Glucosyl hesperidin with effects of resisting aging and removing dark circles as well as preparation method and application of glucosyl hesperidin

Abstract

The invention relates to glucosyl hesperidin with anti-aging and dark eye removing effects and a preparation method and application thereof.The preparation method comprises the steps that orange peel and water are mixed and subjected to enzymolysis under first compound enzyme, and enzymatic hydrolysate is obtained; adding ethanol into the enzymatic hydrolysate, and sequentially carrying out reflux extraction and concentration to obtain a hesperidin concentrated solution; mixing the hesperidin concentrated solution with maltodextrin, and carrying out glucosylation reaction under a second compound enzyme to obtain a glucosyl hesperidin crude product; sequentially performing ultrafiltration, activated carbon first adsorption, cation exchange resin purification and activated carbon second adsorption on the glucosyl hesperidin crude product to obtain the glucosyl hesperidin. According to the preparation method of glucosyl hesperidin provided by the invention, hesperidin is extracted and modified under the action of multiple complex enzymes, and meanwhile, a multiple purification process is combined, so that the solubility and stability of hesperidin are improved, the yield and purity of glucosyl hesperidin are improved, the biological activity is retained, and the glucosyl hesperidin can be directly applied to cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic raw materials, and in particular to glucosyl hesperidin having anti-aging and dark circle removing effects, and a preparation method and application thereof. Background Art

[0002] Hesperidin is a glycoside formed by hesperidin and rutin. It is a dihydroflavonoid derivative and is widely found in plants of the Leguminosae, Lamiaceae, Scutellariae, and Rutaceae families. Hesperidin has the effects of anti-oxidation, strengthening capillaries, improving vascular permeability, and regulating cholesterol. It has certain applications in the fields of medicine, food, and daily chemical industry.

[0003] Although hesperidin has good biological activity, its water solubility and fat solubility are poor, which limits its efficacy and application range. Through the modification of hesperidin, its water solubility, absorbability and drug efficacy have been improved to a certain extent, expanding its application prospects in the fields of food, medicine and daily chemicals. For example, the glycosylation modification product of hesperidin, glucosyl hesperidin, can be used as a high-end cosmetic raw material in skin care products such as face cream, eye cream, toner, etc. due to its good water solubility and biological activity, and has high economic value.

[0004] CN113264965A discloses a method for preparing hesperidin from orange peel, comprising the following steps: spraying fresh orange peel with lactic acid bacteria liquid evenly for fermentation for 12-16 hours, soaking the orange peel in a weak alkaline aqueous solution for 4-6 hours, and then ultrasonic treatment; crushing the orange peel into 1-10 μm particles using a colloid mill and suspending them in an alkaline aqueous solution; adding a composite enzyme to the orange peel powder water suspension for enzymatic hydrolysis for 2-3 hours; distilling the enzymatically hydrolyzed orange peel water suspension for 2-4 hours under microwave conditions; separating the obtained liquid from oil and water and filtering, adjusting the pH of the aqueous phase to 4-6, filtering, washing, and drying under reduced pressure to obtain hesperidin. The above-mentioned method for preparing hesperidin has a complex process and does not involve modification of hesperidin.

[0005] Therefore, providing a method for preparing glucosyl hesperidin with anti-aging and dark circle removal effects, while improving the extraction efficiency of hesperidin and the synthesis efficiency of glucosyl hesperidin, and efficiently preparing high-purity and highly biologically active glucosyl hesperidin are technical problems that need to be solved urgently in the art. Summary of the invention

[0006] In order to solve the above technical problems, the present invention relates to a glucosyl hesperidin with anti-aging and dark circles removal effects, and a preparation method and application thereof. The preparation method of glucosyl hesperidin provided by the present invention extracts and modifies hesperidin under the action of multiple complex enzymes, and combines multiple purification processes at the same time, which not only improves the solubility and stability of hesperidin, but also increases the yield and purity of glucosyl hesperidin, and also retains its biological activity, so that it has excellent antioxidant properties, scavenges free radicals, improves dark circles and dull skin, reduces wrinkles, and anti-aging effects, and can be directly used in the cosmetics industry.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing glucosyl hesperidin, the preparation method comprising the following steps:

[0009] (1) mixing orange peel and water, and performing enzymolysis under a first composite enzyme to obtain an enzymolysis solution;

[0010] (2) adding ethanol to the enzymatic hydrolyzate, and sequentially performing reflux extraction and concentration to obtain a hesperidin concentrate;

[0011] (3) mixing the hesperidin concentrate and maltodextrin, and subjecting the mixture to a glucosylation reaction under the presence of a second complex enzyme to obtain a crude glucosyl hesperidin product;

[0012] (4) The crude glucosyl hesperidin is subjected to ultrafiltration, first adsorption on activated carbon, purification by cation exchange resin and second adsorption on activated carbon in sequence to obtain glucosyl hesperidin.

