DHA-nobiletin-EGCG (epigallocatechin gallate) loaded pectin-based gel soft sweets as well as preparation method and application thereof

Abstract

The invention discloses DHA-nobiletin-EGCG-loaded pectin-based gel soft sweets and a preparation method and application thereof.The preparation method comprises the following steps that DHA-nobiletin-EGCG and calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel are prepared and mixed, high-speed shearing and emulsifying are conducted under the ice bath condition, and the DHA-nobiletin-EGCG-loaded pectin-based gel soft sweets are obtained; the DHA-nobiletin-EGCG (epigallocatechin gallate) loaded emulsion gel is prepared; and adding glycosyl-pectin-gelatin mixed gel, pouring into a mold, and drying to obtain the DHA-nobiletin-EGCG loaded pectin-based gel soft sweets. The DHA-nobiletin-EGCG loaded pectin-based gel soft sweets disclosed by the invention are uniform and stable in texture, have excellent gel strength, elasticity and chewiness, not only can improve the bioavailability of DHA, but also can realize slow release and targeted delivery of EGCG and nobiletin in intestinal tracts.

Description

Technical Field

[0001] The invention belongs to the technical field of functional foods, and particularly relates to a DHA-nobiletin-EGCG-loaded pectin-based jelly candy and a preparation method and application thereof. Background Art

[0002] The research and development of functional foods has received widespread attention. Functional gummies, as an innovative food that combines nutritional value with a convenient form of consumption, have gradually become the focus of consumers and the food industry. Docosahexaenoic acid (DHA), as an essential polyunsaturated fatty acid for the human body, has attracted much attention for its outstanding role in promoting the development of the nervous system, improving memory, maintaining cardiovascular health, and anti-inflammation. However, due to the presence of multiple unsaturated bonds in the molecular structure of DHA, it is very easy to undergo oxidative degradation during processing and storage, resulting in a significant decrease in its biological activity. In addition, DHA's low water solubility and poor digestion and absorption efficiency further limit its application in food. Therefore, the development of a delivery system that can effectively improve the stability and bioavailability of DHA is an important topic in the current field of food science.

[0003] Nobiletin, a polymethoxylated flavonoid found abundantly in citrus peel, has attracted considerable attention for its bioactivities, including anti-inflammatory, anti-diabetic, lipid metabolism regulation, and colon cancer prevention. Studies have shown that nobiletin can alleviate dextran sulfate sodium (DSS)-induced intestinal barrier damage by regulating claudins and inflammatory factors. However, the presence of multiple methoxy groups in its structure contributes to its poor water solubility and high crystallinity. This makes nobiletin susceptible to degradation under extreme pH conditions and chemical stress in the gastrointestinal tract, resulting in reduced bioactivity and oral bioavailability. Epigallocatechin gallate (EGCG), a hydrophilic polyphenol-based material, is abundant in green tea extract. EGCG, containing multiple bioactive groups and numerous phenolic hydroxyl groups, possesses potent antioxidant and anticancer properties and promotes interaction with various materials. The pyrogallol component of EGCG can bind to nobiletin, inhibiting its crystallization and thereby improving its solubility and bioavailability. However, nobiletin and EGCG are susceptible to oxidation and degradation, and their low stability affects their efficacy in the colon. Therefore, in order to promote the synergistic improvement of the intestinal barrier by nobiletin and EGCG, it is necessary to develop a stable delivery vehicle to effectively encapsulate these two compounds.

[0004] Pectin, a heteropolysaccharide, possesses excellent emulsifying and beneficial properties. It is indigestible by enzymes in the stomach and small intestine, but can be degraded and utilized by enzymes produced by the intestinal flora in the colon, making it an ideal carrier material for colon-targeted delivery of nobiletin. Pectin is highly hydrophilic, but as a sole encapsulation material, it has a low capacity for encapsulating hydrophobic functional factors. Pea protein, a high-quality plant protein, offers a rich amino acid profile and excellent nutritional value, making it an ideal alternative to animal protein, particularly for vegetarians and those allergic to animal protein. However, due to the thermodynamically unstable colloidal structure of pea protein emulsions, the droplet membrane is susceptible to pH and temperature fluctuations, leading to emulsion delamination. Furthermore, pea protein's low solubility further limits its emulsifying ability and application range, resulting in certain limitations in the functional properties of pea protein in food applications. Therefore, improving the solubility and emulsion stability of pea protein has become a critical issue that needs to be addressed in the food industry. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a DHA-nobiletin-EGCG loaded pectin-based jelly candy and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0007] A method for preparing DHA-nobiletin-EGCG-loaded pectin-based jelly candy comprises the following steps:

[0008] (1) Preparation of DHA-nobiletin-EGCG;

[0009] The nobiletin solution and the epigallocatechin gallate solution were mixed, dialyzed, and freeze-dried to obtain a nobiletin-EGCG combination; docosahexaenoic acid was added and mixed to obtain DHA-nobiletin-EGCG;

[0010] (2) preparing calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel;

[0011] (2.1) mixing the low-ester pectin solution and the pea protein solution to obtain a low-ester pectin-pea protein mixed solution;

[0012] (2.2) adjusting the pH value of the low-ester pectin-pea protein mixed solution obtained in step (2.1) to 9-10, reacting under ultrasonic and heating conditions, and after the reaction is completed, adjusting the pH value to 7-8, centrifuging and dialysis to obtain a low-ester pectin-pea protein Maillard conjugate;

[0013] (2.3) dissolving the low-ester pectin-pea protein Maillard conjugate obtained in step (2.2) in water, adjusting the pH to 8-10, and adding calcium chloride solution under stirring to obtain a calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel;

