A method for extracting polyphenols from red heart pomelo based on enzymatic extraction

Abstract

The present application belongs to the technical field of natural product extraction and enzyme engineering, and particularly relates to a method for extracting red heart pomelo polyphenols based on enzyme method; in view of the problems that enzymes are prone to inactivation and difficult to recover in the process of red heart pomelo polyphenol extraction, resulting in high cost, etc.; the peel, pomelo seeds and other wastes generated in the processing of red heart pomelo are used as carbon source, and the biological carbon material with multi-stage pore structure is prepared through programmed temperature charring activation; the pectinase, cellulase and naringinase are fixed by using the sequential immobilization strategy of spatial partitioning, and then react with red heart pomelo; the immobilized enzymes are rapidly recovered by magnetic separation to realize the reuse of enzymes; finally, the red heart pomelo polyphenol powder is obtained through multiple processing, the recycling is realized, and the yield of polyphenol extracted by enzyme method is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of natural product extraction and enzyme engineering, and particularly relates to a method for extracting polyphenols from red heart pomelo based on enzyme method. BACKGROUND

[0002] Red heart pomelo pulp is rich in natural antioxidant ingredients such as lycopene and beta-carotene. The peel and seeds of red heart pomelo also contain a large amount of polyphenols, mainly flavonoids such as naringin and neohesperidin. These polyphenols have significant antioxidant and anti-inflammatory biological activities and have application value in functional foods and drugs. However, the by-products such as peel and seeds generated during the processing of red heart pomelo are often treated as waste, and the rich polyphenols contained therein are not effectively extracted, which not only causes resource waste but also may bring environmental burden.

[0003] Traditional polyphenol extraction methods mainly include organic solvent extraction and water extraction (CN120732918A). For example, although polyphenols can be extracted from pomelo peel with ethanol or ethyl acetate to obtain a certain yield, there are problems of solvent residue, toxicity and environmental pollution, and the extraction rate is limited; direct water extraction is safe and environmentally friendly but the yield of polyphenols is very low. In addition, pomelo peel also contains dietary fibers such as pectin and cellulose, which form a dense cell wall structure, hindering solvent penetration and polyphenol release. It is difficult to fully break down this structure by using conventional physical or chemical methods, so the polyphenol leaching is incomplete.

[0004] Enzymatic extraction as a green and efficient method has been used to improve the release of active ingredients in plant raw materials. By adding hydrolytic enzymes such as cellulase and pectinase, cellulose, hemicellulose and pectin in the cell wall can be selectively degraded, the mass transfer resistance is significantly reduced, and the leaching of polyphenols and other active ingredients is accelerated. However, traditional enzyme-assisted extraction usually uses free enzymes, which has the problems of large enzyme dosage and high cost. Free enzymes are difficult to recover after use in aqueous solution and are often discarded with waste liquid, resulting in poor reusability of enzymes. At the same time, free enzymes are sensitive to the environment and are easily inactivated at higher temperatures or non-optimal pH, which limits the application of enzyme method in industrial extraction.

[0005] To overcome the aforementioned shortcomings, enzyme immobilization technology has been introduced to improve enzyme stability and reusability. By immobilizing enzymes on solid supports, they can be easily separated and recovered from the reaction solution while maintaining enzyme activity, enabling multiple recycling of enzymes. Currently, many types of support materials are used for enzyme immobilization, including activated carbon, silica gel, chitosan, and resins. However, existing supports generally suffer from limitations such as limited enzyme loading capacity, complex preparation processes, or separation difficulties. For example, most traditional activated carbons are predominantly microporous, making it difficult for large enzyme molecules to enter the pores and forcing them to adhere only to the surface, resulting in low enzyme activity recovery rates. Inorganic porous materials such as mesoporous silica can provide ordered pore structures, but their preparation processes are cumbersome and costly. Furthermore, research on immobilized multi-enzyme systems is relatively limited. If different enzymes are mixed and immobilized on the same support, uneven enzyme distribution or mutual interference may occur, affecting the synergistic effect.

[0006] In conclusion, it is necessary to develop a novel magnetic enzyme carrier prepared from pomelo processing waste, and to synergistically immobilize multiple hydrolytic enzymes on it for the extraction of pomelo polyphenols, so as to improve the polyphenol recovery rate and realize the resource utilization of waste and the green and efficient process. Summary of the Invention

[0007] This invention provides a method for extracting polyphenols from red pomelo using an enzymatic process, aiming to significantly improve the efficient release and separation and recovery of polyphenols from red pomelo raw materials. It addresses the problems of low polyphenol extraction rates, non-reusable enzymes, and high process costs associated with current red pomelo polyphenol extraction methods.

