Insoluble dietary fiber-EGCG (epigallocatechin gallate) compound as well as preparation method and application thereof

Abstract

The invention discloses an insoluble dietary fiber-EGCG (epigallocatechin gallate) compound as well as a preparation method and application thereof, and belongs to the field of food processing. The preparation method of the insoluble dietary fiber-EGCG compound comprises the following steps: dispersing insoluble dietary fibers in a phosphate buffer solution, fully expanding, uniformly mixing with an EGCG solution dissolved in the phosphate buffer solution, standing at room temperature for a period of time, centrifuging, taking precipitate, and drying to obtain the insoluble dietary fiber-EGCG compound. The insoluble dietary fiber-EGCG compound disclosed by the invention has the advantages that the intestinal inflammation in a mouse colitis model is obviously relieved, and the effect is far better than that of independently using HPSIDF or EGCG; and the HPSIDF-EGCG compound can synergistically regulate and control intestinal flora-immune axis and improve the intestinal barrier function, has mild adsorption reaction conditions, is suitable for industrial production, and is expected to be developed into a product with high additional value.

Description

Technical Field

[0001] The present invention relates to the field of food processing, and in particular to an insoluble dietary fiber-epigallocatechin gallate (EGCG) complex, a preparation method and application thereof. Background Art

[0002] As a nutritious and safe food ingredient, soybean dregs (soya bean okara) represent an underutilized and valuable resource with promising development prospects and a large market potential. The dietary fiber contained in soybean dregs, known as the "seventh nutrient," is a functional food ingredient with unique nutritional properties. Insoluble dietary fiber accounts for over 90% of the total dietary fiber content. Insoluble dietary fiber, consisting of cellulose, hemicellulose, lignin, and resistant starch, possesses unique functional and physiological properties, resulting in beneficial health benefits, including promoting intestinal motility, improving diabetic conditions, enhancing cardiovascular health, reducing obesity, and delaying the onset of chronic diseases.

[0003] Dietary fiber has a loose, porous structure and excellent adsorption properties. Due to its unique structural characteristics, it is often used to adsorb amylase, thereby achieving its lipid-lowering effect. It can even be used to adsorb heavy metal ions such as lead nitrate, zinc nitrate, copper nitrate, and chromium nitrate. Dietary fiber also has a certain ability to adsorb harmful substances, such as glucose, cholesterol, sodium cholate, and acrylamide. Okara-insoluble dietary fiber extracted from bean dregs has excellent water-holding, swelling, and oil-retention properties, which has a positive effect on increasing the added value of okara dietary fiber and promoting its comprehensive utilization.

[0004] Insoluble dietary fiber from bean dregs can improve intestinal inflammation for both acute and chronic enteritis. If insoluble dietary fiber from bean dregs is used to prevent enteritis, experiments have shown that long-term intake of insoluble dietary fiber from bean dregs can, to a certain extent, resist and prevent damage when colitis occurs, but the effects are not significant enough.

[0005] Epigallocatechin gallate (EGCG) is a polyphenol and the most potent bioactive component of green tea polyphenols. It belongs to the catechin group and is widely used in the application and production of health supplements. It is the primary active and water-soluble component in green tea and the catechin component with the highest content. Due to its unique stereochemical structure, EGCG possesses very strong antioxidant activity, at least 100 times that of vitamin C, and can protect cells and DNA from damage. In healthcare, EGCG has been shown to prevent and treat various diseases, including cancer, and to enhance immunity. In the food industry, it is used as an antioxidant.

[0006] EGCG has a strong anti-inflammatory effect. Studies have found that EGCG exerts anti-inflammatory effects in vivo and in vitro by inhibiting the generation and activity of NF-κB; EGCG inhibits inflammatory responses by inhibiting the IL-1β signal transduction pathway and inhibiting IL-1β secretion; in addition, EGCG can block the release of IL-8 through TNF-α, exerting potential anti-inflammatory and antioxidant effects. However, the use of EGCG alone is prone to the following problems: (1) Hepatotoxicity: High doses of EGCG may cause liver and kidney damage. For example, studies have shown that long-term or high-dose intake of EGCG may cause liver damage and renal failure, which is related to the dose, purity and individual differences. (2) Gastrointestinal reactions: EGCG may cause gastrointestinal discomfort symptoms such as nausea, vomiting, diarrhea, and constipation. These side effects are usually dose-related, especially when the daily intake exceeds 800 mg. (3) Hypoglycemia and anemia: EGCG inhibits gluconeogenesis in the liver and may cause hypoglycemia symptoms. In addition, due to its inhibitory effect on iron absorption, long-term use may lead to anemia. (4) Nervous system effects: EGCG may produce anxiolytic effects similar to benzodiazepines, leading to central nervous system symptoms such as dizziness and insomnia. (5) Cytotoxicity: At high concentrations, EGCG has toxic effects on cells, which may affect the cell cycle and cause cell death. For example, studies have shown that when the concentration of EGCG exceeds 100 μM, it will cause oxidative DNA damage and cytotoxicity. These disadvantages have limited the use and development of EGCG.