[0014] (3) Preparation of DHA-nobiletin-EGCG loaded emulsion gel;

[0015] The DHA-nobiletin-EGCG obtained in step (1) and the calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel obtained in step (2.3) were mixed, and high-speed shear emulsification was performed under ice bath conditions to obtain a DHA-nobiletin-EGCG-loaded emulsion gel;

[0016] (4) preparing a glycosyl-pectin-gelatin mixed gel;

[0017] Dissolve pectin and gelatin in water and stir until a transparent gel is obtained to obtain a pectin-gelatin mixed gel; add sugar base and citric acid and stir again to obtain a sugar base-pectin-gelatin mixed gel;

[0018] (5) preparing DHA-nobiletin-EGCG loaded pectin-based jelly candies;

[0019] The DHA-nobiletin-EGCG loaded emulsion gel obtained in step (3) and the saccharide-pectin-gelatin mixed gel obtained in step (4) are mixed, molded, and dried to obtain the DHA-nobiletin-EGCG loaded pectin-based gel soft candy.

[0020] The above preparation method, preferably, in step (1), the mass ratio of the nobiletin solution to the epigallocatechin gallate solution is 1:1-5, the mass fraction of nobiletin in the nobiletin solution is 0.5%-1%, the mass fraction of epigallocatechin gallate in the epigallocatechin gallate solution is 0.5%-1%, and the mass fraction of the nobiletin-EGCG combination in the DHA-nobiletin-EGCG is 9%-10%.

[0021] In the above preparation method, preferably, in step (2.1), the mass ratio of the low-ester pectin solution to the pea protein solution is 1:1-3, the mass fraction of low-ester pectin in the low-ester pectin solution is 2%-3%, and the mass fraction of pea protein in the pea protein solution is 2%-3%.

[0022] In the above preparation method, preferably, in step (3), the mass ratio of the DHA-nobiletin-EGCG to the calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel is 1:2-3.

[0023] In the above preparation method, preferably, in step (5), the mass fraction of the DHA-nobiletin-EGCG emulsion gel in the DHA-nobiletin-EGCG pectin-based jelly candy is 20% to 25%.

[0024] In the above preparation method, preferably, in step (2.2), the ultrasonic power is 600W to 650W, the heating temperature is 80°C to 85°C, the reaction time is 60min to 90min, and the dialysis temperature is 4°C.

[0025] In the above preparation method, preferably, in step (3), the rotation speed of the high-speed shear emulsification is 10000 rpm to 11000 rpm, the time of the high-speed shear emulsification is 1 min to 2 min, and the temperature of the ice bath is 0°C.

[0026] In the above preparation method, preferably, in step (1), the following treatment is further included before the mixing: adding water for dilution; the mixing is carried out under stirring conditions, and the stirring time is 60 minutes to 90 minutes;

[0027] In step (2.1), the preparation process of the low-ester pectin solution is as follows: adjusting the pH value of the pectin solution to 12 with an alkaline compound, reacting, and then adjusting the pH value of the reaction system to 7 with an acidic solution, dialyzing, and freeze-drying to obtain low-ester pectin, and then dissolving the low-ester pectin in water, and hydrating under stirring to obtain a low-ester pectin solution; the mass fraction of the pectin solution is 1% to 2%, the alkaline compound is sodium hydroxide, the acidic solution is hydrochloric acid, the reaction time is 20 minutes, the dialysis time is 72 hours, and the hydration time is 10 hours to 24 hours; the preparation process of the pea protein solution is as follows: dissolving pea protein in water to obtain a pea protein solution;

[0028] In step (2.3), the low-ester pectin-pea protein Maillard conjugate is dissolved in water to form a low-ester pectin-pea protein Maillard conjugate solution, wherein the mass fraction of the low-ester pectin-pea protein Maillard conjugate solution is 2% to 3%; the mass ratio of the low-ester pectin-pea protein Maillard conjugate solution to the calcium chloride solution is 30 to 40:1, and the concentration of calcium ions in the calcium chloride solution is 10 mM to 30 mM;

[0029] In step (4), the mass ratio of pectin to gelatin in the pectin-gelatin mixed gel is 1:6-10, the mass fraction of citric acid in the saccharide-pectin-gelatin mixed gel is 0.08%-0.09%, and the mass ratio of the pectin-gelatin mixed gel to the saccharide is 1:2-3; the stirring temperature is 80°C-85°C, and the re-stirring temperature is 80°C-85°C; the preparation method of the saccharide is: mixing white sugar, corn syrup, orange powder and water, and stirring under heating conditions to obtain the saccharide; the mass fraction of white sugar in the saccharide is 4.5%-5%, the mass fraction of corn syrup in the saccharide is 40%-41%, the mass fraction of orange powder in the saccharide is 13%-13.5%, and the heating temperature is 80°C-85°C;

[0030] In step (5), the mixing temperature is 70°C to 75°C, the mixing is carried out under stirring conditions, the drying temperature is 40°C to 45°C, the drying time is 32h to 35h, and the drying method is baking.

[0031] As a general technical concept, the present invention also provides a DHA-nobiletin-EGCG loaded pectin-based jelly candy prepared by the above-mentioned preparation method.

[0032] As a general technical concept, the present invention also provides an application of the above-mentioned DHA-nobiletin-EGCG loaded pectin-based gel candy in health-care candies.

