Ultrasonic-assisted red pomelo polyphenol extraction method and application

Abstract

The invention provides an ultrasonic-assisted red pomelo polyphenol extraction method and application. The polyphenol component in the red pomelo is extracted through the following steps: firstly, performing activation pretreatment on red pomelo dry powder by adopting a low-temperature plasma device, and destroying a cell wall structure so as to improve the accessibility of combined polyphenol; then cellulase, pectinase, hemicellulase and beta-glucosidase are added into a biodegradable deep eutectic solvent to synergistically hydrolyze the cell wall polysaccharide, and combined polyphenol is released; then applying pulse type ultrasonic wave to assist extraction under a low-temperature condition, so that polyphenol is fully dissolved out and enters the eutectic solvent; and finally, carrying out solid-liquid separation to obtain the red pomelo polyphenol extract. Compared with a conventional heat extraction method, the method has the advantages that the extraction rate of the polyphenol of the red pomelo is remarkably increased at a lower temperature, the degradation of the polyphenol is effectively avoided, and the content of the total polyphenol and the antioxidant activity are increased; the method is green and environment-friendly, and can be applied to the health industry as an antioxidant.

Description

Technical Field

[0001] The present invention belongs to the field of natural product extraction and bioengineering technology, and particularly relates to a method for ultrasonic-assisted extraction of red pomelo polyphenols, and application of the extract obtained by the method as an antioxidant in the health industry. Background Art

[0002] Red pomelo (a red-fleshed grapefruit variety) is rich in polyphenolic active substances, such as naringin, neohesperidin, and flavonoids, which have excellent antioxidant and anti-inflammatory health benefits. However, the extraction of red pomelo polyphenols faces many technical challenges: (1) Thermosensitive properties: Red pomelo polyphenols are sensitive to temperature. Traditional high-temperature extraction can easily lead to degradation of the polyphenol structure or reduction of activity. Polyphenols are generally considered to be thermally unstable components. High temperature and long-term treatment will accelerate their oxidative degradation. Therefore, conventional heating and reflux extraction often leads to the loss of some active polyphenols.

[0003] (2) Bound state: Most of the polyphenols in red pomelo exist in the plant cell wall matrix in a bound state, for example, they are bound to cellulose, hemicellulose, pectin and lignin through ester bonds or ether bonds. Bound polyphenols cannot be directly extracted by solvents and need to be released by breaking the bonds with cell wall components. The fruit tissue of red pomelo is tough and the cell wall structure is intact. Traditional mechanical crushing and organic solvent extraction methods are difficult to effectively release the bound polyphenols.

[0004] (3) Low extraction efficiency: Affected by the above factors, the direct extraction of red pomelo polyphenols using solvents such as water or ethanol often has a very low yield, and the concentration of polyphenols in the extract is not high. In addition, high-temperature treatment may also induce isomerization or browning reactions of polyphenols, further reducing the content of effective ingredients.

[0005] Given the above reasons, there is an urgent need to develop a gentle and efficient method for extracting polyphenols from red pomelo to avoid high-temperature damage to the active ingredients and fully release the polyphenols bound to the cell walls. Emerging green extraction technologies, such as low-temperature plasma pretreatment, enzymatic extraction, ultrasound-assisted extraction, and the use of green solvents, offer solutions to these challenges. Low-temperature plasma treatment (CN117100805A) can disrupt plant surface tissue, creating micropores and cracks without increasing the temperature, thereby improving the extraction rate of active ingredients. Microwave-enzymatic hydrolysis (CN112043762A) utilizes cell wall-degrading enzymes (such as cellulases and pectinases) to specifically hydrolyze cell wall polysaccharides, breaking the bonds that bind the polyphenols to them and releasing the bound polyphenols from the food matrix. Ultrasound-assisted extraction accelerates solvent penetration and cell disruption through acoustic cavitation, achieving high extraction efficiency at low temperatures and shortening extraction time. Deep eutectic solvents (CN115192615A), as new green solvents, offer advantages such as biodegradability, low toxicity, and good solubility.