[0007] With the rapid development of the food industry, a single food system can no longer meet people's needs for taste, function and even health. Complex food systems (such as polysaccharides, polyphenols and proteins) have emerged. However, how to make polyphenols and dietary fiber interact with each other is still a blank in the industry today and needs further research. Summary of the Invention

[0008] In view of this, the main object of the present invention is to provide an insoluble dietary fiber-EGCG complex and a preparation method thereof, in order to at least partially solve the above technical problems.

[0009] In order to achieve the above object, as a first aspect of the present invention, a method for preparing an insoluble dietary fiber-EGCG complex is proposed, comprising the following steps:

[0010] The insoluble dietary fiber is dispersed in a phosphate buffer solution, mixed evenly with an EGCG solution dissolved in the phosphate buffer solution, left at room temperature for a period of time, centrifuged, and the precipitate is dried to obtain the insoluble dietary fiber-EGCG complex.

[0011] As a second aspect of the present invention, an insoluble dietary fiber-EGCG complex prepared by the above-mentioned preparation method is also proposed.

[0012] As a third aspect of the present invention, a use of the insoluble dietary fiber-EGCG complex as described above in a food additive is also proposed.

[0013] Based on the above technical solutions, the insoluble dietary fiber-EGCG complex of the present invention and its preparation method have at least one of the following beneficial effects compared with the prior art:

[0014] 1. The HPSIDF-EGCG complex of the present invention significantly alleviates intestinal inflammation in a mouse colitis model by inhibiting the release of inflammatory factors such as IL-1β, IL-6, IL-8, and TNF-α, with an effect far superior to that of HPSIDF or EGCG alone. Furthermore, the HPSIDF-EGCG complex synergistically regulates the intestinal flora-immune axis. HPSIDF, as an insoluble dietary fiber, has been shown by the team to promote the production of short-chain fats, improve intestinal barrier function, and synergistically inhibit the release of inflammatory factors.

[0015] 2. The retention rates of the HPSIDF-EGCG complex in the present invention in simulated gastric and intestinal fluids reached 80.1% and 86.6%, respectively. EGCG was adsorbed through the pores of HPSIDF to form a stable structure, avoiding degradation by gastric acid. In particular, when insoluble dietary fiber from soybean dregs was combined with EGCG to form a complex, the bottleneck of EGCG bioavailability was broken through. Because HPSIDF has a porous structure, it can physically adsorb and embed EGCG, thereby delaying the release of EGCG.

[0016] 3. The HPSIDF-EGCG complex of the present invention increases the efficiency of scavenging reactive oxygen species (ROS) by 30%, and significantly improves the activity of superoxide dismutase (SOD), effectively alleviating oxidative stress;

[0017] 4. The present invention uses soybean dregs as raw materials and combines them with EGCG to construct a complex, thereby enhancing the physiological function of soybean dregs dietary fiber, achieving a functional upgrade of soybean by-products, promoting a win-win situation for environmental protection and economic benefits, and ultimately effectively realizing the upgrade and transformation of food processing by-products into healthy food raw materials;

[0018] 5. The process of extracting HPSIDF using the composite enzyme method in the present invention is mature, and the adsorption reaction conditions are mild, making it suitable for industrial production. In addition, HPSIDF and EGCG, two functional food ingredients, have unique satiety, anti-inflammatory and antioxidant properties, and promote intestinal motility, and are expected to be developed into high-value-added products, such as intestinal health foods, sports nutrition foods, and foods for the elderly. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments.

[0020] Figure 1 Schematic diagram of the process for preparing the HPSIDF-EGCG complex of the present invention;

[0021] Figure 2 is a bar graph of tumor necrosis factor-α (TNF-α) levels in mice in different experimental groups, Figure 2 A is the serum inflammation level, Figure 2 B is the level of tissue inflammation;

[0022] Figure 3 The bar graph shows the interleukin-1β (IL-1β) levels of mice in different experimental groups, where Figure 3 A is the serum inflammation level, Figure 3 B is the level of tissue inflammation;

[0023] Figure 4 The bar graph shows the interleukin-6 (IL-6) levels of mice in different experimental groups, where Figure 4 A is the serum inflammation level, Figure 4 B is the level of tissue inflammation;

[0024] Figure 5 The bar graph shows the interleukin-8 (IL-8) levels of mice in different experimental groups, where Figure 5 A is the serum inflammation level, Figure 5 B is the level of tissue inflammation;

[0025] Figure 6 It is a bar graph of MPO levels in tissues of different experimental groups;

[0026] Figure 7 It is a bar graph of ROS levels in tissues of different experimental groups;

[0027] Figure 8 It is a bar graph of SOD levels in tissues of different experimental groups;

[0028] Figure 9 The figure is a bar graph showing the intestinal length of mice with acute ulcerative colitis in different experimental groups.