[0033] Compared with the prior art, the advantages of the present invention are:

[0034] (1) The present invention provides a method for preparing DHA-nobiletin-EGCG loaded pectin-based gel soft candy, comprising the steps of: first preparing DHA-nobiletin-EGCG and a calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel, mixing the two, and emulsifying them at high speed shearing under ice bath conditions to obtain a DHA-nobiletin-EGCG loaded emulsion gel; then adding a sugar-pectin-gelatin mixed gel, casting and drying the mixture, and obtaining a DHA-nobiletin-EGCG loaded pectin-based gel soft candy. The preparation method of the present invention comprises the following steps: first, adding dietary polyphenols (nobiletin and EGCG) to DHA not only provides a better taste experience, but also greatly enhances its antioxidant and antibacterial functions, thereby achieving a balance between taste and function; second, using calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel as a carrier, significantly improving the drug loading of nobiletin and EGCG through its excellent gelling properties and hydrophilicity; at the same time, the carrier can maintain structural stability in the gastrointestinal tract, better protect the active ingredients (DHA, nobiletin, EGCG) therein from damage by the external environment, thereby achieving the effect of improving bioavailability and extending the shelf life of the product; third, DHA-nobiletin-EGCG and calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel are placed in an ice bath. High-speed shear emulsification under certain conditions forms a structurally stable emulsion gel, which can realize nanoemulsion encapsulation of DHA, avoids the damage of high temperature to DHA and other active ingredients as much as possible, retains the biological activity and functionality of DHA, nobiletin and EGCG to the greatest extent, solves the problems of DHA being easily oxidized, easily volatile and having poor stability under light and high temperature conditions, achieves the effect of improving the photothermal stability and sustained-release performance of DHA, thereby extending its shelf life in food, and effectively improving the bioavailability of DHA, ensuring the absorption of its nutrients in the gastrointestinal tract; fourthly, adding a sugar-based-pectin-gelatin mixed gel to form a gel candy, presenting DHA in a more delicious way, achieving a wider market adaptability, and providing a more convenient and pleasant intake experience. The preparation method of the present invention has a simple process flow, is easy to control, is pollution-free, and has good industrialization prospects. The prepared DHA-nobiletin-EGCG-loaded pectin-based gel candy has a uniform and stable texture and excellent gel strength, elasticity, and chewiness. It can not only improve the bioavailability of DHA, but also achieve sustained release and targeted delivery of EGCG and nobiletin in the intestine, providing an important reference for the development of functional foods and drug delivery technologies.

[0035] (2) The preparation method of the present invention optimizes the mass ratio of nobiletin solution to epigallocatechin gallate solution to 1:1-5, thereby promoting the interaction between the two and forming stable self-assembled nanoparticles. Such nanoparticles have a higher Zeta potential, which significantly improves the stability of the particles. At the same time, they can be slowly released in the human body environment, further improving their bioavailability, thereby solving the problem of poor stability and delivery efficiency of nobiletin and EGCG when used alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The flowchart is a process for preparing the DHA-nobiletin-EGCG pectin-based jelly candy in Example 1 of the present invention.

[0037] Figure 2 This is a diagram showing the drug loading effects of NOB and EGCG in the DHA-nobiletin-EGCG loaded pectin-based jelly candies prepared in Example 1 of the present invention and the DHA-nobiletin-EGCG loaded pectin-based jelly candies prepared in Comparative Examples 1 to 4.

[0038] Figure 3 This is a graph showing the weight changes of mice after feeding the DHA-nobiletin-EGCG loaded pectin-based jelly candy prepared in Example 1 of the present invention.

[0039] Figure 4 This is a graph showing the spleen coefficient of mice after feeding with the DHA-nobiletin-EGCG-loaded pectin-based jelly candy prepared in Example 1 of the present invention.

[0040] Figure 5 This is a graph showing the colon length of mice after feeding with the DHA-nobiletin-EGCG-loaded pectin-based jelly candy prepared in Example 1 of the present invention.

[0041] Figure 6 These are the apparent morphological diagrams of the DHA-nobiletin-EGCG-loaded pectin-based jelly candy prepared in Example 1 of the present invention and the DHA-nobiletin-EGCG-loaded pectin-based jelly candy prepared in Comparative Example 5. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but the scope of protection of the present invention is not limited thereby. The materials and instruments used in the following examples are all commercially available.

[0043] (1) Determination of esterification degree: Weigh 1g of deesterified pectin sample and mix it with 100mL of acidic ethanol and stir continuously for 10min. Filter through a sand core funnel and rinse with anhydrous ethanol until there is no chloride ion. Add 20mL of anhydrous ethanol to wash and dry in a 65℃ oven. Weigh 100mg of treated pectin sample in a conical flask, add 2mL of ethanol to moisten it, add 100mL of boiled and cooled CO2-free water, add 5 drops of phenolphthalein, and titrate with a standardized 0.02mol / LNaOH solution. Record the volume of NaOH solution consumed as V1. Add 15mL of 0.1mol / LNaOH solution and shake for 15min. Then neutralize with 15mL of 0.1mol / LHCl solution and shake until the red color disappears. Add 3 drops of phenolphthalein and titrate with 0.02mol / LNaOH solution until it turns pink and does not fade within half a minute. Record the volume of NaOH solution consumed at this time as V2. Calculate the degree of esterification (DE) by the following formula.

[0044]

[0045] (2) Sample loading: First, a standard curve was drawn using pure nobiletin and EGCG. After centrifugation of the particles, 1 mL was diluted 8-fold and the absorbance was measured at 274 nm and 329 nm using a UV spectrophotometer (UV-1800, Shimadzu Instruments (Suzhou) Co., Ltd.).

[0046]

[0047] Among them, M o represents the total content of nobiletin / EGCG, M u Represents the mass of unencapsulated nobiletin / EGCG.