[0008] The specific technical solution is as follows:

[0009] A method for extracting polyphenols from red grapefruit using an enzymatic method is as follows:

[0010] S1: Carrier preparation.

[0011] S11: Dry and pulverize the peel and seeds of red pomelo to obtain red pomelo powder; mix the red pomelo powder with K2CO3, add deionized water to immerse it, stir, dry at 105℃, perform programmed pyrolysis under nitrogen protection, cool to room temperature, add HCl, stir, wash, and dry to obtain hierarchical pore biochar.

[0012] S12: The hierarchical porous biochar prepared in S11 was ultrasonically dispersed in deionized water to obtain a hierarchical porous biochar dispersion; ferric chloride and ferrous sulfate were added to deionized water to prepare an iron salt solution; the hierarchical porous biochar dispersion was mixed with the iron salt solution, stirred, and the pH was adjusted to 10. After the reaction was completed, solid-liquid separation was performed using a magnet, the supernatant was discarded, and the solid product was washed, dried, and ground to obtain a magnetic hierarchical porous biochar carrier.

[0013] S2: Preparation of immobilized complex enzymes.

[0014] S21: Dissolve pectinase and cellulase in phosphate buffer, then add the magnetic hierarchical porous biochar carrier prepared in S12, oscillate the reaction, after the reaction is completed, use a magnet to separate the immobilized carrier from the solution, wash with pre-cooled phosphate buffer of the same pH, and obtain the primary immobilized enzyme.

[0015] S22: Dissolve naringinase in phosphate buffer, and add the primary immobilized enzyme prepared in S21, oscillate the reaction, after the reaction is completed, use a magnet to separate the immobilized carrier from the solution, wash with pre-cooled phosphate buffer of the same pH, and obtain the immobilized composite enzyme.

[0016] S3: Redheart pomelo polyphenol extraction and purification.

[0017] S31: Take fresh redheart pomelo pulp, add pH 4.5 citric acid-sodium citrate buffer, stir to prepare a uniform slurry; add the immobilized composite enzyme prepared in S22 to the uniform slurry, oscillate at a constant temperature, after the reaction is completed, place the reaction container next to a magnet to separate the immobilized composite enzyme; concentrate the supernatant to one-fifth of the original volume to obtain a concentrated solution.

[0018] S32: Pass the concentrated solution prepared in S31 through a resin column, then flush the resin column with deionized water, and then elute with an ethanol aqueous solution to obtain a polyphenol-rich solution.

[0019] S33: Take the polyphenol-rich solution as a feed liquid, and perform spray drying to obtain redheart pomelo polyphenol powder.

[0020] Further, the K2CO3 in S11 has a mass ratio of 1:1 to 1:3 to the redheart pomelo powder.

[0021] The programmed pyrolysis in S11 has the following parameter settings: a temperature increase rate of 4-6°C / min, a temperature of 600-800°C, and a holding time of 2-4 h.

[0022] The ferric chloride and ferrous sulfate in S12 have a molar ratio of 1.5:1 to 2.5:1 between the ferric chloride and the ferrous sulfate.

[0023] The stirring in S12 has the following parameter settings: a temperature of 60-80°C and a rate of 100-300 rpm.

[0024] The drying in S12 has the following parameter settings: a temperature of 60-80°C.

[0025] Further, the pectinase and cellulase in S21 have a mass ratio of 1:1 to 1:2, and the mass ratio of the two to the magnetic hierarchical porous biochar carrier is 0.05:1 to 0.15:1.

[0026] The phosphate buffer in S21 is set as follows: pH 5.0 and concentration 0.1 mol / L.

[0027] The oscillation reaction in S21 is set as follows: temperature 35-45 DEG C, rotation speed 100-200 rpm, and time length 2-4 h.

[0028] The naringinase in S22 is set as follows: mass ratio of naringinase to magnetic hierarchical-pore biochar carrier 0.02:1-0.08:1.

[0029] The phosphate buffer in S22 is set as follows: pH 4.0 and concentration 0.1 mol / L.

[0030] The oscillation reaction in S22 is set as follows: temperature 45-55 DEG C, rotation speed 100-200 rpm, and time length 1-3 h.

[0031] Further, the fresh red-heart pomelo pulp in S31 is set as follows: solid-liquid ratio of fresh red-heart pomelo pulp to citric acid-sodium citrate buffer 1:3-1:6 (m:v).

[0032] The immobilized complex enzyme in S31 is set as follows: solid-liquid ratio of immobilized complex enzyme to slurry 1:10-1:50.

