solved by the present invention is to provide a method for preparing the above microbial oil.
  • the third technical problem to be solved by the present invention is to provide a microcapsule having a good embedding effect and a low surface oil content.
  • the present invention provides a microbial oil having a polyunsaturated fatty acid content of more than 30% by weight, a triglyceride content of less than 90% by weight, a diglyceride content of not less than 5% by weight and not more than 20% by weight.
  • the content of the diglyceride in the microbial oil is not less than 10% by weight, and the content of the triglyceride is not less than 75% by weight.
  • the polyunsaturated fatty acid is arachidonic acid, docosahexaenoic acid or eicosapentaenoic acid.
  • the microbial oil is a crude oil.
  • the invention also provides a method for preparing a microbial oil, comprising the following steps:
  • Enzymatic hydrolysis is carried out by adding lipase and water to the mixed oil, and the microbial oil is obtained after purification.
  • the enzymatic hydrolysis parameter comprises: stirring the reaction at a suitable temperature of the lipase for 0.5 to 2 hours, and the amount of the lipase is 0.25 wt% to 2 wt% of the mass of the mixed oil, and the amount of the water is the quality of the mixed oil. 15% to 30% by weight.
  • the specific step of purification in the step (3) comprises: the mixed oil is allowed to stand, after the oil phase is separated from the aqueous phase, the water layer is removed, the lipase is removed by filtration, the solvent is removed by evaporation, and removed by a molecular distillation apparatus. Free fatty acids give microbial crude oil.
  • the method for preparing the microbial oil further comprises refining the microbial oil after the step (3).
  • the invention also provides a method for preparing another microbial oil, comprising the following steps:
  • a mixture containing diglyceride is added to the mixed oil, and after mixing, the solvent is removed to obtain a microbial oil.
  • the method for preparing the microbial oil comprises refining the microbial oil after the step (3).
  • microcapsules provided by the present invention comprise the above-mentioned microbial oil as a core material and a wall material encapsulating microbial oil.
  • the microbial oil of the present invention has the following beneficial effects: the microbial oil contains an appropriate amount of diglyceride, and since the diglyceride has good emulsifying properties, it is advantageous for the microbial oil to form a stable emulsion.
  • the microbial oil can be better embedded, thereby reducing the surface oil content of the microcapsules, improving the antioxidant capacity of the microcapsules, and prolonging the shelf life of the microcapsules, which is beneficial for subsequent Further production and utilization.
  • Example 2 is a spray drying parameter table of the first method for preparing microcapsules of Example 1.
  • Fig. 3 is a table showing the surface oil content of each microcapsule product by the first method for preparing microcapsules in the first embodiment.
  • Figure 4 is a list of ingredients for the second method of preparing microcapsules of Example 1.
  • Figure 5 is a spray drying parameter table of the second method for preparing microcapsules of Example 1.
  • Fig. 6 is a table showing the surface oil content of each microcapsule product by the second method for preparing microcapsules in the first embodiment.
  • Figure 7 is a list of ingredients for the third method of preparing microcapsules of Example 1.
  • Figure 8 is a spray drying parameter table of the third method for preparing microcapsules of Example 1.
  • Fig. 9 is a table showing the surface oil content of each microcapsule product by the third method for preparing microcapsules in the first embodiment.
  • Figure 10 is a list of ingredients for preparing milk powder in the first embodiment.
  • Figure 11 is a table showing the spray drying parameters of the preparation of the milk powder of the first embodiment.
  • Figure 12 is a table showing the surface oil content of the milk powder measured by the preparation of the milk powder of the first embodiment.
  • Figure 13 is a list of ingredients for preparing microcapsules in Example 2.
  • Figure 14 is a spray drying parameter table of the preparation of the microcapsules of Example 2.
  • Figure 15 is a table showing the surface oil content of the microcapsule product obtained by preparing the microcapsules in the second embodiment.
  • Figure 16 is a list of ingredients for preparing milk powder of Example 2.
  • Figure 17 is a table showing the spray drying parameters of the preparation of the milk powder of the second embodiment.
  • Figure 18 is a table showing the surface oil content of the milk powder measured by preparing the milk powder in the second embodiment.
  • Figure 19 is a list of ingredients for preparing microcapsules in Example 3.
  • Figure 20 is a spray drying parameter table of the preparation of the microcapsules of Example 3.
  • Figure 21 is a table showing the surface oil content of the microcapsule product measured by the microcapsules of Example 3.
  • Figure 22 is a list of ingredients for preparing milk powder of Example 3.
  • Figure 23 is a table showing the spray drying parameters of the preparation of the milk powder of the third embodiment.
  • Figure 24 is a table showing the surface oil content of the milk powder measured by the preparation of the milk powder of the third embodiment.
  • a medium solution with a glucose content of 0.03g/mL and a yeast powder content of 0.02g/mL in a 500ml shake flask Multiple bottles can be prepared. After sterilization, the appropriate amount of Mortierella alpina mycelium and spores are placed and placed at 28 °C. A constant temperature shaker, 150 rpm, 2 days later, the shake flask was combined, and the sterilized 1 m3 fermenter containing the medium solution having a glucose content of 0.03 g/mL and a yeast powder content of 0.02 g/mL was transferred. In the seed tank), sterile air is continuously supplied, and the culture temperature is maintained at 28 ⁇ 2 °C.
  • the whole culture solution was transferred to a sterilized 10 m3 fermenter containing a medium solution having a glucose content of 0.03 g/mL and a yeast powder content of 0.02 g/mL (secondary In the seed tank), sterile air is continuously supplied, and the culture temperature is maintained at 28 ⁇ 2 °C.
  • the whole culture solution was transferred to a sterilized 45 m3 fermenter containing a medium solution having a glucose content of 0.05 g/mL and a yeast powder content of 0.02 g/mL.
  • the sterile air is introduced, the culture temperature is maintained at 28 ⁇ 2° C., and the sterilized glucose solution of the total amount of about 0.02 to 0.05 g/mL of the culture solution is added in batches according to the glucose consumption rate, and the fermentation product is obtained after further culture for 7 days, wherein
  • the biomass content was 32 g/L
  • the total oil content in the bacterial stem was 51.9 wt%
  • the arachidonic acid content in the total oil was 50.4 wt%.
  • the microbial oil of the present invention can be prepared by the following different process means.
  • the fermentation product is subjected to solid-liquid separation by centrifugation or pressure filtration, and the wet cells are collected, crushed by a pulverizer, and dried by a boiling drying tower to obtain dried cells.
  • the dried cells are extracted by mixing with an organic solvent such as butane or hexane, and filtered to obtain a mixed oil.
  • the enzymatic treatment parameters include: the amount of the lipase is 0.25 wt% of the weight of the microbial mixed oil; the amount of water is 15 wt% of the weight of the microbial mixed oil, the reaction temperature is about 37 ° C, and the stirring reaction time is 0.5 h. After the end of the enzymatic reaction, the mixed oil is purified.
  • the specific steps of the purification include: the mixed oil is allowed to stand, after the oil phase is separated from the aqueous phase, the aqueous layer is removed, the enzyme is removed by filtration, the solvent is removed by evaporation, and the molecular distillation device is passed through the apparatus.
  • the free fatty acid is removed to obtain a microbial crude oil.
  • the crude oil was found to have the following indexes: polyunsaturated fatty acid content of 61.5 wt%, TG content of 88.7 wt%, and DG content of 5.5 wt%.
