Application of PGA and NHDC or NH compound embedded in PGA in preparation of anti-allergic food or drug

Abstract

The invention discloses polygalacturonic acid (PGA) and application of a neohesperidin dihydrochalcone (NHDC) or neohesperidin (NH) embedded compound of the polygalacturonic acid (PGA) in preparation of anti-allergic food or drugs. The PGA has good biocompatibility and immune regulation and intestinal barrier repair functions. Experiments show that the PGA can significantly reduce IgE-mediated food anaphylaxis, alleviate allergic symptoms and improve intestinal structures. PGA is adopted as a carrier, and the PGA and NHDC / NH form non-covalent binding through intermolecular force to prepare the PGA-NHDC / PGA-NH compound, so that the solubility and the stability of the NHDC / NH are improved, and a synergistic anti-allergic effect is also generated. The PGA-NHDC / PGA-NH compound has the advantages of small molecular weight, fast absorption and non-immunogenicity, and can release NHDC / NH in colon in a targeted manner. On the basis, the invention further provides the application of the PGA-NHDC / PGA-NH compound in preparation of the orange juice, the obtained juice system is in a stable suspension state, has no precipitation layering phenomenon, has targeted synergistic antianaphylaxis and high storage stability, and remarkably improves the functionality and the storage stability of the juice.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food nutrition and medicine, in particular to the application of PGA and its embedding NHDC or NH compound in the preparation of anti-allergic food or medicine. BACKGROUND

[0002] Allergic reaction is a type of disease caused by overreaction of the immune system, common forms include food allergy, drug allergy, pollen allergy and contact dermatitis, etc. Its core mechanism mainly involves the activation of mast cells, basophils and the overexpression of immunoglobulin E (IgE), leading to the release of inflammatory mediators such as histamine, prostaglandin and leukotriene. The commonly used anti-allergic drugs in clinical practice, such as antihistamines and glucocorticoids, can relieve symptoms, but have problems such as large side effects, strong dependence or short-acting. Food allergy refers to the abnormal immune response of the body to specific food proteins, which belongs to adverse food reactions mediated by immune mechanisms. In recent years, food allergy has become a major public health problem worldwide, especially in the pediatric population, with a significantly increasing incidence. Clinical studies have shown that immunoglobulin E (IgE)-mediated food allergy can cause symptoms from mild itching, skin rash to severe gastrointestinal reactions and even respiratory tract restriction, among which egg allergy is one of the most common types of food allergy, with a global prevalence of about 1-9%. In recent years, the research on natural active substances in regulating immune response and relieving allergic symptoms has gradually increased, especially plant-derived polysaccharides, flavonoids and their derivatives. Polysaccharides have good biocompatibility and immunoregulatory potential, while flavonoids exhibit significant activity in anti-inflammatory, antioxidant and immune balance regulation. At present, there are limited treatment options for food allergy in clinical practice, and safe and effective natural anti-allergic ingredients and preparations need to be developed.

[0003] Polygalacturonic acid (PGA) is the main structural unit of pectin, which belongs to plant-derived polysaccharides with low degree of polymerization. It has attracted people's attention due to its potential in regulating immune response and its wide availability in plant cell walls. Studies have shown that PGA is a structural polysaccharide with unique physical and chemical properties, allowing it to interact with immune cells and regulate their activation state. PGA has good biocompatibility and adhesion, can form a protective film in the gut, and exhibits physiological activities such as anti-inflammatory, immune regulation and promotion of intestinal barrier repair. Therefore, it is of great significance to explore the potential application of PGA in the field of anti-allergy. Compared with complex pectin (PC), PGA has the advantages of clear structure, low molecular weight and easy modification, which is more conducive to development and utilization in food and medicine.

[0004] Neohesperidin dihydrochalcone (NHDC) is a citrus dihydrochalcone, a natural functional sweetener derived from the chemical modification of the citrus flavonoid neohesperidin, which is rarely found in nature. It can inhibit bitterness and improve flavor, so it is often used as a food additive or functional ingredient. In 2022, the European Food Safety Authority (EFSA) set the acceptable daily intake (ADI) of NHDC at 20 mg / kg BW per day. It is usually synthesized by structural modification of the precursor neohesperidin (NH) 1,2. Its synthetic precursor neohesperidin (NH) is a natural bitter flavonoid mainly found in Rutaceae plants. Neohesperidin is composed of rhamnose 1→2 linked glucose, which is an important structure that causes the bitterness of NH. Small differences in the rhamnose and glucose linkage sites can cause different tastes of the compounds. Interestingly, treating NH with hot alkali causes the heterocycle to open and produce flavanone glycosides in the form of chalcone. Then, the olefin double bond of chalcone undergoes hydrogenation to produce the corresponding dihydrochalcone, i.e., NHDC, which has high desulfurization potential. From the molecular structure, they have the same basic skeleton, with different sugar moieties attached. Natural flavonoids are safe for the human body and have similar physiological activities, such as anti-fibrosis, regulation of sugar and lipid metabolism, neuroprotective activity, and anticancer activity. These activities have been extensively studied, but these natural molecules may have a broader therapeutic potential than currently known. NHDC not only improves food flavor, but also has certain physiological activities, such as anti-inflammatory, antioxidant, regulation of lipid metabolism and immune function. Studies have shown that NHDC can affect the PI3K / AKT / mTOR signaling pathway, thereby participating in the regulation of immune homeostasis of the body.

[0005] In the prior art, existing antiallergic drugs such as antihistamines, glucocorticoids and the like can alleviate symptoms, but have problems of large side effects, high risk of long-term use and the like, and lack of safe nutritional intervention means for food allergy. Neohesperidin (NH) and its derivative neohesperidin dihydrochalcone (NHDC) as citrus flavonoids have antioxidant, anti-inflammatory and potential immunomodulatory activities. However, NH is limited in food and drug applications due to poor solubility, strong bitterness and insufficient stability; and neohesperidin dihydrochalcone (NHDC) generally has poor stability, low water solubility and poor bioavailability in practical application. These defects directly restrict the large-scale promotion and application of NH / NHDC in the fields of functional food, medicine and the like. Therefore, there is currently a lack of a carrier system that can simultaneously improve the stability and bioavailability of NH / NHDC and endow it with synergistic antiallergic function. How to use natural polysaccharide PGA with immunomodulatory activity to compound with NH or NHDC to improve its solubility, stability and antiallergic effect, and effectively improve its absorption and utilization rate in vivo has become a technical problem to be broken through in the fields of food and medicine. SUMMARY

[0006] In view of the technical problems that existing antiallergic drugs such as antihistamines, glucocorticoids and the like can alleviate symptoms, but have problems of large side effects, high risk of long-term use and the like, and lack of safe nutritional intervention means for food allergy, one of the purposes of the present application is to provide an application of polygalacturonic acid (PGA) in preparation of antiallergic food or medicine.

[0007] PGA is natural, has low toxic side effects and has an antiallergic function of immunomodulatory mechanism, and the intestinal immune response and barrier function are regulated by PGA to relieve or prevent the occurrence of food allergy from the source.

[0008] The present application first discovers that polygalacturonic acid has a significant effect in relieving IgE-mediated food allergy. Through animal experiment verification, oral PGA can effectively reduce the allergic symptoms of chicken ovalbumin (OVA)-induced mice, reduce the levels of OVA-specific IgE and IgG1 antibodies in serum, and significantly improve the intestinal tissue damage and barrier function disorder. PGA is an oligomeric structural unit of pectin, containing abundant carboxyl and hydroxyl groups, and has good adhesion and water solubility. The molecule can form a protective mucous layer in the intestinal tract, reducing the penetration of allergens through the intestinal epithelial barrier; at the same time, by up-regulating the expression of tight junction proteins (such as ZO-1, Claudin-1, Occludin, JAM-1), the damaged intestinal barrier is repaired, thereby reducing the immune overactivation induced by sensitizing substances. In addition, PGA can regulate Th2 immune balance, inhibit the excessive secretion of Th2-type cytokines (IL-4, IL-13), and indirectly reduce the production of IgE mediated by B cells, achieving the effect of systemic anti-allergy.

