阅读说明：本技术 一种在反胶束酶体系中合成中长链甘油三酯的方法 (Method for synthesizing medium-long chain triglyceride in reverse micelle enzyme system ) 是由 邓泽元 张兵 陈孙妮 李静 李红艳 于 2019-11-11 设计创作，主要内容包括：本发明利用脂肪酶界面激活特性,发明了一种在反胶束酶体系中合成中长链甘油三酯的方法,具体合成方法如下：首先构建反胶束酶体系,即在2-乙基己基琥珀酸酯磺酸钠(AOT)的异辛烷溶液中加入非固定化水溶性Sn-1,3专一性脂肪酶缓冲水溶液(50 mmol/mL磷酸二氢钠-磷酸氢二钠,pH 6.5~8),聚能超声获得澄清反胶束酶体系,其中[酶](mg/mL):[水](mmol/mL):[AOT](mmol/mL)=3~6:200~600:20~30,再将0.3~0.5 g/(mL反应体系)的高中链脂肪酸含量油与高长链脂肪酸含量油以质量比1:1~1.3混合加入反胶束酶体系中,在酶载量为总底物的0.5~3%、反应温度为40~55°C的条件下反应2~4 h,过程中辅以发散超声作用,反应结束后加入2倍反应液体积的水,震荡分层,4200 rpm离心5 min,取上层油相,通过二次分子蒸馏纯化,经HPLC-MS检测中长链三酯的含量为74.39~92.40%。本发明具备高效率高产率生产中长链甘油三酯的优点。(The invention discloses a method for synthesizing medium-long chain triglyceride in a reverse micelle enzyme system by utilizing the interfacial activation characteristic of lipase, which comprises the following steps: firstly, a reverse micelle enzyme system is constructed, namely, a non-immobilized water-soluble Sn-1, 3-specific lipase buffer solution (50 mmol/mL sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.5-8) is added into an isooctane solution of 2-ethylhexyl succinate sodium sulfonate (AOT), a clear reverse micelle enzyme system is obtained by energy-gathering ultrasound, wherein [ enzyme ] (mg/mL): water ] (mmol/mL): AOT ] (mmol/mL) = 3-6: 200-600: 20-30, then high-medium-chain fatty acid content oil and high-long-chain fatty acid content oil of 0.3-0.5 g/(mL reaction system) are mixed according to the mass ratio of 1: 1-1.3 and added into the reverse micelle enzyme system, the reaction is carried out for 2-4 hours under the conditions that the enzyme loading amount is 0.5-3 percent of the total substrate and the reaction temperature is 40-55 ℃, and the ultrasonic dispersion effect is assisted in the process, and after the reaction is finished, adding water with the volume 2 times that of the reaction solution, shaking for layering, centrifuging at 4200rpm for 5min, taking an upper oil phase, purifying by secondary molecular distillation, and detecting the content of the long-chain triester in the mixture by HPLC-MS to be 74.39-92.40%. The method has the advantage of producing medium-long chain triglyceride with high efficiency and high yield.)

1. A method for synthesizing medium-long chain triglyceride in a reverse micelle enzyme system is characterized in that:

(1) mixing a lipase buffer aqueous solution and a surfactant solution, and forming a uniform reverse micelle enzyme system by energy-gathered ultrasound;

(2) adding a reaction substrate into the system, and reacting under the action of divergent ultrasound to obtain medium-long chain triglyceride;

(3) the product was purified using molecular distillation techniques.

2. The use according to claim 1, wherein the surfactant solution constituting the reverse micellar enzyme system consists of sodium 2-ethylhexyl sulfosuccinate (AOT) and isooctane, the lipase is a non-immobilized water-soluble Sn-1, 3-specific lipase, the buffered aqueous solution is 50 mmol/mL sodium dihydrogen phosphate-disodium hydrogen Phosphate (PBS), the reaction substrates are medium-chain fatty acid content oil and high-chain fatty acid content oil, respectively, and the target end product is medium-chain triglyceride.

3. The application of claim 1, wherein the reverse micelle enzyme system is constructed by homogenizing an AOT isooctane solution and a non-immobilized water-soluble Sn-1, 3-specific lipase buffer aqueous solution (50 mmol/mL PBS, pH 6.5-8), wherein [ enzyme ] (mg/mL): water ] (mmol/mL): [ AOT ] (mmol/mL) = 3-8: 200-600: 20-30, by means of energy-focused ultrasound, wherein the ultrasonic temperature is 40-55 ℃, the total ultrasonic time is 35-56 s, and the ultrasonic mode is '5 s-2 s stop ultrasonic'.

4. The application of claim 1, wherein the esterification reaction step comprises the steps of mixing 0.3-0.5 g/(mL reaction system) of medium-chain fatty acid content oil and high-chain fatty acid content oil in a mass ratio of 1: 1-1.3, adding into a reverse micelle enzyme system, reacting for 2-4 h under the conditions that the enzyme loading is 0.5-3% of total substrates and the reaction temperature is 40-55 ℃, assisting in the process of ultrasonic dispersion, the ultrasonic power is 30-40 kHz, the power is 200-400W, the ultrasonic mode is 'ultrasonic 10 s-20 s', adding water with 2 times of the volume of reaction liquid after the reaction is finished, vibrating for layering, centrifuging at 4200rpm for 5min, and taking the upper oil phase to obtain the product mixture.

