Method for efficiently extracting bromelain based on magnetic chitosan microspheres

Abstract

The invention discloses a method for efficiently extracting bromelain based on magnetic chitosan microspheres, and relates to the technical field of biochemical engineering, and the method comprises the following steps: preparation of magnetic chitosan microspheres, extraction of bromelain, and purification and detection of bromelain. According to the preparation method disclosed by the invention, the magnetic chitosan microspheres with excellent performance are prepared by performing surface modification on the nanoscale ferroferric oxide particles and accurately controlling glutaraldehyde reaction and ultrasonic dispersion, and the adsorption performance is improved by 60%. The microspheres are used for specifically adsorbing bromelain, the extraction efficiency is improved by more than 30% in combination with optimized conditions, the operation time is shortened by 50% through magnetic separation, and the cost is reduced. An adsorption mechanism is defined, conditions are optimized, and the purity of bromelain is improved by 20% or above. A protective agent is added during adsorption and desorption, the retention rate of enzyme activity reaches 90% or above, the product quality and the application value are guaranteed, the application range is expanded, and good application prospects and economic values are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, in particular to a method for efficiently extracting bromelain based on magnetic chitosan microspheres. Background Art

[0002] In the field of bromelain extraction, there are many existing extraction methods based on the interaction between adsorbent and bromelain (such as "Research Progress of Bromelain Extraction Technology" which elaborates on various adsorption extraction principles), and the separation technology using magnetic materials in the field of biological separation is also relatively common. However, these existing technologies face a series of interrelated and urgent problems in practical applications.

[0003] First, in the preparation of magnetic chitosan microspheres, due to the large differences in the properties of magnetic materials and chitosan, it is difficult to accurately control the ratio of magnetic materials to chitosan using conventional material mixing methods. When the proportion of magnetic materials is too high, it will damage the biocompatibility of chitosan and destroy the specific adsorption sites for bromelain; if the proportion is too low, the magnetism will be too weak, which is not conducive to subsequent separation operations. In addition, it is difficult to achieve uniform dispersion of magnetic materials in chitosan microspheres, which will lead to uneven adsorption performance and magnetism of the microspheres, thereby affecting the subsequent extraction of bromelain. And this preparation defect further has an adverse effect on specific adsorption.

[0004] In terms of specific adsorption mechanism and optimization, due to the less than ideal preparation of magnetic chitosan microspheres, some technologies that emphasize the specific adsorption of bromelain have difficulty in clarifying their exact specific interaction mechanism. At the same time, the structure and properties of bromelain are easily affected by the extraction environment (such as temperature, pH value, ionic strength, etc.). When the performance of the microspheres is unstable, the specific adsorption effect is even more difficult to stabilize. Determining this specific interaction mechanism requires in-depth knowledge of biochemistry and materials science, and optimizing environmental factors to adapt to specific adsorption involves complex interactions of multiple factors, coupled with the uncertainty of the performance of the microspheres themselves, and it is almost impossible to find the best solution through limited experiments and conventional logical reasoning.

[0005] In terms of the active protection of bromelain, the above-mentioned magnetic chitosan microsphere preparation problem and the instability of specific adsorption cause that in the process of utilizing adsorbent to extract bromelain, the influence of adsorption and desorption process on enzyme activity is difficult to control. Due to the microsphere adsorption performance and magnetic inhomogeneity, and the instability of specific adsorption, the adsorption and desorption conditions may be strengthened to ensure the extraction efficiency, but this easily causes the desorption reagent to denature and inactivate the enzyme, or the interaction between the adsorbent and the enzyme in the adsorption process is too strong, destroying the spatial structure and active center of the enzyme. And if the relevant conditions are weakened in order to protect the enzyme activity, it is difficult to ensure the extraction efficiency. The relationship between the balance extraction efficiency and the protection of enzyme activity, under these problems of the prior art intertwined, can not be achieved simply by adjusting the conventional experimental conditions.

[0006] In summary, the problems in the prior art such as the preparation of magnetic chitosan microspheres, specific adsorption and protection of bromelain activity are interrelated and influence each other, which together lead to the dilemma of low extraction efficiency and insufficient purity of bromelain, complicated separation process and difficulty in effectively protecting enzyme activity. A new method is urgently needed to systematically solve these problems, so as to achieve the goal of efficient extraction of bromelain, simplify the separation process and effectively protect the activity of bromelain.

[0007] In view of this, a method for efficiently extracting bromelain based on magnetic chitosan microspheres is provided to overcome the above problems. Summary of the invention

[0008] The object of the present invention is to provide a method for efficiently extracting bromelain based on magnetic chitosan microspheres to solve the problems raised in the above background technology.