[0033] The constant-temperature oscillation in S31 is set as follows: temperature 45-50 DEG C, rotation speed 150-250 rpm, and time length 3-4 h.

[0034] The concentration in S31 is set as follows: temperature 40-60 DEG C and vacuum degree 0.09-0.12 MPa.

[0035] The spray drying in S33 is set as follows: inlet air temperature 180 DEG C and outlet air temperature 85 DEG C.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] 1. The present application greatly reduces the carrier cost by using the processing waste of red-heart pomelo as a raw material to prepare a biochar carrier, and conforms to the green chemistry and circular economy concept.

[0038] 2. The present application positions different functional enzymes at the most suitable spatial positions of the carrier through sequential immobilization, protects pectinase and cellulase in the internal mesopores, reduces the inactivation of the enzymes in a complex system, fixes naringinase in the external macropores, reduces the mass transfer resistance, and enables the naringinase to efficiently act on glycoside substrates in a solution.

[0039] 3. The present application can synergistically and efficiently destroy plant cell walls and convert glycosides through a complex enzyme system, and significantly improves the yield and purity of polyphenols. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is a process flow diagram of extracting polyphenols from red heart pomelo based on enzyme method.

[0041] Figure 2 is a standard curve diagram of polyphenol extraction of examples 1-4 and comparative examples 1-3.

[0042] Figure 3 is a data comparison diagram of enzyme activity recovery, enzyme activity retention after 5 times and polyphenol extraction rate of examples 1-4 and comparative examples 1-3. DETAILED DESCRIPTION

[0043] The following examples further explain and illustrate the technical solutions of the present application. It is particularly pointed out that each specific embodiment is a specific embodiment and explanation of the technical solution, and should not be regarded as a limitation on the protection scope of the present application. Those skilled in the art still have the right to modify the technical solutions of these examples, to equivalently replace part or all of the technical features, and these modifications or replacements do not change the essence of the corresponding technical solution, and do not make the essence of the corresponding technical solution deviate from the scope of the technical solution described in the present application.

[0044] The present application proposes a method for extracting polyphenols from red heart pomelo based on enzyme method, which effectively solves the problems of enzyme activity loss, carrier cost and operation stability in the application of immobilized enzyme by designing a new type of magnetic biochar carrier and innovative partition immobilization strategy. As shown in the accompanying Figure 1 Fig. 1 is a preparation process flow diagram of extracting polyphenols from red heart pomelo based on enzyme method, and the detailed technical solutions are as follows:

[0045] 1. Carrier preparation

[0046] Dry the red heart pomelo peel and pomelo seeds, crush them to obtain red heart pomelo powder; mix the red heart pomelo powder with potassium carbonate, immerse it in deionized water, stir, dry at 105℃, program pyrolysis, cool to room temperature, then add HCl, stir, wash, dry to obtain hierarchical pore biochar. Disperse the hierarchical pore biochar in deionized water to obtain a hierarchical pore biochar dispersion; prepare an iron salt solution by adding ferric chloride and ferrous sulfate to deionized water; mix the hierarchical pore biochar dispersion with the iron salt solution, stir, adjust the pH to 10, after the reaction is completed, perform solid-liquid separation with a magnet, discard the supernatant, wash the solid product, dry, grind to obtain a magnetic hierarchical pore biochar carrier.

[0047] Red heart pomelo processing waste peel, pomelo seeds, these raw materials are rich in cellulose, hemicellulose and lignin, which are excellent precursors for preparing porous carbon, and can achieve "waste treatment with waste". K2CO3 will react with carbon in biomass during pyrolysis, on the one hand, etching the carbon skeleton to form pores, on the other hand, its decomposition product (K2O) and released gas (CO2) can further promote the formation and expansion of pores, thus creating a hierarchical pore structure including micropores, mesopores and macropores. Under alkaline conditions, Fe 2+ and Fe 3+ will undergo coprecipitation reaction to generate black Fe3O4 particles, these nanoscale magnetic particles will deposit on the surface and inside of the pores of biochar, so that the final composite material can be attracted by a magnet, realizing fast solid-liquid separation.

[0048] 2. Preparation of immobilized composite enzyme

[0049] Dissolve pectinase and cellulase in phosphate buffer, then add magnetic hierarchical porous biochar carrier, shake the reaction, after the reaction is completed, use a magnet to adsorb and separate the immobilized carrier from the solution, wash with pre-cooled phosphate buffer of the same pH, and obtain the primary immobilized enzyme; Dissolve naringinase in phosphate buffer and add the primary immobilized enzyme, shake the reaction, after the reaction is completed, use a magnet to perform solid-liquid separation again, wash with pre-cooled phosphate buffer of the same pH, and obtain the immobilized composite enzyme.