[0006] In summary, the integration and application of these new technologies in the extraction of red pomelo polyphenols is expected to overcome the shortcomings of traditional processes and significantly improve the extraction rate and product quality. Summary of the Invention

[0007] The main purpose of this invention is to provide an efficient, low-temperature method for extracting polyphenols from red pomelo, addressing existing issues such as low extraction efficiency and thermal degradation of active ingredients. This method utilizes a combination of low-temperature plasma pretreatment, complex enzyme hydrolysis, pulsed ultrasound-assisted extraction, and a food-grade deep eutectic solvent to maximize the release and extraction of polyphenols from red pomelo under mild conditions.

[0008] The specific technical solutions are as follows: Disclosed are a method and application for ultrasonic-assisted extraction of red pomelo polyphenols. The raw materials comprise the following components: red pomelo peel and pomace, pectinase, cellulase, hemicellulase, β-glucosidase, choline chloride (ChCl, food grade, purity ≥98%, moisture ≤1%), lactic acid (LAC, food grade, purity ≥80%, moisture ≤5%), glycerol (GLY, food grade, purity ≥99.5%, moisture ≤0.5%), deionized water, ethanol, and macroporous adsorption resin.

[0009] S1: Low-temperature plasma pretreatment: evenly spread the red pomelo powder (particle size 2-5 mm) in a low-temperature plasma generator, introduce inert gas (such as argon) or a mixture of air and inert gas, activate the raw material for 1-3 minutes, the plasma power is 100-300W, and the temperature is <40°C.

[0010] S2: Enzymatically hydrolyze the bound polyphenols. The product from S1 pretreatment is premixed with a food-grade DES aqueous solution to form a feed-liquid mixture. The enzyme complex is added to the mixture, and the reaction parameters are controlled: the temperature is set at 35-45°C, and the mixture is gently stirred for 1-2 hours at a stirring rate of 100-300 rpm. The pH is maintained at 4.8 ± 0.1.

[0011] S3: Pulsed ultrasound-assisted extraction: The product prepared in S2 was mixed with food-grade DES, and then 5-15% water was added. Ultrasonic extraction was performed using a pulsed ultrasound mode with a 1:1 duty cycle. The parameters were: 2 seconds on / 2 seconds off, a power density of 100-300 W / L, and a duration of 10-30 minutes. An ice-water bath was added to the outside of the ultrasonic reactor to maintain the temperature below 40°C.

[0012] S4: DES recovery and polyphenol enrichment and purification: An antisolvent (preferably ethanol) is added to the extract to precipitate the polyphenols. The precipitate and supernatant are centrifuged and separated. An ultrafiltration membrane is used to separate macromolecular impurities from the small DES components, retaining the polyphenols. The added water is evaporated at -0.09 MPa and 42°C, and the concentrated DES is recovered. The precipitated crude polyphenols or the membrane retentate can be further purified and enriched using a macroporous adsorption resin, eluted with an ethanol-water solution, and then concentrated and freeze-dried to obtain a high-purity red pomelo polyphenol product.

[0013] Furthermore, the red-heart pomelo dry powder described in S1 is prepared from the peel or pomace of the red-heart pomelo, which is washed and chopped, and then freeze-dried at -40°C to a moisture content of ≤8%.

[0014] Furthermore, the complex enzyme preparation described in S2 has the following composition and total enzyme activity ratio: pectinase 40-55%, cellulase 20-30%, hemicellulase 15-25%, and β-glucosidase 5-10%.

[0015] Furthermore, the food-grade DES aqueous solution described in S2 has the following components: the hydrogen bond acceptor is choline chloride (ChCl), the hydrogen bond donor is lactic acid (LAC) and glycerol (GLY), the ratio of ChCl:LAC:GLY is in the range of 1:0.5:0.5 to 1:1.5:1.5, and the rest is deionized water.

[0016] Furthermore, in the mixing described in S3, the ratio of the product to food-grade DES is 1:10 to 1:30 w / v.