[0009] In order to solve the above technical problems, the present invention provides a method for efficiently extracting bromelain based on magnetic chitosan microspheres, comprising the following steps:

[0010] Preparation of magnetic chitosan microspheres:

[0011] Material preparation: Nano-sized ferroferric oxide particles with a particle size of 20-30 nanometers, chitosan with a deacetylation degree of 90% and a molecular weight of 100,000-150,000, glutaraldehyde with a mass fraction of 25%, and analytically pure glacial acetic acid, anhydrous ethanol, sodium hydroxide and other reagents;

[0012] Preparation steps: Disperse nano-sized ferroferric oxide particles in anhydrous ethanol by ultrasonic dispersion for 30 minutes, add 3-

[0013] Aminopropyl triethoxysilane is stirred and reacted at 60° C. for 4 hours, separated by an external magnetic field and washed with anhydrous ethanol for 3 times to obtain surface-aminated ferrosoferric oxide particles; chitosan is dissolved in a 2% glacial acetic acid solution, stirred until completely dissolved to obtain a chitosan solution, the surface-aminated ferrosoferric oxide particles are added to the chitosan solution for ultrasonic dispersion for 60 minutes, glutaraldehyde solution is slowly added dropwise under stirring, the addition time is controlled within 30 minutes, and the stirring reaction is continued for 4 hours after the addition is completed, separated by an external magnetic field, the pH value is adjusted to 7-8 with a 0.1 mol / L sodium hydroxide solution, and then washed with deionized water and anhydrous ethanol alternately for 3 times to obtain magnetic chitosan microspheres;

[0014] Extraction of bromelain: preparation of extract, adsorption process, separation process, desorption process;

[0015] Purification and detection of bromelain: purification process, detection process.

[0016] Furthermore, in the preparation of magnetic chitosan microspheres, the specific surface area of ​​nanometer-sized ferroferric oxide particles is based on the formula:

[0017]

[0018] Calculation, where S is the specific surface area, d is the particle size, ρ is the density, Fe 3 O 4

[0019] Density is about 5.18g / cm 3 .

[0020] Furthermore, in the preparation of the extract:

[0021] Wash, peel and cut fresh pineapple pulp into pieces, add deionized water at a mass-to-volume ratio of 1:3, homogenize in a high-speed tissue grinder for 5 minutes, and then centrifuge at 8000 r / min at 4°C for 30 minutes, take the supernatant and obtain a crude bromelain extract.

[0022] Furthermore, during the adsorption process:

[0023] The prepared magnetic chitosan microspheres were added to the crude bromelain extract, with the mass volume ratio of the magnetic chitosan microspheres to the crude extract being 1:10, and the adsorption was carried out at 25°C and a rotation speed of 150 r / min for 2 hours.

[0024] Furthermore, during the separation process:

[0025] After the adsorption is completed, the magnetic chitosan microspheres adsorbed with bromelain are quickly separated using an external magnetic field, and the supernatant is removed.

[0026] Furthermore, during the desorption process:

[0027] The separated magnetic chitosan microspheres adsorbed with bromelain were added to a 0.1 mol / L acetic acid-sodium acetate buffer solution with pH = 4.5, and the mass volume ratio of the microspheres to the buffer solution was 1:5. The microspheres were oscillated and desorbed at a speed of 100 r / min at 30°C for 1.5 hours. After the desorption was completed, the microspheres were separated again using an external magnetic field, and the supernatant was collected to obtain a desorption solution rich in bromelain.

[0028] Further, during the purification process:

[0029] The desorbed solution was further purified by a gel filtration chromatography column, using a 0.05 mol / L phosphate buffer solution as an eluent, the flow rate was controlled at 0.5 mL / min, and the solution corresponding to the elution peak was collected to obtain a high-purity bromelain solution.

[0030] Furthermore, during the detection process:

[0031] The activity of bromelain was determined by Folin-phenol reagent method, and the purity of bromelain was detected by SDS-PAGE electrophoresis.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] Preparation of magnetic chitosan microspheres:

[0034] The present invention modifies the surface of nano-sized ferroferric oxide particles by adding 3-

[0035] Aminopropyltriethoxysilane aminated its surface, enhancing the binding force with chitosan, making the prepared magnetic chitosan microspheres more stable, effectively avoiding the shedding of magnetic materials during subsequent extraction, and improving the service life and reliability of the adsorbent. After experimental comparison, the magnetic chitosan microspheres obtained after the optimized preparation method can still maintain good structural integrity and adsorption performance after multiple extractions, while the microspheres prepared by the traditional method have the problem of magnetic material shedding and reduced adsorption performance under the same number of uses.

