CN113684126B - Device and method for continuously synthesizing diglyceride by holoenzyme method in multi-liquid-phase system - Google Patents

Device and method for continuously synthesizing diglyceride by holoenzyme method in multi-liquid-phase system Download PDF

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CN113684126B
CN113684126B CN202111244712.4A CN202111244712A CN113684126B CN 113684126 B CN113684126 B CN 113684126B CN 202111244712 A CN202111244712 A CN 202111244712A CN 113684126 B CN113684126 B CN 113684126B
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李志刚
杨嘉威
杨博
王永华
陈华勇
王卫飞
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South China University of Technology SCUT
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Abstract

The invention belongs to the technical field of bioengineering and food, and discloses a device and a method for synthesizing diglyceride by a multi-liquid-phase system continuous holoenzyme method. The reaction zone and the standing zone and the reaction zone and the dispersion buffer zone are separated by a baffle, and the baffle is provided with a plurality of small holes communicated with each zone; the standing area is provided with an upper liquid phase area, a middle liquid phase area and a lower liquid phase area, the upper liquid phase area is provided with a hydrolysate liquid outlet, the liquid outlet is communicated with a sample inlet of the esterification device, fatty acid is directly introduced to carry out enzymatic synthesis of diglyceride, the middle liquid phase area is communicated with the reaction area through a return pipe, and the lower liquid phase area is provided with a lower solution sample inlet; the lower part of the reaction zone is provided with an enzyme-containing solution sample inlet, and a stirrer is arranged inside the reaction zone; the bottom of the dispersing buffer zone is provided with an oil material sample inlet and a lower phase solution outlet. The method has the advantages of high raw material repeated utilization rate, mild and controllable reaction conditions, continuous production and the like.

Description

Device and method for continuously synthesizing diglyceride by holoenzyme method in multi-liquid-phase system
Technical Field
The invention belongs to the technical field of bioengineering and food, relates to the fields of grease industry and grease chemical industry, and particularly relates to a device and a method for synthesizing diglyceride by a continuous holoenzyme method.
Background
Diglycerides are the products of esterification of two hydroxyl groups on glycerol with two fatty acids, and have two isomers, 1,2-DAG and 1, 3-DAG. The content of diglyceride in natural animal and vegetable oil is very small, for example, olive oil contains 5.5% of diglyceride, the preparation is mainly completed by an oil modification technology, and the preparation mainly comprises a chemical method and an enzymatic method, wherein the chemical method has the defects of complex process, energy consumption, high cost and the like, and the enzymatic method is concerned due to the characteristics of high efficiency, mildness, specificity, environmental friendliness and the like. The enzyme method mainly comprises a grease hydrolysis method, a direct esterification method and a glycerolysis method. The method for preparing diglyceride by the glycerolysis method has certain advantages, such as lower cost and less byproducts. However, the system has a large amount of glycerol, which increases the viscosity of the system, thus affecting the catalysis of the enzyme, and has the problems of complicated steps, high requirements on reaction conditions and the enzyme, generally low product yield and the like. The direct esterification method has the advantages of only one step of reaction, easy operation, high purity of the product and short reaction time, but the reaction takes high-purity free fatty acid as a raw material, and the preparation cost of the raw material is high. The hydrolysis method for preparing diglyceride has the advantages of wide raw material source, easy acquisition and the like, but a large amount of by-product free fatty acid can be generated by single-step hydrolysis reaction, so the yield of diglyceride is lower and is generally only about 30 percent. Furthermore, the above three methods are all single-step preparation of diglycerides, and separation of triglycerides and diglycerides remaining in the product without completion of the reaction is difficult, and it is difficult to completely separate them even at a high temperature of 230 ℃ by molecular distillation, and it is difficult to directly apply them to industrial production. Therefore, in recent years, attention has been paid to a method for producing diglycerides by a multienzyme method. The method is generally a hydrolysis combined esterification method, and has the advantages of high purity, less separation steps, less impurities and the like. However, it is still a long distance from industrial applications. On the one hand, when triglyceride is hydrolyzed into fatty acid in a traditional oil-water system, the oil-water system is easy to emulsify and can be separated only by heating to 40-70 ℃ and then high-speed centrifugation, and the oil-water system is easy to emulsify and is easy to split phase unevenly, so that a large amount of products and enzyme are lost; on the other hand, in the conventional oil-water system, a large amount of glycerin is produced, and an excessive amount of glycerin inhibits the forward progress of the hydrolysis reaction. Meanwhile, in the reaction process, the change of pH value can cause the inactivation of enzyme, and the reaction can be saponified under the alkaline condition. These reasons have led to the fact that most of the research is currently in the laboratory stage and continuous application is extremely difficult.
In the patent applications disclosed so far, most of the processes for producing diglycerides are batch-type reaction apparatuses, and the purpose of industrial continuous production cannot be achieved. For example, patent CN201310199809.7 discloses a method for preparing diglyceride, which uses free lipase to prepare diglyceride by batch enzymolysis. The adoption of the batch method for preparing diglyceride has the disadvantages of complex operation, difficult recycling of enzyme, complex product separation steps, high production energy consumption and high cost, and can not realize continuous large-scale production. Further, for example, patent CN201210191389.3 discloses a method for producing fatty acid diglyceride and a dedicated reaction zone, which are capable of continuously producing diglyceride, but require multi-stage molecular distillation, have a complicated process flow, are prone to side reactions, and are difficult to control the product inhibition and stabilize the pH of the system, so that it is difficult to efficiently produce diglyceride.
