Gas-liquid-solid three-phase fluidized bed enzyme reactor and method for synchronously preparing grease enzymatic deacidification and diglyceride by using gas-liquid-solid three-phase fluidized bed enzyme reactor

Abstract

The invention belongs to the technical field of food and chemical industry, and particularly relates to a gas-liquid-solid three-phase fluidized bed enzyme reactor and a synchronous preparation method of grease enzyme method deacidification and diglyceride by using the same. According to the method, the gas phase of the three-phase fluidized bed is high-purity dry inert gas, the liquid phase is a uniform solution formed by mixing reaction substrate high-acid-value oil and an acyl acceptor, and the solid phase is immobilized enzyme. The method comprises the following steps: mixing high-acid-value oil and an acyl acceptor to prepare a raw material solution, preheating a feeding tank to a reaction temperature, adding the raw material solution into a gas-liquid-solid three-phase fluidized bed reactor, and carrying out esterification and / or transesterification under the bubbling action of inert gas to realize the deacidification of the high-acid-value oil and the synchronous preparation of the functional lipid. The mixture of the deacidified grease and the functional lipid is obtained. The method has the characteristics of high catalytic efficiency, easiness in separation of the product from enzyme, high nutritional value of the product, no need of adding an organic solvent, difficulty in oxidation of grease and suitability for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of food and chemical industry, and particularly relates to a gas-liquid-solid three-phase fluidized bed enzyme reactor and a method for synchronously preparing diglyceride by enzymatic deacidification of oil and fat using the same. Background Art

[0002] Since rice bran oil, rapeseed oil, soybean oil, corn oil, sesame oil, sunflower oil, linseed oil and other vegetable oils and microbial oils such as DHA and ARA algae oil are prone to hydrolysis of triglycerides and generate a large amount of free fatty acids during the extraction, storage and processing, the acid value of the oil is too high, odor is generated, and the quality of the oil is reduced. Rancid rapeseed oil is not only inedible, but also harmful to human health. It can only be used to make low-value soap or be discarded, causing great waste of oil resources, environmental pressure and economic losses.

[0003] CN 114736739 A discloses a method for enzymatic deacidification of oil and grease and simultaneous preparation of functional lipids. However, the viscosity of the oil and grease is too high, and an organic solvent needs to be added additionally. A water absorbent such as a molecular sieve is used, but the molecular sieve is easily saturated with water, resulting in reduced water removal efficiency. In addition, the solid molecular sieve particles are mixed with the immobilized enzyme and are difficult to separate from the immobilized enzyme. The continuous flow reactor is easy to clog and difficult to scale up, and needs to be continuously activated and regenerated before it can be reused. The disadvantages make it difficult to meet the needs of large-scale production.

[0004] Therefore, providing a method for the simultaneous preparation of enzymatic deacidification of oils and diglycerides that does not require the addition of organic solvents and is suitable for large-scale production has become a technical problem that needs to be urgently solved by those skilled in the art. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for enzymatic deacidification of oils and fats and simultaneous preparation of diglycerides based on a gas-liquid-solid three-phase fluidized bed to address the problems existing in the prior art. The method has the characteristics of high catalytic efficiency, easy separation of product and enzyme, high nutritional value of the product, no need to add organic solvents, and is suitable for large-scale production.

[0006] In order to achieve the above-mentioned object, the first object of the present invention is to provide a gas-liquid-solid three-phase fluidized bed enzyme reactor. The following technical scheme is adopted:

[0007] A gas-liquid-solid three-phase fluidized bed enzyme reactor comprises an inert gas control device 1, a main reaction column 2 and a water bath heating circulation device 3; the main reaction column 2 comprises a gas-liquid distributor composed of a porous sieve plate 207 and a wire mesh demister 208, and the porous sieve plate 207 is at least arranged above the wire mesh demister 208; the porous sieve plate 207 is connected to an inert gas inlet control valve 109.

[0008] It is worth mentioning that the gas-liquid distributor refers to the closed cavity that the gas and liquid enter before entering the main reaction column 2. The cavity is filled with a wire mesh demister 208 and is equipped with porous sieve plates 207 above and below to ensure that the gas and liquid are evenly dispersed before entering the upper reaction column.

[0009] The present invention uses a wire mesh demister 208 and a porous sieve plate 207 in combination to achieve inert gas dispersion into the fluidized bed column, promote gas-liquid-solid multiphase mixing, and prevent immobilized enzyme particles from falling into the gas-liquid distributor to block the gas-liquid branch pipe. Among them, the porous sieve plate 207 can achieve uniform dispersion of inert gas into the reaction column, reduce bubble aggregation, promote gas-liquid-solid multiphase mixing and enzymatic catalysis. The porous sieve plate aperture is smaller than the immobilized enzyme particle size, which can prevent the immobilized enzyme particles from entering the gas-liquid distributor from the sieve plate and blocking the gas-liquid inlet.

[0010] In some embodiments, the porous sieve plate 207 in the gas-liquid distributor is arranged above and below the wire mesh demister 208, and the pore size of the porous sieve plate 207 is 10-50 μm. The lower porous sieve plate 207 is connected to the inert gas inlet control valve 109.

[0011] Furthermore, the main reaction column 2 includes a lower part consisting of a finished product outlet control valve 209, an inert gas inlet control valve 109, a gas-liquid branch pipe, a porous sieve plate 207 and a wire mesh demister 208, a middle part with end face fixing bolts 206 and a heating jacket 305, and an upper part consisting of an oil inlet control valve 204, a finished product outlet control valve 209 and an inert gas outlet control valve 110; the upper part, the middle part and the lower part of the main reaction column 2 are connected in sequence through a flange disc, and the flange disc has two circular grooves that match the double-layer reaction column and are sealed with an O-ring.

