WO2017084241A1 - 一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法 - Google Patents

一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法 Download PDF

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WO2017084241A1
WO2017084241A1 PCT/CN2016/079818 CN2016079818W WO2017084241A1 WO 2017084241 A1 WO2017084241 A1 WO 2017084241A1 CN 2016079818 W CN2016079818 W CN 2016079818W WO 2017084241 A1 WO2017084241 A1 WO 2017084241A1
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lipase
raw material
palmitic acid
subcritical
preparing
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PCT/CN2016/079818
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French (fr)
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黄凤洪
郑明明
万楚筠
汤虎
郭萍梅
时杰
邓乾春
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中国农业科学院油料作物研究所
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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

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  • the invention relates to a food additive-breast milk fat substitute, in particular to a preparation method of 1,3-dioleic acid-2-palmitic acid triglyceride, belonging to the field of food additive and structural grease preparation.
  • OPO 1,3-Dioleic acid-2-palmitic acid triglyceride
  • OPO is a nutritional fortifier approved by the Ministry of Health of China in 2008. It is mainly used in infant formula and is already commercialized human milk. Fat substitute products such as betapol45TM and InfatTM are important components. OPO is similar to human milk fat structure. Because of its unique fatty acid structure, it is difficult to form calcium soap and cause constipation when participating in digestion. It not only helps to promote the absorption of calcium and other minerals by infants and young children, but also enhances the bone density of infants and young children. It can also improve the absorption capacity of infants and young children. In the national standard (opinion draft), OPO is defined as the lipase-catalyzed transesterification of edible vegetable oil as raw material and oleic acid (from edible vegetable oil).
  • the synthesis method is not complicated in principle and is synthesized by enzyme catalysis. That is, using Sn-1, a specific lipase catalyzes the oily raw material rich in palmitic acid (C16:0) at the Sn-2 position of the glycerol skeleton, and the acyl donor (free fatty acid and fatty acid ester) A transesterification reaction product is produced.
  • the most representative Betapol TM product is a mimicking the fatty acid composition and distribution of breast milk.
  • Patent 201110139857.8 discloses a method for preparing OPO structural grease by using lard as a raw material enzymatic method
  • Patent 201310264723.8 discloses a method for enriching OPO structural fat in lard by solvent extraction
  • Patent 201110022672.9 discloses a chemical method
  • OPO structural grease is prepared by a combination of enzymatic methods, but the steps are cumbersome.
  • the technical problem to be solved by the present invention is to provide a method for preparing 1,3-dioleic acid-2-palmitic acid triglyceride by enzymatic method in the subcritical system in view of the above-mentioned deficiencies of the prior art, for the first time in a subcritical system.
  • the use of lipase to catalyze the preparation of OPO structural lipids effectively increases the reaction rate and conversion rate without causing damage to lipase; the enzymatic reaction has high selectivity and is not prone to solvent residues.
  • a method for enzymatically preparing 1,3-dioleic acid-2-palmitic acid triglyceride in a subcritical system comprising the steps of:
  • Subcritical enzyme catalyzed transesterification in a subcritical system, under the catalysis of lipase, a selective enzymatic transesterification reaction between starting material A and starting material B in a reaction solvent yields 1,3-dioleic acid- 2-palmitic acid triglyceride crude product.
  • the step 2) further comprises the step 3), wherein the step 3) is the separation and purification of the crude product, and the raw material B remaining in the crude enzyme in the crude product is removed to obtain the refined 1,3-two.
  • Oleic acid-2-palmitic acid triglyceride product In the purified 1,3-dioleate-2-palmitic acid triglyceride product, the 1,3-dioleic acid-2-palmitic acid triglyceride content is 41% or more.
  • the method for preparing the lard having a cholesterol content of less than 0.05% is as follows: the lard is solvent-extracted to remove most of the cholesterol, and the low-cholesterol lard raw material is obtained.
  • a more specific method is: heating the lard to 50-70 ° C, adding a 70-80% aqueous solution of ethanol to extract, stirring, and then removing the lower aqueous phase, and repeating the extraction process by taking the oil phase portion, wherein
  • the amount of the aqueous ethanol solution added is preferably from 30 to 40% by weight based on the mass of the lard.
  • the sl ⁇ 2 palmitic acid-rich recombinant palm oil refers to a recombinant palm oil having a mass content of palmitic acid of not less than 60% at the sn-2 position, and a chemical random transesterification by 52 degree palm oil stearin.
  • the reaction is obtained.
