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CN-122012487-A - Foam SiO2Enzyme array and application thereof in preparation of diglyceride edible oil

CN122012487ACN 122012487 ACN122012487 ACN 122012487ACN-122012487-A

Abstract

The invention discloses a foam SiO 2 enzyme array and application thereof in preparation of diglyceride edible oil, belonging to the technical field of grease chemistry. The preparation method comprises the steps of preparing a hydrophobic foam SiO 2 carrier (MCF-C 8 ), respectively loading lipase ANL, lipase NE and lipase B by a physical adsorption method, preparing three functional specific enzyme arrays, constructing a double-coupling reaction strategy of hydrolysis-esterification and glycerolysis-esterification, catalyzing proper hydrolysis of vegetable oil by ANL@MCF-C 8 or glycerolysis of vegetable oil by NE@MCF-C 8 , obtaining an initial diglyceride mixture, recovering a byproduct fatty acid generated by the reaction, and carrying out esterification reaction with glycerol under the catalysis of CALB@MCF-C 8 to obtain a secondary diglyceride mixture. And purifying to obtain the edible oil product with 40-82% of diglyceride and 90% of 1, 3-diglyceride. The invention realizes the high-efficiency recycling of the byproduct fatty acid, improves the product yield, has strong enzyme array stability, is easy to separate and recycle, can be reused, and effectively reduces the production cost.

Inventors

  • ZHENG MINGMING
  • ZHANG YUFEI
  • LU JINMEI
  • WU LIUMEI

Assignees

  • 湖北洪山实验室

Dates

Publication Date
20260512
Application Date
20260416

Claims (9)

