CN120436336A - Insoluble dietary fiber-EGCG complex, preparation method and application thereof - Google Patents

Abstract

An insoluble dietary fiber-EGCG compound, a preparation method and application thereof, belonging to the field of food processing. The preparation method of the insoluble dietary fiber-EGCG compound comprises dispersing insoluble dietary fiber in phosphate buffer, fully expanding, mixing with EGCG solution dissolved in phosphate buffer, standing at room temperature for a period of time, centrifuging, collecting precipitate, and drying to obtain insoluble dietary fiber-EGCG compound. The insoluble dietary fiber-EGCG compound provided by the invention can obviously relieve intestinal inflammation in a mouse colonitis model, has an effect far better than that of HPSIDF or EGCG which is singly used, and has the advantages that HPSIDF-EGCG compound can synergistically regulate and control intestinal flora-immune axes, improve intestinal barrier function, has mild adsorption reaction conditions, is suitable for industrial production, and is hopefully developed into a product with high added value.

Description

Insoluble dietary fiber-EGCG compound, preparation method and application thereof

Technical Field

The invention relates to the field of food processing, in particular to an insoluble dietary fiber-epigallocatechin gallate (EGCG) compound, a preparation method and application thereof.

Background

The bean dregs are used as food raw and auxiliary materials rich in nutrition and good in safety, belong to precious resources which are not fully utilized, and have good development trend and large market development space. Dietary fiber contained in the bean dregs is called a seventh nutrient, and is a functional food ingredient with unique nutrition. Wherein the insoluble dietary fiber accounts for more than 90% of the total dietary fiber. The insoluble dietary fiber comprises cellulose, hemicellulose, lignin, resistant starch and the like, and has good health care functions due to unique functions and physiological actions, including promoting intestinal peristalsis, improving diabetes, improving cardiovascular health, reducing obesity, delaying occurrence of chronic diseases and the like.

Dietary fiber has a loose and porous structure and good adsorptivity, and is usually used for adsorbing amylase due to the special structural characteristics, so that the effect of reducing blood fat of the dietary fiber is achieved, and even heavy metal ions such as lead nitrate, zinc nitrate, copper nitrate, chromium nitrate and the like are adsorbed. At the same time, the dietary fiber has certain capability of adsorbing harmful substances, such as glucose, cholesterol, sodium cholate, acrylamide and the like. The bean dreg insoluble dietary fiber extracted from the bean dreg has good water retention, expansibility and oil retention, and has positive effects of improving the added value of the bean dreg dietary fiber and promoting comprehensive utilization.

The bean dreg insoluble dietary fiber has the effect of improving intestinal inflammation aiming at both acute enteritis and chronic enteritis, and if the bean dreg insoluble dietary fiber is used for preventing enteritis, experiments prove that after long-term intake of the bean dreg insoluble dietary fiber, the bean dreg insoluble dietary fiber has the effect of resisting and preventing injury to a certain extent when colonitis occurs, but the effect is not obvious enough.

Epigallocatechin gallate (EGCG) is polyphenol, is the most effective bioactive component in tea polyphenol (Green Tea Polyphenols), belongs to catechin, and is widely applied to the application and production of health care products. The green tea is a main active and water-soluble component in green tea, is the component with the highest catechin content, has a special stereochemical structure, has very strong antioxidant activity of EGCG, has the antioxidant activity which is at least 100 times that of vitamin C, and can protect cells and DNA from damage. In the aspect of medical health care, EGCG has the functions of preventing and treating various diseases such as cancers, enhancing immunity and the like, and can be used as an antioxidant in the food industry.

EGCG has a strong effect on anti-inflammatory. The research shows that EGCG can inhibit NF- κB generation and activity in vivo and in vitro to inhibit inflammation, inhibit IL-1β secretion to inhibit inflammatory reaction, and block IL-8 release via TNF- α to exert potential anti-inflammatory and antioxidant effects. However, EGCG alone is prone to (1) hepatorenal toxicity, which can be caused by high doses of EGCG. For example, there are studies showing that long-term or high dose intake of EGCG may cause liver damage and renal failure, which is related to dose, purity and individual differences, (2) gastrointestinal reactions, in which EGCG may cause symptoms of gastrointestinal discomfort such as nausea, vomiting, diarrhea, constipation, etc., which are usually associated with dose, especially more than 800 mg per day, (3) hypoglycemia and anemia, in which EGCG inhibits gluconeogenesis of liver, possibly causing hypoglycemia symptoms, and furthermore, long-term use may cause anemia due to its effect of inhibiting iron absorption, (4) nervous system effects, in which EGCG may cause anxiolytic effects like benzodiazepines, causing symptoms of central nervous system such as dizziness, insomnia, etc., and (5) cytotoxicity, in which EGCG has toxic effects on cells and may affect cell cycle and cause cell death at high concentrations, for example, studies show that oxidized DNA damage and cytotoxicity may be caused when EGCG concentration exceeds 100. Mu.M. Both of these drawbacks limit the use and development of EGCG.