  • the step of purifying the above-mentioned microbial crude oil comprises the steps of: degumming the microbial crude oil with 2.5 wt% citric acid and 5 wt% hot water, deacidifying excess sodium hydroxide solution, separating and separating, and filtering and then using 5 wt% of silica and 3 wt% of activated carbon were decolorized, and after filtration again, direct steam deodorization was carried out at 200 ° C, and Vc palmitate and Ve were added as an antioxidant to obtain a microbial refined oil containing arachidonic acid.
  • the refined oil was determined to have a total content of polyunsaturated fatty acids of 62.0% by weight, a TG content of 89.8 wt%, and a DG content of 6.3 wt%.
  • the process is basically the same as the method, and the difference is that the enzymatic treatment process parameter: the amount of the lipase is 0.5 wt% of the weight of the microbial mixed oil, and the amount of water is 20 wt% of the weight of the microbial mixed oil, and the reaction time is 1h.
  • the obtained microbial crude oil had a polyunsaturated fatty acid content of 61.7 wt%, a TG content of 87.0 wt%, and a DG content of 7.2 wt%.
  • the microbial crude oil is refined by the same refining process as in the first method, and the total content of the polyunsaturated fatty acid is 61.5 wt%, the TG content is 88.4 wt%, the DG content is 8.8 wt%, and other physical and chemical indicators and means are obtained. The physical and chemical indicators obtained are close.
  • the process is basically the same as the method, and the difference is that the enzymatic treatment process parameters: the amount of lipase is 1 wt% of the weight of the microbial mixed oil, the amount of water is 20 wt% of the weight of the microbial mixed oil, and the reaction time is 1.5 h.
  • the obtained microbial crude oil had a polyunsaturated fatty acid content of 60.0% by weight, a TG content of 84.0% by weight, and a DG content of 10.5% by weight.
  • the microbial crude oil is purified by the same refining process as in the first method, and the total micro-unsaturated fatty acid content is 61 wt%, the TG content is 85.3 wt%, the DG content is 11.4 wt%, and other physical and chemical indicators are The physical and chemical indicators obtained by means 1 are close.
  • the process is basically the same as the method, and the difference is that the enzymatic treatment process parameters: the amount of lipase is 1 wt% of the weight of the microbial mixed oil, the amount of water is 25 wt% of the weight of the microbial mixed oil, and the reaction time is 2 h.
  • the obtained microbial crude oil had a polyunsaturated fatty acid content of 65.0% by weight, a TG content of 78.4% by weight, and a DG content of 13.7% by weight.
  • the microbial crude oil was purified by the same refining process as in the first method, and the total content of polyunsaturated fatty acids in the microbial refined oil was 63.8 wt%, the TG content was 80.9 wt%, and the DG content was 15.1 wt%.
  • the indicators are close to the physical and chemical indicators obtained by means 1.
  • the process is basically the same as the method, and the difference is that the enzymatic treatment process parameters: the amount of lipase is 2 wt% of the weight of the microbial mixed oil, the water amount is 30 wt% of the weight of the microbial mixed oil, and the reaction time is 2 h.
  • the obtained microbial crude oil had a polyunsaturated fatty acid content of 57.9 wt%, a TG content of 75.3 wt%, and a DG content of 17.8 wt%.
  • the microbial crude oil is refined by the same refining process as in the first method, and the total micro-unsaturated fatty acid content is 60 wt%, the TG content is 77.2 wt%, the DG content is 19.1 wt%, and other physical and chemical indicators are The physical and chemical indicators obtained by means 1 are close.
  • the fermentation product is subjected to solid-liquid separation by centrifugation or pressure filtration, and the wet cells are collected, crushed by a pulverizer, and dried by a boiling drying tower to obtain dried cells.
  • the dried cells are extracted by mixing with an organic solvent such as butane or hexane, and filtered to obtain a mixed oil.
  • the mixed oil is desolvated and uniformly mixed to obtain a microbial crude oil.
  • the microbial crude oil has the following characteristic characteristics: a polyunsaturated fatty acid content of 38.0% by weight, a TG content of 86.7 wt%, and a DG content of 5.1 wt%.
  • the step of purifying the above-mentioned microbial crude oil comprises the steps of: degumming the microbial crude oil with 2.5 wt% citric acid and 5 wt% hot water, deacidifying excess sodium hydroxide solution, separating and separating, and filtering and then using 5 wt% of silica and 3 wt% of activated carbon were decolorized, and after filtration, direct steam deodorization was carried out at 200 ° C, and Vc palmitate and Ve were added as an antioxidant to obtain a microbial refined oil containing arachidonic acid.
  • the total content of polyunsaturated fatty acids in the refined oil was determined to be 37.0% by weight, and the TG content was 89.2% by weight, the DG content was 6.4% by weight.
  • This process is basically the same as the means 6 except that the diglyceride-containing mixture has a diglyceride content of 50.8 wt% and the added mixture has a specific gravity of 17.5 wt% of the total mixed oil.
  • the obtained microbial crude oil had a polyunsaturated fatty acid content of 41.4% by weight, a TG content of 81.2% by weight, and a DG content of 10.4% by weight.
  • the microbial crude oil is purified by the same refining process as that of the sixth method, and the obtained microbial refined oil has a total content of polyunsaturated fatty acids of 40.4 wt%, a TG content of 83.6 wt%, and a DG content of 12.2 wt%.
  • Other physical and chemical indicators are close to the physical and chemical indicators obtained by means 6.
  • the process is basically the same as the means 6 except that the diglyceride-containing mixture has a diglyceride content of 72.2% by weight and the addition ratio is 22.6% by weight of the total mixed oil.
  • the obtained microbial crude oil had a polyunsaturated fatty acid content of 55.7 wt%, a TG content of 75.6 wt%, and a DG content of 17.8 wt%.
  • the microbial crude oil is purified by the same refining process as that of the sixth method, and the obtained microbial refined oil has a total polyunsaturated fatty acid content of 55.9 wt%, a TG content of 77.0 wt%, and a DG content of 19.0 wt%.
  • Other physical and chemical indicators are close to the physical and chemical indicators obtained by means 6.
  • the arachidonic acid refined oil prepared by using the commercially available arachidonic acid oil, the above-mentioned means 1, the second means and the means three was respectively used according to the ingredient list shown in FIG.
  • the above-mentioned stock solution was sheared at 8,000 rpm for 10 minutes, and then homogenized at 40 MPa to obtain an emulsion.
  • the emulsion was subjected to pressure spray drying, and the spray drying parameters are shown in Fig. 2.
  • the surface oil content of each microcapsule product was measured as shown in FIG.
  • the above liquid was sheared at a number of revolutions of 9000 rpm for 15 minutes, and then homogenized at 45 MPa to obtain an emulsion.
  • the emulsion was subjected to centrifugal spray drying, and the spray drying parameters are shown in Fig. 5.
  • the surface oil content of the microcapsules was measured as shown in Fig. 6.
  • the arachidonic acid refined oil prepared by using the commercially available arachidonic acid oil, the above-mentioned means 6, the method 7, and the means 8 was respectively used according to the ingredient list shown in FIG.
  • the above-mentioned stock solution was sheared at a rotational speed of 10,000 rpm for 15 minutes, and then homogenized at 50 MPa to obtain an emulsion.
  • the emulsion was spray-granulated and dried.
  • 15 kg of maltodextrin was used as a primer.
  • the spray drying parameters are shown in Fig. 8.
  • the surface oil content of the microcapsules was measured as shown in FIG.
  • the surface oil content of the microcapsules is an important indicator of the quality of the microcapsules, which represents the proportion of unencapsulated fat on the surface of the microcapsules.
  • the microbial oil of the present invention contains more diglyceride, which can help the microbial oil to form a more stable emulsion, so that the microbial oil is better embedded, thereby reducing the surface oil content of the microcapsules and the milk powder. Improve the antioxidant capacity of microcapsules and milk powder, and extend the shelf life of microcapsules and milk powder.