[0009] On this basis, the present application further proposes a polygalacturonic acid (PGA) embedded neohesperidin dihydrochalcone (NHDC) complex, which is used for the preparation of anti-allergy food or medicine.

[0010] In the complex system, PGA acts as a carrier material, and forms a non-covalent interaction with NHDC through intermolecular forces, realizing the effective embedding and stable protection of NHDC. On the one hand, the addition of PGA significantly improves the solubility and stability of NHDC in water system; on the other hand, PGA itself has anti-allergy and immune regulation activity, and the combination of the two can produce a synergistic anti-allergy effect, jointly regulate Th1 / Th2 immune balance, and reduce the intensity of IgE-mediated allergic reaction, thereby enhancing the overall anti-allergy effect.

[0011] The PGA embedded NHDC complex has significant advantages in structure and physiological characteristics compared with traditional carriers (pectin, chitosan):

[0012] 1. Smaller molecular weight and stronger diffusion: the molecular weight of the complex system formed after PGA embedding is lower than that of the pectin and chitosan system, which is easier to penetrate the gastrointestinal barrier, thereby accelerating the entry into the intestinal system;

[0013] 2. High targeting and absorption efficiency: PGA is partially degraded and absorbed by intestinal flora in the colon, which can promote the release of NHDC in the colon and improve the local immune regulation efficiency;

[0014] 3. Non-immunogenicity: PGA is not recognized as a foreign body or antigen by the immune system of the body, has good biocompatibility and low immunogenicity, and can be used as a safe active carrier for drugs and food;

[0015] 4. Synergistic anti-allergic mechanism: PGA reduces sensitization by improving intestinal barrier and immune balance, NHDC relieves tissue inflammation by inhibiting inflammatory mediators and signaling pathways (such as PI3K / AKT / mTOR), and the NHDC group can slow the trend of increasing serum IgE and IgG1 concentrations caused by food allergy, the intake of NHDC can better alleviate food allergy, and the two can achieve more significant anti-allergic effect in synergy.

[0016] 5. Significantly improve the solubility and stability of NHDC: PGA forms non-covalent interaction with NHDC through intermolecular force, significantly improves the dispersibility, solubility and photothermal stability of NHDC in water system, avoids the problem of degradation, and NHDC can also be used as a sweetener, thereby improving the activity retention rate and flavor compatibility in food or drug system.

[0017] The application also relates to application of the polygalacturonate-embedded neohesperidine dihydrochalcone complex to preparation of citrus juice.

[0018] Further, the polygalacturonate-embedded neohesperidine dihydrochalcone complex is formed by embedding the neohesperidine dihydrochalcone in the polygalacturonate through establishing an oil-in-water Pickering emulsion system.

[0019] Further, the preparation method of the citrus juice comprises the following steps:

[0020] S1, preparing a citrus juice stock solution;

[0021] S2, preparation of the polygalacturonate-embedded neohesperidine dihydrochalcone complex Pickering emulsion, comprising the following steps:

[0022] St1, preparing a water phase of polygalacturonate nanoparticles-egg phospholipid

[0023] The PGA is dissolved in deionized water, stirred to completely dissolve after ultrasonic treatment, calcium salt solution is continuously added to the PGA solution after stirring, and the PGA is induced to form nanoparticles through ionic crosslinking, the solution is centrifuged, the supernatant is discarded, the precipitated nanoparticles are collected and then placed, and the nanoparticles are dispersed in pure water again to adjust the concentration to obtain a polygalacturonate nanoparticle solution; the polygalacturonate nanoparticle solution and the egg phospholipid solution are mixed and stirred to obtain a water phase;

[0024] St2, preparing an oil phase of neohesperidine dihydrochalcone-plant oil

[0025] The NHDC is dissolved in preheated plant oil, ultrasonic treatment is performed until complete dissolution to obtain a neohesperidine dihydrochalcone-plant oil oil phase;

[0026] St3, preparing a complex Pickering emulsion

[0027] The oil phase prepared in step St2 is added to the water phase prepared in step St1, and homogenized to form a refined emulsion, and the emulsion is subjected to ultrasonic disruption to disperse aggregates, thereby obtaining a Pickering compound emulsion;

[0028] S3, continuously adding the Pickering compound emulsion to the citrus juice stock prepared in step S1, and mixing uniformly.

[0029] Preferably, the calcium salt solution in step St1 can be selected from one or more of calcium chloride, calcium lactate, calcium acetate, calcium gluconate, calcium sulfate or calcium citrate; and the vegetable oil in step St2 can be selected from one or more of coconut oil, medium-chain triglyceride oil, palm kernel oil, sunflower oil, olive oil, corn oil or soybean oil.

[0030] Further, in step St1: the mass-volume concentration of polygalacturonate nanoparticles in the polygalacturonate nanoparticle solution is 40%, the mass concentration of lecithin solution is 4%, and the volume ratio of polygalacturonate nanoparticles to lecithin is 1:1;

[0031] In step St2: the vegetable oil is coconut oil, and the mass-volume concentration of neohesperidin dihydrochalcone in the neohesperidin dihydrochalcone-vegetable oil oil phase is 0.1%;

[0032] In step St3: the volume ratio of oil phase to water phase is 1:6.

[0033] In the citrus juice preparation method of the present application, experiments have shown that when the concentration of PGA nanoparticles is 40% (40g / 100ml), the concentration of lecithin is 4% (4g / 100ml), the volume ratio of PGA to lecithin is 1:1, and the volume ratio of oil phase to water phase is 1:6, the emulsion particle size is the smallest, the absolute value of potential is the largest (|ZP|>50mV), and the emulsion stability is the best. This parameter set is integrated in the method steps, which can significantly improve the dispersion uniformity and long-term storage stability of the emulsion in the juice system. By Ca 2+ The PGA nanoparticles formed by ion cross-linking induction can maintain the NHDC in a stable state in the juice system, prolong the flavor and physiological activity maintenance time, and achieve high-efficiency embedding and slow-release performance.

[0034] It is shown by experiments that after the new hesperidine dihydrochalcone complex Pickering emulsion is added to the polygalacturonate embedding, the total flavonoid content of the citrus juice is significantly improved, and after one week of cold storage, the appearance is stable, without stratification or precipitation, and has the function of suspending agent, the method not only improves the antioxidant and health performance of the juice, but also improves the storage quality of the product. Meanwhile, the Pickering emulsion system constructed by the PGA-NHDC realizes efficient embedding and stable release of the NHDC, significantly improves the stability of the citrus juice, and the PGA-NHDC complex can quickly and target reach the intestine for decomposition and release, producing mutual synergistic anti-allergic health effects.

[0035] The application also relates to application of the polygalacturonate-embedded new hesperidine complex in preparation of anti-allergic food or medicine.

[0036] Further, the polygalacturonate-embedded new hesperidine complex is formed by embedding new hesperidine in the polygalacturonate through establishment of an oil-in-water Pickering emulsion system.

[0037] Still further, the preparation method of the polygalacturonate-embedded new hesperidine complex comprises the following steps:

[0038] Stp1, preparation of an aqueous phase by polygalacturonate nanoparticles-egg lecithin

[0039] The polygalacturonate is dissolved in deionized water, and after ultrasonic treatment, stirring is performed until complete dissolution; after continuous addition of a calcium salt solution to the polygalacturonate solution and stirring, the PGA is induced to form nanoparticles through ionic crosslinking; the solution is centrifuged, and the supernatant is discarded; the collected nanoparticle precipitate is allowed to stand; the nanoparticles are then redispersed in pure water to adjust the concentration to obtain a polygalacturonate nanoparticle solution; and the polygalacturonate nanoparticle solution and the egg lecithin solution are mixed and stirred to obtain an aqueous phase;

[0040] Stp2, preparation of an oil phase by new hesperidine-plant oil

[0041] The new hesperidine is dissolved in preheated plant oil, and ultrasonic treatment is performed until complete dissolution to obtain a new hesperidine-plant oil oil phase;

[0042] Stp3, preparation of a complex Pickering emulsion

[0043] The oil phase prepared in step Stp2 is added to the aqueous phase prepared in step Stp1, and homogenization is performed to form a refined emulsion; after homogenization, the emulsion is subjected to ultrasonic crushing to disperse the aggregates, and thus the polygalacturonate-embedded new hesperidine complex Pickering emulsion is obtained.