With the rapid development of the food industry, a single food system can not meet the demands of people on taste, functions and even health, and a composite food system (such as the composite of polysaccharide, polyphenol and protein) is generated, but how to interact polyphenol and dietary fiber is still blank in the current industry, and needs to be studied intensively.

Disclosure of Invention

Accordingly, the present invention is directed to an insoluble dietary fiber-EGCG complex and a preparation method thereof, so as to at least partially solve the above-mentioned problems.

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

Dispersing insoluble dietary fiber in phosphate buffer solution, uniformly mixing with EGCG solution dissolved in phosphate buffer solution, standing at room temperature for a period of time, centrifuging, and drying precipitate to obtain the insoluble dietary fiber-EGCG compound.

As a second aspect of the present invention, there is also provided an insoluble dietary fiber-EGCG complex prepared by the preparation method as described above.

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

Based on the technical scheme, the insoluble dietary fiber-EGCG compound and the preparation method thereof have at least one of the following beneficial effects compared with the prior art:

1. The HPSIDF-EGCG compound in the invention obviously relieves intestinal inflammation in a colonitis model of mice by inhibiting the release of IL-1 beta, IL-6, IL-8 and TNF-alpha inflammatory factors, the effect is far better than that of single use of HPSIDF or EGCG, and HPSIDF-EGCG compound is used for the cooperative regulation and control of intestinal flora-immune axis, HPSIDF is used as an insoluble dietary fiber, and the team proves that the compound can promote the generation of short chain fat, improve the barrier function of the intestinal tract and synergistically inhibit the release of inflammatory factors;

2. According to the invention, the retention rate of the HPSIDF-EGCG compound in simulated gastric fluid and intestinal fluid respectively reaches 80.1% and 86.6%, EGCG is adsorbed through pores of HPSIDF to form a stable structure, so that gastric acid degradation is avoided;

3. The efficiency of scavenging Reactive Oxygen Species (ROS) of HPSIDF-EGCG compound in the invention is improved by 30%, and the activity of superoxide dismutase (SOD) is obviously improved, so that the oxidative stress is effectively relieved;

4. in the invention, bean dregs are used as raw materials, and EGCG is combined to construct a compound, so that the physiological function of dietary fiber of the bean dregs is improved, the functional upgrading of soybean byproducts is realized, the win-win effect of environmental protection and economic benefit is promoted, and finally, the upgrading and transformation of food processing byproducts to healthy food raw materials are effectively realized;

5. The invention has the advantages that the process of extracting HPSIDF by utilizing the complex enzyme method is mature, the adsorption reaction condition is mild, the invention is suitable for industrial production, in addition, the HPSIDF and EGCG functional food raw materials have unique satiety, anti-inflammatory and antioxidant properties, the functions of promoting intestinal peristalsis and the like, and the invention is expected to be developed into products with high added value, such as intestinal health food, sports nutrition food and hair food.

Drawings

In order to more clearly illustrate the technical solution of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described.

FIG. 1 is a schematic flow chart of a process for preparing HPSIDF-EGCG complex of the invention;

FIG. 2 is a bar graph of tumor necrosis factor-alpha (TNF-alpha) levels in mice from different experimental groups, wherein FIG. 2A is serum inflammation level and FIG. 2B is tissue inflammation level;

FIG. 3 is a bar graph of interleukin 1 beta (IL-1 beta) levels in mice from different experimental groups, wherein FIG. 3A is serum inflammation level and FIG. 3B is tissue inflammation level;

FIG. 4 is a bar graph of interleukin 6 (IL-6) levels in mice from different experimental groups, wherein FIG. 4A is serum inflammation level and FIG. 4B is tissue inflammation level;

FIG. 5 is a bar graph of interleukin 8 (IL-8) levels in mice from different experimental groups, wherein FIG. 5A is serum inflammation level and FIG. 5B is tissue inflammation level;

FIG. 6 is a bar graph of MPO levels in tissues of different experimental groups;

FIG. 7 is a bar graph of ROS levels in tissues of different experimental groups;

FIG. 8 is a bar graph of SOD levels in tissues of different experimental groups;

fig. 9 is a bar graph of intestinal length of acute ulcerative colitis mice in different experimental groups;

fig. 10 is an H & E staining of colon tissue of mice with acute ulcerative colitis from different experimental groups.

Detailed Description

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

The terminology used in the present invention is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments of the invention. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Insoluble dietary fibers are widely used in the food industry for their good water retention, expansion and structural stability.