  • Schizochytrium is used as a starting species, and the production and application of the docosahexaenoic acid-containing microbial oil of the present invention are described in detail below.
  • a medium solution with a glucose content of 0.04g/mL and a yeast extract content of 0.02g/mL in a 1000ml shake flask Multiple bottles can be prepared. After sterilization, the appropriate amount of cryopreserved broth can be added and shaken at 28 °C. The bed was activated at 180 rpm. After 2 days, access to the secondary expansion shake flask for culture. After 2 days, the shake flask was combined and transferred to a sterilized 1 m3 fermenter (primary seed tank) containing 5 wt% glucose and 2 wt% yeast extract. Sterile air was placed and the culture temperature was maintained at 29 ⁇ 1 °C.
  • the culture solution was transferred to a sterilized 10 m3 fermenter (secondary seed tank) containing a glucose content of 0.03 g/mL and a yeast extract content of 0.02 g/mL. Sterile air was introduced and the culture temperature was maintained at 29 ⁇ 1 °C.
  • the fermentation product can be obtained, wherein the biomass is 89.7 g/L and the total oil content is 38.5. g/L, total docosahexaenoic acid content of 51.0 wt%.
  • the fermentation product is subjected to solid-liquid separation by centrifugation or pressure filtration, and the wet cells are collected, crushed by a pulverizer, and dried by a boiling drying tower to obtain dried cells.
  • the dried cells are extracted by mixing with an organic solvent such as butane or hexane, and filtered to obtain a mixed oil.
  • the enzymatic treatment parameters include: the amount of the lipase is 0.25 wt% of the weight of the microbial mixed oil; the amount of water is 30 wt% of the weight of the microbial mixed oil, the reaction temperature is about 37 ° C, and the stirring reaction time is 0.5 h. After the end of the enzymatic reaction, the mixed oil is purified.
  • the specific steps of the purification include: the mixed oil is allowed to stand, after the oil phase is separated from the aqueous phase, the aqueous layer is removed, the enzyme is removed by filtration, the solvent is removed by evaporation, and the molecular distillation device is passed through the apparatus.
  • the free fatty acid is removed to obtain a microbial crude oil.
  • the crude oil was found to have the following indexes: polyunsaturated fatty acid content of 64.0% by weight, TG content of 86.0% by weight, and DG content of 9.8% by weight.
  • the step of purifying the above-mentioned microbial crude oil comprises the steps of: degumming the microbial crude oil with 2.5 wt% citric acid and 5 wt% hot water, deacidifying excess sodium hydroxide solution, separating and separating, and filtering and then using 5 wt% of silica and 3 wt% of activated carbon were decolorized, and after filtration again, direct steam deodorization was carried out at 200 ° C, and Vc palmitate and Ve were added as an antioxidant to obtain a microbial refined oil containing docosahexaenoic acid.
  • the total content of polyunsaturated fatty acids in the refined oil was determined to be 64.0% by weight, and the TG content was 88.7 wt%, the DG content was 11.5% by weight.
  • the fermentation product is subjected to solid-liquid separation by centrifugation or pressure filtration, and the wet cells are collected, crushed by a pulverizer, and dried by a boiling drying tower to obtain dried cells.
  • the dried cells are extracted by mixing with an organic solvent such as butane or hexane, and filtered to obtain a mixed oil.
  • a diglyceride-containing mixture such as a fatty acid mono-diglyceride or the like is added to the mixed oil, the mixture has a diglyceride content of 54.9 wt%, and the added mixture has a specific gravity of 16.2 wt% of the total mixed oil.
  • the mixed oil is desolvated and uniformly mixed to obtain a microbial crude oil.
  • the microbial crude oil has the following characteristic characteristics: a polyunsaturated fatty acid content of 47.2% by weight, a TG content of 82.1% by weight, and a DG content of 10.4% by weight.
  • the step of purifying the above-mentioned microbial crude oil comprises the steps of: degumming the microbial crude oil with 2.5 wt% citric acid and 5 wt% hot water, deacidifying excess sodium hydroxide solution, separating and separating, and filtering and then using 5 wt% of silica and 3 wt% of activated carbon were decolorized, and after filtration again, direct steam deodorization was carried out at 200 ° C, and Vc palmitate and Ve were added as an antioxidant to obtain a microbial refined oil containing docosahexaenoic acid.
  • the total content of polyunsaturated fatty acids in the refined oil was determined to be 48.1% by weight, and the TG content was 84.8 wt%, the DG content was 12.0 wt%.
  • Other physical and chemical indicators are close to the physical and chemical indicators obtained by means.
  • the above docosahexaenoic acid-containing microbial oil can be prepared by using microcapsules, using commercially available docosahexaenoic acid oil and the above-mentioned means one and two means of docosahexaenoic acid oil, according to The ingredient list shown in Figure 13 is compounded.
  • the above-mentioned stock solution was sheared at 8,000 rpm for 10 minutes, and then homogenized at 40 MPa to obtain an emulsion.
  • the emulsion was subjected to pressure spray drying, and the spray drying parameters are shown in FIG.
  • the surface oil content of the microcapsules was measured as shown in Fig. 15.
  • the above docosahexaenoic acid-containing microorganism can prepare milk powder by using oil, using commercially available docosahexaenoic acid oil and the above-mentioned means one and two means of docosahexaenoic acid oil, as shown in the figure.
  • the ingredient list shown in Figure 16 is used for the ingredients.
  • the microcapsules and milk powder prepared by the docosahexaenoic acid-containing microbial oil of the present invention have a lower surface oil content under the same process conditions.
  • the microbial oil of the present invention contains more diglyceride, which can help the microbial oil to form a more stable emulsion, so that the microbial oil is better embedded, thereby reducing the surface oil content of the microcapsules and the milk powder.
  • Improve the antioxidant capacity of microcapsules and milk powder and extend the shelf life of microcapsules and milk powder.
  • the sterile air is continuously supplied, and the culture temperature is maintained at 28 ⁇ 1 °C.
  • the culture solution was transferred into a 10 cubic meter fermenter (secondary seed tank) with sterilized sucrose content of 0.05 g/mL and yeast powder content of 0.005 g/mL. Sterile air was added and the culture temperature was maintained at 28 ⁇ 1 °C.
  • the fermentation product is subjected to solid-liquid separation by centrifugation or pressure filtration, and the wet cells are collected, crushed by a pulverizer, and dried by a boiling drying tower to obtain dried cells.
  • the dried cells are extracted by mixing with an organic solvent such as butane or hexane, and filtered to obtain a mixed oil.
  • the enzymatic treatment parameters include: the amount of the lipase is 0.25 wt% of the weight of the microbial mixed oil; the amount of water is 30 wt% of the weight of the microbial mixed oil, the reaction temperature is about 37 ° C, and the stirring reaction time is 0.5 h. After the end of the enzymatic reaction, the mixed oil is purified.
  • the specific steps of the purification include: the mixed oil is allowed to stand, after the oil phase is separated from the aqueous phase, the aqueous layer is removed, the enzyme is removed by filtration, the solvent is removed by evaporation, and the molecular distillation device is passed through the apparatus.
  • the free fatty acid is removed to obtain a microbial crude oil.
  • the crude oil has the following characteristics: a polyunsaturated fatty acid content of 59.8 wt%, a TG content of 85.1 wt%, and a DG content of 8.5 wt%.
