Modified shaddock peel fiber gel with high naringenin content and preparation method of modified shaddock peel fiber gel
Modified grapefruit peel fiber gel with high naringenin content was prepared by electrochemical-assisted gradient enzymatic hydrolysis and cross-linking reaction, which solved the problem of poor dispersibility and stability of naringenin in aqueous solution, and achieved efficient loading of naringenin and improved stability of gel system.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WUHAN SANJI FOOD TECH CO LTD
- Filing Date
- 2026-02-10
- Publication Date
- 2026-05-05
AI Technical Summary
In existing technologies, naringenin exhibits poor dispersibility and stability in aqueous solutions, making it difficult to load efficiently. Furthermore, the stability and loading capacity of the naringenin fiber gel system are insufficient, resulting in its functionality not being fully realized and resources not being utilized effectively.
An electrochemically assisted gradient enzymatic hydrolysis combined with immobilized naringinase and cross-linking reaction was employed to disrupt the dense structure of grapefruit peel fibers, forming a multi-layer cross-linked network that stabilized the loading of naringin, thereby improving its bioavailability and activity.
It significantly improved the loading rate and stability of naringenin, enhanced the mechanical properties of the gel, achieved efficient enrichment and activity of naringenin, and solved the problems of dispersibility and stability of naringenin in aqueous solution.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural product by-product processing technology, specifically relating to a modified grapefruit peel fiber gel with high naringenin content and its preparation method. Background Technology
[0002] Grapefruit peel is a major byproduct of citrus processing, produced in large quantities and rich in various functional components, possessing significant development potential. Grapefruit peel is rich in dietary fiber and contains a large amount of naringenin, a natural dihydroflavonoid widely found in grapefruit and other citrus peels. Naringenin possesses various effects, including antibacterial, anti-inflammatory, antioxidant, antitussive, and expectorant properties, and can also regulate metabolism and enhance immune function, making it valuable in pharmaceuticals and functional food additives. However, naringenin has inherent drawbacks such as strong hydrophobicity and poor water solubility, making it difficult to disperse in aqueous solutions and resulting in low bioavailability. Furthermore, it is easily degraded by external environmental factors, exhibiting poor stability, which greatly limits its practical application and effectiveness. Grapefruit peel fiber, a major component of grapefruit peel, is a natural dietary fiber with advantages such as wide availability, renewability, non-toxicity, and good biocompatibility. It also possesses certain water absorption, water retention, and gelation potential, making it a potential natural functional carrier material. However, natural grapefruit peel fiber has problems such as dense structure, low surface activity and insufficient functional properties. When applied directly, it has a weak loading capacity for active ingredients such as naringenin and is difficult to form a stable gel system, thus failing to fully play its role as a carrier.
[0003] Currently, existing technologies include research on using modified grapefruit peel fibers to prepare gels or load active ingredients. Modification methods mainly include chemical and physical modifications, which can disrupt the dense structure of grapefruit peel fibers, increase their specific surface area and surface active sites, thereby improving loading and gel performance. Simultaneously, some studies have attempted to combine naringenin with grapefruit peel fibers to prepare composite functional materials, aiming to achieve synergistic effects and improve the solubility and stability of naringenin. However, existing modified grapefruit peel fibers have low loading rates for naringenin, making it difficult to achieve high-content loading and fully utilize the activity of naringenin. Furthermore, the loaded naringenin is prone to desorption and degradation, exhibiting poor stability. In addition, existing preparation processes often suffer from complex procedures, cumbersome operations, and harsh conditions, affecting product safety and biocompatibility. Simultaneously, the resulting gel systems exhibit poor stability, unsatisfactory mechanical properties, and are prone to aging, failing to meet practical application requirements. Moreover, existing technologies have a low degree of comprehensive utilization of grapefruit peel, often only extracting naringenin or utilizing grapefruit peel fibers, resulting in the underutilization of its functional components and resource waste. Summary of the Invention
[0004] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to provide a modified grapefruit peel fiber gel with high naringenin content and its preparation method. The steps are as follows: Grapefruit peel pith is quick-frozen and pulverized, pretreated using an electrochemical-assisted gradient enzymatic hydrolysis process, and then treated with a customized immobilized naringinase to obtain grapefruit peel fiber with high naringenin content. Finally, the fiber is mixed evenly with oxalic acid and soybean saponins, and magnesium chloride is added for cross-linking and maturation to obtain the modified grapefruit peel fiber gel. The present invention utilizes electrochemical-assisted gradient enzymatic hydrolysis to disrupt the dense network structure of the fiber, thereby increasing active sites. The immobilized naringinase not only efficiently converts naringin and precisely enriches naringenin, but also effectively improves bitterness. Magnesium ions regulate the multiple cross-linking networks formed by the components, making the gel structure more stable. Simultaneously, it stably loads naringenin to prevent its migration and aggregation, significantly improving the bioavailability and active efficacy of naringenin.
[0005] Technical solution: A method for preparing modified grapefruit peel fiber gel with high naringenin content, comprising the following steps: S1. Select the white pith of the grapefruit peel after the yellow outer peel has been removed, quick-freeze and pulverize it to obtain grapefruit peel white pith powder; S2. Disperse the pomelo peel white pulp powder in water, treat the pomelo peel white pulp solution with electrochemical-assisted gradient enzymatic hydrolysis, and filter to obtain pretreated pomelo peel white pulp fiber; S3. Immobilized naringinase was added to the pretreated grapefruit peel white pulp fiber for treatment, the enzyme was inactivated by centrifugation, and the fiber was dried to obtain grapefruit peel fiber with high naringin content; S4. Disperse grapefruit peel fiber with high naringenin content in water, then add sarsaparilla acid and soybean saponins, mix well, add magnesium chloride solution to carry out cross-linking reaction, and let stand to mature to obtain modified grapefruit peel fiber gel.
[0006] Preferably, the conditions for quick-freezing and pulverizing in step S1 are: quick-freezing temperature -40 to 25°C, quick-freezing time 1 to 2 hours, and pulverized particle size 100 to 300 mesh.
