WO2021196881A1 - 一种甘油三酯型多不饱和脂肪酸及其制备方法和应用 - Google Patents

一种甘油三酯型多不饱和脂肪酸及其制备方法和应用 Download PDF

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WO2021196881A1
WO2021196881A1 PCT/CN2021/075077 CN2021075077W WO2021196881A1 WO 2021196881 A1 WO2021196881 A1 WO 2021196881A1 CN 2021075077 W CN2021075077 W CN 2021075077W WO 2021196881 A1 WO2021196881 A1 WO 2021196881A1
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fatty acid
lipase
polyunsaturated fatty
fatty acids
hydrolysis
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French (fr)
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梁云
曹晟
王身健
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瞿瀚鹏
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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
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
    • C11C1/045—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis using enzymes or microorganisms, living or dead
    • 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
    • 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
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/08—Refining
    • C11C1/10—Refining by distillation
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
    • C11C3/10—Ester interchange
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • 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
    • 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
    • A23V2250/00—Food ingredients
    • A23V2250/18—Lipids
    • A23V2250/186—Fatty acids
    • A23V2250/1882—Polyunsaturated fatty acids

Definitions

  • the invention relates to the field of microbial oils and fats, in particular to a triglyceride type polyunsaturated fatty acid and a preparation method and application thereof.
  • Polyunsaturated fatty acids contain more than two double bonds, have special biological activities, and have a wide range of functions in biological systems. They are useful for stabilizing cell membrane function, regulating gene expression, maintaining cytokine and lipoprotein balance, resisting cardiovascular diseases, and promoting Growth and development play an important role.
  • Polyunsaturated fatty acids mainly include n-3, n-6 and n-9 series fatty acids, among which the biological activity of n-6 and n-3 series fatty acids has been paid more attention.
  • the polyunsaturated fatty acids are usually obtained by the method of fish oil refining or fermentation of oleaginous microorganisms.
  • triglyceride type polyunsaturated fatty acids are mainly obtained by the method of fermentation of oleaginous microorganisms.
  • the triglyceride-type polyunsaturated fatty acids obtained by fermentation of oleaginous microorganisms are usually polyunsaturated fatty acids with medium and low purity, which are mainly used in the food industry. Among them, it is widely used in product categories such as infant formula food, middle-aged and elderly formula food, and health food. When used in the pharmaceutical industry, higher purity polyunsaturated fatty acids are required.
  • the production of high-purity polyunsaturated fatty acids mainly uses medium and low-purity polyunsaturated fatty acids as raw materials, and uses chromatography, distillation, solvent extraction, low-temperature crystallization, supercritical extraction, and urea inclusion methods for purification.
  • the product form is mainly ethyl ester fatty acid products.
  • Ethyl fatty acids are inferior to glyceride fatty acids in terms of safety and human absorption, and the human absorption rate is only about 20%.
  • the technical problem to be solved by the present invention is to provide a method for preparing triglyceride-type polyunsaturated fatty acids, which has less oxidation loss of polyunsaturated fatty acids, high production efficiency, and purity of triglyceride-type polyunsaturated fatty acids High, and no solvent residue.
  • the further technical problem to be solved by the present invention is to increase the content of polyunsaturated fatty acids in the triglyceride type oil, and remove the saturated fatty acids in the triglyceride structure.
  • the triglyceride type polyunsaturated fatty acids have high purity and are polyunsaturated.
  • Fatty acids have high biological activity and low safety risks.
  • the technical problem to be solved by the present invention is to provide an application of triglyceride type polyunsaturated fatty acid in the preparation of formula foods for infants and young children, health foods, health foods or medical supplies.
  • the first aspect of the present invention provides a method for preparing triglyceride-type polyunsaturated fatty acids, which includes the following steps: (1) Homogenizing treatment is carried out after mixing raw oils and fats containing polyunsaturated fatty acids with water Free fatty acid salt and glycerol are obtained by lipase-catalyzed hydrolysis; (2) the fatty acid salt obtained in step (1) is separated from glycerin to obtain fatty acid salt; (3) the fat obtained in step (2) is obtained Acidification treatment of acid salt to obtain free fatty acids; (4) After drying the free fatty acids obtained in step (3), molecular distillation is performed to remove short-chain fatty acids and enrich them to obtain long-chain polyunsaturated fatty acids; (5) The polyunsaturated fatty acid obtained in step (4) is mixed with glycerin and subjected to esterification reaction catalyzed by lipase to obtain the triglyceride type polyunsaturated fatty acid
  • the mass ratio of the raw fat and water is 1:0.7-1.3; the temperature of the homogenization treatment is 45-65°C, and the pressure is 20-100 MPa; and/or, Use 10-15% sodium hydroxide aqueous solution or potassium hydroxide aqueous solution to adjust the pH of the lipase-catalyzed hydrolysis system to be 6-7.5.
  • the oil-water contact area of the homogeneously formed emulsion is larger; the homogenization temperature of 45-65°C and the homogenization pressure of 20-100MPa can be
  • the homogenized raw fats and oils are fully mixed with water to form particles with a smaller particle size and a larger contact area with water.
  • the large oil-water contact area is conducive to the contact and hydrolysis of the raw fats and oils with the lipase dissolved in water, the hydrolysis speed is faster, and the production efficiency is higher.
  • the free fatty acids produced by the hydrolysis of the raw oil will gradually reduce the PH value of the hydrolysis system, which exceeds the PH value range of the enzyme activity of the lipase. Therefore, it is necessary to add alkaline substances in real time to control the pH value of the hydrolysis system.
  • various alkaline substances commonly used in the art can be used to control the pH value of the hydrolysis system, and it is preferable to use sodium hydroxide aqueous solution or potassium hydroxide aqueous solution to control the pH value of the hydrolysis system.
  • the concentration of the sodium hydroxide aqueous solution or the potassium hydroxide aqueous solution used is 10-15%, and the pH adjustment range is 6-7.5 to maintain the hydrolytic activity of the lipase.