[0029] Figure 10 H&E staining images of colon tissues of mice with acute ulcerative colitis in different experimental groups. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0031] The terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. The singular forms "a", "an", "the" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0032] Insoluble dietary fiber is widely used in the food industry due to its good water holding capacity, swelling properties and structural stability.

[0033] For example: Insoluble dietary fiber can improve the texture and structure of dough, allowing baked goods to form better shape and texture; or In yogurt, adding insoluble dietary fiber can increase the viscosity of yogurt, shorten the fermentation time, and improve the taste; or Insoluble dietary fiber is an important functional ingredient in foods such as bread, biscuits, beverages, etc. by providing bulk and improving intestinal health function, etc.

[0034] One of the more important applications is combining it with other substances to achieve synergistic effects. For example, insoluble dietary fiber can form complexes with metal ions to adsorb harmful substances, such as scavenging nitric oxide (NO). Studies have shown that the carboxyl groups of dietary fiber and metal ions can form a bridging effect, forming a dietary fiber-metal ion-NO complex, which in turn scavenges NO.

[0035] Antioxidant function: Insoluble dietary fiber is used to encapsulate antioxidants (such as puerarin), improving their release efficiency and bioavailability in the body through microencapsulation technology. For example, Hericium erinaceus insoluble dietary fiber was used as a wall material to successfully encapsulate puerarin, significantly improving the sustained release of the antioxidant.

[0036] Lipid-lowering function: Insoluble dietary fiber inhibits pancreatic lipase activity by binding to cholesterol micelles and bile salts, thereby exerting its lipid-lowering effect. For example, insoluble dietary fiber from orange peel has a strong adsorption capacity for soybean oil and can significantly inhibit pancreatic lipase activity.

[0037] However, a search revealed that insoluble dietary fiber is commonly used to adsorb substances such as metal ions and cholesterol, but its combination with polyphenols such as EGCG is largely unexplored. This suggests that while dietary fiber is commonly used to adsorb other components, its combination with EGCG has not been explored. Furthermore, due to its relatively large molecular weight, EGCG can be used directly, and it is not typically considered to be combined with dietary fiber.

[0038] In order to solve the problems of single function of dietary fiber prepared by traditional methods, lack of development of composite system of dietary fiber and polyphenols, and immature technology of stable combination of polyphenols and fiber, which leads to low retention rate of composite in vivo and insignificant effect, the inventors have conducted in-depth research on insoluble dietary fiber, especially soybean dregs insoluble dietary fiber (HPSIDF) and polyphenol epigallocatechin gallate (EGCG), as well as the composite of the two, and proposed the following preferred scheme. After multiple experimental verifications, it was proved that the composite can give full play to the synergistic effect of the two, and the effect was confirmed to be far beyond expectations. Its retention rate in simulated gastrointestinal fluid significantly overcomes the problem that EGCG is easily decomposed under acidic conditions. To solve the problem, the results of in vivo animal tests showed the synergistic effect of the complex in anti-inflammatory and antioxidant effects. In the determination of inflammatory factor levels, the effects of the complex groups were significantly better than those of the single components. These data show that the effect of the complex is not a simple superposition, but a synergistic enhancement, which is a synergistic effect that cannot be predicted by existing technologies. In addition, the addition of EGCG to the HPSIDF overall system effectively promoted the production of beneficial bacteria Akkermansia. In the comparative example, the adsorption effect of rice bran dietary fiber on blueberry polyphenols was greatly affected by temperature, while the insoluble dietary fiber from soybean dregs was more stable under the same conditions, further proving that the combination of insoluble dietary fiber from soybean dregs and EGCG has specific advantages.

[0039] Specifically, the present invention provides a method for preparing an insoluble dietary fiber-epigallocatechin gallate (EGCG) complex, comprising the following steps:

[0040] Insoluble dietary fiber was dispersed in phosphate buffer, fully expanded, and then evenly mixed with an epigallocatechin gallate (EGCG) solution dissolved in phosphate buffer. The mixture was allowed to stand at room temperature for a period of time. After centrifugation, the precipitate was dried to prepare an insoluble dietary fiber-epigallocatechin gallate (EGCG) complex.

[0041] In the above preparation steps, the insoluble dietary fiber is dispersed in the phosphate buffer and allowed to fully swell for 20-60 minutes, preferably 30 minutes. "Fully swell" herein refers to the phenomenon that, under specific conditions, the insoluble fiber, upon contact with water or other solvents, can absorb and retain a large amount of water, thereby significantly increasing its volume, and is to be understood as having a meaning commonly known in the art.

[0042] In the above preparation steps, the insoluble dietary fiber and the EGCG solution dissolved in phosphate buffer are preferably placed at room temperature for 1-4 hours, preferably 2 hours. In vitro characterization results show that during the placement period, the HPSIDF-EGCG complex reacts and binds in a non-covalent manner, such as strong hydrogen bonds, hydrophobic interactions, and weak electrostatic interactions. Therefore, the placement only needs to ensure that the two are mixed together, which can be static, slowly stirred, or vigorously stirred.