[0012] It is worth noting that, in the prior art, fluidized bed reactors are mostly monolithic, which is not conducive to assembly or disassembly according to actual needs. The present invention uses upper and lower flange discs to connect the upper part (gas-liquid outlet, water bath circulation outlet, raw material tank, etc.), the middle part (fluidized bed reaction column body heated by water bath jacket) and the lower part (gas-liquid inlet control device, water bath circulation device, gas-liquid distributor, etc.) of the fluidized bed, which is convenient for assembly and disassembly of fluidized bed reaction equipment, and then convenient for adding and replacing immobilized enzymes during use, and is conducive to subsequent maintenance and replacement of reaction equipment parts, and prolongs service life. In addition, the upper and lower flange discs are provided with two circular grooves and four through holes, the circular grooves are equipped with sealing rings for assembling the reaction column, and the four through holes are connected by screws and nuts, so that the upper and lower flange discs are pressed, thereby avoiding gas-liquid leakage and grease oxidation in the reactor.

[0013] Furthermore, considering the full contact of the three phases of gas, liquid and solid in actual production, the present invention arranges a finished product outlet control valve 209 at the lower and upper parts of the main reaction column 2, and arranges an oil inlet control valve 204 at the upper part of the main reaction column 2, so as to adjust the material inlet and outlet paths according to actual needs.

[0014] Furthermore, a through hole is provided in the middle of the flange disc, and the inner diameter of the through hole is consistent with the inner diameter of the main reaction column; an oil inlet control valve 204, a hot water inlet control valve 302 and a hot water outlet control valve 303 are provided on the side of the flange disc, and are connected to the reactor body. Thus, the present invention realizes the assembly and disassembly of the core components of the reactor through the flange clamping mechanism.

[0015] In the present invention, the main reaction column 2 has a height of 0.5-1.5 m and an inner diameter of 0.2-0.5 m, which is suitable for laboratory preparation and pilot scale-up.

[0016] Furthermore, in the present invention, the main reaction column 2 also includes an auxiliary module consisting of a feed tank 201, a heating coil 202, an oil level controller 203, and a pressure difference detector 205. The heating coil 202 is surrounded by the outside of the feed tank 201, and the feed tank 201 is connected to the oil inlet control valve 204 through a pipeline.

[0017] Furthermore, the inert gas control device 1 includes, in order according to the gas passage, an air compressor 101, an air compression tank 102, a refrigerated dryer 103, an inert gas manufacturer 104, and a drain valve 106 arranged in the pipeline between the air compression tank 102, the refrigerated dryer 103, and the inert gas manufacturer 104, an inert gas purity monitor 107 arranged in the inert gas manufacturer 104, and a flow meter 105 and a pressure gauge 108 arranged in the connecting pipeline between the inert gas manufacturer 104 and the inert gas inlet control valve 109.

[0018] It is worth noting that the introduction of inert gas takes away the byproduct water produced by the esterification reaction of diglycerides generated simultaneously with the deacidification reaction, promotes the forward progress of the esterification reaction, accelerates the reaction rate, and provides antioxidant protection for oil enzymatic processing. Compared with traditional mechanical stirring, gas flexible bubbling causes less mechanical damage to immobilized enzymes and can effectively reduce enzyme damage and inactivation.

[0019] In the present invention, the inert gas includes one of nitrogen, argon and helium, and the purity is above 99.999%.

[0020] Furthermore, the water bath heating circulation device 3 includes a water bath heating device 301, a hot water inlet control valve 302, a hot water outlet control valve 303, and a PLC control system 304 connected by signals in sequence, the hot water inlet control valve 302 is arranged at the lower part of the main reaction column 2, the hot water outlet control valve 303 is arranged at the upper part of the main reaction column 2 and is connected to the water bath heating device 301 through a return pipe; the water bath heating temperature is 30-90°C.

[0021] It is worth noting that the water bath circulation system uses jacket heating to ensure precise temperature control in the reaction column, achieving high activity and catalytic efficiency of the immobilized enzyme at a suitable reaction temperature, so that the temperature of the entire reaction column can accurately reach the temperature of the immobilized enzyme or commercial enzyme.

[0022] Furthermore, the PLC control system 304 is also connected to the flow meter 105 , the inert gas purity monitor 107 , the pressure gauge 108 , the oil level controller 203 , and the differential pressure detector 205 via signals.

[0023] The differential pressure detector 205 can monitor and control the column pressure changes in the reactor in real time, and the PLC control system 304 can online adjust the reaction parameters such as gas-liquid flow rate and reaction temperature.

[0024] Thus, the gas-liquid-solid three-phase fluidized bed enzyme reactor disclosed in the present invention introduces inert gas into the traditional fluidized bed reaction through an inert gas control device, and adopts gas flexible bubbling to replace the stirring blades of the transmission mechanical stirring, so as to avoid the reduction of the activity of the immobilized enzyme or even inactivation caused by the mechanical stress and shear force caused by stirring. In addition, the inert gas bubbling can synchronously take away the byproducts generated by esterification deacidification, accelerate the reaction forward, and do not need to add traditional dehydrating agents such as molecular sieves. Thus, the fluidization movement of the immobilized enzyme is driven by gas bubbling, which can promote gas-liquid-solid multiphase mixing, improve the effect of the immobilized enzyme contacting and catalyzing with the substrate under the high viscosity oil phase reaction system, and there is no need to add organic solvents to reduce the viscosity of the oil, and there is no subsequent desolventizing step, and the safety is also higher. In particular, the inert gas bubbling can effectively remove the dissolved oxygen in the oil, provide antioxidant protection for the oil enzymatic deacidification, and avoid oil oxidation.