  • sodium hydroxide aqueous solution or potassium hydroxide aqueous solution to control the pH value of the hydrolysis system can also make the free fatty acids produced by the hydrolysis of raw oils and fats become sodium or potassium salts, hindering the occurrence of the reverse reaction of the hydrolysis reaction.
  • the lipase is a non-specific lipase, preferably Penicillium cyclopium (PG37 lipase), Candida lipase (candida lipase) rugosalipase (CRL), Aspergillus niger lipase (Aspergillus niger lipase), Novozymes lipase 435 (Novozym435), Lipozyme RM M and C. rugosa lipase (CRL) .
  • PG37 lipase Penicillium cyclopium
  • Candida lipase candida lipase
  • rugosalipase CRL
  • Aspergillus niger lipase Aspergillus niger lipase
  • Novozymes lipase 435 Novozym435
  • Lipozyme RM M Lipozyme RM M
  • C. rugosa lipase CRL
  • the non-specific lipase can hydrolyze the fatty acids in each position of the raw oil and fat at the same time, which overcomes the fact that the specific lipase can only hydrolyze the fatty acids at the 1st and 3rd positions of the raw oil and the fatty acids at the 2nd position need to be rearranged. Defects that can be hydrolyzed at position 1 or 3 speed up the rate of hydrolysis reaction.
  • the method for catalyzing the lipase is directly adding the immobilized lipase to the hydrolysis kettle or making the immobilized lipase into a packed bed for catalytic hydrolysis, preferably Packed beds connected in series or in parallel carry out catalytic reactions.
  • the lipase is solidified, it is directly put into the hydrolysis kettle, or after the lipase is solidified, it is made into a packed bed for catalytic hydrolysis, which can fix the lipase at the position where it is in full contact with the hydrolyzed substrate, and expand the lipase and the hydrolyzed substrate.
  • the contact area of the substance accelerates the speed of the hydrolysis reaction, and can reduce the loss of lipase during the hydrolysis process and improve the utilization rate of lipase.
  • the use of series or parallel filled reactors can further expand the contact area of the enzymatic reaction, and the contact between the lipase and the hydrolysis substrate is more sufficient, and the speed of the hydrolysis reaction can be further accelerated.
  • the amount of added enzyme for the lipase-catalyzed hydrolysis is 100-500 units of lipase per gram of raw oil and fat, the hydrolysis temperature is 35-65°C, and the hydrolysis time is 8-72 hours.
  • the amount of enzyme added for the lipase-catalyzed hydrolysis directly affects the hydrolysis rate of the raw oil, but the increase of the amount of enzyme added will significantly increase the production cost.
  • the proper hydrolysis temperature can effectively increase the hydrolysis rate of lipase, and the proper hydrolysis time can make the raw oil and fat fully hydrolyzed and have higher production efficiency.
  • step (5) the amount of enzyme added in the esterification reaction is 5-20% of the amount of free fatty acids, the reaction temperature is 35-55°C, the stirring speed is 100-180 rpm, and the reaction time is 30-50 hours.
  • the above reaction conditions are more conducive to the esterification reaction.
  • the pressure of the molecular distillation treatment is less than 0.5 Pa
  • the temperature is 120-185° C.
  • the feed rate is 15-20 kg/h
  • the film scraping speed is 110-180 rpm.
  • a pressure of less than 0.5 Pa not only reduces the boiling temperature of free fatty acids and prevents thermal decomposition of free fatty acids, but also increases the mean free path of vapor molecules, which is more conducive for gas molecules to reach the condensation surface and condense.
  • the temperature of 130-185°C can gradually evaporate the short and medium-chain fatty acids with twelve to twenty carbons, and condense on the condensation surface to separate from the long-chain polyunsaturated fatty acids.
  • the purity of the separated polyunsaturated fatty acids is higher. .
  • the feed rate of 15-20 kg/h and the film scraping speed of 120-180 rpm can make the free fatty acid form a very thin, turbulent liquid film, which is more conducive to the temperature increase and evaporation of the free fatty acid.
  • the preparation process is all carried out in an oxygen barrier state.
  • the oxygen barrier state can be the storage of raw materials, intermediate substances, and reaction products, or the reaction vessel being vacuumed, or the storage or reaction vessel can be filled with an inert atmosphere such as nitrogen to isolate oxygen.
  • the raw material oil is DHA algae oil and/or ARA algae oil.
  • DHA algae oil and ARA algae oil are rich in DHA and ARA, respectively.
  • DHA and ARA have strong biological activity, wide application range and great market demand.
  • the second aspect of the present invention provides a triglyceride type polyunsaturated fatty acid obtained according to the method provided in the first aspect of the present invention; wherein the content of triglyceride type DHA or triglyceride type ARA is not less than 72%; and The monoglyceride content is not more than 15%, the diglyceride content is not more than 10%, and the triglyceride content is not less than 75%.
  • the third aspect of the present invention provides an application of the triglyceride type polyunsaturated fatty acid provided by the present invention in the preparation of formula foods for infants and young children, health foods, health foods or medical supplies.
  • molecular distillation technology is used to separate short and medium-chain fatty acids in free fatty acids, and polyunsaturated fatty acids with higher purity can be obtained. It avoids the use of traditional short-chain alcohol esterification chemical treatment and separation methods, when short-chain alcohols are used to remove short-chain and medium-chain fatty acids under the condition of using organic solvents, the part of polyunsaturated fatty acids in the production process caused Concomitant loss of esterification or residual organic solvents.
  • the separation process of the present invention does not use chemical solvents, does not add new chemicals, and avoids food safety risks caused by the reaction loss of polyunsaturated fatty acids and short-chain alcohols and organic solvent residues.
  • the raw oil and fat are mixed with water and then homogenized, so that the raw oil and fat form tiny particles in the water, increasing the contact area of the raw oil and fat with water and lipase, and at the same time
  • the distorted molecular structure of long-chain polyunsaturated fatty acids leads to steric hindrance during lipase hydrolysis, which speeds up the hydrolysis reaction of raw oil and water under the action of lipase, and improves the efficiency of the hydrolysis reaction.