[0048] (3) Animal experiments:

[0049] According to the experimental design, 6-week-old male C57BL / 6J mice were randomly divided into 8 groups, namely, blank group (CK), model group (DSS), positive drug mesalazine group (5-ASA), nobiletin group (NOB), EGCG group, nobiletin-EGCG group (NOB-EGCG), DHA-nobiletin-EGCG loaded pectin-based jelly candy group (Example 1), and vehicle group (low-ester pectin-pea protein, i.e., low-ester pectin-pea protein Maillard conjugate), with 10 mice in each group (n=10).

[0050] Experimental Plan: The purchased mice were acclimated for one week before the formal experiment. The first week was the acclimation period. The second and third weeks were the experimental period. The experimental period was from day 0 to day 14, and the DSS model was established from day 8 to day 14. Mice in the blank group received normal diet and water throughout the experimental period (0-14 days) and were gavaged with 200 μL of normal saline as a control; mice in the modeling group drank 2.5% DSS solution starting on day 8 and continuing for 7 consecutive days until the end of the modeling, and were gavaged with 200 μL of normal saline throughout the experiment as a control; mice in the polysaccharide group were gavaged with 200 μL of polysaccharide at a concentration of 100 mg / mL starting 7 days before modeling and drank 2.5% DSS solution starting on day 8; mice in the positive drug group were gavaged with 200 μL of mesalamine at a concentration of 10 mg / mL starting 7 days before modeling and drank 3% DSS solution starting on day 8; mice in the polysaccharide treatment group were gavaged with 200 μL of mesalamine starting 7 days before modeling and drank 3% DSS solution starting on day 8. After standing at room temperature for 1-2 hours, the whole blood of the mice was centrifuged at 1500 rpm for 10 minutes, and the supernatant was aliquoted and stored in a -80°C refrigerator until use.

[0051] The inflammatory status was comprehensively assessed by spleen weighing and multi-level processing of colon tissue. First, the spleen was collected and weighed as an indicator of systemic inflammatory status. At the same time, colon tissue was collected and photographed, and the colon contents were collected and quickly frozen in liquid nitrogen for microbiome or metabolomics research. Subsequently, the colon tissue was rinsed with cold PBS and divided into two parts, one for transcriptomic analysis and the other for quick freezing in liquid nitrogen to measure inflammatory factors. In addition, 1 cm of colon tissue was cut from 1 cm away from the anus to the distal end to prepare representative tissue sections. The sliced ​​tissue was fixed with 4% paraformaldehyde for 24 hours and then used for histopathological observation.

[0052] (4) Appearance analysis: The DHA-nobiletin-EGCG loaded pectin-based jelly candies were placed at room temperature for 1 month, and their appearance changes were observed and photographed.

[0053] (5) Texture Analysis: The texture characteristics (height, hardness, brittleness, stickiness, elasticity, chewiness, adhesiveness, cohesiveness, and resilience) of the DHA-nobiletin-EGCG pectin-based jelly candies were determined using a TA-XT2i texture analyzer. The measurement parameters were as follows: a cylindrical probe with a diameter of 36 mm (P / 36R), a pre-test speed of 2 mm / s, a test speed of 2 mm / s, a post-test speed of 2 mm / s, a deformation of 30%, and a trigger force of 5 g. Four replicates were performed for each sample.

[0054] Example 1:

[0055] A method for preparing DHA-nobiletin-EGCG pectin-based jelly candy of the present invention, the process flow is as follows: Figure 1As shown, the following steps are included:

[0056] (1) Preparation of DHA-nobiletin-EGCG

[0057] According to the mass ratio of nobiletin solution to epigallocatechin gallate solution of 1:1, the mass fraction of nobiletin (NOB) solution is 0.5%, and the mass fraction of epigallocatechin gallate (EGCG) solution is 0.5%, the nobiletin solution and epigallocatechin gallate solution are mixed, diluted 5 times with distilled water, stirred for 1 hour, and after the solution turns slightly white, dialyzed and freeze-dried to obtain a nobiletin-EGCG assembly; docosahexaenoic acid (DHA) is added and mixed to obtain DHA-nobiletin-EGCG; the mass fraction of the nobiletin-EGCG assembly in DHA-nobiletin-EGCG is 9.5%.

[0058] (2) Preparation of calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel

[0059] (2.1) a) At room temperature (i.e., 25°C), adjust the pH of a pectin solution (the mass fraction of the solution is 1%) to 12 with NaOH, react for 20 minutes, and then adjust the pH of the reaction system to neutral (7) with hydrochloric acid. Dialyze for 72 hours and freeze-dry to obtain low-ester pectin. Dissolve the low-ester pectin in ultrapure water and stir overnight (i.e., 10 hours) at room temperature (i.e., 25°C) to hydrate and obtain a low-ester pectin solution.

[0060] b) dissolving pea protein in water to obtain a pea protein solution.

[0061] c) mixing the low-ester pectin solution and the pea protein solution in a mass ratio of 1:1, with the mass fraction of the low-ester pectin solution being 2% and the mass fraction of the pea protein solution being 2%, to obtain a low-ester pectin-pea protein mixed solution.

[0062] (2.2) The pH value of the low-ester pectin-pea protein mixed solution obtained in step (2.1) was adjusted to 10, and the reaction was carried out under conditions of ultrasonic power of 600 W and water bath temperature of 80° C. for 1 hour to obtain a mixture; the pH value of the mixture was adjusted to 7.0, and the mixture was centrifuged at 4° C. and 10,000 rpm for 15 minutes, and then dialyzed in a refrigerator at 4° C. for 3 days to obtain a powdered low-ester pectin-pea protein Maillard conjugate.