[0044] Preferably, the calcium salt solution in step Stpl can be selected from one or more of calcium chloride, calcium lactate, calcium acetate, calcium gluconate, calcium sulfate or calcium citrate; and the vegetable oil in step Stp2 can be selected from one or more of coconut oil, medium-chain triglyceride oil, palm kernel oil, sunflower seed oil, olive oil, corn oil or soybean oil.

[0045] The present application realizes efficient embedding of neohesperidin (NH) by constructing a stable Pickering emulsion system with polygalacturonic acid (PGA) as a carrier. The technical system utilizes the synergistic interfacial action of PGA nanoparticles and lecithin to form a dense particle adsorption layer, thereby significantly improving the physical stability of the emulsion system and the dispersibility of neohesperidin. The oligomeric structure of PGA makes the composite system have a smaller molecular weight and higher fluidity, and the composite particles can maintain a good dispersion state in the gastrointestinal environment, avoid rapid release of bitterness, and gradually release active ingredients in the colon section, thereby improving the bioavailability and in vivo absorption efficiency of NH.

[0046] In terms of anti-allergy function, the PGA-embedded neohesperidin composite system exhibits a significant synergistic effect. PGA can effectively alleviate IgE-mediated allergic reactions by improving the expression of intestinal tight junction proteins, reducing mucosal permeability, and regulating Th1 / Th2 immune balance; NH can reduce allergic inflammatory damage by inhibiting the secretion of inflammatory cytokines and oxidative stress. The PGA-NH complex Pickering emulsion formed by the combination of the two has higher targeting and sustained-release performance in vivo, and can gradually release NH in the colon to maintain sustained anti-allergic effects. This system not only overcomes the technical bottlenecks of poor water solubility, low stability, and bitter taste of neohesperidin, but also endows the composite with excellent suspensibility and layered resistance, allowing it to be stably applied in citrus juice, functional drinks or oral drug formulations, achieving the unification of structural stability, flavor coordination and targeted anti-allergic effect. BRIEF DESCRIPTION OF DRAWINGS

[0047] Brief description of the content expressed by each figure of the specification and the markings in the figures:

[0048] Figure 1 The animal experiment design process for Example 1 is as follows:

[0049] Figure 2 The influence of citrus pectin and polygalacturonic acid on the allergic characterization of OVA-induced food allergy mice in Example 1 is shown in (A) the allergic symptoms of mice, (B) the body weight changes of mice during dietary intervention, (C) the allergic score, (D) the anal temperature changes, and (E) the immune organ index; *p<0.05; **p<0.01; ***p<0.001, n=6.

[0050] Figure 3Effect of citrus pectin and polygalacturonic acid on the contents of OVA-sIgE and OVA-sIgG1 in serum of OVA-induced food allergy mice in Example 1;

[0051] Figure 4 Effect of citrus pectin and polygalacturonic acid on the intestinal barrier function of OVA-induced food allergy mice in Example 1; (A) HE staining of mouse jejunum; (B) mRNA expression of tight junction factors ZO-1, JAM-1, Claduin-1 and Occudin-1 in mouse jejunum; *p<0.05; **p<0.01; ***p<0.001, n=6;

[0052] Figure 5 Effect of citrus pectin and polygalacturonic acid on the balance of Th1 / Th2 cells in mice in Example 1; *p<0.05; **p<0.01; ***p<0.001, n=6;

[0053] Figure 6 Particle size and zeta potential of PGA-NHDC Pickering emulsion under different concentrations of PGA in Example 2;

[0054] Figure 7 Particle size and zeta potential of PGA-NHDC Pickering emulsion under different concentrations of lecithin in Example 2;

[0055] Figure 8 Particle size and zeta potential of PGA-NHDC Pickering emulsion under different ratios of PGA to lecithin in Example 2;

[0056] Figure 9 Particle size and zeta potential of PGA-NHDC Pickering emulsion under different ratios of oil phase to water phase in Example 2;

[0057] Figure 10 Particle size and zeta potential of pectin gel particles and PGA-NHDC Pickering emulsion in Example 2;

[0058] Figure 11 Particle size and zeta potential of chitosan gel particles and PGA-NHDC Pickering emulsion in Example 2;

[0059] Figure 12 Particle size and zeta potential of each emulsion under the original solution, strong acid and strong base environment in Example 2;

[0060] Figure 13 PGA-NHDC Pickering emulsion under strong acid and strong base environment in the first week in Example 2; A: the left beaker is strong acid and the right beaker is strong base; B: rear view of the two beakers;

[0061] Figure 14 This is the Pickering emulsion of the PGA-NHDC complex under strong acid and strong alkaline conditions in the second week of Example 2; in A, the left beaker is a strong acid and the right beaker is a strong base; B is a rear view of the two beakers;

[0062] Figure 15 This is the Pickering emulsion of the PGA-NHDC complex under strong acid and strong alkaline conditions in the third week of Example 2; in A, the left beaker is a strong base and the right beaker is a strong acid; B is a rear view of the two beakers;

[0063] Figure 16 Example 2 shows the total flavonoid content of orange juice containing Pickering emulsions with different concentrations of PGA-NHDC complex;

[0064] Figure 17 Example 2, Day 1: Pickering emulsions with different PGA-NHDC complex contents in orange juice state; A is a front view, B is a rear view;

[0065] Figure 18 The orange juice state of different PGA-NHDC complex Pickering emulsion contents one week after Example 2; A is the front view, B is the rear view;

[0066] Figure 19 This is the animal experiment design diagram for Example 3;

[0067] Figure 20 This is a line graph showing the change in mouse body weight in Example 3;

[0068] Figure 21 The allergy score of mice after challenge in Example 3;

[0069] Figure 22 The image shows the change in rectal temperature of the mice during the last challenge in Example 3.

[0070] Figure 23 Example 3 shows the immune organ indices of mice; A is the spleen index, and B is the thymus index.

[0071] Figure 24 The table shows the serum IgE and IgG1 levels in mice in Example 3; A represents the OVA-sIgE level, and B represents the OVA-sIgG1 level.

[0072] Figure 25 HE staining and toluidine blue staining of mouse jejunal tissue in Example 3. Detailed Implementation

[0073] The application will be further described by non-limiting examples with reference to the accompanying drawings. It is to be understood that the description is merely exemplary and is not intended to limit the scope of the application. Furthermore, in the following description, well-known functions or constructions are not described in detail since they would obscure the application in unnecessary detail.

[0074] Example 1 Anti-allergic experiment of polygalacturonic acid

[0075] 1. Test materials

[0076] 1.1 Test reagents

[0077] The reagents used are shown in Table 1.

[0078] Table 1 Test reagents

[0079]

[0080] 1.2 Test instruments,

[0081] See Table 2.