  • the lipase used in the present invention adopts non-specific lipase.
  • the specific lipase used in the traditional hydrolysis process is only sensitive to fatty acids at positions 1 and 3.
  • fatty acids at positions 1 and 3 can be directly hydrolyzed, but fatty acids at position 2 cannot be directly hydrolyzed.
  • the fatty acids at position 2 need to wait for the hydrolysis of fatty acids at positions 1 and 3 to be hydrolyzed, and then shift to position 1 or 3.
  • the rate of hydrolysis of oils and fats is slow.
  • the non-specific lipase used in the present invention can hydrolyze fatty acids from multiple positions during hydrolysis, accelerate the hydrolysis reaction speed, and improve the production efficiency.
  • the entire preparation process in the present invention is carried out in an oxygen-isolated environment to avoid oxidation of the polyunsaturated fatty acid during the reaction and storage process, and improve the final product quality of the triglyceride-type polyunsaturated fatty acid.
  • the DHA algae oil used in the following examples is the DHA oil fermented by Schizochytrium sp. produced by Linyi Youkang Biotechnology Co., Ltd.; the ARA algae oil is the ARA oil produced by Linyi Youkang Biotechnology Co., Ltd. that is fermented with mildew from the mountains; water use Purified water for reverse osmosis; the lipase used is produced by Novozymes or Hangzhou Weikang Technology Co., Ltd.; the nitrogen filled is produced by the JH-PN49-10 nitrogen generator produced by Guangzhou Zhongshan Extreme High-Tech Co., Ltd.
  • step (2) Put the mixture obtained in step (1) into a temporary storage tank with nitrogen pressure, filter and recover the lipase with a butterfly filter, and obtain a mixture of free fatty acid sodium salt and glycerol, and fill the temporary storage tank with nitrogen Under the conditions, let it stand for 4-6 hours to separate and remove the glycerin and water to obtain fatty acid sodium.
  • step (3) Acidify the fatty acid sodium obtained in step (2) with a 10% sulfuric acid solution, leave it for 2-3 hours and then separate and remove the water to obtain free fatty acids;
  • the heavy phase After being processed by a three-stage short-path molecular distillation system, the heavy phase is collected to obtain high-purity long-chain polyunsaturated fatty acids.
  • step (4) Take 20kg of the polyunsaturated fatty acid obtained in step (4), add 2.8Kg of glycerol, and then add 3Kg of Novozym 435 esterification enzyme for esterification reaction, keep the reaction temperature at 45°C, and stir at 140rpm. After 40 hours of reaction, 4Kg of 65°C hot water was added to wash, and then left to stand for 2 hours to separate the mixture of excess glycerin and water to obtain triglyceride type polyunsaturated fatty acid.
  • step (2) Put the mixture obtained in step (1) into a temporary storage tank with nitrogen pressure, filter and recover the lipase with a butterfly filter, and obtain a mixture of free fatty acid potassium salt and glycerol, and fill the temporary storage tank with nitrogen Leave it for 4-6 hours under the circumstance that the glycerin and water are separated and removed to obtain fatty acid sodium.
  • step (3) Acidify the fatty acid sodium obtained in step (2) with a 10% sulfuric acid solution, leave it for 2-3 hours and then separate and remove the water to obtain free fatty acids;
  • step (3) Heat the free fatty acid obtained in step (3) to 80-90°C and vacuum to -0.09MPa, then dehydrate for 30-50 minutes; turn on the three-stage short-path molecular distillation system and the working parameters of the three-stage molecular distillation equipment As shown in table 2:
  • the heavy phase After being processed by a three-stage short-path molecular distillation system, the heavy phase is collected to obtain high-purity long-chain polyunsaturated fatty acids.
  • step (4) Take 20kg of the polyunsaturated fatty acid obtained in step (4), add 2Kg of glycerol, and then add 1Kg of Novozym435 esterase to carry out the esterification reaction, keep the reaction temperature at 35°C, and stir at 100rpm for a 50% reaction. After hours, add 4Kg of 65°C hot water to wash, and then stand for 2 hours to separate the mixture of excess glycerin and water to obtain triglyceride type polyunsaturated fatty acid.
  • step (2) Put the mixture obtained in step (1) into a temporary storage tank with nitrogen pressure, filter and recover the lipase with a butterfly filter, and obtain a mixture of free fatty acid sodium salt and glycerin, and fill the temporary storage tank with nitrogen Under the conditions, let it stand for 4-6 hours to separate and remove the glycerin to obtain fatty acid sodium.
  • step (3) The fatty acid sodium obtained in step (2) is acidified with a 10% sulfuric acid solution, and after standing for 2-3 hours, the water is separated and removed to obtain free fatty acids;
  • the heavy phase After being processed by a three-stage short-path molecular distillation system, the heavy phase is collected to obtain high-purity long-chain polyunsaturated fatty acids.
  • step (4) Take 20kg of the polyunsaturated fatty acid obtained in step (4), add 3.6Kg of glycerol, and then add 4Kg of Lipozyme RM M for esterification reaction, keep the reaction temperature at 55°C, and stir at 180rpm, reaction 30 After hours, add 4Kg of 65°C hot water to wash, and then stand for 2 hours to separate the mixture of excess glycerin and water to obtain triglyceride type polyunsaturated fatty acid.
  • step (2) Put the mixture obtained in step (1) into a temporary storage tank with nitrogen pressure, filter and recover the lipase with a butterfly filter, and obtain a mixture of free fatty acid sodium salt and glycerol, and fill the temporary storage tank with nitrogen Under the conditions, let it stand for 4-6 hours to separate and remove the glycerin and water to obtain fatty acid sodium.