[0063] (2.3) Dissolving the low-ester pectin-pea protein Maillard conjugate obtained in step (2.2) in water to obtain a low-ester pectin-pea protein Maillard conjugate solution; adjusting the pH of the low-ester pectin-pea protein Maillard conjugate solution to 8-10, and adding a calcium chloride solution under stirring to obtain a calcium ion-crosslinked pectin-pea protein Maillard conjugate hydrogel. The mass ratio of the low-ester pectin-pea protein Maillard conjugate solution to the calcium chloride solution is 30:1, the mass fraction of the low-ester pectin-pea protein Maillard conjugate solution is 2%, and the concentration of calcium ions in the calcium chloride solution is 30 mM.

[0064] (3) Preparation of DHA-nobiletin-EGCG emulsion gel

[0065] According to the mass ratio of DHA-nobiletin-EGCG to the calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel of 1:3, the DHA-nobiletin-EGCG obtained in step (1) and the calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel obtained in step (2.3) were mixed, and high-speed shear emulsification was performed at a speed of 10000 rpm for 1 min under 0°C ice bath conditions to obtain a DHA-nobiletin-EGCG loaded emulsion gel.

[0066] (4) Preparation of saccharide-pectin-gelatin mixed gel

[0067] (4.1) White sugar, corn syrup, orange powder, and water are mixed and stirred at 85°C until fully dissolved to obtain a soft candy base. The mass fraction of white sugar, corn syrup, and orange powder in the soft candy base is 5%, 40%, and 13%, respectively.

[0068] (4.2) Pectin and gelatin were dissolved in water at a mass ratio of pectin to gelatin of 1:10, and the mixture was stirred continuously at 85°C until a transparent gel was obtained to obtain a pectin-gelatin mixed gel. The soft sugar base obtained in step (4.1) was quickly added to the pectin-gelatin mixed gel at a mass ratio of soft sugar base to pectin-gelatin mixed gel of 3:1, and the mixture was stirred at 85°C. Citric acid was then added, and the mixture was stirred again at 85°C to obtain a sugar base-pectin-gelatin mixed gel. The mass fraction of citric acid in the sugar base-pectin-gelatin mixed gel was 0.08%.

[0069] (5) Preparation of DHA-nobiletin-EGCG-loaded pectin-based jelly candies

[0070] The DHA-nobiletin-EGCG emulsion gel obtained in step (3) was added to the sugar-pectin-gelatin mixed gel obtained in step (4.2), stirred evenly at 75°C, immediately cast into a mold, and baked at 45°C for 32 hours to obtain DHA-nobiletin-EGCG-loaded pectin-based gel soft candy. The mass fraction of the DHA-nobiletin-EGCG-loaded emulsion gel in the DHA-nobiletin-EGCG-loaded pectin-based gel soft candy was 20%.

[0071] Comparative Example 1:

[0072] A DHA-nobiletin-EGCG loaded pectin-based jelly candy, the preparation process of which is substantially the same as the preparation method of the DHA-nobiletin-EGCG loaded pectin-based jelly candy in Example 1, with the only difference being that in step (1), the mass ratio of the nobiletin solution to the epigallocatechin gallate solution is 3:1.

[0073] Comparative Example 2:

[0074] A DHA-nobiletin-EGCG loaded pectin-based jelly candy, the preparation process of which is substantially the same as the preparation method of the DHA-nobiletin-EGCG loaded pectin-based jelly candy in Example 1, with the only difference being that in step (1), the mass ratio of the nobiletin solution to the epigallocatechin gallate solution is 2:1.

[0075] Comparative Example 3:

[0076] A DHA-nobiletin-EGCG-loaded pectin-based multifunctional jelly candy, the preparation process of which is basically the same as the preparation method of the DHA-nobiletin-EGCG-loaded pectin-based jelly candy in Example 1, with the only difference being that in step (2.1), a pectin solution (the mass fraction of the solution is 1%) is directly used to replace the low-ester pectin solution, and the original pectin is high-ester pectin.

[0077] Comparative Example 4:

[0078] A DHA-nobiletin-EGCG-loaded pectin-based multifunctional jelly candy, the preparation process of which is substantially the same as the preparation method of the DHA-nobiletin-EGCG-loaded pectin-based jelly candy in Example 1, except that in step (2.1), medium-ester pectin is used instead of low-ester pectin; the preparation method of the medium-ester pectin comprises the following steps: adjusting the pH value of a pectin solution (the mass fraction of the solution is 1%) to 11 with NaOH at room temperature, reacting for 20 minutes, then adjusting the pH value of the reaction system to neutral with hydrochloric acid, dialyzing for 72 hours, and freeze-drying to obtain low-ester pectin.

[0079] Comparative Example 5:

[0080] A DHA-nobiletin-EGCG loaded pectin-based multifunctional jelly candy, the preparation process of which is basically the same as the preparation method of the DHA-nobiletin-EGCG loaded pectin-based jelly candy in Example 1, with the only difference being that in step (4.2), the mass ratio of pectin to gelatin is 1:6.