[0036] By precisely controlling the dripping speed and reaction time of glutaraldehyde, the ratio of magnetic material to chitosan is precisely controlled, and the prepared magnetic chitosan microspheres have the best adsorption performance and magnetic strength. The moderate ratio of magnetic material not only ensures that the biocompatibility of chitosan and the specific adsorption sites for bromelain are not affected, but also provides strong enough magnetism to facilitate subsequent separation operations. Experimental data show that the adsorption performance of the optimized magnetic chitosan microspheres is 60% higher than that before optimization.

[0037] The ultrasonic dispersion step effectively ensures the uniform dispersion of the magnetic material in the chitosan microspheres, making the adsorption performance and magnetism of the microspheres uniform, improving the stability and reliability of the extraction effect, and reducing the experimental error. In repeated experiments, the magnetic chitosan microspheres prepared by the present invention are used to extract bromelain, and the results obtained have a small deviation, while the traditional method has a large fluctuation in the experimental results each time due to the uneven dispersion of the magnetic material.

[0038] Bromelain extraction process:

[0039] By using the specific adsorption of bromelain by magnetic chitosan microspheres and combining optimized adsorption separation and desorption conditions, efficient extraction of bromelain is achieved, and the extraction efficiency is increased by more than 30% compared with the traditional method. Through comparative experiments, under the same time and raw material input, the method of the present invention can extract more bromelain.

[0040] The magnetic separation method is simple and quick to operate, greatly simplifying the separation process. Compared with traditional separation methods such as centrifugation and filtration, the operation time is shortened by more than 50%, which improves production efficiency and reduces production costs. According to the magnetic response principle of magnetic materials, under the action of the magnetic field, the magnetic chitosan microspheres can quickly aggregate and separate from the solution. The separation time can be shortened to a few minutes, while traditional centrifugation or filtration may take dozens of minutes or even longer.

[0041] Specific adsorption mechanism and optimization:

[0042] Through Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and other analytical methods as well as molecular docking technology, it was clarified that the specific adsorption mechanism of hydrogen bonds and electrostatic interactions was formed between the functional groups such as amino and hydroxyl groups on the surface of magnetic chitosan microspheres and the specific amino acid residues on the surface of bromelain molecules, providing a theoretical basis for optimizing the adsorption conditions.

[0043] Adsorption experiments were conducted under different temperature, pH value and ionic strength conditions. Using orthogonal experimental design and other methods, the optimal adsorption conditions were determined to be 25°C, pH=4.5, and ionic strength 0.1 mol / L, which improved the stability and selectivity of adsorption, and increased the purity of the extracted bromelain by more than 20% compared with the traditional method, meeting the demand for high-purity bromelain in the fields of food and medicine. In practical applications, the bromelain extracted by the method of the present invention has significantly reduced impurity content and higher purity after detection.

[0044] Bromelain's active protection aspects:

[0045] During the adsorption process, 0.1% bovine serum albumin is added to the crude bromelain extract as a bioactive protective agent, which can form a protective film around the bromelain, reduce the nonspecific interaction between the magnetic chitosan microspheres and the bromelain, avoid the destruction of the active center of the bromelain during the adsorption process, and thus protect the activity of the bromelain.

[0046] 0.05 mol / L of glycerol is added to the desorption buffer solution. Glycerol has the function of moisturizing and stabilizing the protein structure, and can protect the activity of bromelain during the desorption process and prevent the desorption reagent from denaturing and inactivating the enzyme. By taking these effective activity protection measures, the activity retention rate of the extracted bromelain reaches more than 90%, which ensures the quality and application value of the product and expands the application range of bromelain.

[0047] In summary, the method of the present invention has significant advantages in improving extraction efficiency and purity, simplifying the separation process and protecting the activity of bromelain, and has good application prospects and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The present invention is a schematic diagram of a method for efficiently extracting bromelain based on magnetic chitosan microspheres. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0050] See also Figure 1 , the present invention provides a technical solution:

[0051] See also Figure 1 As shown, an embodiment of a method for efficiently extracting bromelain based on magnetic chitosan microspheres:

[0052] (I) Preparation of magnetic chitosan microspheres

[0053] Material preparation

[0054] Magnetic material: Nano-sized ferroferric oxide (Fe 3 O 4 ) particles, with a particle size of 20-30 nanometers. According to the specific surface area formula:

[0055]

[0056] Where S is the specific surface area, d is the particle size, ρ is the density, Fe 3 O 4

[0057] Density is about 5.18g / cm 3 , at the nanometer particle size, the specific surface area is larger, which is beneficial to improve the magnetic strength, and it is easier to disperse when combined with chitosan in the subsequent process, ensuring the uniformity of the microspheres. For example, when the particle size is 25 nanometers, the specific surface area is:

[0058]

[0059] The larger specific surface area enhances magnetism and dispersibility.

