A deep processing method for extracting flavonoids and citrine from citrus

Abstract

The application discloses a deep processing method for extracting flavonoids and citrate in citrus, adopts a process route of raw material pretreatment, ultrasonic-enzymatic hydrolysis synergistic cell wall breaking, gradient solvent synchronous extraction, liquid-liquid gradient extraction separation, membrane separation coupling purification and low-temperature refining and drying, synergistically breaks cell walls through composite enzymes and ultrasonic, and improves the release rate of active ingredients; two types of components are synchronously extracted through low-temperature ultrasonic using ethanol-ethyl acetate green composite extractant; preliminary separation is realized through pH regulation and polarity gradient extraction; high-purity products are obtained by coupling nanofiltration, microfiltration and macroporous resin purification; the flavonoid extraction rate reaches 92%-96% and the purity is greater than or equal to 95%, the citrate extraction rate reaches 88%-92% and the purity is greater than or equal to 90%, the process is green, low in consumption, high in continuous degree, strong in industrialization adaptability, and realizes high-value utilization of all components of citrus by-products.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product deep processing technology, specifically relating to a deep processing method for extracting flavonoids and limonene from citrus fruits. Background Technology

[0002] Citrus byproducts are rich in two high-value active ingredients: flavonoids and limonene. Flavonoids are important natural antioxidants with effects such as scavenging free radicals, protecting the cardiovascular system, and regulating immunity. They are widely used in health products, pharmaceuticals, and food. Limonene (a limonene analogue) is a tetracyclic triterpenoid compound with significant antitumor, antiviral, antibacterial, and anti-inflammatory activities. It is a high-quality raw material for natural medicines and functional foods.

[0003] Currently, the extraction technology for citrus flavonoids and limonene suffers from core technical defects: it cannot achieve simultaneous and efficient extraction and separation of the two substances, and the process suffers from problems such as low extraction rate, poor selectivity, solvent contamination, high energy consumption, and difficulty in industrialization. Specifically:

[0004] Stepwise extraction is inefficient: Traditional processes often use stepwise extraction methods, first extracting flavonoids with methanol and ethanol, and then extracting limonene with acetone and dichloromethane. The process is cumbersome and time-consuming (single-step extraction ≥8h). Repeated processing of raw materials leads to degradation of active ingredients, with flavonoid extraction rate of only 60%-75% and limonene extraction rate of less than 65%.

[0005] Poor solvent safety: Commonly used toxic organic solvents such as methanol and dichloromethane not only have high recycling costs, but also easily cause environmental pollution. The solvent residue in the extract exceeds the standard and cannot meet the safety requirements of food and pharmaceutical products.

[0006] Separation and purification are difficult: flavonoids and limonene are mixed in the extract, requiring multiple extractions and column chromatography for separation. The loss rate during the separation process exceeds 20%, resulting in low product purity (flavonoid purity ≤70%, limonene purity ≤60%).

[0007] High temperature leads to loss of activity: Traditional hot reflux and Soxhlet extraction require long-term high-temperature heating (80-100℃), which leads to oxidation and isomerization of heat-sensitive flavonoids and limonene, resulting in an activity loss rate of 15%-30%.

[0008] To address the aforementioned issues, a deep processing method is needed that combines ultrasonic-enzymatic hydrolysis for synergistic cell disruption, gradient solvent extraction, liquid-liquid gradient extraction and separation, and membrane separation coupled with purification. This method would fundamentally solve the problem of simultaneous extraction and separation, achieving high yield, high purity, green and low-consumption production, and industrial-scale production. Summary of the Invention