  • the catalyst is sodium methoxide or sodium ethoxide
  • the catalyst addition amount is 1-2% of the mass of 52 degree palm oil stearin
  • the reaction temperature is 80-110 ° C
  • the reaction time is 0.5-1 h.
  • the oleic acid is a product obtained by hydrolysis of oleic acid-rich vegetable oil
  • the oleic acid ester is a product obtained by enriching methyl ester/ethyl esterification of oleic acid vegetable oil.
  • the oleic acid-rich vegetable oil is a vegetable oil having an oleic acid content of more than 60%, and one or more selected from the group consisting of canola oil, olive oil, tea seed oil, and high oleic sunflower oil. Mixtures in any ratio.
  • the mass ratio of the raw material A to the raw material B is 1: (1 to 3), and the lipase is 3% to 10% of the total mass of the raw material A and the raw material B.
  • the temperature of the subcritical system is 40 to 60 ° C
  • the pressure is 0.5 to 4 MPa
  • the reaction time is 1 to 4 hours.
  • the reaction solvent of the subcritical system is selected from a mixture of one or more solvents of propane, butane, R134a, R600a, DME and sulfur hexafluoride in any ratio.
  • the reaction solvent is added in an amount of from 2 to 5 times the total volume of the raw materials.
  • the lipase is a 1,3 specific lipase selected from Candida antarctica lipase, Rhizopus lipase, Mucor lipase, Candida lipolytica lipase, and A mixture of one or more of Thermosporum lipase, Antarctic Candida lipase, Aspergillus sp. lipase, or Rhizomucor miehei lipase in any ratio.
  • the lipase comprises a lipase in a free form and an immobilized form, preferably a lipase in an immobilized form.
  • the carrier for immobilizing the enzyme is selected from one or a mixture of a macroporous resin, silica gel, diatomaceous earth, attapulgite, or the like, in any ratio.
  • the means for separating in the step 3) is suction filtration to remove the catalyst lipase; the means for purifying is distillation or extraction to remove the raw material B remaining in the crude product. More specifically, the temperature of the suction filtration is 50 to 60 ° C, and the number of times is preferably 2 to 4 times. More specifically, the distillation is a short-range molecular distillation with a degree of vacuum of 1 to 40 Pa and an evaporation temperature of 180 to 220 °C.
  • the present invention utilizes lipase to catalyze the preparation of OPO structural grease in a subcritical system for the first time.
  • the subcritical fluid acts as a reaction medium, which can reduce the stagnation resistance, has strong permeability, has good solubility to the reaction raw materials, and has less enzyme dosage, which is beneficial to Reducing the diffusion restriction of substrate-enzyme contact in the enzyme-catalyzed reaction, improving the enzymatic reaction kinetics to increase the reaction rate and conversion rate;
  • the invention adopts a subcritical reaction system to make the preparation process at a lower temperature, which is beneficial to protecting the activity of the enzyme, and the subcritical fluid is mostly a non-polar solvent, and does not cause damage to the lipase;
  • the enzymatic reaction has high selectivity and low acyl transfer rate;
  • the preparation method of the present invention is carried out in a subcritical reaction system, and the solvent can be separated and recovered from the product conveniently and completely by pressure change, and solvent residue is not easily generated; and the subcritical equipment pressure is low. It can greatly reduce the input and operation cost of disposable equipment, and has good production continuity, which should be suitable for modern large-scale continuous production.
  • the immobilized form of lipase may be commercially available Lipozyme RM IM, Lipozyme TL IM, etc., or may be prepared by itself, and the preparation method comprises the following steps:
  • the technical solution of the present invention can be realized by any device capable of realizing a subcritical system, and the embodiments 1 to 11 employ the device disclosed in Patent No. 200910034263.3.
  • the specific operation process is as follows: 1) The reaction tank is heated to the set reaction temperature and the temperature is stabilized by the circulating water bath; 2) the raw material and the immobilized lipase are weighed in an autoclave, and after the system is sealed, the solvent storage tank is opened, and the solvent gas is opened. After liquefaction through the condenser, pressurize into the reaction kettle, pressurize to the set different pressure, after reaching the selected pressure, stop the plunger pump, close the storage tank, and then close the stirring reaction to the set time, then open the valve. The pressure is then turned on to recover the solvent from the separation vessel, the lipase is recovered from the reaction tank, and the crude product is recovered from the separation tank.