  • step (3) The fatty acid sodium obtained in step (2) is acidified with a 10% sulfuric acid solution, and after standing for 2-3 hours, the water is separated and removed to obtain free fatty acids;
  • step (3) Heat the free fatty acids obtained in step (3) to 80-90°C, vacuum to -0.09MPa, and vacuum dehydration for 30-50 minutes; then slowly cool down according to the parameters shown in Table 4, and perform winterization on the free fatty acids.
  • Chemical treatment makes the saturated fatty acids crystallize out, and then use a plate and frame filter to filter out the crystallized saturated fatty acids to obtain polyunsaturated fatty acids.
  • the filtrate is collected to obtain high-purity long-chain polyunsaturated fatty acids.
  • step (4) Take 20kg of polyunsaturated fatty acids obtained in step (4), add 2.3Kg of glycerol, and then add 3.5Kg of Rhizomucor miehei lipase (RML) for esterification reaction, keep the reaction temperature at 45°C, and Stir at a speed of 140 rpm. After reacting for 40 hours, add 4Kg of hot water at 65°C for washing, and then stand for 2 hours to separate the mixture of excess glycerin and water to obtain triglyceride-type polyunsaturated fatty acids.
  • RML Rhizomucor miehei lipase
  • Example 1 87.1 8.5 6.2 85.3
  • Example 2 85.8 11.3 6.6 82.1
  • Example 3 84.3 14.3 6.9 78.8 Control example 62.3 17.9 11.3 70.8
  • the production time of triglyceride-type polyunsaturated fatty acids is shorter and the efficiency is higher.
  • the preparation method of the triglyceride type polyunsaturated fatty acid of the present invention has higher economic value.
  • the obtained triglyceride type polyunsaturated fatty acid has higher purity of the polyunsaturated fatty acid and higher medicinal and edible value.

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Abstract

本发明公开了一种甘油三酯型多不饱和脂肪酸的制备方法，包括以下步骤：(1)将原料油脂与水混合后进行均质处理，经脂肪酶催化水解，得到游离脂肪酸盐、甘油；(2)将脂肪酸盐与甘油分离，得到脂肪酸盐；(3)将脂肪酸盐进行酸化处理得到游离脂肪酸；(4)将游离脂肪酸干燥后，进行分子蒸馏处理，得到长链的多不饱和脂肪酸；(5)将多不饱和脂肪酸与甘油混合，经脂肪酶催化进行酯化反应，得到甘油三酯型多不饱和脂肪酸。该方法得到的甘油三酯型多不饱和脂肪酸纯度高，多不饱和脂肪酸的氧化损失小，无化学物质残留。本发明还公开了一种甘油三酯型多不饱和脂肪酸及其在制备婴幼儿配方食品、健康食品、保健食品或医药用品中的应用。

Description

一种甘油三酯型多不饱和脂肪酸及其制备方法和应用 技术领域
本发明涉及微生物油脂领域，具体涉及一种甘油三酯型多不饱和脂肪酸及其制备方法和应用。
背景技术
多不饱和脂肪酸含有两个以上的双键，具有特殊的生物活性，在生物系统中有着广泛的功能，对于稳定细胞膜功能、调控基因表达、维持细胞因子和脂蛋白平衡、抗心血管疾病以及促进生长发育等方面起着重要作用。多不饱和脂肪酸主要包括n-3、n-6和n-9系列脂肪酸，其中n-6和n-3系列脂肪酸的生物活性更加受到重视。
