Self-assembly microsphere reactor for dairy product processing as well as preparation method and application of self-assembly microsphere reactor

Abstract

The invention discloses a self-assembly microsphere reactor for dairy product processing and a preparation method and application thereof. The preparation method comprises the following steps: preparing silicon dioxide colloid nanoparticles as a water phase and n-hexadecane as an oil phase, and preparing a receiving solution containing a surfactant; preparing a water-in-oil emulsion from the prepared oil phase and water phase, shearing the water-in-oil emulsion into liquid drops, and collecting the liquid drops into a receiving liquid containing a surfactant; evaporating a water phase in the formed liquid drops to obtain self-assembled silicon dioxide microspheres; and finally, filling the obtained self-assembled silicon dioxide microspheres into a quartz column to obtain the self-assembled silicon dioxide microspheres. The self-assembly microsphere reactor provides an efficient reactor model for lactase hydrolysis reaction, and has a promoting effect on improving lactase hydrolysis efficiency and stability; meanwhile, the risk of activity loss caused by lactase falling is reduced, and the reusability of a catalytic system is greatly enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of food materials, and in particular relates to a self-assembling microsphere reactor for dairy product processing, a preparation method and an application thereof. Background Art

[0002] With increasing concerns about lactose intolerance and excessive sugar intake, sugar-free milk has become a popular healthy drink.

[0003] Lactose is the main sugar in milk, with a content of more than 99%. Enzymatic hydrolysis is currently an effective technology for producing sugar-free milk. Enzymes are powerful biocatalysts that mediate all biological processes in organisms. Lactase, also known as β-galactosidase, can hydrolyze lactose into galactose and glucose. Therefore, effective enzyme microcarriers are essential for enzymatic reactions. A key step in the evolution of eukaryotic organisms is the development and positional assembly capabilities of multi-compartment subcellular organelles, which improve the overall efficiency and specificity of biological reactions. The multiple enzymes in these unique internal structures provide eukaryotic cells with the ability to control multi-step metabolic reactions in time and space. Inspired by the multi-enzyme structure in eukaryotic cells, researchers are committed to creating engineered enzyme-linked reaction systems with synergistic and complementary functions, which are based on liposomes or polymers, polymer-based capsules and Pickering emulsions. These systems have important applications in enzyme replacement therapy, biodetoxification, chemical synthesis and sensors. However, due to the relatively simple structure of these enzyme microcarriers, it is difficult to control the number, type and spatial arrangement of enzymes in the system, which also brings difficulties to the quantitative control of cascade reactions and reduces the efficiency of biocatalysts. In addition, most enzyme microcarrier systems still have the problem of insufficient enzymatic hydrolysis capacity. Therefore, it is still worth looking forward to construct robust microcarriers as sustainable multi-enzyme systems with spatiotemporal control functions.

[0004] Opal structures are colloidal crystals grown by self-assembly of monodisperse colloidal particles of nanometer to micrometer size, usually with a face-centered cubic structure. Due to their three-dimensional ordered close-packed structure and huge specific surface area, they are ideal enzyme microcarriers, which can greatly improve the enzyme degradation rate and stability. Despite great success, the application of such self-assembled opal-structured microspheres in sugar degradation has not been studied, and it is difficult to evaluate their role in the development of sugar-free buffalo milk. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a self-assembled microsphere reactor for dairy product processing and a preparation method thereof in view of the deficiencies in the prior art, which provides an efficient reactor model for lactase hydrolysis reaction, while reducing the risk of activity loss caused by lactase shedding, and greatly enhancing the reusability of the catalytic system.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing a self-assembled microsphere reactor for dairy product processing comprises the following steps: Step 1: Prepare silica colloidal nanoparticles as a water phase and n-hexadecane as an oil phase; Step 2, preparing a receiving solution containing a surfactant; Step 3: The oil phase and the water phase prepared in step 1 are prepared into a water-in-oil emulsion, and the emulsion is sheared into droplets and collected into a receiving liquid containing a surfactant; Step 4, evaporating the water phase in the droplets formed in step 3 to obtain self-assembled silica microspheres; Step 5: Fill the self-assembled silica microspheres obtained in step 4 into a quartz column.