  • a method for enzymatically preparing 1,3-dioleic acid-2-palmitic acid triglyceride in a subcritical state comprising the following steps:
  • Raw material B selects oleic acid, which is derived from high oleic rapeseed oil hydrolysis
  • the crude product obtained in the step (2) is subjected to hot filtration filtration to recover the lipase to obtain a lipase filter cake and a liquid crude product, and then the liquid crude product is repeatedly filtered through a lipase filter cake at 50 ° C. Then, the product after suction filtration is obtained; the product after filtration is subjected to molecular distillation at a temperature of 180 ° C and a vacuum of 1 Pa to remove excess oleic acid in the crude product to obtain purified 1,3-dioleic acid-2.
  • Palmitic acid triglyceride product yield 88%, purity greater than 41%.
  • a method for enzymatically preparing 1,3-dioleic acid-2-palmitic acid triglyceride in a subcritical state comprising the following steps:
  • Raw material B selects oleic acid and is derived from the hydrolysis of olive oil
  • the crude product obtained in the step (2) is subjected to hot filtration filtration to recover the lipase to obtain a lipase filter cake and a liquid crude product, and then the liquid crude product is repeatedly filtered through a lipase filter cake at 50 ° C. Then, the product after suction filtration is obtained; the product after filtration is subjected to molecular distillation at a temperature of 190 ° C and a vacuum of 10 Pa to remove excess oleic acid in the crude product to obtain purified 1,3-dioleic acid-2. a palmitic acid triglyceride product with a yield of 89% and a purity greater than 42%.
  • a method for enzymatically preparing 1,3-dioleic acid-2-palmitic acid triglyceride in a subcritical state comprising the following steps:
  • Raw material B selects oleic acid and is derived from hydrolysis of tea seed oil
  • the crude product obtained in the step (2) is subjected to hot filtration to recover the lipase to obtain a lipase filter cake and a liquid crude product, and then the liquid crude product is repeatedly filtered through a lipase filter cake at 60 ° C. Then, the product after suction filtration is obtained; the product after filtration is subjected to molecular distillation at a temperature of 200 ° C and a vacuum of 20 Pa to remove excess oleic acid in the crude product to obtain purified 1,3-dioleic acid-2.
  • Palmitic acid triglyceride product yield 86%, purity greater than 45%.
  • a method for enzymatically preparing 1,3-dioleic acid-2-palmitic acid triglyceride in a subcritical state comprising the following steps:
  • Raw material B selects oleic acid, which is derived from high oleic sunflower oil hydrolysis
  • the crude product obtained in the step (2) is subjected to hot filtration filtration to recover the lipase to obtain a lipase filter cake and a liquid crude product, and then the liquid crude product is repeatedly filtered through a lipase filter cake at 50 ° C. Then, the product after suction filtration is obtained; the product after filtration is subjected to molecular distillation at a temperature of 220 ° C and a vacuum of 40 Pa to remove excess oleic acid in the crude product to obtain purified 1,3-dioleic acid-2.
  • - Palmitic acid triglyceride product yield 95%, purity greater than 45%.
  • Example 2 It is basically the same as in Example 1, except that in the step (1), the raw material oleic acid is replaced by oleic acid derived from hydrolysis of peanut oil.
  • a purified 1,3-dioleic acid-2-palmitic acid triglyceride product having a yield of 90% and a purity of more than 41% can be obtained.
  • Example 2 It is basically the same as Example 1, except that in the step (1), the raw material oleic acid is replaced by methyl oleate.
  • a purified 1,3-dioleic acid-2-palmitic acid triglyceride product having a yield of 92% and a purity of more than 41% can be obtained.
  • Example 2 It is basically the same as Example 1, except that in the step (1), the raw material oleic acid is replaced by ethyl oleate.
  • a purified 1,3-dioleic acid-2-palmitic acid triglyceride product having a yield of 88% and a purity of more than 41% can be obtained.
  • step (2) the diatomaceous earth immobilized Candida lipolytic enzyme is replaced by diatomaceous earth immobilized Rhizopus lipase, and propane is replaced by dimethyl ether, which can be refined.
  • the 1,3-dioleic acid-2-palmitic acid triglyceride product has a yield of 89% and a purity of more than 41%.
  • step (2) the diatomaceous earth immobilized Candida lipolytic enzyme is replaced by diatomaceous earth immobilized Mucor lipase, and propane is replaced by liquefied petroleum gas, which can be refined.
  • the 1,3-dioleic acid-2-palmitic acid triglyceride product has a yield of 93% and a purity of more than 41%.
  • step (2) the diatomaceous earth immobilized Candida lipolytic lipase is replaced by diatomaceous earth immobilized Aspergillus sp. lipase, and propane is replaced by sulfur hexafluoride.