[0025] The second object of the present invention is to provide a method for the simultaneous preparation of diglycerides by enzymatic deacidification of oils and fats. The following technical scheme is adopted:

[0026] A method for the simultaneous enzymatic deacidification of oil and grease and preparation of diacylglycerol comprises the following steps: mixing high acid value oil and acyl acceptor in a solvent-free condition to obtain a raw material liquid; continuously bubbling inert gas to fluidize immobilized enzyme particles in a three-phase system of inert gas-high acid value oil-immobilized enzyme; and causing esterification / ester exchange reaction in the gas-liquid-solid three-phase fluidized bed enzyme reactor as described above, thereby achieving the simultaneous deacidification of high acid value oil and preparation of diacylglycerol.

[0027] It is worth noting that inert gas bubbling can promote the contact between glycerol and free fatty acids in the mixed raw materials, and fully contact with the immobilized enzyme under the action of fluidization movement, resulting in esterification / ester exchange reaction, thereby achieving deacidification of high acid value oil and simultaneous preparation of diglycerides.

[0028] In the prior art, due to the high viscosity of oils and fats, the contact and catalysis of "oils and fats-immobilized enzymes" are limited. Traditional enzymatic deacidification requires the addition of organic solvents such as n-hexane and tert-butanol to the oils and fats to reduce the viscosity of the oils and fats and promote the contact and mass transfer between the immobilized enzymes and the substrates. However, subsequent separation and purification steps are required to remove the organic solvents, which has the disadvantages of high energy consumption, cumbersome operation, and solvent residues. Based on this, the present invention adopts a "gas-liquid-solid" three-phase fluidized bed reactor to achieve fluidized motion of the immobilized enzymes through gas bubbling, which is conducive to mixing with oil and liquid, improves mass transfer efficiency, and does not need to use organic solvents to reduce the viscosity of the oils and fats to improve substrate mass transfer. Therefore, it has the characteristics of being green, safe, and easy to operate.

[0029] Furthermore, one or a combination of glycerol and monoglyceride, the high acid value oil is a vegetable oil or microbial oil with an acid value of 5-50 mgKOH / g, and the molar ratio of the acyl acceptor to the free fatty acids in the high acid value oil is 0.5:1 to 8:1.

[0030] Furthermore, the vegetable oil is rice bran oil, rapeseed oil, soybean oil, corn oil, sesame oil, sunflower seed oil or linseed oil, and the microbial oil includes DHA or ARA algae oil.

[0031] In the present invention, the immobilized enzyme is prepared by fixing a free enzyme to a carrier such as hydrophobic mesoporous silica gel, macroporous resin, carbon particles, hydrophobic cellulose, etc.; or, the immobilized enzyme is a commercial immobilized enzyme.

[0032] Preferably, the free enzyme is one or more combinations of phospholipase A1, Candida antarctica lipase, Rhizomucor miehei lipase, Pseudomonas cepacia lipase, Aspergillus niger lipase, and Candida rugosa lipase.

[0033] Preferably, the particle size of the immobilized enzyme is 10-500 μm, the mesopore size is 3-11 nm, the enzyme loading is 80-200 mg / g, and the loading rate is 40-80%.

[0034] Preferably, the immobilized enzyme used in the present invention is a self-made immobilized enzyme with high activity and high reusability, and can still maintain more than 70% relative activity after being reused 20 times in the enzymatic deacidification of oils and the simultaneous preparation of diglycerides.

[0035] In order to fully disclose the technical solution of the present invention, a method for preparing a homemade immobilized enzyme is now disclosed. It should be acknowledged that all methods for preparing immobilized enzymes based on the prior art without creative labor can be applied to prepare the "homemade immobilized enzyme" of the present invention.

[0036] The preparation method of the immobilized enzyme is as follows: free lipase is prepared into an enzyme solution with a pH value of 5.0-9.0 of a phosphate buffer, and then the hydrophobic mesoporous silica carrier and the enzyme solution are mixed uniformly in a certain solid-liquid ratio for immobilization, and the immobilized enzyme is obtained by freeze-drying; wherein the concentration of the enzyme solution is 10-100 mg / mL, the solid-liquid ratio is 1:1-1:5, and the immobilization time is 30-90 min.

[0037] In the present invention, the "solid-to-liquid ratio" is the ratio of the mass of the hydrophobic mesoporous carrier to the volume of the enzyme solution.

[0038] The hydrophobic mesoporous silica gel is obtained by the reaction of a hydrophobic modifier and mesoporous silica gel; the hydrophobic modifier mainly adopts alkyl siloxanes with different carbon chain lengths.

[0039] Furthermore, the specific reaction steps of the method for simultaneous enzymatic deacidification of oils and greases and preparation of diglycerides in a gas-liquid-solid three-phase fluidized bed enzyme reactor are as follows:

[0040] The preheated mixed raw material liquid of acyl acceptor and high acid value oil is added to a fluidized bed reactor equipped with a water bath heating device; the inert gas control device 1 is started, and high-purity dry inert gas is introduced into the inert gas inlet 109 at the lower end of the fluidized bed, and the inert gas is dispersed by the wire mesh demister 208 and the porous sieve plate 207 in the gas-liquid distributor and then enters the main reaction column 2 to form a continuous bubbling gas; after the immobilized lipase particles are added into the reaction column, the immobilized enzyme is made to move upward disorderly by gas bubbling, and the gas pressure gauge 108 and the flow meter 105 are adjusted to control the gas pressure and gas velocity, so as to realize the fluidized movement of the immobilized enzyme particles in the three-phase system of inert gas-high acid value oil-immobilized enzyme, and then the esterification / ester exchange reaction occurs, so as to realize the enzymatic deacidification of oil and fat and the synchronous preparation of diglyceride.