[0009] This invention addresses the core problem of existing technologies being unable to simultaneously and efficiently extract and separate flavonoids and limonene from citrus fruits. It provides a deep-processing method for extracting flavonoids and limonene from citrus fruits. Through synergistic enhanced extraction, precise gradient separation, and efficient membrane purification technology, it achieves simultaneous high-yield extraction and efficient separation and purification of the two types of active ingredients. It also has the advantages of being green and environmentally friendly, low-energy consumption, and highly adaptable to industrial applications.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A deep processing method for extracting flavonoids and limonene from citrus fruits includes the following steps: Step 1, raw material pretreatment: Wash and remove impurities from citrus peel residue and seeds, dry them at a low temperature of 40-50℃ until the moisture content is ≤8%, pulverize them and pass them through a 60-80 mesh sieve to obtain citrus compound powder; Step 2, Ultrasonic-Enzymatic Synergistic Cell Wall Disruption: Add a compound enzymatic hydrolysate to the citrus compound powder at a material-to-liquid ratio of 1:15-20 (g / mL). The compound enzymatic hydrolysate contains cellulase, pectinase, and xylanase, with an enzyme activity ratio of 3:2:1 and a total enzyme concentration of 0.5%-0.8%. Adjust the pH to 4.5-5.5 and enzymatically hydrolyze for 30-50 minutes at 40-50℃ and an ultrasonic power of 200-300W to obtain the enzymatically hydrolyzed material. Step 3, gradient solvent simultaneous extraction: Add 70%-80% (v / v) of ethanol-ethyl acetate composite extractant to the enzymatic hydrolysate, with an ethanol to ethyl acetate volume ratio of 5:1-3:1 and a material-to-liquid ratio of 1:25-30 (g / mL). Adjust the pH to 6.0-7.0 and extract for 20-40 min at 50-60℃ and ultrasonic power of 150-250W. After filtration, obtain the mixed extract. Step 4, liquid-liquid gradient extraction separation: Concentrate the mixed extract under reduced pressure to 1 / 4-1 / 3 of its original volume, adjust the pH to 3.0-4.0, add 1 / 2 volume of petroleum ether for extraction for 15-20 min, and separate the petroleum ether phase (crude limonene extract phase) and the aqueous phase; then add an equal volume of ethyl acetate to the aqueous phase for extraction for 20-30 min, and separate the ethyl acetate phase (crude flavonoid extract phase) and the raffinate aqueous phase; Step 5, membrane separation coupled purification: The petroleum ether phase is concentrated under reduced pressure until no solvent odor is detected, and desalted and impurities are removed using a nanofiltration membrane with a molecular weight cutoff of 300-500 Da. The operating pressure is 0.6-0.8 MPa and the temperature is 35-45℃ to obtain the citric acid purified solution. The ethyl acetate phase is concentrated under reduced pressure and then impurities are removed by a 0.22 μm microfiltration membrane. The filtrate is passed through an AB-8 macroporous adsorption resin at a flow rate of 1-2 BV / h. Impurities are first washed away with water, and then eluted with 60%-70% ethanol (v / v). The eluent is collected to obtain the flavonoid purified solution. Step 6, Low-temperature purification and drying: The citric acid purified solution is concentrated under vacuum, recrystallized with n-hexane, and dried under vacuum at 35-45℃ for 4-6 hours to obtain the citric acid product; the flavonoid purified solution is concentrated under vacuum, recrystallized with ethanol, and dried under vacuum at 40-50℃ for 3-5 hours to obtain the flavonoid product.

[0011] In a preferred embodiment of the present invention, in step 1, the citrus peel residue and seeds are fresh by-products of processing sweet oranges, Wenzhou mandarins, pomelos, and lemons, free from mold and rot.

[0012] In a preferred embodiment of the present invention, in step 2, the cellulase activity is ≥10000U / g, the pectinase activity is ≥8000U / g, and the xylanase activity is ≥6000U / g.

[0013] In a preferred embodiment of the present invention, in step 2, the ultrasound is pulsed ultrasound with a pulse frequency of 20-30kHz, a working time of 3s, and an interval of 1s.

[0014] In a preferred embodiment of the present invention, in step 3, 0.1%-0.3% of vitamin C is added to the compound extract as an antioxidant.

[0015] In a preferred embodiment of the present invention, in step 4, the extraction process is carried out under constant temperature and stirring conditions, with a stirring speed of 150-200 r / min.

[0016] In a preferred embodiment of the present invention, in step 5, the diameter-to-height ratio of the AB-8 macroporous adsorption resin is 1:8-1:10, and the adsorption temperature is 25-30℃.