[0007] Preferably, in step S2, the solid-liquid ratio of grapefruit peel white pulp powder to water is 1:(5-15); the electrochemical auxiliary conditions are a voltage of 10-30V, a pulse frequency of 50-200Hz, and a processing time of 10-30min.
[0008] Preferably, the gradient enzymatic hydrolysis in step S2 consists of a first-stage enzymatic hydrolysis and a second-stage enzymatic hydrolysis. The conditions for the first-stage enzymatic hydrolysis are: 0.2-0.5 vt% of mixed enzyme added, 40-60℃ of hydrolysis temperature, and 0.5-2 h of hydrolysis time. The mixed enzyme is prepared by mixing pectinase and polygalacturonase at a mass ratio of 1:(0.2-1). The conditions for the second-stage enzymatic hydrolysis are: 0.3-0.6 vt% of cellulose endopeptidase added, 45-55℃ of hydrolysis temperature, and 1.5-4 h of hydrolysis time.
[0009] Preferably, the conditions for immobilized naringinase treatment in step S3 are: immobilized naringinase addition amount 1-5wt%, enzymatic hydrolysis temperature 45-65℃, and enzymatic hydrolysis time 3-8h.
[0010] Preferably, the method for preparing the immobilized naringinase in step S3 is as follows: ① Add 0.2-0.6% 3-aminophenylboronic acid to chitosan at a mass fraction of 1-4%, stir evenly, and react at a constant temperature of 45-65℃ for 3-6 hours, then dry to obtain borate-modified chitosan; ② Dissolve hydroxypropyl-β-cyclodextrin and borate-modified chitosan at a mass ratio of (1-3.5):5 to prepare a mixed solution, and uniformly drop it into a 1-3% calcium chloride solution to solidify for 15-30 minutes, then wash and dry to obtain composite microspheres; ③ Disperse the composite microspheres at a solid-liquid ratio of 1:(5-10) in a 2-8 g / L naringin template molecule solution and treat at pH 8.5-9.0 for 5-15 minutes, centrifuge, wash with an ethanol solution containing acetic acid, and dry to obtain naringin molecularly imprinted microspheres; ④ Immerse the naringin molecularly imprinted microspheres at a solid-liquid ratio of 1:(5-10) in a solution with a concentration of 0.1-0.5 g / L. Immobilized naringinase was adsorbed into a mg / mL naringinase solution for 3-8 hours, washed, and dried to obtain immobilized naringinase.
[0011] Preferably, in step S4, the mass ratio of naringenin-containing grapefruit peel fiber, sarsaponin, and soybean saponins is 10:(0.5-2):(1-3); the concentration of magnesium chloride solution is 0.1-0.5 mol / L; and the amount of magnesium chloride solution added is 5-12%.
[0012] Modified grapefruit peel fiber gel prepared by any of the methods described above.
[0013] Beneficial effects: 1. This invention utilizes the redox reaction occurring on the electrode surface to generate active groups such as hydroxyl radicals and hydrogen ions, which break the glycosidic bonds, hydrogen bonds, and hydrophobic interactions between cellulose, hemicellulose, and pectin in the cell walls of grapefruit peel fibers, thereby disrupting the dense cross-linked network formed by the three components and creating micron-sized initial pores in the fibers inside the white pith of the grapefruit peel. At the same time, the current effect changes the charge distribution on the fiber surface, reduces the exposed proportion of hydrophobic groups, significantly improves the hydrophilicity and reactivity of the fiber surface, and provides more accessible sites for subsequent gradient enzymatic hydrolysis. Subsequently, a combination of pectinase and polygalacturonase was used to efficiently break the α-1,4-glycosidic bonds of pectin, thereby disrupting the pectin layer surrounding the cellulose backbone and releasing the physical binding of pectin to cellulose. This fully exposed the previously encapsulated cellulose backbone, creating conditions for the second stage of cellulase degradation. Then, cellulase was used to break only the glycosidic bonds within the cellulose molecular chains in the white pith of grapefruit peel and perform limited hydrolysis. This approach did not damage the overall structural integrity of the fiber but also formed a large number of nanoscale micropores inside and on the surface of the fiber, significantly increasing the specific surface area and exposing more active sites.
[0014] 2. This invention uses chitosan modified with boric acid groups and hydroxypropyl-β-cyclodextrin as microsphere packaging materials. Naringin serves as the template molecule, and the vicinal diol groups on its aglycone and sugar chains can form stable borate ester bonds with the boric acid groups, achieving precise anchoring of the naringin template molecule to the packaging material. After elution, a conformationally stable molecular imprinted cavity is left, which facilitates the specific binding of naringin in the grapefruit peel fiber matrix. In addition, the positive charge of chitosan can generate a strong electrostatic attraction with the negative charge of the carboxyl group of naringinase, so that naringinase is firmly fixed on the surface of the imprinted microspheres. The abundant hydroxyl and amino groups on the surface of the naringinase molecule can form abundant hydrogen bond crosslinks with the hydroxyl and amino groups on the surface of the microspheres, thereby achieving stable fixation of naringinase, which is conducive to the efficient decomposition of naringin in the grapefruit peel fiber matrix to generate naringenin.
[0015] 3. The loose and porous grapefruit peel fiber matrix of the present invention exposes a large number of active sites on its surface, which can form hydrogen bonds with the hydroxyl groups of chitosan, the wall material of the immobilized enzyme microspheres, allowing it to quickly penetrate into the interior of the grapefruit peel fiber matrix. Furthermore, the molecular recognition effect of the naringin molecularly imprinted microspheres can efficiently and specifically bind to naringin in the grapefruit peel fiber matrix. At the same time, the naringinase immobilized by the microspheres decomposes the glucose-rhamnoside glycosidic bond of the naringin molecule, converting it into naringenin aglycone and greatly improving the bitterness of the grapefruit peel white pulp. In addition, naringenin can first be adsorbed onto the molecular imprinted sites of the microspheres through weak hydrophobic interactions and hydrogen bonds. The hydrophobic cavity of hydroxypropyl-β-cyclodextrin in the microspheres is firmly enriched in its hydrophobic cavity through strong hydrophobic interactions with the hydrophobic aglycone of naringenin, achieving the purpose of precise decomposition of naringin and efficient enrichment of naringenin.