[0041] It is worth noting that, considering the shortcomings of the existing reactions that require the addition of solvents, the reactor is easily blocked, and molecular sieves need to be added, the present invention combines an immobilized enzyme or a commercial enzyme through a gas-liquid-solid three-phase fluidized bed, and the synchronous preparation of enzymatic deacidification and diglyceride does not require the addition of solvents and water absorbents, and the reactor does not have problems such as blockage, and the gas-liquid-solid three-phase fluidized bed has a small shear force, and the enzyme damage is small, and the immobilized enzyme or commercial enzyme can be reused many times. In particular, the present invention uses inert gas bubbling to take away the byproduct water generated by enzymatic esterification deacidification and provide antioxidant protection for oil deacidification, without the need to use a dehydrating agent, thereby avoiding the technical problem of the difficulty in separating the water absorbent from the immobilized enzyme.

[0042] Furthermore, the inert gas inlet pressure is 0.1-5MPa, the gas flow rate is 0-1000mL / s; the immobilized enzyme addition amount is 1%-5% of the mass of the high acid value oil, and the reaction time of the enzymatic deacidification of oil and fat and the simultaneous preparation of diglyceride is 0.5h to 6h.

[0043] It is worth noting that the acid value of the oil product obtained by the method for simultaneous preparation of diglyceride and enzymatic deacidification of oil disclosed in the present invention is in the range of 0.4-2.5 mg KOH / g, and the mass percentage of diglyceride in the oil product is 5%-50%, wherein the content of Sn-1,3 diglyceride is greater than 70%. The moisture and volatile content of the oil product is less than 0.02%, the peroxide value is less than 0.1 g / 100 g, the shelf life is greater than 18 months, and all indicators meet the edible oil standards.

[0044] Furthermore, the method for the simultaneous enzymatic deacidification of oils and fats and preparation of diglycerides is carried out in a gas-liquid-solid three-phase fluidized bed enzyme reactor in a semi-continuous manner.

[0045] It is worth noting that in the semi-continuous production process, the method for the simultaneous preparation of diglycerides by enzymatic deacidification of oils and fats disclosed in the present invention does not require the removal of the immobilized enzyme. After one reaction is completed, the oil product is discharged from the outlet of the gas-liquid-solid three-phase fluidized bed enzyme reactor, and the raw liquid can continue to be added to the inlet of the reactor to achieve semi-continuous production.

[0046] The present invention discloses a method for enzymatic deacidification of oils and fats and preparation of diglycerides based on a gas-liquid-solid three-phase fluidized bed, wherein the gas phase of the three-phase fluidized bed is a high-purity dry inert gas, the liquid phase is a uniform solution of a reaction substrate high acid value oil mixed with an acyl acceptor, and the solid phase is an immobilized enzyme. The present invention mixes high acid value oil and acyl acceptor to prepare a raw material liquid, and after the feed tank is preheated to the reaction temperature, it is added to the gas-liquid-solid three-phase fluidized bed reactor, and esterification and / or transesterification reactions occur under the action of inert gas bubbling, thereby achieving the deacidification of high acid value oil and the simultaneous preparation of functional lipids, and obtaining a mixture of deacidified oils and functional lipids. Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. The present invention uses monoglyceride, glycerol, etc. as acyl acceptors, and achieves deacidification of high acid value oil and simultaneous preparation of functional lipids by esterification reaction with high acid oil under solvent-free conditions, killing two birds with one stone; and the obtained product is a fat product rich in functional lipids, the proportion of functional lipids such as diglyceride in the fat product is between 5% and 50%, and the acid value range of the fat product is between 0.4 and 2.5 mg KOH / g. The fat product rich in functional lipids meets the edible oil standard.

[0048] 2. The present invention adopts the method of fixing free lipase to a hydrophobic carrier to prepare the immobilized enzyme, and its activity and stability are significantly improved compared with the commercial immobilized enzyme. The mesoporous structure of the carrier can not only load the enzyme efficiently, but also facilitate the macroscopic flow and microscopic mass transfer of the reaction substrate in the gas-liquid-solid three-phase enzyme reactor, and has the advantages of fast flow rate, low pressure, high mass transfer and heat transfer efficiency, etc.

[0049] 3. The present invention replaces traditional stirring with inert gas bubbling, which has low energy consumption, small shear, reduces enzyme damage, is easy to control, and improves the reuse effect of the enzyme; the introduction of inert gas takes away the water generated by the esterification reaction and provides antioxidant protection for oil deacidification, without adding a water absorbent (such as molecular sieve), avoiding the problem of difficult separation of the immobilized enzyme and the water absorbent in the reaction column, and keeping the moisture and volatile content in the oil within 0.02%.

[0050] 4. The present invention adopts a gas-liquid-solid three-phase fluidized bed reactor. The fluidized motion in the gas-liquid-solid three-phase fluidized bed reactor is relatively static compared to the enzyme in the packed bed enzyme reactor, and will not cause blockage of the reaction column. It has a large mass transfer efficiency, high efficiency and low energy consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work.

[0052] Figure 1 The full flow chart of the gas-liquid-solid three-phase fluidized bed reactor of the present invention, wherein the gas-liquid-solid three-phase fluidized bed enzyme reactor includes 1-inert gas control device, 101-air compressor, 102-air compression tank, 103-freeze dryer, 104-inert gas manufacturing machine, 105-flow meter, 106-drain valve, 107-inert gas purity monitoring, 108-pressure gauge, 109-inert gas inlet control valve, 110-inert gas outlet control valve; 2 -main reaction column, 201-feeding tank, 202-heating coil, 203-oil level controller, 204-oil inlet control valve, 205-differential pressure detector, 206-end face fixing bolts, 207-porous sieve plate, 208-wire mesh demister, 209-finished product outlet control valve; 3-water bath heating circulation device, 301-water bath heating device, 302-hot water inlet control valve, 303-hot water outlet control valve, 304-PLC control system, 305-heating jacket.