[0043] In the above preparation steps, the precipitate after centrifugation can be dried, for example, by vacuum filtration followed by air drying, vacuum freeze drying, or the like.

[0044] In the above preparation steps, the main source of insoluble dietary fiber can be plant-based foods, especially the shells or fiber parts of grains, beans, nuts, vegetables and fruits, such as extracted from Polygonatum sibiricum residue or lettuce leaves, etc. Preferably, the insoluble dietary fiber is soybean dregs insoluble dietary fiber (HPSIDF), which can form a more optimal composite system with EGCG and has room for synergistic development.

[0045] Specifically, the okara insoluble dietary fiber can be prepared from defatted soybean meal by a composite enzyme method, for example.

[0046] It should be noted that the dissolution of defatted soybean meal refers to the process of mixing defatted soybean meal with a solvent so that the components in the soybean meal are dispersed in the solvent to form a uniform system.

[0047] Preferably, the specific steps of the composite enzyme preparation method include:

[0048] After defatted soybean meal is dissolved, α-amylase, neutral protease and amyloglucosidase are added in sequence, and after enzymatic hydrolysis, washing is performed, and insoluble solid matter is selected, specifically, by water washing, centrifugation, alcohol precipitation, suction filtration and vacuum freeze drying to obtain the soybean dregs insoluble dietary fiber.

[0049] Preferably, the mass concentration of okara insoluble dietary fiber in the reaction system is, for example, 2 mg / mL, and the mass concentration of epigallocatechin gallate (EGCG) is, for example, 0.2-0.6 mg / mL, so that the mass ratio of okara insoluble dietary fiber to epigallocatechin gallate (EGCG) is, for example, 3.3-10:1, preferably 5:1.

[0050] Preferably, in the step of centrifuging to obtain the precipitate after the reaction, the centrifugation conditions are, for example, a rotation speed of 7500 r / min and a centrifugation time of 10-15 min.

[0051] Preferably, the adsorption rate of EGCG by the insoluble dietary fiber-EGCG complex is 15-20%, and the adsorption amount is 0.03-0.04 g / g.

[0052] Preferably, the retention rate of the insoluble dietary fiber-EGCG complex in simulated gastric fluid at a pH of 1.2 is ≥80%, and the retention rate in simulated intestinal fluid at a pH of 6.8 is ≥85%.

[0053] The present invention also provides an insoluble dietary fiber-EGCG complex prepared by the above preparation method.

[0054] The present invention also proposes an application of the insoluble dietary fiber-EGCG complex as described above in food additives.

[0055] The present invention will be further described below through specific examples. It should be noted that the following examples are merely illustrative and are not intended to limit the present invention. Based on the embodiments of the present invention shown below, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the embodiments of the present invention.

[0056] Example 1

[0057] Screening of the compound ratio of soybean dregs insoluble dietary fiber (HPSIDF) and epigallocatechin gallate (EGCG).

[0058] Reference Figure 1 Two portions (40 mg) of HPSIDF extracted by the complex enzymatic method were weighed and dissolved in 1 portion (20 mL) of PBS buffer (pH 7.0-7.2) to obtain a HPSIDF concentration of 2 mg / mL (w / v). Four mg of epigallocatechin gallate (EGCG) was weighed and dissolved in 20 mL of PBS buffer to obtain a solution with an EGCG concentration of 0.2 mg / mL (w / v). The two solutions were mixed and allowed to react at room temperature for 2 h, at which point the EGCG concentration in the total reaction system reached 0.1 mg / mL (w / v). The mixture was centrifuged at 7500 r / min, and the precipitate was freeze-dried in vacuo to obtain the HPSIDF-EGCG complex.

[0059] Example 2

[0060] 6 mg of epigallocatechin gallate (EGCG) was weighed and dissolved in 20 mL of PBS buffer to obtain a solution with an EGCG concentration of 0.3 mg / mL (m / v). The two solutions were mixed and allowed to react at room temperature for 2 h, resulting in an EGCG concentration of 0.15 mg / mL (w / v). The mixture was centrifuged at 7500 r / min, and the precipitate was freeze-dried in vacuo to obtain okara insoluble dietary fiber (HPSIDF)-EGCG complexes with different compounding ratios.

[0061] Example 3

[0062] 8 mg of epigallocatechin gallate (EGCG) was weighed and dissolved in 20 mL of PBS buffer to obtain a solution with an EGCG concentration of 0.4 mg / mL (m / v). The two solutions were mixed and allowed to react at room temperature for 2 hours, resulting in an EGCG concentration of 0.2 mg / mL (w / v). The mixture was centrifuged at 7500 r / min, and the precipitate was freeze-dried in vacuo to obtain okara insoluble dietary fiber (HPSIDF)-EGCG complexes with different compounding ratios.

