Passion fruit peel extract and extraction process thereof

Abstract

The invention relates to the technical field of extracts, in particular to a passion fruit peel extract and an extraction process thereof. The passion fruit peel extract comprises a flavonoid extract and a pectin extract, an extraction process of the flavonoid extract comprises the following steps: extracting flavonoid substances in passion fruit peels by using an enzymolysis method, and obtaining peel residues; the extraction process of the pectin extract comprises the following steps: mixing fruit peel residues, water and a eutectic solvent, and extracting pectin in the residues at 140-150 DEG C and 2-3 MPa; according to the extraction process, an enzymolysis method is combined with high-temperature and high-pressure eutectic solvent extraction, a flavonoid extract and a pectin extract are obtained, efficient separation and high-purity extraction of flavones and pectin in passion fruit peels are achieved, and maximization of the value of raw materials is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of extracts, and in particular to a guava peel extract and an extraction process thereof. BACKGROUND

[0002] Guava is an economic crop with characteristics in tropical and subtropical regions. Its processing by-product, guava peel, accounts for more than 50% of the total mass of fresh fruit, and up to 60% for some varieties, which is a considerable biological resource. For a long time, guava peel has been directly discarded after processing due to its high water content, which makes it difficult to store and prone to spoilage, and its low utilization value. This not only wastes functional ingredients such as pectin, flavonoid extracts, and polysaccharides, but also easily causes environmental pollution problems. Studies have shown that guava peel is rich in high-value ingredients: the pectin content can reach 14.60% (dry basis), and galacturonic acid (GalA) is the main monomer; flavonoid extracts include rutin, quercetin derivatives, and luteolin glycosides; it also contains polysaccharides, polyphenols, and minerals such as zinc, manganese, and phosphorus. These ingredients endow guava peel with biological activities such as antioxidant, hypoglycemic, and antibacterial, and have core potential for high-value development in the fields of food, medicine, and daily chemicals.

[0003] The existing extraction process of functional ingredients (such as pectin and flavonoids) in guava peel has many technical bottlenecks, making it difficult to achieve efficient utilization of resources:

[0004] Traditional extraction methods are low in efficiency and not environmentally friendly: the yield of traditional hot reflux extraction of polysaccharides is only 3.57%, and traditional acid method (CAE, such as using hydrochloric acid or nitric acid to extract pectin) requires treatment at high temperature (88-90℃) for more than 6h. This not only easily leads to the breakage of pectin glycosidic bonds and the reduction of GalA content (as low as 68.94%), but also causes corrosion of equipment and generation of toxic wastewater due to strong acid, and the yield of pectin is only 8.68%; the conventional industrial pectin extraction also has a "static extraction mode", which consumes a large amount of solvent and has low pectin diffusion efficiency, further limiting its industrial application.

[0005] In addition, most existing processes focus on the extraction of a single component (such as only extracting pectin or polysaccharides). If enzyme extraction is used alone to extract flavonoids, the enzyme solution penetration is blocked due to the integrity of the guava peel cell wall (pectin as a "adhesive component" makes the fiber network dense), and the enzyme hydrolysis time needs to be extended. If pectin is not removed in advance, flavonoids will co-precipitate with pectin, reducing the yield of flavonoids.

[0006] Therefore, the present application provides a guava peel extract and an extraction process thereof. SUMMARY

[0007] In order to overcome the deficiencies of the prior art, the application provides a guava peel extract and an extraction process thereof, the extraction process is combined with enzymatic hydrolysis and high-temperature and high-pressure eutectic solvent extraction to obtain flavonoid extracts and pectin extracts, realizes efficient separation and high-purity extraction of flavones and pectin in guava peel, and realizes maximization of raw material value.

[0008] The technical scheme adopted by the application to solve the technical problems is:

[0009] According to a first aspect of the application, a guava peel extract is provided, comprising flavonoid extracts and pectin extracts.

[0010] The extraction process of the flavonoid extracts is to extract flavonoid substances in the guava peel by using enzymatic hydrolysis, and obtain peel residues.

[0011] The extraction process of the pectin extracts is to mix the peel residues, water and eutectic solvent, and extract pectin in the residues under the conditions of 140-150 DEG C and 2-3 MPa.

[0012] In the technical scheme, the enzymatic hydrolysis destroys the cell wall structure of the peel, realizes efficient dissolution of flavonoid substances and preservation of biological activity, the peel residues are extracted twice to obtain pectin, and the recycling utilization of the raw materials is achieved with nearly zero waste; the eutectic solvent is combined with high-temperature and high-pressure conditions to significantly accelerate the release of pectin and reduce solvent consumption. Finally, flavone extracts with a purity of ≥85% and pectin extracts with a purity of ≥90% are obtained, which reduces the comprehensive cost and environmental load, completely preserves the biological activity of flavones and the excellent properties of high-galacturonic acid content pectin, and meets the strict requirements of the food and medicine fields for high-grade natural extracts.

[0013] The above technical scheme realizes efficient separation and high-purity extraction of flavones and pectin in guava peel, and maximizes the value of raw materials through recycling utilization of residues.

[0014] In some embodiments, the enzymes used in the enzymatic hydrolysis include cellulase and hemicellulase, and the added mass of the cellulase and the hemicellulase is 0.3%-0.4% and 0.1%-0.2% of the mass of the dried guava peel, respectively.

[0015] In some embodiments, the mass ratio of the dried peel residues, the eutectic solvent and water is (70-80):(140-160):(1400-1600).

[0016] In some embodiments, the eutectic solvent includes choline chloride, citric acid, proline and EDTA-2Na, and the molar ratio of choline chloride, citric acid, proline and EDTA-2Na is 1:1:1:0.4-0.5.

[0017] In some embodiments, a first auxiliary agent and a second auxiliary agent are also added during the extraction of the pectin;

[0018] In some embodiments, the first auxiliary agent is polyethylene glycol, and the added mass of the polyethylene glycol is 1.0% to 1.5% of the mass of the dried pomace residue;

[0019] In some embodiments, the second auxiliary agent is L-ascorbic acid, and the added mass of the L-ascorbic acid is 0.08% to 0.1% of the mass of the dried pomace residue.

[0020] In some embodiments, the passion fruit peel extract is applied in the fields of food and medicine.

[0021] According to a second aspect of the present application, an extraction process of the passion fruit peel extract of the first aspect is provided, including the following steps:

[0022] S1. Pre-treating passion fruit peels to obtain passion fruit peel powder;

[0023] S2. Enzymatically hydrolyzing the passion fruit peel powder to obtain supernatant and pomace residue;

[0024] S3. Purifying the flavonoid extract from the supernatant;

[0025] S4. Mixing the pomace residue, water, and eutectic solvent, and extracting the pectin extract from the pomace residue at 140°C to 150°C and 2 MPa to 3 MPa.

[0026] Optionally, in step S2, passion fruit peel powder is weighed, citric acid-sodium citrate buffer solution with pH = 4.5 is added, and cellulase and hemicellulase are added, and enzymatic hydrolysis is performed at 45°C to 50°C for 120 min to 150 min.

[0027] After the enzymatic hydrolysis is completed, inactivation treatment is performed, centrifugal separation is performed, the supernatant is collected, and the pomace residue is reserved.

[0028] In some embodiments, in step S3, the supernatant is diluted to a flavonoid concentration of 1.0 mg / mL, elution is performed through an AB-8 type macroporous resin column, eluate with an absorbance at 280 nm of ≥0.2 to ≤0.05 is collected, and the eluate is concentrated and dried to obtain flavonoid extract powder.