[0017] In a preferred embodiment of the present invention, in step 5, the vacuum concentration temperature of both the limonene purification solution and the flavonoid purification solution is ≤50℃, and the vacuum degree is -0.08 to -0.06MPa.

[0018] In a preferred embodiment of the present invention, in step 6, the recrystallization process is repeated 2-3 times until the purity of the limonene product is ≥90% and the purity of the flavonoid product is ≥95%.

[0019] A citrus active ingredient extract, comprising flavonoid products and limonene products, prepared by the above-mentioned deep processing method; wherein, the flavonoid products are mainly composed of hesperidin, naringin, and nobiletin, with a total purity ≥95%; and the limonene products are mainly composed of limonin and nomiline, with a total purity ≥90%.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention solves the technical problem of simultaneous extraction of two types of active ingredients by integrating ultrasonic-enzymatic hydrolysis for synergistic cell wall disruption, simultaneous extraction with polar gradient composite solvents, pH-controlled gradient extraction and fractionation, membrane separation and macroporous resin coupling purification, and low-temperature controlled drying throughout the entire process. This significantly improves extraction efficiency. Utilizing the polarity difference between flavonoids and limonene, and combining it with a food-grade ethanol-ethyl acetate dual-component green solvent, this invention can simultaneously dissolve both target substances in one step, eliminating the traditional step-by-step, cumbersome process of first extracting flavonoids and then limonene. Combined with ultrasonic-enzymatic hydrolysis for synergistic cell wall disruption, it specifically degrades cellulose, pectin, and hemicellulose in the cell walls of citrus peel residue and seeds. The ultrasonic cavitation effect enhances mass transfer, achieving a cell wall disruption rate of over 95%, fully releasing bound flavonoids and limonene. Compared to traditional processes, the flavonoid extraction rate increases from 60%–75% to 92%–96%, and the limonene extraction rate increases from less than 65% to 88%–92%, shortening the extraction cycle by more than 60%. This completely solves the problems of repeated raw material processing, active ingredient degradation, and lengthy, inefficient procedures inherent in traditional processes.

[0021] 2. High selectivity in fractional separation, high product purity, and extremely low separation loss. This invention achieves directional fractional enrichment of limonene and flavonoids through precise pH control combined with low- to medium-polarity gradient liquid-liquid extraction, based on dissociation characteristics and polarity differences. This eliminates the need for repeated extractions and column chromatography elutions. Nanofiltration membranes remove small molecule impurities, solvent residues, and inorganic salts, while macroporous resins provide selective adsorption purification. This dual purification process achieves a total purity of 95%–98% for flavonoids and 90%–93% for limonene. The overall material loss rate is controlled below 5%, far superior to the over 20% loss of traditional processes. The product quality meets food-grade and pharmaceutical-grade application standards.

[0022] 3. The entire process is green and low in toxicity, with no harmful solvent residues and high safety. This invention uses food-grade green solvents such as ethanol, ethyl acetate, and petroleum ether throughout the process, eliminating the use of toxic and easily residual organic solvents such as methanol, dichloromethane, and acetone commonly used in traditional processes. At the same time, it is combined with nanofiltration membrane desolvation and low-temperature vacuum concentration processes to completely eliminate the problem of harmful solvent residues in the extract. There is no toxic waste liquid discharged during the production process, making it environmentally friendly.

[0023] 4. Low-temperature process throughout, maximizing the preservation of bioactivity. This invention abandons the traditional 80-100℃ high-temperature reflux and Soxhlet extraction methods. The extraction, concentration, and drying temperatures are all controlled below 60℃. Low-temperature ultrasonic extraction, low-temperature vacuum concentration, and low-temperature vacuum drying processes are adopted, effectively avoiding oxidation, isomerization, and structural damage of heat-sensitive flavonoids and limonene. The retention rate of active ingredients can reach over 97%, solving the defects of traditional high-temperature processes, such as significant loss of activity and substantial reduction in efficacy. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments or prior art, the drawings used in the description of the embodiments or prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic flowchart illustrating a deep processing method for extracting flavonoids and limonene from citrus fruits, as provided in an embodiment of the present invention.