[0007] Specifically, in step 1, the concentration of the silica colloidal nanoparticles in the aqueous phase is 10-15wt%; the silica colloidal nanoparticles are uniformly dispersed by ultrasound; and the silica colloidal nanoparticles refer to lactase-loaded silica colloidal nanoparticles.

[0008] Specifically, the preparation process of the lactase-loaded silica colloidal nanoparticles is as follows: S1. Preparation of high quality silica nanoparticles: Tetraethoxysilane is dissolved in ethanol, and water and ammonia catalyst are slowly added to perform hydrolysis and condensation reactions to form nanoparticles with uniform particle size and good dispersibility; S2. Functionalization treatment: Functionalizing the surface of the silica nanoparticles to obtain amino-functionalized silica nanoparticles; S3, enzyme coupling: Lactose hydrolase molecules are combined with amino-functionalized silica nanoparticles by using a cross-linking agent to obtain stable lactase-loaded silica colloidal nanoparticles.

[0009] Specifically, in step 2, the surfactant is hypermer 2296, the receiving liquid is n-hexadecane, and the concentration of the surfactant in the receiving liquid is 2-3 wt%.

[0010] Specifically, in step three, the water-in-oil emulsion prepared is an emulsion of silica colloidal nanoparticles encapsulated by n-hexadecane.

[0011] Specifically, in step three, the specific method for shearing the oil-in-water emulsion into droplets is: pushing the oil phase and the water phase to the oil-water interface by a peristaltic pump, and utilizing the combined effect of the oil-water interfacial tension and the oil phase shear force to shear the liquid into droplets.

[0012] Specifically, in step 4, the evaporation temperature is 40-50° C.; self-assembled silica microspheres refer to silica colloidal nanoparticles that are orderly assembled into spherical aggregates in three-dimensional space, which have a tightly arranged hexagonal close-packed structure.

[0013] Furthermore, in step five, the specific method of loading the self-assembled silica microspheres into the quartz column is as follows: loading the self-assembled silica microspheres into the quartz column, filling the entire column without leaving dead volume, and sealing to obtain a self-assembled microsphere reactor for dairy product processing.

[0014] Specifically, the sealing adopts a filter membrane with a pore size of 0.2-0.22 microns to seal both ends of the quartz column.

[0015] Furthermore, the self-assembled microsphere reactor prepared by the above preparation method is also within the protection scope of the present invention.

[0016] Furthermore, the present invention also claims to protect the use of the self-assembled microsphere reactor in the lactose-reducing process of dairy products. Beneficial Effects

[0017] (1) The self-assembled microsphere reactor for dairy product processing of the present invention is prepared by filling a quartz column with self-assembled silica microspheres. The self-assembled silica microspheres have an ordered structure arranged in three-dimensional space, which provides a huge specific surface area for the mass transfer of enzymes; since the size of silica colloidal nanoparticles is adjustable, the specific surface area of ​​the self-assembled silica microspheres can be adjusted according to actual needs, which is conducive to accurately controlling the amount and spatial distribution of the loaded enzyme.

[0018] (2) The self-assembled microsphere reactor for dairy product processing of the present invention provides an efficient reactor model for lactase hydrolysis reaction, which promotes the improvement of lactase hydrolysis efficiency and stability; at the same time, it reduces the risk of activity loss caused by lactase shedding, and greatly enhances the reusability of the catalytic system.

[0019] (3) The self-assembled microsphere reactor for dairy product processing of the present invention can achieve high-throughput production without increasing equipment investment. It can decompose lactose without destroying the nutrients in milk, and the decomposition products are easily absorbed by the human body. These advantages make the self-assembled microsphere reactor for dairy product processing of the present invention show good performance in the application of buffalo milk glucose reduction.