[0029] In some embodiments, in step S4, the specific preparation process is as follows:

[0030] S41: The dried peel residues, part of the water, and the eutectic solvent are mixed in a reaction kettle, and part of the first auxiliary agent is added and uniformly mixed; the temperature is raised to 140-150 DEG C at a rate of 5 DEG C / min, and the pressure is increased to 2-3 MPa, and the reaction is carried out at 140-150 DEG C and 2-3 MPa for 60-70 min;

[0031] S42: The remaining deionized water and the remaining first auxiliary agent are added to the reaction kettle, the temperature is maintained at 140-150 DEG C, the pressure is maintained at 2-3 MPa, and the reaction is continued for 60-70 min;

[0032] S43: The second auxiliary agent is added to the reaction kettle, and the temperature is lowered to 100 DEG C, and the pressure is maintained at 2-3 MPa for 15-20 min; after the reaction is completed, the temperature is lowered to room temperature, and the pressure is released to normal pressure.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The technical solution described above first extracts flavonoids by enzymatic hydrolysis, and then extracts pectin from the residues, realizing the full-component high-value conversion of agricultural processing by-products with a fresh fruit quality ratio of more than 50%. The obtained extracts have high purity and excellent biological activity: the purity of flavonoids is greater than or equal to 85%, and the purity of pectin is greater than or equal to 90%; the mild enzymatic hydrolysis process effectively preserves the antioxidant activity of flavonoids, and the pectin has excellent gel properties and emulsion stability due to its high galacturonic acid content, which is significantly better than traditional acid methods (which have defects such as low pectin extraction rate and easy hydrolysis of glycosidic bonds).

[0035] Moreover, the extraction process has the characteristics of green environmental protection and high efficiency: the use of eutectic solvent instead of strong acid medium avoids equipment corrosion and the generation of toxic wastewater, and the enzymatic hydrolysis process significantly reduces pollution load; the pectin extraction is completed at 140-150 DEG C and 2-3 MPa, which is about 50% shorter than the traditional acid method, which improves efficiency while ensuring industrial feasibility, and provides a source of high-purity natural raw materials for the food and pharmaceutical industries. DETAILED DESCRIPTION

[0036] In order to facilitate the understanding of those skilled in the art, the present application will be further described below in conjunction with examples, and the content mentioned in the embodiments is not a limitation of the present application.

[0037] As used herein, the term "and / or", includes any and all combinations of one or more of the associated listed items. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising", when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0038] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an overly literal or overly formal sense unless expressly so defined herein.

[0039] The exemplary applications described herein can suitably lack any one or more of the elements described herein, and such applications should not be deemed outside the scope of the present application. Thus, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms "comprise", "comprising", "comprised of", "comprising of", "comprise of", and the like can be used synonymously with "including", "including

[0040] According to a first aspect of the present application, there is provided a guava peel extract, comprising a flavonoid extract, a pectin extract;

[0041] The extraction process of the flavonoid extract is to extract flavonoids in the guava peel using enzymatic hydrolysis, and obtain a peel residue;

[0042] The extraction process of the pectin extract is to mix the peel residue, water, and a eutectic solvent, and extract the pectin extract in the residue at 140-150°C and 2-3 MPa. For example, the extraction can be performed at 140°C and 2 MPa, 145°C and 2.5 MPa, 150°C and 3 MPa, or 140°C and 3 MPa;

[0043] By the technical scheme, compared with the traditional solvent extraction method, the enzymatic hydrolysis method has milder reaction conditions, can effectively avoid structural damage of flavonoids due to high temperature or strong solvent action, significantly improves the extraction rate of flavonoids, and retains the biological activities such as antioxidant and anti-inflammatory of flavonoids; and the peel residue after extracting flavonoids is further used for pectin extraction, realizing the "zero waste" recycling of raw materials and reducing the total production cost.

[0044] Moreover, the eutectic solvent as a green extraction medium can significantly enhance the solubility of pectin molecules in the mixed system with water, and in combination with high temperature and high pressure conditions of 140-150 DEG C and 2-3 MPa, the release process of pectin from the cell wall is accelerated, and the extraction time is shortened (only 2-3 hours, saving half the time compared with the traditional acid extraction method), and the extracted pectin extract has high purity, good gelation and emulsification properties.

[0045] It can be understood that the eutectic solvent is a low eutectic mixture formed by mixing a hydrogen bond donor and a hydrogen bond acceptor in a specific molar ratio, and has good biocompatibility, low volatility and high solubility.

[0046] For example, common hydrogen bond donors include polyols (such as glycerol, ethylene glycol), organic acids (such as citric acid, lactic acid), etc., and hydrogen bond acceptors are mostly quaternary ammonium salts (such as choline chloride, choline bromide) or amide compounds (such as urea, thiourea).

[0047] In the present application, the extraction processes of flavonoids and pectin are organically combined, which not only improves the utilization rate of raw materials, but also ensures the quality of the two extracts, and provides a feasible path for the high-value utilization of passion fruit peel.

[0048] In some embodiments, the purity of the flavonoid extract is ≥85%, and the purity of the pectin extract is ≥90%. By the above technical scheme, not only the efficient separation and high-purity extraction of flavonoids and pectin in passion fruit peel are realized, but also the maximum exploitation of the value of raw materials is achieved through the recycling of residues; moreover, the biological activities such as antioxidant and anti-inflammatory of flavonoids can be completely retained, and the pectin has excellent gel stability and emulsification performance due to high galacturonic acid content, which can meet the needs of food thickening, pharmaceutical carriers and other fields for high-quality natural extracts; at the same time, the "zero waste" mode of raw materials greatly reduces the total production cost and significantly reduces waste emissions, providing a replicable technical path for the green and sustainable development of passion fruit industry and the high-value utilization of peel.

[0049] In some embodiments, the enzymes used in the enzymatic hydrolysis include cellulase and hemicellulase, and the added mass of cellulase and hemicellulase is 0.3% to 0.4% and 0.1% to 0.2% of the mass of the dried passion fruit peel, respectively. For example, the added mass of cellulase is 0.3%, 0.33%, 0.36%, 0.38%, 0.39%, or 0.40% of the mass of the dried passion fruit peel, or is within a range defined by any two of the above values; and the added mass of hemicellulase is 0.1%, 0.13%, 0.16%, 0.18%, 0.19%, or 0.20% of the mass of the dried passion fruit peel, or is within a range defined by any two of the above values.

[0050] By the above technical solution, cellulase and hemicellulase synergistically act in the above proportions to destroy the cell wall cellulose-hemicellulose skeleton structure of the passion fruit peel, effectively weaken the wrapping effect of the intercellular substance on flavonoids, and make the target components quickly dissolve from the cells, thereby laying a foundation of high-quality raw materials for subsequent separation and purification processes, and reducing the cost of enzymatic hydrolysis compared with the conventional enzyme dosage, achieving a balance between efficiency and cost.

[0051] It can be understood that cellulase is an enzyme complex capable of hydrolyzing the β-1, 4-glucosidic bond in cellulose (the main structural component of plant cell walls), which decomposes cellulose into small molecules such as glucose and cellobiose.

[0052] Hemicellulase is a general term for a class of enzymes capable of degrading hemicellulose (a polysaccharide interwoven with cellulose in plant cell walls).

[0053] In this application, cellulase and hemicellulase synergistically act like “molecular scissors” to destroy the cellulose and hemicellulose network constituting the tough cell wall of the passion fruit peel, thereby releasing the flavonoids wrapped inside the cells.

[0054] In some embodiments, the mass ratio of the dried peel residue, the eutectic solvent, and water is (70-80):(140-160):(1400-1600); for example, the mass ratio of the peel residue, water, and eutectic solvent can be 70:140:1400, 72:145:1450, 75:150:1500, 78:155:1550, 80:160:1600, 70:150:1500, 75:140:1600, 80:150:1400, or any two of the above numerical combinations.

[0055] Through the technical scheme, the dried peel residue can be fully dispersed in the mixed system of water and the eutectic solvent, the problem of uneven enzymatic reaction caused by material aggregation can be avoided, and the system has suitable viscosity, so that the subsequent stirring and mass transfer process can be smoothly performed.

[0056] Further, in the eutectic solvent, choline chloride as a hydrogen bond donor framework of the eutectic solvent, cooperates with citric acid and proline to form a strong polar solvent environment, which can effectively destroy the cellulose-hemicellulose structure of the peel cell wall, enhance the solubility of flavonoids and pectin, and EDTA-2Na can inhibit the degradation of flavonoids caused by oxidation by chelating metal ions in the system.