[0013] In step (1), the first composite enzyme is used for enzymatic hydrolysis, and multiple enzymes work together, which, combined with the reflux extraction in step (2), improves the extraction efficiency of hesperidin in orange peel.

[0014] In step (3), maltodextrin is used to modify hesperidin in the presence of a second complex enzyme, and multiple enzymes cooperate with each other to jointly promote the progress of the glucosylation reaction.

[0015] In step (4), multiple purification processes are adopted, wherein ultrafiltration removes large molecular weight impurities, activated carbon first adsorbs small molecular weight impurities; cation exchange resin separates, purifies and concentrates ions, and activated carbon secondly adsorbs to achieve decolorization. Multiple purification operations are performed layer by layer and are linked together to improve the yield and purity of glucosyl hesperidin.

[0016] The preparation method of glucosyl hesperidin provided by the present invention not only improves the extraction efficiency of hesperidin by introducing the first composite enzyme, but also modifies hesperidin under the action of the second composite enzyme, and combines multiple purification processes at the same time, not only improves the solubility and stability of hesperidin, but also improves the yield and purity of glucosyl hesperidin, and also retains its biological activity, so that it has excellent antioxidant properties, free radical scavenging, improving dark circles and dull skin, fading wrinkles, anti-aging and other effects, and can be directly used in the cosmetics industry.

[0017] Preferably, in step (1), the orange peel is orange peel powder obtained by drying, sterilizing and crushing.

[0018] Preferably, in step (1), the first complex enzyme comprises a combination of cellobiase, cellulase and protease.

[0019] In the present invention, cellulase hydrolyzes or degrades the components of the cell wall and impurities such as mucus, destroys the plant cell wall, exposes the effective components, dissolves them in the solvent, increases the solubility of the effective components, and thus extracts the effective components in the cell; cellobiase promotes cellulase hydrolysis, acts on the glucosidic bond and rhamnosidic bond in the structure of glycoside compounds, effectively cuts off the glycosidic bond and converts them into aglycone compounds; protease hydrolyzes protein molecules into polypeptides or amino acids, and has the effect of promoting fermentation and clarifying the liquid.

[0020] The invention improves the extraction efficiency of hesperidin by rationally compounding three enzymes.

[0021] Preferably, the mass ratio of cellobiase, cellulase and protease is 1:(0.5-1.5):(0.5-1.5).

[0022] When the composite enzyme in the present invention is within the above ratio range, the extraction effect of hesperidin is better.

[0023] The numerical value in the above (0.5-1.5) may be, for example, 0.6, 0.8, 1, 1.2, 1.4, etc.

[0024] Preferably, in step (1), the mass ratio of the orange peel to water is 1:(1.5-2.5), for example, it can be 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, etc.

[0025] Preferably, in step (1), the dosage of the first complex enzyme is 3.2-4.8% of the substrate weight, for example, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, etc.

[0026] Preferably, in step (1), the temperature of the enzymatic hydrolysis is 40-55°C (for example, it can be 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, etc.), and the time of the enzymatic hydrolysis is 60-84h (for example, it can be 64h, 68h, 72h, 76h, 80h, etc.).

[0027] Preferably, in step (2), the mass concentration of ethanol added to the system is 25-35%, for example, 26%, 28%, 30%, 32%, 34%, etc.

[0028] Preferably, in step (2), the reflux extraction time is 55-65 min (for example, it can be 56 min, 58 min, 60 min, 62 min, 64 min, etc.).

[0029] Preferably, in step (2), the concentration temperature is 95-105°C (for example, 96°C, 98°C, 100°C, 102°C, 104°C, etc.), and the concentration time is 1.2-1.8h (for example, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, etc.).

[0030] Preferably, in step (3), the second complex enzyme comprises a combination of sucrose phosphorylase and transglucosidase.

[0031] In the present invention, sucrose phosphorylase and transfer glucosidase are both indispensable and have a synergistic technical effect in promoting the glucosylation reaction.

[0032] Preferably, the mass ratio of sucrose phosphorylase to transglucosidase is 1:(0.5-1.5), for example, it can be 1:0.6, 1:0.8, 1:1, 1:1.2, 1:1.4, etc.