[0020] (4) The present invention The self-assembled microsphere reactor for dairy product processing provided by the present invention is simple to prepare. The formed self-assembled microsphere reactor for dairy product processing can provide a large specific surface area and active sites, has excellent practicality and promotion, and will have broad application prospects in the food field in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0022] Figure 1 It is a schematic diagram of the preparation process of the self-assembled microsphere reactor for dairy product processing of the present invention, wherein 1 is a lactase-loaded silica colloidal nanoparticle solution, 2 is a self-assembled silica microsphere, and 3 is a self-assembled microsphere reactor for dairy product processing.

[0023] Figure 2 Electron micrographs of the interior and cross-section of self-assembled silica microspheres in a self-assembled microsphere reactor for dairy processing.

[0024] Figure 3 Light microscopy images of self-assembled silica microspheres prepared from lactase-loaded silica colloidal nanoparticles of different sizes. DETAILED DESCRIPTION

[0025] The present invention can be better understood with reference to the following examples. Example 1

[0026] Preparation of Lactase-loaded Silica Colloidal Nanoparticles: (1) Solution preparation: Mixture A: Dissolve 2 ml of tetraethyl orthosilicate (TEOS) in 50 ml of anhydrous ethanol. Mixture B: Dissolve 10 ml of aqueous ammonia in 50 ml of anhydrous ethanol. Mixture C: Dissolve 2 ml of tetraethyl orthosilicate in 100 ml of anhydrous ethanol. Aminosilane solution: 3-aminopropyltriethoxysilane (APTES), concentration is 1-5% (w / v). Activation solution: N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride crystals (EDC) and N-hydroxysuccinimide (NHS), concentrations are 10 mM and 5 mM, respectively. Crosslinker solution: glutaraldehyde, concentration is 1% (w / v). Buffer solution: phosphate buffer solution (PBS), pH 7.4.

[0027] (2) Prepare reaction vessels and instruments: 1 three-necked flask, 500 ml. Several centrifuge tubes, 50 ml. 1 centrifuge, 10,000 rpm. 1 vacuum drying oven, adjustable temperature. 1 microfluidic chip, for preparing monodisperse emulsion droplets. 1 stirrer, adjustable speed. 1 oven, adjustable temperature.

[0028] (3) Preparation of silica nanoparticles: Preheat the mixed solutions A, B, and C in an oven to make them the same temperature as the reaction system. Add solution A and solution B to a three-necked flask with a mechanical stirrer and start stirring at the same time at a speed of about 800 rpm. After 30 minutes of reaction, add 10 ml of ammonia water from the side of the three-necked flask. After another 5-10 minutes of reaction, add solution C to the system and continue the reaction for 2 hours before stopping. High-quality silica nanoparticles can be obtained by centrifugal washing and purification.

[0029] (4) Functionalization of nanoparticles: Disperse silica nanoparticles in anhydrous ethanol to form a uniform suspension. Add aminosilane (3-aminopropyltriethoxysilane, APTES) to the suspension and stir the reaction at room temperature for 4 hours. After the reaction is completed, remove the unreacted aminosilane and solvent by centrifugation. Wash the obtained amino-functionalized silica nanoparticles with anhydrous ethanol three times to remove residual reactants. Dry the washed amino-functionalized silica nanoparticles in a vacuum drying oven to obtain stable functionalized nanoparticles.