[0017] Furthermore, an antisolvent is added to the extract described in S4, and the volume ratio parameter thereof is: DES extract: antisolvent is 1:2 to 1:4.

[0018] The centrifugal separation described in S4 has the following parameter settings: speed 3000-8000 rpm, duration 10-30 min, temperature 4-25°C.

[0019] The elution parameters described in S4 are set as follows: temperature ≤ 30°C, primary elution and secondary elution, primary elution with 20-30% ethanol aqueous solution, flow rate 4-5BV / h (BV / h: the distance a particle in a liquid moves per unit time), and dosage 3-5BV (BV: column bed volume); secondary elution with 60-80% ethanol aqueous solution, flow rate 2-3BV / h, and dosage 4-6BV.

[0020] The concentration described in S4 has the following parameter settings: temperature 40 to 45°C, vacuum degree -0.08 to -0.095 MPa.

[0021] The freeze drying described in S4 has the following parameter settings: pre-freezing temperature -40°C, duration 2 to 3 hours, sublimation temperature -25°C, pressure 0.1 kPa, duration 24 to 30 hours, decomposition temperature 25°C, pressure 0.01 kPa, duration 4 to 8 hours.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. Significantly improve the extraction rate: Low-temperature plasma pretreatment combined with enzymatic hydrolysis and ultrasound can greatly break the constraints of cell walls on polyphenols, prompting more bound polyphenols to be converted into free state and extracted by solvents.

[0023] 2. Protect active ingredients and enhance antioxidant activity: The entire extraction process is maintained at a low temperature, preventing high temperatures from damaging the polyphenol structure and causing oxidation. Pulsed ultrasound shortens the extraction time, further reducing the polyphenols' exposure to adverse conditions. The resulting extract retains the polyphenols intact and significantly enhances their antioxidant activity.

[0024] 3. Green solvent and environmentally friendly: The low eutectic solvent used in the present invention is a degradable food-grade solvent, and no toxic organic solvent is used, which complies with the principles of green chemistry.

[0025] 4. High Product Application Value: The red pomelo polyphenol extract obtained by the present invention is rich in natural antioxidants. Derived from the fruit, the extraction process is free of harmful substances, resulting in excellent safety. This extract can be directly used in the development of health products such as functional beverages, dietary supplements, and natural preservatives and antioxidants, meeting current consumer demand for natural, safe, and highly effective antioxidant products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a flow chart of a method for preparing ultrasonic-assisted extraction of red pomelo polyphenols.

[0027] Figure 2The standard curve of Example 1, a method for ultrasonic-assisted extraction of red pomelo polyphenols, was drawn using gallic acid as the standard.

[0028] Figure 3 This is a data comparison chart of polyphenol extraction rate and free radical scavenging rate of a method for ultrasonic-assisted extraction of red pomelo polyphenols from Example 1-5 to Comparative Example 1-3. DETAILED DESCRIPTION

[0029] The following examples further illustrate and describe the technical solutions of the present invention. It is particularly noted that each specific embodiment is intended to be a concretization and explanation of the technical solutions and should not be construed as limiting the scope of protection of the present invention. Persons of ordinary skill in the art are entitled to modify the technical solutions of these embodiments and to substitute equivalent features for some or all of the technical features. Such modifications or substitutions do not alter the essence of the corresponding technical solutions and do not deviate from the scope of the technical solutions described in the present invention.

[0030] The present invention proposes a method and application of ultrasonic-assisted extraction of red pomelo polyphenols, wherein the raw materials include the following components: red pomelo peel or pomace, pectinase, cellulase, hemicellulase, β-glucosidase, choline chloride (ChCl, food grade, purity ≥98%, moisture ≤1%), lactic acid (LAC, food grade, purity ≥80%, moisture ≤5%), glycerol (GLY, food grade, purity ≥99.5%, moisture ≤0.5%), deionized water, ethanol, and macroporous adsorption resin. Figure 1 The figure shows a method for ultrasonic-assisted extraction of red pomelo polyphenols. The detailed preparation steps are as follows: 1. Raw material processing 1.1 Preparation of red pomelo powder The peel or pomace of the red-heart pomelo is washed, chopped, and freeze-dried at -40°C until the moisture content is ≤8% to prepare the red-heart pomelo dry powder.