  • the step of purifying the above-mentioned microbial crude oil comprises the steps of: degumming the microbial crude oil with 2.5 wt% citric acid and 5 wt% hot water, deacidifying excess sodium hydroxide solution, separating and separating, and filtering and then using 5 wt% of silica and 3 wt% of activated carbon were decolorized, and after again filtering, direct steam deodorization was carried out at 200 ° C, and Vc palmitate and Ve were added as an antioxidant to obtain a microbial refined oil containing eicosapentaenoic acid.
  • the total content of polyunsaturated fatty acids in the refined oil was determined to be 59.4% by weight, and the TG content was 87.5 wt%, the DG content was 10.4 wt%.
  • the fermentation product is subjected to solid-liquid separation by centrifugation or pressure filtration, and the wet cells are collected, crushed by a pulverizer, and dried by a boiling drying tower to obtain dried cells.
  • the dried cells are extracted by mixing with an organic solvent such as butane or hexane, and filtered to obtain a mixed oil.
  • a diglyceride-containing mixture such as a fatty acid mono-diglyceride or the like is added to the mixed oil, the mixture has a diglyceride content of 56.1% by weight, and the added mixture has a specific gravity of 15.0% by weight of the total mixed oil.
  • the mixed oil is desolvated and uniformly mixed to obtain a microbial crude oil.
  • the crude oil had the following characteristics: polyunsaturated fatty acid content of 45.0% by weight, TG content of 82.0% by weight, and DG content of 9.9% by weight.
  • the step of purifying the above-mentioned microbial crude oil comprises the steps of: degumming the microbial crude oil with 2.5 wt% citric acid and 5 wt% hot water, deacidifying excess sodium hydroxide solution, separating and separating, and filtering and then using 5 wt% of silica and 3 wt% of activated carbon were decolorized, and after again filtering, direct steam deodorization was carried out at 200 ° C, and Vc palmitate and Ve were added as an antioxidant to obtain a microbial refined oil containing eicosapentaenoic acid.
  • the total content of polyunsaturated fatty acids in the refined oil was determined to be 44.9 wt%, and the TG content was 84.3 wt%, the DG content was 11.9% by weight.
  • Other physical and chemical indicators are close to the physical and chemical indicators obtained by means.
  • the above microbial oil containing eicosapentaenoic acid is used to prepare microcapsules, and the commercially available eicosapentaenoic acid oil, the above-mentioned means I and the second method of the eicosapentaenoic acid oil are used, as shown in FIG.
  • the ingredients list is used for ingredients.
  • the above-mentioned stock solution was sheared at 8,000 rpm for 10 minutes, and then homogenized at 40 MPa to obtain an emulsion.
  • the emulsion was spray dried, and the spray drying parameters are shown in FIG.
  • the surface oil content of the microcapsules was measured as shown in FIG.
  • the above-mentioned microbial oil containing eicosapentaenoic acid is used to prepare milk powder, and the commercially available eicosapentaenoic acid oil, the above-mentioned means 1 and the second method of the eicosapentaenoic acid oil are used, as shown in FIG.
  • the ingredients list is used for the ingredients.