[0050] Micropores (less than 2 nm) provide a large specific surface area (specific surface area greater than 800 m 2 / g), mesopores (2-50 nm) are suitable for immobilizing pectinase and cellulase with large molecular weight (10 kDa-150 kDa), and macropores (greater than 50 nm) act as transport channels. The high specific surface area and rich surface functional groups of biochar provide strong adsorption force for enzyme molecules, and this adsorption is firm and can better maintain the natural conformation of the enzyme, thereby maintaining high enzyme activity. First immobilize pectinase and cellulase, and then immobilize naringinase, which avoids the spatial steric hindrance of the latter affecting the entry of the former two into the mesopores. Pectinase and cellulase, which have relatively small molecular weight and are used to break down cell walls, are immobilized in the internal mesopores, making them more stable when breaking down cell walls; while naringinase, which has a large molecular weight or needs to directly act on glycoside substrates in the liquid phase, is immobilized in the external and macropores, making it more fully contact with the substrate and having higher reaction efficiency.

[0051] 3. Red heart pomelo polyphenol extraction and purification

[0052] Fresh red heart pomelo pulp is added into a citric acid-sodium citrate buffer solution with pH 4.5, stirred to prepare a uniform slurry; the immobilized complex enzyme is added into the uniform slurry, constant-temperature oscillation is performed, after the reaction is completed, the reaction container is placed beside a magnet to separate the immobilized complex enzyme; the supernatant is concentrated to obtain a concentrated solution; the concentrated solution is passed through a resin column, then the resin column is washed with deionized water, and then an ethanol aqueous solution is used for elution to obtain a polyphenol enrichment solution, which is subjected to spray drying to obtain a red heart pomelo polyphenol powder.

[0053] The pectinase and cellulase in the immobilized complex enzyme synergistically degrade pectin and cellulose in the cell wall of the red heart pomelo, destroy the cell structure, and fully release the internal polyphenol substances. At the same time, the bitter precursor substances released are hydrolyzed by the naringinase, which can not only remove bitterness, but also generate some aglycones with higher biological activity, so as to realize synchronous extraction and conversion. The macroporous adsorption resin method is used, which is a physical separation technology based on intermolecular forces. There is a strong adsorption force between the benzene ring structure of the polyphenol substances and the resin skeleton, while the water-soluble impurities such as sugars and proteins are washed away by water. Subsequently, ethanol is used as an eluent to destroy the adsorption force, and the high-purity polyphenols are selectively eluted.

[0054] Example 1

[0055] A method for extracting red heart pomelo polyphenols based on enzyme method is as follows:

[0056] Table 1 main raw material table

[0057]

[0058] S1: carrier preparation.

[0059] S11: The red heart pomelo peel and pomelo seeds are dried and crushed to obtain red heart pomelo powder; the red heart pomelo powder is mixed with K2CO3, immersed in deionized water, stirred, dried at 105℃, programmed pyrolysis under nitrogen protection, cooled to room temperature, then HCl is added, stirred, washed, and dried to obtain hierarchical pore biochar. The mass ratio of K2CO3 to red heart pomelo powder is 1:2, the programmed pyrolysis heating rate is 5℃ / min, the temperature is 700℃, and the holding time is 3h;

[0060] S12: ultrasonic dispersion of the hierarchical-pore biochar prepared in S11 in deionized water to obtain a hierarchical-pore biochar dispersion; preparation of a ferric salt solution by adding ferric chloride and ferrous sulfate to deionized water; mixing of the hierarchical-pore biochar dispersion with the ferric salt solution, stirring, adjustment of pH to 10, solid-liquid separation by a magnet after the reaction is completed, discarding of the supernatant, washing of the solid product, drying at 70°C, grinding, and obtaining of 100 g of a magnetic hierarchical-pore biochar carrier. The molar ratio between the ferric chloride and the ferrous sulfate is 2.0:1; the stirring temperature is 70°C, and the stirring rate is 200 rpm.

[0061] S2: preparation of immobilized composite enzymes.

[0062] S21: dissolution of pectinase and cellulase in a phosphate buffer, then addition of the magnetic hierarchical-pore biochar carrier (10 g) prepared in S12, oscillation reaction at 30°C and at 150 rpm for 3 h, adsorption and separation of the immobilized carrier from the solution by a magnet after the reaction is completed, washing with pre-cooled phosphate buffer of the same pH, and obtaining of primary immobilized enzymes. The mass ratio of the pectinase and the cellulase is 1:1.5, and the mass ratio of the two to the magnetic hierarchical-pore biochar carrier is 0.1:1.