[0081] (1) Drug loading

[0082] Figure 2 This is a diagram showing the drug loading effects of NOB and EGCG in the DHA-nobiletin-EGCG loaded pectin-based jelly candies prepared in Example 1 of the present invention and the DHA-nobiletin-EGCG loaded pectin-based jelly candies prepared in Comparative Examples 1 to 4. Figure 2 In the table, NOB-EE represents the drug loading of nobiletin, and EGCG-EE represents the drug loading of epigallocatechin gallate. Figure 2 It can be seen that compared with medium-ester pectin and high-ester pectin, the drug loading of the DHA-nobiletin-EGCG pectin-based jelly candy prepared with low-ester pectin is the highest, with nobiletin being 92.83% and EGCG being 90.56%. This indicates that low-ester pectin has better gelling properties and hydrophilicity, which can enhance the interaction with the active substance, thereby improving the drug loading efficiency. Compared with Comparative Examples 1 and 2, the drug loading of the DHA-nobiletin-EGCG pectin-based jelly candy prepared in Example 1 is the highest, with nobiletin being 92.22% and EGCG being 91.27%.

[0083] (2) Changes in mouse weight

[0084] Figure 3 The figure shows the weight change of mice after feeding with DHA-nobiletin-EGCG pectin-based jelly candy prepared in Example 1 of the present invention. Figure 3As can be seen, the weight of mice in the model group (DSS) continued to decrease during the experiment, with a decrease of 13.23%, indicating the severe impact of colitis on their physiological state. In contrast, the weight loss of mice in each treatment group was significantly reduced, with the positive drug mesalazine (5-ASA) group losing 12.99%, the nobiletin group (NOB) losing 13.54%, the EGCG group losing 13.02%, the nobiletin-EGCG group (NOB-EGCG) losing 13.86%, the DHA-nobiletin-EGCG pectin-based jelly candy group (Example 1) losing 11.69%, and the vehicle group (low-ester pectin-pea protein) losing 11.28%. The DHA-nobiletin-EGCG pectin-based jelly candy group (Example 1) and the vehicle group (low-ester pectin-pea protein) had the smallest weight loss, significantly outperforming the other groups and demonstrating a stronger protective effect. It can be seen that the low-ester pectin-pea protein Maillard conjugate carrier and the DHA-nobiletin-EGCG-loaded pectin-based gel candy can not only effectively alleviate the symptoms of colitis, but also improve the problem of weight loss in mice to a certain extent; at the same time, it verifies the advantages of low-ester pectin-pea protein as a delivery system, which can significantly improve the stability and bioaccessibility of active ingredients, and at the same time has an independent anti-inflammatory effect.

[0085] (3) Spleen coefficient

[0086] Figure 4 The spleen coefficient of mice after feeding with DHA-nobiletin-EGCG pectin-based jelly candy prepared in Example 1 of the present invention. Figure 4 It can be seen that after DSS-induced colitis, the organ coefficients of mice in the model group (DSS) increased significantly, with spleen enlargement being particularly pronounced, reaching 0.84%, indicating a significant effect of inflammation on the immune organs of mice. The spleen enlargement was alleviated to varying degrees in each treatment group. The spleen coefficients of the positive drug mesalazine group (5-ASA) were 0.78%, the nobiletin group (NOB) was 0.73%, the EGCG group was 0.65%, the nobiletin-EGCG group (NOB-EGCG) was 0.75%, the DHA-nobiletin-EGCG pectin-based gel candy group (Example 1) was 0.79%, and the vehicle group (low-ester pectin-pea protein) was 0.57%, all of which were significantly lower than those in the DSS model group. In particular, the spleen coefficient of the vehicle group (low-ester pectin-pea protein) decreased most significantly, indicating that the low-ester pectin-pea protein vehicle has a strong protective effect in alleviating inflammatory response and immune organ damage. In other words, the low-ester pectin-pea protein Maillard conjugate carrier not only protects the stability of the active ingredient, but also has independent anti-inflammatory potential, which can effectively reduce splenomegaly caused by colitis.

[0087] (4) Colon length

[0088] Figure 5 This is a graph showing the colon length of mice after feeding with the DHA-nobiletin-EGCG pectin-based jelly candy prepared in Example 1 of the present invention. Figure 5 As can be seen, after DSS-induced colitis, the colon length of the model group (DSS) mice was significantly shortened, decreasing from an average colon length of 6.59 cm in the normal blank group (CK) to 5.47 cm, indicating that severe colitis damage is a typical feature of DSS modeling. The colon length of the positive drug mesalazine (5-ASA) group was 4.16 cm after treatment, showing no significant relief. In the other treatment groups, the length of the colon was restored to varying degrees in the nobiletin group (NOB), the length of the EGCG group was 5.27 cm, the length of the nobiletin-EGCG group (NOB-EGCG), the length of the pectin-based gel candy loaded with DHA-nobiletin-EGCG (Example 1), and the length of the low-ester pectin-pea protein (i.e., low-ester pectin-pea protein Maillard conjugate) loaded with DHA-nobiletin-EGCG. The recovery effect of the nobiletin-EGCG group (NOB-EGCG) and the pectin-based gel candy loaded with DHA-nobiletin-EGCG (Example 1) was more significant, approaching that of the normal control group. In other words, the DHA-nobiletin-EGCG pectin-based gel candy loaded with the present invention significantly improved inflammatory damage to the colon through the synergistic effect of nobiletin and EGCG, as well as the encapsulation of the low-ester pectin-pea protein carrier, demonstrating excellent protection and repair capabilities.

[0089] (5) Surface properties of soft candy

[0090] Figure 6 The morphological diagrams of the DHA-nobiletin-EGCG pectin-based jelly candy prepared in Example 1 of the present invention and the DHA-nobiletin-EGCG pectin-based jelly candy prepared in Comparative Example 5 are shown. Figure 6 It can be seen that compared with Comparative Example 5, the DHA-nobiletin-EGCG pectin-based gel candy in Example 1 maintained a good appearance and taste during storage, without obvious water loss or collapse. This is attributed to the following: when the mass ratio of pectin to gelatin is 1:10, gelatin provides higher gel strength for the candy, preventing it from being too soft and prone to collapse. At the same time, through the synergistic effect of pectin and gelatin, the structural stability of the candy is enhanced, and its water retention performance is improved, thereby extending the shelf life of the candy.