  1. 1. The preparation method of the foam SiO 2 enzyme array is characterized by comprising the following steps of: S1, constructing a framework by copolycondensation of tetraethyl orthosilicate and 1, 2-bis (triethoxysilyl) ethane serving as composite silicon sources, and extracting, washing and drying by a mixed solution of hydrochloric acid and ethanol to obtain a foam SiO 2 carrier MCF with a hierarchical pore structure, wherein the specific surface area of the MCF is 800-952 m 2 /g, the mesoporous aperture is 11-14 nm, and the contact angle is 27-30 degrees; S2, dispersing MCF in n-hexane solution containing n-octyl trichlorosilane, carrying out ultrasonic treatment and oscillation, carrying out surface hydrophobization modification, filtering, washing and drying to obtain a hydrophobic foam SiO 2 carrier MCF-C 8 with a hierarchical pore structure, wherein the specific surface area of the MCF-C 8 is 432-500 m 2 /g, the mesoporous aperture is 11-13 nm, and the contact angle is 99-103 degrees; S3, taking MCF-C 8 as a carrier, respectively loading Aspergillus niger lipase, thermomyces lanuginosus lipase and candida antarctica lipase B by a physical adsorption method, and correspondingly preparing the foam SiO 2 enzyme array ANL@MCF-C 8 with high-efficiency hydrolytic activity, the foam SiO 2 enzyme array NE@MCF-C 8 with high-efficiency glycerolysis activity and the foam SiO 2 enzyme array CALB@MCF-C 8 ; ANL@MCF-C 8 、NE@MCF-C 8 and CALB@MCF-C 8 with high-efficiency esterification activity, wherein the solid contents are 132-142 mg/g, 100-156 mg/g and 130-150 mg/g respectively.
  2. 2. The foam SiO 2 enzyme array according to claim 1, wherein the copolycondensation building skeleton in the step S1 is characterized in that polyether P123, 1,3, 5-trimethylbenzene, ammonium fluoride and hydrochloric acid are stirred for 1h at 37 ℃, tetraethyl orthosilicate and 1, 2-bis (triethoxysilyl) ethane are sequentially added, stirring is continued for 12h, then the mixture is transferred into an autoclave, subjected to static hydrothermal aging for 24h, cooled, washed by absolute ethyl alcohol and dried; The volume ratio of the polyether P123 to the 1,3, 5-trimethylbenzene is 3-4 g/3.45-4.6 mL; the mass ratio of the polyether P123 to the ammonium fluoride is 3-4:0.035-0.046; the volume ratio of the 1,3, 5-trimethylbenzene to the hydrochloric acid is 3.45-4.6:15-20, and the concentration of the hydrochloric acid is 1.6mol/L; the mass ratio of the tetraethyl orthosilicate to the 1, 2-bis (triethoxysilyl) ethane is 3-1:1-3; The mass ratio of the polyether P123 to the tetraethyl orthosilicate is 3-4:2.2-6.6; The static hydrothermal aging temperature is 100 ℃, the drying temperature is 60 ℃ and the time is 12h.
  3. 3. The foam SiO 2 enzyme array according to claim 1, wherein the volume fraction of hydrochloric acid in the mixed solution of hydrochloric acid and ethanol in the step S1 is 1-2%, and the concentration of the used hydrochloric acid is 12mol/L; The extraction time is 6 hours, and the process is repeated for three times; The ethanol for washing; The drying temperature is 60 ℃ and the time is 12h.
  4. 4. The foam SiO 2 enzyme array according to claim 1, wherein the volume ratio of n-octyl trichlorosilane to n-hexane in the n-hexane solution containing n-octyl trichlorosilane in step S2 is 0.46:20; The volume ratio of the mass of the MCF to the n-octyl trichlorosilane is 1 g:0.46-1.30 mL; The ultrasonic treatment time is 10min, the oscillating speed is 250rpm, the temperature is 25 ℃, and the ultrasonic treatment time is 2h; the drying temperature is 60 ℃ and the time is 12 hours.
  5. 5. The foam SiO 2 enzyme array according to claim 1, wherein the physical adsorption method in step S3 is specifically: preparing enzyme solutions of Aspergillus niger lipase, thermomyces lanuginosus lipase and candida antarctica lipase B by using a phosphate buffer solution respectively, then mixing MCF-C 8 soaked by ethanol with the enzyme solutions, carrying out ultrasonic treatment, vacuumizing, removing gas in a pore canal, incubating by a shaker, centrifuging, collecting precipitate, and freeze-drying to obtain an enzyme array; the concentration of the enzyme solution of the Aspergillus niger lipase is 125-150 mg/mL, the concentration of the phosphate buffer solution in the preparation is 50mM/L, and the pH is 5; The concentration of the enzyme solution of the thermomyces lanuginosus lipase is 20-50 mg/mL, the concentration of the phosphate buffer solution in the preparation is 50mM/L, and the pH is 7; the concentration of the enzyme solution of the candida antarctica lipase B is 40-60 mg/mL, the concentration of the phosphate buffer solution in the preparation is 50mM/L, and the pH value is 7; The ratio of the MCF-C 8 to the enzyme solution is 1g to 100mL, the ultrasonic time is 5min, the vacuumizing time is 5min, the temperature of shaking table incubation is 30 ℃, the rotating speed is 220rpm, and the time is 40min; The rotational speed of the centrifugation is 8000rpm and the time is 10min.
  6. 6. A method for preparing edible oil rich in diglyceride by using a foam SiO 2 enzyme array, which is characterized in that the foam SiO 2 enzyme array ANL@MCF-C 8 and CALB@MCF-C 8 as claimed in claim 1 are applied, and the method comprises the following steps: S1, carrying out hydrolysis reaction, namely uniformly mixing vegetable oil, water and ANL@MCF-C 8 , carrying out hydrolysis reaction, obtaining a primary diglyceride mixture through centrifugal separation after the reaction is finished, recovering ANL@MCF-C 8 , purifying the primary diglyceride mixture through molecular distillation, obtaining edible oil rich in diglycerides, and recovering free fatty acid; S2, carrying out esterification reaction, namely uniformly mixing the recovered free fatty acid, glycerol and CALB@MCF-C 8 , carrying out esterification reaction, obtaining a secondary diglyceride mixture through centrifugal separation after the reaction is finished, and obtaining the edible oil rich in diglyceride after molecular distillation and purification.
  7. 7. The method of claim 6, wherein the addition amount of ANL@MCF-C 8 in the step S1 is 0.5-3% of the mass of vegetable oil, the mass ratio of the vegetable oil to water is 1:0.4-1:1, the hydrolysis reaction temperature is 25-45 ℃ and the time is 1-8 hours, and the molecular distillation purification conditions are that the heating temperature is 170-190 ℃, the feeding speed is 10-15 kg/h, the vacuum degree is 0.1-10 Pa and the scraper rotating speed is 200-300 r/min; In the step S2, the addition amount of CALB@MCF-C 8 is 1-5% of the total mass of free fatty acid and glycerin, the molar ratio of the free fatty acid to the glycerin is 1:1-1:3, the esterification reaction temperature is 40-80 ℃ and the time is 2-12 hours, the molecular distillation purification condition is that the heating temperature is 170-190 ℃, the feeding speed is 10-15 kg/h, the vacuum degree is 0.1-10 Pa, and the scraper rotating speed is 200-300 r/min.
  8. 8. A method for preparing edible oil rich in diglyceride by using a foam SiO 2 enzyme array, which is characterized in that the foam SiO 2 enzyme array NE@MCF-C 8 and CALB@MCF-C 8 as claimed in claim 1 are applied, and the method comprises the following steps: S1, glycerolysis reaction, namely uniformly mixing vegetable oil, glycerol, water and NE@MCF-C 8 , stirring for reaction, obtaining a primary diglyceride mixture through centrifugal separation after the reaction is finished, recovering NE@MCF-C 8 , purifying the primary diglyceride mixture through molecular distillation, obtaining edible oil rich in diglycerides, and recovering free fatty acid; S2, carrying out esterification reaction, namely uniformly mixing the recovered free fatty acid, glycerol and CALB@MCF-C 8 , carrying out esterification reaction, obtaining a secondary diglyceride mixture through centrifugal separation after the reaction is finished, and obtaining the edible oil rich in diglyceride after molecular distillation and purification.
  9. 9. The method of claim 8, wherein in the step S1, the addition amount of NE@MCF-C 8 is 1-3% of the mass of vegetable oil, the addition amount of water is 1-2% of the mass of vegetable oil, the molar ratio of vegetable oil to glycerin is 1:1-1:4, the stirring reaction temperature is 45-65 ℃, the reaction time is 6-12 h, the molecular distillation purification condition is that the heating temperature is 170-190 ℃, the feeding speed is 10-15 kg/h, the vacuum degree is 0.1-10 Pa, and the scraper rotating speed is 200-300 r/min; In the step S2, the addition amount of CALB@MCF-C 8 is 1-5% of the total mass of free fatty acid and glycerin, the molar ratio of the free fatty acid to the glycerin is 1:1-1:3, the esterification reaction temperature is 40-80 ℃ and the time is 2-12 hours, the molecular distillation purification condition is that the heating temperature is 170-190 ℃, the feeding speed is 10-15 kg/h, the vacuum degree is 0.1-10 Pa, and the scraper rotating speed is 200-300 r/min.