For example, insoluble dietary fiber can improve the texture and texture of dough, give baked goods better shape and texture, or

The insoluble dietary fiber can increase viscosity of yogurt, shorten fermentation time, and improve taste, or

In foods such as bread, biscuits, beverages, etc., insoluble dietary fibers become important functional components by providing a volume of food and improving intestinal health functions, etc.

One of the important applications is to combine them with other substances to exert a synergistic effect on each other, e.g. adsorption and scavenging, in which insoluble dietary fibres can adsorb harmful substances, e.g. scavenge Nitric Oxide (NO), by forming a complex with metal ions. Studies show that bridging action can be formed between carboxyl groups and metal ions of the dietary fiber, so that a dietary fiber-metal ion-NO complex is generated, and NO is further removed.

Antioxidant function, insoluble dietary fiber is used for embedding antioxidant (such as puerarin), and the release efficiency and bioavailability of the insoluble dietary fiber in vivo are improved by microcapsule technology. For example, the Hericium erinaceus insoluble dietary fiber is used as a wall material, and puerarin is successfully embedded, so that the slow release effect of the antioxidant is remarkably improved.

Lipid-lowering function the insoluble dietary fiber inhibits pancreatic lipase activity by combining cholesterol micelles and cholate, thereby exerting lipid-lowering effect. For example, orange peel insoluble dietary fiber has strong adsorption capacity to soybean oil and can significantly inhibit pancreatic lipase activity.

However, it has been found that the insoluble dietary fiber is usually used for adsorbing substances such as metal ions and cholesterol in the prior art, but the combination with polyphenols such as EGCG is still blank, and no existing cases exist, which indicate that the combination with EGCG is not explored although the dietary fiber is usually used for adsorbing other components. And EGCG can be directly used by itself because of the relatively large molecular weight, and is not generally thought to be compounded with dietary fiber.

In order to solve the problems that the dietary fiber prepared by the traditional method is single in function, a composite system of dietary fiber and polyphenol is not developed, and a stable combination technology of polyphenol and fiber is not mature, so that the composite has low in vivo retention rate, insignificant effect and the like, the inventor conducts intensive research on insoluble dietary fiber, especially bean dreg insoluble dietary fiber (HPSIDF) and polyphenol epigallocatechin gallate (EGCG), and the composite of the insoluble dietary fiber and the polyphenol epigallocatechin gallate (EGCG), and provides a preferable scheme, and experiments prove that the composite can fully exert the synergistic effect of the insoluble dietary fiber and the polyphenol, the effect proves that the retention rate of the composite in simulated gastric and intestinal fluids is far beyond expectation, the problem that EGCG is easy to decompose under the acidic condition is remarkably overcome, the result of in vivo animal experiments shows that the synergistic effect of the composite is remarkably superior to that of a single component in the measurement of inflammatory factor level, the data show that the effect of the composite is not simply overlapped, but is the synergistic effect unpredictable in the prior art, and the EGCG is beneficial for HPSIDF overall addition of beneficial gram bacteria is promoted; in the comparative example, the adsorption effect of the rice bran dietary fiber on the blueberry polyphenol is greatly influenced by temperature, and the bean dreg insoluble dietary fiber is more stable under the same condition, so that the combination of the bean dreg insoluble dietary fiber and EGCG is further proved to have specific advantages.

Specifically, the invention provides a preparation method of an insoluble dietary fiber-epigallocatechin gallate (EGCG) compound, which comprises the following steps:

Dispersing insoluble dietary fiber in phosphate buffer solution, fully expanding, mixing with epigallocatechin gallate (EGCG) solution dissolved in phosphate buffer solution, standing at room temperature for a period of time, centrifuging, collecting precipitate, and drying to obtain insoluble dietary fiber-epigallocatechin gallate (EGCG) complex.

In the preparation step, insoluble dietary fiber is dispersed in phosphate buffer solution and fully swelled as much as possible by 20-60 min, preferably 30 min. By "substantially expanded" is meant herein the phenomenon that under certain conditions, the insoluble fiber is able to absorb and retain a substantial amount of moisture upon contact with water or other solvents, thereby substantially increasing its volume, as understood in the art.

In the above preparation steps, the insoluble dietary fiber and EGCG solution dissolved in phosphate buffer are preferably left for 1-4 hours, preferably 2 hours, at room temperature. The in vitro characterization result shows that the HPSIDF-EGCG compound is in a non-covalent form, such as strong hydrogen bond, hydrophobic interaction and weak electrostatic interaction for reaction combination during the placement, so that the placement is only required to ensure that the two are mixed together, and the placement can be standing, slow stirring or intense stirring.

In the above preparation steps, the centrifuged precipitate may be dried, for example, by vacuum filtration, drying in the shade, vacuum freeze-drying, or the like.