[0031] 1.2 Preparation of complex enzyme The following ratio is used: pectinase 40-55%, cellulase 20-30%, hemicellulase 15-25%, and β-glucosidase 5-10%. Pectinase and hemicellulase are first mixed, followed by cellulase, and finally β-glucosidase. The complex enzyme preparation is prepared by carrier loading, low-temperature drying, and stabilization.

[0032] 1.3 Preparation of food-grade DES The hydrogen bond acceptor is choline chloride (ChCl), and the hydrogen bond donors are lactic acid (LAC) and glycerol (GLY). The ratio of ChCl:LAC:GLY ranges from 1:0.5:0.5 to 1:1.5:1.5. ChCl and GLY are first mixed, then LAC is added. The reaction is then constant temperature reacted and vacuum dehydrated to prepare food-grade DES.

[0033] 2. Activation and hydrolysis 2.1 Low-temperature plasma activation Evenly spread the red pomelo powder in a low-temperature plasma generator for treatment. Inert gas (such as argon) or a mixture of air and inert gas is introduced for 1 to 3 minutes. The plasma power is 100 to 300 W and the temperature is less than 40°C.

[0034] 2.2 Complex enzyme hydrolysis The treated pomelo powder was premixed with a food-grade DES aqueous solution to form a feed-liquid mixture. The enzyme complex was added to the mixture, and the reaction parameters were controlled, with the temperature set at 35-45°C and gentle stirring for 1-2 hours, to a pH of 4.8±0.1.

[0035] 3. Pulsed Ultrasound Assisted Extraction 3.1 Mixing The hydrolyzed product is mixed with food-grade DES at a material-liquid ratio of 1:10 to 1:30 w / v, and then 5 to 15% water is added to improve the polyphenol extraction rate.

[0036] 3.2 Extraction Ultrasonic extraction was performed using a pulsed ultrasound mode with a duty cycle of 1:1. The parameters were: 2s on / 2s off, a power density of 100-300W / L, and a duration of 10-30min. An ice-water bath was added to the outside of the ultrasonic reactor to maintain the temperature below 40°C.

[0037] 4. DES recovery and polyphenol enrichment and purification 4.1DES Recycling An antisolvent is added to the extract with a volume ratio of DES extract to antisolvent of 1:2 to 1:4 to precipitate the polyphenols. The precipitate and the supernatant are separated by centrifugation. The parameters are set as follows: speed of 3000 to 8000 rpm, time of 10 to 30 minutes, temperature of 4 to 25°C, ultrafiltration membrane is used to separate macromolecular impurities and DES small molecular components, and polyphenols are retained; the added water is evaporated at -0.09 MPa and 42°C to recover the concentrated DES.

[0038] 4.2 Polyphenol enrichment and purification The crude polyphenol product or membrane retentate can be further purified and enriched by macroporous adsorption resin, eluted with ethanol aqueous solution, the temperature is required to be ≤30°C, and two elutions are adopted, and the parameter settings are: the first elution is 20-30% ethanol aqueous solution, the flow rate is 4-5BV / h, and the amount is 3-5BV; the second elution is 60-80% ethanol aqueous solution, the flow rate is 2-3BV / h, and the amount is 4-6BV; and then concentrated at 40-45°C, vacuum degree -0.08 to -0.095MPa, and freeze-dried (pre-freezing temperature -40°C, time 2-3h, sublimation temperature -25°C, pressure 0.1kPa, time 24-30h, desorption temperature 25°C, pressure 0.01kPa, time 4-8h) to obtain a high-purity red pomelo polyphenol product.