  • the microcapsules and milk powder prepared by the microbial oil have a lower surface oil content. This is mainly because: the microbial oil of the present invention contains more diglyceride, which can help the microbial oil to form a more stable emulsion, so that the microbial oil is better embedded, thereby reducing the surface oil content of the microcapsules and the milk powder. Improve the antioxidant capacity of microcapsules and milk powder, and extend the shelf life of microcapsules and milk powder.

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Abstract

提供了一种微生物油及其制备方法，还提供了一种微胶囊。所述微生物油的多不饱和脂肪酸含量大于30wt%，甘油三酯含量小于90wt%，甘油二酯含量不低于5wt%且不高于20wt%。

Description

一种微生物油及其制备方法、一种微胶囊 技术领域
本发明涉及本发明涉及一种微生物油及其制备方法，并涉及一种微胶囊。本申请是基于申请日为2014年3月14日，申请号为CN201410096097.0的中国发明专利申请，该申请的内容引入本文作为参考。
 背景技术
多不饱和脂肪酸（polyunsaturated fatty acid，PUFA）是指含有两个或两个以上双键的脂肪酸，一般含18至22个碳原子。工业化的PUFA大多由真菌、藻类等单细胞微生物产生，故也称为微生物油。
多不饱和脂肪酸因其结构特点主要分为ω-3及ω-6两个系列。ω-3系列包括十八碳三烯酸（俗称α-亚麻酸，ALA）、二十碳五烯酸（EPA）、二十二碳六烯酸（DHA）。ω-6系列包括十八碳二烯酸（俗称亚油酸，LA）、十八碳三烯酸（俗称γ-亚麻酸，GLA）、二十碳四烯酸（俗称花生四烯酸）。多不饱和脂肪酸是人体细胞膜磷脂的主要成分，对细胞膜功能有决定性影响。一些特定的多不饱和脂肪酸如花生四烯酸和DHA是脑和视网膜中两种主要的多不饱和脂肪酸，特别对于胎儿和婴幼儿影响显著，摄入不足可能导致脑功能和视神经发育障碍。
工业化生产得到的微生物油主要是以甘油酯的形式存在。甘油酯是甘油与脂肪酸酯化而成的化合物，根据反应程度的不同，分为甘油一酯（单甘酯、MG）、甘油二酯（双甘酯、DG）、甘油三酯（甘三酯、TG）。其中，甘油三酯（TG）由3分子脂肪酸和1分子甘油酯化而成，是体内能量的主要来源，也是自然界各类生物体内油脂储存的主要形式。甘油二酯（DG）是由2分子脂肪酸与1分子甘油酯化得到的产物，是油脂的天然成分，也是油脂在人体内代谢的中间产物。同时，甘油二酯还是细胞内肌醇磷脂信号传递途径的中间物质。
工业化生产得到的微生物油很多都是功能性或针对性很强的油脂，一般用于大众消费品如乳制品的添加剂或营养强化剂，很少直接食用。由于其富含多不饱和脂肪酸，很容易被氧化而导致风味恶化，因此其在用作食品添加剂或营养强化剂时，通常需要先进行微胶囊包埋处理。微胶囊包埋主要是将微生物油芯材与适当材料及水混合、剪切、均质、乳化后，在喷雾干燥的同时加入壁材（例如麦芽糊精等）进行包埋，使油脂被紧密包裹于壁材中。这样的微胶囊产品既可以防止油脂被氧化，又能改善产品的风味和口感。通常情况下，油脂的乳化性能越强，则包埋效果越好，所生产出来的微胶囊风味和稳定性也越好。
公开号为CN1662642A的专利申请公开了一种含有至少40%多不饱和脂肪酸的微生物油，该微生物油中的甘油三酯含量大于90%。该微生物油存在以下缺陷：由于甘油三酯无亲水基团，无乳化性能，因此，该微生物油的乳化性能较差。在后续微胶囊生产的过程中，微生物油不能形成良好的包埋，最终得到的微胶囊产品，其表面油含量较高，不利于后续进一步的生产及应用。
因此，提供一种改进的微生物油实为必要。
 技术问题
本发明所要解决的技术问题之一在于提供一种具有良好乳化性能、利于包埋的微生物油。
本发明所要解决的技术问题之二在于提供一种制备上述微生物油的方法。
本发明所要解决的技术问题之三在于提供一种具有良好包埋效果、表面油含量低的微胶囊。
 技术解决方案
为了解决上述技术问题，本发明提供一种微生物油，其多不饱和脂肪酸的含量大于30wt%，甘油三酯含量小于90wt%，甘油二酯含量不低于5wt%且不高于20wt%。
上述方案中，微生物油中甘油二酯的含量不低于10wt%，甘油三酯的含量不低于75wt%。
上述方案中，多不饱和脂肪酸为花生四烯酸、二十二碳六烯酸或者二十碳五烯酸。可选地，该微生物油为粗油。
本发明还提供一种微生物油的制备方法，包括以下步骤：
(1) 利用产油微生物发酵得到富含PUFA微生物油的发酵液；
(2) 收集富含PUFA微生物油的菌体，萃取过滤后，得到混合油；
(3) 在混合油中加入脂肪酶和水进行酶解，纯化后得到微生物油。
上述方案中，步骤（3）中，酶解参数包括：在脂肪酶最适温度下搅拌反应0.5至2小时，脂肪酶用量为混合油质量的0.25wt%至2wt%，水用量为混合油质量的15wt%至30wt%。
上述方案中，步骤（3）中纯化的具体步骤包括：将混合油静置，待油相与水相分层后，除去水层，过滤除去脂肪酶，蒸发脱除溶剂，通过分子蒸馏设备除去游离脂肪酸，得到微生物粗油。
上述方案中，微生物油的制备方法还包括在步骤（3）之后对微生物油进行精制。
本发明还提供另外一种微生物油的制备方法，包括以下步骤：
(1) 利用产油微生物发酵得到富含PUFA微生物油的发酵液；
(2) 收集富含PUFA微生物油的菌体，萃取过滤后得到混合油；
(3) 向混合油中添加含有甘油二酯的混合物，混合均匀后去除溶剂得到微生物油。
上述方案中，微生物油的制备方法包括在步骤（3）之后对微生物油进行精制。
本发明提供的微胶囊包括作为芯材的上述微生物油及包裹微生物油的壁材。
 有益效果
本发明的微生物油具有如下有益效果：微生物油中含有适量的甘油二酯，由于甘油二脂具有较好的乳化性能，其有利于微生物油形成稳定的乳化液。在制备微胶囊的过程中，能使微生物油更好地被包埋，进而可降低微胶囊的表面油含量，提高微胶囊的抗氧化能力，并可适度延长微胶囊的货架期，有利于后续进一步的生产及利用。
 附图说明
图1是实施例一制备微胶囊第一种方法的配料表。
图2是实施例一制备微胶囊第一种方法的喷雾干燥参数表。
图3是实施例一制备微胶囊第一种方法测得各微胶囊产品的表面油含量表格。
图4是实施例一制备微胶囊第二种方法的配料表。
图5是实施例一制备微胶囊第二种方法的喷雾干燥参数表。
图6是实施例一制备微胶囊第二种方法测得各微胶囊产品的表面油含量表格。
图7是实施例一制备微胶囊第三种方法的配料表。
图8是实施例一制备微胶囊第三种方法的喷雾干燥参数表。
图9是实施例一制备微胶囊第三种方法测得各微胶囊产品的表面油含量表格。
图10是实施例一制备奶粉的配料表。
图11是实施例一制备奶粉的喷雾干燥参数表。
图12是实施例一制备奶粉测得奶粉的表面油含量表格。
图13是实施例二制备微胶囊的配料表。
图14是实施例二制备微胶囊的喷雾干燥参数表。
图15是实施例二制备微胶囊测得微胶囊产品的表面油含量表格。
图16是实施例二制备奶粉的配料表。
图17是实施例二制备奶粉的喷雾干燥参数表。
图18是实施例二制备奶粉测得奶粉的表面油含量表格。