[0033] When the composite enzyme in the present invention is within the above ratio range, the effect of promoting the glucosylation reaction is better.

[0034] Preferably, in step (3), the mass ratio of the hesperidin concentrate to maltodextrin is 1:(0.16-0.28), for example, it can be 1:0.18, 1:0.2, 1:0.22, 1:0.24, 1:0.26, etc.

[0035] Preferably, in step (3), the dosage of the second complex enzyme is 1.5-2.5% of the substrate weight, for example, 1.6%, 1.8%, 2%, 2.2%, 2.4%, etc.

[0036] Preferably, in step (3), the pH of the glucosylation reaction is 6.5-7.5, for example, 6.6, 6.8, 7, 7.2, 7.4, etc.

[0037] Preferably, in step (3), the temperature of the glucosylation reaction is 50-55°C (for example, 51°C, 52°C, 53°C, 54°C, etc.), and the time of the glucosylation reaction is 23-25h (for example, 23.5h, 24h, 24.5h, etc.).

[0038] Preferably, in step (3), after the glucosylation reaction, the step further comprises: heating the reaction solution to inactivate the enzyme, allowing the reaction solution to stand and collecting the supernatant to obtain a crude glucosyl hesperidin.

[0039] Preferably, the heating temperature is 80-85°C (for example, 81°C, 82°C, 83°C, 84°C, etc.), and the heating time is 25-35min (for example, 26min, 28min, 30min, 32min, 34min, etc.).

[0040] Preferably, the standing time is 20-28 hours, for example, it can be 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, etc.

[0041] Preferably, in step (4), the filter membrane used in the ultrafiltration comprises a polyethersulfone filter membrane.

[0042] Preferably, in step (4), the molecular weight cut-off of the ultrafiltration is 8000-12000, for example, it can be 8500, 9000, 9500, 10000, 10500, 11000, 11500, etc.

[0043] Preferably, in step (4), in the first adsorption of activated carbon, the particle size of the activated carbon is 4.5-6 mm, for example, it can be 4.6 mm, 4.8 mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, etc.

[0044] Preferably, in step (4), in the first adsorption of the activated carbon, the adsorption time is 30-40 min, for example, it can be 32 min, 34 min, 35 min, 36 min, 38 min, etc.

[0045] Preferably, in step (4), in the first adsorption of the activated carbon, based on 100 mL of the system after ultrafiltration, the amount of the activated carbon used is 0.5-1 g, for example, 0.6 g, 0.7 g, 0.8 g, 0.9 g, etc.

[0046] Preferably, in step (4), the structure of the cation exchange resin comprises a sulfonic acid group-modified styrene divinylbenzene copolymer.

[0047] Preferably, in step (4), the eluent used for the cation exchange resin purification comprises water.

[0048] Preferably, in step (4), in the second adsorption of activated carbon, the particle size of the activated carbon is 1.5-2.5 mm, for example, it can be 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, etc.

[0049] Preferably, in step (4), in the second adsorption of the activated carbon, the adsorption time is 30-50 min, for example, it can be 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, etc.

[0050] Preferably, in step (4), in the second adsorption of the activated carbon, based on 100 mL of the system purified by the cation exchange resin, the amount of the activated carbon used is 2-3 g, for example, 2.2 g, 2.4 g, 2.5 g, 2.6 g, 2.8 g, etc.

[0051] Preferably, in step (4), the activated carbon further includes post-treatment after the second adsorption.

[0052] Preferably, the post-treatment comprises concentration and spray drying performed sequentially.

[0053] Preferably, the preparation method comprises the following steps:

[0054] (1) orange peel and water are mixed in a mass ratio of 1:(1.5-2.5), a first composite enzyme of 3.2-4.8% of the substrate mass is added, and enzymolysis is performed at 40-55° C. for 60-84 hours to obtain an enzymatic solution; the first composite enzyme comprises a combination of cellobiase, cellulase and protease in a mass ratio of 1:(0.5-1.5):(0.5-1.5);

[0055] (2) adding ethanol to the enzymatic hydrolyzate until the mass concentration of ethanol in the system is 25-35%, reflux extraction for 55-65 minutes, and then concentrating at 95-105° C. for 1.2-1.8 hours to obtain a hesperidin concentrate;

[0056] (3) mixing the hesperidin concentrate and maltodextrin in a mass ratio of 1:(0.16-0.28), adding a second composite enzyme of 1.5-2.5% of the substrate mass, and reacting at a pH of 6.5-7.5 and a temperature of 50-55° C. for 23-25 ​​hours to obtain a crude glucosyl hesperidin; the second composite enzyme comprises a combination of sucrose phosphorylase and transglucosidase in a mass ratio of 1:(0.5-1.5);