[0026] Figure 2 This invention provides a schematic diagram of the process of ultrasonic-enzymatic hydrolysis synergistic cell disruption in step 2.

[0027] Figure 3 This invention provides a schematic diagram of the process for simultaneous extraction of gradient solvents in step 3, as described in an embodiment of the invention.

[0028] Figure 4 This invention provides a schematic diagram of the liquid-liquid gradient extraction separation process in step 4 of the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0030] like Figure 1 As shown, this invention provides a deep processing method for extracting flavonoids and limonene from citrus fruits, comprising the following steps: Step 1, Raw Material Pretreatment: Wash and remove impurities from citrus peels and seeds, dry them at a low temperature of 40-50℃ until the moisture content is ≤8%, pulverize them, and pass them through a 60-80 mesh sieve to obtain citrus compound powder. Specifically, Step 1 involves selecting fresh peels and seeds from processed citrus fruits (sweet orange, Satsuma mandarin orange, pomelo, lemon), removing mud and rotten parts, and rinsing them 2-3 times with clean water; placing them in a hot air circulating oven and drying them at a low temperature of 40-50℃ until the moisture content is ≤8% to avoid damaging the active ingredients with high temperatures; pulverizing them using an ultra-micro pulverizer and passing them through a 60-80 mesh standard sieve to obtain uniformly sized citrus compound powder, which is then sealed and refrigerated for later use.

[0031] Step 2, Ultrasonic-Enzymatic Synergistic Cell Wall Disruption: Add a compound enzymatic hydrolysate to the citrus compound powder at a material-to-liquid ratio of 1:15-20 (g / mL). The compound enzymatic hydrolysate contains cellulase, pectinase, and xylanase, with an enzyme activity ratio of 3:2:1 and a total enzyme concentration of 0.5%-0.8%. Adjust the pH to 4.5-5.5 and perform enzymatic hydrolysis for 30-50 minutes at 40-50℃ and an ultrasonic power of 200-300W to obtain the enzymatically hydrolyzed material.

[0032] like Figure 2 As shown, step 2 specifically involves the following: Citrus flavonoids and limonene exist in a bound state within the cell wall and intercellular matrix, making them difficult to fully release through simple solvent extraction. Step 2 utilizes a combination of enzymes and ultrasound to efficiently disrupt plant cell walls. Add the compound enzymatic hydrolysate to the citrus compound powder at a material-to-liquid ratio of 1:15-20 (g / mL). The compound enzymatic hydrolysate uses acetate-sodium acetate buffer as the solvent and contains cellulase, pectinase, and xylanase, with an enzyme activity ratio of 3:2:1 and a total enzyme concentration of 0.5%-0.8%. Adjust the pH of the system to 4.5-5.5 (the optimal pH for the compound enzyme), place it in an ultrasonic enzymatic hydrolysis reactor, control the temperature at 40-50℃, the ultrasonic power at 200-300W, use pulsed ultrasound (frequency 20-30kHz, 3s operation, 1s interval), and enzymatic hydrolysis for 30-50 minutes. Principle: The compound enzyme specifically degrades cellulose, pectin, and hemicellulose components of the cell wall. The cavitation effect generated by ultrasound enhances the enzyme's mass transfer efficiency, and the synergistic effect results in a cell wall breakage rate ≥95%, significantly increasing the release rate of bound active ingredients.

[0033] Step 3, gradient solvent simultaneous extraction: Add 70%-80% by volume of ethanol-ethyl acetate composite extractant to the enzymatic hydrolysate, with the volume ratio of ethanol to ethyl acetate being 5:1-3:1 and the material-to-liquid ratio being 1:25-30 (g / mL). Adjust the pH to 6.0-7.0 and extract for 20-40 minutes at 50-60℃ and ultrasonic power of 150-250W. After filtration, obtain the mixed extract.