多不包含脂肪酸通常通过鱼油提炼或产油微生物的发酵的方法获得，其中，甘油三酯型多不饱和脂肪酸主要通过产油微生物的发酵的方法获得。但通过产油微生物的发酵的方法获得的甘油三酯型多不饱和脂肪酸通常为中低纯度的多不饱和脂肪酸，主要应用于食品行业。其中在婴幼儿配方食品、中老年配方食品、保健食品等产品类别上有广泛的应用。而应用于医药行业时，则需要更高纯度的多不饱和脂肪酸。
目前，高纯度的多不饱和脂肪酸的生产，主要采用中低纯度的多不饱和脂肪酸为原料，采用色谱法、蒸馏法、溶剂提取法、低温结晶法、超临界萃取法以及尿素包合法来提纯，但产品形式主要是乙酯型的脂肪酸产品居多。乙酯型脂肪酸在安全性和人体吸收性方面不如甘油酯型脂肪酸，人体吸收率只有20％左右。
近来出现了将中低纯度的多不饱和脂肪酸油脂水解成游离脂肪酸，去掉其中的中短链脂肪酸，在重新酯化得到高纯度多不饱和脂肪酸的方法。但通常采用短链醇与短链脂肪酸反应的方式来去掉短链脂肪酸，但这样的处理方式一方面引入新的化学品，导致生产成本的升高，同时又产生食品安全风险。现有的多不饱和脂肪酸油脂的水解工艺中所使用的脂肪酶多为特异性酶，对油脂不同位上的脂肪酸的敏感性不同，油脂的水解速度缓慢，影响了生产的效率。
发明内容
本发明所要解决的技术问题是提供一种甘油三酯型多不饱和脂肪酸的制备方法，该制备方法的多不饱和脂肪酸的氧化损失少，生产效率高，甘油三酯型多不饱和脂肪酸的纯度高，且无溶剂残留。
本发明进一步要解决的技术问题是提高甘油三酯型油脂中多不饱和脂肪酸的含量，将甘油三酯结构中的饱和脂肪酸去除，该甘油三酯型多不饱和脂肪酸的纯度高，多不饱和脂肪酸的生物活性高，安全风险低。
本发明还要解决的技术问题是提供一种甘油三酯型多不饱和脂肪酸在制备婴幼儿配方食品、健康食品、保健食品或医药用品中的应用。
为了解决上述技术问题，本发明第一方面提供一种甘油三酯型多不饱和脂肪酸的制备方法，包括以下步骤：(1)将含多不饱和脂肪酸的原料油脂与水混合后进行均质处理，经脂肪酶催化水解，得到游离脂肪酸盐、甘油；(2)将步骤(1)得到的脂肪酸盐与甘油分离，得到脂肪酸盐；(3)将步骤(2)得到的所述脂肪酸盐进行酸化处理得到游离脂肪酸；(4)将步骤(3)得到的游离脂肪酸干燥后，进行分子蒸馏处理，去除短链的脂肪酸，富集得到长链的多不饱和脂肪酸；(5)将步骤(4)得到的所述多不饱和脂肪酸与甘油混合，经脂肪酶催化进行酯化反应，得到所述甘油三酯型多不饱和脂肪酸。
根据本发明，在步骤(1)中，所述原料油脂与水的质量比为1:0.7-1.3；所述均质处理的温度为45-65℃、压力为20-100MPa；和/或，使用10-15％的氢氧化钠水溶液或氢氧化钾水溶液调控脂肪酶催化水解体系的PH为6-7.5。通过该优选技术方案，原料油脂与水的质量比为1:0.7-1.3时均质形成的乳液的油水接触面积更大；45-65℃的均质温度和20-100MPa的均质压力为能够使均质后的原料油脂与水充分混合，形成粒径更小的颗粒，与水的接触面积更大。大的油水接触面积有利于原料油脂与溶解在水中的脂肪酶的接触和水解，水解的速度更快，生产效率更高。而原料油的水解产生的游离脂肪酸会使得水解体系的PH值逐渐降低，超出脂肪酶发挥酶活性的PH值范围，因而需要实时添加碱性物质调控水解体系的PH值。在本发明中，可以使用本领域常用的各种碱性物质来调控水解体系的PH值，优选使用氢氧化钠水溶液或氢氧化钾水溶液来调控水解体系的PH值。使用的氢氧化钠水溶液或氢氧化钾水溶液的浓度在10-15％，PH值的调控范围为6-7.5，以保持脂肪酶的水解活性。使用氢氧化钠水溶液或氢氧化钾水溶液来调控水解体系的PH值还能够使原料油脂水解产生的游离脂肪酸变成钠盐或钾盐，阻碍水解反应的逆反应的发生。
根据本发明，在步骤(1)和步骤(5)中，所述脂肪酶为非特异性脂肪酶，优选为圆弧青酶脂肪酶(Penicillium cyclopium，PG37脂肪酶)、假丝酵母脂肪酶(candida rugosalipase，CRL)、黑曲霉脂肪酶(Aspergillus niger lipase)、诺维信脂肪酶435(Novozym435)、Lipozyme RM M和柱状假丝酵母脂肪酶(C.rugosa lipase,CRL)中的一种或多种。在该优选技术方案中，非特异性脂肪酶能够同时对原料油脂各个位上的脂肪酸进行水解，克服了特异脂肪酶水解时仅能水解原料油脂1、3位上的脂肪酸，2位脂肪酸需要重排到1位或3位才能水解的缺陷，加快水解反应速度。
根据本发明，在步骤(1)和步骤(5)中，所述脂肪酶催化的方法为将固定化脂肪酶直接加入水解釜中或将固定化脂肪酶制作成填充床进行催化水解，优选采用串联或并联的填充床进行催化反应。将脂肪酶固化处理后，直接投入水解釜中，或者将脂肪酶固化处理后，制作成填充床进行催化水解，能够使得脂肪酶固定在与水解底物充分接触的位置，扩大脂肪酶与水解底物的接触面积，加速水解反应的速度，并且能够减少水解过程中脂肪酶的损失，提高脂肪酶的利用率。采用串联或并联的填充式反应器能够进一步扩大酶促反应的接触面积，脂肪酶与水解底物的接触更加充分，能够进一步 加快水解反应的速度。
根据本发明，在步骤(1)中，所述脂肪酶催化水解的加酶量为每克原料油脂添加100-500单位脂肪酶、水解温度为35-65℃、水解时间为8-72小时。在该优选技术方案中，所述脂肪酶催化水解的加酶量直接影响原料油脂的水解速度，但加酶量的增加又会显著提高生产成本。适宜的水解温度能够有效提高脂肪酶的水解速度，合适的水解时间能够使得原料油脂充分水解而又有较高的生产效率。在步骤(5)中，所述酯化反应的加酶量为游离脂肪酸量的5-20％、反应温度为35-55℃、搅拌速度为100-180rpm、反应时间为30-50小时。通过该优选技术方案，在无水的环境下，上述反应条件更加有利于酯化反应的进行。