[0082] Table 2 Test instruments

[0083]

[0084] 2. Test method

[0085] Female BALB / c mice (5-6 weeks) were randomly divided into 5 groups: normal group (CD group), model group (MD group), citrus pectin diet intervention group (MD+PC group) (40 mg / kg), polygalacturonic acid diet intervention group (MD+PGA group) (40 mg / kg) and positive control cetirizine hydrochloride group (MD+CT group) (10 mg / kg), 6-8 in each group. On day 0 and day 14, the CD group was injected intraperitoneally with 200 μL of phosphate buffered saline (PBS), the MD group, the MD+PC group and the MD+PGA group were injected with 2 mg of alum and 100 μg of OVA. From day 28 to day 45, the mice were challenged every 3 days with 50 mg of OVA (200 μL of PBS). The intervention group mice were fed with PC, PGA every day from day 1. The specific operation is as shown in Figure 1

[0086] 2.1 Measurement of mouse body weight: the body weight of the mice was measured and recorded after each challenge, and the body weight was measured 10 times.

[0087] 2.2 Recording of mouse allergy score: the mouse allergy score within 1 hour after each attack was recorded according to Table 3.

[0088] Table 3 Allergy score table

[0089] ​

[0090]

[0091] 2.3 Rectal temperature measurement in mice: Rectal temperature was recorded every 20 minutes within 1 hour after the last OVA challenge using a rectal thermometer.

[0092] 2.4 Preparation of mouse serum: After the last challenge, the body temperature was measured, and the eyeball was bled. After standing at room temperature for 1 h, the blood was centrifuged at 4 °C and 3000 r / min for 15 min. The supernatant was aliquoted and stored at -80 °C for later use.

[0093] 2.5 Measurement of IgE and IgG1 in serum: The procedure was carried out according to the ELISA kit instructions.

[0094] 2.6 Extraction of mouse tissue RNA: About 50 mg of tissue was placed in an Eppendorf tube containing 1 mL Trizol and grinding beads, ground in a tissue grinder, and then placed at low temperature for 15 min. After centrifugation at 4 °C and 12000 rpm for 15 min, 200 ul of chloroform was added to the supernatant, shaken and mixed, and then placed at low temperature for 15 min. After centrifugation, 500 uL of isopropanol was added to the supernatant, mixed gently, and then placed at low temperature for 15 min. After centrifugation, the supernatant was removed, and the precipitate was washed with 1 mL of 75% ethanol. Immediately after centrifugation at 4 °C and 7600 rpm for 5 min, the supernatant was removed, and the precipitate was dissolved in an appropriate amount of DEPC water after air-drying. After standing for 5 min, the mixture was mixed with a vortex mixer. 1 ul was used to determine the concentration with a nucleic acid concentration detector, and reverse transcription was performed according to the reverse transcription kit.

[0095] 2.7 RT-qPCR: RT-qPCR was performed on Quantagene q225 using SYBR Green Master (ROX). Beta-Actin was used as an internal control, and the relative expression of mRNA in jejunum tissue was determined by 2 -ΔΔCt RT-PCR primer sequences are shown in Table 4.

[0096] Table 4 Real-time PCR primer sequences

[0097]

[0098] 2.8 Histopathology: Jejunum tissue was fixed with 4% paraformaldehyde solution, then dehydrated and embedded in paraffin. After cutting 5 um, H&E staining was performed for each sample. Then all digital slice images were visualized using an inverted optical microscope at room temperature.

[0099] 2.9 Data analysis and processing: Prism 8 and SPSS 22.0 software were used for data processing in this experiment, and the test level a = 0.05. P < 0.05 was considered to have a statistically significant difference between groups, P < 0.01 was considered to have a significant statistically significant difference between groups, and P < 0.001 was considered to have a very significant statistically significant difference between groups.

[0100] 3 Experimental results and analysis

[0101] 3.1 Effect of citrus PC and PGA on allergic symptoms of OVA-induced allergic mice

[0102] In order to study whether citrus pectin and polygalacturonic acid can alleviate OVA-induced food allergy, OVA-induced food allergy model in mice was established using OVA as the allergen and dietary intervention with citrus pectin and polygalacturonic acid. The results showed that OVA-induced food allergy mice showed typical allergic symptoms, such as red and swollen mouth, erect hair, weakness, etc., as shown in Figure 2 -A. However, the weight loss of allergic mice did not show significant difference, as shown in Figure 2 -B, which is consistent with the results of Han et al. (2024). In general, citrus PC and PGA can effectively alleviate the phenotype of food allergy mice, and the anal temperature trend and allergic score within 1 hour after each stimulation are significantly reduced compared with allergic mice, as shown in Figure 2 -C, D. The immune organ index can reflect the size of the immune organs, and its value can reflect the immune status of the body. Compared with the normal group, the spleen index and thymus index of the model group increased, but decreased after dietary intervention with citrus pectin and polygalacturonic acid, as shown in Figure 2 -E. Combined with the above results, the OVA-induced food allergy model was successfully established.

[0103] 3.2 Effect of citrus PC and PGA on antibody content in mouse serum

[0104] Allergen-specific IgE and IgG mediate allergic reactions. When mice ingest OVA, if the immune system recognizes it as a foreign allergen, B cells will produce OVA-specific IgE with the help of T cells. At the same time, Th2 cells will secrete cytokines such as IL-4 and IL-13, which will promote B cells to class switch to produce IgG1. The effect of PC and PGA on antibody levels in mouse serum was detected at the molecular level using an Elisa kit. As shown in Figures 2-3It can be seen that the specific antibody content of normal mice is very low, and the antibody content of the model group is the highest, with IgE and IgG1 contents as high as 28.93 ng / mL and 37.21 ng / mL, which are significantly higher than those of the normal group and the dietary intervention group. Oral administration of citrus pectin and polygalacturonic acid significantly reduced the level of systemic immune markers in OVA allergic mice, and the serum OVA specific IgE and IgG1 levels were significantly reduced. Among the two polysaccharide substances, dietary PGA is more effective in alleviating food allergy.

[0105] 3.3 Effect of citrus PC and PGA on intestinal barrier function of mice

[0106] As shown in Figure 4 HE staining was used to observe the pathological condition of the jejunum of mice. Food allergy mice showed obvious intestinal damage, manifested as obvious broken and damaged villi, villi swelling caused by increased vascular permeability, and partial infiltration of inflammatory cells. After intervention with citrus pectin, polygalacturonic acid and positive drug, the damage phenomenon was alleviated, and polygalacturonic acid was already close to the normal group.

[0107] When the intestinal barrier function is impaired, allergens are more likely to cross the intestinal barrier and enter the body, triggering an allergic reaction. Tight junction factors (TJ) located between intestinal epithelial cells are an important part of the intestinal barrier. TJ structure is composed of transmembrane proteins (Claudins family and Occludin family), connecting adhesion molecules JAM family and intracellular plaque proteins (such as ZO family of occludin). Claudins family is located in the cytoplasm, responsible for forming tight junctions between cells and participating in intracellular signal transduction. Studies have found that the absence of Claudin-1 protein can cause severe dehydration and even death in mice. ZO can regulate the assembly and stability of TJ structure. Therefore, we evaluated the expression of TJ in the jejunum tissue of mice Figure 4 -B), and it was observed that the mRNA expression of tight junction factors ZO-1, JAM-1, Claudin-1 and Occudin-1 in allergic mice was significantly decreased, and the mRNA expression of TJ proteins (ZO-1, Occudin and JAM-1) in PC and PGA groups was significantly up-regulated, and the effect was better than that of the positive control. Therefore, citrus PC and PGA can positively regulate the function of colonic mucosal barrier and promote the integrity of intestinal barrier. In contrast, the effects of PC and PGA are not significantly different.