[0034] like Figure 3 As shown, step 3 specifically involves: based on the polarity difference between flavonoids (moderately polar) and limonene (weakly polar), a green composite extractant is used to simultaneously extract both types of components. Add 70%-80% (v / v) of ethanol-ethyl acetate composite extractant to the enzymatic hydrolysate, with an ethanol-ethyl acetate volume ratio of 5:1-3:1 and a material-liquid ratio of 1:25-30 (g / mL). Add 0.1%-0.3% vitamin C as an antioxidant and adjust the pH to 6.0-7.0; Place the sample in an ultrasonic extractor, control the temperature at 50-60℃, and the ultrasonic power at 150-250W for 20-40 minutes. Vacuum filter to obtain the mixed extract. Advantages: The ethanol-ethyl acetate composite system balances the solubility of flavonoids and limonene; low-temperature ultrasonication enhances mass transfer, shortens extraction time, reduces energy consumption, and avoids degradation of active ingredients.

[0035] Step 4, liquid-liquid gradient extraction separation: Concentrate the mixed extract under reduced pressure to 1 / 4-1 / 3 of the original volume, adjust the pH to 3.0-4.0, add 1 / 2 volume of petroleum ether for extraction for 15-20 min, and separate the petroleum ether phase (crude limonene extract phase) and the aqueous phase; then add an equal volume of ethyl acetate to the aqueous phase for extraction for 20-30 min, and separate the ethyl acetate phase (crude flavonoid extract phase) and the raffinate aqueous phase.

[0036] like Figure 4 As shown, step 3 specifically involves: achieving preliminary separation of the two types of components through pH adjustment and polar gradient extraction. The mixed extract was concentrated under reduced pressure (temperature ≤50℃, vacuum degree -0.08~-0.06MPa) to 1 / 4-1 / 3 of its original volume, reducing the amount of solvent used and increasing the concentration of components; Adjust the pH of the concentrate to 3.0-4.0 (limonene stabilizes pH), add 1 / 2 volume of petroleum ether (low polarity), stir at constant temperature (150-200 r / min) and extract for 15-20 min, let stand to separate layers, the petroleum ether phase is enriched with limonene, and the aqueous phase contains residual flavonoids; Add an equal volume of ethyl acetate (medium polar) to the aqueous phase, and extract with stirring at a constant temperature for 20-30 minutes. After standing and separating the layers, the ethyl acetate phase is enriched with flavonoids, while the remaining aqueous phase contains a small amount of water-soluble impurities. Principle: pH adjustment alters the dissociation state of the two types of substances, combined with polar gradient extraction to achieve efficient separation with a selectivity ≥90%.

[0037] Step 5, membrane separation coupled purification: The petroleum ether phase is concentrated under reduced pressure until no solvent odor is detected, and desalted and impurities are removed using a nanofiltration membrane with a molecular weight cutoff of 300-500 Da. The operating pressure is 0.6-0.8 MPa and the temperature is 35-45℃ to obtain the citric acid purified solution. The ethyl acetate phase is concentrated under reduced pressure and then impurities are removed by a 0.22 μm microfiltration membrane. The filtrate is passed through an AB-8 macroporous adsorption resin at a flow rate of 1-2 BV / h. Impurities are first washed away with water, and then eluted with 60%-70% ethanol (v / v). The eluent is collected to obtain the flavonoid purified solution. Step 5 specifically involves using membrane separation coupled with macroporous resin technology to achieve efficient purification of the two types of components: Limonene purification: The petroleum ether phase is concentrated under reduced pressure until there is no solvent odor. A nanofiltration membrane with a molecular weight cutoff of 300-500 Da is used at an operating pressure of 0.6-0.8 MPa and a temperature of 35-45℃ to remove small molecule impurities and solvent residues, thus obtaining a purified limonene solution. Flavonoid purification: After concentration under reduced pressure in the ethyl acetate phase, solid impurities are removed by filtration through a 0.22 μm microfiltration membrane. The filtrate is then passed through AB-8 macroporous adsorption resin (diameter-to-height ratio 1:8-1:10, adsorption temperature 25-30℃) at a flow rate of 1-2 BV / h. Water-soluble impurities are first removed by washing with 2-3 BV of deionized water, followed by elution with 60%-70% ethanol (v / v). 3-5 BV of eluent is collected to obtain the purified flavonoid solution. Advantages: Membrane separation provides highly efficient impurity removal without secondary pollution; macroporous resin enables selective adsorption and purification; and the product purity is significantly improved after coupling.