[0063] Example 4

[0064] 10 mg of epigallocatechin gallate (EGCG) was weighed and dissolved in 20 mL of PBS buffer to obtain a solution with an EGCG concentration of 0.5 mg / mL (m / v). The two solutions were mixed and allowed to react at room temperature for 2 hours, at which point the EGCG concentration in the total reaction system reached 0.25 mg / mL (w / v). The mixture was centrifuged at 7500 r / min, and the precipitate was freeze-dried in vacuo to obtain okara insoluble dietary fiber (HPSIDF)-EGCG complexes at different ratios.

[0065] Example 5

[0066] 12 mg of epigallocatechin gallate (EGCG) was weighed and dissolved in 20 mL of PBS buffer to obtain a solution with an EGCG concentration of 0.6 mg / mL (m / v). The two solutions were mixed and allowed to react at room temperature for 2 hours, resulting in an EGCG concentration of 0.3 mg / mL (w / v). The mixture was centrifuged at 7500 r / min, and the precipitate was freeze-dried in vacuo to obtain okara insoluble dietary fiber (HPSIDF)-EGCG complexes with different compounding ratios.

[0067] The following measurements were performed on the okara insoluble dietary fiber-epigallocatechin gallate complex prepared in Examples 1-5:

[0068] Determination of the adsorption capacity of okara insoluble dietary fiber (HPSIDF) for epigallocatechin gallate (EGCG)

[0069] The epigallocatechin gallate (EGCG) content of the resulting HPSIDF-epigallocatechin gallate (EGCG) complex was determined using the Folin-Ciocalteu (FC) method. A 0.5 mL sample (0.2 mg / mL) of the complex was reacted with 2.5 mL of 0.2N FC reagent for 10 minutes. 2 mL of 75 mg / mL Na₂CO₃ was added, vortexed, and incubated at room temperature in the dark for 2 hours. The absorbance was measured at 760 nm using a microplate reader. The adsorbed EGCG content was calculated by plotting a standard curve against the EGCG concentration.

[0070] The adsorption rate of epigallocatechin gallate (EGCG) by okara insoluble dietary fiber (HPSIDF) was calculated according to formula (1):

[0071] Adsorption rate of epigallocatechin gallate (EGCG) (%) = ×100 (1).

[0072] The adsorption capacity of epigallocatechin gallate (EGCG) by okara insoluble dietary fiber (HPSIDF) was calculated according to formula (2):

[0073] Epigallocatechin gallate (EGCG) adsorption capacity (g / g) = (2).

[0074] Where: C0 is the mass concentration of epigallocatechin gallate (EGCG) in the solution system before adsorption (mg / mL); Ct is the mass concentration of epigallocatechin gallate (EGCG) in the supernatant after adsorption (mg / mL); V is the total volume of the solution when the adsorption reaction occurs (mL); m is the mass of insoluble dietary fiber in okara (mg).

[0075] The results are shown in Table 1. As can be seen, at a 20:4 ratio of HPSIDF to epigallocatechin gallate (EGCG), the adsorption rate reached 18%, and the adsorption capacity (the amount of HPSIDF bound to EGCG per gram) reached 0.035 g / g. For ease of description, only the adsorption capacity is used as the range-limiting criterion to determine the protection scope in this invention.

[0076] Table 1 Adsorption characteristics results list

[0077]

[0078] Comparative Example 1

[0079] The specific preparation steps are the same as those in Example 1, except that rice bran insoluble dietary fiber is used instead of bean dregs insoluble dietary fiber. The blueberry polyphenol adsorption capacity of the two is compared, and the following conclusions are obtained:

[0080] Insoluble rice bran dietary fiber can adsorb blueberry polyphenols. The amount of blueberry polyphenol adsorption by rice bran insoluble dietary fiber varies under different environmental conditions. Under neutral conditions (pH 7.0-7.2), the amount of blueberry polyphenol adsorbed by rice bran insoluble dietary fiber ranges from 0.03 to 0.04 g / g. However, temperature is negatively correlated with the amount of blueberry polyphenol adsorbed by rice bran insoluble dietary fiber. As the temperature increases from room temperature to 55°C, the amount of blueberry polyphenol adsorbed by rice bran insoluble dietary fiber decreases from 0.05-0.10 to 0.00-0.05. This demonstrates that okara insoluble dietary fiber has excellent adsorption capacity.

[0081] In vitro simulated digestion of soybean okara insoluble dietary fiber (HPSIDF)-epigallocatechin gallate (EGCG) complex.

[0082] 1. Preparation of simulated digestive fluid.

[0083] The preparation method of the simulated digestive fluid was prepared according to the United States Pharmacopoeia (USP) method. The specific method is as follows:

[0084] Preparation of simulated gastric fluid (SGF): Take 2g of sodium chloride (NaCl) and 3.2g of pepsin, add 7mL of hydrochloric acid (HCl), and dilute to 1L with sterile water to obtain simulated gastric fluid. The pH of the simulated gastric fluid should be around 1.2.