[0016] 4. In the process of preparing modified grapefruit peel fiber gel, magnesium ions act as coordination centers to form stable coordination structures with the carboxyl and hydroxyl groups of adjacent molecular chains on grapefruit peel fibers, thereby constructing an ion bridge connection network in grapefruit peel fibers. Furthermore, the carboxyl groups contained in soybean saponins and oxalic acid form coordination crosslinks with magnesium ions through metal coordination. The hydrophobic triterpenoid saponins contained in soybean saponins and the catechol aromatic ring of oxalic acid form a hydrophobic core through π-π stacking, which is embedded in the micropores of grapefruit peel fibers to form grapefruit peel fiber aggregates. In addition, the polyhydroxy structure on the hydrophilic sugar chain of soybean saponins, the phenolic hydroxyl groups of oxalic acid, and the cellulose molecular chains of grapefruit peel fibers intertwine with each other through the formation of an intermolecular hydrogen bond network, so that the grapefruit peel fiber aggregates are further assembled into modified grapefruit peel fiber gel with a stable structure. Magnesium ions can form weak coordination chelates with the hydroxyl and carbonyl groups in naringenin molecules. Due to its hydrophobic flavonoid skeleton, naringenin molecules can bind to the hydrophobic core region formed by soybean saponins and oxalic acid. Through hydrophobic interactions and π-π stacking, the stable loading of naringenin in the modified grapefruit peel fiber gel is further ensured. This avoids the migration and aggregation problems caused by naringenin detaching from the composite molecularly imprinted microspheres, and does not excessively bind the molecular structure of naringenin, ensuring that naringenin can still exert its biological activity better after release. Detailed Implementation
[0017] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: Example 1
[0018] A method for preparing immobilized naringinase includes the following steps: Step 1. Weigh 2.0g of chitosan powder and dissolve it in 98mL of acetic acid solution to obtain a 2% chitosan solution; add 0.5g of 3-aminophenylboronic acid to 100mL of chitosan solution, stir at 300r / min until homogeneous, and then stir at 55℃ and 250r / min for 4.5h; place in a vacuum drying oven at 60℃ and dry for 2h to obtain borate-treated chitosan powder; Step 2. Weigh 2.0g of hydroxypropyl-β-cyclodextrin and 5.0g of borate-modified chitosan powder, dissolve them in 200mL of acetic acid solution to prepare a mixture; add the mixture dropwise at a uniform rate of 2mL / min to 500mL of 1% calcium chloride solution and stir to solidify for 15min, then filter and wash the filter residue three times with water, and dry in a vacuum drying oven at 60℃ for 1.5h to obtain composite microspheres; Step 3. Weigh 2g of naringin and add it to 1L of water. Dissolve it using an ultrasonic power of 200W for 15min to obtain a 2g / L naringin template molecule solution. Adjust the pH of the solution to 8.5. Weigh 20g of composite microspheres and disperse them in 100mL of naringin template molecule solution. Stir at 250r / min for 5min and centrifuge at 3000r / min for 10min to collect the precipitate. Then wash the precipitate three times with a 50mL volume fraction of 95% ethanol solution containing 5% acetic acid for 5min each time. Place the precipitate in a vacuum dryer at 60℃ for 1h to obtain naringin molecularly imprinted microspheres. Step 4. Weigh 20g of naringin molecularly imprinted microspheres and immerse them in 100mL of 0.1mg / mL naringinase solution. Static adsorption is carried out at 25℃ for 3h. After filtration, the microspheres are vacuum dried at 50℃ for 1h to obtain the immobilized naringinase product. Example 2
[0019] A method for preparing immobilized naringinase includes the following steps: Step 1. Weigh 1.0 g of chitosan powder and dissolve it in 99 mL of acetic acid solution to obtain a 1% chitosan solution; add 0.3 g of 3-aminophenylboronic acid to 100 mL of chitosan solution, stir at 300 r / min until homogeneous, and then stir at 45 °C and 300 r / min for 3 h; place in a vacuum drying oven at 60 °C and dry for 2 h to obtain borate-treated chitosan powder; Step 2. Weigh 1.0 g of hydroxypropyl-β-cyclodextrin and 5.0 g of borate-modified chitosan powder, dissolve them in 200 mL of acetic acid solution to prepare a mixture; add the mixture dropwise at a uniform rate of 2 mL / min to 500 mL of 1.5% calcium chloride solution and stir to solidify for 15 min, then filter and wash the filter residue three times with water, and dry in a vacuum drying oven at 60 °C for 2 h to obtain composite microspheres; Step 3. Weigh 2g of naringin and add it to 1L of water. Dissolve it using an ultrasonic power of 200W for 15min to obtain a 2g / L naringin template molecule solution. Adjust the pH of the solution to 8.5. Weigh 20g of composite microspheres and disperse them in 120mL of naringin template molecule solution. Stir at 300r / min for 5min and centrifuge at 3500r / min for 10min to collect the precipitate. Then wash the precipitate three times with a 50mL volume fraction of 95% ethanol solution containing 5% acetic acid for 5min each time. Place the precipitate in a vacuum dryer at 60℃ for 1h to obtain naringin molecularly imprinted microspheres. Step 4. Weigh 30g of naringin molecularly imprinted microspheres and immerse them in 150mL of 0.1mg / mL naringinase solution. Static adsorption is carried out at 25℃ for 3h. After filtration, the microspheres are vacuum dried at 50℃ for 1.5h to obtain immobilized naringinase. Example 3
[0020] A method for preparing immobilized naringinase includes the following steps: Step 1. Weigh 4.0g of chitosan powder and dissolve it in 96mL of acetic acid solution to obtain a 4% chitosan solution; add 0.6g of 3-aminophenylboronic acid to 100mL of chitosan solution, stir at 300r / min until homogeneous, and then stir at 65℃ and 200r / min for 5h; place in a vacuum drying oven at 60℃ and dry for 2h to obtain borate-treated chitosan powder; Step 2. Weigh 3.5g of hydroxypropyl-β-cyclodextrin and 5.0g of borate-modified chitosan powder, dissolve them in 200mL of acetic acid solution to prepare a mixture; add the mixture dropwise at a uniform rate of 1mL / min to 500mL of 3% calcium chloride solution and stir to solidify for 10min, then filter and wash the filter residue with water 3 times, and dry in a vacuum drying oven at 60℃ for 1.5h to obtain composite microspheres; Step 3. Weigh 8g of naringin and add it to 1L of water. Dissolve it using an ultrasonic power of 200W for 15min to obtain a naringin template molecule solution with a concentration of 8g / L. Adjust the pH of the solution to 9.0. Weigh 10g of composite microspheres and disperse them in 100mL of naringin template molecule solution. Stir at 300r / min for 10min and centrifuge at 4500r / min for 5min to collect the precipitate. Then wash the precipitate three times with a 5% ethanol solution containing 5% acetic acid, each time using 50mL of ethanol for 5min. Place the precipitate in a vacuum dryer at 60℃ for 1h to obtain naringin molecularly imprinted microspheres. Step 4. Weigh 10g of naringin molecularly imprinted microspheres and immerse them in 100mL of 0.5mg / mL naringinase solution. Static adsorption is carried out at 25℃ for 5h. After filtration, the microspheres are vacuum dried at 50℃ for 1.5h to obtain immobilized naringinase.