图19是实施例三制备微胶囊的配料表。
图20是实施例三制备微胶囊的喷雾干燥参数表。
图21是实施例三制备微胶囊测得微胶囊产品的表面油含量表格。
图22是实施例三制备奶粉的配料表。
图23是实施例三制备奶粉的喷雾干燥参数表。
图24是实施例三制备奶粉测得奶粉的表面油含量表格。
以下结合附图及实施例对发明作进一步说明。
 本发明的实施方式
以下实施例更详细的说明本发明的微生物油生产及应用方法。
实施例一
以高山被孢霉为出发菌种，详细描述本发明含有花生四烯酸的微生物油的生产及应用。
1.发酵
配制葡萄糖含量为0.03g/mL、酵母粉含量为0.02g/mL的培养基溶液于500ml摇瓶中，可配制多瓶，灭菌后接入适量高山被孢霉菌丝及孢子，置于28℃恒温摇床，150rpm，2天后合并摇瓶，移入已灭菌的、盛放有葡萄糖含量为0.03g/mL、酵母粉含量为0.02g/mL的培养基溶液的1立方米发酵罐（一级种子罐）中，持续通入无菌空气，保持培养温度28±2℃。一级种子罐培养2天后，将全部培养液移入已灭菌的、盛放有葡萄糖含量为0.03g/mL、酵母粉含量为0.02g/mL的培养基溶液的10立方米发酵罐（二级种子罐）中，持续通入无菌空气，保持培养温度28±2℃。二级种子罐培养1天后，将全部培养液移入已灭菌的、盛放有葡萄糖含量为0.05g/mL、酵母粉含量为0.02g/mL的培养基溶液的45立方米发酵罐中，持续通入无菌空气，保持培养温度28±2℃，根据葡萄糖消耗速度分批补加总量约0.02至0.05g/mL培养基溶液的灭菌葡萄糖溶液，再培养7天后可获得发酵产物，其中生物质含量32g/L、菌体干基中总油含量51.9wt%、总油中花生四烯酸含量50.4wt%。
2.制备富含花生四烯酸的微生物油
可采用以下不同的工艺手段制备本发明的微生物油。
手段一
将发酵产物通过离心或压滤方式实现固液分离，收集湿菌体，利用粉碎机破碎，再通过沸腾干燥塔烘干，得到干菌体。将干菌体与有机溶剂如丁烷或己烷混合浸泡萃取，过滤后得到混合油。
向混合油中加入市售脂肪酶和水进行酶解处理，该市售脂肪酶具有将甘油酯水解的功能，下述实施例均与此相同。所述的酶解处理参数包括：脂肪酶用量为微生物混合油重量的0.25wt%；水用量为微生物混合油重量的15wt%，反应温度约37℃，搅拌反应时间0.5h。酶解反应结束后对混合油进行纯化，纯化的具体步骤包括：将混合油静置，待油相与水相分层后，除去水层，过滤除去酶，蒸发脱除溶剂，通过分子蒸馏设备除去游离脂肪酸，得到微生物粗油。测得该粗油具有如下指标：多不饱和脂肪酸含量61.5wt%，TG含量88.7wt%，DG含量5.5wt%。
更进一步的，对上述微生物粗油进行精制，精制的步骤包括：将该微生物粗油经2.5wt%柠檬酸和5wt%热水脱胶、过量氢氧化钠溶液脱酸，沉降分离，过滤后再用5wt%二氧化硅及3wt%活性炭脱色，再次过滤后，在200℃的条件下直接蒸汽脱臭，添加Vc棕榈酸酯和Ve作为抗氧化剂，得到含有花生四烯酸的微生物精制油。经测定，该精制油中多不饱和脂肪酸总含量62.0wt%，TG含量为89.8wt%，DG含量为6.3wt%。
进一步测定该微生物精制油的部分理化指标：不皂化物1.1wt%，水分0.01wt%，不溶性杂质0.01wt%，溶剂残留<1.0mg/kg，酸价0.1mgKOH/g，过氧化值0.03meq/kg，反式脂肪酸0.06wt%，黄曲霉毒素B1<5.0μg/kg，总砷（以As计）<0.1mg/kg，铅<0.1mg/kg。
手段二
该工艺手段与手段一基本相同，不同之处在于酶解处理工艺参数：脂肪酶用量为微生物混合油重量的0.5wt%，水量为微生物混合油重量的20wt%，反应时间为 1h。得到的微生物粗油中，多不饱和脂肪酸含量61.7wt%，TG含量87.0wt%，DG含量7.2wt%。
使用与手段一中相同的精制工艺对本微生物粗油进行精制，得到的微生物精制油中，多不饱和脂肪酸总含量61.5wt%，TG含量88.4wt%，DG含量8.8wt%，其他理化指标与手段一获得的理化指标接近。
手段三
该工艺手段与手段一基本相同，不同之处在于酶解处理工艺参数：脂肪酶用量为微生物混合油重的1wt%，水量为微生物混合油重的20wt%，反应时间为1.5h。得到的微生物粗油中，多不饱和脂肪酸含量60.0wt%，TG含量84.0wt%，DG含量10.5wt%。
使用与手段一中相同的精制工艺对本微生物粗油进行精制，得到的微生物精制油中，多不饱和脂肪酸总含量61wt%，TG含量为85.3wt%，DG含量为11.4wt%，其他理化指标与手段一获得的理化指标接近。
手段四
该工艺手段与手段一基本相同，不同之处在于酶解处理工艺参数：脂肪酶用量为微生物混合油重的1wt%，水量为微生物混合油重的25wt%，反应时间为2h。得到的微生物粗油中，多不饱和脂肪酸含量65.0wt%，TG含量78.4wt%，DG含量13.7wt%。
使用与手段一中相同的精制工艺对本微生物粗油进行精制，得到的微生物精制油中，多不饱和脂肪酸总含量为63.8wt%，TG含量为80.9wt%，DG含量为15.1wt%，其他理化指标与手段一获得的理化指标接近。
手段五
该工艺手段与手段一基本相同，不同之处在于酶解处理工艺参数：脂肪酶用量为微生物混合油重的2wt%，水量为微生物混合油重的30wt%，反应时间为2h。得到的微生物粗油中，多不饱和脂肪酸含量57.9wt%，TG含量75.3wt%，DG含量17.8wt%。
使用与手段一中相同的精制工艺对本微生物粗油进行精制，得到的微生物精制油中，多不饱和脂肪酸总含量60wt%，TG含量为77.2wt%，DG含量为19.1wt%，其他理化指标与手段一获得的理化指标接近。
手段六
将发酵产物通过离心或压滤方式实现固液分离，收集湿菌体，利用粉碎机破碎，再通过沸腾干燥塔烘干，得到干菌体。将干菌体与有机溶剂如丁烷或己烷混合浸泡萃取，过滤后得到混合油。
向混合油中添加含甘油二酯的混合物，例如脂肪酸单双甘酯或其类似物，该混合物中甘油二酯含量为31.4wt%，添加混合物的比重为总混合油的11.5wt%。对混合油脱溶，混合均匀后得到微生物粗油。该微生物粗油具有如下指标特征：多不饱和脂肪酸含量38.0wt%，TG含量86.7wt%，DG含量5.1wt%。
更进一步的，对上述微生物粗油进行精制，精制的步骤包括：将该微生物粗油经2.5wt%柠檬酸和5wt%热水脱胶、过量氢氧化钠溶液脱酸，沉降分离，过滤后再用5wt%二氧化硅及3wt%活性炭脱色，过滤后，在200℃的条件下直接蒸汽脱臭，添加Vc棕榈酸酯和Ve作为抗氧化剂，得到含有花生四烯酸的微生物精制油。经测定，该精制油中多不饱和脂肪酸总含量达37.0wt%，TG含量为 89.2wt%，DG含量为6.4wt%。
进一步测定该微生物精制油的部分理化指标：不皂化物1.0wt%，水分0.01wt%，不溶性杂质0.01wt%，溶剂残留<1.0mg/kg，酸价0.1mgKOH/g，过氧化值0.03meq/kg，反式脂肪酸0.04wt%，黄曲霉毒素B1<5.0μg/kg，总砷（以As计）<0.1mg/kg，铅<0.1mg/kg。
手段七
该工艺手段与手段六基本相同，不同之处在于：含甘油二酯混合物中，甘油二酯含量为50.8wt%，添加的混合物的比重为总混合油的17.5wt%。得到的微生物粗油中，多不饱和脂肪酸含量41.4wt%，TG含量81.2wt%，DG含量10.4wt%。
更进一步的，使用与手段六相同的精制工艺对本微生物粗油进行精制，得到的微生物精制油中，多不饱和脂肪酸总含量40.4wt%，TG含量为83.6wt%，DG含量为12.2wt%，其他理化指标与手段六获得的理化指标接近。
手段八
该工艺手段与手段六基本相同，不同之处在于：含甘油二酯混合物中，甘油二酯含量为72.2wt%，添加比例为占总混合油的22.6wt%。得到的微生物粗油中，多不饱和脂肪酸含量55.7wt%，TG含量75.6wt%，DG含量17.8wt%。