In the above preparation steps, the main source of insoluble dietary fiber may be vegetable food, especially hulls or fibrous parts of cereals, legumes, nuts, vegetables and fruits, e.g. extracted from rhizoma Polygonati residues, or from lettuce leaves, etc. Preferably, the insoluble dietary fiber adopts bean dreg insoluble dietary fiber (HPSIDF), which can construct a better composite system with EGCG and has a synergistic development space.

Specifically, the bean dreg insoluble dietary fiber can be prepared from defatted bean dreg by a complex enzyme method.

The dissolution of defatted soybean meal refers to a process in which defatted soybean meal is mixed with a solvent to disperse components in the soybean meal in the solvent to form a uniform system.

Preferably, the specific steps of the complex enzyme method comprise:

Dissolving defatted soybean meal, sequentially adding alpha-amylase, neutral protease and amyloglucosidase, carrying out enzymolysis, washing, selecting insoluble solid substances, and specifically carrying out washing, centrifugation, alcohol precipitation, suction filtration and vacuum freeze drying to obtain the soybean meal insoluble dietary fiber.

Preferably, the mass concentration of the bean dreg insoluble dietary fiber in the reaction system is 2 mg/mL, the mass concentration of the epigallocatechin gallate (EGCG) is 0.2-0.6 mg/mL, and the mass ratio of the bean dreg insoluble dietary fiber to the epigallocatechin gallate (EGCG) is 3.3-10:1, preferably 5:1.

Preferably, in the step of taking the precipitate after the reaction, the centrifugation condition is, for example, 7500 r/min, and the centrifugation time is 10-15 min.

Preferably, the adsorption rate of the insoluble dietary fiber-EGCG compound for adsorbing EGCG is 15-20%, and the adsorption amount is 0.03-0.04 g/g.

Preferably, the retention rate of the insoluble dietary fiber-EGCG compound in simulated gastric fluid with the pH value of 1.2 is more than or equal to 80 percent, and the retention rate of the insoluble dietary fiber-EGCG compound in simulated intestinal fluid with the pH value of 6.8 is more than or equal to 85 percent.

The invention also provides an insoluble dietary fiber-EGCG compound prepared by the preparation method.

The invention also provides application of the insoluble dietary fiber-EGCG compound in food additives.

The invention will be further illustrated by the following examples. It should be noted that the following examples are illustrative only and are not intended to limit the present invention. All other embodiments, which can be made by a person of ordinary skill in the art without any inventive effort, are within the scope of the embodiments of the present invention based on the embodiments presented below.

Example 1

Screening the compounding proportion of bean dreg insoluble dietary fiber (HPSIDF) and epigallocatechin gallate (EGCG).

Referring to FIG. 1,2 parts (40 mg) of bean dreg insoluble dietary fiber (HPSIDF) extracted by a complex enzyme method is weighed and dissolved in 1 part (20 mL) of PBS buffer (pH 7.0-7.2) to obtain HPSIDF with a mass concentration of 2 mg/mL (w/v), epigallocatechin gallate (EGCG) of 4mg is weighed and dissolved in 20 mL PBS buffer to obtain a solution with a mass concentration of 0.2 mg/mL (w/v) of epigallocatechin gallate (EGCG), the two solutions are uniformly mixed and fully reacted for 2 hours at room temperature, and the mass concentration of epigallocatechin gallate (EGCG) in the total reaction system is 0.1mg/mL (w/v). Centrifuging at 7500 r/min, vacuum lyophilizing the precipitate to obtain bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) complex.

Example 2

And weighing 6 mg of epigallocatechin gallate (EGCG) and dissolving in 20 mL PBS buffer to obtain a solution with the mass concentration of the epigallocatechin gallate (EGCG) of 0.3 mg/mL (m/v), uniformly mixing the two solutions, and fully reacting for 2 hours at room temperature, wherein the mass concentration of the epigallocatechin gallate (EGCG) in the total reaction system is 0.15 mg/mL (w/v). Centrifuging at 7500 r/min, vacuum lyophilizing the precipitate to obtain insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound with different compounding ratio.

Example 3

And weighing and dissolving the epigallocatechin gallate (EGCG) of 8mg in a 20 mL PBS buffer solution to obtain a solution with the mass concentration of the epigallocatechin gallate (EGCG) of 0.4 mg/mL (m/v), uniformly mixing the two solutions, and fully reacting for 2 hours at room temperature, wherein the mass concentration of the epigallocatechin gallate (EGCG) accounts for 0.2 mg/mL (w/v) of the total reaction system. Centrifuging at 7500 r/min, vacuum lyophilizing the precipitate to obtain insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound with different compounding ratio.

Example 4

And weighing and dissolving 10 mg of epigallocatechin gallate (EGCG) in a 20mL PBS buffer solution to obtain a solution with the mass concentration of the epigallocatechin gallate (EGCG) of 0.5 mg/mL (m/v), uniformly mixing the two solutions, and fully reacting for 2 hours at room temperature, wherein the mass concentration of the epigallocatechin gallate (EGCG) in a total reaction system is 0.25 mg/mL (w/v). Centrifuging at 7500 r/min, vacuum lyophilizing the precipitate to obtain insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound with different compounding ratio.