[0063] S22: dissolution of naringinase in a phosphate buffer, addition of the primary immobilized enzymes prepared in S21, oscillation reaction, solid-liquid separation by a magnet again after the reaction is completed, washing with pre-cooled phosphate buffer of the same pH, and obtaining of immobilized composite enzymes. The mass ratio of the naringinase to the magnetic hierarchical-pore biochar carrier is 0.05:1; the oscillation reaction parameters are set as follows: temperature 50°C, rotation speed 150 rpm, and time length 2 h.

[0064] S3: extraction and purification of red-heart pomelo polyphenols.

[0065] S31: addition of fresh red-heart pomelo pulp to a citric acid-sodium citrate buffer solution with pH 4.5, stirring, preparation of a uniform slurry, addition of the immobilized composite enzymes prepared in S22 to the uniform slurry, constant-temperature oscillation, separation of the immobilized composite enzymes by placing the reaction container next to a magnet after the reaction is completed, concentration of the supernatant to one-fifth of the original volume to obtain a concentrated solution. The solid-liquid ratio of the fresh red-heart pomelo pulp to the citric acid-sodium citrate buffer solution is 1:4.5 (m:v); the constant-temperature oscillation temperature is 48°C, the rotation speed is 200 rpm, the time length is 3.5 h, the mass-volume ratio of the immobilized composite enzymes to the slurry is 1:25; and the concentration temperature is 50°C, and the vacuum degree is 0.11 MPa.

[0066] S32: passage of the concentrated solution prepared in S31 through a resin column, then flushing of the resin column with deionized water, and then elution with an ethanol aqueous solution to obtain a polyphenol-rich solution.

[0067] S33: The polyphenol-rich liquid is used as a feed liquid for spray drying to obtain red heart pomelo polyphenol powder.

[0068] Example 2

[0069] Referring to the composition and preparation process of Example 1, the difference is that:

[0070] In S11 of the preparation process, the mass ratio of K2CO3 to red heart pomelo powder is 1:1, and the other components are the same.

[0071] In S11 of the preparation process, the temperature ramping rate of programmed pyrolysis is 4℃ / min, the temperature is 600℃, and the holding time is 2h, and the other steps are the same.

[0072] In S12 of the preparation process, the molar ratio of ferric chloride to ferrous sulfate between ferric chloride and ferrous sulfate is 1.5:1, and the other components are the same.

[0073] In S12 of the preparation process, the stirring temperature is 60℃, the rate is 100rpm, the drying temperature is 60℃, and the other steps are the same.

[0074] In S21 of the preparation process, the mass ratio of pectinase and cellulase is 1:1, and the mass ratio of the two to the magnetic hierarchical pore biochar carrier is 0.05:1, and the other components are the same.

[0075] In S21 of the preparation process, the oscillation reaction temperature is 35℃, the rotation speed is 100rpm, and the time is 2h, and the other steps are the same.

[0076] In S22 of the preparation process, the mass ratio of naringinase to magnetic hierarchical pore biochar carrier is 0.02:1, and the other steps are the same.

[0077] In S22 of the preparation process, the oscillation reaction temperature is 45℃, the rotation speed is 100rpm, and the time is 1h, and the other steps are the same.

[0078] In S31 of the preparation process, the fresh red heart pomelo pulp has a feed liquid ratio of 1:3 (m:v) with citric acid-sodium citrate buffer; the constant temperature oscillation temperature is 45℃, the rotation speed is 150rpm, and the time is 3h; the concentration temperature is 40℃, the vacuum degree is 0.09MPa, and the other steps are the same.

[0079] In S31 of the preparation process, the immobilized complex enzyme has a mass-volume ratio of 1:10 with the slurry, and the other components are the same.

[0080] Example 3

[0081] Referring to the composition and preparation process of Example 1, the difference is that:

[0082] In S11 of the preparation process, the mass ratio of K2CO3 to red heart pomelo powder is 1:3, and the other components are the same.

[0083] In S11 of the preparation process, the temperature rising rate of programmed pyrolysis is 6℃ / min, the temperature is 800℃, the holding time is 4h, and the other steps are the same.

[0084] In S12 of the preparation process, the molar ratio of iron chloride to ferrous sulfate between iron chloride and ferrous sulfate is 2.5:1, and the other components are the same.

[0085] In S12 of the preparation process, the stirring temperature is 80℃, the rate is 300rpm, the drying temperature is 80℃, and the other steps are the same.