[0021] Example 4 A method for preparing immobilized naringinase includes the following steps: Step 1. Weigh 1.5g of chitosan powder and dissolve it in 98.5mL of acetic acid solution to obtain a 1.5% chitosan solution by mass fraction; add 0.4g of 3-aminophenylboronic acid to 100mL of chitosan solution, stir evenly at 350r / min, and then stir at a constant temperature of 50℃ and 300r / min for 5h; place in a vacuum drying oven at 60℃ and dry for 2h to obtain borate-treated chitosan powder; Step 2. Weigh 1.5g of hydroxypropyl-β-cyclodextrin and 5.0g of borate-modified chitosan powder, dissolve them in 200mL of acetic acid solution to prepare a mixture; add the mixture dropwise at a uniform rate of 1mL / min to 500mL of 1.5% calcium chloride solution and stir to solidify for 20min, then filter and wash the filter residue with water 3 times, and dry in a vacuum drying oven at 60℃ for 1.5h to obtain composite microspheres; Step 3. Weigh 3g of naringin and add it to 1L of water. Dissolve it using an ultrasonic power of 200W for 15min to obtain a 3g / L naringin template molecule solution. Adjust the pH of the solution to 9.0. Weigh 25g of composite microspheres and disperse them in 150mL of naringin template molecule solution. Stir at 250r / min for 10min and centrifuge at 3000r / min for 10min to collect the precipitate. Then wash the precipitate three times with a 50mL volume fraction of 95% ethanol solution containing 5% acetic acid for 5min each time. Place the precipitate in a vacuum dryer at 60℃ for 1h to obtain naringin molecularly imprinted microspheres. Step 4. Weigh 20g of naringin molecularly imprinted microspheres and immerse them in 120mL of 0.2mg / mL naringinase solution. Static adsorption is carried out at 25℃ for 4h. After filtration, the microspheres are vacuum dried at 50℃ for 1.5h to obtain the immobilized naringinase product. Example 5
[0022] A method for preparing immobilized naringinase includes the following steps: Step 1. Weigh 3.0g of chitosan powder and dissolve it in 97mL of acetic acid solution to obtain a 3% chitosan solution; add 0.5g of 3-aminophenylboronic acid to 100mL of chitosan solution, stir at 300r / min until homogeneous, and then stir at 60℃ and 300r / min for 5h; place in a vacuum drying oven at 60℃ and dry for 2h to obtain borate-treated chitosan powder; Step 2. Weigh 3.0g of hydroxypropyl-β-cyclodextrin and 5.0g of borate-modified chitosan powder, dissolve them in 200mL of acetic acid solution to prepare a mixture; add the mixture dropwise at a uniform rate of 2mL / min to 500mL of 2.5% calcium chloride solution and stir to solidify for 10min, then filter and wash the filter residue with water 3 times, and dry in a vacuum drying oven at 60℃ for 2h to obtain composite microspheres; Step 3. Weigh 7g of naringin and add it to 1L of water. Dissolve it using an ultrasonic power of 200W for 15min to obtain a 7g / L naringin template molecule solution. Adjust the pH of the solution to 9.0. Weigh 20g of composite microspheres and disperse them in 180mL of naringin template molecule solution. Stir at 300r / min for 15min and centrifuge at 4500r / min for 5min to collect the precipitate. Then wash the precipitate three times with a 50mL volume fraction of 95% ethanol solution containing 5% acetic acid for 5min each time. Place the precipitate in a vacuum dryer at 60℃ for 1h to obtain naringin molecularly imprinted microspheres. Step 4. Weigh 20g of naringin molecularly imprinted microspheres and immerse them in 180mL of 0.4mg / mL naringinase solution. Static adsorption is carried out at 25℃ for 7h. After filtration, the microspheres are vacuum dried at 50℃ for 1.5h to obtain immobilized naringinase.
[0023] Example 6 A method for preparing immobilized naringinase includes the following steps: Step 1. Weigh 2.5g of chitosan powder and dissolve it in 97.5mL of acetic acid solution to obtain a 2.5% chitosan solution; add 0.4g of 3-aminophenylboronic acid to 100mL of chitosan solution, stir evenly at 300r / min, and then stir at 55℃ and 200r / min for 4h; place at 60℃ and vacuum dry for 2h to obtain borate-treated chitosan powder; Step 2. Weigh 2.5g of hydroxypropyl-β-cyclodextrin and 5.0g of borate-modified chitosan powder, dissolve them in 200mL of acetic acid solution to prepare a mixture; add the mixture dropwise at a uniform rate of 2mL / min to 500mL of 2% calcium chloride solution and stir to solidify for 20min, then filter and wash the filter residue with water 3 times, and dry in a vacuum drying oven at 60℃ for 1.5h to obtain composite microspheres; Step 3. Weigh 6g of naringin and add it to 1L of water. Dissolve it using an ultrasonic power of 200W for 20min to obtain a 6g / L naringin template molecule solution. Adjust the pH of the solution to 9.0. Weigh 20g of composite microspheres and disperse them in 140mL of naringin template molecule solution. Stir at 300r / min for 10min and centrifuge at 4500r / min for 5min to collect the precipitate. Then wash the precipitate three times with 50mL of 95% ethanol solution containing 5% acetic acid for 5min each time. Place the precipitate in a vacuum dryer at 60℃ for 1.5h to obtain naringin molecularly imprinted microspheres. Step 4. Weigh 20g of naringin molecularly imprinted microspheres and immerse them in 160mL of 0.3mg / mL naringinase solution. Static adsorption is carried out at 25℃ for 6h. After filtration, the microspheres are vacuum dried at 50℃ for 1.5h to obtain immobilized naringinase. Comparative Example 1
[0024] The difference between this comparative example and Example 6 is that hydroxypropyl-β-cyclodextrin is not added; the remaining operations are the same as in Example 6.