Example 5

And weighing 12 mg of epigallocatechin gallate (EGCG) and dissolving in 20 mL PBS of buffer solution to obtain a solution with the mass concentration of the epigallocatechin gallate (EGCG) of 0.6 mg/mL (m/v), uniformly mixing the two solutions, and fully reacting for 2 hours at room temperature, wherein the mass concentration of the epigallocatechin gallate (EGCG) in the total reaction system is 0.3 mg/mL (w/v). Centrifuging at 7500 r/min, vacuum lyophilizing the precipitate to obtain insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound with different compounding ratio.

The insoluble dietary fiber-epigallocatechin gallate complexes prepared in examples 1 to 5 were measured as follows:

determination of adsorption Capacity of Bean dreg insoluble dietary fiber (HPSIDF) to epigallocatechin gallate (EGCG)

The content of epigallocatechin gallate (EGCG) in the obtained bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) complex was determined by a Folin-Ciocalteu (FC) method. A sample of the complex 0.5 mL (0.2 mg/mL) was taken and reacted with FC reagent (0.2N) of 2.5 mL for 10 min, na ₂CO₃ (75 mg/mL) of 2mL was added, vortexed and mixed well, and the mixture was left to react at room temperature for 2 hours in the absence of light, and the absorbance was measured at 760 nm using an enzyme-labeled instrument. The adsorbed epigallocatechin gallate (EGCG) content was calculated by plotting a standard curve of epigallocatechin gallate concentration.

The adsorption rate of the bean dreg insoluble dietary fiber (HPSIDF) to epigallocatechin gallate (EGCG) is calculated according to formula (1):

Epigallocatechin gallate (EGCG) adsorption rate (%) =

×100 (1)。

The adsorption amount of the bean dreg insoluble dietary fiber (HPSIDF) to epigallocatechin gallate (EGCG) is calculated according to the formula (2):

epigallocatechin gallate (EGCG) adsorption amount (g/g) = (2)。

Wherein C0 is the mass concentration (mg/mL) of epigallocatechin gallate (EGCG) in a solution system before adsorption, ct is the mass concentration (mg/mL) of epigallocatechin gallate (EGCG) in a supernatant after adsorption, V is the total volume (mL) of the solution when an adsorption reaction occurs, and m is the mass (mg) of insoluble dietary fiber of bean dregs.

The results are shown in Table 1, and it can be seen from the table that the adsorption rate reaches 18% and the adsorption amount (content of combined EGCG per gram HPSIDF) reaches 0.035 and g/g when the compounding ratio of the bean dreg insoluble dietary fiber (HPSIDF) and the epigallocatechin gallate (EGCG) is 20:4. For convenience of description, only the adsorption amount is selected as a range limiting standard to determine the protection range in the present invention.

Table 1 adsorption characteristic results list

Comparative example 1

The specific preparation procedure was the same as in example 1, except that the insoluble dietary fiber of rice bran was used instead of the insoluble dietary fiber of bean dregs. Comparing the blueberry polyphenol adsorption amounts of the two to obtain the following conclusion:

The rice bran insoluble dietary fiber can adsorb blueberry polyphenol, and under the condition of different environmental factors, the adsorption amount of the rice bran insoluble dietary fiber to the blueberry polyphenol exists in different sections. Under the same neutral condition (pH 7.0-7.2), the adsorption quantity of the rice bran insoluble dietary fiber to the blueberry polyphenol reaches 0.03-0.04 (g/g) range. However, the temperature and the content of blueberry polyphenol adsorbed by the rice bran insoluble dietary fiber are in a negative correlation trend, and the adsorption amount of the blueberry polyphenol by the rice bran insoluble dietary fiber is reduced from 0.05-0.10 to 0.00-0.05 along with the temperature rising from room temperature to 55 ℃. The bean dreg insoluble dietary fiber has good adsorption capacity.

Simulated digestion of the okara insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) complex in vitro.

1. And (5) simulating the preparation of digestive juice.

The preparation method of the simulated digestive juice is prepared according to the method of United states Pharmacopeia (United States Pharmacopoeia, USP), and the specific method is as follows:

the simulated gastric fluid (Simulated gastric fluid, SGF) is prepared by adding 7mL of hydrochloric acid (HCl) into 2g of sodium chloride (NaCl) and 3.2g of pepsin, and fixing the volume to 1L by using sterile water to obtain the simulated gastric fluid, wherein the pH of the simulated gastric fluid is about 1.2.