[0039] Example 1

[0040] A method and application of ultrasonic-assisted extraction of red pomelo polyphenols is as follows: The raw materials include: red pomelo peel and pomace, pectinase, cellulase, hemicellulase, β-glucosidase, choline chloride (ChCl, food grade, purity ≥98%, moisture ≤1%), lactic acid (LAC, food grade, purity ≥80%, moisture ≤5%), glycerol (GLY, food grade, purity ≥99.5%, moisture ≤0.5%), deionized water, ethanol, and macroporous adsorption resin.

[0041] S1: Wash and chop the peel or pomace of red pomelo, then freeze-dry at -40°C to 6% moisture to prepare red pomelo powder (particle size 3 mm). Spread the powder evenly in a low-temperature plasma generator and activate it with argon for 2 minutes at a plasma power of 300 W and a temperature of 35°C.

[0042] S2: Enzymatic hydrolysis to release bound polyphenols. The product from S1 was premixed with a 12% food-grade DES aqueous solution to form a feed-liquid mixture with a feed-liquid ratio of 1:20. A composite enzyme preparation (3% by weight of the raw materials) consisting of 55% pectinase, 25% cellulase, 15% hemicellulase, and 5% β-glucosidase was added to the mixture. The reaction parameters were controlled: temperature at 40°C, stirring at 200 rpm for 1.5 hours, and pH 4.8. During this time, the enzyme preparation gradually hydrolyzed the cell wall polysaccharides in the red pomelo powder, releasing a faint pomelo aroma and slightly reducing the viscosity of the liquid.

[0043] S3: The product prepared in S2 was mixed with food-grade DES at a ratio of 1:15 w / v (ChCl:LAC:GLY = 1:1.5:1). 10% water was then added. Ultrasonic extraction was performed using pulsed ultrasonic mode with the following parameters: 2s on / 2s off, 300W / L power density, and 20min duration. The temperature was strictly controlled at 35°C using an ice-water bath outside the ultrasonic reactor.

[0044] S4: Add antisolvent ethanol to the extract at a volume ratio of 1:3 to ethanol to precipitate the polyphenols. Centrifuge (7000 rpm, 20 min, 10°C) to separate the precipitate and supernatant. Use an ultrafiltration membrane to separate large molecular impurities and small molecular components of DES, and retain the polyphenols. Evaporate the added water under low temperature and reduced pressure (-0.09 MPa, 42°C) to recover the concentrated DES. The crude polyphenols or membrane retentate obtained by precipitation can be further purified and enriched by macroporous adsorption resin and eluted with ethanol-water solution. The parameter settings are: first elution with 25% ethanol, 4.5BV / h, 4BV, second elution with 70% ethanol, 2.5BV / h, 5BV, and then concentrated (-0.09MPa, 42℃), and then freeze-dried. The method is pre-freezing at -40℃ for 2.5h, sublimation at 0.1kPa and -25℃ for 28h, and analysis at 0.01kPa and 25℃ for 6h to obtain high-purity red pomelo polyphenols.

[0045] S5: Detection, the extraction rate of red pomelo polyphenols was measured by the Folin phenol method to measure the absorbance. Use a pipette to take 0.1, 0.2, 0.3, 0.4, and 0.5 mL of 0.1 mg / mL gallic acid standard solution, add 1 mL of Folin phenol reagent and 2 mL of 12% Na2CO3 solution, respectively, dilute to 10 mL with distilled water and shake well, and repeat 3 groups. At room temperature 25 ° C, place the volumetric flask in the dark. After reacting for 2 hours, use a UV-visible spectrophotometer to measure the absorbance at 765 nm. With the concentration of the gallic acid standard solution as the horizontal axis and the absorbance as the vertical axis, as shown in the attached figure. Figure 2 As shown, the standard curve equation is y=13.41x±0.0031, and the correlation coefficient is R 2 >0.99. Add 0.01 g of red pomelo polyphenols to 1 mL of Folin's phenol reagent and 2 mL of 12% Na2CO3 solution, dilute to 10 mL with distilled water, and shake well. Place the volumetric flask in the dark at room temperature (25°C). After reacting for 2 h, measure the absorbance at 765 nm using a UV-visible spectrophotometer.