  • a purified 1,3-dioleic acid-2-palmitic acid triglyceride product having a yield of 92% and a purity of more than 41% can be obtained.
  • step (2) the diatomaceous earth immobilized Candida lipolytic enzyme is replaced by the free type Rhizomucor miehei lipase, and the propane is replaced by sulfur hexafluoride, which can be refined.
  • the 1,3-dioleic acid-2-palmitic acid triglyceride product has a yield of 91% and a purity of more than 41%.
  • the method for preparing 1,3-dioleic acid-2-palmitic acid triglyceride by the enzymatic method in the subcritical state of the present invention has a reaction time of 1 to 4 hours, and the reaction is compared with the prior art.
  • the rate is greatly improved, and the amount of the catalyst lipase is only 3% to 10% of the total mass of the raw material, and the yield of the target product 1,3-dioleic acid-2-palmitic acid triglyceride can reach 85%.
  • the purity can reach 41% or more.

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Abstract

提供了一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，包括如下步骤：(1)原料准备：选取胆固醇质量含量低于0.05％的猪油作为原料A，或者选取sn-2棕榈酸质量含量不低于60％的重组棕榈油作为原料A；选取油酸、油酸酯中的至少一种作为原料B；(2)亚临界酶催化酯交换反应：在亚临界体系中，在脂肪酶的催化下，原料A与原料B在反应溶剂中发生选择性酶促酯交换反应，得到1,3-二油酸-2-棕榈酸甘油三酯。该方法反应速率快，选择性高，酶用量少，无溶剂残留，易于大规模连续性生产。

Description

一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法 技术领域
本发明涉及一种食品添加剂-母乳脂肪代用品，尤其涉及1,3-二油酸-2-棕榈酸甘油三酯的制备方法，属于食品添加剂与结构脂制备领域。
背景技术
1,3-二油酸-2-棕榈酸甘油三酯(以下简称OPO)是2008年我国卫生部第13号公告批准的营养强化剂，主要用于婴幼儿配方食品，是已经商业化人乳脂肪替代品产品如betapol45TM及InfatTM中的重要组分。OPO与人乳脂肪结构相似，因其特有的脂肪酸结构组成，在参与消化时不易形成钙皂而产生便秘，不仅有助于促进婴幼儿对钙等矿物质的吸收，增强婴幼儿的骨骼密度，还能提高婴幼儿对营养素的吸收能力。国标(意见稿)中对OPO定义为以食用植物油为原料与油酸(来自于食用植物油)经脂肪酶催化酯交换制得。
对于酶法合成人乳脂替代品的研究，国内外已有很多，并且在国外的市场上已经有了比较成熟的商业化的产品，其合成方法在原理上并不复杂，采用酶催化的方法合成，即采用Sn-1,3位专一性脂肪酶催化甘油骨架的Sn-2位上含有丰富的棕榈酸(C16:0)的油脂原料，与酰基供体(游离的脂肪酸和脂肪酸酯)发生酯交换反应合成产品。最具代表性的Betapol TM产品，是模仿母乳的脂肪酸组成和分布，将富含Sn-2位棕榈酸的植物油与富含油酸的向日葵油混合，经专一性脂肪酶的催化作用而研制的一种植物油脂混合物，是营养价值及棕榈酸含量与位置分布接近母乳脂肪的新型脂肪。专利201110139857.8公开了一种采用猪油为原料酶法制取OPO结构脂的方法；专利201310264723.8公开了一种采用溶剂分提富集猪油中OPO结构脂的方法；专利201110022672.9公开了一种化学法和酶法相结合的方法制取OPO结构脂，但是步骤比较繁琐。