[0057] In some embodiments, the eutectic solvent includes choline chloride, citric acid, proline and EDTA-2Na, and the molar ratio of choline chloride, citric acid, proline and EDTA-2Na is 1:1:1:0.4-0.5; for example, the molar ratio of choline chloride, citric acid, proline and EDTA-2Na can be 1:1:1:0.4, 1:1:1:0.42, 1:1:1:0.45, 1:1:1:0.48, 1:1:1:0.5, or any two between the molar ratios formed by the above numerical combinations.

[0058] In the above technical scheme, through the four-component eutectic solvent system with a specific molar ratio, choline chloride as a hydrogen bond donor framework cooperates with citric acid and proline to form a strong polar solvent environment, which can efficiently destroy the cellulose-hemicellulose structure of the peel cell wall, solve the technical problems of weak penetration ability and low extraction rate of traditional solvents for pectin; and the molar ratio of 1:1:1:0.4-0.5 can realize the synergistic balance of hydrogen bond donors and acceptors, which not only ensures that the solvent polarity is suitable for pectin dissolution, but also avoids the abnormal viscosity caused by excessive single component, optimizes the mass transfer efficiency of the extraction system, and improves the extraction rate of pectin.

[0059] In some embodiments, a first auxiliary agent and a second auxiliary agent are further added during the extraction of pectin;

[0060] In some embodiments, the first auxiliary agent is polyethylene glycol, and the added mass of polyethylene glycol is 1.0%-1.5% of the mass of the dried peel residue; for example, the added mass of polyethylene glycol is 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5% of the mass of the dried peel residue, or any two between the above numbers.

[0061] In the technical solution, the polyethylene glycol is used as a hydrophilic additive. When the content of the polyethylene glycol is controlled within the range, the viscosity and surface activity of the system are balanced. Meanwhile, the hydroxyl groups of the polyethylene glycol can form hydrogen bond interaction with the pectin molecules, inhibit the β-elimination reaction of the pectin under high temperature and high pressure, and retain the linear structure and esterification groups of the pectin (solve the technical problem that the structure of the pectin is easy to degrade). In addition, the polyethylene glycol can reduce the aggregation degree of the pectin molecules by means of the steric hindrance effect, and further improve the solubility of the pectin in the extraction system by cooperating with the hydrogen bond network formed by the citric acid and proline in the eutectic solvent.

[0062] In some embodiments, the second additive is L-ascorbic acid, and the mass of the added L-ascorbic acid is 0.08% to 0.1% of the mass of the dried peel residue. For example, the mass of the added L-ascorbic acid is 0.08%, 0.085%, 0.09%, 0.095% or 0.1% of the mass of the dried peel residue, or any value between the above-mentioned values.

[0063] In the technical solution, the L-ascorbic acid is used as a strong antioxidant. On the one hand, the L-ascorbic acid can neutralize free radicals by providing hydrogen atoms, and inhibit the oxidation and rupture process of the hydroxyl groups in the pectin molecules. On the other hand, the reducing property of the L-ascorbic acid can reduce the thermodynamic driving force of the β-elimination reaction, and maintain the linear structure and esterification groups of the galacturonic acid chain, so as to meet the application requirements of high structural integrity pectin in the food and medical fields.

[0064] It can be understood that the β-elimination reaction of the pectin refers to the elimination reaction of the C4 hydroxyl group of the galacturonic acid unit in the pectin molecules and the C5 hydrogen of the adjacent unit under high temperature, strong acid or strong base conditions, which can cause the rupture of the glycosidic bond of the pectin main chain, shorten the pectin molecular chain, reduce the molecular weight, and further lose the excellent gelation and emulsification stability. In the document, the polyethylene glycol (hydrogen bond interaction) and L-ascorbic acid (antioxidant) are added to inhibit the reaction and ensure that the extracted pectin retains high galacturonic acid content and complete molecular structure.

[0065] In some embodiments, the guava peel extract is applied in the food field and the medical field. According to the technical solution, the pectin in the extract retains high galacturonic acid content and complete molecular structure, and the gelation performance, emulsification performance and physiological activity of the pectin are effectively maintained, so that the pectin has more extensive application value in various fields.

[0066] In the food field, pectin can be used as a natural food additive in the production of jam, jelly, milk beverage, etc.: for example, in jam making, its good gelling properties can form a uniform and stable gel structure, improving the taste and reducing the use of artificial gelling agents; in milk beverage, its emulsifying properties can improve the compatibility of milk fat and water phase, prevent stratification, prolong the shelf life of the product, and meet the needs of consumers for natural and healthy food. In the medical field, pectin has better physiological function because the hydroxyl groups are not excessively oxidized: as a dietary fiber, it can promote the proliferation of beneficial bacteria in the intestine, regulate the balance of intestinal microecology, and help improve constipation and intestinal health; as a drug carrier, its water solubility and molecular structure can encapsulate easily oxidized active ingredients (such as curcumin, anthocyanin), protect the active ingredients from being destroyed by the gastrointestinal environment, and improve the bioavailability; in addition, its natural source characteristics also reduce the irritation of drug preparations, improving patient medication compliance.

[0067] According to the second aspect of the present application, the extraction process of the passion fruit peel extract of the first aspect is provided, comprising the following steps:

[0068] S1. Preparing passion fruit peel powder by pretreating passion fruit peel;

[0069] S2. Enzymatic hydrolysis of passion fruit peel powder to obtain supernatant and peel residue;

[0070] S3. Purifying flavonoid extract from the supernatant;

[0071] S4. Mixing the peel residue, water, and eutectic solvent to extract pectin from the residue at 140-150°C and 2-3MPa.

[0072] Through the above technical solution, first, S1 pretreatment turns passion fruit peel into powder, effectively increasing the specific surface area of the material, providing sufficient contact sites for subsequent enzymatic reaction and improving the reaction rate; S2 enzymatic hydrolysis step, with the specific catalytic action of enzymes, selectively decomposes the cell wall structure of cellulose, hemicellulose, etc. in the peel, allowing flavonoids to be fully released into the supernatant, while avoiding excessive degradation of pectin molecules, ensuring the structural integrity of pectin in the residue, and achieving efficient separation of flavonoids and pectin; S3 purifies the flavonoid extract in the supernatant, which can obtain high-purity flavonoid active ingredients, meeting the requirements of food antioxidants or pharmaceutical active materials; and S4, for the peel residue after enzymatic hydrolysis, uses a mixed system of eutectic solvent and water under high temperature and high pressure conditions of 140-150°C and 2-3MPa, the eutectic solvent enters the residue interior quickly due to its excellent solubility and penetration ability, and forms hydrogen bond interactions with pectin molecules, significantly improving the extraction rate of pectin.

[0073] The whole process realizes step-by-step extraction of flavonoids and pectin in passion fruit peel and multi-component utilization, avoids waste of raw materials, and improves product added value; meanwhile, the recyclability of the eutectic solvent and the mildness of the enzymatic process make the whole extraction process more green and environmentally friendly, meeting the high-quality requirements of the current food and pharmaceutical fields for natural extracts.

[0074] In some embodiments, in step S2, passion fruit peel powder is weighed, citric acid-sodium citrate buffer with pH=4.5 is added, and cellulase and hemicellulase are added, and enzymolysis is carried out at 45-50°C for 120-150 min.

[0075] After the enzymolysis is completed, inactivation treatment is carried out, centrifugal separation is carried out, the supernatant is collected, and the peel residue is reserved.

[0076] Through the above technical solution, the cell wall structure composed of cellulose and hemicellulose in passion fruit peel is effectively decomposed, the wrapping barrier of flavonoids in the cells is broken, the flavonoid active ingredients are fully dissolved into the buffer, and the extraction efficiency of flavonoids is significantly improved; at the same time, the precisely controlled pH value 4.5 and temperature (45-50°C) environment avoid the degradation of pectin molecules due to over-acid or over-heat conditions, retain the original structure of pectin substances in the residue after enzymolysis, and avoid the premature dissolution of pectin, which provides a structural basis for the subsequent step S4 of using the eutectic solvent to efficiently extract pectin.

[0077] In some embodiments, in step S3, the supernatant is diluted to a flavonoid concentration of 1.0 mg / mL, eluted through an AB-8 type macroporous resin column, and the eluate with an absorbance of 280 nm of ≥0.2 to ≤0.05 is collected. The eluate is concentrated and dried to obtain a flavonoid extract powder.