[0108] 3.4 Effect of citrus PC and PGA on immune function of allergic mice

[0109] Food allergy is associated with Th2 immune response overproduction, and the imbalance of Th1 and Th2 immune responses is one of the main causes of food allergy. In order to further determine whether citrus PC and PGA can regulate Th1 and Th2 cell responses, the gene expressions of Th1 and Th2 cytokines and their key transcription factors T-bet and GATA-3 in the jejunum and spleen were determined. The results are shown in Figure 5 As compared with normal group mice, the OVA-sensitized mice had a very significant decrease in IFN-γ level in the jejunum (p<0.001), and a very significant increase in Th2 cytokines IL-4 and IL-5 expression (p<0.001), and the ratio of IFN-γ / IL-4 also indicated that OVA sensitization caused a serious Th1 / Th2 immune imbalance in the mice. This imbalance was alleviated after the PC and PGA diet Figure 5 A-C). At the same time, Th2 cell polarization occurred in the OVA-sensitized mice, as shown by the significant increase in the relative expression of IL-4, IL-5 and their transcription factor GATA-3 in the mouse spleen. In contrast, the relative expression of IFN-γ and T-bet was significantly increased in the intervention group. IFN-γ / IL-4 explains this result Figure 5 D-E). These results show that citrus PC and PGA can increase the Th1 series and inhibit the Th2 cell immune response to reduce the allergic reaction.

[0110] Example 2 Preparation of citrus juice with polygalacturonic acid-embedded neohesperidin dihydrochalcone complex (PGA-NHDC complex)

[0111] 1. Materials and methods

[0112] 1.1 Materials and chemicals

[0113] Polygalacturonic acid, soy lecithin, neohesperidin dihydrochalcone, coconut oil, calcium chloride, NFC orange juice, sodium hydroxide, sulfuric acid, sodium nitrite, aluminum nitrate, anhydrous ethanol, ultrasonic cleaner, magnetic stirrer, centrifuge, homogenizer, ultrasonic cell disruptor, Malvern nanoparticle size and zeta potential analyzer, microplate reader.

[0114] 1.2 Experimental methods

[0115] 1.2.1 Preparation of aqueous phase of polygalacturonic acid nanoparticles (PGA) / pectin gel particles / chitosan gel particles-lecithin, respectively

[0116] PGA (0.25% w / v) was dissolved in deionized water and sonicated for 2 h followed by magnetic stirring overnight until complete dissolution. The PGA was induced to form nanoparticles by ionic crosslinking by adding 0.1 M CaCl2solution dropwise into the PGA at a volume ratio of 1 : 10 followed by magnetic stirring for 1 h. The solution was centrifuged at 12000 rpm for 20 min, the supernatant was discarded, and the precipitated nanoparticles were collected and left overnight. The nanoparticles were then re-dispersed in pure water to obtain a PGA nanoparticle solution to adjust the concentration.

[0117] Pectin solution (2 mg / mL) was prepared by dissolving 100 mg pectin in 50 mL deionized water with constant stirring overnight. Pectin gel particles were formed by adding 0.1 M CaCl2solution into the pectin solution at a volume ratio of 1 : 10 followed by magnetic stirring for 1 h.

[0118] Chitosan solution (2 mg / mL) was prepared by completely dissolving 60 mg chitosan in 30 mL 1% acetic acid, at which time the pH value was about 3.0. The obtained solution was centrifuged at 12000 rpm for 10 min, and the supernatant was taken to remove impurities before use. The pH value was changed by adding NaOH (0.1 mol / L) until the transparent chitosan solution became slightly milky white, at which time the pH value was about 6.6, and chitosan gel particles were obtained.

[0119] The above PGA nanoparticle solution, pectin gel particles, and chitosan gel particles were mixed with lecithin solution, respectively, and then magnetically stirred for 30 min to obtain different kinds of water phases.

[0120] 1.2.2 New Hesperidin Dihydrochalcone (NHDC) - Coconut Oil Preparation of Oil Phase

[0121] NHDC was dissolved in preheated 50 °C coconut oil with a concentration of 0.1% w / v (0.1 g / 100 ml), and sonicated until complete dissolution to obtain NHDC-coconut oil oil phase, which was stored in the dark.

[0122] 1.2.3 Preparation of Pickering Emulsion Stabilized by Complexes

[0123] The NHDC-coconut oil oil phase was slowly added to the above different water phases at different ratios. A homogenizer was used to initially homogenize the mixture at 8000 rpm for 2 min to form a coarse emulsion, and then further homogenized at 13000 rpm for 5 min to refine the emulsion. After homogenization, the emulsion was transferred to a centrifuge tube for ultrasonic disruption (200 W, pulse mode: 2 seconds on / 1 second off) for 10 min to disperse the aggregates, with ice bath cooling (<30 °C) during the process.

[0124] PGA nanoparticle Pickering emulsion (PGA-NHDC complex Pickering emulsion), pectin gel particle Pickering emulsion and chitosan gel particle Pickering emulsion were obtained respectively in this way.

[0125] 1.2.4 Single factor test of PGA nanoparticle Pickering emulsion to optimize emulsion component concentration and proportion

[0126] 1.2.4.1 Effect of PGA nanoparticle concentration (w / v) on Pickering emulsion stability

[0127] The concentration of lecithin solution was fixed at 3% (3 g / 100 ml, w / v), the volume ratio of PGA nanoparticle:lecithin was 1:1, and the volume ratio of oil phase:aqueous phase was 1:6. Pickering emulsions were prepared with PGA nanoparticle solution concentrations of 5% (5 g / 100 ml, w / v, same below), 10%, 20%, 30%, 40% and 50% respectively. The particle size and zeta potential of the emulsions were measured, and the average value was taken from three parallel experiments in each group.

[0128] 1.2.4.2 Effect of lecithin solution concentration (w / v) on Pickering emulsion stability

[0129] The concentration of PGA nanoparticle solution was fixed at 40% (40 g / 100 ml, w / v), the volume ratio of PGA nanoparticle:lecithin was 1:1, and the volume ratio of oil phase:aqueous phase was 1:6. Pickering emulsions were prepared with lecithin solution concentrations of 1% (1 g / 100 ml, w / v, same below), 2%, 3%, 4% and 5% respectively. The particle size and zeta potential of the emulsions were measured, and the average value was taken from three parallel experiments in each group.

[0130] 1.2.4.3 Effect of PGA nanoparticle:lecithin ratio on Pickering emulsion stability

[0131] The concentration of PGA nanoparticle solution was fixed at 40% (40 g / 100 ml, w / v), the concentration of lecithin solution was fixed at 4% (4 g / 100 ml, w / v), and the volume ratio of oil phase:aqueous phase was 1:6. Pickering emulsions were prepared with the volume ratio of PGA nanoparticle:lecithin being 1:0, 0:1, 1:1, 2:1 and 3:1 respectively. The particle size and zeta potential of the emulsions were measured, and the average value was taken from three parallel experiments in each group.

[0132] 1.2.4.4 Effect of oil phase:aqueous phase ratio on Pickering emulsion stability

[0133] The concentration of the fixed PGA nanoparticle solution was 40% (40 g / 100 ml, w / v), the concentration of the lecithin solution was 4% (4 g / 100 ml, w / v), and the volume ratio of PGA nanoparticles to lecithin was 1:1. Pickering emulsions were prepared under the volume ratio of oil phase to water phase of 1:4, 1:5, 1:6, 1:7, and 1:8, and the particle size and potential of the emulsions were measured, respectively. The average value was obtained by repeating each experiment in triplicate.

[0134] 1.2.5 Characterization of Pickering emulsion stabilized by complex

[0135] The particle size and potential of different types of Pickering emulsions were measured by dynamic light scattering and laser Doppler microelectrophoresis using a Malvern laser particle size potential instrument, which was used to preliminarily judge the stability of the emulsion.

[0136] 1.2.6 Characterization of PGA nanoparticle Pickering emulsion in strong acid and strong base environment

[0137] A group of PGA nanoparticle Pickering emulsions were adjusted to PH 12 using concentrated NaOH, and another group of PGA nanoparticles were adjusted to PH 1 using concentrated H2SO4. The particle size and potential of the emulsions were detected, respectively, and the changes of the emulsions were captured by a camera within three weeks, so as to judge the stability of the Pickering emulsion in strong acid and strong base environment.