[0046] Example 2

[0047] The composition and preparation process are the same as in Example 1, except that: In S1 of the preparation process, a mixture of air and argon was introduced to activate the raw materials for 1 min, with a plasma power of 200 W and a temperature of 38° C., while other components remained unchanged.

[0048] The composite enzyme preparation in S2 of the preparation process has the following components: pectinase 50%, cellulase 25%, hemicellulase 20%, β-glucosidase 5%, enzymatic hydrolysis temperature 40°C, pH 4.9, and other components are the same.

[0049] In the preparation process S3, the DES components are: ChCl: LAC: GLY = 1:1:1, the product is mixed with food-grade DES at a ratio of 1:20 w / v, the ultrasonic power density is 200 W / L, the duration is 25 min, and the other steps are the same.

[0050] In the preparation process, the volume ratio of the purified liquid in S4 to ethanol is 1:2.5, the ethanol elution concentration is 30% for the first time and 60% for the second time, and the other components are the same.

[0051] Example 3

[0052] The composition and preparation process are the same as in Example 1, except that: In S1 of the preparation process, argon gas was introduced to activate the raw materials for 3 minutes with a plasma power of 200 W. The other steps were the same.

[0053] The composite enzyme preparation in S2 of the preparation process has the following components: pectinase 45%, cellulase 30%, hemicellulase 20%, β-glucosidase 5%, enzymatic hydrolysis temperature 35°C, stirring time 2h, and other steps are the same.

[0054] In the preparation process S3, the DES components are: ChCl: LAC: GLY = 1: 0.5: 0.5, the product is mixed with food-grade DES at a ratio of 1: 10 w / v, the ultrasonic power density is 150 W / L, the duration is 30 min, and the other steps are the same.

[0055] In the preparation process, the volume ratio of the purified liquid in S4 to ethanol is 1:4, the ethanol elution concentration is 20% for the first time and 80% for the second time, and the other components are the same.

[0056] Example 4

[0057] The composition and preparation process are the same as in Example 1, except that: In S1 of the preparation process, air was introduced to activate the raw materials for 1 min, and the other steps remained unchanged.

[0058] The composite enzyme preparation in S2 of the preparation process has the following components: pectinase 55%, cellulase 20%, hemicellulase 15%, β-glucosidase 10%, enzymatic hydrolysis temperature 45°C, and other components are the same.

[0059] In the preparation process S3, the DES components are: ChCl: LAC: GLY = 1: 1.2: 1.2, the product is mixed with food-grade DES at a ratio of 1: 20 w / v, the ultrasonic power density is 250 W / L, the duration is 15 min, and the other steps are the same.

[0060] In S4 of the preparation process, centrifugal separation was performed at a speed of 6000 rpm for 15 min, and the other steps were the same.

[0061] Example 5

[0062] The composition and preparation process are the same as in Example 1, except that: In the preparation process S2, the concentration of food-grade DES aqueous solution was premixed at 10%, and the other steps were the same.

[0063] In the preparation process S3, the DES components are: ChCl: LAC: GLY = 1:1:1, and the product is mixed with food-grade DES at a ratio of 1:25 w / v. The other steps are the same.

[0064] The volume ratio of the purified liquid in S4 to ethanol in the preparation process is 1:3.5, and the other steps are the same.

[0065] Comparative Example 1 The composition and preparation process are the same as in Example 1, except that: In S1 of the preparation process, plasma treatment was skipped and dried fruit powder was used directly, and the other steps were the same.

[0066] Comparative Example 2 The composition and preparation process are the same as in Example 1, except that: In S2 of the preparation process, a 30% ethanol-water solution was used to replace the 12% food-grade DES aqueous solution in S2, and the other steps were the same.

[0067] In S3 of the preparation process, 30% ethanol-water solution was used instead of food-grade DES and mixed at a ratio of 1:15 w / v. The other steps were the same.