[0078] In the technical scheme, the supernatant is diluted to a flavone concentration of 1.0 mg / mL, the adsorption environment of the AB-8 type macroporous resin is optimized, the excessive occupation of the resin adsorption sites by high-concentration flavones is avoided, and the adsorption efficiency of the resin for flavones is ensured; meanwhile, the AB-8 type macroporous resin has moderate polarity and specific surface area, shows good selectivity for flavone active ingredients, can effectively adsorb flavones in the supernatant, and has weak adsorption capacity for residual citric acid buffer, sugars, proteins and other impurities, so that the flavone active ingredients and impurities are preliminarily separated; in addition, the absorbance at 280 nm is used as a monitoring index of the elution process, and the eluate with an absorbance of 0.2 or more to 0.05 or less is collected, so that the concentration of the flavone active ingredients in the eluate reaches the effective extraction requirement, and the eluate with low concentration or many impurities is avoided, and the purity of the flavone extract is further improved; finally, the flavone extract powder obtained after concentration and drying has low impurity content and good stability, can meet the purity and quality requirements of flavone raw materials in the fields of food, medicine, cosmetics and the like, and provides high-quality active ingredient basis for subsequent product research and application.

[0079] In some embodiments, in step S4, the specific preparation process is as follows:

[0080] S41: In a reaction kettle, the dried peel residue, part of the water, and the eutectic solvent are mixed, and part of the first auxiliary agent is added and uniformly mixed; the temperature is raised to 140-150 DEG C at a rate of 5 DEG C / min, and the pressure is increased to 2-3 MPa; the reaction is carried out at 140-150 DEG C and 2-3 MPa for 60-70 min;

[0081] S42: The remaining deionized water and the remaining first auxiliary agent are added to the reaction kettle, the temperature is maintained at 140-150 DEG C, the pressure is maintained at 2-3 MPa, and the reaction is continued for 60-70 min;

[0082] S43: The second auxiliary agent is added to the reaction kettle, the temperature is lowered to 100 DEG C, the pressure is maintained at 2-3 MPa, and the reaction is carried out for 15-20 min; after the reaction is completed, the temperature is lowered to room temperature, and the pressure is released to normal pressure.

[0083] S44: The reaction liquid is filtered, the pH is adjusted to 2.0, anhydrous ethanol is used for alcohol precipitation treatment, and then dried to obtain a pectin extract powder.

[0084] By the technical scheme, the high selectivity dissolution characteristics of the pectin component in the peel residue by the eutectic solvent are utilized, and the gradient reaction design of step-by-step addition of water and the auxiliary agent is combined, so that the lignin-cellulose network structure of the peel cell is gradually broken under the high-temperature and high-pressure conditions of 140-150°C and 2-3 MPa, and the pectin molecules are pushed out from the cell wall matrix with high efficiency; the addition of the second auxiliary agent in the S53 and the cooling operation further inhibit the β-elimination reaction of the pectin at high temperature, and effectively retain the linear structure and esterification groups of the pectin. The process design of step-by-step pressurization and temperature rise also improves the stability of the reaction system, avoids the local overheating or pectin degradation problem caused by one-time temperature rise and pressurization, and ensures the consistency of the product quality. In addition, the combination steps of pH adjustment and alcohol precipitation effectively remove the impurities such as polysaccharides and proteins in the extract, so that the purity of the pectin powder is further improved to more than 92%, and the application requirements of high-purity pectin in the fields of medicine, food and the like are met.

[0085] The guava peel extract and the extraction process thereof are further described through the following preparation examples, examples and comparative examples.

[0086] Test method and equipment

[0087] 1. Content test of flavonoids and content test of pectin in the dried guava peel

[0088] 1.1 Content test of flavonoids

[0089] (1) Reagents and instruments:

[0090] Rutin standard, deionized water, anhydrous ethanol, ultraviolet spectrophotometer (such as Shimadzu UV-1800), analytical balance;

[0091] (2) Test steps:

[0092] Sample solution preparation:

[0093] 1.0 g of dried guava peel powder was taken, 50 mL of 70% ethanol solution was added, and extraction was carried out at 60°C water bath for 60 min. After cooling, the filtrate was filtered, the filtrate was repeatedly extracted three times, the filtrate was combined, and the volume was adjusted to 200 mL to obtain a sample solution to be tested.

[0094] Rutin standard curve drawing:

[0095] Take rutin standard solution (0-0.05 mg / mL), add 0.3 mL 5% sodium nitrite solution (stand for 6 min)→0.3 mL 10% aluminum nitrate solution (stand for 6 min)→4 mL 4% sodium hydroxide solution, and dilute with 70% ethanol to 10 mL, avoid light and stand for 15 min, determine the absorbance of each standard solution at 510 nm wavelength with 70% ethanol as blank. Draw the standard curve with rutin concentration as abscissa and absorbance as ordinate, and obtain the regression equation:

[0096] y=0.0241x+0.152 (R 2 =0.9993).

[0097] (3) Sample determination

[0098] Take 1.0 mL sample solution, and determine its absorbance at 510 nm under the same conditions as above, and calculate the flavone concentration in the sample by substituting into the standard curve.

[0099] (4) Content calculation

[0100] The flavone content (mg / g) is calculated according to the following formula:

[0101] Flavone content=(C×V×N) / W;

[0102] Wherein: C: the flavone concentration (mg / mL) calculated from the standard curve; V: sample constant volume (mL); N: dilution multiple; W: sample mass (g).

[0103] 1.2 Pectin content test

[0104] (1) Reagents and instruments

[0105] Galacturonic acid standard (purity≥98%);

[0106] Concentrated sulfuric acid;

[0107] Anhydrous ethanol;

[0108] Carbazole, prepared into 1 g / L ethanol solution (freshly prepared or stored in dark);

[0109] Deionized water;

[0110] UV-visible spectrophotometer (such as Shimadzu UV-1800)

[0111] Analytical balance;

[0112] (2) Test steps

[0113] Sample solution preparation:

[0114] Take 1.0 g (accurate to 0.0001 g) of dried passion fruit peel powder and place it in a 150 mL conical flask. Add 50 mL of citric acid-sodium citrate buffer with pH = 4.5, extract in a 90°C water bath for 60 min, cool and filter, repeat the extraction three times with the filter residue, combine the filtrate, and dilute to 200 mL as the sample solution to be tested.

[0115] Galacturonic acid standard curve drawing

[0116] Weigh 10 mg of galacturonic acid standard and dissolve it in deionized water and dilute to 100 mL to obtain a 0.1 mg / mL standard stock solution.

[0117] Take 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of the standard stock solution into 10 mL stoppered graduated test tubes, respectively, and make up to 1.0 mL with deionized water to obtain a series of standard solutions of 0, 0.02, 0.04, 0.06, 0.08, 0.10 mg / mL. Slowly add 5 mL of concentrated sulfuric acid to each test tube (operate under ice water bath conditions), shake evenly, and then place in a 85°C water bath for 20 min.

[0118] After taking out, quickly cool to room temperature with ice water, add 0.25 mL of 1 g / L carbazole-ethanol solution, shake well, and stand at room temperature for 30 min in the dark. With 0 tube as blank, measure the absorbance of each standard solution at 528 nm wavelength.

[0119] With the concentration of galacturonic acid as the abscissa and the absorbance as the ordinate, draw the standard curve to obtain the regression equation:

[0120] y = 0.0241x + 0.2157 (R 2 = 0.9991).

[0121] (3) Sample determination

[0122] Take 1.0 mL of sample solution and operate under the same conditions as above (add sulfuric acid, water bath, add carbazole, color development) to measure the absorbance at 528 nm. Substitute the standard curve to calculate the concentration of galacturonic acid in the sample.

[0123] (4) Content determination

[0124] The pectin content (mg / g) is calculated according to the following formula:

[0125] Pectin content = (C x V x N) / W;

[0126] Where: C is the concentration of galacturonic acid calculated from the standard curve (mg / mL); V is the sample volume (mL); N is the dilution factor; W is the sample mass (g).

[0127] Example 1 to Example 5, the same batch of fresh passion fruit, the flavonoids content in the dried passion fruit peel was 2.51 mg / g, and the pectin content was 163 mg / g.