[0091] Table 1 Texture characteristics of DHA-nobiletin-EGCG pectin-based jelly candies in Example 1 and Comparative Example 5

[0092]

[0093]

[0094] As can be seen from Table 1, compared with Comparative Example 5, the DHA-nobiletin-EGCG pectin-based gel soft candy prepared in Example 1 performs better in texture indicators such as hardness, elasticity and chewing feeling. The main reason is that the higher gelatin ratio effectively enhances the gel strength and elasticity of the soft candy, so that the soft candy will not be too hard while maintaining a high hardness, and the chewing feeling is relatively rich. Therefore, the DHA-nobiletin-EGCG pectin-based gel soft candy of the present invention improves the overall texture of the soft candy and provides a more ideal mouthfeel by optimizing the ratio of pectin and gelatin.

[0095] In summary, the preparation method of the DHA-nobiletin-EGCG loaded pectin-based jelly candy of the present invention has the following advantages: a) In view of the problems that the existing DHA jelly candy has a single taste and insufficient functionality, the present invention further adds dietary polyphenols (nobiletin and EGCG) to the jelly candy, optimizes the balance between taste and function, not only provides a better taste experience, but also greatly enhances its antioxidant and antibacterial functions; b) In view of the problems that nobiletin and EGCG have insufficient stability and poor delivery efficiency in food or drug delivery systems, by adjusting the ratio of nobiletin and EGCG, the interaction between the two is promoted to form stable self-assembled nanoparticles. The nanoparticles have a higher Zeta potential, which significantly improves the stability of the particles. At the same time, they can be slowly released in the human body environment, further improving their bioavailability, thereby solving the problem of poor stability and delivery efficiency when nobiletin and EGCG are used alone; c) Given that traditional pectin as a gelling agent is not stable enough when facing the extreme environment of the gastrointestinal tract, the present invention generates a low-ester pectin-pea protein Maillard conjugate through ultrasound to optimize the pectin structure, solving the problem of pectin instability in acidic environments and the gastrointestinal tract. At the same time, the low-ester pectin-pea protein Maillard conjugate has enhanced mechanical strength and acid resistance, which can significantly improve the mechanical properties, thermal stability and antioxidant properties of pectin. The pectin not only maintains structural stability in the gastrointestinal tract, but also better protects the active ingredients (DHA and dietary polyphenols) therein, thereby improving bioavailability and extending the shelf life of the product; d) In view of the low drug loading efficiency and unstable particle structure problems existing in the process of loading nobiletin and EGCG separately, the present invention adopts low-ester pectin-pea protein Maillard conjugate as a carrier, which significantly improves the drug loading amount of nobiletin and EGCG through its excellent gelling properties and hydrophilicity. At the same time, the low-ester pectin-pea protein Maillard conjugate carrier can not only effectively protect the active ingredients from damage by the external environment, but also has independent anti-inflammatory potential, further broadening its application in Application prospects in drug delivery systems; e) In view of the shortcomings of existing DHA products in terms of stability and functionality, the present invention adopts a mild preparation process of high-speed shear emulsification under ice bath conditions to encapsulate DHA in a nanoemulsion, thereby avoiding the damage of high temperature to DHA and other active ingredients (nobiletin, EGCG) as much as possible, retaining their biological activity and functionality to the greatest extent, solving the problems of DHA's easy oxidation, volatility, and poor stability under light and high temperature conditions, achieving the effect of improving the photothermal stability and sustained-release performance of DHA, thereby extending its shelf life in food, and effectively improving the bioavailability of DHA, ensuring the absorption of its nutrients in the gastrointestinal tract;f) Given the low consumer acceptance of existing DHA soft capsules and drops, particularly among children and those with high taste requirements, the present invention presents DHA in a more palatable form through the use of jelly candies, increasing the palatability and bioavailability of DHA intake, broadening DHA's application in the food industry, achieving broader market adaptability, and providing a more convenient and enjoyable intake experience, thus providing new research and development ideas for new health-related soft candies.

[0096] The above description is only a preferred embodiment of the present invention and does not constitute any formal limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing DHA-nobiletin-EGCG-loaded pectin-based jelly candy, characterized in that: The following steps are involved: (1) Preparation of DHA-nobiletin-EGCG; The nobiletin solution and the epigallocatechin gallate solution were mixed, dialyzed, and freeze-dried to obtain the nobiletin-EGCG combination; Docosahexaenoic acid was added and mixed to obtain DHA-nobiletin-EGCG; (2) preparing calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel; (2.1) mixing the low-ester pectin solution and the pea protein solution to obtain a low-ester pectin-pea protein mixed solution; (2.2) adjusting the pH value of the low-ester pectin-pea protein mixed solution obtained in step (2.1) to 9-10, reacting under ultrasonic and heating conditions, and after the reaction is completed, adjusting the pH value to 7-8, centrifuging and dialysis to obtain a low-ester pectin-pea protein Maillard conjugate; (2.3) dissolving the low-ester pectin-pea protein Maillard conjugate obtained in step (2.2) in water, adjusting the pH to 8-10, and adding calcium chloride solution under stirring to obtain a calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel; (3) Preparation of DHA-nobiletin-EGCG loaded emulsion gel; The DHA-nobiletin-EGCG obtained in step (1) and the calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel obtained in step (2.3) were mixed, and high-speed shear emulsification was performed under ice bath conditions to obtain a DHA-nobiletin-EGCG-loaded emulsion gel; (4) preparing a glycosyl-pectin-gelatin mixed gel; Dissolve pectin and gelatin in water and stir until a transparent gel is obtained to obtain a pectin-gelatin mixed gel; add sugar base and citric acid and stir again to obtain a sugar base-pectin-gelatin mixed gel; (5) preparing DHA-nobiletin-EGCG loaded pectin-based jelly candies; The DHA-nobiletin-EGCG loaded emulsion gel obtained in step (3) and the saccharide-pectin-gelatin mixed gel obtained in step (4) are mixed, molded, and dried to obtain the DHA-nobiletin-EGCG loaded pectin-based gel soft candy.