[0068] Comparative Example 3 The composition and preparation process are the same as in Example 1, except that: In S3 of the preparation process, pulse ultrasound is eliminated and continuous ultrasound is adopted, and the other steps are the same.

[0069] Combining Examples 1-5 and Comparative Examples 1-3, a method and application of ultrasonic-assisted extraction of red pomelo polyphenols was developed. The polyphenol extraction rate was measured by the Folin-phenol method to measure absorbance, and the DPPH free radical scavenging rate was tested according to the GB / T 23193-2015 "Test Method for Food Antioxidants". The specific test results are shown in Table 1: Table 1 Comparison of results of Examples 1-5 and Comparative Examples 1-3

[0070] From the above comparison results, it can be seen that Example 1 has the highest polyphenol extraction rate and the highest DPPH free radical scavenging rate; the polyphenol extraction rates of Examples 2 to 5 are slightly lower than those of Example 1, which shows that excellent extraction effects are still achieved under a wide range of parameter changes; Comparative Example 1 skips plasma treatment, resulting in insufficient cell wall destruction and insufficient release of bound polyphenols, so that the polyphenol extraction rate is reduced by 50% compared with Example 1; the polyphenol extraction rate and DPPH free radical scavenging rate of Comparative Example 2 are lower than those of Example 1, indicating that DES has a higher solubility efficiency for bound polyphenols than ethanol; the low polyphenol extraction rate and DPPH free radical scavenging rate of Comparative Example 3 are due to the oxidative inactivation of antioxidant components caused by continuous high temperature.

[0071] In summary, the ultrasonic-assisted low-temperature extraction method for pomelo polyphenols proposed in this invention offers significant advantages over traditional processes, including high extraction yield, reduced extraction time, lower temperatures, and improved activity retention. This method increases the total amount of pomelo polyphenols extracted by over 50%, and enhances the antioxidant properties of the extract by approximately 20-40%. This method has demonstrated excellent results under laboratory conditions and is suitable for developing natural antioxidant products for the health industry, such as preparing a powder rich in pomelo polyphenols. Adding this active powder to functional beverage formulations, at a dose of 50 mg per bottle, significantly improves the antioxidant capacity and nutritional value of the beverage. Sensory evaluations revealed that beverages containing this extract exhibited a light pomelo aroma and a slightly bitter aftertaste (derived from pomelo flavonoids), demonstrating good consumer acceptance. In vitro antioxidant studies demonstrated that beverages containing pomelo polyphenols increased the DPPH free radical scavenging rate by over 30%. The extract can also be formulated into capsules or tablets for use as a dietary supplement; daily intake can help enhance the body's antioxidant defenses. In the cosmetics field, this polyphenol can be incorporated into lotions or serums, leveraging its free radical scavenging and anti-aging properties to become a natural skincare active ingredient. Thus, the red pomelo polyphenol extract obtained by this invention has broad application prospects in health-related industries, meeting people's demand for natural, highly effective, and safe antioxidant products.

Claims

1. A method for ultrasonic-assisted extraction of red pomelo polyphenols, characterized in that: The following steps are involved: S1: Low-temperature plasma pretreatment: the red pomelo powder is evenly spread in a low-temperature plasma generator, and an inert gas or a mixture of air and inert gas is introduced to activate the raw material; S2: The compound enzyme hydrolyzes and releases bound polyphenols. The product pretreated in S1 is added to a food-grade DES aqueous solution and premixed to form a feed-liquid mixture. The compound enzyme preparation is added to the mixture, and the reaction parameters are controlled. The temperature is set at 35-45°C, and the mixture is gently stirred for 1-2 hours at a stirring rate of 100-300 rpm and a pH of 4.8±0.