[0025] Performance testing (1) Average particle size Accurately weigh 5 mg of immobilized naringinase and add it to 2 mL of phosphate buffer. Disperse the mixture by sonication for 3 min. Set aside for later use. Then, use a dynamic light scattering particle size analyzer to determine the average value. Test conditions: temperature 25℃, scattering angle 90°.
[0026] (2) Enzyme immobilization efficiency Protein concentration was determined using the Coomassie Brilliant Blue method. Enzyme immobilization efficiency was calculated using the formula: Enzyme immobilization efficiency (%) = (C0V0 - C1V1) / C0V0 × 100%, where C0 is the initial naringinase concentration, V0 is the initial enzyme solution volume, C1 is the protein concentration in the supernatant after immobilization, and V1 is the supernatant volume.
[0027] Table 1. Average particle size and enzyme immobilization efficiency of the immobilized naringinase prepared in Examples 1-6 and Comparative Example 1
[0028] As shown in Table 1, the average particle size of the immobilized naringinase prepared in Examples 1-6 is smaller than that in Comparative Example 1, and the enzyme immobilization efficiency is significantly lower than that in Comparative Example 1. This indicates that the introduction of hydroxypropyl-β-cyclodextrin can tightly bind to chitosan, increase the specific surface area of the microspheres and the degree of exposure of active sites, thereby significantly enhancing the binding ability of naringinase molecules. Example 7
[0029] A method for preparing a modified grapefruit peel fiber gel with high naringenin content includes the following steps: S1. Select the white pith of the grapefruit peel after removing the yellow outer peel, cut it into small pieces and quick-freeze it at -35℃ for 1.5 hours; then crush the quick-frozen white pith of the grapefruit peel at high speed, and collect the sieve material through a 200-mesh standard sieve, which is the grapefruit peel white pith powder. S2. Weigh 100g of grapefruit peel white pulp powder, add 1L of water and stir for 30min to obtain grapefruit peel white pulp solution. Then, electrochemically treat it for 20min under the conditions of voltage 20V and pulse frequency 120Hz to obtain pretreated oily peel white pulp solution. S3. Mix 10g of pectinase and 6g of polygalacturonase to obtain a mixed enzyme. Add 3g of the mixed enzyme to 1L of pretreated grapefruit peel white pulp solution and enzymatically hydrolyze at 50℃ for 1h. Then add 4g of cellulase and enzymatically hydrolyze at 50℃ for 2.5h. After enzymatic hydrolysis, heat at 95℃ for 5min to inactivate the enzyme. Use a 0.45μm filter membrane for vacuum filtration to collect the filter residue, which is the pretreated grapefruit peel white pulp fiber. S4. Disperse 50g of pretreated grapefruit peel white pulp fiber in 500mL of water, add 1.5g of immobilized naringinase prepared in Example 6 and treat at 55℃ and 120r / min for 5h; after treatment, heat at 95℃ for 10min to inactivate the enzyme, filter and collect the precipitate, and vacuum dry at 60℃ to obtain grapefruit peel fiber with high naringin content. S5. Weigh 50g of grapefruit peel fiber with high naringenin content, add 1L of water and stir for 20min. Then add 6g of oxalic acid and 10g of soybean saponins and continue stirring for 15min. Add 80mL of 0.3mol / L magnesium chloride solution at a uniform speed and let stand for 4h to mature. Dry to obtain modified grapefruit peel fiber gel.