[0138] 1.2.7 Preparation of PGA nanoparticle Pickering emulsion-orange juice and detection of total flavonoid content

[0139] The experimental group and the control group of orange juice were set to 35 mL each. The control group of orange juice was not added with emulsion, and the experimental group of orange juice was added with 8 mL, 5 mL, and 3 mL of PGA nanoparticle Pickering emulsion (i.e. the emulsion concentration was 22.9%, 14.3%, and 8.6%, respectively, v / v), respectively. After shaking well, the centrifuge was used at 12000 rpm for 10 min, and 1 mL of sample was accurately sucked into a 10 mL volumetric flask using a pipette. 0.3 mL of 5% sodium nitrite solution was added, shaken well, and placed for 6 min. Then 0.3 mL of 10% aluminum nitrate solution was added, shaken well, and placed for 6 min. Then 4 mL of 4% sodium hydroxide solution was added, and then anhydrous ethanol was added to the mark line. After shaking, the wavelength was measured at 505 nm using an enzyme marker, and the related concentration was converted according to the total flavonoid standard curve. At the same time, the appearance change of the finished solution was captured by a camera within one week.

[0140] 2 Results and analysis

[0141] 2.1 Determination of the optimal conditions of PGA nanoparticle Pickering emulsion

[0142] 2.1.1 Single factor experiment Set different concentrations of PGA nanoparticle solution, lecithin solution, PGA nanoparticle:lecithin ratio, oil phase:water phase ratio, each single factor change and particle size, potential change plot as follows:

[0143] From Figure 6 It can be seen that the concentration of PGA nanoparticle solution in the emulsion is in the range of 5%-50%, and there is no significant difference in each potential, but the |ZP| value is all > 50mV, which proves that the emulsion has stability, and the particle size is obviously the smallest when the concentration of PGA nanoparticle solution is 40%, and the emulsion stability is the best, so the single factor PGA nanoparticle solution concentration is selected as 40%. From Figure 7 It can be seen that the concentration of lecithin solution is in the range of 1%-5%, and there is no significant difference in each potential, but all > 50mV, and the particle size is the smallest when the concentration is 4%, which proves that the emulsion stability is the best at this time, so the single factor lecithin solution concentration is selected as 4%. From Figure 8 It can be seen that the particle size of the emulsion decreases significantly when it has lecithin, but there is no significant difference in the particle size when the volume ratio of PGA nanoparticles is 0-3, and the absolute value of the potential increases significantly when the volume ratio of PGA nanoparticles:lecithin is 1:1, which proves that the emulsion stability is the best at this time, so the single factor PGA nanoparticle:lecithin ratio is selected as 1:1. From Figure 9 It can be seen that the particle size of the emulsion is obviously the smallest when the oil phase:water phase ratio is 1:6, and the absolute value of the potential decreases slightly at this time, but the |ZP| value is > 50mV, and the comprehensive analysis proves that the emulsion stability is the best at this time, so the single factor oil phase:water phase ratio is selected as 1:6.

[0144] 2.2 Comparison between pectin gel particle Pickering emulsion and PGA nanoparticle Pickering emulsion

[0145] By comparing the Pickering emulsion prepared by pectin gel particles and PGA nanoparticles respectively, the particle size and potential were detected.

[0146] From Figure 10 It can be seen that the Pickering emulsion prepared by PGA nanoparticles has a smaller particle size and a larger absolute value of potential than the Pickering emulsion prepared by pectin gel particles, which proves that the stability of PGA nanoparticle Pickering emulsion is better than that of pectin gel particle Pickering emulsion.

[0147] 2.3 Comparison between chitosan gel particle Pickering emulsion and PGA nanoparticle Pickering emulsion

[0148] By comparing the Pickering emulsion prepared by chitosan gel particles and PGA nanoparticles respectively, the particle size and potential were detected.

[0149] FromFigure 11 It can be seen that the Pickering emulsion prepared from PGA nanoparticles has a slightly larger absolute potential value than that prepared from chitosan gel particles, but there is no significant difference. However, the Pickering emulsion prepared from PGA nanoparticles has a significantly smaller particle size, which proves that the stability of the Pickering emulsion prepared from PGA nanoparticles is better than that prepared from chitosan gel particles.

[0150] 2.4 Stability of PGA nanoparticle Pickering emulsion under strong acid and alkali environments

[0151] 2.4.1 Emulsion Characterization and Detection: The particle size and potential of the original solution and strong acid / alkaline emulsions were measured, and data were obtained and plotted.

[0152] Depend on Figure 12 It can be seen that the particle size of the emulsion increases under strong acid and strong alkali conditions, but all are <500nm, which belongs to nanoparticles. Under strong alkaline conditions, the absolute value of the potential increases significantly (|ZP|>80mV), while under strong acid conditions, the absolute value of the potential decreases slightly but is >50mV, proving that the emulsion can remain stable under strong acid and strong alkali conditions.

[0153] 2.4.2 Camera captures changes in Pickering emulsion after three weeks of storage.

[0154] Depend on Figure 13 , Figure 14 , Figure 15 It can be seen that the Pickering emulsion remained stable in the first week under strong acid and alkaline conditions, with no visible difference; in the second week, the fluidity decreased compared to the first week, but there was no significant difference visible to the naked eye; in the third week, obvious white lumpy aggregates began to appear in the emulsion; this proves that the emulsion has storage stability for two weeks under strong acid and alkaline conditions.

[0155] Total flavonoid content and storage stability in 2.5PGA nanoparticle Pickering emulsion-orange juice

[0156] 2.5.1 Determination of Total Flavonoid Content in Pickering Emulsion-Orange Juice containing PGA Nanoparticles

[0157] The total flavonoid content of orange juice containing different concentrations of Pickering emulsion was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the data are as follows: Figure 16 As shown, the higher the concentration of Pickering emulsion in orange juice, the higher the total flavonoid content. The highest total flavonoid content was found in orange juice containing 8 mL of Pickering emulsion (i.e., an emulsion concentration of 22.9%).

[0158] 2.5.2 Storage stability of PGA nanoparticle Pickering emulsion-orange juice

[0159] Changes in the finished orange juice during cold storage for one week were captured by camera, Figure 17 , Figure 18 It can be seen that the Pickering emulsion added to the orange juice has no obvious changes in appearance after cold storage for one week, proving that the orange juice has apparent stability.

[0160] Example 3 Anti-allergy experiment of polygalacturonic acid-embedded neohesperidine dihydrochalcone complex (PGA-NHDC complex) and polygalacturonic acid-embedded neohesperidine complex (PGA-NH complex)

[0161] 1. Test materials

[0162] 1.1 Test reagents

[0163] The reagents used are shown in Table 5.

[0164] Table 5 Test reagents

[0165]

[0166] 1.2 Test instruments

[0167] See Table 6.

[0168] Table 6 Experimental instruments

[0169]

[0170]

[0171] 2. Test method

[0172] Female BALB / c mice (5-6 weeks) were randomly divided into 5 groups: normal group (CD group), model group (MD group), polygalacturonic acid-neohesperidine administration group (PGA-NH group) (200 mg / kg), polygalacturonic acid-neohesperidine dihydrochalcone group (PGA-NHDC group) (200 mg / kg), 6-8 in each group. On day 0 and day 14, the CD group was intraperitoneally sensitized with 200 μL phosphate buffered saline (PBS), and the MD group, PGA-NH group and PGA-NHDC group were injected with 2 mg of alum and 100 μg of OVA. From day 28 to day 45, the mice were challenged every 3 days with 50 mg of OVA (200 μL PBS). The intervention group (PGA-NH group, PGA-NHDC group) of mice was gavaged with the PGA-NH complex Pickering emulsion, the PGA-NHDC complex Pickering emulsion every day from day 1. The specific operation is shown in Figure 19 .