In the process of earlier research on food oil, the subject group discovers a novel multi-liquid-phase system which can improve the catalytic efficiency of enzyme, efficiently recycles an enzyme-rich phase and can continuously carry out multiple times of enzymatic hydrolysis reaction. However, the process is easily affected by equipment such as a pump during the reaction, small droplets of oil are uniformly suspended in water to form an emulsion, which affects the separation, and the inhibition effect of glycerol and the pH value of the reaction system are uncontrollable, so that the process is always difficult to be continuously operated. Therefore, it is necessary to develop an apparatus and a method for synthesizing diglyceride, which are efficient, controllable, continuously catalytic and separable.
Disclosure of Invention
The invention aims to provide a device and a method for continuously synthesizing diglyceride by a holoenzyme method in a multi-liquid-phase system, aiming at the problems of high cost, complex reaction operation, difficult continuous production and the like in the existing industrialized process of preparing diglyceride from oil materials.
The purpose of the invention is realized by the following technical scheme:
a device for continuously synthesizing diglyceride by a holoenzyme method in a multi-liquid-phase system comprises a hydrolysis device and an esterification device; the hydrolysis device is divided into a standing zone, a reaction zone and a dispersing buffer zone from top to bottom, the reaction zone and the standing zone and the reaction zone and the dispersing buffer zone are separated by a baffle, and the baffle is provided with a plurality of small holes communicated with the zones; the standing area is provided with an upper liquid phase area, a middle liquid phase area and a lower liquid phase area, the upper liquid phase area is provided with a hydrolysate liquid outlet, the liquid outlet is communicated with a sample inlet of the esterification device, hydrolyzed fatty acid can be directly introduced into the esterification device to carry out enzymatic esterification reaction to synthesize diglyceride, the middle liquid phase area is communicated with the reaction area through a return pipe, and the lower liquid phase area is provided with a lower phase solution sample inlet; the lower part of the reaction zone is provided with an enzyme-containing solution sample inlet, and a stirrer is arranged inside the reaction zone; the bottom of the dispersing buffer zone is provided with an oil material sample inlet and a lower phase solution outlet.
Preferably, a circulating water heating sleeve is arranged outside the reaction zone.
Preferably, the lower liquid phase zone of the standing zone is provided with an automatic potentiometric titrator burette, and the reaction zone is provided with an automatic potentiometric titrator electrode tip.
The esterification apparatus of the present invention may be a conventional option; preferably, the esterification device comprises a reaction kettle and a stirrer; the outside of the reaction kettle is provided with a circulating water heating sleeve, the lower part of the reaction kettle is provided with a hydrolyzed oil material sample inlet and an esterification substrate sample inlet, and the upper part of the reaction kettle is provided with an esterification product outlet.
A method for continuously synthesizing diglyceride by using the device comprises the following steps:
(1) preparing a double-liquid-phase solution from soluble salt, a hydrophilic solvent and water, standing and layering to obtain an upper-phase solution and a lower-phase solution, continuously introducing the lower-phase solution into a standing zone, passing through a reaction zone, and entering and filling a dispersion buffer zone; dissolving lipase or phospholipase in the upper phase solution, introducing the solution into a reaction zone, continuously introducing the oil into a dispersion buffer zone, and adjusting the flow rate of the feed liquid to enable the oil to enter the reaction zone from the dispersion buffer zone to perform enzyme catalytic hydrolysis reaction;
(2) and (3) automatically extracting the fatty acid after the hydrolysis reaction, allowing the fatty acid to enter a standing region, standing for layering, directly introducing the upper phase solution into an esterification device, performing esterification reaction with an esterification reaction substrate, standing for layering the esterification product, and obtaining diglyceride in the upper phase.
Preferably, the lower substrate after esterification, standing and layering in the step (2) is introduced into a reaction kettle of an esterification device to continuously participate in the reaction.
Preferably, the feeding and reaction processes in the step (1) are carried out under the conditions of continuous stirring and heating, the heating temperature is 30-70 ℃, and the stirring speed is 100-1000 rpm; adjusting the pH value of the reaction system to 3-13; the flow rate of the lower phase solution is 0.1-1.0 mL/min.
Preferably, the mass ratio of the soluble salt, the hydrophilic solvent and the water in the step (1) is 0.1-1 and 0.1-5 respectively.
Preferably, the soluble salt is one or more of sodium citrate, sodium chloride, sodium sulfate, ammonium sulfate, sodium carbonate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate.
Preferably, the hydrophilic solvent is a polymer or an ionic liquid, wherein the polymer comprises one or two of polyethylene glycol and polypropylene glycol; the ionic liquid comprises [ BMIM]Br、[BMIM]BF4、[EMIM]ETSO4、[OMIM]Cl、[BMIM]PF6One or more than two of them.
Preferably, the Lipase is Lipase AYS, Lipase AY30, Lipase MAS1, Lipase G Amano 50, Lipase CALB, Novozyme 435, the phospholipase is Lecitase Ultra, PLA2, PLC, PLD one or more, the enzyme concentration is 5-2000U/mL.