[0060] Chitosan: Use chitosan with a deacetylation degree of 90% and a molecular weight of 100,000-150,000. A higher deacetylation degree can enhance the adsorption performance of chitosan because more amino groups are produced after deacetylation, which can interact with more substances. The appropriate molecular weight can ensure good sphericity and stability during the preparation process, which is based on a large number of experiments and related literature reports, such as:

[0061] The basis for the influence of deacetylation degree on adsorption performance: Chitosan is composed of N-acetyl-D-glucosamine and D-glucosamine through β-1,4-

[0062] The deacetylation process is to remove the acetylamino group (-NHCOCH 3 ) is converted to amino group (-NH 2 ). From the perspective of chemical reaction principles, amino groups have strong nucleophilicity and can react chemically with a variety of substances, such as complexation reactions with metal ions, and condensation reactions with substances with functional groups such as carboxyl and aldehyde groups. When the degree of deacetylation increases, the number of amino groups increases, allowing chitosan to interact with more types and quantities of substances, thereby enhancing its adsorption performance. This principle is explained in many literatures on the adsorption performance of chitosan, such as "Adsorption Performance and Application Research Progress of Chitosan", which analyzes in detail the relationship between the degree of deacetylation and adsorption performance of chitosan. A large amount of experimental data shows that with the increase of the degree of deacetylation, the adsorption of chitosan on various pollutants such as heavy metal ions and dye molecules is significantly improved.

[0063] The basis for the molecular weight to affect the ball-forming property and stability: from the perspective of the material structure characteristics, the chitosan with a lower molecular weight has a shorter molecular chain and weaker intermolecular forces, which makes it difficult to form a stable three-dimensional network structure during the preparation of microspheres, resulting in poor ball-forming property, and the prepared microspheres are easily broken or deformed, and have poor stability. However, the chitosan with a high molecular weight has a long molecular chain and serious entanglement, and the solution viscosity is too large, which is not conducive to the formation and dispersion of the microspheres, and also affects the quality of the microspheres. In a large number of experiments, it was found that the chitosan with a molecular weight of 100,000-150,000 has a moderate molecular chain length and a more appropriate intermolecular force. Under specific preparation conditions, such as under the action of a suitable solvent (such as 2% glacial acetic acid solution) and a cross-linking agent (such as glutaraldehyde), a stable cross-linked network structure can be formed to ensure that the microspheres have good ball-forming property and stability. [Specific document name] "Preparation and Characterization of Chitosan Microspheres with Different Molecular Weights" clearly points out that under specific conditions, microspheres prepared from chitosan with a molecular weight range of 100,000-150,000 have a regular spherical appearance, high mechanical strength and good dispersibility, which strongly supports this view.

[0064] Cross-linking agent: glutaraldehyde, mass fraction is 25%. Glutaraldehyde as a cross-linking agent can form a cross-linking structure between chitosan molecules. According to the cross-linking reaction principle, the aldehyde group of glutaraldehyde reacts with the amino group of chitosan to improve the mechanical strength and stability of the microspheres.

[0065] Other reagents: glacial acetic acid, anhydrous ethanol, sodium hydroxide, etc. are all analytically pure. Analytically pure reagents can ensure the accuracy and repeatability of the experiment and reduce the impact of impurities on the experimental results.

[0066] Preparation steps

[0067] Fe 3 O 4 Surface modification of particles: Nano-sized Fe 3 O 4

[0068] The particles were dispersed in 100 mL of anhydrous ethanol and ultrasonically dispersed for 30 minutes to make them uniformly dispersed. Then 5 mL of 3-

[0069] Aminopropyltriethoxysilane (APTES) was stirred and reacted at 60°C for 4 hours. After the reaction, it was separated by an external magnetic field and washed with anhydrous ethanol three times to obtain Fe 3 O 4

[0070] Ultrasonic dispersion can use the cavitation effect of ultrasound to make Fe 3 O 4

[0071] The particles were evenly dispersed in anhydrous ethanol to avoid agglomeration. APTES was added for surface amination because the ethoxy groups of APTES can be hydrolyzed to form silanol groups, which react with Fe 3 O 4 The hydroxyl groups on the surface undergo condensation reaction, thereby 3 O 4

[0072] The surface is introduced with amino groups to enhance the binding force with chitosan. The reaction temperature is controlled at 60°C. After many experiments, the reaction rate is faster and the product stability is better at this temperature.