[0086] In S21 of the preparation process, the mass ratio of pectinase to cellulase is 1:2, and the mass ratio of the two to the magnetic hierarchical porous biochar carrier is 0.15:1, and the other components are the same.

[0087] In S21 of the preparation process, the oscillation reaction temperature is 45℃, the rotation speed is 200rpm, and the time is 4h, and the other steps are the same.

[0088] In S22 of the preparation process, the mass ratio of naringinase to magnetic hierarchical porous biochar carrier is 0.08:1, and the other steps are the same.

[0089] In S22 of the preparation process, the oscillation reaction temperature is 55℃, the rotation speed is 200rpm, and the time is 3h, and the other steps are the same.

[0090] In S31 of the preparation process, the fresh red heart pomelo pulp has a solid-liquid ratio of 1:6 (m:v) with citric acid-sodium citrate buffer solution; constant temperature oscillation, temperature 50℃, rotation speed 250rpm, time 4h; concentration temperature 60℃, vacuum degree 0.12MPa, and the other steps are the same.

[0091] In S31 of the preparation process, the mass volume ratio of immobilized complex enzyme to slurry is 1:50, and the other components are the same.

[0092] Example 4

[0093] Referring to the composition and preparation process of Example 1, the difference is that:

[0094] In S11 of the preparation process, the mass ratio of K2CO3 to red heart pomelo powder is 1:2.5, and the other components are the same.

[0095] In S11 of the preparation process, the temperature rising rate of programmed pyrolysis is 5.5℃ / min, the temperature is 750℃, the holding time is 3.5h, and the other steps are the same.

[0096] In S12 of the preparation process, the molar ratio of iron chloride to ferrous sulfate between iron chloride and ferrous sulfate is 1.8:1, and the other components are the same.

[0097] The stirring temperature in S12 of the preparation process is 72°C, the stirring rate is 180 rpm, the drying temperature is 66°C, and other steps are the same.

[0098] In S21 of the preparation process, the mass ratio of pectinase and cellulase is 1:1.3, and the mass ratio of the two to the magnetic hierarchical-pore biochar carrier is 0.06:1, and other components are the same.

[0099] In S21 of the preparation process, the oscillation reaction temperature is 36°C, the rotation speed is 190 rpm, the time length is 2.5 h, and other steps are the same.

[0100] In S22 of the preparation process, the mass ratio of naringinase to the magnetic hierarchical-pore biochar carrier is 0.07:1, and other steps are the same.

[0101] In S22 of the preparation process, the oscillation reaction temperature is 52°C, the rotation speed is 120 rpm, the time length is 2.5 h, and other steps are the same.

[0102] In S31 of the preparation process, the fresh red heart pomelo pulp has a material-liquid ratio of 1:5 (m:v) to the citric acid-sodium citrate buffer solution; constant temperature oscillation is performed at a temperature of 49°C and a rotation speed of 180 rpm for a time length of 3.5 h; the concentration temperature is 42°C, and the vacuum degree is 0.1 MPa, and other steps are the same.

[0103] In S31 of the preparation process, the immobilized composite enzyme has a mass-volume ratio of 1:30 to the slurry, and other components are the same.

[0104] Comparative Example 1

[0105] Referring to the composition and preparation process of Example 1, the difference is that:

[0106] The enzymes are not immobilized, and the pectinase, cellulase, and naringinase with enzyme activity units are directly mixed and added to the red heart pomelo homogenate for enzymolysis.

[0107] Comparative Example 2

[0108] Referring to the composition and preparation process of Example 1, the difference is that:

[0109] The carrier is changed to a commercialized amino-functionalized magnetic nanoparticle, the three enzymes are mixed, and then immobilized on the carrier by a glutaraldehyde cross-linking method, and the amount of immobilized enzyme and the enzyme hydrolysis conditions are the same as in Example 1.

[0110] Comparative Example 3

[0111] Referring to the composition and preparation process of Example 1, the difference is that:

[0112] In S2 of the preparation process, the pectinase, cellulase, and naringinase are directly mixed and then immobilized on the carrier at one time, and other steps are the same.

[0113] In combination of Examples 1-4 and Comparative Examples 1-3, the immobilized complex enzyme was sampled to test the enzyme activity recovery rate: the immobilized enzyme was added to a mixed substrate solution (buffer containing 1.0% pectin sodium acid and 1.0% carboxymethyl cellulose sodium), the reaction was shaken, after the reaction was completed, the carrier was quickly separated from the reaction solution on a magnetic separation rack, the supernatant was aspirated, color development and determination were performed, and an equal amount of immobilized carrier (without enzyme) was used to react with the substrate as a blank to exclude the interference of the carrier itself adsorption. Similarly, naringinase was tested by the above method, the substrate was 0.5% naringin solution, and the product generation amount per unit time of an equal amount of pectinase, cellulase and naringinase was tested by the above method. The calculation formula is: .