[0128] 2. Flavonoids antioxidant activity test

[0129] (1) Reagents and instruments

[0130] DPPH (1,1-diphenyl-2-trinitrobenzene hydrazine) was prepared into an ethanol solution of 0.2 mmol / L (stored in the dark, prepared fresh);

[0131] Flavonoids extract sample solution (concentration 1 mg / mL, filtered through a 0.45 μm filter membrane);

[0132] Vitamin C (positive control, concentration 1 mg / mL);

[0133] Anhydrous ethanol, deionized water;

[0134] UV spectrophotometer (such as Shimadzu UV-1800)

[0135] (2) Test steps

[0136] Sample preparation:

[0137] Take flavonoids extract powder, dissolve with deionized water and dilute to 1 mg / mL, filter and reserve.

[0138] Reaction system:

[0139] Sample tube (S): mix 2.0 mL sample solution (1.0 mg / mL) with 2.0 mL DPPH solution.

[0140] Sample background tube (S0): mix 2.0 mL sample solution (1.0 mg / mL) with 2.0 mL anhydrous ethanol.

[0141] DPPH blank tube (A0): mix 2.0 mL anhydrous ethanol with 2.0 mL DPPH solution.

[0142] Positive control tube (C): mix 2.0 mL vitamin C solution (1.0 mg / mL) with 2.0 mL DPPH solution.

[0143] Reaction conditions:

[0144] Place the above mixture in a 25°C dark environment for 30 min.

[0145] Absorbance determination:

[0146] After the reaction, the absorbance values of each group were measured at 517 nm.

[0147] (3) Result calculation

[0148] Clearance = (1 - (S - S0) / A0) x 100%;

[0149] Wherein, S is the absorbance of sample tube; S0 is the absorbance of sample background tube; A0 is the absorbance of DPPH blank tube.

[0150] 3. Purity test of flavonoids

[0151] (1) Reagents and instruments

[0152] The same as "(1) Reagents and instruments" in "1.1 Content test of flavonoids".

[0153] (2) Test procedure

[0154] Standard curve drawing: directly quote the whole process and the regression equation (y = 0.0241x + 0.152, R 2 = 0.9993) obtained therefrom under "(2) Test procedure" in "1.1 Content test of flavonoids".

[0155] Sample solution preparation: weigh 10 mg of the prepared flavonoid extract powder, place it in a 100 mL volumetric flask, dissolve with 70% ethanol and dilute to the mark, shake well, and use it as the sample stock solution. Take 1.0 mL of the stock solution into a 10 mL volumetric flask, add 70% ethanol to the mark, shake well, and it is the sample solution to be tested.

[0156] Sample determination: the same as "(3) Sample determination" in "1.1 Content test of flavonoids", determine the absorbance of the sample solution to be tested at 510 nm.

[0157] (3) Purity calculation

[0158] Substitute the measured absorbance value of the sample solution into the above rutin standard curve regression equation to calculate the concentration of flavonoids in the sample solution (C, mg / mL).

[0159] The purity P1 of the flavonoid extract is calculated as follows:

[0160] P1 (%) = (C x V x D x 10 -3 / M) 100%;

[0161] Wherein: C: the concentration of flavonoids in the sample solution to be tested calculated from the standard curve (mg / mL);

[0162] V: the constant volume of the sample stock solution (100 mL);

[0163] D: dilution factor (in this case, 10, i.e. 1 mL of the stock solution was diluted to 10 mL); M: the mass of the flavonoid extract powder taken (mg).

[0164] 4. Pectin extract purity test

[0165] (1) Reagents and instruments

[0166] The same as the "(1) Reagents and instruments" section in "1.2 Pectin content test".

[0167] (2) Test procedure

[0168] Standard curve preparation: Directly refer to the standard curve preparation process and the regression equation (y = 0.0325x + 0.2157, R 2 = 0.9991) obtained in the "(2) Test procedure" section in "1.2 Pectin content test".

[0169] Sample solution preparation: Take 10 mg of the pectin extract powder (i.e. the final product) prepared in this case and place it in a 100 mL volumetric flask. Dissolve it with a citric acid-sodium citrate buffer solution with pH = 4.5 and make up to the mark. Shake well to obtain the sample stock solution. Take 1.0 mL of the stock solution and place it in a 10 mL stoppered graduated test tube as the sample solution.

[0170] Sample determination: The same as the "(3) Sample determination" procedure in "1.2 Pectin content test". Perform the operations of adding concentrated sulfuric acid, water bath heating, adding carbazole reagent, and color development on the sample solution, and finally determine its absorbance at 528 nm.

[0171] (3) Purity calculation

[0172] Substitute the measured sample solution absorbance value into the above galacturonic acid standard curve regression equation to calculate the concentration (C, mg / mL) of galacturonic acid in the sample solution.

[0173] The purity P2 of the pectin extract is calculated as follows:

[0174] P2 (%) = (C x V x D x 10 -3 / M) 100%;

[0175] Where: C: the concentration of the pectin extract in the sample solution calculated from the standard curve (mg / mL);

[0176] V: the constant volume of the sample stock solution (100 mL);

[0177] D: dilution factor (in this case, 10, i.e. 1 mL of the stock solution was diluted to 10 mL);

[0178] M: mass of the pectin extract powder (mg).

[0179] In the examples or comparative examples, the raw materials are all from the market; wherein, choline chloride CAS: 67-48-1; citric acid CAS: 77-92-9; proline CAS: 147-85-3; EDTA-2Na CAS: 6381-92-6; AB-8 type macroporous resin column from Langfang Shengquan Chemical Co., Ltd.

[0180] Example 1

[0181] An extraction process of a guava peel extract, comprising the following steps:

[0182] S1: Take 1000g of fresh guava peel, wash 3 times with deionized water after removing the rotten part, remove the damaged and moldy peel, and transfer to a freeze dryer at -50℃ for 48h until the moisture content is 7.8%. After crushing by a pulverizer, pass through a 80 mesh standard sieve to obtain a uniform powder for standby.

[0183] S2: Take 100g of guava peel powder, add 1800g of pH = 4.5 citric acid-sodium citrate buffer (50mmol / L), mix and then magnetically stir for 10min. Add 0.35g of cellulase and 0.15g of hemicellulase, and enzymatically hydrolyze at 48℃ for 135min. After the enzymatic hydrolysis is completed, heat to 90℃ for 15min to inactivate the enzyme, centrifuge at 8000r / min for 15min to collect the supernatant, and reserve the residue.

[0184] S3: Dilute the supernatant to a flavonoid concentration of 1.0mg / mL, and pass through an AB-8 type macroporous resin column (column diameter 1.5cm, column height 20cm, adsorption temperature 25℃) at a flow rate of 2BV / h. Elute with deionized water at a flow rate of 3BV / h until the pH of the effluent stabilizes at 4.5-5.0, then elute with 70% ethanol at a flow rate of 1BV / h, and collect the eluate in the range of 280nm absorbance ≥0.2 to ≤0.05. Concentrate the eluate to 1 / 10 of the original volume at 60℃ and 0.08MPa, and freeze-dry at -50℃ for 24h to obtain a flavonoid extract powder.

[0185] S4: Take the residue obtained by centrifugation in step S2, wash twice with deionized water (500ml each time, centrifuge at 5000r / min for 10min), and freeze-dry the residue at -50℃ for 24h after washing until the moisture content = 10%.

[0186] S5: Weigh 80 g of S4 dried residue, add 150 g of quaternary solvent (its composition is choline chloride: citric acid: proline: EDTA-2Na molar ratio 1:1:1:0.45), 800 g of deionized water and 0.6 g of PEG-4000, mix uniformly and then transfer to a high-pressure reaction kettle. Seal the reaction kettle, heat to 145°C at a rate of 5°C / min, simultaneously pressurize to 2.5 MPa, and stir at 300 r / min for 65 min.

[0187] S6: Add 700 g of deionized water and 0.6 g of PEG-4000 to the reaction solution of S5, stir uniformly at 300 r / min, and continue to react under the conditions of 145°C, 2.5 MPa and 300 r / min for 65 min.