[0057] (4) ultrafiltration of the crude glucosyl hesperidin using a filter membrane with a molecular weight cutoff of 8000-12000; treating the filtrate with activated carbon having a particle size of 4.5-6 mm for 30-40 min to complete the first adsorption of the activated carbon; then purifying the filtrate with a styrene divinylbenzene copolymer having a structure modified with a sulfonic acid group; then treating the filtrate with activated carbon having a particle size of 1.5-2.5 mm for 30-50 min to complete the second adsorption of the activated carbon, then heating at 80-85° C. for 25-35 min to inactivate the enzyme, standing for 20-28 h, and taking the supernatant to obtain glucosyl hesperidin;

[0058] In the first activated carbon adsorption, the amount of the activated carbon is 0.5-1g based on 100mL of the system after ultrafiltration. In the second activated carbon adsorption, the amount of the activated carbon is 2-3g based on 100mL of the system after purification by the cation exchange resin.

[0059] In a second aspect, the present invention provides a glucosyl hesperidin, wherein the glucosyl hesperidin is prepared by the method for preparing the glucosyl hesperidin described in the first aspect.

[0060] In a third aspect, the present invention provides a use of the glucosyl hesperidin according to the second aspect in the preparation of cosmetics.

[0061] Preferably, the cosmetics include facial mask essence, facial mask, lotion, cream or toner.

[0062] Compared with the prior art, the present invention has at least the following beneficial effects:

[0063] The preparation method of glucosyl hesperidin provided by the present invention not only improves the extraction efficiency of hesperidin by introducing the first composite enzyme, but also modifies hesperidin under the action of the second composite enzyme, and combines multiple purification processes at the same time, not only improves the solubility and stability of hesperidin, but also improves the yield and purity of glucosyl hesperidin, and also retains its biological activity, so that it has excellent antioxidant properties, free radical scavenging, improving dark circles and dull skin, fading wrinkles, anti-aging and other effects, and can be directly used in the cosmetics industry. DETAILED DESCRIPTION

[0064] For the convenience of understanding the present invention, the present invention lists the following embodiments. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0065] The sources of materials used in the following examples are as follows:

[0066] Maltodextrin, DE value 15.3, purchased from Suixin;

[0067] The cellobiose enzyme model is SPE-007A and the brand is Xiasheng;

[0068] The cellulase model is CEL-01 and the brand is Xiasheng;

[0069] The protease product number is FDG-2202, and the brand is Xiasheng;

[0070] The product number of sucrose phosphorylase is A-PJ1164, and the brand is Fusheng Biotechnology;

[0071] The product number of the transfer glucosidase is 308, and the brand is Jiangsu Caiwei;

[0072] Activated carbon 1, particle size 6 mm, model BH0012, brand Boheng;

[0073] Activated carbon 2, particle size 2mm, model BH0012, brand Boheng.

[0074] Example 1

[0075] This embodiment provides a method for preparing glucosyl hesperidin, and the preparation method comprises the following steps:

[0076] (1) drying, sterilizing, and crushing orange peel to obtain orange peel powder; mixing the orange peel powder and water in a mass ratio of 1:2, adding a first complex enzyme (cellulobiase, cellulase, and protease in a mass ratio of 1:1:1) with a substrate mass ratio of 4%, and performing enzymolysis at a normal pressure of 50° C. for 72 hours, and continuously venting and releasing the pressure of the enzymolysis pot once a day to obtain an enzymolysis solution;

[0077] (2) adding 95% ethanol to the enzymatic hydrolyzate until the mass concentration of ethanol in the system is 30%, reflux extraction for 60 minutes, and then concentrating at 100° C. for 1.5 hours to obtain a hesperidin concentrate;

[0078] (3) The hesperidin concentrate and maltodextrin were mixed at a mass ratio of 1:0.2, and a second complex enzyme (sucrose phosphorylase and transglucosidase at a mass ratio of 1:1) of 2% of the substrate mass was added, and the mixture was reacted at a pH of 7 and a temperature of 52° C. for 24 h, and then heated at 82° C. for 30 min to inactivate the enzyme. The mixture was allowed to stand for 24 h and the supernatant was collected to obtain a crude glucosyl hesperidin;

[0079] (4) The crude glucosyl hesperidin was ultrafiltered using a PES membrane with a molecular weight cutoff of 10,000; the filtrate was treated with 0.7% (w / v) activated carbon 1 for 35 minutes to complete the first adsorption of activated carbon; then it was purified using a cation exchange resin HPR1200Na and eluted with deionized water; finally, it was treated with 2.5% (w / v) activated carbon 2 for 40 minutes to complete the second adsorption of activated carbon, and after concentration and spray drying, glucosyl hesperidin powder was obtained.