[0030] (6) Coupling of enzyme and functionalized nanoparticles: N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride crystals (EDC) and N-hydroxysuccinimide (NHS) are used to activate silica colloidal nanoparticles. Covalent bonding is performed using a cross-linking agent (such as glutaraldehyde). Lactose hydrolase is mixed with functionalized silica colloidal nanoparticles in the presence of a cross-linking agent and reacted overnight. After gentle washing with a buffer solution, the result is obtained. Example 2

[0031] This embodiment provides a self-assembled microsphere reactor for dairy product processing, such as Figure 1 As shown, the preparation method thereof comprises the following steps: Step 1: preparing silica colloidal nanoparticles as a water phase and n-hexadecane as an oil phase, comprising: The silica colloidal nanoparticles loaded with lactase are uniformly dispersed in the aqueous phase solution by ultrasound, and the concentration thereof is configured to be 10wt%. The silica colloidal nanoparticles loaded with lactase of different sizes are selected to configure 10wt% silica colloidal nanoparticle solutions of different sizes. The silica colloidal nanoparticles loaded with lactase are used as the aqueous phase, and n-hexadecane is used as the oil phase.

[0032] Step 2: preparing a receiving solution containing a surfactant, comprising: 2 wt% of the surfactant hypermer 2296 was added to n-hexadecane and mixed evenly. The obtained receiving liquid containing the surfactant was poured into a collection container, and the collection container was placed on a hot table for preheating.

[0033] Step 3, preparation of oil-in-water droplets: The oil phase and water phase configured in step 1 are prepared into an oil-in-water emulsion of lactase-loaded silica colloidal nanoparticles encapsulated in n-hexadecane, and the oil phase and the water phase are pushed to the oil-water interface by a peristaltic pump. The liquid is sheared into droplets by the combined action of the oil-water interfacial tension and the shear force of the oil phase, and the droplets are collected by a preheated receiving liquid containing a surfactant.

[0034] Step 4, preparing self-assembled silica microspheres: evaporating the water phase in the water-in-oil droplets formed in step 3 at a temperature of 40°C. During the gradual evaporation of the water phase, the lactase-loaded silica colloidal nanoparticles gradually self-assemble into spherical aggregates in a three-dimensional space. After the water phase is completely evaporated, it exhibits a tightly arranged hexagonal close-packed structure, thereby obtaining self-assembled silica microspheres.

[0035] Step 5. Preparation of a self-assembled microsphere reactor for dairy product processing: The self-assembled silica microspheres obtained in step 4 are loaded into a quartz column, filling the entire column without leaving any dead volume, and sealed with a filter membrane with a pore size of 0.2 microns to obtain a self-assembled microsphere reactor for dairy product processing.

[0036] The structure of the self-assembled silica microspheres in the self-assembled microsphere reactor for dairy processing was characterized. The internal structure and cross-section of the self-assembled silica microspheres were characterized by scanning electron microscopy. The results are as follows Figure 2 The results show that the surface of the silica microspheres presents a large area of ​​particles arranged in a long-range orderly hexagonal close-packed structure ( Figure 2 A), reflecting the high-quality particle assembly effect. This ordered structure exists not only on the surface of the microspheres, but also inside the microspheres, such as Figure 2 B shows a scanning electron microscope photograph of a cross-section of a silica microsphere. From the photograph, one can not only observe the tight and orderly arrangement of the particles inside the microsphere, but also clearly see the different levels of the particle opal structure.

[0037] The specific surface area of ​​the self-assembled silica microspheres in the self-assembled microsphere reactor for dairy product processing was adjusted as follows: lactase-loaded silica colloidal nanoparticles with sizes of 210 nm, 250 nm and 290 nm were used to prepare self-assembled silica microspheres of different sizes using the above method. The results are as follows: Figure 3 As shown. Figure 3It was observed from the light microscopy images that the self-assembled silica microspheres of different sizes all showed a uniform spherical morphology, and the surface was very smooth without obvious cracks, indicating the high-quality particle assembly effect. In addition, as the size of lactase-loaded silica colloidal nanoparticles increased, the self-assembled silica microspheres showed different structural colors, from blue to green to red. These results indicate that the specific surface area of ​​the self-assembled silica microspheres can be controlled by adjusting the size of lactase-loaded silica colloidal nanoparticles, which is conducive to the precise control of the amount and spatial distribution of the loaded enzyme. Example 3

[0038] Application of self-assembled microsphere reactor for dairy processing in glucose reduction of buffalo milk.