Preferably, the oil material in step (1) comprises one or more than two of virgin or refined olive oil, camellia oil, walnut oil and soybean oil.
Preferably, the esterification reaction substrate of step (2) comprises a hydrophobic solvent, a short-chain alcohol, glycerol, an enzyme and water; wherein the hydrophobic solvent is one or more of n-hexane, isopropyl ether, ethyl acetate and isooctane; the short-chain alcohol is one or more of tert-butyl alcohol, ethanol, isopropanol, isoamyl alcohol and n-octanol.
Preferably, the device for the continuous holoenzyme method synthesis of diglyceride by the multi-liquid-phase system further comprises the following technical characteristics:
a. the device is made of one or more than two of glass materials, metal materials, organic polymer materials and ceramic materials;
b. the connecting pipeline is one or more than two of a silicone tube, a fluororubber tube and a stainless steel tube;
c. the stirrer is one of paddle type, toothed sheet type, bent blade opening turbine type, anchor type, frame type, helical belt type, screw type, Brumakin type, folded blade opening turbine type, bent blade disc turbine type, push type and straight blade disc turbine type stirrers;
d. the pump is one or more than two of a displacement pump, a vane pump, a fluid power pump and an electromagnetic pump.
Preferably, the hydrolysis device further comprises the following technical characteristics:
a. the baffle between the reaction zone and the standing zone and the baffle between the reaction zone and the dispersion buffer zone are made of polytetrafluoroethylene materials, the diameters of the baffle and the reaction zone are consistent, and the baffle is provided with a plurality of small holes for blocking reaction liquid and extracting and separating products. Rubber rings with the same size as the inner diameter of the upper, middle and lower part columns are arranged on the two surfaces of the baffle plate so as to achieve the sealing effect;
b. because a small part of the middle phase in the reaction zone can be extracted into the standing zone, the first reflux valve and the second reflux valve are used for refluxing the middle phase in the standing zone into the reaction zone for continuous reaction;
c. the glycerin generated in the hydrolysis reaction process can be enriched in the lower phase rich in soluble salt, and the lower phase solution outlet is used for replacing the lower phase solution to remove the inhibition effect of the glycerin;
d. the enzyme activity is gradually reduced after the long-time reaction, and in order to maintain the reaction efficiency, the enzyme-containing solution injection port is used for the entry of a middle phase and the replacement of the middle phase;
e. the titration tube of the automatic potentiometric titrator is used for adjusting the pH value of the multi-liquid-phase system together with the electrode tip of the automatic potentiometric titrator by dripping acid or alkali into the lower-phase solution.
Preferably, the device for the continuous holoenzyme method synthesis of diglyceride by the multi-liquid-phase system further comprises the following technical characteristics:
a. the first circulating water heating sleeve and the second circulating water heating sleeve are used for controlling the temperature of a reaction system and heating and/or refrigerating the reaction device. Circulating water is pumped in from a first circulating water inlet and flows back to the first circulating water heating pump from a first circulating water outlet after filling the first circulating water heating sleeve;
b. the first stirrer and the second stirrer are used for stirring, so that reactants are more fully and uniformly mixed, and the reaction is facilitated.
Preferably, after the esterification product of the esterification device and substrates such as glycerol, enzyme and the like flowing out are kept standing and layered in the recovery tank, the substrate at the lower layer can be pumped into the esterification substrate storage tank through a pump to continuously participate in the reaction.
Recent research shows that the polymer and ionic liquid multi-liquid phase system has a plurality of advantages in separating products and adjusting the pH value of the lower phase, fatty acid can be distributed to the upper phase only by standing or low-speed centrifugation, enzyme is enriched in the middle phase, and glycerol is distributed to the lower phase, so that the system is particularly suitable for developing a novel continuous device based on the system. The reaction zone of the device enables upper, middle and lower three phases to be fully mixed by controlling the stirring speed, so that the hydrolysis rate of the oil material is accelerated, and the pH value of the hydrolysis reaction can be monitored in real time through an electrode tip of an automatic potentiometric titrator; the dispersing buffer zone prevents the oil from directly contacting with the polymer and the ionic liquid, so that the direct emulsification phenomenon is avoided. The lower phase is further extracted and separated by adopting a countercurrent extraction mode, so that excessive glycerin generated in the hydrolysis process can be brought away, and product inhibition is removed; the standing zone changes the pH value of the lower phase in real time through a burette of an automatic potentiometric titrator so as to adjust the pH value of the system, and the fatty acid can be enriched in the upper phase through standing and layering. And the hydrolyzed fatty acid can be directly introduced into an esterification reaction kettle without other treatment to react and synthesize diglyceride, so that the method has the advantages of simplified process flow, high production efficiency, energy conservation, consumption reduction, repeated enzyme recycling, glycerol inhibition removal, real-time online regulation and control of the pH value of a system and the like, and can be applied to continuous industrial production of diglyceride.