[0038] Step 6, Low-temperature purification and drying: The citric acid purified solution is concentrated under vacuum, recrystallized with n-hexane, and dried under vacuum at 35-45℃ for 4-6 hours to obtain the citric acid product; the flavonoid purified solution is concentrated under vacuum, recrystallized with ethanol, and dried under vacuum at 40-50℃ for 3-5 hours to obtain the flavonoid product.

[0039] Step 6 is as follows: Limonene product: The purified limonene solution is concentrated under vacuum to a paste state, and then recrystallized 2-3 times with 5-8 times the amount of n-hexane. It is then dried under vacuum at 35-45℃ for 4-6 hours to obtain white limonene crystals (purity ≥90%). Flavonoid finished product: Vacuum concentrate the flavonoid purified solution to a paste state, add 3-5 times the amount of 70% ethanol for recrystallization 2-3 times, and vacuum dry at 40-50℃ for 3-5 hours to obtain a light yellow flavonoid powder (purity ≥95%).

[0040] Preferably, in step 1, the citrus peel residue and seeds are fresh by-products from the processing of sweet oranges, Satsuma mandarins, pomelos, and lemons, free from mold and rot. In step 2, the cellulase activity is ≥10000 U / g, the pectinase activity is ≥8000 U / g, and the xylanase activity is ≥6000 U / g. In step 2, the ultrasound is pulsed ultrasound with a pulse frequency of 20-30 kHz, a working time of 3 seconds, and an interval of 1 second. In step 3, 0.1%-0.3% vitamin C is added to the compound extractant as an antioxidant. In step 4, the extraction process is carried out under constant temperature and stirring conditions at a stirring speed of 150-200 r / min. In step 5, the diameter-to-height ratio of the AB-8 macroporous adsorption resin is 1:8-1:10, and the adsorption temperature is 25-30℃. In step 5, the vacuum concentration temperature of both the limonene purification solution and the flavonoid purification solution is ≤50℃, and the vacuum degree is -0.08~-0.06MPa.

[0041] In step 6, the recrystallization process is repeated 2-3 times until the purity of the limonene product is ≥90% and the purity of the flavonoid product is ≥95%.

[0042] The present invention also provides a citrus active ingredient extract, the extract comprising flavonoid products and limonene products, prepared by the above-mentioned deep processing method; wherein, the flavonoid products are mainly composed of hesperidin, naringin and nobiletin, with a total purity ≥95%; the limonene products are mainly composed of limonene and nomiline, with a total purity ≥90%.

[0043] Example 1 Raw material pretreatment: Select sweet orange peel and pulp, wash and remove impurities, dry at 45℃ to 7.2% moisture content, pulverize and pass through a 60-mesh sieve to obtain citrus compound powder.

[0044] Ultrasonic-enzymatic cell disruption: material-liquid ratio 1:18 (g / mL), compound enzymatic hydrolysate (cellulase: pectinase: xylanase = 3:2:1, total enzyme concentration 0.6%), pH 5.0, 45℃, 250W pulsed ultrasound (25kHz, 3s / 1s), enzymatic hydrolysis for 40min.

[0045] Gradient extraction: 75% ethanol-ethyl acetate (4:1), solid-liquid ratio 1:28 (g / mL), add 0.2% vitamin C, pH 6.5, 55℃, 200W ultrasonic extraction for 30 min, filter to obtain mixed extract.

[0046] Gradient extraction: Concentrate to 1 / 3 volume, adjust pH to 3.5, extract with petroleum ether for 18 min, extract with ethyl acetate for 25 min, and separate into limonene phase and flavonoid phase.

[0047] Membrane separation and purification: citric acid phase nanofiltration (400 Da, 0.7 MPa, 40 °C); flavonoid phase microfiltration followed by AB-8 resin (1:9), washing with water, and eluting with 70% ethanol.