[0030] Example 8 A method for preparing a modified grapefruit peel fiber gel with high naringenin content includes the following steps: S1. Select the white pith of the grapefruit peel after removing the yellow outer peel, cut it into small pieces and quick-freeze it at -40℃ for 1 hour; then crush the quick-frozen white pith of the grapefruit peel at high speed, and collect the sieve material through a 300-mesh standard sieve, which is grapefruit peel white pith powder. S2. Weigh 100g of grapefruit peel white pulp powder, add 0.5L of water and stir for 20min to obtain grapefruit peel white pulp solution. Then, electrochemically treat it for 10min under the conditions of voltage 30V and pulse frequency 50Hz to obtain pretreated oil peel white pulp solution. S3. Mix 10g of pectinase and 2g of polygalacturonase to obtain a mixed enzyme. Add 2g of the mixed enzyme to 1L of pretreated grapefruit peel white pulp solution and hydrolyze at 40℃ for 1.5h. Then add 3g of cellulase and hydrolyze at 45℃ for 1.5h. After hydrolysis, heat at 95℃ for 5min to inactivate the enzyme. Collect the filter residue by vacuum filtration using a 0.45μm filter membrane. This residue is the pretreated grapefruit peel white pulp fiber. S4. Disperse 100g of pretreated grapefruit peel white pulp fiber in 1L of water, add 2g of immobilized naringinase prepared in Example 6 and treat at 45℃ and 120r / min for 3h; after treatment, heat at 95℃ for 10min to inactivate the enzyme, filter and collect the precipitate, and vacuum dry at 60℃ to obtain grapefruit peel fiber with high naringin content. S5. Weigh 100g of grapefruit peel fiber with high naringenin content, add 1L of water and stir for 20min, then add 5g of sarsaparilla acid and 10g of soybean saponins and continue stirring for 20min. Add 50mL of 0.1mol / L magnesium chloride solution at a uniform speed, let stand and mature for 3.5h, and dry to obtain modified grapefruit peel fiber gel. Example 9
[0031] A method for preparing a modified grapefruit peel fiber gel with high naringenin content includes the following steps: S1. Select the white pith of the grapefruit peel after removing the yellow outer peel, cut it into small pieces and quick-freeze it at -25℃ for 2 hours; then crush the quick-frozen white pith of the grapefruit peel at high speed, and collect the sieve material through a 100-mesh standard sieve, which is the grapefruit peel white pith powder. S2. Weigh 100g of grapefruit peel white pulp powder, add 1.5L of water and stir for 30min to obtain grapefruit peel white pulp solution. Then, electrochemically treat it for 30min under the conditions of voltage 10V and pulse frequency 200Hz to obtain pretreated oily peel white pulp solution. S3. Mix 10g of pectinase and 10g of polygalacturonase to obtain a mixed enzyme. Add 5g of the mixed enzyme to 1L of pretreated grapefruit peel white pulp solution and enzymatically hydrolyze at 60℃ for 2h. Then add 6g of cellulase and enzymatically hydrolyze at 55℃ for 4h. After enzymatic hydrolysis, heat at 95℃ for 5min to inactivate the enzyme. Use a 0.45μm filter membrane for vacuum filtration to collect the filter residue, which is the pretreated grapefruit peel white pulp fiber. S4. Disperse 50g of pretreated grapefruit peel white pulp fiber in 400mL of water, add 2.5g of immobilized naringinase prepared in Example 6, and treat at 65℃ and 120r / min for 6.5h; after treatment, heat at 95℃ for 15min to inactivate the enzyme, filter and collect the precipitate, and vacuum dry at 60℃ to obtain grapefruit peel fiber with high naringin content. S5. Weigh 50g of grapefruit peel fiber with high naringenin content, add 1L of water and stir for 20min. Then add 10g of sarsaparilla acid and 15g of soybean saponins and continue stirring for 20min. Add 120mL of 0.5mol / L magnesium chloride solution at a uniform speed and let stand for 4.5h to mature. Dry to obtain modified grapefruit peel fiber gel.
[0032] Example 10 A method for preparing a modified grapefruit peel fiber gel with high naringenin content includes the following steps: S1. Select the white pith of the grapefruit peel after removing the yellow outer peel, cut it into small pieces and quick-freeze it at -35℃ for 2 hours; then crush the quick-frozen white pith of the grapefruit peel at high speed, and collect the sieve material through a 250-mesh standard sieve, which is the grapefruit peel white pith powder. S2. Weigh 100g of grapefruit peel white pulp powder, add 0.8L of water and stir for 30min to obtain grapefruit peel white pulp solution. Then, electrochemically treat it for 15min under the conditions of voltage 25V and pulse frequency 100Hz to obtain pretreated oily peel white pulp solution. S3. Mix 10g of pectinase and 4g of polygalacturonase to obtain a mixed enzyme. Add 4g of the mixed enzyme to 1L of pretreated grapefruit peel white pulp solution and hydrolyze at 45℃ for 1.5h. Then add 5g of cellulase and hydrolyze at 50℃ for 3h. After hydrolysis, heat at 95℃ for 10min to inactivate the enzyme. Use a 0.45μm filter membrane for vacuum filtration to collect the filter residue, which is the pretreated grapefruit peel white pulp fiber. S4. Disperse 50g of pretreated grapefruit peel white pulp fiber in 500mL of water, add 1g of immobilized naringinase prepared in Example 6 and treat at 50℃ and 120r / min for 4h; after treatment, heat at 95℃ for 10min to inactivate the enzyme, filter and collect the precipitate, and vacuum dry at 60℃ to obtain grapefruit peel fiber with high naringin content. S5. Weigh 50g of grapefruit peel fiber with high naringenin content, add 1L of water and stir for 20min. Then add 4g of sarsaparilla acid and 7.5g of soybean saponins and continue stirring for 30min. Add 60mL of 0.2mol / L magnesium chloride solution at a uniform speed and let stand for 4h to mature. Dry to obtain modified grapefruit peel fiber gel. Example 11
[0033] A method for preparing a modified grapefruit peel fiber gel with high naringenin content includes the following steps: S1. Select the white pith of the grapefruit peel after removing the yellow outer peel, cut it into small pieces and quick-freeze it at -30℃ for 1.5 hours; then crush the quick-frozen white pith of the grapefruit peel at high speed, and collect the sieve material through a 200-mesh standard sieve, which is the grapefruit peel white pith powder. S2. Weigh 100g of grapefruit peel white pulp powder, add 1.2L of water and stir for 30min to obtain grapefruit peel white pulp solution. Then, electrochemically treat it for 25min under the conditions of voltage 15V and pulse frequency 150Hz to obtain pretreated oily peel white pulp solution. S3. Mix 10g of pectinase and 8g of polygalacturonase to obtain a mixed enzyme. Add 3.5g of the mixed enzyme to 1L of pretreated grapefruit peel white pulp solution and hydrolyze at 55℃ for 1.5h. Then add 4.5g of cellulase and hydrolyze at 50℃ for 3.5h. After hydrolysis, heat at 95℃ for 10min to inactivate the enzyme. Collect the filter residue by vacuum filtration through a 0.45μm filter membrane. This residue is the pretreated grapefruit peel white pulp fiber. S4. Disperse 50g of pretreated grapefruit peel white pulp fiber in 500mL of water, add 2g of immobilized naringinase prepared in Example 6 and treat at 60℃ and 120r / min for 6h; after treatment, heat at 95℃ for 15min to inactivate the enzyme, filter and collect the precipitate, and vacuum dry at 60℃ to obtain grapefruit peel fiber with high naringin content. S5. Weigh 60g of grapefruit peel fiber with high naringenin content, add 1L of water and stir for 20min. Then add 9g of oxalic acid and 15g of soybean saponins and continue stirring for 25min. Add 100mL of 0.4mol / L magnesium chloride solution at a uniform speed and let stand for 4.5h to mature. Dry to obtain modified grapefruit peel fiber gel. Comparative Example 2
[0034] The difference between this comparative example and Example 9 is that the immobilized naringinase prepared in Comparative Example 1 was used; the remaining operations are the same as in Example 9. Comparative Example 3
[0035] The difference between this comparative example and Example 9 is that no electrochemical-assisted treatment is used; the remaining operations are the same as in Example 9. Comparative Example 4
[0036] The difference between this comparative example and Example 9 is that the pectinase, polygalacturonase and cellulase are directly mixed before use, without gradient enzymatic hydrolysis. The remaining operations are the same as in Example 9. Comparative Example 5
[0037] The difference between this comparative example and Example 9 is that the immobilized naringinase prepared in Example 6 is replaced with naringinase, and the remaining operations are the same as in Example 9.