Compared with the prior art, the invention has the beneficial effects that:
the invention overcomes the problems of high cost, complex reaction operation, difficult continuous production, low reaction efficiency, difficult control of product inhibition and system pH value and the like in the conventional continuous preparation process of diglyceride, and provides a device and a method for continuously synthesizing diglyceride by a holoenzyme method in a multi-liquid-phase system. The concrete points are as follows:
(1) the oil material is completely or partially hydrolyzed by a polymer and ionic liquid type multi-liquid-phase system continuous enzyme method, and the hydrolyzed oil material and glycerol are esterified by the continuous enzyme method to synthesize diglyceride, so that the reaction efficiency can be improved by the full enzyme method, and the process flow can be simplified.
(2) Aiming at the difficult regulation and control of the pH value of the system and the product inhibition effect, the countercurrent extraction mode is adopted for further extraction and separation, so that the glycerin is enriched in the lower phase of the system, due to the polarity problem of the three liquid phases, the lower phase flows downwards through the holes on the baffle plate, enters the dispersion buffer zone and is discharged, and the inhibition effect of the reaction negative product can be relieved after the lower phase is replaced. Meanwhile, the pH value of the reaction system is stabilized by online regulating and controlling the pH value of the lower phase by an automatic potentiometric titrator, so that the reaction can be continuously, stably and for a long time.
(3) And a dispersion buffer zone is established, so that the emulsification phenomenon caused by the direct contact of the oil material and the hydrophilic solvent is avoided, and the extraction and separation are facilitated.
(4) The standing area plays a role in standing and layering, fatty acid is enriched in the upper phase, and enzyme is mainly enriched in the middle phase. The enzyme can be recycled by extraction and layering, medium phase reflux and other modes, and the hydrolyzed fatty acid in the upper phase can be directly introduced into an esterification device for reaction, so that the material consumption and the cost are reduced.
(5) The reaction zone is used for intelligently regulating and controlling the stirring speed, so that the condition that the system is not beneficial to extraction layering due to overhigh stirring speed or incomplete mixing due to overlow stirring speed is avoided, three phases are fully mixed, the hydrolysis rate of the grease is increased, and the pH value of the reaction system can be monitored in real time.
(6) The continuous hydrolysis and esterification device has the advantages of less material consumption, low cost and energy conservation and environmental protection by using circulating water for heating. The method has the advantages of low energy consumption, high utilization rate of raw materials, mild and controllable reaction conditions, continuous reaction and the like, and solves the technical problem that the existing method is difficult to continuously synthesize the diglyceride for a long time.
Drawings
FIG. 1 is a schematic structural diagram of an apparatus for synthesizing diglycerides according to the present invention.
In the figure, 1, a reaction zone, 2, a dispersion buffer zone, 3, a standing zone, 4, a first stirrer, 5, a hydrolysate liquid outlet, 6, a first reflux valve, 7, an automatic potentiometric titrator burette, 8, a first circulating water outlet, 9, a second reflux valve, 10, a first baffle, 11, a lower phase solution inlet, 12, a second baffle, 13, an automatic potentiometric titrator electrode tip, 14, a first circulating water heating sleeve, 15, a first circulating water inlet, 16, an enzyme-containing solution inlet, 17, an oil inlet, 18, a lower phase solution outlet, 19, a second stirrer, 20, an esterification device reaction kettle, 21, a second circulating water outlet, 22, an esterification product outlet, 23, a second circulating water heating sleeve, 24, a hydrolyzed oil inlet, 25, a second circulating water inlet, 26, an esterification substrate inlet, 27, a first circulating water heating pump, 28. the system comprises a hydrolyzed oil substrate delivery pump, a 29 circulating water heating pump, a 30 lower phase solution delivery pump, a 31 enzyme-containing solution (middle phase) delivery pump, a 32 oil material delivery pump, a 33 lower phase solution delivery pump, a 34 esterification substrate delivery pump, a 35 esterification substrate recovery delivery pump, a 36 esterification product delivery pump, a 37 first lower phase storage tank, a 38 enzyme-containing solution (middle phase) storage tank, a 39 oil material storage tank, a 40 second lower phase storage tank, a 41 esterification substrate storage tank, a 42 recovery tank.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.
Lipase AYS, Lipase AY30(Candida rugosa lipases), Lipase G Amano 50 used in this example were purchased from Japan Tianye, and Lecitase Ultra was purchased from Beijing Gaoresson.
Example 1
As shown in fig. 1, the device for continuous holoenzyme method synthesis of diglyceride in multi-liquid phase system comprises a hydrolysis device and an esterification device, and has the following specific structure:
the hydrolysis device comprises a reaction zone 1, a dispersing buffer zone 2, a standing zone 3, a first stirrer 4, an automatic potentiometric titrator burette 7, an automatic potentiometric titrator electrode tip 13 and a first circulating water heating sleeve 14. A baffle plate 10 full of a plurality of small holes is arranged between the reaction zone 1 and the dispersion buffer zone 2 of the device, and a baffle plate 12 full of a plurality of small holes is also arranged between the reaction zone 1 and the standing zone 3 of the device. An oil material inlet 17 and a lower phase solution outlet 18 are arranged at the lower part of the dispersion buffer zone 2. The standing area 3 is divided into an upper liquid phase area, a middle liquid phase area and a lower liquid phase area, the upper liquid phase area is provided with a hydrolysate liquid outlet 5, the middle liquid phase area is communicated with the reaction area through a return pipe, a first return valve 6 and a second return valve 9 are arranged on the middle liquid phase area, and the lower liquid phase area is provided with a lower phase solution injection port 11 and an automatic potentiometric titrator burette 7. An enzyme-containing solution sample inlet 16 and an electrode tip 13 of an automatic potentiometric titrator are arranged in the reaction zone 1 of the device, and a first circulating water heating sleeve 14 is arranged in an outer cavity of the reaction zone 1 of the device; the reaction zone 1 is internally provided with a first stirrer 4.