[0053] Figure 2 It is a cross-sectional view of the main reaction column 2 of the gas-liquid-solid three-phase fluidized bed reactor of the present invention.

[0054] Figure 3 Schematic diagram of enzymatic deacidification and diglyceride preparation in a gas-liquid-solid three-phase fluidized bed reactor of the present invention, taking Example 1 as an example.

[0055] Figure 4 Examples 1 and 3 of the present invention are SEM characterization pictures of hydrophobic mesoporous silica gel.

[0056] Figure 5 This is a diagram showing the deacidification effect of rapeseed oil with different acid values ​​in Example 2 of the present invention.

[0057] Figure 6 The content changes of fatty acids and functional lipids and the content of main components in rice bran oil before and after deacidification in Example 2 of the present invention are shown.

[0058] Figure 7 This is a diagram showing the effect of repeatedly using the immobilized enzyme ten times in a gas-liquid-solid three-phase fluidized bed reactor in Example 5 of the present invention.

[0059] Figure 8 This is a comparison chart of the reaction effects of the gas-liquid-solid three-phase fluidized bed reactor and the magnetic stirring reactor in Example 6 of the present invention. DETAILED DESCRIPTION

[0060] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0062] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0063] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0064] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0065] The present invention discloses a method for enzymatic deacidification of oil and grease and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, wherein the gas phase of the three-phase fluidized bed is a high-purity dry inert gas, the liquid phase is a uniform solution after the reaction substrate high acid value oil and the acyl acceptor are mixed, and the solid phase is an immobilized enzyme. The present invention mixes the high acid value oil and the acyl acceptor to prepare a raw material liquid, and after the feed tank is preheated to the reaction temperature, it is added to the gas-liquid-solid three-phase fluidized bed reactor, and esterification and / or transesterification reaction occurs under the action of inert gas bubbling, realizing the deacidification of the high acid value oil and the synchronous preparation of functional lipids, and obtaining a mixture of oil and grease after deacidification and functional lipids. The method has the characteristics of high catalytic efficiency, easy separation of product and enzyme, high nutritional value of product, no need to add organic solvent, and is suitable for large-scale production.

[0066] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0067] In an embodiment of the present invention, the preparation method of hydrophobic mesoporous silica gel is as follows: 0.5 g of mesoporous silica gel and 231 μL of hydrophobic modifier are added to 5 mL of n-hexane, and after ultrasonication for 10 minutes, the mixture is shaken at room temperature for 1.5-2 hours in a 220 rpm shaker, washed with anhydrous ethanol, and dried in an oven at 60°C to obtain modified hydrophobic mesoporous silica gel. Among them, the hydrophobic modifier is methyltrimethoxysilane, propyltrimethoxysilane, octyltrimethoxysilane, n-octyltrichlorosilane, octadecyltrimethoxysilane, and other alkyl alkoxysilanes, and hydrophobic mesoporous silica gel modified with hydrophobic groups such as methyl, propyl, octyl, and octadecyl can be prepared accordingly, and the preparation can be scaled up according to needs.

[0068] In the embodiment of the present invention, for a gas-liquid-solid three-phase fluidized bed reactor, after the immobilized enzyme or commercial enzyme is added, no new immobilized enzyme or commercial enzyme needs to be added for subsequent reactions, and the post-reaction product can be removed to carry out a cyclic reaction.

[0069] Example 1

[0070] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0071] (1) Construction of gas-liquid-solid three-phase fluidized bed reactor: Dissolve the lipase (CALB) from Candida antarctica in phosphate buffer (50 mM, pH = 7) to prepare a 90 mg / mL enzyme solution, then add the hydrophobic mesoporous silica modified with n-octylsilane to a ground-mouth conical flask at a ratio of 10 mg / mL to the enzyme solution, react on a shaker (200 rpm, 30°C) for 30 min, centrifuge, and freeze-dry the precipitate to obtain the immobilized enzyme. A certain amount of immobilized enzyme was added to the gas-liquid-solid three-phase fluidized bed reactor, dry nitrogen was introduced, and the reactor was preheated to 65°C.

[0072] (2) Enzymatic deacidification reaction: Glycerol was added to rapeseed oil with an acid value of 4.2 mg KOH / g in a molar ratio of free fatty acids to glycerol of 1:1. The mixture was homogenized for 1 min and preheated to 65° C. before being added to the reactor. After the reaction was completed, the rapeseed oil was collected at the reactor outlet.

[0073] The collected rapeseed oil is a mixture of deacidified rapeseed oil and diglyceride prepared simultaneously, i.e., a rapeseed oil product rich in diglyceride. According to the measurement, the acid value of the rapeseed oil product is 0.4 mg KOH / g, and the content of diglyceride is 5.7%.

[0074] Table 1 is a comparison of various indicators of the high acid value rapeseed oil raw material initially used in Example 1, the deacidified rapeseed oil prepared in Example 1 (i.e., the rapeseed oil product rich in diglycerides), and commercial rapeseed oil, which proves that the various indicators of the enzymatic deacidified rapeseed oil are significantly higher than those of the high acid value rapeseed oil.