[0073] Preparation of magnetic chitosan microspheres: Dissolve 2 g of chitosan in 100 mL of 2% glacial acetic acid solution and stir until completely dissolved to obtain a chitosan solution. 3 O 4

[0074] The particles were added to the chitosan solution and ultrasonically dispersed for 60 minutes to make Fe 3 O 4

[0075] The particles are evenly dispersed in the chitosan solution. Then, under stirring conditions, 5 mL of 25% glutaraldehyde solution is slowly added dropwise, and the addition time is controlled within 30 minutes. After the addition is completed, stirring and reacting is continued for 4 hours. After the reaction is completed, it is separated by an external magnetic field, and the pH value is adjusted to 7-8 with 0.1 mol / L sodium hydroxide solution, and then washed alternately with deionized water and anhydrous ethanol for 3 times to obtain magnetic chitosan microspheres. Chitosan can be fully dissolved in 2% glacial acetic acid solution because the amino group in the chitosan molecule can combine with the hydrogen ion of acetic acid to form a soluble salt. Ultrasonic dispersion of Fe 3 O 4

[0076] The particles are in the chitosan solution to further ensure uniform dispersion. Slowly dripping glutaraldehyde and controlling the time is to accurately control the degree of cross-linking reaction. According to the cross-linking reaction kinetics, slow dripping can make glutaraldehyde evenly distributed and fully react with chitosan, accurately control the ratio of magnetic material to chitosan, and ensure that the magnetic chitosan microspheres have good adsorption performance and magnetic strength. Adjusting the pH value is to terminate the cross-linking reaction and stabilize the surface charge of the microspheres. The washing step can remove unreacted reagents and impurities.

[0077] Through Fe 3 O 4

[0078] The surface of the particles is modified to enhance their binding force with chitosan, making the prepared magnetic chitosan microspheres more stable and less likely to cause the magnetic material to fall off during the subsequent extraction process, thereby improving the service life and reliability of the adsorbent.

[0079] The precise control of the dripping speed and reaction time of glutaraldehyde achieves the precise control of the ratio of magnetic material to chitosan, and the prepared magnetic chitosan microspheres have the best adsorption performance and magnetic strength. The moderate ratio of magnetic material not only ensures that the biocompatibility of chitosan and the specific adsorption sites for bromelain are not affected, but also provides strong enough magnetism to facilitate the subsequent separation operation and improve the separation efficiency.

[0080] The ultrasonic dispersion step effectively ensures the uniform dispersion of the magnetic material in the chitosan microspheres, making the adsorption performance and magnetism of the microspheres uniform, improving the stability and reliability of the extraction effect, and reducing experimental errors.

[0081] (II) Extraction of bromelain

[0082] Extraction solution preparation

[0083] Wash, peel and cut fresh pineapple pulp, add deionized water in a ratio of 1:3 (mass to volume ratio), homogenize in a high-speed tissue crusher for 5 minutes, and then centrifuge at 8000r / min for 30 minutes at 4°C, take the supernatant, and obtain a crude bromelain extract. Fresh pineapple pulp is selected because its bromelain activity is high and can ensure the initial enzyme activity of the extraction. Adding deionized water in a ratio of 1:3 is determined based on the solubility of bromelain in water and experimental experience. This ratio can better extract bromelain. The homogenization time and centrifugation conditions have been optimized through multiple experiments. Homogenization for 5 minutes can fully break the cells and release the enzyme. Low-temperature centrifugation at 4°C can reduce the inactivation of the enzyme. The speed of 8000r / min and the centrifugation time of 30 minutes can effectively remove impurities, providing better raw materials for subsequent specific adsorption.

[0084] Adsorption process

[0085] The prepared magnetic chitosan microspheres were added to the crude extract of bromelain, with a mass volume ratio of 1:10 between the magnetic chitosan microspheres and the crude extract. The adsorption was carried out at 25°C and a speed of 150 r / min for 2 hours. During the adsorption process, the specific functional groups on the surface of the magnetic chitosan microspheres interacted specifically with bromelain to achieve specific adsorption of bromelain. This mass volume ratio was determined by experimentally measuring the adsorption amount and adsorption selectivity at different ratios, and the adsorption effect was best at 1:10. The adsorption temperature was controlled at 25°C because this temperature was close to the optimal activity temperature of bromelain, which could ensure the activity of the enzyme and facilitate the adsorption reaction. The speed and time were also optimized. The speed of 150 r / min could make the microspheres fully contact with the enzyme, and the adsorption time of 2 hours could achieve a good adsorption balance.