[0173] The PGA-NHDC complex Pickering emulsion used in the polygalacturonic acid-neohesperidine dihydrochalcone administration group (PGA-NHDC group) (200 mg / kg) was prepared according to the following proportions: the mass-volume concentration of the polygalacturonic acid nanoparticle solution was 40%, the mass-volume concentration of the lecithin solution was 4%, and the volume ratio of the polygalacturonic acid nanoparticles to lecithin was 1:1; the mass-volume concentration of neohesperidine dihydrochalcone in the neohesperidine dihydrochalcone-coconut oil oil phase was 0.1%; and the volume ratio of the oil phase to the water phase was 1:6.

[0174] The preparation method and preparation conditions of the PGA-NH complex Pickering emulsion used in the polygalacturonic acid-neohesperidine administration group (PGA-NH group) were the same as those of the PGA-NHDC complex Pickering emulsion described above.

[0175] The preparation method of the PGA-NH complex Pickering emulsion was as follows:

[0176] 1) Preparation of the water phase of polygalacturonic acid nanoparticles-lecithin

[0177] PGA (0.25% w / v) was dissolved in deionized water, and after ultrasonic treatment for 2 h, magnetic stirring was performed overnight until complete dissolution. After adding 0.1 M CaCl2 solution dropwise to the PGA at a volume ratio of 1:10 and magnetic stirring for 1 h, the PGA was induced to form nanoparticles by ionic crosslinking. The solution was centrifuged at 12000 rpm for 20 min, and the supernatant was discarded. The collected nanoparticle precipitate was allowed to stand overnight, and the nanoparticles were then redispersed in pure water to obtain a PGA nanoparticle solution to adjust the concentration, so that the concentration of the PGA nanoparticle solution was 40% (40 g / 100 ml, w / v). The PGA nanoparticle solution was mixed with a lecithin solution with a concentration of 4% (4 g / ml, w / v), and then magnetic stirring was performed for 30 min to obtain the water phase.

[0178] 2) Preparation of the oil phase of neohesperidine-coconut oil

[0179] NH was dissolved in preheated 50°C coconut oil, and the concentration of NH was 0.1% w / v (0.1 g / 100 ml). Ultrasonic treatment was performed until complete dissolution to obtain an NH-coconut oil oil phase, which was stored in the dark.

[0180] 3) Preparation of the PGA-NH complex stabilized Pickering emulsion

[0181] The NH-coconut oil oil phase was slowly added to the water phase at a ratio of 1:6, and a homogenizer was used to homogenize the mixture at 8000 rpm for 2 min to form a coarse emulsion. The emulsion was then homogenized again at 13000 rpm for 5 min to further refine the emulsion. After homogenization, the emulsion was transferred to a centrifuge tube for ultrasonic disruption (200 W, pulse mode: 2 seconds on / 1 second off) for 10 min to disperse the aggregates, with ice bath cooling (<30°C) during the process. The PGA-NH complex Pickering emulsion was obtained.

[0182] 2.1 Mouse weight determination: The mice were weighed and recorded on days 0, 4, 11, 18, 25, 32, and 39, for a total of seven times.

[0183] 2.2 Mouse allergy score recording: The mouse allergy scores were recorded within 1 hour after each episode according to Table 3.

[0184] 2.3 Mouse rectal temperature determination: The rectal temperature was recorded every 20 minutes within 1 hour after the last OVA challenge using a rectal thermometer.

[0185] 2.4 Mouse serum preparation: After the last challenge, the mice were taken by the eyes, and the blood was placed at room temperature for 1 hour. Then, the blood was centrifuged at 3000 r / min for 15 min at 4°C. The supernatant was aliquoted and stored at -80°C for future use.

[0186] 2.5 IgE and IgG1 determination in serum: The determination was performed according to the ELISA kit instructions.

[0187] 2.6 Mouse tissue RNA extraction: About 50 mg of tissue was placed in an Ep tube containing 1 mL Trizol and grinding beads, and then ground in a tissue grinder. After low-temperature standing for 15 min, the mixture was centrifuged at 12000 rpm for 15 min at 4°C. Then, 200 ul of chloroform was added to the supernatant, and the mixture was shaken and mixed at low temperature for 15 min. The mixture was centrifuged, and the supernatant was added to 500 uL of isopropanol. The mixture was mixed gently and stood at low temperature for 15 min. The supernatant was removed, and the precipitate was washed with 1 mL of 75% ethanol. The mixture was immediately centrifuged at 7600 rpm for 5 min at 4°C, and the supernatant was removed. After drying, an appropriate amount of DEPC water was added to dissolve the precipitate, and the mixture was stood for 5 min and mixed using a vortex mixer. 1 ul of the mixture was used to determine the concentration using a nucleic acid concentration detector, and the sample was prepared for the next step of reverse transcription.

[0188] 2.7 Histopathology: The jejunum tissue was fixed with 4% paraformaldehyde solution, then dehydrated and embedded in paraffin. After cutting 5 um, H&E and toluidine blue staining were performed on each sample. Then, all digital slice images were visualized using an inverted optical microscope at room temperature.

[0189] 2.8 Data analysis and processing: Prism8 and SPSS22.0 software were used for data processing, and the test level a = 0.05. P < 0.05 was considered to have statistical significance between groups, P < 0.01 was considered to have significant statistical significance between groups, and P < 0.001 was considered to have extremely significant statistical difference between groups.

[0190] 3. Test results

[0191] PGA-NH complex and PGA-NHDC complex can effectively alleviate the phenotype of food allergy mice. The change of mouse body weight is as shown in Figure 20 From the figure, it can be seen that compared with the normal group, the body weight of the model group mice has a downward trend, but the trend change is not significant. Intuitively, PGA-NH and PGA-NHDC complex have equivalent weight loss effect.

[0192] The allergic score of mice within 1h after each challenge was recorded, and the results are shown in Figure 21 Compared with the normal group, the allergic score of OVA sensitized mice was significantly increased. But with the increase of allergic challenge times, the allergic symptoms of the model group mice became more and more serious, but the allergic symptoms of the mice were effectively controlled after the administration of PGA-NH and PGA-NHDC complex. Among them, the effect of PGA-NHDC complex is obviously better than that of PGA-NH complex.

[0193] From the Figure 22 mouse anal temperature change graph, it can be seen that from 0min to 60min, the body temperature of mice in each group changed to varying degrees. The anal temperature of the sensitized model group mice rapidly decreased within 20min of OVA sensitization, lower than the other three groups, and then slowly rose. Combined with the observation of allergic symptoms, the OVA food allergy model of mice was successfully established. The anal temperature of PGA-NH group and PGA-NHDC group decreased after challenge, and the anal temperature of PGA-NHDC group rapidly rose to near normal level at 40min. It can be seen that oral administration of PGA-NH and PGA-NHDC complex can slow down the trend of body temperature decrease caused by food allergy in mice. Among them, feeding PGA-NHDC complex can better alleviate the symptoms of body temperature decrease caused by food allergy reaction in mice.

[0194] The spleen is the largest immune organ in the human body, and during OVA sensitization, the spleen is an important place for antigen presentation, lymphocyte activation and antibody production. The thymus is the place where T lymphocytes mature. Under normal circumstances, the thymus will gradually atrophy with age, but under immune stress, its size and cell composition may change. Figure 23The spleen weight and thymus weight of the mice were measured after dissection, and the organ index was calculated. Compared with the normal group, the spleen index and thymus index of the sensitized model group of mice increased significantly, thereby proving that the food allergy model was successfully established. Compared with the normal group, the spleen index and thymus index after administration of the PGA-NH complex decreased, and interestingly, the spleen index after administration of the PGA-NHDC complex decreased significantly. Thus, it is proved that the administration of the PGA-NH and PGA-NHDC complexes can slow down the immune organ changes caused by allergic reactions in the body.