[0080] Example 2

[0081] This embodiment provides a method for preparing glucosyl hesperidin, and the preparation method comprises the following steps:

[0082] (1) drying, sterilizing, and crushing orange peel to obtain orange peel powder; mixing the orange peel powder and water in a mass ratio of 1:1.5, adding a first composite enzyme (cellulobiase, cellulase, and protease in a mass ratio of 1:0.5:1.5) with a substrate mass ratio of 3.2%, performing enzymolysis at a normal pressure of 40° C. for 84 hours, and continuously venting and releasing the pressure of the enzymolysis pot once a day to obtain an enzymolysis solution;

[0083] (2) adding 95% ethanol to the enzymatic hydrolyzate until the mass concentration of ethanol in the system is 25%, reflux extraction for 65 minutes, and then concentrating at 95° C. for 1.8 hours to obtain a hesperidin concentrate;

[0084] (3) The hesperidin concentrate and maltodextrin were mixed at a mass ratio of 1:0.16, and a second composite enzyme (sucrose phosphorylase and transglucosidase at a mass ratio of 1:0.5) of 1.5% of the substrate mass was added, and the mixture was reacted at a pH of 6.5 and a temperature of 50° C. for 25 h, and then heated at 80° C. for 35 min to inactivate the enzyme, and allowed to stand for 20 h, and the supernatant was collected to obtain a crude glucosyl hesperidin;

[0085] (4) The crude glucosyl hesperidin was ultrafiltered using a PES membrane with a molecular weight cutoff of 12,000; the filtrate was treated with 1% (w / v) activated carbon 1 for 30 min to complete the first adsorption of activated carbon; then purified with a cation exchange resin HPR1200Na and eluted with deionized water; finally, treated with 3% (w / v) activated carbon 2 for 50 min to complete the second adsorption of activated carbon, and after concentration and spray drying, glucosyl hesperidin powder was obtained.

[0086] Example 3

[0087] This embodiment provides a method for preparing glucosyl hesperidin, and the preparation method comprises the following steps:

[0088] (1) drying, sterilizing, and crushing orange peel to obtain orange peel powder; mixing the orange peel powder and water in a mass ratio of 1:2.5, adding a first composite enzyme (cellulobiase, cellulase, and protease in a mass ratio of 1:1:1) with a substrate mass of 4.8%, performing enzymolysis at a normal pressure of 55° C. for 60 hours, and continuously venting and releasing the pressure of the enzymolysis pot once a day to obtain an enzymolysis solution;

[0089] (2) adding 95% ethanol to the enzymatic hydrolyzate until the mass concentration of ethanol in the system is 35%, reflux extraction for 55 minutes, and then concentrating at 105° C. for 1.2 hours to obtain a hesperidin concentrate;

[0090] (3) The hesperidin concentrate and maltodextrin were mixed at a mass ratio of 1:0.28, and a second composite enzyme (sucrose phosphorylase and transglucosidase at a mass ratio of 1:1.5) of 2.5% of the substrate mass was added, and the mixture was reacted at a pH of 7.5 and a temperature of 55° C. for 23 h, and then heated at 85° C. for 25 min to inactivate the enzyme, and allowed to stand for 28 h, and the supernatant was collected to obtain a crude glucosyl hesperidin;

[0091] (4) The crude glucosyl hesperidin was ultrafiltered using a PES membrane with a molecular weight cutoff of 8000; the filtrate was treated with 0.5% (w / v) activated carbon 1 for 40 min to complete the first adsorption of activated carbon; then purified with a cation exchange resin HPR1200Na and eluted with deionized water; finally, treated with 2% (w / v) activated carbon 2 for 30 min to complete the second adsorption of activated carbon, and after concentration and spray drying, glucosyl hesperidin powder was obtained.

[0092] Example 4

[0093] This embodiment provides a method for preparing glucosyl hesperidin, which differs from Embodiment 1 only in that, in step (1), the first composite enzyme is a combination of cellobiase and cellulase in a mass ratio of 1:1, and other operations refer to Embodiment 1.