以往的研究多在无溶剂条件下进行，由于传滞阻力的限制，反应速度较慢，反应时间较长，酶用量大，并且容易出现酰基转移，目标产物产率不高；而采用正己烷、异辛烷等长链烷烃做溶剂时，反应完毕后难以去除干净，容易产生溶剂残留。而且，已报道的1,3-选择性脂肪酶主要有Lipozyme RMIM(诺维信公司)、RhizopusDelamar等，脂肪酶高昂的价格和缺乏高效酶促反应体系是导致制备OPO的成本居高不下的主要原因，已经成为困扰OPO规模化生产的瓶颈问题。
发明内容
本发明所要解决的技术问题是针对上述现有技术存在的不足而提供一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，首次在亚临界体系中利用脂肪酶催化制备OPO结构脂，有效提高反应速率及转化率，不会对脂肪酶产生破坏作用；酶促反应选择性高，且不易产生溶剂残留。
本发明为解决上述提出的问题所采用的技术方案为：
一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，它包括如下步骤：
1)原料准备：选取胆固醇质量含量低于0.05％的猪油作为原料A，或者选取sn-2位富含棕榈酸的重组棕榈油作为原料A；选取油酸、油酸酯中的至少一种作为原料B；
2)亚临界酶催化酯交换反应：在亚临界体系中，在脂肪酶的催化下，原料A与原料B在反应溶剂中发生选择性酶促酯交换反应，得到1,3-二油酸-2-棕榈酸甘油三酯粗产品。
按上述方案，所述步骤2)后续还包括步骤3)，该步骤3)为粗产品的分离和纯化，将粗产品中的脂肪酶残留的原料B除去，以得到精制的1,3-二油酸-2-棕榈酸甘油三酯产品。该精制的1,3-二油酸-2-棕榈酸甘油三酯产品中，1,3-二油酸-2-棕榈酸甘油三酯含量在41％以上。
按上述方案，所述胆固醇质量含量低于0.05％的猪油的制备方法如下：猪油采用溶剂萃取除去大部分胆固醇，获得低胆固醇含量的猪油原料。更具体的方法为：将猪油加热至50～70℃，加入体积浓度为70～80％的乙醇水溶液进行萃取，搅拌后静置去除下层水相，取油相部分再重复该萃取过程，其中乙醇水溶液的加入量优选为猪油质量的30～40wt％。
按上述方案，所述sn～2位富含棕榈酸的重组棕榈油是指sn-2位棕榈酸质量含量不低于60％的重组棕榈油，由52度棕榈油硬脂通过化学随机酯交换反应得到的。其中，所述的化学随机酯交换反应中，催化剂为甲醇钠或乙醇钠，催化剂添加量为52度棕榈油硬脂质量的1～2％，反应温度80～110℃，反应时间0.5～1h。
按上述方案，所述油酸为富含油酸植物油水解得到的产物；油酸酯为富含油酸植物油甲酯/乙酯化反应得到的产物。其中，所述富含油酸植物油为油酸含量大于60％的植物油，选自低芥酸菜籽油、橄榄油、茶籽油、高油酸葵花籽油等中的一种或多种按任意比例的混合物。
按上述方案，所述步骤2)中，原料A与原料B的质量比为1：(1～3)，脂肪酶为原料A与原料B总质量的3％～10％。
按上述方案，所述步骤2)中，亚临界体系的温度为40～60℃，压力为0.5～4MPa，反应时间为1～4h。
按上述方案，所述步骤2)中，亚临界体系的反应溶剂选自丙烷、丁烷、R134a、R600a、DME和六氟化硫等中的一种或多种溶剂按任意比例的混合物。优选地，反应溶剂的加入量为原料总体积的2～5倍。
按上述方案，所述步骤2)中，脂肪酶为1，3特异性脂肪酶，选自南极假丝酵母脂肪酶、根霉脂肪酶、毛霉脂肪酶、解脂假丝酵母脂肪酶、嗜热丝孢菌脂肪酶、南极洲念珠菌属脂肪酶、黑曲霉菌属脂肪酶或米黑根毛霉脂肪酶等中的一种或几种按任意比例的混合物。
按上述方案，所述脂肪酶包括游离型和固定化形式的脂肪酶，优选固定化形式的脂肪酶。其中，固定化酶的载体选自大孔树脂、硅胶、硅藻土、凹凸棒土等中的一种或几种按任意比例的混合物。
按上述方案，所述步骤3)中分离的手段为抽滤，以除去催化剂脂肪酶；提纯的手段为蒸馏或者萃取，以除去粗产品中残留的原料B。更具体地，所述抽滤的温度为50～60℃，次数优选2～4次。更具体地，所述蒸馏为短程分子蒸馏，真空度1～40Pa，蒸发温度180～220℃。
与现有技术相比，本发明的有益效果是：