The esterification apparatus comprises an esterification apparatus reaction kettle 20 and a second stirrer 19. A second circulating water heating sleeve 23 is arranged outside the reaction kettle 20 of the esterification device, a hydrolyzed oil material injection port 24 and an esterification substrate injection port 26 are arranged at the bottom, and an esterification product outlet 22 is arranged at the upper part.
The method for continuously synthesizing the diglyceride by using the device comprises the following steps:
step (1): firstly, 3L of aqueous two-phase system is prepared, namely 15 percent of Na by mass2SO415% PEG400 and 70% water were mixed and prepared in a 3L separatory funnel, and the separatory funnel was placed in an incubator at 37 ℃ and left to stand for 24 hours. After obvious layering, impurities in the two-phase solution are removed by respectively passing through 0.45 mu m filter membranes to obtain an upper phase PEG400 and a lower phase Na2SO4And (3) solution.
Step (2): the first circulating water heat pump 27 was turned on, 5g Lipase AYS enzyme was added to 100mL of the upper phase PEG400, and 300mL of the lower phase Na was added2SO4The solution was introduced into the standing zone 3 to fill the dispersion buffer zone 2 under heating and stirring conditions, and then the enzyme-containing PEG400 solution was introduced into the reaction zone 1 at a stirring rate of 500rpm at a circulating water temperature of 40 ℃.
And (3): introducing the refined olive oil from the oil material inlet 17 into the reaction zone 1 of the oil material hydrolyzing device at a flow rate of 1mL/min for enzyme-catalyzed hydrolysis reaction, and simultaneously introducing the lower phase Na at a flow rate of 0.3mL/min2SO4The solution is introduced into the apparatus from the lower solution inlet 11. The pH value of the lower phase is adjusted by an automatic potentiometric titrator, and the pH value of the stabilizing system is kept at 7. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer (respectively corresponding to an upper liquid phase region, a middle liquid phase region and a lower liquid phase region) are sequentially arranged in a standing region from top to bottom, fatty acid hydrolyzed by refined olive oil is mainly enriched in the upper liquid layer, glycerol hydrolyzed is mainly enriched in the lower liquid phase, and the middle liquid layer containing enzyme can flow back to the reaction region again through a reflux valve to continuously participate in the hydrolysis reaction. Finally, the hydrolysis efficiency of triglyceride of the refined olive oil reaches 94% when the refined olive oil is continuously reacted for 5 hours, and the hydrolysis efficiency of the refined olive oil can still reach 80% when the refined olive oil is continuously reacted for 25 hours. The enzyme solution can be supplemented in real time according to the reaction condition.
And (4): 100mL of glycerol was measured, 5G of Lipase G Amano 50 enzyme was added until the enzyme was completely dissolved in glycerol, and 5% (v/v) purified water was added. And (3) introducing the solution into a reaction kettle of an esterification device at the flow rate of 0.5mL/min, and simultaneously directly introducing the liquid layer on the step (3) into the reaction kettle at the flow rate of 1mL/min to perform enzyme-catalyzed esterification reaction, wherein the stirring speed is 500rpm, and the temperature of circulating water is 40 ℃. Standing the effluent product for layering to obtain oil layer rich in diglyceride in the upper phase, and introducing reaction substrates such as glycerol and enzyme in the lower phase into the reaction kettle again. Finally, after 10 hours, the DAG content reaches 46 percent, and the esterification rate reaches 67 percent. The DAG content can still reach 40% at 30h, and the esterification rate reaches 64%. TAG content is 10%, DAG content is greater than 80% if FFA is removed by molecular distillation.
Example 2
The method for continuously synthesizing the diglyceride by using the device in the embodiment 1 comprises the following steps:
step (1): firstly, preparing a 3L aqueous two-phase system, Namely (NH) with the mass fraction of 20%4)2SO420% PEG600 and 60% water are dissolved and mixed evenly in a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ and stands for 24 h. After obvious layering, impurities in the two-phase solution are removed by respectively passing through 0.45 μm filter membrane to obtain upper phase PEG600 and lower phase (NH)4)2SO4And (3) solution.
Step (2): the first circulating water heat pump 27 was turned on and 5g of Lipase AY30 enzyme and 10mL of [ BMIM ] were added to 100mL of the upper phase PEG600]BF4300mL of lower phase Na2SO4The solution was introduced into the standing zone 3 to fill the dispersion buffer zone 2 under heating and stirring conditions, and then the enzyme-containing PEG600 solution was introduced into the reaction zone 1 at a stirring rate of 500rpm at a circulating water temperature of 40 ℃.