[0094] Example 5

[0095] This embodiment provides a method for preparing glucosyl hesperidin, which differs from Embodiment 1 only in that, in step (1), the first composite enzyme is a combination of cellobiase and protease in a mass ratio of 1:1, and other operations refer to Embodiment 1.

[0096] Example 6

[0097] This embodiment provides a method for preparing glucosyl hesperidin, which differs from Embodiment 1 only in that, in step (1), the first composite enzyme is a combination of cellulase and protease in a mass ratio of 1:1, and other operations refer to Embodiment 1.

[0098] Comparative Example 1

[0099] This comparative example provides a method for preparing glucosyl hesperidin, which differs from Example 1 only in that, in step (3), sucrose phosphorylase is not added, and its amount is allocated to transfer glucosidase, and other operations refer to Example 1.

[0100] Comparative Example 2

[0101] This comparative example provides a method for preparing glucosyl hesperidin, which differs from Example 1 only in that, in step (3), glucosidase is not added, and its amount is allocated to sucrose phosphorylase, and other operations refer to Example 1.

[0102] Comparative Example 3

[0103] This comparative example provides a method for preparing glucosyl hesperidin, which differs from Example 1 only in that ultrafiltration is not performed in step (4), and other operations refer to Example 1.

[0104] Comparative Example 4

[0105] This comparative example provides a method for preparing glucosyl hesperidin, which differs from Example 1 only in that the first adsorption of activated carbon is not performed in step (4), and other operations refer to Example 1.

[0106] Comparative Example 5

[0107] This comparative example provides a method for preparing glucosyl hesperidin, which differs from Example 1 only in that no cation exchange resin purification is performed in step (4), and other operations refer to Example 1.

[0108] Comparative Example 6

[0109] This comparative example provides a method for preparing glucosyl hesperidin, which differs from Example 1 only in that no second adsorption of activated carbon is performed in step (4), and other operations refer to Example 1.

[0110] Test Example 1

[0111] The extraction rates of hesperidin and the yields and purity of glucosyl hesperidin in Examples 1-6 and Comparative Examples 1-6 are shown in Table 1.

[0112] Table 1

[0113]

[0114] The test results show that:

[0115] (1) It can be seen from Examples 1 to 6 that the present invention uses a variety of composite enzymes to enzymatically hydrolyze orange peel, which not only reduces the molecular weight of hesperidin, making it more conducive to human absorption, but also improves the extraction efficiency of hesperidin; at the same time, the present invention uses composite enzymes not only to promote the progress of glucosylation reaction, but also combines with multiple purification procedures to jointly improve the yield and purity of the product.

[0116] (2) By comparing Example 1 with Examples 4-6, it can be seen that when any one of the enzymes is missing in the first composite enzyme, the extraction efficiency of the obtained hesperidin is reduced, and the yield and purity of the subsequent glucosylation reaction are affected.

[0117] (3) By comparing Example 1 with Comparative Examples 1-2, it can be seen that when one of the second complex enzymes is missing, the yield and purity of the obtained glucosyl hesperidin are both reduced; this indicates that the present invention has a synergistic technical effect in promoting the reaction by rationally compounding glucosidase and sucrose phosphorylase.

[0118] By comparing Example 1 with Comparative Examples 3-6, it can be seen that when any one of the steps in the purification process is missing, the yield and purity of the obtained glucosyl hesperidin are reduced, indicating that the multiple purification steps in the present invention are progressive, and have a synergistic technical effect in improving purity and yield.

[0119] Test Example 2

[0120] Antioxidant Test

[0121] Test method:

[0122] Prepare the sample into the test solution of the corresponding concentration, prepare the reaction system according to the instructions of the hydroxyl radical determination kit (Nanjing Jiancheng, A018-1-1), mix well, set up 3 replicates for each concentration, and use 0.3 mg / mL vitamin C as a positive control. Mix well, leave at room temperature for 20 minutes, take 200 μL from each tube and transfer to a 96-well plate, measure the absorbance value of each tube at a wavelength of 550 nm, and calculate the scavenging rate of the sample for hydroxyl free radicals according to the following formula:

[0123]

[0124] The product obtained by the preparation method provided in Example 1 was tested, and the results are shown in Table 2.

[0125] Table 2

[0126]

[0127]

[0128] Conclusion: The sample glucosyl hesperidin has excellent scavenging ability for hydroxyl free radicals at a concentration of 2-5 mg / mL, and there is a statistical difference compared with the control group (p<0.01), indicating that the glucosyl hesperidin prepared by the specific preparation method of the present invention has excellent antioxidant capacity.