[0086] Separation process

[0087] After the adsorption is completed, the magnetic chitosan microspheres adsorbed with bromelain are quickly separated using an external magnetic field, and the supernatant is removed, achieving the initial separation of bromelain and impurities. The magnetic separation method is simple and quick to operate, greatly simplifying the separation process, saving time and energy compared to traditional separation methods such as centrifugation and filtration. According to the magnetic response principle of magnetic materials, under the action of a magnetic field, the magnetic chitosan microspheres can quickly aggregate and separate from the solution, and the separation time can be shortened to a few minutes, while traditional centrifugation or filtration may take dozens of minutes or even longer.

[0088] Desorption process

[0089] The separated magnetic chitosan microspheres adsorbed with bromelain were added to 0.1mol / L acetic acid-sodium acetate buffer solution (pH=4.5), with a mass volume ratio of microspheres to buffer solution of 1:5, and desorbed at 30°C at a speed of 100r / min for 1.5 hours. After desorption, the microspheres were separated again using an external magnetic field, and the supernatant was collected to obtain a desorption solution rich in bromelain. The 0.1mol / L acetic acid-sodium acetate buffer solution (pH=4.5) was selected because this buffer system can change the interaction between the microspheres and bromelain, allowing the enzyme to be desorbed, and the pH value is close to the isoelectric point of bromelain, which is conducive to the stability of the enzyme. The mass volume ratio, temperature, rotation speed and time are all determined through experimental optimization, which can ensure the efficient desorption of bromelain and protect the activity of bromelain to the greatest extent.

[0090] (III) Purification and detection of bromelain

[0091] Purification process

[0092] The desorbed solution is further purified by gel filtration chromatography column (Sephadex G-75), using 0.05 mol / L phosphate buffer solution (pH=7.0) as eluent, flow rate control at 0.5 mL / min, collecting the solution corresponding to the elution peak, and obtaining a high-purity bromelain solution. The gel filtration chromatography column separates substances according to molecular size, and the separation range of Sephadex G-75 is suitable for bromelain, which can effectively remove impurities larger or smaller than bromelain molecules. The phosphate buffer solution (pH=7.0) of 0.05 mol / L is selected as eluent because this condition can ensure the activity of bromelain, and the flow rate control at 0.5 mL / min can fully separate substances of different molecular sizes and improve the purification effect.

[0093] Testing process

[0094] The activity of bromelain was determined by Folin-phenol reagent method, and the purity of bromelain was detected by SDS-PAGE electrophoresis.

[0095] The phenol reagent method uses the product generated by the enzyme-catalyzed substrate reaction to react with fulin-

[0096] The phenol reagent reaction produces a color change, and the enzyme activity is calculated by comparing the absorbance with the standard curve. The principle is based on the reaction of tyrosine and tryptophan residues in proteins with the Folin-phenol reagent. SDS-PAGE electrophoresis separates proteins of different molecular weights based on the relationship between the mobility of protein molecules in an electric field and the molecular weight, thereby detecting the purity of bromelain. The protein bands can be visually observed to determine whether there are impurities.

[0097] (IV) Specific adsorption mechanism and optimization

[0098] Exploration of specific adsorption mechanism

[0099] The functional groups on the surface of magnetic chitosan microspheres were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and other analytical methods. At the same time, molecular docking technology was used to simulate the interaction between the functional groups on the surface of magnetic chitosan microspheres and bromelain molecules. Combined with the experimental results, it was determined that hydrogen bonds and electrostatic interactions were formed between the functional groups such as amino and hydroxyl groups on the surface of magnetic chitosan microspheres and specific amino acid residues on the surface of bromelain molecules, thereby achieving specific adsorption. FT-IR can determine chemical bonds and functional groups through characteristic absorption peaks, XPS can analyze the chemical state and surface composition of elements, and molecular docking technology simulates intermolecular interactions based on the principles of geometric complementarity and energy matching between molecules. The combination of these methods can provide a deep understanding of the specific adsorption mechanism and provide a theoretical basis for optimizing adsorption conditions.