[0188] S7: Add 0.07 g of L-ascorbic acid to the reaction solution of S6, stir uniformly at 300 r / min. Cool to 100°C at a rate of 3°C / min, maintain 2.5 MPa pressure and stir for 18 min, turn off the heating device and naturally cool to 25°C, and slowly release the pressure to normal pressure.

[0189] S8: Vacuum suction filter the reaction solution of S7 through a 0.45 μm filter membrane, adjust the pH of the filtrate to 2.0 with 1 mol / L hydrochloric acid, and stir for 10 min. Slowly add 900 ml of anhydrous ethanol to the adjusted filtrate, stir at 200 r / min and stand at 4°C for 12 h. Centrifuge at 10000 r / min for 20 min to collect the precipitate, wash it twice with 70% ethanol and once with anhydrous ethanol. Freeze-dry the precipitate at -50°C for 48 h to a moisture content of 6%, crush it and pass it through a 60 mesh sieve to obtain pectin powder.

[0190] Example 2

[0191] An extraction process of a passion fruit peel extract includes the following steps:

[0192] S1: Take 1000 g of fresh passion fruit peel, wash it 3 times with deionized water to remove damaged and moldy peel, transfer it to a freeze dryer and dry it at -50°C for 48 h until the moisture content is 7.8%. Crush it with a crusher and pass it through an 80 mesh standard sieve to obtain uniform powder for use.

[0193] S2: Weigh 100 g of passion fruit peel powder, add 1500 g of pH = 4.5 citric acid-sodium citrate buffer (50 mmol / L), mix, and then magnetically stir for 10 min. Add 0.3 g of cellulase and 0.1 g of hemicellulase, and enzymatically hydrolyze at 45°C for 150 min. After the enzymatic hydrolysis is completed, heat to 90°C and keep for 15 min to inactivate the enzymes, centrifuge at 8000 r / min for 15 min to collect the supernatant, and reserve the residue.

[0194] S3: The supernatant was diluted to a flavonoid concentration of 1.0 mg / mL and passed through an AB-8 macroporous resin column (column diameter 1.5 cm, column height 20 cm, adsorption temperature 25 °C) at a flow rate of 2 BV / h. Elution was performed with deionized water at a flow rate of 3 BV / h until the pH of the effluent was stable at 4.5-5.0, followed by elution with 70% ethanol at a flow rate of 1 BV / h, and the eluate was collected in sections with an absorbance at 280 nm of ≥0.2 to ≤0.05. The eluate was concentrated to 1 / 10 of the original volume at 60 °C and 0.08 MPa, and freeze-dried at -50 °C for 24 h to obtain a flavonoid extract powder.

[0195] S4: The residue obtained in step S2 was centrifuged, and the residue was washed twice with deionized water (500 mL each time, centrifuged at 5000 r / min for 10 min). After washing, the residue was freeze-dried at -50 °C for 24 h until the water content was 7.3%.

[0196] S5: 80 g of the dried residue from step S4 was weighed, and a quaternary solvent (140 g, consisting of choline chloride: citric acid: proline: EDTA-2Na at a molar ratio of 1:1:1:0.4), 800 g of deionized water, and 0.5 g of PEG-4000 were added. The mixture was mixed uniformly and then transferred to a high-pressure reaction kettle. The kettle was sealed, heated to 140 °C at a rate of 5 °C / min, pressurized to 2 MPa simultaneously, and stirred at 300 r / min for 60 min.

[0197] S6: To the reaction solution from step S5, 600 g of deionized water and 0.5 g of PEG-4000 were added, and the mixture was stirred uniformly at 300 r / min. The reaction was continued at 140 °C, 2 MPa, and 300 r / min for 60 min.

[0198] S7: To the reaction solution from step S6, 0.064 g of L-ascorbic acid was added, and the mixture was stirred uniformly at 300 r / min. The temperature was decreased to 100 °C at a rate of 3 °C / min, and the reaction was continued at 2 MPa for 15 min. The heating device was turned off, and the mixture was naturally cooled to 25 °C. The pressure was slowly released to atmospheric pressure.

[0199] S8: The reaction solution from step S7 was vacuum filtered through a 0.45 μm filter membrane. The pH of the filtrate was adjusted to 2.0 with 1 mol / L hydrochloric acid, and the mixture was stirred for 10 min. The adjusted filtrate was slowly added to 900 mL of anhydrous ethanol, and the mixture was stirred at 200 r / min and allowed to stand at 4 °C for 12 h. The precipitate was collected by centrifugation at 10,000 r / min for 20 min, washed twice with 70% ethanol and once with anhydrous ethanol. The precipitate was freeze-dried at -50 °C for 48 h until the water content was 4.7%, crushed, and sieved through a 60 mesh sieve to obtain a pectin powder.

[0200] Example 3

[0201] An extraction process for a passion fruit peel extract, comprising the following steps:

[0202] S1: Take fresh passion fruit peel 1000g, remove the rotten part and rinse with deionized water for 3 times to remove the damaged and mildewed peel, then transfer to a freeze dryer and dry at -50℃ for 48h until the moisture content is 7.8%. After crushing by a pulverizer, pass through an 80 mesh standard sieve to obtain a uniform powder for standby.

[0203] S2: Take 100g of passion fruit peel powder, add 2000g of pH = 4.5 citric acid-sodium citrate buffer (50mmol / L), mix and then magnetically stir for 10min. Add 0.4g of cellulase and 0.2g of hemicellulase, and enzymatically hydrolyze at 50℃ for 150min. After the enzymatic hydrolysis is completed, heat to 90℃ for 15min to inactivate the enzyme, centrifuge at 8000r / min for 15min to collect the supernatant, and reserve the residue for standby.

[0204] S3: Dilute the supernatant to a flavonoid concentration of 1.0mg / mL, and pass through an AB-8 type macroporous resin column (column diameter 1.5cm, column height 20cm, adsorption temperature 25℃) at a flow rate of 2BV / h. Elute with deionized water at a flow rate of 3BV / h until the pH of the effluent stabilizes at 4.5-5.0, then elute with 70% ethanol at a flow rate of 1BV / h, and collect the eluate in the range where the absorbance at 280nm is ≥0.2 to ≤0.05. Concentrate the eluate to 1 / 10 of the original volume at 60℃ and 0.08MPa, and freeze-dry at -50℃ for 24h to obtain a flavonoid extract powder.

[0205] S4: Take the residue obtained by centrifugation in step S2, wash twice with deionized water (500ml each time, centrifuge at 5000r / min for 10min), and freeze-dry the washed residue at -50℃ for 24h until the moisture content is 9.5%.

[0206] S5: Take 80g of the dried residue from S4, add 143g of a quaternary solvent (composition: choline chloride: citric acid: proline: EDTA-2Na molar ratio 1:1:1:0.5), 900g of deionized water, and 0.75g of PEG-4000, mix well, and then transfer to a high-pressure reaction kettle. Seal the reaction kettle, heat to 150℃ at a rate of 5℃ / min, simultaneously pressurize to 3MPa, and stir at 300r / min for 70min.

[0207] S6: To the reaction solution of S5, add 700g of deionized water and 0.75g of PEG-4000, stir uniformly at 300r / min, and continue to react at 150℃, 3MPa, and 300r / min for 70min.

[0208] S7: To the reaction solution of S6, 0.08 g of L-ascorbic acid was added and stirred uniformly at 300 r / min. The temperature was decreased to 100℃ at a rate of 3℃ / min, and the stirring reaction was carried out for 20 min under a pressure of 3 MPa. The heating device was turned off, and the solution was naturally cooled to 25℃. The pressure was slowly released to normal pressure.

[0209] S8: The reaction solution of S7 was filtered through a 0.45 μm filter membrane under vacuum. The pH of the filtrate was adjusted to 2.0 with 1 mol / L hydrochloric acid, and the solution was stirred for 10 min. The adjusted filtrate was slowly added to 900 ml of anhydrous ethanol, and the solution was stirred at 200 r / min and placed at 4℃ for 12 h. The precipitate was collected by centrifugation at 10,000 r / min for 20 min, and then washed twice with 70% ethanol and once with anhydrous ethanol. The precipitate was freeze-dried at -50℃ for 48 h until the moisture content was 4.2%. The precipitate was ground and sieved through a 60 mesh sieve to obtain a pectin powder.