根据本发明，在步骤(4)中，所述分子蒸馏处理的压力小于0.5Pa、温度为120-185℃、进料速度为15-20千克/小时、刮膜转速110-180rpm。在该优选技术方案中，小于0.5Pa的压力不仅降低了游离脂肪酸的沸腾温度，防止游离脂肪酸的受热分解，还增加了蒸汽分子的平均自由程，更有利于气体分子到达冷凝表面而冷凝。130-185℃的温度能够使十二到二十碳的短、中链脂肪酸渐次蒸发，并在冷凝表面冷凝而与长链的多不饱和脂肪酸分离，分离得到的多不饱和脂肪酸的纯度更高。15-20千克/小时的进料速度和120-180rpm的刮膜转速能够使得游离脂肪酸形成一层极薄、呈湍流状的液膜，更有利于游离脂肪酸温度的升高和蒸发。
根据本发明，所述制备的过程均在隔氧状态下进行。所述隔氧状态，可以是将原料、中间态物质和反应产物的存放或者反应容器抽成真空状态，也可以是在存放或者反应容器中充入氮气等惰性气氛以隔绝氧气。
根据本发明，所述原料油脂为DHA藻油和/或ARA藻油。DHA藻油和ARA藻油分别富含DHA和ARA，DHA和ARA的生物活性强，应用范围广，市场需求大。
本发明第二方面提供了一种根据本发明第一方面提供的方法得到的甘油三酯型多不饱和脂肪酸；其中，甘油三酯型DHA或甘油三酯型ARA含量不低于72％；且甘油一酯含量不高于15％，甘油二酯含量不高于10％，甘油三酯含量不低于75％。
本发明第三方面提供了一种本发明提供的甘油三酯型多不饱和脂肪酸在制备婴幼儿配方食品、健康食品、保健食品或医药用品中的应用。
通过上述技术方案，本发明的有益效果为：
1、本发明中采用分子蒸馏的技术分离游离脂肪酸中的短、中链脂肪酸，能够得到纯度更高的多不饱和脂肪酸。避免了采用传统的短链醇酯化的化学处理分离方式时，在使用有机溶剂的条件下用短链醇来去除短链与中链脂肪酸时，所导致的生产过程中多不饱和脂肪酸的部分连带酯化损失或者有机溶剂的残留。本发明的分离过程不使用化学溶剂，不添加新的化学品，避免了多不饱和脂肪 酸与短链醇反应损失和有机溶剂残留所带来的食品安全风险。
2、本发明中在原料油脂进行水解反应前，将原料油脂与水混合后进行均质处理，使得原料油脂在水中形成微小的颗粒，增加了原料油脂与水和脂肪酶的接触面积，同时能够服长链多不饱和脂肪酸的扭曲分子结构所导致脂肪酶水解时的位阻效应，加快原料油脂与水在脂肪酶的作用下发生水解反应的速度，提高水解反应的效率。
3、本发明中所使用的脂肪酶采用了采用非特异性脂肪酶。传统的水解工艺中所使用的特异脂肪酶仅仅对1、3位脂肪酸敏感，在水解过程中仅能直接水解1、3位上的脂肪酸，而不能直接水解2位上的脂肪酸。在水解过程中，2位脂肪酸需要等1、3位脂肪酸水解完成后，再换位到1位或3位后才能水解，油脂的水解速度缓慢。本发明所采用的非特异性脂肪酶水解时能够从多位对脂肪酸进行水解，加快水解反应速度，提高了生产的效率。
4、本发明中的整个制备过程均在隔氧环境下进行，避免反应和存储过程中多不饱和脂肪酸发生氧化，提高了甘油三酯型多不饱和脂肪酸的最终产品品质。
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。
在本发明中，在未作特别说明的情况下，所使用的百分比值均表示重量百分比。
以下通过具体实施例对本发明的技术方案进行详细描述。以下实施例中所使用的DHA藻油是临沂友康生物科技有限公司生产的裂壶藻发酵的DHA油脂；ARA藻油是临沂友康生物科技有限公司生产的高山被饱霉发酵的ARA油脂；水使用反渗透的纯净水；所使用的脂肪酶为诺维信公司或杭州维康科技有限公司生产；充入的氮气是广州中山极高科技有限公司生产的JH-PN49-10型制氮机生产的纯度为99.99％的氮气；分子蒸馏设备是四川久远化工技术有限公司生产的DZ-80型“三级短程分子蒸馏设备”；其他设备均为常规化工设备，试剂均为市售分析纯制剂。产品成分的检测均按照国标中的规定进行测定。
实施例1
(1)取100Kg藻油来源的DHA精炼油(DHA含量43％)，置于水解釜中，加入100Kg的水，在55℃的温度、60MPa的压力下均质处理10分钟。按200单位/克油脂的比例用固定化假丝酵母脂肪酶和200单位/克油脂的比例用固定化柱状假丝酵母脂肪酶设置串联的填充床，将均质好的原料加入填充床，用氢氧化钠水溶液调节PH值至4.8，抽负压到-0.09MPa后充入氮气至釜内压力 0.05-0.09MPa。在50℃的温度下水解45小时，在水解过程进行4.5小时后在线自动添加12％氢氧化钠水溶液，将水解体系的PH值维持在6.8-7。45小时后结束水解反应，得到含游离脂肪酸钠和甘油的混合物。
(2)将步骤(1)得到的混合物置用氮气压送到暂存罐中，用蝶式过滤机过滤回收脂肪酶，并得到游离脂肪酸钠盐和甘油的混合物，在暂存罐中充氮气的条件下，静置4-6小时，分离并去掉甘油和水，得到脂肪酸钠。
(3)将步骤(2)得到的脂肪酸钠用10％的硫酸溶液酸化，静置2-3小时后分离去掉水得到游离脂肪酸；
(4)将步骤(3)得到的游离脂肪酸加热到80-90℃，并抽真空至-0.09MPa，真空脱水30-50分钟；开启三级短程分子蒸馏系统，三级分子蒸馏设备的工作参数如表1所示：
表1 实施例1分子蒸馏参数表
  一级蒸馏 二级蒸馏 三级蒸馏
蒸发器温度(℃) 125-135 150-165 170-180
冷凝面温度(℃) 50-65 65-75 75--85
真空度(Pa) 0.3-0.4 0.3-0.1 0.1-0.06
进料速度(kg/h) 16-18 16-18 16-18
刮板转速(rpm) 165 145 125
经过三级短程分子蒸馏系统的处理后，收集重相得到高纯度长链多不饱和脂肪酸。