[0114] In combination of Examples 1-4 and Comparative Examples 2-3, the separated immobilized enzyme was sampled to test the enzyme activity retention rate after 5 times: a portion of the immobilized enzyme was not subjected to long-term enzymolysis and was directly used to determine the initial activity. The immobilized enzyme after reaction and activity determination was separated by a magnet, the reaction solution was discarded, the carrier was gently washed with pre-cooled buffer for 3 times, and then was re-put into a new prepared, equal amount of red heart pomelo pulp solution, and was cycled for 5 times. Then, the activity of the recovered immobilized enzyme after the 5th use was determined, and the calculation formula is: .

[0115] In combination of Examples 1-4 and Comparative Examples 1-3, the supernatant prepared by S31 was sampled to test the polyphenol extraction rate: first, 5g of fresh red heart pomelo pulp of the same batch was taken, 50mL of pre-cooled 70% acetone aqueous solution was added, and was thoroughly ground under ice bath condition, was transferred to a centrifugal tube, and was ultrasonically extracted for 30min (power 300W, water temperature was kept at 4°C). Then, it was centrifuged at 8000rpm for 15min at 4°C, the supernatant was aspirated, and 30mL of 70% acetone was added again. The above operation was repeated, and the supernatants of two times were combined to obtain a theoretical test solution. 10mg of gallic acid was accurately weighed, dissolved in 100mL of deionized water to prepare a standard stock solution. 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0mL of the stock solution was respectively taken into a series of 10mL test tubes, Folin-Ciocalteu reagent and Na2CO3 solution were added, and the reaction was carried out in the dark at room temperature for 90min. The absorbance value of each tube was measured by ultraviolet spectrophotometer at 760nm wavelength, taking the blank tube as zero. As shown in Figure 2 , the standard curve was drawn with gallic acid mass (μg) as the abscissa and the absorbance value as the ordinate, and the linear regression equation was obtained. The supernatant and the theoretical test solution were titrated respectively to obtain the total phenol mass, and the polyphenol extraction rate was obtained by comparison.

[0116] The above specific test results are shown in Table 2,Figure 2 、 Figure 3 as shown in Table 2:

[0117] Table 2 Comparison table of core performance of Examples 1-4 and Comparative Examples 1-3

[0118]

[0119] From the above comparison results, it can be seen that the comprehensive performance of Example 1 is the best, the optimized activator ratio, pyrolysis temperature and stepwise immobilization strategy maximize the protection of the active center of each enzyme, use cheap agricultural waste as raw material, low cost, and because the magnetic biochar carrier has a stable hierarchical pore structure, it is firmly combined with the enzyme, and its operation stability is excellent; the comprehensive performance of Examples 2 to 4 is slightly lower than that of Example 1 but still maintains a high level, which shows that excellent extraction effect is achieved under a larger range of parameter changes; Comparative Example 1 has no carrier cost, the enzyme cannot be recovered, the stability is zero, and new enzymes need to be invested each time, so the total cost is the highest in the long run; Comparative Example 2 has a mismatch between the surface properties of the commercial carrier and the enzyme, and the glutaraldehyde crosslinking damages the active center of the enzyme, resulting in the lowest recovery rate; Comparative Example 3 has one-time immobilization, which leads to steric hindrance and competition between enzyme molecules, and the recovery rate is lower than that of all examples.

[0120] In summary, through the above examples and comparative examples, it can be clearly seen that the enzyme method for extracting polyphenols from red heart pomelo provided by the present application is significantly better than the traditional scheme in terms of enzyme activity recovery rate, enzyme activity retention rate after 5 times, and polyphenol extraction rate, which is due to the construction of a hierarchical pore biochar carrier, stepwise immobilization, and complex enzyme cooperation, thereby solving the problems of enzyme activity loss, carrier cost and operation stability of immobilized enzymes in application.