1. S3: Pulsed ultrasound-assisted extraction: The product of S2 enzymatic hydrolysis was mixed with food-grade DES, and then 5-15% water was added. The extraction was performed using a pulsed ultrasound mode with a duty cycle of 1:

1. The parameters were: duty cycle of 2s / duration of 2s, power density of 100-300W / L, and duration of 10-30min. An ice-water bath was added to the outside of the ultrasonic reactor to keep the temperature below 40°C. S4: DES recovery and polyphenol enrichment and purification: antisolvent is added to the extract to precipitate the polyphenols, the precipitate and supernatant are centrifuged, and an ultrafiltration membrane is used to separate macromolecular impurities and DES small molecular components, and the polyphenols are retained; the added water is evaporated under -0.09 MPa and 42°C, and the concentrated DES is recovered; the precipitated crude polyphenols and membrane retentate are further purified and enriched by macroporous adsorption resin, eluted with ethanol aqueous solution, and then concentrated and freeze-dried to obtain high-purity red pomelo polyphenols.

2. The method for ultrasonic-assisted extraction of red pomelo polyphenols according to claim 1, characterized in that: The low-temperature plasma generator described in S1 has the following parameter settings: duration 1 to 3 minutes, power 100 to 300 W, and temperature <40°C.

3. The method for ultrasonic-assisted extraction of red pomelo polyphenols according to claim 1, characterized in that: The food-grade DES aqueous solution described in S2 has a mass ratio of DES of 10-20%, and its components are: a hydrogen bond acceptor of choline chloride, a hydrogen bond donor of lactic acid and glycerol, a ratio of choline chloride:lactic acid:glycerol in the range of 1:0.5:0.5 to 1:1.5:1.5, and the rest is deionized water; The feed-liquid mixture described in S2 has a feed-liquid ratio of 1:15 to 1:

30.

4. The method for ultrasonic-assisted extraction of red pomelo polyphenols according to claim 1, characterized in that: The composite enzyme preparation described in S2 has a total enzyme addition amount of 3-5% of the mass of the raw material dry powder, and its components are: pectinase 40-55%, cellulase 20-30%, hemicellulase 15-25%, and β-glucosidase 5-10%.

5. The method for ultrasonic-assisted extraction of red pomelo polyphenols according to claim 1, characterized in that: The ratio of the mixture described in S3 to food-grade DES is 1:10 to 1:30 w / v.

6. The method for ultrasonic-assisted extraction of red pomelo polyphenols according to claim 1, characterized in that: The antisolvent described in S4 has a volume ratio of DES extract to antisolvent of 1:2 to 1:

4.

7. The method for ultrasonic-assisted extraction of red pomelo polyphenols according to claim 1, characterized in that: The centrifugal separation described in S4 has the following parameters: speed 3000-8000 rpm, duration 10-30 min, temperature 4-25°C; The elution parameters of S4 are as follows: temperature ≤ 30°C, two elutions, one elution with 20-30% ethanol aqueous solution, flow rate 4-5BV / h, dosage 3-5BV; the second elution with 60-80% ethanol aqueous solution, flow rate 2-3BV / h, dosage 4-6BV.

8. The method for ultrasonic-assisted extraction of red pomelo polyphenols according to claim 1, characterized in that: The concentration described in S4 has the following parameters: temperature 40-45°C, vacuum degree -0.08-0.095 MPa; The freeze drying described in S4 has the following parameter settings: pre-freezing temperature -40°C, duration 2 to 3 hours, sublimation temperature -25°C, pressure 0.1 kPa, duration 24 to 30 hours, decomposition temperature 25°C, pressure 0.01 kPa, duration 4 to 8 hours.

9. The ultrasonic-assisted extraction method for red pomelo polyphenols according to any one of claims 1 to 8, characterized in that: The red pomelo polyphenol is rich in polyphenol active substances derived from red pomelo, and its DPPH free radical scavenging rate is greater than 20%.

10. The use of ultrasonic-assisted extraction of red pomelo polyphenols in the preparation of antioxidant products according to claim 9, characterized in that: The red pomelo polyphenol is used in functional foods, nutritional health products or cosmetics as a natural active ingredient for improving antioxidant performance.

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