1、本发明首次在亚临界体系中利用脂肪酶催化制备OPO结构脂，亚临界流体作为反应介质，可以降低传滞阻力，渗透性强，对反应原料溶解度好，酶用量少，加有利于降低酶催化反应中底物与酶接触的扩散限制，改善酶促反应动力学提高反应速率及转化率；
2、本发明通过采用亚临界反应体系中，使制备过程在较低的温度下进行，有利于保护酶的活力，而且亚临界流体多为非极性溶剂，不会对脂肪酶产生破坏作用；酶促反应选择性高，酰基转移率低；
3、本发明所述制备方法在亚临界反应体系中进行，通过压力改变可方便且较为完全地将溶剂从产物中分离并回收再利用，且不易产生溶剂残留；并且，亚临界设备压力较低，可大大降低一次性设备投入及运行费用，生产连续性好，应能适于现代化大规模连续性生产。
具体实施方式
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实施例。
本发明中，所述固定化形式的脂肪酶，简称固定化脂肪酶，可以是市售的Lipozyme RM IM，Lipozyme TL IM等，也可以自行制备，其制备方法包含以下几个步骤：
1)载体的活化：用1mol/L盐酸溶液浸泡除去载体中金属离子等杂质，然后纯水清洗至中性；2)吸附固定化：配制缓冲溶液，将游离脂肪酶溶解到缓冲溶液中，然后加入硅胶、树 脂、硅藻土等载体，抽真空搅拌吸附0.5～4h，抽滤收集固定化脂肪酶粗品；3)洗涤干燥：将固定化脂肪酶粗品用缓冲溶液清洗3次后，冷冻干燥或冷风干燥即可得到固定化脂肪酶产品。
采用任何能够实现亚临界体系的装置均可实现本发明的技术方案，实施例1～11采用的是专利200910034263.3公开的装置。具体操作过程如下：1)反应罐升温至设定反应温度并通过循环水浴保持温度稳定；2)称取原料和固定化脂肪酶置于高压釜中，体系密闭后，打开溶剂储罐，溶剂气体经冷凝器液化后经加压进入反应釜，加压至设定的不同压力，达到选定的压力后，停止柱塞泵，关闭储罐，然后密闭搅拌反应至设定时间后，打开阀门减压，继而打开压缩机从分离釜回收溶剂，从反应罐中回收脂肪酶，从分离罐中回收粗产品。
实施例1
一种亚临界状态下酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，包括如下步骤：
(1)原料A的制备：采用52度棕榈油硬脂为原料，甲醇钠为催化剂，甲醇钠添加量为52度棕榈油硬脂质量的1％，于反应温度80℃反应1h，然后采用柠檬酸水溶液除去甲醇钠，旋转蒸发除去水分，即可得到sn-2位棕榈酸质量含量不低于60％的重组棕榈油，即原料A；
原料B选择油酸，来源于高油酸菜籽油水解；
(2)亚临界酶催化酯交换反应：将500g原料A、500g原料B、30g硅藻土固定化解脂假丝酵母脂肪酶加入高压反应釜中，加入5L液化的丙烷溶剂，在亚临界丙烷体系中，在40℃、压力0.5MPa的条件下，搅拌反应4h，反应完毕后打开阀门将产物转入分离釜，通过压缩机和阀门控制分离，从分离釜回收反应溶剂丙烷，从反应罐中回收脂肪酶，从分离罐中回收1,3-二油酸-2-棕榈酸甘油三酯粗产品；
(3)产品的分离纯化：步骤(2)得到的粗产品趁热抽滤回收脂肪酶得到脂肪酶滤饼和液态粗品，然后将液态粗品在50℃下再经过脂肪酶滤饼反复抽滤2次，得到抽滤后的产物；抽滤后产物在温度180℃、真空度1Pa的条件下进行采用分子蒸馏，除去粗产品中过量的油酸，得到精制的1,3-二油酸-2-棕榈酸甘油三酯产品，产率为88％，纯度大于41％。
实施例2
一种亚临界状态下酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，包括如下步骤：
(1)原料A的制备：采用52度棕榈油硬脂为原料，乙醇钠为催化剂，乙醇钠添加量为52度棕榈油硬脂质量的2％，于反应温度100℃反应0.5h，然后采用柠檬酸水溶液除去乙醇钠，旋转蒸发除去水分，即可得到sn-2位棕榈酸质量含量不低于60％的重组棕榈油，即原 料A；
原料B选择油酸，来源于橄榄油水解；
(2)亚临界酶催化酯交换反应：将500g原料A、1000g原料B、75g硅胶固定化南极假丝酵母脂肪酶(原料总质量的5％)加入高压反应釜中，加入4.5L液化的丁烷溶剂，在亚临界丁烷体系中，在50℃、压力1.4MPa的条件下，搅拌反应3h，反应完毕后打开阀门将产物转入分离釜，通过压缩机和阀门控制分离，从分离釜回收反应溶剂丙烷，从反应罐中回收脂肪酶，从分离罐中回收1,3-二油酸-2-棕榈酸甘油三酯粗产品；