[0038] Performance testing (1) Content of naringin and naringenin Accurately weigh 0.25 g of sample into an iodine flask, add 25 mL of methanol, and accurately weigh the sample. Extract the sample using an ultrasonic processor at 40 kHz and 400 W for 1 hour. Remove the sample and allow it to cool to room temperature. Accurately weigh the sample and replenish the lost mass with methanol. Shake thoroughly to mix, then filter the solution. Filter the filtrate through a 0.45 μm microporous membrane for later use. Accurately weigh 4.5 mg of naringin reference standard and 2.5 mg of naringenin reference standard, dissolve them in methanol, and prepare stock solutions with concentrations of 0.18 mg / mL and 0.1 mg / mL, respectively. Analyze the samples using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Hypersil ODS2C column. 18 The chromatographic column (4.6 mm × 250 mm), the mobile phase was acetonitrile A and water B, the elution gradient was (0-10 min, 20%-60% A; 10-20 min, 60% A), the detection wavelength was 283 nm, the injection volume was 10 μL, the flow rate was 1 mL / min, and the column temperature was 25 ℃.
[0039] (2) Water-holding and oil-holding properties Accurately weigh 0.5000 g of the modified grapefruit peel fiber gel sample dried to constant weight and place it in a 50 mL centrifuge tube. Add 30 mL of distilled water and let it stand at room temperature for 2 hours. During this period, gently shake the tube once every 30 minutes to allow the sample to fully absorb water and swell. After soaking, centrifuge at 4000 r / min for 20 minutes. Carefully pour off the supernatant and blot the remaining water droplets on the tube wall with filter paper. Weigh the sample immediately. Then, place the centrifuge tube and its contents at 60℃ to dry to constant weight. Calculate the water-holding capacity by dividing the difference between the wet sample mass and the dry sample mass after centrifugation by the dry sample mass.
[0040] Accurately weigh 0.5000 g of the modified grapefruit peel fiber gel sample dried to constant weight and place it in a 50 mL centrifuge tube. Add 30 mL of soybean oil and let it stand at room temperature for 2 hours, gently shaking it once every 30 minutes. After soaking, centrifuge at 4000 r / min for 20 minutes, carefully pour off the upper oil phase and blot the remaining oil droplets on the tube wall with filter paper, and weigh it immediately. Then, place the centrifuge tube and its contents at 60℃ to dry to constant weight. Calculate the oil retention by dividing the difference between the mass of the oil-soaked sample and the mass of the dried sample by the mass of the dried sample.
[0041] Table 2. Naringin, naringenin, water-holding capacity, and oil-holding capacity of the modified grapefruit peel fiber gels prepared in Examples 7-11 and Comparative Examples 2-5.
[0042] As shown in Table 2, the naringenin content of the modified grapefruit peel fiber gels prepared in Examples 7-11 was significantly higher than that in Comparative Examples 2-5. At the same time, the water-holding and oil-holding properties of the fiber gels were also better than those in Comparative Examples 2-5. This indicates that the modified grapefruit peel fiber gel prepared by the method described in this invention can promote the efficient decomposition of naringin and the efficient enrichment of naringenin, while significantly improving the hydrophilic and lipophilic amphiphilicity and the stability of the three-dimensional pore structure of the fiber network, thereby endowing the gel with excellent water-holding and oil-holding synergistic properties.
[0043] (3) Simulated in vitro digestion stability Accurately weigh 1.0000g of modified grapefruit peel fiber gel sample and place it in a 100mL Erlenmeyer flask with a stopper. Add 10mL of simulated saliva and incubate in a water bath at 37℃ and 100r / min for 2min to complete oral digestion. Immediately afterward, add 20mL of simulated gastric juice (pH 1.5) and continue incubating in a water bath at 37℃ for 2h to complete gastric digestion. Immediately after gastric digestion, take 5mL of the digestion solution and add 20mL of methanol to terminate the reaction. After centrifugation at 8000r / min for 10min, take the supernatant and filter it through a 0.45μm filter membrane. HPLC is used to determine the naringenin content as the amount released after simulated gastric digestion. Subsequently, add 30mL of simulated intestinal juice (pH 6.8) to the remaining digestion solution and incubate in a water bath at 37℃ for 2h to complete intestinal digestion. Take a sample to terminate the reaction, centrifuge and filter, and then determine the naringenin content by HPLC as the amount released after simulated intestinal digestion.
[0044] (4) Antioxidant activity Take a stoppered test tube and add 1.0 mL of 9 mmol / L ferrous sulfate solution, 1.0 mL of 9 mmol / L salicylic acid-ethanol solution, and 1.0 mL of sample solution (concentration 2 mg / mL) in sequence. Finally, add 1.0 mL of 8.8 mmol / L hydrogen peroxide solution to initiate the Fenton reaction to generate hydroxyl radicals. Shake well and react in a water bath at 37°C for 30 min. Immediately after removing the tube, zero the absorbance with distilled water and measure the absorbance at 510 nm. At the same time, measure the blank absorbance with distilled water instead of the sample absorbance, and measure the control absorbance with distilled water instead of hydrogen peroxide to eliminate the color interference of the sample itself. Calculate the scavenging rate of the sample for hydroxyl radicals based on the degree of absorbance reduction.