[0085] Preparation of simulated intestinal fluid (SIF): Dissolve 6.8 g of potassium dihydrogen phosphate (KH2PO4) in 250 mL of sterile water. Add 77 mL of 0.2 mol / L sodium chloride (NaCl) solution and 500 mL of sterile water. Dissolve 10 g of trypsin. Adjust the pH to approximately 6.8 with 0.2 mol / L sodium hydroxide (NaOH) or dilute hydrochloric acid (HCl) solution. Dose the solution to 1 L with sterile water to obtain simulated intestinal fluid.

[0086] 2. In vitro simulated digestion method.

[0087] During simulated gastric digestion, an appropriate volume of simulated gastric fluid was added to make the mass concentration (w / v) of epigallocatechin gallate (EGCG) reach 1 mg / mL, and the mixture was mixed in a water bath at 37°C for 2 h.

[0088] During the simulated intestinal fluid digestion, an appropriate volume of simulated intestinal fluid was added to make the mass concentration (w / v) of epigallocatechin gallate (EGCG) reach 1 mg / mL, and the mixture was mixed in a water bath at 37°C for 4 h.

[0089] 3. Determination of retention rate.

[0090] The Folin-Ciocalteu (FC) method revealed that HPSIDF exhibited a relatively stable adsorption of epigallocatechin gallate (EGCG). Calculations based on a standard curve of EGCG concentrations showed that, after digestion in simulated gastric fluid, the HPSIDF-EGCG complex partially decomposed under a strong acidic environment, releasing unstable free phenols that diffused into the solution, resulting in a retention rate of 80.1%. Furthermore, the retention rate of the HPSIDF-EGCG complex after digestion in simulated intestinal fluid was 86.6%. Therefore, it is speculated that the retention rate of the HPSIDF-EGCG complex in the intestine after digestion and decomposition in the stomach can reach 69.3%, and the pores of the HPSIDF adsorb EGCG to form a stable structure under neutral conditions.

[0091] Animal tests were conducted on the okara insoluble dietary fiber-epigallocatechin gallate complexes prepared in Examples 1-5.

[0092] 1. Selection of animals.

[0093] Sixty healthy adult male C57BL / 6 mice were randomly divided into five groups, with 12 mice in each group, and fed with different diets:

[0094] Group A: normal control group (Control): ordinary feed;

[0095] Group B: Model control group (DSS): normal feed + dextran sulfate modeling;

[0096] Group C: intervention group (DSS+HPSIDF): ordinary feed + dextran sulfate modeling + soybean dregs insoluble dietary fiber;

[0097] Group D: intervention group (DSS+EGCG): normal feed + dextran sulfate modeling + epigallocatechin gallate;

[0098] Group E: Intervention group (DSS+HPSIDF-EGCG): ordinary feed + dextran sulfate modeling + soybean dregs insoluble dietary fiber-epigallocatechin gallate complex.

[0099] exist Figure 9Group A: normal control group (Control); Group B: model control group (DSS); Group C: intervention group (DSS+HPSIDF); Group D: intervention group: (DSS+EGCG); Group E: intervention group (DSS+HPSIDF-EGCG).

[0100] Before modeling, mice were fed a basal diet for one week. After their metabolic status stabilized, they were randomly divided into five groups: one group received drinking water and a normal diet as the normal control group; one group received a dextran sulfate solution and a normal diet as the model control group; one group received a dextran sulfate solution, a normal diet, and an additional 10 mg / day of soybean okara insoluble dietary fiber as the intervention group; one group received a dextran sulfate solution, a normal diet, and an additional 10 mg / day of soybean okara insoluble dietary fiber-epigallocatechin gallate complex as the intervention group; and one group received a dextran sulfate solution, a normal diet, and an additional 0.35 g / day of epigallocatechin gallate as the intervention group. Six mice were housed in cages. The light intensity was 12 h per day, and the temperature was maintained at 20 ± 2°C and the humidity was 50 ± 5%. After 7 days of feeding, the mice were exsanguinated from their eyeballs and sacrificed. The mice were weighed before being sacrificed. After being sacrificed, the abdominal cavity of the mice was opened and the colon was immediately isolated. 0.5 cm colon tissue was taken from the distal and proximal ends of the colon for hematoxylin-eosin (H&E) staining.

[0101] 2. Preparation of mouse tissue samples.

[0102] Immediately after killing the mouse, the abdominal cavity was opened. 0.2 g of colon tissue was removed from the intact colon and placed in a 5 mL centrifuge tube. 2 mL of PBS buffer was added using a pipette, and the bottom of the 5 mL centrifuge tube was placed in a container filled with ice. While holding a portable homogenizer in the left hand at a speed of 10,000-15,000 rpm, the pestle with the right hand was inserted vertically into the 5 mL centrifuge tube. Triturate the colon tissue several times (6-8 minutes) until it is thoroughly homogenized. The resulting tissue fluid was centrifuged at 3,000 rpm for 15 minutes at 4°C. The supernatant was then collected and retained for analysis.