(3)产品的分离纯化：步骤(2)得到的粗产品趁热抽滤回收脂肪酶得到脂肪酶滤饼和液态粗品，然后将液态粗品在50℃下再经过脂肪酶滤饼反复抽滤2次，得到抽滤后的产物；抽滤后产物在温度190℃、真空度10pa的条件下进行采用分子蒸馏，除去粗产品中过量的油酸，得到精制的1,3-二油酸-2-棕榈酸甘油三酯产品，产率为89％，纯度大于42％。
实施例3
一种亚临界状态下酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，包括如下步骤：
(1)原料A的制备：采用52度棕榈油硬脂为原料，乙醇钠为催化剂，乙醇钠添加量为52度棕榈油硬脂质量的2％，于反应温度110℃反应0.5h，然后采用柠檬酸水溶液除去乙醇钠，旋转蒸发除去水分，即可得到sn-2位富含棕榈酸的重组棕榈油，即原料A；
原料B选择油酸，来源于茶籽油水解；
(2)亚临界酶催化酯交换反应：将500g原料A、1500g原料B、160g大孔树脂固定化嗜热丝孢菌脂肪酶(原料总质量的8％)加入高压反应釜中，加入4L液化的R134a溶剂，在亚临界R134a体系中，在60℃、压力4MPa的条件下，搅拌反应1h，反应完毕后打开阀门将产物转入分离釜，通过压缩机和阀门控制分离，从分离釜回收反应溶剂丙烷，从反应罐中回收脂肪酶，从分离罐中回收1,3-二油酸-2-棕榈酸甘油三酯粗产品；
(3)产品的分离纯化：步骤(2)得到的粗产品趁热抽滤回收脂肪酶得到脂肪酶滤饼和液态粗品，然后将液态粗品在60℃下再经过脂肪酶滤饼反复抽滤3次，得到抽滤后的产物；抽滤后产物在温度200℃、真空度20pa的条件下进行采用分子蒸馏，除去粗产品中过量的油酸，得到精制的1,3-二油酸-2-棕榈酸甘油三酯产品，产率为86％，纯度大于45％。
实施例4
一种亚临界状态下酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，包括如下步骤：
(1)原料A的制备：猪油为原料，将猪油加热至50℃，加入体积浓度为80％的乙醇水溶液进行萃取，搅拌后静置去除下层水相，取油相部分再重复上述过程萃取1次，旋蒸除去溶 剂乙醇，即可得到低胆固醇的猪油，即原料A；其中乙醇水溶液的加入量为猪油质量的40wt％；
原料B选择油酸，来源于高油酸葵花籽油水解；
(2)亚临界酶催化酯交换反应：将500g原料A、500g原料B、100g凹凸棒土固定化嗜热丝孢菌脂肪酶(原料总质量的10％)加入高压反应釜中，加入5L液化的丁烷溶剂，在亚临界丁烷体系中，在60℃、压力1.5MPa的条件下，搅拌反应1h，反应完毕后打开阀门将产物转入分离釜，通过压缩机和阀门控制分离，从分离釜回收反应溶剂丙烷，从反应罐中回收脂肪酶，从分离罐中回收1,3-二油酸-2-棕榈酸甘油三酯粗产品；
(3)产品的分离纯化：步骤(2)得到的粗产品趁热抽滤回收脂肪酶得到脂肪酶滤饼和液态粗品，然后将液态粗品在50℃下再经过脂肪酶滤饼反复抽滤2次，得到抽滤后的产物；抽滤后产物在温度220℃、真空度40pa的条件下进行采用分子蒸馏，除去粗产品中过量的油酸，得到精制的1,3-二油酸-2-棕榈酸甘油三酯产品，产率为95％，纯度大于45％。
实施例5
与实施例1基本相同，不同之处在于步骤(1)中：原料油酸由来源于花生油水解的油酸替代。可得到精制1,3-二油酸-2-棕榈酸甘油三酯产品，产率为90％，纯度大于41％。
实施例6
与实施例1基本相同，不同之处在于步骤(1)中：原料油酸由油酸甲酯替代。可得到精制1,3-二油酸-2-棕榈酸甘油三酯产品，产率为92％，纯度大于41％。
实施例7
与实施例1基本相同，不同之处在于步骤(1)中：原料油酸由油酸乙酯替代。可得到精制1,3-二油酸-2-棕榈酸甘油三酯产品，产率为88％，纯度大于41％。
实施例8
与实施例1基本相同，不同之处在于步骤(2)中：硅藻土固定化解脂假丝酵母脂肪酶由硅藻土固定化根霉脂肪酶替代，丙烷由二甲醚替代，可得到精制1,3-二油酸-2-棕榈酸甘油三酯产品，产率为89％，纯度大于41％。
实施例9
与实施例1基本相同，不同之处在于步骤(2)中：硅藻土固定化解脂假丝酵母脂肪酶由硅藻土固定化毛霉脂肪酶替代，丙烷由液化石油气替代，可得到精制1,3-二油酸-2-棕榈酸甘油三酯产品，产率为93％，纯度大于41％。
实施例10
与实施例1基本相同，不同之处在于步骤(2)中：硅藻土固定化解脂假丝酵母脂肪酶由硅藻土固定化黑曲霉菌属脂肪酶替代，丙烷由六氟化硫替代，可得到精制1,3-二油酸-2-棕榈酸甘油三酯产品，产率为92％，纯度大于41％。
实施例11
与实施例1基本相同，不同之处在于步骤(2)中：硅藻土固定化解脂假丝酵母脂肪酶由游离型米黑根毛霉脂肪酶替代，丙烷由六氟化硫替代，可得到精制1,3-二油酸-2-棕榈酸甘油三酯产品，产率为91％，纯度大于41％。