Preparation of simulated intestinal fluid (Simulated Intestinal fluid, SIF) comprises taking 6.8g of potassium dihydrogen phosphate (KH ₂PO₄), adding sterile water 250 mL for dissolution, adding 0.2 mol/L sodium chloride (NaCl) solution 77 mL and 500 mL sterile water, adding 10g of trypsin for dissolution, adjusting pH to about 6.8 with 0.2 mol/L sodium hydroxide (NaOH) or dilute hydrochloric acid (HCl) solution, and fixing volume to 1L with sterile water to obtain simulated intestinal fluid.

2. Methods for simulating digestion in vitro.

When the simulated gastric juice is digested, a proper volume of simulated gastric juice is added to enable the mass concentration (w/v) of epigallocatechin gallate (EGCG) to reach 1 mg/mL, and water bath mixing is carried out for 2h at 37 ℃.

When the simulated intestinal juice is digested, a proper volume of the simulated intestinal juice is added, so that the mass concentration (w/v) of epigallocatechin gallate (EGCG) reaches 1 mg/mL, and water bath mixing is carried out for 4 hours at 37 ℃.

3. Measurement of retention.

The bean dreg insoluble dietary fiber (HPSIDF) has certain stability on the adsorption of epigallocatechin gallate (EGCG) as measured by a Folin-Ciocalteu (FC) method. According to the standard curve of the concentration of the epigallocatechin gallate (EGCG), after the digestion of simulated gastric fluid, the bean dreg insoluble dietary fiber-epigallocatechin gallate (EGCG) compound is digested by strong acid environment to decompose a part of bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound, the digested and decomposed unstable free phenol is diffused in the solution, the retention rate is 80.1%, and the retention rate is 86.6% after the digestion of simulated intestinal fluid. Therefore, it is presumed that the retention rate of the epigallocatechin gallate (EGCG) complex reaching the intestinal tract after digestion and decomposition in the stomach can reach 69.3%, and the hole adsorption epigallocatechin gallate (EGCG) of the bean dreg insoluble dietary fiber (HPSIDF) forms a stable structure under neutral conditions.

Animal tests were performed on the bean dreg insoluble dietary fiber-epigallocatechin gallate complexes prepared in examples 1-5.

1. Selection of animals.

Healthy adult male C57BL/6 mice were selected 60, randomly assigned to 5 groups of 12 mice each, fed different diets, respectively:

group A is normal Control group (Control) common feed;

Group B is model control group (DSS) and is prepared by molding common feed and dextran sulfate;

group C, intervention group (DSS+ HPSIDF), common feed, dextran sulfate modeling, and bean dreg insoluble dietary fiber;

Group D, intervention group (DSS+EGCG) of common feed, dextran sulfate modeling, and epigallocatechin gallate;

group E, intervention group (DSS+ HPSIDF-EGCG) common feed, dextran sulfate modeling, and insoluble dietary fiber of bean dreg-epigallocatechin gallate complex.

In FIG. 9, group A is a normal Control group (Control), group B is a model Control group (DSS), group C is an intervention group (DSS+ HPSIDF), group D is an intervention group (DSS+EGCG), and group E is an intervention group (DSS+ HPSIDF-EGCG).

The mice were fed with basic feed for 1 week before molding, and were randomly divided into 5 groups after their metabolic states were stabilized, one group was fed with drinking water and normal feed as normal control groups, one group was fed with dextran sulfate solution and normal feed as model control groups, one group was fed with dextran sulfate solution, normal feed and additional intake of 10 mg/day okara insoluble dietary fiber as intervention groups, one group was fed with dextran sulfate solution, normal feed and additional intake of 10 mg/day okara insoluble dietary fiber-epigallocatechin gallate complex as intervention groups, one group was fed with dextran sulfate solution, and additional intake of 0.35 g/day epigallocatechin gallate as intervention groups. Every 6 cages. The illumination time is 12 hours per day, the temperature is controlled at 20+/-2 ℃ and the humidity is 50+/-5%. After 7 days of mice feeding, the eyeballs were sacrificed by blood collection. The mice were weighed before death, the abdominal cavity was opened after sacrifice, the colon of the mice was immediately isolated, and 0.5cm of colon tissue was taken from each of the distal and proximal ends of the colon for hematoxylin-eosin (H & E) staining.

2. Preparation of mouse tissue samples.

Immediately after mice were sacrificed, the abdominal cavity was opened, 0.2g of colon tissue was removed from the intact colon tissue and placed into a 5mL centrifuge tube, 2 mL PBS buffer was added with a pipette, and the bottom of the centrifuge tube of 5mL was placed into a container containing ice, the left hand was held with a portable homogenizer at a speed of 10000-15000 r/min, the right hand was used to vertically insert the tamper into the 5mL centrifuge tube, and grinding was performed several tens of times (6-8 min), and sufficient grinding was performed to allow the colon tissue to be sufficiently homogenized. The homogenized tissue fluid was centrifuged at 3000 r/min at 4℃for 15: 15min, and the supernatant was then taken for examination.