[0103] 3. Determination of inflammatory factor levels.

[0104] The indicators of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), myeloperoxidase antibody (MPO), reactive oxygen species (ROS), superoxide dismutase (SOD) and tumor necrosis factor (TNF-α) in the colon tissue of mice were strictly operated according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit.

[0105] Results and analysis:

[0106] Figure 2 A and 2B are bar graphs of tumor necrosis factor-α (TNF-α) levels in mice from different experimental groups. Different letters above different bars indicate significant differences (p < 0.05). Figure 2 A is the serum inflammation level, Figure 2 B is the level of tissue inflammation.

[0107] Figure 3 A and 3B are bar graphs of interleukin-1β (IL-1β) levels in mice from different experimental groups. Different letters above different bars indicate significant differences (p < 0.05). Figure 3 A is the serum inflammation level, Figure 3 B is the level of tissue inflammation.

[0108] Figure 4 The figure is a line graph showing the mass concentration and adsorption rate of the insoluble dietary fiber of bean dregs for epigallocatechin gallate of the present invention.

[0109] Figure 5 A and 5B are bar graphs of interleukin-8 (IL-8) levels in mice from different experimental groups. Different lowercase letters above different bars indicate significant differences (p < 0.05). Figure 5 A is the serum inflammation level, Figure 5 B is the level of tissue inflammation.

[0110] Figure 6 The figure shows the histogram of MPO levels in tissues of different experimental groups. Different letters above different bars indicate significant differences (p<0.05).

[0111] Figure 7 The bar graph shows the ROS levels in tissues of different experimental groups. Different letters indicate significant differences (p<0.05).

[0112] Figure 8 The bar graph shows the SOD levels in tissues of different experimental groups. Different letters indicate significant differences (p<0.05).

[0113] Figure 9 The figure shows the intestinal length of mice with acute ulcerative colitis in different experimental groups. Different letters indicate significant differences (p<0.05).

[0114] From the above Figure 2 A- Figure 9It can be seen that compared with the model control group of group B, the normal control group of group A and the intervention group of group E were significantly lower than the model control group of group B. The intake of soybean dregs insoluble dietary fiber (HPSIDF)-epigallocatechin gallate (EGCG) complex can reduce the release of IL-1β by the body's immune cells to a certain extent and alleviate the damage to the body; compared with the model control group of group B, the intervention group of group E was significantly lower than the model control group of group B. The intake of soybean dregs insoluble dietary fiber (HPSIDF)-epigallocatechin gallate (EGCG) complex effectively alleviated the excessive release of IL-6; compared with the normal control group of group A and the model control group of group B, the intervention group of group E had significant changes. The intake of soybean dregs insoluble dietary fiber (HPSIDF)-epigallocatechin gallate (EGCG) complex inhibited the release of IL-8 to a certain extent, avoided the aggregation of neutrophils, and alleviated the inflammatory response; compared with the model control group of group B, the intervention group of group E was significantly reduced. The intake of soybean dregs insoluble dietary fiber (HPSIDF)-epigallocatechin gallate (EGCG) complex Compared with the model control group in group B, the values of intervention groups E and D were very close and significantly lower. Intake of HPSIDF-EGCG complex can gradually develop the body's ROS level to the normal control group in group A. Compared with the normal control group in group A, the SOD level of intervention group E tended to approach the inflammatory level of the healthy group. Intake of HPSIDF-EGCG complex can inhibit the generation of oxygen free radicals in the body and alleviate oxidative stress. Compared with the model control group in group B, the TNF-α level of intervention group E was significantly reduced. Generally speaking, the higher the TNF-α level, the more severe the inflammation in the body. Intake of HPSIDF-EGCG complex can effectively alleviate inflammation.

[0115] The above results show that the soybean dregs insoluble dietary fiber (HPSIDF)-epigallocatechin gallate (EGCG) complex has good anti-inflammatory ability. At the same time, as a polysaccharide-polyphenol complex with prebiotic potential, it has a protective effect on DSS-induced acute enteritis. At the same time, the two intervention methods of soybean dregs insoluble dietary fiber (HPSIDF) alone and epigallocatechin gallate (EGCG) alone were compared. The soybean dregs insoluble dietary fiber (HPSIDF)-epigallocatechin gallate (EGCG) complex had better indicators in mice than the single intervention group, proving that this polysaccharide-polyphenol complex played a synergistic role to a certain extent.

[0116] 4. Determination of colon tissue status and hematoxylin-eosin (H&E) staining in mice with acute ulcerative colitis.

[0117] Immediately after sacrifice, the mice were opened and the entire colon tissue was removed for length measurement. The colon was flushed with PBS to remove feces. Tissue samples of approximately 0.5 cm were taken from both the distal and proximal ends of the colon and stained with hematoxylin and eosin (H&E).