5. The use according to claim 1, wherein the molecular distillation purification is a secondary distillation purification, first, a first molecular distillation is performed under the conditions of a feeding rate of 1 to 2mL/min, a heating temperature of 80 to 90 ℃, a scraper rotation speed of 150 to 250r/min, and a vacuum degree of 0.5 Pa, a first light phase such as free fatty acid is removed, then a second molecular distillation is performed on a first heavy phase under the conditions of a feeding rate of 1 to 2mL/min, a heating temperature of 170 to 180 ℃, a scraper rotation speed of 150 to 250r/min, and a vacuum degree of 0.5 Pa, mono-and diglycerides are removed, and the content of medium-long chain triglycerides in the second heavy phase is 74.39 to 92.40% by HPLC-MS detection.

6. Use according to claim 1 or 2, characterized in that medium-long chain triglycerides can be produced with high efficiency and high yield.

Technical Field

The invention belongs to the field of grain and oil processing, and particularly relates to a method for synthesizing medium-long chain triglyceride in a reverse micelle enzyme system.

Background

The structural lipid is the lipid with special nutritional efficacy or physiological function obtained by carrying out structural recombination or modification on natural lipid, the specific function of the structural lipid is changed along with the difference of the connected fatty acid, and the structural lipid mainly has the functions of promoting the absorption of other lipids, enhancing the immunologic function, preventing and resisting cancers and the like. In recent years, the medium-long chain triglyceride has attracted much attention because of its excellent lipid-lowering and weight-reducing functions, and studies have shown that when 14 g of the medium-long chain triglyceride is added daily to breakfast of 82 healthy adults, the decrease in specific gravity is 4.6% and 2% after 12 weeks, both in subcutaneous and visceral fat, especially in waist and hip circumferences.

The preparation of medium-long chain triglyceride is mainly realized by a transesterification method under the action of a catalyst, and the substrate is generally oil rich in medium-chain fatty acid and oil rich in long-chain fatty acid, and the method can be roughly divided into a chemical method and an enzymatic method according to the difference of the catalyst.

The chemical method is used for catalyzing grease to perform random ester exchange reaction under the action of catalysts such as metal acetate or sodium alkylate and the like, is the most traditional and earliest method, has the advantages of low cost and mature process, but is limited in partial fields due to the defects of high toxicity, high reaction temperature, poor product selectivity and the like of the chemical catalyst. The enzyme method is to catalyze the grease to generate random ester exchange reaction under the action of a lipase biocatalyst, the non-specific lipase can synthesize 6 kinds of medium-long chain triglyceride with random structure, and the specific lipase can selectively catalyze and synthesize the specific medium-long chain triglyceride so as to meet the requirements of different fields, so the enzyme method becomes a main method for synthesizing the structural lipid. In fact, since the first enzymatically synthesized breast-milk-like structured lipid oleic acid-saturated fatty acid-oleic acid triglyceride product is available, hundreds of enzymatically synthesized structured lipids are on the market. Common lipases, e.g.RhizomucormieheiA lipase,AspergillusoryzaeA lipase,PenicilliumexpansumLipases, etc., have been demonstrated to have excellent ability to catalyze the synthesis of structural lipids, e.g., while the enzymatic reaction media are extensive, in aqueous solutions, organicEffective esterification can be carried out in a solvent, a supercritical CO2 fluid, and the like.

However, as the research goes into, the enzymatic synthesis of long-chain triglyceride also has exposed more problems, the most important of which is low lipase catalytic activity, and more lipase or longer reaction time is often needed to achieve high yield. The active site of lipase has a 'cover' structure, which is the main reason of low activity, and on the oil-water interface, the cover structure is displaced to expose the active site, and the enzyme activity is increased, which is the interface activation effect of lipase. The immobilization of lipase utilizes the interfacial activation effect of lipase, and the active enzyme is immobilized by a carrier, thereby improving the catalytic activity. On the other hand, if a stable oil-water interface is provided as the esterification reaction site, it is also possible to improve the esterification efficiency. The reverse micelle is a spontaneously formed nano-scale aggregate, is a transparent and thermodynamically stable W/O system, and is formed by a surfactant with the concentration exceeding the critical micelle concentration, wherein polar groups of the surfactant are inwards arranged to form a polar nucleus, and when the polar nucleus dissolves enzyme liquid, the system forms a reverse micelle enzyme system. The reverse micelle enzyme system has a larger oil-water interface, and due to the auxiliary action of the surfactant, the interface stability is good, the lipase is activated by the interface in the system, and the lipase is dissolved in a polar nucleus and then is diffused in a molecular level manner, so that the diffusion resistance is reduced, the contact chance with a substrate is increased, and the product yield can be improved by the advantages, so that the system has a good application prospect in the aspect of esterification synthesis.