[0075] Table 1. Comparison of moisture, volatiles, peroxide value, and oxidative stability of high acid value rapeseed oil raw material, deacidified rapeseed oil, and commercial rapeseed oil

[0076]

[0077] Example 2

[0078] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0079] (1) Construction of a gas-liquid-solid three-phase fluidized bed reactor: Commercial immobilized enzyme NE-100 was added into a reactor preheated to 65°C and continuously supplied with dry nitrogen.

[0080] (2) Reactor enzymatic deacidification system: In rice bran oil with an acid value of 10 mg KOH / g, glycerol was added according to the molar ratio of free fatty acids and glycerol in the rice bran oil of 1:2 and homogenized for 1 min to form a uniform solution, which was preheated to 65° C. and then added to a gas-liquid-solid three-phase fluidized bed reaction. After the reaction was completed, the reacted rice bran oil was collected at the reactor outlet.

[0081] The collected rice bran oil is a mixture of deacidified rice bran oil and diglyceride prepared synchronously, i.e. a rice bran oil product rich in diglyceride. The acid value of the rice bran oil product is measured to be 0.6 mg KOH / g, and the content of diglyceride is 30%.

[0082] Example 3

[0083] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0084] (1) Construction of gas-liquid-solid three-phase fluidized bed reactor: Lipase (CALB) from Candida antarctica was dissolved in phosphate buffer (50 mM, pH = 7) to prepare an 80 mg / mL enzyme solution, and then the hydrophobic mesoporous silica modified with n-octylsilane was added to a ground-mouth conical flask at a ratio of 10 mg / mL to the enzyme solution. After reacting on a shaker (200 rpm, 30°C) for 30 min, the solution was centrifuged and the precipitate was freeze-dried to obtain the immobilized enzyme. A certain amount of immobilized enzyme was added to the gas-liquid-solid three-phase fluidized bed reactor, dry nitrogen was introduced, and the reactor was preheated to 65°C.

[0085] (2) Reactor enzymatic deacidification system: In tea seed oil with an acid value of 17 mg KOH / g, glycerol was added according to the molar ratio of free fatty acids and glycerol in the tea seed oil of 1:2 and homogenized for 1 min to form a uniform solution, which was preheated to 65°C and added to a gas-liquid-solid three-phase fluidized bed reactor. After the reaction was completed, the tea seed oil after the reaction was collected at the reactor outlet.

[0086] The collected tea seed oil is a mixture of deacidified tea seed oil and diglyceride prepared simultaneously, i.e. a tea seed oil product rich in diglyceride. According to the measurement, the acid value of the tea seed oil product is 1.3 mg KOH / g, and the content of diglyceride is 15.4%.

[0087] Example 4

[0088] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0089] (1) Construction of gas-liquid-solid three-phase fluidized bed reactor and magnetic stirring reaction bottle: Dissolve the lipase (CALB) from Candida antarctica in phosphate buffer (50mM, pH=7) to prepare an 80mg / mL enzyme solution, then add the hydrophobic mesoporous silica modified with n-octylsilane to the enzyme solution at a ratio of 10mg / mL into a ground-mouth conical bottle, react on a shaker (200rpm, 30℃) for 30min, take out and centrifuge, precipitate and freeze-dry to obtain the immobilized enzyme. A certain amount of immobilized enzyme was added to the gas-liquid-solid three-phase fluidized bed reactor, dry nitrogen was introduced, and the reactor was preheated to 65℃.

[0090] The immobilized enzyme prepared according to the above steps was added into the reaction bottle, a magnet was added, and the reaction was preheated to 65° C. at a rotation speed of 400 rpm.

[0091] (2) Enzymatic deacidification reaction: In rapeseed oil with an acid value of 15.3 mg KOH / g, glycerol was added according to the molar ratio of free fatty acids and glycerol in rapeseed oil of 1:1, homogenized for 1 min and preheated to 65°C, and then added to the reactor. After the reaction was completed, the rapeseed oil after the reaction was collected at the reactor outlet. Under the same conditions, in a magnetic stirring reaction bottle, the same amount of high acid value rapeseed oil and glycerol were added to the uniformly mixed solution, and after the same reaction time, the rapeseed oil in the stirring reactor was collected.

[0092] The rapeseed oil collected in the gas-liquid-solid three-phase fluidized bed reactor and during stirring is a mixture of deacidified rapeseed oil and diglyceride prepared synchronously, i.e., a rapeseed oil product rich in diglyceride. According to measurement, the acid value of the rapeseed oil product in the gas-liquid-solid three-phase fluidized bed reactor is 1.67 mg KOH / g, wherein the content of diglyceride is 30.2%, and the acid value of the rapeseed oil product in the stirred reaction bottle is 2.11 mg KOH / g, wherein the content of diglyceride is 12.2%.

[0093] Example 5

[0094] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0095] (1) Construction of a gas-liquid-solid three-phase fluidized bed reactor and a magnetic stirring reaction flask: Same as step (1) in Example 4.

[0096] (2) Enzymatic deacidification reaction: The same as step (2) in Example 4. After the reaction is completed, a new mixed solution of high acid value rapeseed oil and glycerol homogenized in proportion is continuously added to the gas-liquid-solid three-phase fluidized bed reactor and the magnetic stirring reaction bottle, and the reaction is continued for 10 times without further adding the immobilized enzyme.

[0097] In the 10 deacidification reactions, the rapeseed oil in the gas-liquid-solid three-phase fluidized bed reactor was a mixture of the deacidified rapeseed oil and the diglyceride prepared simultaneously, that is, a rapeseed oil product rich in diglyceride. It was determined that the acid value of the rapeseed oil product after 10 reactions in the gas-liquid-solid three-phase fluidized bed reactor was between 1.0 and 2.0 mg KOH / g, and the content of diglyceride was between 28% and 32%; after repeating the reaction 3 times, the acid value of the rapeseed oil product in the stirred reaction bottle could not be reduced.