[0195] Immunoglobulin E (IgE) is a key antibody that mediates type I hypersensitivity. When mice are sensitized by OVA, the immune system recognizes OVA as a foreign antigen. Antigen-presenting cells (such as dendritic cells) take up and process OVA antigens, and present antigen information to T cells. With the help of T cells, B cells are activated and differentiated into plasma cells, which produce OVA-IgE antibodies. In addition to IgE, IgG1 is also involved in immediate allergic reactions, and IgG1 antibodies in the serum bind to the FcεR III receptor of mast cells, leading to mast cell degranulation in the intestine. Figure 24 The IgE and IgG1 concentration data graphs in the serum of mice are shown. The IgE concentrations in the serum of the normal group, the sensitized model group, the PGA-NH group, and the PGA-NHDC group were 14.47 ± 1.13 ug / L, 34.14 ± 5.28 ug / L, 21.11 ± 1.28 ug / L, and 17.26 ± 1.20 ug / L, respectively, and the IgG1 concentrations were 32.21 ± 1.17 ug / L, 43.15 ± 2.00 ug / L, 37.81 ± 1.22 ug / L, and 37.14 ± 2.60 ug / L, respectively. Compared with the normal group, the IgE and IgG1 concentrations in the sensitized model group increased significantly. Compared with the sensitized model group, the IgE concentrations in the PGA-NH group and the PGA-NHDC group decreased significantly, and there was a statistical difference, and the IgG1 concentrations also showed a downward trend. Thus, it is proved that oral administration of PGA-NH and PGA-NHDC complexes can slow down the increase in the IgE and IgG1 concentrations in the serum of mice caused by food allergy, and the intake of PGA-NHDC complex can better alleviate food allergy in mice.

[0196] As Figure 25As shown, by observing the HE staining results, it can be found that the jejunum villi of the normal group of mice are arranged in order, and the muscle layer and serosa layer structure is relatively complete. The model group appears obvious villi rupture damage or shedding phenomenon, villi swelling caused by increased vascular permeability and partial infiltration of inflammatory cells. After the PGA-NH complex, PGA-NHDC complex administration, the damage phenomenon is alleviated, and the PGA-NHDC group has obviously approached the normal group state. Inflammatory cells (such as eosinophils) will gather at the inflammation site, and toluidine blue staining can show the existence of these inflammatory cells. From the toluidine blue staining results, it can be found that compared with the normal group, the jejunum tissue of the model group is slightly congested, and the inflammatory cells increase, and the PGA-NH group and the PGA-NHDC group effectively restore the damage of the jejunum tissue.

[0197] The present application is not limited to the above-mentioned embodiments, and it should be understood that those skilled in the art can make many modifications and variations without creative labor according to the concept of the present application. Therefore, any technical solution that can be obtained by logical analysis, reasoning or limited experiment on the basis of the prior art according to the concept of the present application should be within the protection scope determined by the claims.

Claims

1. Application of polygalacturonic acid in the preparation of anti-allergic foods or medicines.

2. The application of the polygalacturonic acid-encapsulated neohesperidin dihydrochalcone complex according to claim 1 in the preparation of anti-allergic foods or pharmaceuticals.

3. The application according to claim 2, characterized in that, The polygalacturonic acid-encapsulated neohesperidin dihydrochalcone complex is formed by encapsulating neohesperidin dihydrochalcone in polygalacturonic acid through an oil-in-water Pickering emulsion system.

4. The application of the polygalacturonic acid-encapsulated neohesperidin dihydrochalcone complex according to claim 2 in the preparation of citrus juice.

5. The application according to claim 4, characterized in that, The polygalacturonic acid-encapsulated neohesperidin dihydrochalcone complex is formed by encapsulating neohesperidin dihydrochalcone in polygalacturonic acid through an oil-in-water Pickering emulsion system.

6. The application according to claim 4 or 5, characterized in that, The preparation method of citrus juice includes the following steps: S1. Pre-preparing citrus juice concentrate; The preparation of S2, Pickering emulsion containing polygalacturonic acid-encapsulated neohesperidin dihydrochalcone complex, includes the following steps: St1, preparation of aqueous phase from polygalacturonic acid nanoparticles-lecithin Polygalacturonic acid (PCA) was dissolved in deionized water and stirred until completely dissolved. A calcium salt solution was continuously added to the PCA solution while stirring to induce PCA to form nanoparticles through ionic cross-linking. The solution was centrifuged, the supernatant was discarded, and the precipitated nanoparticles were collected and allowed to stand. The nanoparticles were then redispersed in pure water to adjust the concentration, resulting in a PCA nanoparticle solution. The PCA nanoparticle solution was mixed with a lecithin solution and stirred to obtain an aqueous phase. The calcium salt solution was selected from one or more of calcium chloride, calcium lactate, calcium acetate, calcium gluconate, calcium sulfate, or calcium citrate. St2, neohesperidin dihydrochalcone-vegetable oil phase preparation Neohesperidin dihydrochalcone was dissolved in preheated vegetable oil until completely dissolved to obtain the neohesperidin dihydrochalcone-vegetable oil phase; the vegetable oil was selected from one or more of coconut oil, medium-chain triglyceride oil, palm kernel oil, sunflower seed oil, olive oil, corn oil or soybean oil. Step 3: Preparation of the Pickering complex emulsion The oil phase prepared in step St2 is added to the aqueous phase prepared in step St1, and homogenized to form a refined emulsion. After homogenization, the emulsion is broken to disperse the aggregates, and the Pickering emulsion containing polygalacturonic acid-embedded neohesperidin dihydrochalcone complex is obtained. S3. Continuously add the Pickering complex emulsion to the citrus juice concentrate from step S1 and mix well.

7. The application according to claim 6, characterized in that, In step St1: The polygalacturonic acid nanoparticle solution has a polygalacturonic acid nanoparticle mass-to-volume concentration of 40%, and the lecithin solution has a lecithin mass-to-volume concentration of 4%. The volume ratio of polygalacturonic acid nanoparticles to lecithin is 1:1; In step St2: The vegetable oil is coconut oil, and the mass-volume concentration of neohesperidin dihydrochalcone in the vegetable oil phase is 0.1%. In step St3: The oil phase:water phase volume ratio is 1:

6.

8. The application of the polygalacturonic acid-encapsulated neohesperidin complex according to claim 1 in the preparation of anti-allergic foods or pharmaceuticals.

9. The application according to claim 8, characterized in that, The polygalacturonic acid-encapsulated neohesperidin complex is formed by encapsulating neohesperidin in polygalacturonic acid through an oil-in-water Pickering emulsion system.

10. The application according to claim 9, characterized in that, The preparation method of the polygalacturonic acid-encapsulated neohesperidin complex includes the following steps: Stp1, polygalacturonic acid nanoparticles-lecithin preparation aqueous phase Polygalacturonic acid (PCA) was dissolved in deionized water and stirred until completely dissolved. A calcium salt solution was continuously added to the PCA solution while stirring to induce PCA to form nanoparticles through ionic cross-linking. The solution was centrifuged, the supernatant was discarded, and the precipitated nanoparticles were collected and allowed to stand. The nanoparticles were then redispersed in pure water to adjust the concentration, resulting in a PCA nanoparticle solution. The PCA nanoparticle solution was mixed with a lecithin solution and stirred to obtain an aqueous phase. The calcium salt solution was selected from one or more of calcium chloride, calcium lactate, calcium acetate, calcium gluconate, calcium sulfate, or calcium citrate. Stp2, neohesperidin-vegetable oil preparation oil phase Neohesperidin is dissolved in preheated vegetable oil until completely dissolved to obtain a neohesperidin-vegetable oil phase; the vegetable oil is selected from one or more of coconut oil, medium-chain triglyceride oil, palm kernel oil, sunflower seed oil, olive oil, corn oil or soybean oil; Stp3, Preparation of the Pickering Complex Emulsion The oil phase prepared in step Stp2 is added to the aqueous phase prepared in step Stp1, and homogenized to form a refined emulsion. After homogenization, the emulsion is broken to disperse the aggregates, thus obtaining the Pickering emulsion of polygalacturonic acid-embedded neohesperidin complex.

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