(5)取步骤(4)得到的多不饱和脂肪酸20kg，加入2.8Kg的甘油，再加入3Kg的Novozym 435酯化酶进行酯化反应，保持反应温度在45℃，并以140rpm的速度搅拌，反应40小时后，加入4Kg65℃的热水洗涤，然后静置2小时分离过量的甘油与水的混合物，得到甘油三酯型多不饱和脂肪酸。
实施例2
(1)取100Kg藻油来源的DHA毛油(DHA含量45％)，置于水解釜中，加入70Kg的水，在45℃的温度、20MPa的压力下均质处理10分钟。按100单位/克油脂的比例用固定化圆弧青酶脂肪酶设置填充床，将均质好的原料加入填充床，用氢氧化钾水溶液调节PH值至4.8，抽负压到-0.09MPa后充入氮气至釜内压力0.05-0.09MPa。在35℃的温度下水解72小时，在水解过程进行4.5小时后在线自动添加10％氢氧化钾水溶液，将水解体系的PH值维持在6-6.5。72小时后结束水解反应，得到含游离脂肪酸钠和甘油的混合物。
(2)将步骤(1)得到的混合物置用氮气压送到暂存罐中，用蝶式过滤机过滤回收脂肪酶，并得到游离脂肪酸钾盐和甘油的混合物，在暂存罐中充氮气的条件下，静置4-6小时，分离去掉甘油和水， 得到脂肪酸钠。
(3)将步骤(2)得到的脂肪酸钠用10％的硫酸溶液酸化，静置2-3小时后分离去掉水得到游离脂肪酸；
(4)将步骤(3)得到的游离脂肪酸加热到80-90℃，并抽真空至-0.09MPa后，脱水30-50分钟；开启三级短程分子蒸馏系统，三级分子蒸馏设备的工作参数如表2所示：
表2 实施例2分子蒸馏参数表
  一级分子蒸馏系统 二级分子蒸馏系统 三级分子蒸馏系统
蒸发器温度，℃ 120-128 130-155 160-170
冷凝面温度，℃ 45-55 55-65 65--75
真空度，Pa 0.3-0.4 0.3-0.1 0.1-0.06
进料速度,kg/h 15-17 15-17 15-17
刮板转速,rpm 155 135 110
经过三级短程分子蒸馏系统的处理后，收集重相得到高纯度长链多不饱和脂肪酸。
(5)取步骤(4)得到的多不饱和脂肪酸20kg，加入2Kg的甘油，再加入1Kg的Novozym435酯化酶进行酯化反应，保持反应温度在35℃，并以100rpm的速度搅拌，反应50小时后，加入4Kg 65℃的热水洗涤，然后静置2小时分离过量的甘油与水的混合物，得到甘油三酯型多不饱和脂肪酸。
实施例3
(1)取100Kg高山被饱霉来源的ARA精炼油(ARA含量46％)，置于水解釜中，加入130Kg的水，在65℃的温度、100MPa的压力下均质处理10分钟，按500单位/克油脂的比例加入黑曲霉脂肪酶(Aspergillus niger lipase)，用氢氧化钠水溶液调节PH值至4.8，原料进入水解釜后抽负压到-0.09MPa后充入氮气至釜内压力0.05-0.09MPa。在65℃的温度下水解8小时，在水解过程进行4.5小时后在线自动添加15％氢氧化钠水溶液，将水解体系的PH值维持在7-7.5。8小时后结束水解反应，得到含游离脂肪酸钠和甘油的混合物。
(2)将步骤(1)得到的混合物置用氮气压送到暂存罐中，用蝶式过滤机过滤回收脂肪酶，并得到游离脂肪酸钠盐和甘油的混合物，在暂存罐中充氮气的条件下，静置4-6小时，分离去掉甘油，得到脂肪酸钠。
(3)将步骤(2)得到的脂肪酸钠用10％的硫酸溶液酸化，静置2-3小时后分离去掉水得到游离脂肪酸；
(4)将步骤(3)得到的游离脂肪酸加热到80-90℃，并抽真空至-0.09MPa后，脱水30-50分钟；开启三级短程分子蒸馏系统，分别将三级分子蒸馏系统的工作参数如表3所示：
表3 实施例3分子蒸馏参数表
  一级分子蒸馏系统 二级分子蒸馏系统 三级分子蒸馏系统
蒸发器温度，℃ 130-140 155-170 175-185
冷凝面温度，℃ 50-65 65-75 75--85
真空度，Pa 0.3-0.4 0.3-0.1 0.1-0.06
进料速度,kg/h 18-20 18-20 18-20
刮板转速,rpm 180 160 135
经过三级短程分子蒸馏系统的处理后，收集重相得到高纯度长链多不饱和脂肪酸。
(5)取步骤(4)得到的多不饱和脂肪酸20kg，加入3.6Kg的甘油，再加入4Kg的Lipozyme RM M进行酯化反应，保持反应温度在55℃，并以180rpm的速度搅拌，反应30小时后，加入4Kg 65℃的热水洗涤，然后静置2小时分离过量的甘油与水的混合物，得到甘油三酯型多不饱和脂肪酸。
对照例
(1)取100Kg藻油来源的DHA精炼油(DHA含量43％)，置于水解釜中，加入100Kg的水，在55℃的温度、60MPa的压力下均质处理10分钟，按400单位/克油脂的比例加入米黑根毛霉脂肪酶(RML)这一特异性酶,用氢氧化钠水溶液调节PH值，原料进入水解釜后抽负压到-0.09MPa后充入氮气至釜内压力0.05-0.09MPa。在50℃的温度下水解82小时，在水解过程中使用在线添加12％氢氧化钠水溶液将水解体系的PH值维持在6.8-7。82小时后结束水解反应，得到含游离脂肪酸钠和甘油的混合物。
(2)将步骤(1)得到的混合物置用氮气压送到暂存罐中，用蝶式过滤机过滤回收脂肪酶，并得到游离脂肪酸钠盐和甘油的混合物，在暂存罐中充氮气的条件下，静置4-6小时，分离并去掉甘油和水，得到脂肪酸钠。
(3)将步骤(2)得到的脂肪酸钠用10％的硫酸溶液酸化，静置2-3小时后分离去掉水得到游离脂肪酸；
(4)将步骤(3)得到的游离脂肪酸加热到80-90℃，并抽真空至-0.09MPa，真空脱水30-50分钟；然后按照表4所示的参数缓慢降温，对游离脂肪酸进行冬化处理，使得饱和脂肪酸结晶析出，然后用板框压滤器过滤去掉结晶的饱和脂肪酸，得到多不饱和脂肪酸。
表4 游离脂肪酸冬化参数表
步骤 温度(℃) 时间(min) 转速(Hz) 降温幅度 维持时间(h)
1 70 30 15 0 0.5