[0129] Test Example 3

[0130] Anti-aging efficacy test

[0131] Test method:

[0132] A blank control group, a positive drug group and a sample group (0.1 g / L) were set up respectively. When testing the relative expression of type I collagen and elastin in zebrafish, acetyl hexapeptide-8 (0.1 g / L) was used in the positive drug group, and glutathione-reduced (GSH) (0.1 g / L) was used in the positive drug group when testing the activity of zebrafish SOD. Three biological replicates were set up in each group, and 10 embryos were set up in each replicate. 5.0 mL of the working solution corresponding to each concentration group was added to each well; each test group was placed in a constant temperature incubator at 28.5±0.5℃ and incubated for 24 hours; the embryos of each group were collected, and the total protein was extracted by low-temperature mechanical grinding, and the total protein was quantified using the BCA method; the superoxide dismutase (SOD) activity was determined by the xanthine oxidase method, and the absorbance of each group was determined at 550 nm using an enzyme marker, and the total SOD activity of each group was calculated, and the blank control group, the positive drug group and the sample group were tested for significant differences.

[0133] The product obtained by the preparation method provided in Example 1 was tested. The relative expression results of type I collagen in each group of zebrafish are shown in Table 3, the relative expression results of elastin in each group of zebrafish are shown in Table 4, and the statistical results of SOD activity in each group of zebrafish are shown in Table 5.

[0134] Table 3

[0135] Group project Relative expression Standard Deviation Significance (p) Promotion rate (%) Blank control group Col1a1a 1.0022 0.0820 / / Blank control group Col1a1b 1.0038 0.1061 / / Blank control group Col1a2 1.0042 0.1099 / / Positive drug group Col1a1a 1.5798 0.2557 0.0204 57.64 Positive drug group Col1a1b 1.5223 0.2510 0.0300 51.66 Positive drug group Col1a2 1.5791 0.1344 0.0046 57.25 Sample set Col1a1a 1.8556 0.1171 0.0005 85.16 Sample set Col1a1b 2.8305 0.3071 0.0006 181.99 Sample set Col1a2 1.2231 0.0721 0.0448 21.80

[0136] Table 4

[0137] Group project Relative expression Standard Deviation Significance (p) Promotion rate (%) Blank control group Elastin 1 1.0098 0.1665 / / Positive drug group Elastin 1 1.7393 0.0670 0.0021 72.25 Sample set Elastin 1 1.3240 0.0750 0.0407 31.12

[0138] Table 5

[0139]

[0140]

[0141] Conclusion: Under the above experimental conditions, the glucosyl hesperidin prepared by the preparation method provided by the present invention can significantly promote the expression of type I collagen and elastin genes in zebrafish (p<0.05), and can significantly increase the SOD activity in zebrafish embryos (p<0.05), and has excellent anti-aging effect.

[0142] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing glucosyl hesperidin with anti-aging and dark circle removing effects, characterized in that: The preparation method comprises the following steps: (1) mixing orange peel and water, and performing enzymolysis under a first composite enzyme to obtain an enzymolysis solution; (2) adding ethanol to the enzymatic hydrolyzate, and sequentially performing reflux extraction and concentration to obtain a hesperidin concentrate; (3) mixing the hesperidin concentrate and maltodextrin, and subjecting the mixture to a glucosylation reaction under the presence of a second complex enzyme to obtain a crude glucosyl hesperidin product; (4) The crude glucosyl hesperidin is subjected to ultrafiltration, first adsorption on activated carbon, purification by cation exchange resin and second adsorption on activated carbon in sequence to obtain glucosyl hesperidin.

2. The preparation method according to claim 1, characterized in that: In step (1), the first complex enzyme comprises a combination of cellobiase, cellulase and protease; Preferably, the mass ratio of cellobiase, cellulase and protease is 1:(0.5-1.5):(0.5-1.5).

3. The preparation method according to claim 1 or 2, characterized in that: In step (1), the mass ratio of the orange peel to water is 1:(1.5-2.5); Preferably, in step (1), the amount of the first complex enzyme is 3.2-4.8% of the substrate mass; Preferably, in step (1), the temperature of the enzymatic hydrolysis is 40-55°C, and the time of the enzymatic hydrolysis is 60-84h.

4. The preparation method according to any one of claims 1 to 3, characterized in that In step (2), the mass concentration of ethanol added to the system is 25-35%; Preferably, in step (2), the reflux extraction time is 55-65 min; Preferably, in step (2), the concentration temperature is 95-105° C., and the concentration time is 1.2-1.8 h.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In step (3), the second complex enzyme comprises a combination of sucrose phosphorylase and transglucosidase; Preferably, the mass ratio of sucrose phosphorylase to transglucosidase is 1:(0.5-1.5).