更进一步的，使用与手段六相同的精制工艺对本微生物粗油进行精制，得到的微生物精制油中，多不饱和脂肪酸总含量55.9wt%，TG含量为77.0wt%，DG含量为19.0wt%，其他理化指标与手段六获得的理化指标接近。
下面介绍使用上述含有花生四烯酸的微生物油制备微胶囊的方法。
分别使用市售的花生四烯酸油、上述手段一、手段二、手段三制得的花生四烯酸精制油，按照如图1所示的配料表进行配料。
将上述料液在8000rpm的转速下剪切10分钟后，在40MPa下进行均质，得到乳化液。将乳化液进行压力式喷雾干燥，喷雾干燥参数如图2所示。
测得各微胶囊产品的表面油含量结果如图3所示。
分别使用市售花生四烯酸油、上述手段四、手段五制得的花生四烯酸精制油，按照如图4所示的配料表进行配料。
将上述料液在9000rpm的转速下剪切15分钟后，在45MPa下进行均质，得到乳化液。将此乳化液进行离心式喷雾干燥，喷雾干燥参数如图5所示。
测得微胶囊的表面油含量结果如图6所示。
分别使用市售花生四烯酸油、上述手段六、手段七、手段八制得的花生四烯酸精制油，按照如图7所示的配料表进行配料。
将上述料液在10000rpm的转速下剪切15分钟后，在50MPa下进行均质，得到乳化液。将此乳化液进行喷雾造粒干燥，首先需要投入15kg麦芽糊精做底料，喷雾干燥参数如图8所示。
测得微胶囊的表面油含量结果如图9所示。
下面介绍使用上述含有花生四烯酸的微生物油制备奶粉的方法。
分别使用市售花生四烯酸油、上述手段一至八制得的花生四烯酸精制油，按照如图10所示的配料表进行配料。
按照上述配方比例投料后，在5000rpm的转速下剪切10分钟后，在20MPa下进行均质，得到乳化液。然后经过三级降膜式真空浓缩器将此乳化液浓缩至水分含量50%，最后进行压力式喷雾干燥，喷雾干燥参数如图11所示。
测得奶粉的表面油含量结果如图12所示。
微胶囊的表面油含量是表征微胶囊质量的一项重要指标，其表示未被包埋的油脂在微胶囊表面的比例。微胶囊的表面油含量越高，表明越多表面油脂会被氧化，则产品的质量就越差。通过以上数据对比可以看出，在同等工艺条件下，由本发明的含有花生四烯酸的微生物油所制得的微胶囊及奶粉，其表面油含量更低。这主要是因为：本发明的微生物油含有较多的甘油二酯，它可以帮助微生物油形成更稳定的乳化液，使微生物油脂更好地被包埋，从而降低微胶囊及奶粉的表面油含量，提高微胶囊及奶粉的抗氧化能力，延长微胶囊及奶粉的货架期。
实施例二
本实施例以裂殖壶菌为出发菌种，下面详细描述本发明含有二十二碳六烯酸的微生物油的生产及应用。
1.发酵
配制葡萄糖含量0.04g/mL、酵母浸膏含量0.02g/mL的培养基溶液于1000ml摇瓶中，可配制多瓶，灭菌后接入适量冷藏裂殖壶菌液，置于28℃恒温摇床，180rpm进行活化。2天后接入二级扩大摇瓶进行培养，2天后合并摇瓶，移入已灭菌的、含5wt%葡萄糖及2wt%酵母浸膏的1立方米发酵罐（一级种子罐）中，持续通入无菌空气，保持培养温度29±1℃。一级种子罐培养2天后，将全部培养液移入已灭菌的、含葡萄糖含量为0.03g/mL及酵母浸膏含量0.02g/mL的10立方米发酵罐（二级种子罐）中，持续通入无菌空气，保持培养温度29±1℃。二级种子罐培养1天后，移全部培养液入已灭菌的、含葡萄糖含量0.05g/mL及酵母浸膏含量0.02g/mL的45立方米发酵罐中，持续通入无菌空气，保持培养温度29±1℃，根据葡萄糖消耗速度批次补加总约0.02至0.04g/mL的灭菌葡萄糖溶液，再培养5天后可获得发酵产物，其中生物量89.7g/L、总油含量38.5g/L、总油中二十二碳六烯酸含量51.0wt%。
2.制备富含二十二碳六烯酸的微生物油
手段一
将发酵产物通过离心或压滤方式实现固液分离，收集湿菌体，利用粉碎机破碎，再通过沸腾干燥塔烘干，得到干菌体。将干菌体与有机溶剂如丁烷或己烷混合浸泡萃取，过滤后得到混合油。
向混合油中加入市售脂肪酶和水进行酶解处理，该市售脂肪酶具有将甘油酯水解的功能。所述的酶解处理参数包括：脂肪酶用量为微生物混合油重量的0.25wt%；水用量为微生物混合油重量的30wt%，反应温度约37℃，搅拌反应时间0.5h。酶解反应结束后对混合油进行纯化，纯化的具体步骤包括：将混合油静置，待油相与水相分层后，除去水层，过滤除去酶，蒸发脱除溶剂，通过分子蒸馏设备除去游离脂肪酸，得到微生物粗油。测得该粗油具有如下指标：多不饱和脂肪酸含量64.0wt%，TG含量86.0wt%，DG含量9.8wt%。
更进一步的，对上述微生物粗油进行精制，精制的步骤包括：将该微生物粗油经2.5wt%柠檬酸和5wt%热水脱胶、过量氢氧化钠溶液脱酸，沉降分离，过滤后再用5wt%二氧化硅及3wt%活性炭脱色，再次过滤后，在200℃的条件下直接蒸汽脱臭，添加Vc棕榈酸酯和Ve作为抗氧化剂，得到含有二十二碳六烯酸的微生物精制油。经测定，该精制油中多不饱和脂肪酸总含量达64.0wt%，TG含量为 88.7wt%，DG含量为11.5wt%。
进一步测定该微生物精制油的部分理化指标：不皂化物1.0wt%，水分0.01wt%，不溶性杂质0.01wt%，溶剂残留<1.0mg/kg，酸价0.1mgKOH/g，过氧化值0.03meq/kg，反式脂肪酸0.06wt%，黄曲霉毒素B1<5.0μg/kg，总砷（以As计）<0.1mg/kg，铅<0.1mg/kg。
手段二
将发酵产物通过离心或压滤方式实现固液分离，收集湿菌体，利用粉碎机破碎，再通过沸腾干燥塔烘干，得到干菌体。将干菌体与有机溶剂如丁烷或己烷混合浸泡萃取，过滤后得到混合油。
向混合油中添加含甘油二酯的混合物，例如脂肪酸单双甘酯或其类似物，该混合物中甘油二酯含量为54.9wt%，添加混合物的比重为总混合油的16.2wt%。对混合油脱溶，混合均匀后得到微生物粗油。该微生物粗油具有如下指标特征：多不饱和脂肪酸含量47.2wt%，TG含量82.1wt%，DG含量10.4wt%。
更进一步的，对上述微生物粗油进行精制，精制的步骤包括：将该微生物粗油经2.5wt%柠檬酸和5wt%热水脱胶、过量氢氧化钠溶液脱酸，沉降分离，过滤后再用5wt%二氧化硅及3wt%活性炭脱色，再次过滤后，在200℃的条件下直接蒸汽脱臭，添加Vc棕榈酸酯和Ve作为抗氧化剂，得到含有二十二碳六烯酸的微生物精制油。经测定，该精制油中多不饱和脂肪酸总含量达48.1wt%，TG含量为 84.8wt%，DG含量为12.0wt%。其他理化指标与手段一获得的理化指标接近。
1.应用
上述含有二十二碳六烯酸的微生物油可以制备微胶囊，使用市售的二十二碳六烯酸油以及上述手段一、手段二制得的二十二碳六烯酸油，按照如图13所示的配料表进行配料。
将上述料液在8000rpm的转速下剪切10分钟后，在40MPa下进行均质，得到乳化液。将此乳化液进行压力式喷雾干燥，喷雾干燥参数如图14所示。
测得微胶囊的表面油含量结果如图15所示。
上述含有二十二碳六烯酸的微生物可以油制备奶粉，使用市售的二十二碳六烯酸油以及上述手段一、手段二制得的二十二碳六烯酸油，按照如图16所示的配料表进行配料。
按照上述配方比例投料后，在5000rpm的转速下剪切10分钟后，在20MPa下进行均质，得到乳化液。然后经过三级降膜式真空浓缩器将此乳化液浓缩至水分含量50%，最后进行压力式喷雾干燥，喷雾干燥参数如图17所示。
测得奶粉的表面油含量结果如图18所示。
通过以上实验数据可以看出，在同等工艺条件下，由本发明的含有二十二碳六烯酸的微生物油所制得的微胶囊及奶粉，其表面油含量更低。这主要是因为：本发明的微生物油含有较多的甘油二酯，它可以帮助微生物油形成更稳定的乳化液，使微生物油脂更好地被包埋，从而降低微胶囊及奶粉的表面油含量，提高微胶囊及奶粉的抗氧化能力，延长微胶囊及奶粉的货架期。
实施例三