3. Measurement of inflammatory factor levels.

The indexes of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), interleukin 8 (IL-8), myeloperoxidase antibody (MPO), reactive Oxygen Species (ROS), superoxide dismutase (SOD) and tumor necrosis factor (TNF-alpha) in colon tissues of mice are strictly operated according to the instruction of an enzyme-linked immunosorbent assay (ELISA) kit.

Results and analysis:

FIGS. 2A and 2B are bar graphs of mouse tumor necrosis factor-alpha (TNF-alpha) levels from different experimental groups. Wherein the different letters representing the level above the different columns represent the presence of significant differences (p < 0.05), fig. 2A for serum inflammation levels and fig. 2B for tissue inflammation levels.

FIGS. 3A and 3B are bar graphs of interleukin 1 beta (IL-1 beta) levels in mice from different experimental groups. Wherein the different letters above the different columns represent significant differences (p < 0.05), FIG. 3A is serum inflammation level, and FIG. 3B is tissue inflammation level.

Fig. 4 is a line graph of the mass concentration and adsorption rate of the okara insoluble dietary fiber of the present invention to epigallocatechin gallate.

FIGS. 5A and 5B are bar graphs of the levels of mouse interleukin 8 (IL-8) in different experimental groups. Wherein different lowercase letters above different columns represent significant differences (p < 0.05), fig. 5A for serum inflammation levels and fig. 5B for tissue inflammation levels.

FIG. 6 is a bar graph of MPO levels in tissues of different experimental groups. Wherein the different letters above the different columns represent that there is a significant difference (p < 0.05).

Fig. 7 is a bar graph of ROS levels in tissues of different experimental groups. Wherein the different letters represent the presence of a significant difference (p < 0.05).

FIG. 8 is a bar graph of SOD levels in tissues of different experimental groups. Wherein the different letters represent the presence of a significant difference (p < 0.05).

Fig. 9 is a bar graph of intestinal length of acute ulcerative colitis mice in different experimental groups. Wherein the different letters represent the presence of a significant difference (p < 0.05).

As can be seen from fig. 2A-9, compared with the group B model control group, both the group a normal control group and the group E intervention group are significantly lower than the group B model control group, and intake of the okara insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) complex can reduce release of IL-1β by immune cells of the organism to a certain extent, thereby alleviating damage of the organism; compared with the group B model control group, the group E intervention group is obviously lower than the group B model control group, the excessive release of IL-6 is effectively relieved after the bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound is ingested, compared with the group A normal control group and the group B model control group, the group E intervention group has obvious change, the bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound is ingested to a certain extent, the release of IL-8 is inhibited to a certain extent, the aggregation of neutrophils is avoided, the inflammatory reaction is relieved, compared with the group B model control group, the group E intervention group is obviously reduced, the level of MPO is reduced to a certain extent after the bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound is ingested, the body is promoted to release MPO to take part in the regulation and control of inflammation, compared with the group B model control group, the group E intervention group is quite similar to the value of the group D intervention group, the number of the group E intervention group is obviously reduced, the bean dreg insoluble dietary fiber (34) -epigallocatechin gallate (EGCG) is gradually developed to the normal control group A normal control group, the SOD level of the E group intervention group has a trend of approaching to the inflammation level of the healthy group, the intake of the bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound can inhibit the generation of oxygen free radicals in the body and relieve the condition of oxidative stress, and compared with the B group model control group, the TNF-alpha level of the E group intervention group is obviously reduced, and in general, the higher the TNF-alpha level is, the heavier the body inflammation is represented, and the intake of the bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound effectively relieves the inflammation.

In conclusion, the results show that the bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound has good anti-inflammatory capability, simultaneously, the polysaccharide-polyphenol compound with prebiotic potential has a protective effect on DSS-induced acute enteritis, and simultaneously, compared with two intervention methods of independently intervention of the bean dreg insoluble dietary fiber (HPSIDF) and independently intervention of the epigallocatechin gallate (EGCG), each index of the bean dreg insoluble dietary fiber (HPSIDF) -epigallocatechin gallate (EGCG) compound in a mouse body is superior to that of an independent intervention group, and the polysaccharide-polyphenol compound has a synergistic effect to a certain extent.

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

Immediately after the mice were sacrificed, the abdominal cavity was opened and the whole colon tissue was removed to measure the length. The colon was rinsed with PBS solution to clear the feces therefrom. Tissue samples of approximately 0.5cm each were taken from the distal and proximal ends of the colon and stained with hematoxylin-eosin (H & E).