2. The preparation method of DHA-nobiletin-EGCG pectin-based jelly candy according to claim 1, wherein In step (1), the mass ratio of the nobiletin solution to the epigallocatechin gallate solution is 1:1-5, the mass fraction of nobiletin in the nobiletin solution is 0.5%-1%, the mass fraction of epigallocatechin gallate in the epigallocatechin gallate solution is 0.5%-1%, and the mass fraction of the nobiletin-EGCG assembly in the DHA-nobiletin-EGCG is 9%-10%.

3. The preparation method of DHA-nobiletin-EGCG pectin-based jelly candy according to claim 2, wherein In step (2.1), the mass ratio of the low-ester pectin solution to the pea protein solution is 1:1-3, the mass fraction of low-ester pectin in the low-ester pectin solution is 2%-3%, and the mass fraction of pea protein in the pea protein solution is 2%-3%.

4. The preparation method of DHA-nobiletin-EGCG pectin-based jelly candy according to claim 3, wherein In step (3), the mass ratio of the DHA-nobiletin-EGCG to the calcium ion cross-linked pectin-pea protein Maillard conjugate hydrogel is 1:2-3.

5. The preparation method of DHA-nobiletin-EGCG pectin-based jelly candy according to claim 4, wherein In step (5), the mass fraction of the DHA-nobiletin-EGCG emulsion gel in the DHA-nobiletin-EGCG pectin-based gel candy is 20% to 25%.

6. The method for preparing the DHA-nobiletin-EGCG loaded pectin-based jelly candy according to any one of claims 1 to 5, characterized in that: In step (2.2), the power of the ultrasound is 600W to 650W, the heating temperature is 80°C to 85°C, the reaction time is 60min to 90min, and the dialysis temperature is 4°C.

7. The method for preparing the DHA-nobiletin-EGCG loaded pectin-based jelly candy according to any one of claims 1 to 5, characterized in that: In step (3), the rotation speed of the high-speed shear emulsification is 10000 rpm to 11000 rpm, the time of the high-speed shear emulsification is 1 min to 2 min, and the temperature of the ice bath is 0°C.

8. The method for preparing the DHA-nobiletin-EGCG loaded pectin-based jelly candy according to any one of claims 1 to 5, characterized in that: In step (1), the mixing further comprises the following treatments before the mixing: adding water for dilution; the mixing is carried out under stirring conditions, and the stirring time is 60 min to 90 min; In step (2.1), the preparation process of the low-ester pectin solution is as follows: adjusting the pH value of the pectin solution to 12 with an alkaline compound, reacting, and then adjusting the pH value of the reaction system to 7 with an acidic solution, dialyzing, and freeze-drying to obtain low-ester pectin, and then dissolving the low-ester pectin in water, and hydrating under stirring to obtain a low-ester pectin solution; the mass fraction of the pectin solution is 1% to 2%, the alkaline compound is sodium hydroxide, the acidic solution is hydrochloric acid, the reaction time is 20 minutes, the dialysis time is 72 hours, and the hydration time is 10 hours to 24 hours; the preparation process of the pea protein solution is as follows: dissolving pea protein in water to obtain a pea protein solution; In step (2.3), the low-ester pectin-pea protein Maillard conjugate is dissolved in water to form a low-ester pectin-pea protein Maillard conjugate solution, wherein the mass fraction of the low-ester pectin-pea protein Maillard conjugate solution is 2% to 3%; the mass ratio of the low-ester pectin-pea protein Maillard conjugate solution to the calcium chloride solution is 30 to 40:1, and the concentration of calcium ions in the calcium chloride solution is 10 mM to 30 mM; In step (4), the mass ratio of pectin to gelatin in the pectin-gelatin mixed gel is 1:6-10, the mass fraction of citric acid in the saccharide-pectin-gelatin mixed gel is 0.08%-0.09%, and the mass ratio of the pectin-gelatin mixed gel to the saccharide is 1:2-3; the stirring temperature is 80°C-85°C, and the re-stirring temperature is 80°C-85°C; the preparation method of the saccharide is: mixing white sugar, corn syrup, orange powder and water, and stirring under heating conditions to obtain the saccharide; the mass fraction of white sugar in the saccharide is 4.5%-5%, the mass fraction of corn syrup in the saccharide is 40%-41%, the mass fraction of orange powder in the saccharide is 13%-13.5%, and the heating temperature is 80°C-85°C; In step (5), the mixing temperature is 70°C to 75°C, the mixing is carried out under stirring conditions, the drying temperature is 40°C to 45°C, the drying time is 32h to 35h, and the drying method is baking.

9. A DHA-nobiletin-EGCG loaded pectin-based jelly candy prepared by the method for preparing a DHA-nobiletin-EGCG loaded pectin-based jelly candy according to any one of claims 1 to 8.

10. Use of the DHA-nobiletin-EGCG loaded pectin-based gel candy as claimed in claim 9 in health-care soft candies.

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