2 60 30 15 10 0.5
3 50 30 15 10 0.5
4 44 60 15 6 1
5 41 60 15 3 1
6 38 60 15 3 1
7 35 60 15 3 1
8 32 90 15 3 1.5
9 28 90 15 3 1.5
10 25 60 15 3 1
11 21 120 15 4 2
12 19 60 15 2 1
13 17 60 15 2 1
14 16 60 15 1 1
15 15 60 15 1 1
16 14 60 15 1 1
17 13 60 15 1 1
18 12.8 60 15 0.2 1
19 12.6 60 15 0.2 1
20 13 60 15 -0.4 1
21 14 60 15 -1 1
22 12 60 15 2 1
23 10 60 15 2 1
24 8 60 15 2 1
25 6 60 15 2 1
26 6 60 15 0 20
经过冬化处理后，收集滤液得到高纯度长链多不饱和脂肪酸。
(5)取步骤(4)得到的多不饱和脂肪酸20kg，加入2.3Kg的甘油，再加入3.5Kg的米黑根毛霉脂肪酶(RML)进行酯化反应，保持反应温度在45℃，并以140rpm的速度搅拌，反应40小时后，加入4Kg 65℃的热水洗涤，然后静置2小时分离过量的甘油与水的混合物，得到甘油三酯型多不饱和脂肪酸。
检测各实施例和对照例得到的多不饱和脂肪酸中的DHA或ARA含量、甘油一酯、甘油二酯、甘油三酯的含量。结果如表5所示。
表5 各实施例及对照例得到的多不饱和脂肪酸成分检测结果
实施例编号 DHA/ARA(％) 甘油一酯(％) 甘油二酯(％) 甘油三酯(％)
实施例1 87.1 8.5 6.2 85.3
实施例2 85.8 11.3 6.6 82.1
实施例3 84.3 14.3 6.9 78.8
对照例 62.3 17.9 11.3 70.8
通过表1的结果可以看出，采用本发明的方法的实施例1-3所得到的甘油三酯型多不饱和脂肪酸中的DHA/ARA的含量要明显多于采用对照例的方法所得到的甘油三酯型多不饱和脂肪酸中的DHA/ARA含量。而且，相比于对照例，采用实施例1-3的方法所得到的多不饱和脂肪酸中的甘油三脂型多不饱和脂肪酸的占比更高，甘油一脂型多不饱和脂肪酸或甘油二脂型多不饱和脂肪酸的占比较低。同时，采用本发明的方法的实施例1-3，甘油三酯型多不饱和脂肪酸的生产时间更短，效率更高。本发明的甘油三酯型多不饱和脂肪酸的制备方法具有更高的经济价值。所得到的甘油三酯型多不饱和脂肪酸，多不饱和脂肪酸的纯度更高，具有更高的药用和食用价值。
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 (10)

  1. 一种甘油三酯型多不饱和脂肪酸的制备方法，其特征在于，包括以下步骤：
    (1)将含多不饱和脂肪酸的原料油脂与水混合后进行均质处理，经脂肪酶催化水解，得到游离脂肪酸盐、甘油；
    (2)将步骤(1)得到的脂肪酸盐与甘油分离，得到脂肪酸盐；
    (3)将步骤(2)得到的所述脂肪酸盐进行酸化处理得到游离脂肪酸；
    (4)将步骤(3)得到的游离脂肪酸干燥后，进行分子蒸馏处理，去除短链的脂肪酸，富集得到长链的多不饱和脂肪酸；
    (5)将步骤(4)得到的所述多不饱和脂肪酸与甘油混合，经脂肪酶催化进行酯化反应，得到所述甘油三酯型多不饱和脂肪酸。
  2. 根据权利要求1所述的方法，其特征在于，在步骤(1)中，所述原料油脂与水的质量比为1:0.7-1.3；所述均质处理的温度为45-65℃、压力为20-100MPa；和/或
    使用10-15％的氢氧化钠水溶液或氢氧化钾水溶液调控脂肪酶催化水解体系的PH为6-7.5。
  3. 根据权利要求1所述的方法，其特征在于，在步骤(1)和步骤(5)中，所述脂肪酶为非特异性脂肪酶，优选为圆弧青酶脂肪酶、假丝酵母脂肪酶、黑曲霉脂肪酶、诺维信脂肪酶435、Lipozyme RM M和柱状假丝酵母脂肪酶中的一种或多种。
  4. 根据权利要求1所述的方法，其特征在于，在步骤(1)和步骤(5)中，所述脂肪酶催化的方法为将固定化脂肪酶直接加入水解釜中或将固定化脂肪酶制作成填充床进行催化水解，优选采用串联或并联的填充床进行催化反应。
  5. 根据权利要求1所述的方法，其特征在于，在步骤(1)中，所述脂肪酶催化水解的加酶量为每克原料油脂添加100-500单位脂肪酶、水解温度为35-65℃、水解时间为8-72小时；在步骤(5)中，所述甘油的添加量为多不饱和脂肪酸重量的10-18％，所述酯化反应的加酶量为游离脂肪酸量的5-20％、反应温度为35-55℃、搅拌速度为100-180rpm、反应时间为30-50小时。
  6. 根据权利要求1所述的方法，其特征在于，步骤(4)中，所述分子蒸馏处理的压力小于0.5Pa、温度为120-185℃、进料速度为15-20千克/小时、刮膜转速110-180rpm。
  7. 根据权利要求1至6中任一所述的方法，其特征在于，所述制备的过程均在隔氧状态下进行。
  8. 根据权利要求1至6中任一所述的方法，其特征在于，所述原料油脂为DHA藻油和/或ARA藻油。
  9. 一种甘油三酯型多不饱和脂肪酸，其特征在于：根据权利要求1至8中任一项所述的方法制得；其中，甘油三酯型DHA或甘油三酯型ARA含量不低于72％；且甘油一酯含量不高于15％，甘油二酯含量不高于10％，甘油三酯含量不低于75％。
  10. 权利要求9所述的甘油三酯型多不饱和脂肪酸在制备婴幼儿配方食品、健康食品、保健食品或医药用品中的应用。
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