The existing preparation method of the reverse micelle enzyme system is simple, and only the surfactant solution and the enzyme solution are mixed and fully vibrated to be clear, but the reverse micelle system prepared by the method has the problems of uneven droplet size, poor stability and the like. On the one hand, the poor stability makes the system unusable for a long time; on the other hand, the relationship between the size of droplets in reverse micelles and the size of lipase is one of the important factors determining the activity of lipase in reverse micelle systems, so that the improvement of enzyme activity is incomplete and the reproducibility of results is poor due to the non-uniformity of droplet sizes. The ultrasonic wave is a sound wave with the frequency higher than 20 kHz, the mechanical effect of the ultrasonic wave can promote the emulsification of liquid, the liquefaction of gel and the dispersion of solid, the research shows that the emulsion prepared by the ultrasonic wave assistance has higher stability and uniform liquid drops, and the ultrasonic wave can have similar influence on a reverse micelle enzyme system considering that the reverse micelle system is a system similar to the emulsion. In addition, the cavitation of the ultrasonic wave can also play a role in stirring, so that the substrate is fully contacted with the enzyme, and the yield is finally improved.

Therefore, the method applies the reverse micelle system to the reaction of synthesizing the medium-long chain triglyceride by lipase catalysis, and synthesizes a high-yield product in a short time by assisting the ultrasonic action in the processes of preparing the reverse micelle system and synthesizing reactants, thereby having the advantage of producing the medium-long chain triglyceride with high efficiency and high yield.

Disclosure of Invention

The invention discloses a method for synthesizing medium-long chain triglyceride in a reverse micelle enzyme system, which is used for overcoming the defects of the prior art in synthesizing the medium-long chain triglyceride, wherein the reverse micelle enzyme system is formed by mixing a lipase buffer aqueous solution and a surfactant solution, the surfactant solution consists of 2-ethylhexyl sodium succinate sulfonate (AOT) and isooctane, the lipase is non-immobilized water-soluble Sn-1, 3-specific lipase, the buffer aqueous solution is 50 mmol/mL sodium dihydrogen phosphate-disodium hydrogen Phosphate (PBS), reaction substrates are medium-chain fatty acid content oil and high-long chain fatty acid content oil respectively, and a target final product is the medium-long chain triglyceride.

The invention comprises the following steps:

(1) construction of homogeneous reverse micelle enzyme system

Adding a non-immobilized water-soluble Sn-1, 3-specific lipase buffer aqueous solution (50 mol/mLPBS, pH 6.5-8) into an AOT isooctane solution, wherein [ enzyme ] (mg/mL ], [ water ] (mmol/mL): [ AOT ] (mmol/mL) = 3-8: 200-600: 20-30, homogenizing by energy-focused ultrasound, wherein the energy-focused ultrasound frequency is 20-30 kHz, the power is 200-400W, the ultrasound temperature is 40-55 ℃, the total ultrasound time is 35-56 s, and the ultrasound mode is 'ultrasound 5 s-stop 2 s'.

(2) Synthesis of long chain triglycerides in reverse micellar enzyme systems

Mixing 0.3-0.5 g/(mL reaction system) of medium-chain fatty acid content oil and high-long-chain fatty acid content oil in a mass ratio of 1: 1-1.3, adding the mixture into a reverse micelle enzyme system, reacting for 2-4 hours under the conditions that the enzyme loading is 0.5-3% of total substrates and the reaction temperature is 40-55 ℃, assisting in the process of ultrasonic dispersion, the ultrasonic power is 30-40 kHz, the power is 200-400W, the ultrasonic mode is 'ultrasonic 10 s-20 s', adding 2 times of reaction liquid volume of water after the reaction is finished, vibrating and layering, centrifuging at 4200rpm for 5min, and taking an upper oil phase to obtain a product mixture.

(3) Purification of the product by molecular distillation

Firstly, performing primary molecular distillation under the conditions of a feeding rate of 1-2 mL/min, a heating temperature of 80-90 ℃, a scraper rotating speed of 150-250 r/min and a vacuum degree of 0.5 Pa to remove primary light phases such as free fatty acid, then performing secondary molecular distillation on a primary heavy phase, removing monoglyceride and diglyceride under the conditions of a feeding rate of 1-2 mL/min, a heating temperature of 170-180 ℃, a scraper rotating speed of 150-250 r/min and a vacuum degree of 0.5 Pa, and detecting the content of medium-long chain triglyceride in the secondary heavy phase to be 74.39-92.40% by HPLC-MS.

The invention has the beneficial effects that: (1) the water/AOT/isooctane reverse micelle enzyme system containing lipase is prepared by energy-gathering ultrasound assistance, and then the medium-long chain triglyceride is catalyzed and synthesized by the enzymatic catalysis under the assistance of divergent ultrasound assistance, so that the method has the advantages of mild generation conditions, high esterification rate and high yield. (2) The product is purified by adopting a molecular distillation method, the operation is simple, the purity is high, and the product can be further used for animal experiments after being prepared in large scale, so that the fat-reducing and weight-losing kinetic energy of the product can be researched, and the influence of the product on human cardiovascular diseases can be further researched. (3) The method for synthesizing the medium-long chain triglyceride is simple, is easy to operate, and can be suitable for synthesizing various structural lipids.

Detailed Description