[0210] Example 4

[0211] An extraction process of a passion fruit peel extract includes the following steps:

[0212] S1: 1000 g of fresh passion fruit peel was taken, washed with deionized water for 3 times to remove the rotten and moldy peel, and then transferred to a freeze dryer and dried at -50℃ for 48 h until the moisture content was 7.8%. The peel was ground and sieved through an 80 mesh standard sieve to obtain a uniform powder for use.

[0213] S2: 100 g of the passion fruit peel powder was taken, added to 1600 g of a citric acid-sodium citrate buffer (50 mmol / L) with a pH of 4.5, mixed, and then magnetically stirred for 10 min. 0.33 g of cellulase and 0.13 g of hemicellulase were added, and the solution was enzymatically hydrolyzed at 46℃ for 130 min. After the enzymatic hydrolysis, the temperature was increased to 90℃ for 15 min to inactivate the enzymes. The supernatant was collected by centrifugation at 8000 r / min for 15 min, and the residue was reserved for use.

[0214] S3: The supernatant was diluted to a flavonoid concentration of 1.0 mg / mL, and then passed through an AB-8 type macroporous resin column (column diameter 1.5 cm, column height 20 cm, adsorption temperature 25℃) at a flow rate of 2 BV / h. The column was eluted with deionized water at a flow rate of 3 BV / h until the pH of the effluent was stable at 4.5-5.0. Then, the column was eluted with 70% ethanol at a flow rate of 1 BV / h, and the eluate with an absorbance at 280 nm of ≥0.2 to ≤0.05 was collected. The eluate was concentrated to 1 / 10 of the original volume at 60℃ and 0.08 MPa, and then freeze-dried at -50℃ for 24 h to obtain a flavonoid extract powder.

[0215] S4: The residue obtained in step S2 was centrifuged, washed twice with deionized water (500 ml each time, centrifuged at 5000 r / min for 10 min), and the residue after washing was placed in a freeze dryer at -50°C for 24 h until the water content was 7.6%.

[0216] S5: 80 g of the dried residue in S4 was weighed, 143 g of a quaternary solvent (the composition of choline chloride: citric acid: proline: EDTA-2Na was 1:1:1:0.43), 800 g of deionized water, and 0.5 g of PEG-4000 were added, and the mixture was uniformly mixed and then transferred to a high-pressure reaction kettle. The reaction kettle was sealed, heated to 143°C at a rate of 5°C / min, pressurized to 2.3 MPa synchronously, and stirred at 300 r / min for 62 min.

[0217] S6: To the reaction solution in S5, 650 g of deionized water and 0.6 g of PEG-4000 were added, and the mixture was uniformly stirred at 300 r / min. The reaction was continued at 143°C, 2.3 MPa, and 300 r / min for 62 min.

[0218] S7: To the reaction solution in S6, 0.067 g of L-ascorbic acid was added, and the mixture was uniformly stirred at 300 r / min. The temperature was lowered to 100°C at a rate of 3°C / min, and the reaction was continued at 2.3 MPa for 16 min. The heating device was turned off, and the mixture was naturally cooled to 25°C. The pressure was slowly released to normal pressure.

[0219] S8: The reaction solution in S7 was vacuum filtered through a 0.45 μm filter membrane. The pH of the filtrate was adjusted to 2.0 with 1 mol / L hydrochloric acid, and the mixture was stirred for 10 min. The adjusted filtrate was slowly added to 900 ml of anhydrous ethanol, and the mixture was stirred at 200 r / min and placed at 4°C for 12 h. The precipitate was collected by centrifugation at 10000 r / min for 20 min, washed twice with 70% ethanol and once with anhydrous ethanol, and placed in a freeze dryer at -50°C for 48 h until the water content was 5.6%. The precipitate was ground and passed through a 60 mesh sieve to obtain a pectin powder.

[0220] Example 5

[0221] An extraction process of a passion fruit peel extract includes the following steps:

[0222] S1: 1000 g of fresh passion fruit peel was washed 3 times with deionized water to remove damaged and moldy peel, and then transferred to a freeze dryer and dried at -50°C for 48 h until the water content was 7.8%. The dried peel was ground and passed through an 80 mesh standard sieve to obtain a uniform powder for use.

[0223] S2: 100 g of passion fruit peel powder was weighed and added to 1900 g of citric acid-sodium citrate buffer (50 mmol / L) with pH = 4.5, and after mixing, it was magnetically stirred for 10 min. 0.38 g of cellulase and 0.18 g of hemicellulase were added, and enzymatic hydrolysis was carried out at 48°C for 145 min. After the enzymatic hydrolysis was completed, the temperature was raised to 90°C to inactivate the enzyme, and centrifugation was carried out at 8000 r / min for 15 min to collect the supernatant, and the residue was reserved for later use.

[0224] S3: The supernatant was diluted to a flavonoid concentration of 1.0 mg / mL, and passed through an AB-8 type macroporous resin column (column diameter 1.5 cm, column height 20 cm, adsorption temperature 25°C) at a flow rate of 2 BV / h. Elution was carried out with deionized water at a flow rate of 3 BV / h until the pH of the effluent was stable at 4.5-5.0, and then elution was carried out with 70% ethanol at a flow rate of 1 BV / h, and the eluate with an absorbance at 280 nm ≥ 0.2 to ≤ 0.05 was collected. The eluate was concentrated to 1 / 10 of the original volume at 60°C and 0.08 MPa, and freeze-dried at -50°C for 24 h to obtain flavonoid extract powder.

[0225] S4: The residue obtained by centrifugation in step S2 was washed twice with deionized water (500 ml each time, centrifugation at 5000 r / min for 10 min), and after washing, the residue was placed in a freeze dryer at -50°C for 24 h until the water content was 7.4%.

[0226] S5: 80 g of the dried residue from S4 was weighed, and 143 g of a quaternary solvent (choline chloride: citric acid: proline: EDTA-2Na molar ratio 1:1:1:0.48), 900 g of deionized water, and 0.7 g of PEG-4000 were added, and after mixing well, it was transferred to a high-pressure reaction kettle. The reaction kettle was sealed, heated to 148°C at a rate of 5°C / min, simultaneously pressurized to 2.8 MPa, and stirred at 300 r / min for 68 min.

[0227] S6: To the reaction solution of S5, 650 g of deionized water and 0.7 g of PEG-4000 were added, and stirred uniformly at 300 r / min, and the reaction was continued at 148°C, 2.8 MPa, and 300 r / min for 68 min.

[0228] S7: To the reaction solution of S6, 0.075 g of L-ascorbic acid was added, and stirred uniformly at 300 r / min. The temperature was lowered to 100°C at a rate of 3°C / min, and the reaction was carried out at 2.8 MPa pressure and 300 r / min for 18 min, the heating device was turned off, and the temperature was naturally cooled to 25°C, and the pressure was slowly released to normal pressure.

[0229] S8: The reaction solution of S7 was vacuum filtered through a 0.45 μm filter membrane, and the filtrate was adjusted to pH 2.0 with 1 mol / L hydrochloric acid and stirred for 10 min. The adjusted filtrate was slowly added into 900 ml of anhydrous ethanol, stirred at 200 r / min and placed at 4°C for 12 h. The precipitate was collected by centrifugation at 10 000 r / min for 20 min, washed twice with 70% ethanol and once with anhydrous ethanol. The precipitate was freeze-dried at -50°C for 48 h until the moisture content was 5.6%, ground and passed through a 60 mesh sieve to obtain pectin powder.

[0230] Comparative Example 1

[0231] In step S2, no hemicellulase was added, and the rest was the same as in Example 1.

[0232] Comparative Example 2

[0233] In step S2, no cellulase was added, and the rest was the same as in Example 1.

[0234] Comparative Example 3

[0235] In step S2, no cellulase was added, and no hemicellulase was added, and the rest was the same as in Example 1.

[0236] Comparative Example 4

[0237] In step S2, 0.1 g of cellulase and 0.05 g of hemicellulase were added, and the rest was the same as in Example 1.

[0238] Comparative Example 5

[0239] In step S2, 1 g of cellulase and 0.6 g of hemicellulase were added, and the rest was the same as in Example 1.