[0045] Take 2.0 mL of 0.1 mmol / L DPPH ethanol solution into a stoppered test tube, add 2.0 mL of sample solution (concentration of 1 mg / mL), mix well, and react at room temperature in the dark for 30 min. After zeroing with anhydrous ethanol, measure the absorbance at 517 nm. At the same time, use anhydrous ethanol to replace the sample absorbance to measure the blank absorbance, and use anhydrous ethanol to replace the DPPH solution to measure the control absorbance to eliminate the color interference of the sample itself. Calculate the DPPH free radical scavenging rate of the sample based on the degree of DPPH purple fading, i.e., the decrease in absorbance. The entire operation process must be strictly protected from light to prevent spontaneous decomposition of DPPH.
[0046] Table 3. Gastrointestinal digestibility and antioxidant activity of the modified grapefruit peel fiber gels prepared in Example 9 and Comparative Examples 2-5.
[0047] As shown in Table 3, in the process of simulating in vitro digestion stability, the release rate of naringenin in the gastric digestion stage of Example 9 was significantly lower than that in Comparative Examples 2-5, while the release rate of naringenin in the simulated intestinal digestion stage was significantly higher than that in Comparative Examples 2-5. Moreover, the scavenging rates of hydroxyl radicals and DPPH radicals were both higher than those in Comparative Examples 2-5. This indicates that the modified grapefruit peel fiber gel prepared by the present invention can ensure the stable existence of naringenin in the stomach and its targeted release in the intestine, significantly improving the bioavailability and antioxidant efficacy of naringenin. This fully verifies the synergistic effect of the fiber network structure of the method described in the present invention on the protection and sustained release of active ingredients.
[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.
Claims
1. A method for preparing a modified grapefruit peel fiber gel with high naringenin content, characterized in that, Includes the following steps: S1. Select the white pith of the grapefruit peel after the yellow outer peel has been removed, quick-freeze and pulverize it to obtain grapefruit peel white pith powder; S2. Disperse the pomelo peel white pulp powder in water, treat the pomelo peel white pulp solution with electrochemical-assisted gradient enzymatic hydrolysis, and filter to obtain pretreated pomelo peel white pulp fiber; S3. Immobilized naringinase was added to the pretreated grapefruit peel white pulp fiber for treatment, the enzyme was inactivated by centrifugation, and the fiber was dried to obtain grapefruit peel fiber with high naringin content; S4. Disperse grapefruit peel fiber with high naringenin content in water, then add sarsaparilla acid and soybean saponins, mix well, add magnesium chloride solution to carry out cross-linking reaction, and let stand to mature to obtain modified grapefruit peel fiber gel.
2. The method for preparing a modified grapefruit peel fiber gel with high naringenin content according to claim 1, characterized in that: The conditions for quick-freezing and pulverizing in step S1 are: quick-freezing temperature -40 to 25°C, quick-freezing time 1 to 2 hours, and pulverized particle size 100 to 300 mesh.
3. The method for preparing a modified grapefruit peel fiber gel with high naringenin content according to claim 1, characterized in that: In step S2, the solid-liquid ratio of grapefruit peel white pulp powder to water is 1:(5-15); the electrochemical auxiliary conditions are voltage 10-30V, pulse frequency 50-200Hz, and processing time 10-30min.
4. The method for preparing a modified grapefruit peel fiber gel with high naringenin content according to claim 1, characterized in that: The gradient enzymatic hydrolysis in step S2 consists of a first-stage enzymatic hydrolysis and a second-stage enzymatic hydrolysis. The conditions for the first-stage enzymatic hydrolysis are: 0.2-0.5 vt% of mixed enzyme added, 40-60℃ of enzymatic hydrolysis temperature, and 0.5-2 h of enzymatic hydrolysis time. The mixed enzyme is prepared by mixing pectinase and polygalacturonase at a mass ratio of 1:(0.2-1). The conditions for the second-stage enzymatic hydrolysis are: 0.3-0.6 vt% of cellulose endopeptidase added, 45-55℃ of enzymatic hydrolysis temperature, and 1.5-4 h of enzymatic hydrolysis time.
5. The method for preparing a modified grapefruit peel fiber gel with high naringenin content according to claim 1, characterized in that: The conditions for immobilized naringinase treatment in step S3 are: immobilized naringinase addition amount 1-5wt%, enzymatic hydrolysis temperature 45-65℃, and enzymatic hydrolysis time 3-8h.
6. The method for preparing a modified grapefruit peel fiber gel with high naringenin content according to claim 1, characterized in that, The preparation method of immobilized naringinase in step S3 is as follows: ① Add 0.2-0.6% 3-aminophenylboronic acid to chitosan with a mass fraction of 1-4%, stir evenly, and react at a constant temperature of 45-65℃ for 3-6 hours. Dry to obtain borate-modified chitosan; ② Dissolve hydroxypropyl-β-cyclodextrin and borate-modified chitosan at a mass ratio of (1-3.5):5 to prepare a mixed solution, and uniformly drop it into a 1-3% calcium chloride solution to solidify for 15-30 minutes. Wash and dry to obtain composite microspheres; ③ Disperse the composite microspheres in a 2-8 g / L naringin template molecule solution at a solid-liquid ratio of 1:(5-10) and treat at pH 8.5-9.0 for 5-15 minutes. After centrifugation, wash with an ethanol solution containing acetic acid and dry to obtain naringin molecularly imprinted microspheres; ④ Immerse the naringin molecularly imprinted microspheres in a 0.1-0.5% calcium chloride solution at a solid-liquid ratio of 1:(5-10). Immobilized naringinase was adsorbed into a mg / mL naringinase solution for 3-8 hours, washed, and dried to obtain immobilized naringinase.
7. The method for preparing a modified grapefruit peel fiber gel with high naringenin content according to claim 1, characterized in that, In step S4, the mass ratio of naringenin-containing citrus peel fiber, sarsaponin, and soybean saponins is 10:(0.5-2):(1-3); the concentration of magnesium chloride solution is 0.1-0.5 mol / L; and the amount of magnesium chloride solution added is 5-12%.
8. Modified grapefruit peel fiber gel prepared by the method according to any one of claims 1-7.