[0098] Example 6

[0099] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0100] (1) Construction of liquid-solid three-phase fluidized bed reactor and magnetic stirring reaction bottle: The commercial immobilized enzyme NE-100 was added into a reactor preheated to 65°C and continuously supplied with dry nitrogen.

[0101] The commercial immobilized enzyme NE-100 was added into a magnetic stirring reaction bottle, a magnet was added, and the reaction bottle was preheated to 65°C at a rotation speed of 400 rpm.

[0102] (2) Enzymatic deacidification reaction: In rice bran oil with an acid value of 8 mg KOH / g, glycerol was added according to the molar ratio of free fatty acids and glycerol in the rice bran oil of 1:1.5 and homogenized for 1 min to form a uniform solution, which was preheated to 65° C. and added to a gas-liquid-solid three-phase fluidized bed reactor and a magnetic stirring reaction bottle. After the reaction was completed, the reacted rice bran oil was collected at the reactor outlet and in the magnetic stirring reaction bottle.

[0103] The rice bran oil collected in the reactor is a mixture of deacidified rice bran oil and diglyceride prepared synchronously, i.e., a rice bran oil product rich in diglyceride. The acid value of the rice bran oil product is 1.45 mg KOH / g, wherein the content of diglyceride is 25%. The rice bran oil collected in the magnetic stirring reaction bottle has an acid value of 5.49 mg KOH / g.

[0104] Example 7

[0105] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0106] Step (1) and step (2) are the same as in Example 2.

[0107] (3) Repeated reaction in the reactor: After the rice bran oil in the reactor is collected, a uniform solution of high acid value rice bran oil and glycerol is added without adding new immobilized commercial enzyme, and the reaction is continued. The reaction is repeated 10 times, and the rice bran oil after the reaction is collected.

[0108] The collected rice bran oil is a mixture of deacidified rice bran oil and diglyceride prepared synchronously, i.e. a rice bran oil product rich in diglyceride. The acid value of the rice bran oil product is measured to be between 0.5 and 1.6 mg KOH / g, and the content of diglyceride is 25 to 38%.

[0109] Example 8

[0110] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0111] (1) Construction of gas-liquid-solid three-phase fluidized bed reactor: Lipase (CALB) from Candida antarctica was dissolved in phosphate buffer (50 mM, pH = 7) to prepare an enzyme solution of 80 mg / mL, and then the hydrophobic mesoporous silica modified with n-octylsilane was added to a ground-mouth conical flask at a ratio of 10 mg / mL to the enzyme solution. After reacting on a shaker (200 rpm, 30°C) for 30 min, the mixture was centrifuged and the precipitate was freeze-dried to obtain the immobilized enzyme. A certain amount of immobilized enzyme was added to the gas-liquid-solid three-phase fluidized bed reactor, dry nitrogen was introduced, and the reactor was preheated to 65°C.

[0112] (2) Enzymatic deacidification reaction: Monoglyceride was added to rapeseed oil with an acid value of 6 mg KOH / g according to the molar ratio of free fatty acids and monoglyceride in rapeseed oil of 1:3. After homogenization for 1 min and preheating to 65°C, the mixture was added to the reactor. After the reaction was completed, the rapeseed oil after the reaction was collected at the outlet of the reactor.

[0113] The collected rapeseed oil is a mixture of deacidified rapeseed oil and diglyceride prepared simultaneously, i.e., a rapeseed oil product rich in diglyceride. According to the measurement, the acid value of the rapeseed oil product is 0.7 mg KOH / g, and the content of diglyceride is 8.2%.

[0114] Example 9

[0115] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0116] (1) Construction of gas-liquid-solid three-phase fluidized bed reactor: Phospholipase A1 (LPA1) fermented from Mortierella alpina was diluted 1-fold with phosphate buffer (50 mM, pH = 7) to obtain an enzyme solution, and then added to a ground-mouth conical flask with a macroporous resin washed with acid and alkali at a ratio of 1:50 to the enzyme solution. After reacting on a shaker (200 rpm, 30°C) for 30 minutes, the immobilized enzyme was taken out and filtered, and air-dried with cold air to obtain an immobilized enzyme. A certain amount of immobilized enzyme was added to the gas-liquid-solid three-phase fluidized bed reactor, dry nitrogen was introduced, and the reactor was preheated to 35°C.

[0117] (2) Enzymatic deacidification reaction: Glycerol was added to rapeseed oil with an acid value of 6.9 mg KOH / g at a molar ratio of free fatty acids to glycerol of 1:6. The mixture was homogenized for 1 min and preheated to 35° C. before being added to the reactor. After the reaction was completed, the rapeseed oil was collected at the reactor outlet.

[0118] The collected rapeseed oil is a mixture of deacidified rapeseed oil and diglyceride prepared simultaneously, i.e., a rapeseed oil product rich in diglyceride. According to the measurement, the acid value of the rapeseed oil product is 1.9 mg KOH / g, and the content of diglyceride is 14.6%.

[0119] Example 10

[0120] A method for enzymatic deacidification of oil and fat and preparation of diglyceride based on a gas-liquid-solid three-phase fluidized bed, specifically comprising the following steps:

[0121] Step (1) is the same as in Example 9.

[0122] (2) Enzymatic deacidification reaction: Glycerol was added to rice bran oil with an acid value of 25.6 mg KOH / g at a molar ratio of free fatty acids to glycerol of 1:6. The mixture was homogenized for 1 min and preheated to 35° C. before being added to a reactor. After the reaction was completed, the reacted rice bran oil was collected at the reactor outlet.