Claims

1. A method for extracting polyphenols from red grapefruit using an enzymatic method, characterized in that, Includes the following steps: S1: Carrier preparation; S11: Dry and crush the peel and seeds of red pomelo to obtain red pomelo powder; mix the red pomelo powder with K2CO3, add deionized water to immerse it, stir, dry at 105℃, pyrolyze under nitrogen protection, cool to room temperature, add HCl, stir, wash, and dry to obtain hierarchical pore biochar. S12: The hierarchical porous biochar prepared in S11 was ultrasonically dispersed in deionized water to obtain a hierarchical porous biochar dispersion; ferric chloride and ferrous sulfate were added to deionized water to prepare an iron salt solution; the hierarchical porous biochar dispersion was mixed with the iron salt solution, stirred, and the pH was adjusted to 10. After the reaction was completed, solid-liquid separation was performed using a magnet, the supernatant was discarded, and the solid product was washed, dried, and ground to obtain a magnetic hierarchical porous biochar carrier. S2: Preparation of immobilized complex enzymes; S21: Dissolve pectinase and cellulase in phosphate buffer, then add the magnetic hierarchical pore biochar carrier prepared in S12, shake the reaction, and after the reaction is complete, use a magnet to adsorb and separate the immobilized carrier from the solution, wash with pre-cooled phosphate buffer of the same pH to obtain the primary immobilized enzyme. S22: Dissolve naringinase in phosphate buffer and add the primary immobilized enzyme prepared in S21. Shake the reaction. After the reaction is complete, use a magnet to separate the solid and liquid again. Wash with pre-cooled phosphate buffer of the same pH to obtain the immobilized complex enzyme. S3: Extraction and purification of polyphenols from red grapefruit; S31: Take fresh red grapefruit pulp, add citrate-sodium citrate buffer solution at pH 4.5, stir, and prepare a homogeneous slurry; add the immobilized complex enzyme prepared in S22 to the homogeneous slurry, oscillate at a constant temperature, and after the reaction is complete, place the reaction vessel next to a magnet to separate the immobilized complex enzyme; concentrate the supernatant to one-fifth of the original volume to obtain a concentrated solution. S32: Pass the concentrated solution prepared in S31 through a resin column, then rinse the resin column with deionized water, and then elute with an ethanol aqueous solution to obtain a polyphenol enriched solution. S33: Use the polyphenol enrichment solution as the feed liquid and spray dry it to obtain red grapefruit polyphenol powder.

2. The method for extracting red grapefruit polyphenols based on enzymatic extraction according to claim 1, characterized in that: The K2CO3 mentioned in S11 has a mass ratio of 1:1 to 1:3 with the red grapefruit powder. The pyrolysis procedure described in S11 has the following parameter settings: heating rate 4-6℃ / min, temperature 600-800℃, and holding time 2-4h.

3. The method for extracting red grapefruit polyphenols based on enzymatic extraction according to claim 1, characterized in that: The ferric chloride and ferrous sulfate described in S12 have a molar ratio of ferric chloride to ferrous sulfate of 1.5:1 to 2.5:

1. The stirring described in S12 has the following parameters: temperature 60-80℃, speed 100-300rpm; The drying process described in S12 has the following parameter settings: temperature 60-80℃.

4. The method for extracting red grapefruit polyphenols based on enzymatic extraction according to claim 1, characterized in that: The pectinase and cellulase described in S21 have a mass ratio of 1:1 to 1:2, and the mass ratio of the two to the magnetic hierarchical pore biochar carrier is 0.05:1 to 0.15:

1. The phosphate buffer solution described in S21 has the following parameters: pH 5.0 and concentration 0.1 mol / L. The oscillation reaction described in S21 has the following parameters: temperature 35-45℃, rotation speed 100-200rpm, and duration 2-4h.

5. The method for extracting red grapefruit polyphenols based on enzymatic extraction according to claim 1, characterized in that: The naringinase described in S22 has a mass ratio of 0.02:1 to 0.08:1 with the magnetic hierarchical pore biochar carrier. The phosphate buffer solution described in S22 has the following parameters: pH 4.0 and concentration 0.1 mol / L. The oscillation reaction described in S22 has the following parameters: temperature 45-55℃, rotation speed 100-200rpm, and duration 1-3h.

6. The method for extracting red grapefruit polyphenols based on enzymatic extraction according to claim 1, characterized in that: The fresh red pomelo pulp described in S31 has a material-to-liquid ratio (m:v) of 1:3 to 1:6 with that of citric acid-sodium citrate buffer solution. The immobilized composite enzyme described in S31 has a mass-to-volume ratio of 1:10 to 1:50 with respect to the slurry. The isothermal oscillation described in S31 has the following parameter settings: temperature 45-50℃, rotation speed 150-250rpm, and duration 3-4h. The concentration described in S31 has the following parameter settings: temperature 40~60℃, vacuum degree 0.09~0.12MPa.

7. The method for extracting red grapefruit polyphenols based on enzymatic extraction according to claim 1, characterized in that: The spray drying described in S33 has the following parameter settings: inlet air temperature 180℃, outlet air temperature 85℃.

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