[0048] Refining and drying: Limonene was recrystallized three times with n-hexane and dried under vacuum at 40℃ for 5 h; flavonoids were recrystallized three times with ethanol and dried under vacuum at 45℃ for 4 h. Results: Flavonoid extraction rate was 94.2%, purity was 96.8%; limonene extraction rate was 90.5%, purity was 91.3%.

[0049] Example 2 Raw material pretreatment: Select Wenzhou mandarin orange seeds, wash and remove impurities, dry at 40℃ to 6.8% moisture content, and crush through an 80-mesh sieve.

[0050] Ultrasonic-enzymatic cell disruption: material-liquid ratio 1:15 (g / mL), total enzyme concentration 0.5%, pH 4.5, ultrasonic treatment at 40℃ and 200W for 50 min.

[0051] Gradient extraction: 70% ethanol-ethyl acetate (5:1), solid-liquid ratio 1:25 (g / mL), add 0.1% vitamin C, pH 6.0, 50℃, 150W ultrasonic extraction for 40 min.

[0052] Gradient extraction: Concentrate to 1 / 4 volume, adjust pH to 3.0, extract with petroleum ether for 20 min, and extract with ethyl acetate for 30 min.

[0053] Membrane separation and purification: citric acid phase nanofiltration (300 Da, 0.6 MPa, 35 °C); flavonoid resin elution (60% ethanol).

[0054] Refining and drying: recrystallization twice, followed by low-temperature vacuum drying. Results: Flavonoid extraction rate 92.7%, purity 95.6%; limonene extraction rate 88.9%, purity 90.2%.

[0055] Example 3 Raw material pretreatment: Select pomelo peel residue, wash and remove impurities, dry at 50℃ to 7.5% moisture content, and crush through a 60-mesh sieve.

[0056] Ultrasonic-enzymatic cell disruption: material-liquid ratio 1:20 (g / mL), total enzyme concentration 0.8%, pH 5.5, ultrasonication at 50℃ and 300W for 30 min.

[0057] Gradient extraction: 80% ethanol-ethyl acetate (3:1), solid-liquid ratio 1:30 (g / mL), add 0.3% vitamin C, pH 7.0, ultrasonic extraction at 60℃ and 250W for 20 min.

[0058] Gradient extraction: Concentrate to 1 / 3 volume, adjust pH to 4.0, extract with petroleum ether for 15 min, and extract with ethyl acetate for 20 min.

[0059] Membrane separation and purification: citric acid phase nanofiltration (500 Da, 0.8 MPa, 45 °C); flavonoid resin purification.

[0060] Refining and drying: recrystallization three times, followed by vacuum drying. Results: Flavonoid extraction rate 95.8%, purity 97.9%; Limonene extraction rate 91.7%, purity 92.5%.