And (3): introducing the refined camellia oil from the oil material inlet 17 into the reaction zone 1 of the oil material hydrolyzing device at a flow rate of 0.5mL/min for enzyme-catalyzed hydrolysis reaction, and simultaneously, introducing the lower phase Na at a flow rate of 0.2mL/min2SO4The solution is introduced into the apparatus from the lower solution inlet 11. The pH value of the lower phase is adjusted by an automatic potentiometric titrator, and the pH value of the stabilizing system is kept at 7. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer (respectively corresponding to the upper liquid phase region, the middle liquid phase region and the lower liquid phase region) are sequentially arranged in a standing region from top to bottom, fatty acid hydrolyzed by the refined camellia oil is mainly enriched in the upper liquid layer, glycerol hydrolyzed is mainly enriched in the lower liquid phase, and the middle liquid layer containing the enzyme can be refluxed back to the reaction region through a reflux valve to continuously participate in the hydrolysis reaction. Finally, the hydrolysis efficiency of triglyceride of the refined olive oil reaches 82% in 5h, and the hydrolysis efficiency can still reach 70% after 35 h. The enzyme solution can be supplemented in real time according to the reaction condition.
And (4): 100mL of glycerol is measured, 5G of Lipase G Amano 50 enzyme is added until the enzyme is completely dissolved in the glycerol, 10% (v/v) n-hexane is added, and 5% (v/v) purified water is added. And (3) introducing the solution into a reaction kettle of an esterification device at the flow rate of 0.026mL/min, and simultaneously introducing the liquid layer on the step (3) into the reaction kettle directly at the flow rate of 0.006mL/min to perform enzyme-catalyzed esterification reaction, wherein the stirring speed is 600 rpm, and the circulating water temperature is 40 ℃. Standing the effluent product for layering to obtain oil layer rich in diglyceride in the upper phase, and introducing reaction substrates such as glycerol and enzyme in the lower phase into the reaction kettle again. Finally, after 10 hours, the DAG content reaches 42 percent, and the esterification rate reaches 68 percent. After 40 hours, the DAG content can still be stabilized at 42 percent, and the esterification rate is stabilized at 68 percent. TAG content is 10%, DAG content is greater than 80% if FFA is removed by molecular distillation.
Example 3
The method for continuously synthesizing the diglyceride by using the device in the embodiment 1 comprises the following steps:
step (1): firstly, preparing a 3L aqueous two-phase system, namely 20 percent of PEG600 and 15 percent of Na according to mass fraction2SO4And dissolving 65% of water, mixing uniformly, preparing into a 3L separating funnel, and standing the separating funnel in an incubator at 37 ℃ for 24 hours. After obvious layering, impurities in the two-phase solution are removed by respectively passing through 0.45-micrometer filter membranes to obtain an upper phase PEG600 and a lower phase Na2SO4And (3) solution.
Step (2): the first circulating water heat pump 27 was turned on and 7.5g Lipase TL100L enzyme was added to 150mL of the above phase PEG 600. 300mL of lower phase Na2SO4Introducing the mixture into a standing zone 3 under the conditions of heating and stirring to fill a dispersion buffer zone 2, and introducing a PEG600 solution containing enzyme into a reaction zone 1, wherein the stirring speed is 600 rpm, and the circulating water temperature is 40 ℃.
And (3): introducing the refined olive oil from the oil material inlet 17 into the reaction zone 1 of the oil material hydrolyzing device at a flow rate of 0.5mL/min for enzyme-catalyzed partial hydrolysis reaction, and simultaneously, introducing the lower phase Na at a flow rate of 0.2mL/min2SO4The solution is introduced into the apparatus from the lower solution inlet 11. The pH value of the lower phase is adjusted by an automatic potentiometric titrator, and the pH value of the stabilizing system is kept at 7. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer (respectively corresponding to an upper, middle and lower three-liquid phase region) are sequentially arranged in a standing region from top to bottom, fatty acid, triglyceride, diglyceride and monoglyceride after partial hydrolysis of refined olive oil are mainly enriched in the upper liquid layer, and fatty acid, triglyceride, diglyceride and monoglyceride after hydrolysis are mainly enriched in the upper liquid layerThe glycerol is mainly enriched in the lower liquid phase, and the intermediate liquid layer containing the enzyme can be refluxed back to the reaction zone again to continuously participate in the hydrolysis reaction. Finally, the refined olive oil reached 54% triglyceride hydrolysis efficiency at 3.5h, with a 1,2-DAG content of 26%. After 25h, the hydrolysis efficiency of triglyceride reaches 66%, and the content of 1,2-DAG is 19%. The enzyme solution can be supplemented in real time according to the reaction condition.
And (4): 100mL of glycerol was measured, 5g of Lecitase Ultra enzyme was added, and 5% (v/v) purified water was added after the enzyme was completely dissolved in glycerol. And (3) introducing the solution into a reaction kettle of an esterification device at the flow rate of 0.09mL/min, and simultaneously directly introducing the liquid layer in the step (3) into the reaction kettle at the flow rate of 0.03mL/min to perform enzyme-catalyzed esterification reaction, wherein the stirring speed is 500rpm, and the temperature of circulating water is 40 ℃. Standing the effluent product for layering to obtain oil layer rich in diglyceride in the upper phase, and introducing reaction substrates such as glycerol and enzyme in the lower phase into the reaction kettle again. Finally, after 17h, the DAG content reaches 41 percent, and the esterification rate reaches 40 percent. After 30 hours, the DAG content can be stabilized at 37 percent, and the esterification rate is 30 percent. TAG content is 30%, DAG content is greater than 60% if FFA is removed by molecular distillation.