[0100] Optimization of adsorption conditions

[0101] Adsorption experiments were conducted under different temperatures (20°C, 25°C, 30°C), pH values ​​(4.0, 4.5, 5.0) and ionic strengths (0.05mol / L, 0.1mol / L, 0.15mol / L). By measuring the adsorption capacity and adsorption selectivity, the optimal adsorption conditions were determined to be 25°C, pH = 4.5, and ionic strength 0.1mol / L. Through multi-factor experiments and data analysis, using orthogonal experimental design and other methods, the effects of different factors on adsorption capacity and adsorption selectivity were studied, the optimal conditions were determined, and the stability and efficiency of specific adsorption were improved.

[0102] (V) Active protection of bromelain

[0103] Active protection during adsorption

[0104] During the adsorption process, 0.1% bovine serum albumin (BSA) is added to the crude extract of bromelain as a bioactive protective agent. BSA can form a protective film around bromelain, reduce the nonspecific interaction between the magnetic chitosan microspheres and bromelain, avoid the destruction of the active center of bromelain during the adsorption process, and thus protect the activity of bromelain. This is based on the structure and properties of BSA. There are multiple hydrophilic groups on its molecular surface, which can form a hydration layer with water molecules, wrap around bromelain, and play a protective role.

[0105] Active protection during desorption

[0106] 0.05 mol / L of glycerol is added to the desorption buffer solution. Glycerol has the function of moisturizing and stabilizing the protein structure. It can protect the activity of bromelain during the desorption process and prevent the desorption reagent from denaturing and inactivating the enzyme. The hydroxyl group of glycerol can form hydrogen bonds with protein molecules to stabilize the secondary and tertiary structures of the protein. At the same time, its moisturizing effect can prevent the enzyme from being inactivated due to water loss during the desorption process.

[0107] Summarize

[0108] Preparation of magnetic chitosan microspheres: Through surface modification of magnetic materials and precise control of the preparation process, magnetic chitosan microspheres with good adsorption performance, magnetic strength and uniformity were successfully prepared, providing high-quality adsorbents for the efficient extraction of bromelain. The adsorption performance was improved by 60% compared with that before optimization.

[0109] The following are the basis, tables and related instructions:

[0110] The basis for the value:

[0111] Two groups of experiments were prepared, one group using the conventional preparation method of magnetic chitosan microspheres before optimization, and the other group using the optimized preparation method of the present invention.

[0112] Fifteen parallel experiments were set up in each group to ensure the reliability of the data.

[0113] Under the same experimental conditions, including the same bromelain solution concentration (5 mg / mL), the same solution volume (50 mL), the same temperature (25°C), pH value (4.5) and adsorption time (2 hours), adsorption experiments were carried out using magnetic chitosan microspheres before and after optimization.

[0114] After the adsorption was completed, the concentration of the remaining bromelain in the solution was accurately determined by high performance liquid chromatography (HPLC), and then the adsorption amount was calculated according to the formula: (initial bromelain concentration -

[0115] The adsorption amount of the two microspheres was calculated by (remaining bromelain concentration) × solution volume ÷ microsphere mass.

[0116] The average adsorption capacity of magnetic chitosan microspheres before optimization was 15 mg / g, and the average adsorption capacity after optimization was 24 mg / g. According to the formula:

[0117]

[0118] Right now:

[0119]

[0120] The improvement ratio of adsorption performance was obtained.

[0121] In order to more intuitively show the significant difference in the adsorption performance of magnetic chitosan microspheres before and after optimization, the following are the detailed data statistics of 15 groups of parallel experiments:

[0122]

[0123] In the extraction process: the specific adsorption of bromelain by magnetic chitosan microspheres, combined with optimized adsorption, separation and desorption conditions, has achieved efficient extraction of bromelain, with the extraction efficiency increased by more than 30% compared with traditional methods. At the same time, the use of magnetic separation has greatly simplified the separation process, shortened the operation time by more than 50%, improved production efficiency, and reduced production costs.

[0124] Specific adsorption mechanism and optimization: The specific adsorption mechanism was clarified, and the stability and selectivity of adsorption were improved by optimizing the adsorption conditions. The purity of the extracted bromelain was increased by more than 20% compared with the traditional method, meeting the demand for high-purity bromelain in the fields of food and medicine.

[0125] Activity protection of bromelain: effective activity protection measures are taken during the adsorption and desorption process, so that the activity retention rate of the extracted bromelain reaches more than 90%, ensuring the quality and application value of the product and expanding the application scope of bromelain.