6. The preparation method according to any one of claims 1 to 5, characterized in that: In step (3), the mass ratio of the hesperidin concentrate to maltodextrin is 1:(0.16-0.28); Preferably, in step (3), the amount of the second complex enzyme is 1.5-2.5% of the substrate mass; Preferably, in step (3), the pH of the glucosylation reaction is 6.5-7.5; Preferably, in step (3), the temperature of the glucosylation reaction is 50-55° C., and the time of the glucosylation reaction is 23-25 ​​h.

7. The preparation method according to any one of claims 1 to 6, characterized in that: In step (4), the filter membrane used in the ultrafiltration comprises a polyethersulfone filter membrane; Preferably, in step (4), the molecular weight cut-off of the ultrafiltration is 8000-12000; Preferably, in step (4), in the first adsorption of activated carbon, the particle size of the activated carbon is 4.5-6 mm; Preferably, in step (4), the first adsorption of the activated carbon takes place for 30-40 minutes; Preferably, in step (4), in the first adsorption of the activated carbon, the amount of the activated carbon used is 0.5-1 g based on 100 mL of the system after ultrafiltration; Preferably, in step (4), the structure of the cation exchange resin comprises a sulfonic acid group-modified styrene divinyl benzene copolymer; Preferably, in step (4), the eluent used for the cation exchange resin purification comprises water; Preferably, in step (4), in the second adsorption of activated carbon, the particle size of the activated carbon is 1.5-2.5 mm; Preferably, in step (4), the second adsorption of the activated carbon has an adsorption time of 30-50 min; Preferably, in step (4), in the second adsorption of the activated carbon, the amount of the activated carbon used is 2-3 g based on 100 mL of the system purified by the cation exchange resin; Preferably, in step (4), the activated carbon further comprises post-treatment after the second adsorption; Preferably, the post-treatment comprises concentration and spray drying performed sequentially.

8. The preparation method according to any one of claims 1 to 7, characterized in that: The preparation method comprises the following steps: (1) orange peel and water are mixed in a mass ratio of 1:(1.5-2.5), a first composite enzyme of 3.2-4.8% of the substrate mass is added, and enzymolysis is performed at 40-55° C. for 60-84 hours to obtain an enzymatic solution; the first composite enzyme comprises a combination of cellobiase, cellulase and protease in a mass ratio of 1:(0.5-1.5):(0.5-1.5); (2) adding ethanol to the enzymatic hydrolyzate until the mass concentration of ethanol in the system is 25-35%, reflux extraction for 55-65 minutes, and then concentrating at 95-105° C. for 1.2-1.8 hours to obtain a hesperidin concentrate; (3) mixing the hesperidin concentrate and maltodextrin in a mass ratio of 1:(0.16-0.28), adding a second composite enzyme of 1.5-2.5% of the substrate mass, and reacting at a pH of 6.5-7.5 and a temperature of 50-55° C. for 23-25 ​​hours to obtain a crude glucosyl hesperidin; the second composite enzyme comprises a combination of sucrose phosphorylase and transglucosidase in a mass ratio of 1:(0.5-1.5); (4) ultrafiltration of the crude glucosyl hesperidin using a filter membrane with a molecular weight cutoff of 8000-12000; treating the filtrate with activated carbon having a particle size of 4.5-6 mm for 30-40 min to complete the first adsorption of the activated carbon; then purifying the crude glucosyl hesperidin using a cation exchange resin having a structure including a sulfonic acid group-modified styrene divinylbenzene copolymer; and then treating the crude glucosyl hesperidin with activated carbon having a particle size of 1.5-2.5 mm for 30-50 min to complete the second adsorption of the activated carbon to obtain glucosyl hesperidin; In the first activated carbon adsorption, the amount of the activated carbon is 0.5-1g based on 100mL of the system after ultrafiltration. In the second activated carbon adsorption, the amount of the activated carbon is 2-3g based on 100mL of the system after purification by the cation exchange resin.

9. A glucosyl hesperidin having anti-aging and dark circles removing effects, characterized in that: The glucosyl hesperidin is prepared by the method for preparing glucosyl hesperidin with anti-aging and dark circle removing effects according to any one of claims 1 to 8.

10. Use of the glucosyl hesperidin with anti-aging and dark circle removal effects according to claim 9 in the preparation of cosmetics; Preferably, the cosmetics include facial mask essence, facial mask, lotion, cream or toner.

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