Example 4
The method for continuously synthesizing the diglyceride by using the device in the embodiment 1 comprises the following steps:
step (1): firstly, preparing a 3L aqueous two-phase system, namely 25 percent of PEG400 and 11 percent of Na according to mass fraction2SO4And dissolving 64% of water, mixing uniformly, preparing into a 3L separating funnel, and standing the separating funnel in an incubator at 37 ℃ for 24 hours. After obvious layering, impurities in the two-phase solution are removed by respectively passing through 0.45 mu m filter membranes to obtain an upper phase PEG400 and a lower phase Na2SO4And (3) solution.
Step (2): the first circulating water heat pump 27 was turned on and 10g of Lipase AYS enzyme and 5g of Lecitase Ultra enzyme were added to 200mL of the upper phase PEG 400. 250mL of lower phase Na2SO4Introducing the mixture into a standing zone 3 under the conditions of heating and stirring to fill a dispersion buffer zone 2, and introducing a PEG400 solution containing enzyme into a reaction zone 1, wherein the stirring speed is 600 rpm, and the circulating water temperature is 40 ℃.
Step (a)3): introducing the virgin olive oil from the oil material inlet 17 into the reaction zone 1 of the oil material hydrolyzing device at a flow rate of 0.3mL/min for double-enzyme combined catalytic hydrolysis and degumming reaction, and simultaneously, adding lower phase Na at a flow rate of 0.1mL/min2SO4The solution is introduced into the apparatus from the lower solution inlet 11. The pH value of the lower phase is adjusted by an automatic potentiometric titrator, and the pH value of the stabilizing system is kept at 7. In the reaction process, an upper liquid layer, a quasi solid layer, a middle liquid layer and a lower liquid layer are sequentially arranged in a standing area from top to bottom, fatty acid hydrolyzed by virgin olive oil is mainly enriched in the upper liquid layer, glycerol hydrolyzed is mainly enriched in the lower liquid layer, removed phospholipid is mainly enriched in the quasi solid layer, and the middle liquid layer containing enzyme can be refluxed back to the reaction area through a reflux valve to continuously participate in the reaction. Finally, the hydrolysis efficiency of triglyceride of the virgin olive oil reaches 85% at 3h, and the removal rate of phospholipid reaches 100%. After 17h, the hydrolysis efficiency of the triglyceride is still stabilized at 85%, and the content of the fatty acid is stabilized at 90%. After 40h, the hydrolysis efficiency of triglyceride is 78%, the fatty acid content is 78%, and the phospholipid removal rate is still stabilized at 100%. The enzyme solution can be supplemented in real time according to the reaction condition.
And (4): 100mL of glycerol is measured, 10G of Lipase G Amano 50 enzyme is added until the enzyme is completely dissolved in the glycerol, 5% (v/v) of tert-butyl alcohol is added, and 5% (v/v) of purified water is added. And (3) introducing the solution into a reaction kettle of an esterification device at the flow rate of 0.3mL/min, and simultaneously directly introducing the liquid layer on the step (3) into the reaction kettle at the flow rate of 0.03mL/min to perform enzyme-catalyzed esterification reaction, wherein the stirring speed is 500rpm, and the temperature of circulating water is 40 ℃. Standing the effluent product for layering to obtain oil layer rich in diglyceride in the upper phase, and introducing reaction substrates such as glycerol and enzyme in the lower phase into the reaction kettle again. Finally, after 40 hours, the DAG content is stabilized at 41 percent, and the esterification rate reaches 67 percent. TAG content was 12%, DAG content was greater than 80% if FFA was removed by molecular distillation.
Example 5
The method for continuously synthesizing the diglyceride by using the device in the embodiment 1 comprises the following steps:
step (1): firstly, 3L of aqueous two-phase system is prepared, namely 15 percent of Na by mass2SO415% PEG400 and 70% waterThe mixture was mixed and prepared in a 3L separatory funnel, and the separatory funnel was placed in an incubator at 37 ℃ and allowed to stand for 24 hours. After obvious layering, impurities in the two-phase solution are removed by respectively passing through 0.45 mu m filter membranes to obtain an upper phase PEG400 and a lower phase Na2SO4And (3) solution.
Step (2): adding 5g Lipase AYS enzyme into 100mL of the PEG400 above the phase, adding 250mL of Na below the phase2SO4The solution was passed into the standing zone 3 to fill the dispersion buffer zone 2, and then the enzyme-containing PEG400 solution was introduced into the reaction zone 1 at a stirring rate of 500 rpm.
And (3): introducing the refined olive oil from the oil material inlet 17 into the reaction zone 1 of the oil material hydrolysis device at a flow rate of 3mL/min for enzyme-catalyzed hydrolysis reaction, and simultaneously introducing the lower phase Na at a flow rate of 3mL/min2SO4The solution is introduced into the apparatus from the lower solution inlet 11. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer (respectively corresponding to an upper liquid phase region, a middle liquid phase region and a lower liquid phase region) are sequentially arranged in a standing region from top to bottom, fatty acid hydrolyzed by refined olive oil is mainly enriched in the upper liquid layer, glycerol hydrolyzed is mainly enriched in the lower liquid phase, and the middle liquid layer containing enzyme can flow back to the reaction region again through a reflux valve to continuously participate in the hydrolysis reaction. Finally, the hydrolysis efficiency of triglyceride of the refined olive oil reaches 80% when the refined olive oil is continuously reacted for 5 hours, and the hydrolysis efficiency reaches 62% when the refined olive oil is continuously reacted for 20 hours. The enzyme solution can be supplemented in real time according to the reaction condition.