[0126] In summary, the method for efficiently extracting bromelain based on magnetic chitosan microspheres provided by the present invention has significant advantages in improving extraction efficiency and purity, simplifying the separation process, and protecting the activity of bromelain, and has good application prospects and economic value.

Claims

1. A method for efficiently extracting bromelain based on magnetic chitosan microspheres, characterized in that: The following steps are involved: Preparation of magnetic chitosan microspheres: Material preparation: Use a particle size of 20- 30 nanometer nanometer ferrosoferric oxide particles, chitosan with a deacetylation degree of 90% and a molecular weight of 100,000-150,000, glutaraldehyde with a mass fraction of 25%, and analytically pure glacial acetic acid, anhydrous ethanol, sodium hydroxide and other reagents; Preparation steps: Disperse nano-sized ferroferric oxide particles in anhydrous ethanol by ultrasonic dispersion for 30 minutes, add 3- Aminopropyl triethoxysilane was stirred and reacted at 60° C. for 4 hours, separated by an external magnetic field and washed with anhydrous ethanol for 3 times to obtain surface amino-treated ferroferric oxide particles; chitosan was dissolved in a 2% glacial acetic acid solution and stirred until completely dissolved to obtain a chitosan solution, the surface amino-treated ferroferric oxide particles were added to the chitosan solution and ultrasonically dispersed for 60 minutes, glutaraldehyde solution was slowly added dropwise under stirring, the addition time was controlled within 30 minutes, and the stirring reaction was continued for 4 hours after the addition was completed, separated by an external magnetic field, and the pH value was adjusted to 7-8 with a 0.1 mol / L sodium hydroxide solution.

8. Wash the mixture alternately with deionized water and anhydrous ethanol for 3 times to obtain magnetic chitosan microspheres; Extraction of bromelain: preparation of extract, adsorption process, separation process, desorption process; Purification and detection of bromelain: purification process, detection process.

2. A method for efficiently extracting bromelain based on magnetic chitosan microspheres as claimed in claim 1, characterized in that: In the preparation of magnetic chitosan microspheres, the specific surface area of ​​nanometer-sized ferroferric oxide particles is based on the formula: Calculation, where S is the specific surface area, d is the particle size, ρ is the density, Fe3O4 Density is about 5.18g / cm 3 .

3. A method for efficiently extracting bromelain based on magnetic chitosan microspheres as claimed in claim 1, characterized in that, In extract preparation: Wash, peel and cut fresh pineapple pulp into pieces, add deionized water at a mass-to-volume ratio of 1:3, homogenize in a high-speed tissue grinder for 5 minutes, and then centrifuge at 8000 r / min at 4°C for 30 minutes, take the supernatant and obtain a crude bromelain extract.

4. A method for efficiently extracting bromelain based on magnetic chitosan microspheres as claimed in claim 1, characterized in that, During the adsorption process: The prepared magnetic chitosan microspheres were added to the crude bromelain extract, with the mass volume ratio of the magnetic chitosan microspheres to the crude extract being 1:10, and the adsorption was carried out at 25°C and a rotation speed of 150 r / min for 2 hours.

5. A method for efficiently extracting bromelain based on magnetic chitosan microspheres as claimed in claim 1, characterized in that, During the separation process: After the adsorption is completed, the magnetic chitosan microspheres adsorbed with bromelain are quickly separated using an external magnetic field, and the supernatant is removed.

6. A method for efficiently extracting bromelain based on magnetic chitosan microspheres as claimed in claim 1, characterized in that, During the desorption process: The separated magnetic chitosan microspheres adsorbed with bromelain were added to a 0.1 mol / L acetic acid-sodium acetate buffer solution with pH = 4.5, and the mass volume ratio of the microspheres to the buffer solution was 1:

5. The microspheres were oscillated and desorbed at a speed of 100 r / min at 30°C for 1.5 hours. After the desorption was completed, the microspheres were separated again using an external magnetic field, and the supernatant was collected to obtain a desorption solution rich in bromelain.

7. A method for efficiently extracting bromelain based on magnetic chitosan microspheres as claimed in claim 1, characterized in that: During the purification process: The desorbed solution was further purified by a gel filtration chromatography column, using a 0.05 mol / L phosphate buffer solution as an eluent, the flow rate was controlled at 0.5 mL / min, and the solution corresponding to the elution peak was collected to obtain a high-purity bromelain solution.

8. A method for efficiently extracting bromelain based on magnetic chitosan microspheres as claimed in claim 1, characterized in that: During the test: The activity of bromelain was determined by Folin-phenol reagent method, and the purity of bromelain was detected by SDS-PAGE electrophoresis.

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