[0061] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A deep processing method for extracting flavonoids and limonene from citrus fruits, characterized in that, Includes the following steps: Step 1, raw material pretreatment: Wash and remove impurities from citrus peel residue and seeds, dry them at a low temperature of 40-50℃ until the moisture content is ≤8%, pulverize them and pass them through a 60-80 mesh sieve to obtain citrus compound powder; Step 2, Ultrasonic-Enzymatic Synergistic Cell Wall Disruption: Add a compound enzymatic hydrolysate to the citrus compound powder at a material-to-liquid ratio of 1:15-20 (g / mL). The compound enzymatic hydrolysate contains cellulase, pectinase, and xylanase, with an enzyme activity ratio of 3:2:1 and a total enzyme concentration of 0.5%-0.8%. Adjust the pH to 4.5-5.5 and enzymatically hydrolyze for 30-50 minutes at 40-50℃ and an ultrasonic power of 200-300W to obtain the enzymatically hydrolyzed material. Step 3, gradient solvent simultaneous extraction: Add 70%-80% (v / v) of ethanol-ethyl acetate composite extractant to the enzymatic hydrolysate, with an ethanol to ethyl acetate volume ratio of 5:1-3:1 and a material-to-liquid ratio of 1:25-30 (g / mL). Adjust the pH to 6.0-7.0 and extract for 20-40 min at 50-60℃ and ultrasonic power of 150-250W. After filtration, obtain the mixed extract. Step 4, liquid-liquid gradient extraction separation: Concentrate the mixed extract under reduced pressure to 1 / 4-1 / 3 of the original volume, adjust the pH to 3.0-4.0, add 1 / 2 volume of petroleum ether for extraction for 15-20 min, and separate the crude limonene extract phase and the aqueous phase; then add an equal volume of ethyl acetate to the aqueous phase for extraction for 20-30 min, and separate the crude flavonoid extract phase and the raffinate aqueous phase. Step 5, membrane separation coupled purification: The petroleum ether phase is concentrated under reduced pressure until no solvent odor is detected, and desalted and impurities are removed using a nanofiltration membrane with a molecular weight cutoff of 300-500 Da. The operating pressure is 0.6-0.8 MPa and the temperature is 35-45℃ to obtain the citric acid purified solution. The ethyl acetate phase is concentrated under reduced pressure and then impurities are removed by a 0.22 μm microfiltration membrane. The filtrate is passed through an AB-8 macroporous adsorption resin at a flow rate of 1-2 BV / h. Impurities are first washed away with water, and then eluted with 60%-70% ethanol (v / v). The eluent is collected to obtain the flavonoid purified solution. Step 6, Low-temperature purification and drying: The citric acid purified solution is concentrated under vacuum, recrystallized with n-hexane, and dried under vacuum at 35-45℃ for 4-6 hours to obtain the citric acid product; the flavonoid purified solution is concentrated under vacuum, recrystallized with ethanol, and dried under vacuum at 40-50℃ for 3-5 hours to obtain the flavonoid product.

2. The deep processing method for extracting flavonoids and limonene from citrus fruits according to claim 1, characterized in that, In step 1, the citrus peel residue and seeds are fresh by-products of processing sweet oranges, Wenzhou tangerines, pomelos, and lemons, and are free from mold and rot.

3. The deep processing method for extracting flavonoids and limonene from citrus fruits according to claim 1, characterized in that, In step 2, the cellulase activity is ≥10000U / g, the pectinase activity is ≥8000U / g, and the xylanase activity is ≥6000U / g.

4. The deep processing method for extracting flavonoids and limonene from citrus fruits according to claim 1, characterized in that, In step 2, the ultrasound is pulsed ultrasound with a pulse frequency of 20-30kHz, a working time of 3s, and an interval of 1s.

5. The deep processing method for extracting flavonoids and limonene from citrus fruits according to claim 1, characterized in that, In step 3, 0.1%-0.3% of vitamin C is added to the compound extract as an antioxidant.

6. The deep processing method for extracting flavonoids and limonene from citrus fruits according to claim 1, characterized in that, In step 4, the extraction process was carried out under constant temperature and stirring conditions, with a stirring speed of 150-200 r / min.

7. The deep processing method for extracting flavonoids and limonene from citrus fruits according to claim 1, characterized in that, In step 5, the diameter-to-height ratio of the AB-8 macroporous adsorption resin is 1:8-1:10, and the adsorption temperature is 25-30℃.

8. The deep processing method for extracting flavonoids and limonene from citrus fruits according to claim 1, characterized in that, In step 5, the vacuum concentration temperature of both the limonene purification solution and the flavonoid purification solution is ≤50℃, and the vacuum degree is -0.08~-0.06MPa.

9. The deep processing method for extracting flavonoids and limonene from citrus fruits according to claim 1, characterized in that, In step 6, the recrystallization process is repeated 2-3 times until the purity of the limonene product is ≥90% and the purity of the flavonoid product is ≥95%.

10. A citrus active ingredient extract, characterized in that, The extract comprises flavonoid products and limonene products, prepared by the deep processing method described in any one of claims 1-9; wherein, the flavonoid products are mainly composed of hesperidin, naringin, and nobiletin, with a total purity ≥95%; and the limonene products are mainly composed of limonin and nomiline, with a total purity ≥90%.

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