[0118] Colon H&E staining method:

[0119] (1) Cut an intestinal segment about 1 cm long 2 cm away from the anus and wash it in PBS solution;

[0120] (2) The specimen was fixed in 4% paraformaldehyde for approximately 12 h;

[0121] (3) The specimen was embedded in paraffin and sectioned along the coronal plane of the intestinal tract with a thickness of approximately 4 μm;

[0122] (4) Flatten the slices, place them on a glass slide, and dry them in a 45°C constant temperature oven;

[0123] (5) Remove paraffin wax by xylene three times, 20 min each time;

[0124] (6) Treat the sections with alcohol in descending order: 100% alcohol, 95% alcohol, 85% alcohol, and 70% alcohol for 2 minutes each.

[0125] (7) Wash the slices in distilled water for 5 minutes;

[0126] (8) After hematoxylin staining for 5 minutes, rinse again with distilled water for 1 minute;

[0127] (9) Separate the slides with 1% hydrochloric acid alcohol (prepared with 70% alcohol) for 10 seconds and then rinse with distilled water for 1 minute;

[0128] (10) 0.1% eosin stain mixed with 2 drops of glacial acetic acid for 5 min;

[0129] (11) Treat the slices with alcohol in descending order: 70% alcohol, 85% alcohol, 95% alcohol, and 100% alcohol for 2 minutes each.

[0130] (12) Xylene treatment twice, 5 min each time;

[0131] (13) Sealing: Seal the coverslip with neutral gum;

[0132] (14) Observe under a microscope after HE staining.

[0133] Figure 10 Figure 2 shows hematoxylin and eosin (H&E) staining of colonic tissue from mice with acute ulcerative colitis in different experimental groups. As can be seen, the colonic mucosa of animals in the DSS group was severely damaged, accompanied by significant inflammatory cell infiltration and a decrease in the number of goblet cells. In contrast, the colonic mucosal structure of animals in the DSS+HPSIDF-EGCG group was significantly restored, with a significant decrease in inflammatory cell infiltration and a slight increase in the number of goblet cells. This indicates that the complex helps repair mucosal barrier function and alleviate the inflammatory response. Furthermore, colon tissue length measurements further validated this conclusion. While the colon tissue length of animals in the DSS group was significantly shortened, the colon tissue length of the DSS+HPSIDF-EGCG group was significantly increased compared to the other DSS-treated groups (p<0.05), indicating that the complex effectively reduces colonic atrophy and inhibits intestinal damage.

[0134] The present invention also provides the use of the method described in the above technical solution in preparing a complex of insoluble dietary fiber from soybean dregs and epigallocatechin gallate. This complex enhances the added value and physiological activity of soybean dregs dietary fiber through synergistic effects. The complex alleviates intestinal inflammation by inhibiting the release of inflammatory factors such as IL-1β, IL-6, IL-8, and TNF-α. The complex also mitigates oxidative stress damage by scavenging reactive oxygen species (ROS) and enhancing superoxide dismutase (SOD) activity.

[0135] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing an insoluble dietary fiber-EGCG complex, characterized in that: The following steps are involved: The insoluble dietary fiber is dispersed in a phosphate buffer solution, mixed evenly with an EGCG solution dissolved in the phosphate buffer solution, left at room temperature for a period of time, centrifuged, and the precipitate is dried to obtain the insoluble dietary fiber-EGCG complex.

2. The preparation method according to claim 1, characterized in that When dispersing the insoluble dietary fiber in phosphate buffer, fully expand it for 20 to 60 minutes; and / or The insoluble dietary fiber and the EGCG solution dissolved in phosphate buffer are stirred evenly and then placed at room temperature for 1 to 4 hours; and / or The precipitate after centrifugation is dried by vacuum filtration and then air-dried or vacuum freeze-dried.

3. The preparation method according to claim 2, characterized in that When dispersing the insoluble dietary fiber in phosphate buffer, fully swell for 30 minutes; and / or The insoluble dietary fiber and the EGCG solution dissolved in phosphate buffer were stirred evenly and then placed at room temperature for 2 hours.

4. The preparation method according to claim 3, characterized in that The insoluble dietary fiber is bean dregs insoluble dietary fiber.

5. The preparation method according to claim 4, characterized in that The bean dregs insoluble dietary fiber is prepared from defatted soybean meal by a composite enzyme method; the composite enzyme method specifically comprises the following steps: dissolving the defatted soybean meal, sequentially adding α-amylase, neutral protease and amyloglucosidase, washing with water after enzymolysis, separating out the solid matter and drying.

6. The preparation method according to claim 4, characterized in that The mass ratio of the okara insoluble dietary fiber to EGCG is 3.3-10:1; and / or After standing at room temperature for 1 to 4 hours, the centrifugal conditions are as follows: centrifugal speed 7500 r / min, centrifugal time 10 to 15 minutes.

7. The preparation method according to claim 6, characterized in that The mass ratio of the okara insoluble dietary fiber to EGCG is 5:

1.

8. An insoluble dietary fiber-EGCG complex prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the insoluble dietary fiber-EGCG complex according to claim 8 in food additives.

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