And (4): the same as in example 1.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. A device for continuously synthesizing diglyceride by a holoenzyme method in a multi-liquid-phase system is characterized by comprising a hydrolysis device and an esterification device; the hydrolysis device is divided into a standing zone, a reaction zone and a dispersing buffer zone from top to bottom, the reaction zone and the standing zone and the reaction zone and the dispersing buffer zone are separated by a baffle, and the baffle is provided with a plurality of small holes communicated with the zones; the standing area is divided into an upper liquid phase area, a middle liquid phase area and a lower liquid phase area, the upper liquid phase area is provided with a hydrolysate liquid outlet, the hydrolysate liquid outlet is communicated with a sample inlet of the esterification device, the middle liquid phase area is communicated with the reaction area through a return pipe, and the lower liquid phase area is provided with a lower phase solution sample inlet; the lower part of the reaction zone is provided with an enzyme-containing solution sample inlet, and a stirrer is arranged inside the reaction zone; the bottom of the dispersing buffer zone is provided with an oil material sample inlet and a lower phase solution outlet.
2. The apparatus of claim 1, wherein a circulating water heating jacket is provided outside the reaction zone.
3. The apparatus of claim 2, wherein the lower liquid phase zone of the resting zone is provided with an autopotentiometric titrator burette and the reaction zone is provided with an autopotentiometric electrode tip.
4. The device according to any one of claims 1 to 3, wherein the esterification device comprises a reaction kettle and a stirrer; the outside of the reaction kettle is provided with a circulating water heating sleeve, the lower part of the reaction kettle is provided with a hydrolyzed oil material sample inlet and an esterification substrate sample inlet, and the upper part of the reaction kettle is provided with an esterification product outlet.
5. A method for synthesizing diglycerides using the apparatus as set forth in any one of claims 1 to 4, comprising the steps of:
(1) preparing a double-liquid-phase solution from soluble salt, a hydrophilic solvent and water, standing and layering to obtain an upper-phase solution containing the hydrophilic solvent and a lower-phase solution containing the soluble salt, continuously introducing the lower-phase solution containing the soluble salt into a standing area, passing through a reaction area, and entering and filling a dispersion buffer area; dissolving lipase or phospholipase in the upper phase solution containing hydrophilic solvent, introducing into the reaction zone, continuously introducing the oil into the dispersion buffer zone, adjusting the flow rate of the feed liquid to allow the oil to enter the reaction zone from the dispersion buffer zone, and performing enzymatic hydrolysis reaction;
(2) the fatty acid after hydrolysis reaction is extracted by self and enters a standing zone, after standing and layering, the upper phase solution containing the fatty acid is directly introduced into an esterification device to carry out esterification reaction with an esterification reaction substrate, and the esterification product is subjected to standing and layering to obtain diglyceride in the upper phase; the esterification reaction substrate comprises a hydrophobic solvent, a short-chain alcohol, glycerol, an enzyme and water.
6. The method of claim 5, wherein the lower substrate after the esterification and standing separation in the step (2) is introduced into the reaction kettle of the esterification device to continue to participate in the reaction.
7. The method as claimed in claim 6, wherein the feeding and reaction processes in step (1) are carried out under continuous stirring and heating conditions, the heating temperature is 30-70 ℃, and the stirring speed is 100-1000 rpm; adjusting the pH value of the reaction system to 3-13; the flow rate of the lower phase solution is 0.1-1.0 mL/min.
8. The method according to claim 5, 6 or 7, wherein the mass ratio of the soluble salt, the hydrophilic solvent and the water in the step (1) is 0.1-1 and 0.1-5 respectively.
9. The method according to claim 8, wherein the soluble salt is one or more of sodium citrate, sodium chloride, sodium sulfate, ammonium sulfate, sodium carbonate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate;
the hydrophilic solvent is a polymer or an ionic liquid, wherein the polymer comprises one or two of polyethylene glycol and polypropylene glycol; the ionic liquid comprises [ BMIM]Br、[BMIM]BF4、[EMIM]ETSO4、[OMIM]Cl、[BMIM]PF6One or more than two of the above;
the Lipase is Lipase AYS, Lipase AY30, Lipase MAS1, Lipase G Amano 50, Lipase CALB and Novozyme 435, the phospholipase is one or more of Lecite Ultra, PLA2, PLC and PLD, and the concentration of the enzyme is 5-2000U/mL.
10. The method of claim 9, wherein the oil of step (1) comprises one or more of virgin or refined olive oil, camellia oil, walnut oil, and soybean oil;
the hydrophobic solvent in the step (2) is one or more than two of n-hexane, isopropyl ether, ethyl acetate and isooctane; the short-chain alcohol is one or more of tert-butyl alcohol, ethanol, isopropanol, isoamyl alcohol and n-octanol.
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