[0240] Comparative Example 6

[0241] In steps S5 and S6, no quaternary solvent was added; and the rest was the same as in Example 1.

[0242] Comparative Example 7

[0243] In steps S5 and S6, no PEG-400 was added; and the rest was the same as in Example 1.

[0244] Comparative Example 8

[0245] In step S7, no L-ascorbic acid was added; and the rest was the same as in Example 1.

[0246] Comparative Example 9

[0247] In steps S5 and S6, a binary solvent was added, which was composed of choline chloride and citric acid at a molar ratio of 1:1; and the rest was the same as in Example 1.

[0248] Comparative Example 10

[0249] In steps S5 and S6, a binary solvent is added, with the composition of choline chloride:citric acid:proline in a molar ratio of 1:1:1; the rest is the same as in Example 1.

[0250] Comparative Example 11

[0251] In steps S5 and S6, the temperature is maintained at 120°C and the pressure is maintained at 20 MPa; the rest is the same as in Example 1.

[0252] Comparative Example 12

[0253] In steps S5 and S6, the temperature is maintained at 175°C and the pressure is 1 MPa; the rest is the same as in Example 1.

[0254] The tests were conducted according to the above testing methods, and the test results are summarized in Table 1.

[0255] Table 1

[0256]

[0257]

[0258] As seen in Examples 1 to 5 and Comparative Examples 1 to 12, the extraction process combines enzymatic hydrolysis with co-crystal solvent extraction. By controlling the dosage of cellulase and hemicellulase, the composition of the co-crystal solvent, the addition of adjuvants, and the temperature and pressure parameters within the range specified in this application, the passion fruit peel extract exhibits high DPPH scavenging rate (≥87.8%), flavonoid purity (≥85.5%), pectin extract purity (≥90.8%), flavonoid recovery rate (≥82.3%), and pectin recovery rate (≥85.7%). This indicates that the extract possesses superior antioxidant activity, higher purity, and higher extraction efficiency, achieving high-value utilization of passion fruit peel. Comparative Examples 1 and 2 did not add hemicellulase or cellulase, respectively; Comparative Example 3 did not add any enzymes; Comparative Example 4 used too little enzyme; and Comparative Example 5 used too much enzyme, resulting in lower DPPH scavenging rate, flavonoid purity, pectin extract purity, and recovery rate in their respective extracts. This indicates that when at least one of the enzyme types and dosages is outside the scope of this application, the extraction efficiency and quality of flavonoids and pectin will significantly decrease.

[0259] The composition of the eutectic solvent generally affects the purity and recovery rate of the pectin. As can be seen from Example 1, Comparative Example 9 and Comparative Example 10, when the quaternary eutectic solvent (choline chloride: citric acid: proline: EDTA-2Na, molar ratio 1:1:1:0.4-0.5) within the scope of the present application is selected, the purity of the pectin extract is ≥92.5%, and the recovery rate of the pectin is ≥88.3%; while in Comparative Example 9, the binary solvent (choline chloride: citric acid) is used, and in Comparative Example 10, the ternary solvent (choline chloride: citric acid: proline) is used, the purity of the pectin extract is only 81.5% and 87.6% respectively, and the recovery rate of the pectin is 70.8% and 82.1% respectively. This shows that the quaternary eutectic solvent can more effectively destroy the cell wall structure and improve the solubility and extraction rate of pectin.

[0260] The addition of the auxiliary agent and the process parameters generally affect the purity and biological activity of the extract. As can be seen from Example 1, Comparative Examples 6-8, Comparative Examples 11-12, when the addition amount of polyethylene glycol and L-ascorbic acid and the temperature and pressure conditions are adjusted within the scope of the present application, the extract has a higher purity and recovery rate; while in Comparative Example 6, no eutectic solvent is added, in Comparative Example 7, no polyethylene glycol is added, in Comparative Example 8, no L-ascorbic acid is added, and in Comparative Examples 11 and 12, the temperature and pressure are not within the scope, the purity and recovery rate of the pectin extract are significantly reduced. This shows that the synergistic effect of the addition of the auxiliary agent and the high temperature and pressure conditions can maintain the integrity of the pectin structure and improve the extraction efficiency.

[0261] The above examples are the preferred implementation schemes of the present application, in addition to this, the present application can also be implemented in other ways, without departing from the concept of the present application, any obvious replacement within the scope of the present application.

Claims

1. A guava peel extract, characterized in that, The passion fruit peel extract includes flavonoid extract and pectin extract; The flavonoid extract is extracted from the passion fruit peel by using enzymatic hydrolysis, and peel residue is obtained; The pectin extract is extracted from the peel residue by mixing the peel residue, water and eutectic solvent under the conditions of 140-150 DEG C and 2-3 MPa.

2. The guava peel extract according to claim 1, characterized in that, The purity of the flavonoid extract is greater than or equal to 85%; The purity of the pectin extract is greater than or equal to 90%.

3. The guava peel extract according to claim 1, characterized in that, The enzymes used in the enzymatic hydrolysis include cellulase and hemicellulase, and the added mass of the cellulase and the hemicellulase is 0.3-0.4% and 0.1-0.2% of the mass of the dried passion fruit peel respectively.

4. The guava peel extract according to claim 1, characterized in that, The mass ratio of the dried peel residue, eutectic solvent and water is (70-80):(140-160):(1400-1600); The eutectic solvent includes choline chloride, citric acid, proline and EDTA-2Na, and the molar ratio of choline chloride, citric acid, proline and EDTA-2Na is 1:1:1:0.4-0.

5.

5. The guava peel extract according to claim 1, characterized in that, The first and second additives are added in the extraction process of the pectin; The first additive is polyethylene glycol, and the added mass of the polyethylene glycol is 1.0-1.5% of the mass of the dried peel residue; The second additive is L-ascorbic acid, and the added mass of the L-ascorbic acid is 0.08-0.1% of the mass of the dried peel residue.

6. The guava peel extract according to claim 1, characterized in that, The passion fruit peel extract is applied in the fields of food and medicine.

7. The extraction process of guava peel extract according to claim 1, characterized in that, The method includes the following steps: S1. pretreating passion fruit peel to obtain passion fruit peel powder; S2. enzymatically hydrolyzing the passion fruit peel powder to obtain supernatant and peel residue; S3. purifying the flavonoid extract from the supernatant; S4. mixing the peel residue, water and eutectic solvent, and extracting the pectin extract from the peel residue under the conditions of 140-150 DEG C and 2-3 MPa.

8. The extraction process of claim 7, wherein, In step S2, passion fruit peel powder is weighed, citric acid-sodium citrate buffer solution with pH of 4.5 is added, and then cellulase and hemicellulase are added, and enzymatic hydrolysis is carried out at 45-50 DEG C for 120-150 min; after the enzymatic hydrolysis, inactivation treatment is carried out, centrifugal separation is carried out, the supernatant is collected, and the peel residue is reserved; 9. The extraction process of claim 7, wherein, In step S3, the supernatant is diluted to a flavonoid concentration of 1.0 mg / mL, elution is carried out through an AB-8 type macroporous resin column, eluate with absorbance of 280 nm of greater than or equal to 0.2 to less than or equal to 0.05 is collected, and the eluate is concentrated and dried to obtain flavonoid extract powder.

10. The extraction process of claim 7, wherein, In step S4, the specific preparation process is as follows: S41: in a reaction kettle, the dried peel residue, part of water, eutectic solvent and part of the first additive are mixed, and then mixed uniformly; the temperature is raised to 140-150 DEG C at a rate of 5 DEG C / min, and the pressure is raised to 2-3 MPa, and reaction is carried out under the conditions of 140-150 DEG C and 2-3 MPa for 60-70 min. S42: The remaining deionized water and the remaining first auxiliary agent are added to the reactor, the temperature is maintained at 140-150 DEG C, the pressure is maintained at 2-3 MPa, and the reaction is continued for 60-70 min; S43: The second auxiliary agent is added to the reactor, and the temperature is lowered to 100 DEG C, the pressure is maintained at 2-3 MPa, and the reaction is continued for 15-20 min; after the reaction is completed, the temperature is lowered to room temperature, and the pressure is released to normal pressure.

Images