本实施例以终极腐霉为出发菌种，详细描述含有二十碳五烯酸的微生物油的生产及应用。
1.发酵
配制蔗糖含量为0.05g/mL及酵母粉含量为0.005g/mL的培养基溶液于1000ml摇瓶中，可配制多瓶，灭菌后接入适量终极腐霉，置于28℃恒温摇床，180rpm进行活化。2天后接入二级扩大摇瓶进行培养，2天后合并摇瓶，移入已灭菌的、蔗糖含量为0.05g/mL及酵母粉含量为0.005g/mL的1立方米发酵罐（一级种子罐）中，持续通入无菌空气，保持培养温度28±1℃。一级种子罐培养2天后，移全部培养液入已灭菌的、蔗糖含量为0.05g/mL及酵母粉含量为0.005g/mL的10立方米发酵罐（二级种子罐）中，持续通入无菌空气，保持培养温度28±1℃。二级种子罐培养1天后，移全部培养液入已灭菌的、蔗糖含量为0.05g/mL及酵母粉含量为0.005g/mL的45立方米发酵罐中，持续通入无菌空气，保持培养温度28±1℃，根据蔗糖消耗速度批次补加总约0.02~0.04g/mL的灭菌蔗糖溶液，再培养5天后可获得发酵产物，其中二十碳五烯酸含量207.8mg/L。
2.制备富含二十碳五烯酸的微生物油
手段一
将发酵产物通过离心或压滤方式实现固液分离，收集湿菌体，利用粉碎机破碎，再通过沸腾干燥塔烘干，得到干菌体。将干菌体与有机溶剂如丁烷或己烷混合浸泡萃取，过滤后得到混合油。
向混合油中加入市售脂肪酶和水进行酶解处理，该市售脂肪酶具有将甘油酯水解的功能。所述的酶解处理参数包括：脂肪酶用量为微生物混合油重量的0.25wt%；水用量为微生物混合油重量的30wt%，反应温度约37℃，搅拌反应时间0.5h。酶解反应结束后对混合油进行纯化，纯化的具体步骤包括：将混合油静置，待油相与水相分层后，除去水层，过滤除去酶，蒸发脱除溶剂，通过分子蒸馏设备除去游离脂肪酸，得到微生物粗油。该粗油具有如下指标特征：多不饱和脂肪酸含量59.8wt%，TG含量85.1wt%，DG含量8.5wt%。
更进一步的，对上述微生物粗油进行精制，精制的步骤包括：将该微生物粗油经2.5wt%柠檬酸和5wt%热水脱胶、过量氢氧化钠溶液脱酸，沉降分离，过滤后再用5wt%二氧化硅及3wt%活性炭脱色，再次过滤后，在200℃的条件下直接蒸汽脱臭，添加Vc棕榈酸酯和Ve作为抗氧化剂，得到含有二十碳五烯酸的微生物精制油。经测定，该精制油中多不饱和脂肪酸总含量达59.4wt%，TG含量为 87.5wt%，DG含量为10.4wt%。
进一步测定该微生物精制油的部分理化指标：不皂化物0.8wt%，水分0.01wt%，不溶性杂质0.01wt%，溶剂残留<1.0mg/kg，酸价0.1mgKOH/g，过氧化值0.03meq/kg，反式脂肪酸0.06wt%，黄曲霉毒素B1<5.0μg/kg，总砷（以As计）<0.1mg/kg，铅<0.1 mg/kg。
手段二
将发酵产物通过离心或压滤方式实现固液分离，收集湿菌体，利用粉碎机破碎，再通过沸腾干燥塔烘干，得到干菌体。将干菌体与有机溶剂如丁烷或己烷混合浸泡萃取，过滤后得到混合油。
向混合油中添加含甘油二酯的混合物，例如脂肪酸单双甘酯或其类似物，该混合物中甘油二酯含量为56.1wt%，添加的混合物的比重为总混合油的15.0wt%。对混合油脱溶，混合均匀后得到微生物粗油。该粗油具有如下指标特征：多不饱和脂肪酸含量45.0wt%，TG含量82.0wt%，DG含量9.9wt%。
更进一步的，对上述微生物粗油进行精制，精制的步骤包括：将该微生物粗油经2.5wt%柠檬酸和5wt%热水脱胶、过量氢氧化钠溶液脱酸，沉降分离，过滤后再用5wt%二氧化硅及3wt%活性炭脱色，再次过滤后，在200℃的条件下直接蒸汽脱臭，添加Vc棕榈酸酯和Ve作为抗氧化剂，得到含有二十碳五烯酸的微生物精制油。经测定，该精制油中多不饱和脂肪酸总含量达44.9wt%，TG含量为 84.3wt%，DG含量为11.9wt%。其他理化指标与手段一获得的理化指标接近。
3.应用
上述含有二十碳五烯酸的微生物油制备微胶囊，使用市售的二十碳五烯酸油、上述手段一、手段二制得的二十碳五烯酸油，按照如图19所示的配料表进行配料。
将上述料液在8000rpm的转速下剪切10分钟后，在40MPa下进行均质，得到乳化液。将此乳化液进行喷雾干燥，喷雾干燥参数如图20所示。
测得微胶囊的表面油含量结果如图21所示。
上述含有二十碳五烯酸的微生物油制备奶粉，使用市售的二十碳五烯酸油、上述手段一、手段二制得的二十碳五烯酸油，按照如图22所示的配料表进行配料。
按照上述配方比例投料后，在5000rpm的转速下剪切10分钟后，在20MPa下进行均质，得到乳化液。然后经过三级降膜式真空浓缩器将此乳化液浓缩至水分含量50%，最后进行压力式喷雾干燥，喷雾干燥参数如图23所示。
测得奶粉的表面油含量结果如图24所示。
 工业实用性
微生物油所制得的微胶囊及奶粉，其表面油含量更低。这主要是因为：本发明的微生物油含有较多的甘油二酯，它可以帮助微生物油形成更稳定的乳化液，使微生物油脂更好地被包埋，从而降低微胶囊及奶粉的表面油含量，提高微胶囊及奶粉的抗氧化能力，延长微胶囊及奶粉的货架期。

Claims (13)

  1. 一种微生物油，其特征在于：其多不饱和脂肪酸的含量大于30wt%，甘油三酯含量小于90wt%，甘油二酯含量不低于5wt%且不高于20wt%。
  2. 根据权利要求1所述的微生物油，其特征在于：所述甘油二酯的含量不低于10wt%，所述甘油三酯的含量不低于75wt%。
  3. 根据权利要求1或2所述的微生物油，其特征在于：所述多不饱和脂肪酸为花生四烯酸、二十二碳六烯酸或者二十碳五烯酸。
  4. 根据权利要求1或2所述的微生物油，其特征在于：所述微生物油为粗油。
  5. 制备如权利要求1所述的微生物油的方法，其特征在于：包括以下步骤：
    (1) 利用产油微生物发酵得到富含PUFA微生物油的发酵液；
    (2) 收集富含PUFA微生物油的菌体，萃取过滤后得到混合油；
    (3) 在混合油中加入脂肪酶和水进行酶解，纯化后得到微生物油。
  6. 根据权利要求5所述的微生物油的制备方法，其特征在于：在步骤（3）之后对微生物油进行精制。
  7. 根据权利要求5或6所述的微生物油的制备方法，其特征在于：所述步骤（3）中，酶解参数包括：在脂肪酶最适温度下搅拌反应0.5至2小时，脂肪酶用量为混合油质量的0.25wt%至2wt%，水用量为混合油质量的15wt%至30wt%。
  8. 制备如权利要求1所述的微生物油的方法，其特征在于：包括以下步骤：
    (1) 利用产油微生物发酵得到富含PUFA微生物油的发酵液；
    (2) 收集富含PUFA微生物油的菌体，萃取过滤后得到混合油；
    (3) 向混合油中添加含有甘油二酯的混合物，混合均匀后去除溶剂得到微生物油。
  9. 根据权利要求8所述的微生物油的制备方法，其特征在于：在步骤（3）之后对微生物油进行精制。
  10. 一种微胶囊，其特征在于：该微胶囊包括微生物油芯材及包裹所述微生物油的壁材，所述微生物油的多不饱和脂肪酸的含量大于30wt%，所述微生物油的甘油三酯含量小于90wt%，所述微生物油的甘油二酯含量不低于5wt%且不高于20wt%。
  11. 根据权利要求10所述的微胶囊，其特征在于：所述甘油二酯的含量不低于10wt%，所述甘油三酯的含量不低于75wt%。
  12. 根据权利要求10或11所述的微胶囊，其特征在于：所述多不饱和脂肪酸为花生四烯酸、二十二碳六烯酸或者二十碳五烯酸。
  13. 根据权利要求10或11所述的微胶囊，其特征在于：所述微生物油为粗油。
PCT/CN2015/074205 2014-03-14 2015-03-13 一种微生物油及其制备方法、一种微胶囊 WO2015135501A1 (zh)

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