[0039] The self-assembled microsphere reactor used in dairy processing was used to reduce sugar in buffalo milk as follows: First, buffalo milk is heated to 40°C and then passed into a self-assembled microsphere reactor for dairy processing, where it is fully contacted with silica microspheres self-assembled by lactase loaded in the self-assembled microsphere reactor for dairy processing. The lactose in the buffalo milk treated by the self-assembled microsphere reactor for dairy processing is decomposed into galactose and glucose, and then the treated buffalo milk is filtered through the filter membrane of the self-assembled microsphere reactor for dairy processing, ultimately obtaining sugar-free buffalo milk while retaining its other nutrients.

[0040] The present invention provides a self-assembled microsphere reactor for dairy product processing and a preparation method and application thereof. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a self-assembled microsphere reactor for dairy product processing, characterized in that: The steps include: Step 1: Prepare silica colloidal nanoparticles as a water phase and n-hexadecane as an oil phase; Step 2, preparing a receiving solution containing a surfactant; Step 3: The oil phase and the water phase prepared in step 1 are prepared into a water-in-oil emulsion, and the emulsion is sheared into droplets and collected into a receiving liquid containing a surfactant; Step 4, evaporating the water phase in the droplets formed in step 3 to obtain self-assembled silica microspheres; Step 5: Fill the self-assembled silica microspheres obtained in step 4 into a quartz column.

2. The method for preparing a self-assembled microsphere reactor for dairy product processing according to claim 1, characterized in that: In step 1, the concentration of the silica colloidal nanoparticles in the aqueous phase is 10-15wt%; the silica colloidal nanoparticles are uniformly dispersed by ultrasound; and the silica colloidal nanoparticles refer to lactase-loaded silica colloidal nanoparticles.

3. The method for preparing a self-assembled microsphere reactor for dairy product processing according to claim 1, characterized in that: In step 2, the surfactant is hypermer 2296, the receiving liquid is n-hexadecane, and the concentration of the surfactant in the receiving liquid is 2-3 wt %.

4. The method for preparing a self-assembled microsphere reactor for dairy product processing according to claim 1, characterized in that: In step 3, the water-in-oil emulsion prepared is a silica colloidal nanoparticle emulsion encapsulated by n-hexadecane.

5. The method for preparing a self-assembled microsphere reactor for dairy product processing according to claim 1, characterized in that: In step three, the specific method of shearing the oil-in-water emulsion into droplets is: pushing the oil phase and the water phase to the oil-water interface by a peristaltic pump, and utilizing the combined effect of the oil-water interfacial tension and the oil phase shear force to shear the liquid into droplets.

6. The method for preparing a self-assembled microsphere reactor for dairy product processing according to claim 1, characterized in that: In step 4, the evaporation temperature is 40-50° C.; self-assembled silica microspheres refer to silica colloidal nanoparticles orderly assembled into spherical aggregates in three-dimensional space, which have a tightly arranged hexagonal close-packed structure.

7. The method for preparing a self-assembled microsphere reactor for dairy product processing according to claim 1, characterized in that: In step five, the self-assembled silica microspheres are loaded into a quartz column. The specific method is as follows: the self-assembled silica microspheres are loaded into the quartz column to fill the entire column without leaving any dead volume, and then sealed to obtain a self-assembled microsphere reactor for dairy product processing.

8. The method for preparing a self-assembled microsphere reactor for dairy product processing according to claim 7, characterized in that: The seal uses a filter membrane with a pore size of 0.2-0.22 microns to seal both ends of the quartz column.

9. The self-assembled microsphere reactor prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the self-assembled microsphere reactor according to claim 9 in the lactose-reducing process of dairy products.

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