Description

Foam SiO 2 enzyme array and application thereof in preparation of diglyceride edible oil Technical Field The invention belongs to the technical field of grease chemistry, and particularly relates to a foam SiO 2 enzyme array and application thereof in preparation of diglyceride edible oil. Background The diglyceride (Diacylglycerol, DAG for short) is used as a functional lipid with multiple physiological activities of preventing obesity, improving cardiovascular health and the like, and has wide application prospect in the fields of food, health care products and medicines. At present, the core technology for industrially preparing diglyceride mainly depends on a free enzyme preparation and is realized through a single linear synthesis route such as a hydrolysis method, a glycerolysis method, an esterification method and the like. However, these conventional technical routes have a number of bottlenecks that are difficult to break. In the catalyst layer, free lipase is easy to be deactivated by the factors of temperature, pH value, substrate concentration and the like in a reaction system, and is difficult to recycle after the reaction is finished, so that an enzyme preparation cannot be recycled, the enzyme consumption cost in the production process is obviously increased, meanwhile, the catalytic specificity of the free lipase is insufficient, side reaction is easy to be initiated, and the purity of a product is influenced. In order to alleviate the problem, some prior arts try to use commercial immobilized enzymes (such as Lipozyme TL IM and Novozyme 435), but carrier materials of the commercial enzymes have structural defects that the Lipozyme TL IM takes inorganic silica gel as a carrier, has single pore channel structure and strong surface hydrophilicity, and in an oil-water biphasic reaction system, the interaction between enzyme molecules and the carrier is weak, and is easy to fall off and loss, so that the catalytic efficiency is continuously reduced, the Novozyme 435 adopts organic resin as the carrier, has larger particle size and poor mechanical stability, and is easy to break the carrier in the stirring reaction process, thereby not only influencing the recovery and reuse of the enzyme, but also possibly causing the mixing of carrier impurities in the product and increasing the subsequent separation difficulty. The limitation of a single linear process further aggravates the technical pain point in the aspect of a synthetic route, the hydrolysis method is used for directly hydrolyzing vegetable oil to prepare diglyceride, the hydrolysis degree is difficult to accurately control in the reaction process, excessive hydrolysis is very easy to occur, the content of the diglyceride in the product is low, a large amount of free fatty acid and monoglyceride are generated simultaneously, serious waste of high-quality fatty acid in raw materials is caused, the processes of deacidification, purification and the like are additionally required to be additionally added for the byproducts, the production cost is obviously improved, the vegetable oil and the glycerol are used as substrates for the glycerolysis method for carrying out transesterification reaction, the viscosity of a reaction system is extremely high, the mass transfer efficiency of the substrate is low, the catalytic conversion rate is low, the reaction period is long, the byproducts such as triglyceride and monoglyceride are easy to be produced, the quality of the product is influenced, the esterification method is used for directly esterifying the free fatty acid and the glycerol to synthesize the diglyceride, the free fatty acid is prepared through the multistep processes such as the pre-hydrolysis, the separation and the purification of the vegetable oil, the substrate source route is long, the preparation cost is high, the economy of the whole process is poor, and the large-scale industrial production requirement is difficult to be satisfied. The patent report (CN 105400837A) tries to adopt a two-step process of esterifying a vegetable oil hydrolysis product, which alleviates the problem of excessive hydrolysis to a certain extent, but still has obvious defects that the content of diglyceride in the final product is only 60% -65%, the current market requirement of high-purity diglyceride (the purity is generally more than or equal to 80% and the high-end application scene is more than or equal to 90%) cannot be met, the process depends on the conventional commodity immobilized enzyme, the core problems of poor suitability of a carrier and the enzyme, limited catalytic efficiency and the like are not solved, the ratio of the functional optimal 1, 3-diglyceride isomer in the product is not effectively regulated, and the application of the product in high-end health products is further limited. Therefore, developing a foam SiO 2 enzyme array and a method for application in the preparation of diglyceride edible oil are technical problems that