综上所述，本发明所述亚临界状态下酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，反应时间在1～4h范围内，相对于现有技术，反应速率有了很大提高，而催化剂脂肪酶的用量仅为原料总质量的3％～10％，所得目标产物1,3-二油酸-2-棕榈酸甘油三酯的产率可以达到85％以上，纯度可以达到41％以上。
以上所述仅是本发明的优选实施方式，应当指出，对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干改进和变换，这些都属于本发明的保护范围。

Claims (10)

  1. 一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，其特征在于它包括如下步骤：
    1)原料准备：选取胆固醇质量含量低于0.05％的猪油作为原料A，或者选取sn-2棕榈酸质量含量不低于60％的重组棕榈油作为原料A；选取油酸、油酸酯中的至少一种作为原料B；
    2)亚临界酶催化酯交换反应：在亚临界体系中，在脂肪酶的催化下，原料A与原料B在反应溶剂中发生选择性酶促酯交换反应，得到1,3-二油酸-2-棕榈酸甘油三酯粗产品。
  2. 根据权利要求1所述的一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，其特征在于所述步骤2)后续还包括步骤3)，该步骤3)为粗产品的分离和纯化，将粗产品中的脂肪酶残留的原料B除去，以得到质量含量在41％以上的1,3-二油酸-2-棕榈酸甘油三酯产品。
  3. 根据权利要求2所述的一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，其特征在于所述步骤3)中分离的手段为抽滤，以除去催化剂脂肪酶；提纯的手段为蒸馏或者萃取，以除去粗产品中残留的原料B。
  4. 根据权利要求3所述的一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，其特征在于所述抽滤的温度为50～60℃，次数为2～4次；所述蒸馏为短程分子蒸馏，真空度1～40Pa，蒸发温度180～200℃。
  5. 根据权利要求1所述的一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，其特征在于所述步骤2)中，原料A与原料B的质量比为1：(1～3)，脂肪酶为原料A与原料B总质量的3％～10％。
  6. 根据权利要求1所述的一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，其特征在于所述步骤2)中，亚临界体系的温度为40～60℃，压力为0.5～4MPa，反应时间为1～4h。
  7. 根据权利要求1所述的一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，其特征在于所述步骤2)中，亚临界体系的反应溶剂选自丙烷、丁烷、R134a、R600a、DME和六氟化硫中的一种或多种溶剂按任意比例的混合物。
  8. 根据权利要求1所述的一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，其特征在于所述步骤2)中，脂肪酶为1，3-特异性脂肪酶，选自南极假丝酵母脂肪酶、根霉脂肪酶、毛霉脂肪酶、解脂假丝酵母脂肪酶、嗜热丝孢菌脂肪酶、南极洲念珠菌属脂肪酶、黑曲霉菌属脂肪酶或米黑根毛霉脂肪酶中的一种或几种按任意比例的混合物。
  9. 根据权利要求1所述的一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方 法，其特征在于所述脂肪酶包括游离型和固定化形式的脂肪酶。
  10. 根据权利要求9所述的一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法，其特征在于所述固定化形式的脂肪酶的载体选自大孔树脂、硅胶、硅藻土、凹凸棒土中的一种或几种按任意比例的混合物。
PCT/CN2016/079818 2015-11-20 2016-04-21 一种亚临界体系中酶法制备1,3-二油酸-2-棕榈酸甘油三酯的方法 WO2017084241A1 (zh)

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