Colon H & E staining method:

(1) Cutting an intestinal section with the length of about 1cm from the anus at a position 2cm away from the anus, and cleaning the intestinal section in PBS solution;

(2) The specimens were fixed in 4% paraformaldehyde for about 12 hours;

(3) Embedding the specimen in paraffin, and slicing along the coronal surface of the intestinal canal to a thickness of about 4 μm;

(4) Flattening the slice, placing the slice on a glass slide, and drying the slice in a 45 ℃ incubator;

(5) The xylene is deparaffinized, three times, 20 min times each time;

(6) Sequentially processing slices with alcohol from high concentration to low concentration, wherein each slice is 2 min of 100% alcohol, 95% alcohol, 85% alcohol and 70% alcohol;

(7) Placing into distilled water to clean slices 5 min;

(8) Hematoxylin staining 5 min followed by rinsing with distilled water again 1 min;

(9) Washing the glass slide with distilled water again after 1% hydrochloric acid alcohol (70% alcohol) is used for color separation for 10 s;

(10) Mixing 0.1% eosin dye solution with 2 drops of glacial acetic acid for dyeing 5 min;

(11) Sequentially processing slices with alcohol from low concentration to high concentration, wherein each slice is 2 min of 70% alcohol, 85% alcohol, 95% alcohol and 100% alcohol;

(12) Xylene treatment 2 times, 5 min at a time;

(13) Sealing the cover glass with neutral gum;

(14) HE staining was followed by observation under a microscope.

Fig. 10 is a hematoxylin-eosin (H & E) staining of colon tissue of acute ulcerative colitis mice of different experimental groups. As can be seen from the figure, the colonic mucosa of the DSS group of animals was severely damaged and the goblet cell number was reduced with significant inflammatory cell infiltration. In contrast, the colon mucosa structure of the DSS+ HPSIDF-EGCG group test animals is obviously recovered, inflammatory cell infiltration is obviously reduced, and the number of goblet cells is increased, so that the compound is beneficial to repairing the mucosa barrier function and relieving inflammatory reaction. In addition, colon tissue length measurements further validated this conclusion. The colon tissue length of the DSS group tested animals is obviously shortened, and the colon tissue length of the DSS+ HPSIDF-EGCG group is obviously improved (p is less than 0.05) compared with other DSS treatment groups, so that the compound can effectively relieve colon atrophy and inhibit intestinal damage.

The invention also provides application of the method in preparation of the bean dreg insoluble dietary fiber-epigallocatechin gallate compound. The compound improves the added value and the physiological activity of the bean dreg dietary fiber through the synergistic effect. The complex can relieve intestinal inflammation by inhibiting the release of IL-1 beta, IL-6, IL-8 and TNF-alpha inflammatory factors. The complex reduces oxidative stress damage by scavenging Reactive Oxygen Species (ROS) and enhancing superoxide dismutase (SOD) activity.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, but rather, the foregoing embodiments and description are merely preferred embodiments of the present invention, and that any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. A method for preparing an insoluble dietary fiber-EGCG compound, comprising the steps of:

Dispersing insoluble dietary fiber in phosphate buffer solution, uniformly mixing with EGCG solution dissolved in phosphate buffer solution, standing at room temperature for a period of time, centrifuging, and drying precipitate to obtain the insoluble dietary fiber-EGCG compound.

2. The method according to claim 1, wherein,

Dispersing the insoluble dietary fiber in phosphate buffer solution, and fully expanding for 20-60 min, and/or

The insoluble dietary fiber and EGCG solution dissolved in phosphate buffer solution are stirred uniformly and then placed for 1-4 hours at room temperature, and/or

The precipitate after centrifugation is dried by vacuum filtration and then dried in the shade or vacuum freeze drying.

3. The method according to claim 2, wherein,

The insoluble dietary fiber is fully swelled by 30min when dispersed in phosphate buffer, and/or

After the insoluble dietary fiber and the EGCG solution dissolved in the phosphate buffer solution are uniformly stirred, the mixture is placed for 2 hours at room temperature.

4. The method of claim 3, wherein the insoluble dietary fiber is okara insoluble dietary fiber.

5. The method according to claim 4, wherein the bean dreg insoluble dietary fiber is prepared from defatted soybean meal by a complex enzyme method, and the complex enzyme method specifically comprises the steps of dissolving defatted soybean meal, sequentially adding alpha-amylase, neutral protease and amyloglucosidase, washing with water after enzymolysis, separating out solid matters and drying.

6. The method according to claim 4, wherein the mass ratio of the bean dreg insoluble dietary fiber to EGCG is 3.3-10:1, and/or

And the condition of centrifugation after being placed for 1-4 hours at room temperature is that the centrifugation speed is 7500 r/min and the centrifugation time is 10-15 min.

7. The preparation method according to claim 6, wherein the mass ratio of the bean dreg insoluble dietary fiber to EGCG is 5:1.

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

9. Use of the insoluble dietary fiber-EGCG complex of claim 8 in a food additive.