[0123] The collected rice bran oil is a mixture of deacidified rice bran oil and diglyceride prepared synchronously, i.e. a rapeseed oil product rich in diglyceride. The acid value of the rice bran oil product is measured to be 2.8 mg KOH / g, and the content of diglyceride is 33.2%.

[0124] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A gas-liquid-solid three-phase fluidized bed enzyme reactor, characterized in that: The invention comprises an inert gas control device (1), a main reaction column (2) and a water bath heating circulation device (3); the main reaction column (2) comprises a gas-liquid distributor composed of a porous sieve plate (207) and a wire mesh demister (208), and the porous sieve plate (207) is at least arranged above the wire mesh demister (208); the porous sieve plate (207) is connected to an inert gas inlet control valve (109).

2. The gas-liquid-solid three-phase fluidized bed enzyme reactor according to claim 1, characterized in that: The main reaction column (2) comprises a lower part consisting of a finished product outlet control valve (209), an inert gas inlet control valve (109), a porous sieve plate (207) and a wire mesh demister (208), a middle part with end surface fixing bolts (206) and a heating jacket (305), and an upper part consisting of an oil inlet control valve (204), a finished product outlet control valve (209) and an inert gas outlet control valve (110); the upper part, the middle part and the lower part of the main reaction column (2) are connected in sequence through a flange disc, and the flange disc has two circular grooves matching the double-layer reaction column and is sealed with an O-ring.

3. The gas-liquid-solid three-phase fluidized bed enzyme reactor according to claim 1, characterized in that: The inert gas control device (1) comprises, in order according to the gas passage, an air compressor (101), an air compression tank (102), a refrigerated dryer (103), an inert gas manufacturing machine (104), and a drain valve (106) arranged in the pipelines between the air compression tank (102), the refrigerated dryer (103), and the inert gas manufacturing machine (104), an inert gas purity monitor (107) arranged in the inert gas manufacturing machine (104), and a flow meter (105) and a pressure gauge (108) arranged in the connecting pipeline between the inert gas manufacturing machine (104) and the inert gas inlet control valve (109).

4. The gas-liquid-solid three-phase fluidized bed enzyme reactor according to claim 1, characterized in that: The water bath heating circulation device (3) comprises, in sequence, a water bath heating device (301), a hot water inlet control valve (302), a hot water outlet control valve (303) connected by pipelines, and a PLC control system (304) connected by signals, wherein the hot water inlet control valve (302) is arranged at the lower part of the main reaction column (2), and the hot water outlet control valve (303) is arranged at the upper part of the main reaction column (2) and connected to the water bath heating device (301) through a return water pipeline; the water bath heating temperature is 30-90°C.

5. A method for the simultaneous enzymatic deacidification of oils and greases and the preparation of diglycerides, characterized in that: A high acid value oil and an acyl acceptor are mixed in the absence of a solvent to obtain a raw material liquid, and immobilized enzyme particles are fluidized and moved in a three-phase system of inert gas-high acid value oil-immobilized enzyme by continuous bubbling of an inert gas, and an esterification / ester exchange reaction occurs in a gas-liquid-solid three-phase fluidized bed enzyme reactor as described in any one of claims 1 to 4, thereby achieving deacidification of the high acid value oil and simultaneous preparation of diglyceride.

6. The preparation method according to claim 5, characterized in that: The acyl acceptor is one or a combination of glycerol and monoglyceride, the high acid value oil is a vegetable oil or microbial oil with an acid value of 5-50 mg KOH / g, and the molar ratio of the acyl acceptor to the free fatty acids in the high acid value oil is 0.5:1 to 8:

1.

7. The preparation method according to claim 6, characterized in that: The vegetable oil includes rice bran oil, rapeseed oil, soybean oil, corn oil, sesame oil, sunflower seed oil or linseed oil, and the microbial oil includes DHA or ARA algae oil.

8. The preparation method according to claim 5, characterized in that: The specific reaction steps of the method for simultaneous enzymatic deacidification of oil and fat and preparation of diglyceride in a gas-liquid-solid three-phase fluidized bed enzyme reactor are as follows: A preheated mixed raw material liquid of acyl acceptor and high acid value oil is added to a fluidized bed reactor equipped with a water bath heating device; an inert gas control device (1) is started to introduce high-purity dry inert gas into the inert gas inlet (109) at the lower end of the fluidized bed, and the inert gas is dispersed by a wire mesh demister (208) and a porous sieve plate (207) in a gas-liquid distributor and then enters a main reaction column (2) to form a continuous bubbling gas; after adding immobilized lipase particles into the reaction column, the immobilized enzyme is made to move upward in a disordered manner by gas bubbling, and the gas pressure gauge (108) and the flow meter (105) are adjusted to control the gas pressure and gas velocity, so as to realize the fluidized movement of the immobilized enzyme particles in the three-phase system of inert gas-high acid value oil-immobilized enzyme, thereby causing esterification / ester exchange reaction to occur, thereby realizing the simultaneous preparation of enzymatic deacidification of oil and fat and diglyceride.

9. The preparation method according to claim 8, characterized in that: The inert gas inlet pressure is 0.1-5MPa, and the gas flow rate is 0-1000mL / s; the immobilized enzyme addition amount is 1%-5% of the mass of the high acid value oil, and the reaction time of the oil enzymatic deacidification and synchronous preparation of diglyceride is 0.5h-6h.

10. The preparation method according to claim 5, characterized in that: The method for simultaneous enzymatic deacidification of oil and fat and preparation of diglyceride is carried out in a gas-liquid-solid three-phase fluidized bed enzyme reactor in a semi-continuous manner.

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