Composite particle with prebiotic function and colon-targeted co-delivery performance as well as preparation method and application of composite particle

Abstract

The invention discloses a composite particle with a prebiotic function and colon-targeted co-delivery performance as well as a preparation method and application thereof, and belongs to the technical field of functional foods and intestinal micro-ecological regulators. According to the composite particle, the curcumin and the resveratrol are encapsulated by the peanut protein isolate to form a nano core, the outer layer of the nano core is a composite shell layer composed of the carboxymethyl konjac glucomannan and the prebiotics, a stable core-shell structure is formed, and the composite particle is small in particle size, good in dispersity and stability and good in stability. The stability of a protein-based delivery system in a gastrointestinal tract environment can be obviously improved; the release rate of the composite particles in simulated gastric juice and intestinal juice is low, and the composite particles can be specifically degraded and release active ingredients in simulated colon fluid containing beta-mannase, so that colon-targeted co-delivery can be realized. The composite particle disclosed by the invention is simple in preparation process and stable in function, can be applied to the fields of functional foods, health foods, formula foods for special medical purposes and the like, realizes dual purposes of active component delivery and intestinal microenvironment regulation, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of functional foods and intestinal microecological regulators, specifically relating to a composite particle with prebiotic function and colon-targeted co-delivery performance, its preparation method, and its application. Background Technology

[0002] Curcumin, a natural polyphenol extracted from the rhizome of turmeric, possesses antioxidant, anti-inflammatory, and antibacterial activities, showing great potential in the prevention of colonic diseases. Resveratrol, another natural polyphenol found in numerous plants, has also been shown to have beneficial effects on colitis, colon cancer, and other lower gastrointestinal diseases. Studies have shown that the combined use of curcumin and resveratrol can exert a stronger synergistic effect, enhancing intestinal antioxidant capacity, improving intestinal mucosal integrity, and promoting gut microbiota health, making them promising synergistic bioactive components. However, the poor water solubility, light and heat sensitivity, and easy degradation in the gastrointestinal tract of both curcumin and resveratrol reduce their concentrations in the colon after oral administration, limiting their synergistic beneficial effects in the colon. Based on these issues, there is an urgent need to construct a co-delivery system with colon-targeted co-delivery capabilities for the co-encapsulation of curcumin and resveratrol, achieving targeted release and bioavailability of both in the colon, and maximizing their synergistic effects.

[0003] Protein-based colloidal particles are ideal carriers for encapsulating hydrophobic polyphenolic compounds. Peanut protein isolate, due to its good biocompatibility and biodegradability, as well as its excellent granulation properties, has become a good wall material for preparing nanoparticles and microcapsules. However, as a protein-based colloidal system, peanut protein particles tend to aggregate at pH close to their isoelectric point or at high salt concentrations, and their encapsulation effect and environmental stability are relatively poor, limiting their application in constructing colloidal particles.

[0004] Furthermore, the health of the colon is closely related to the balance of gut microbiota. Prebiotics, as indigestible food components, can selectively stimulate the growth and activity of beneficial bacteria in the colon. Co-delivering prebiotics with curcumin and resveratrol could potentially create a synergistic healthy microenvironment in the colon: prebiotics promote the proliferation of beneficial bacteria and produce beneficial metabolites (such as short-chain fatty acids), which may further enhance the bioavailability and bioactivity of polyphenols, jointly strengthening the prevention and intervention effects on colonic diseases. However, currently, there is a lack of integrated particle systems that can simultaneously achieve efficient colon-targeted delivery of polyphenols and precise supply of prebiotics, especially the synergistic use of prebiotics with carboxymethylated konjac glucomannan to construct a protein-based delivery system with stability, targeting capabilities, and microecological regulation functions. Summary of the Invention

[0005] Based on the above requirements, the purpose of this invention is to provide a composite particle with prebiotic function and colon-targeted co-delivery performance, as well as its preparation method and application. The composite particle prepared by this invention can not only improve the stability of peanut protein-based colloidal particles in the gastrointestinal environment, but also achieve efficient co-encapsulation and colon-targeted release of curcumin and resveratrol, and regulate the intestinal microecology with the help of the outer prebiotic layer, thereby forming a "prebiotic-polyphenol" synergistic intervention system in the colon.

[0006] To achieve the above objectives, the present invention is implemented through the following solution:

[0007] This invention provides a method for preparing composite particles with prebiotic function and colon-targeted co-delivery properties, the method comprising the following steps:

[0008] (1) Dissolve peanut protein isolate in water and heat and sonicate to obtain the treated peanut protein dispersion;

[0009] (2) Add the ethanol aqueous solution containing curcumin and resveratrol dropwise to the peanut protein dispersion, stir continuously and then remove the ethanol by rotary evaporation to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol.

[0010] (3) Disperse konjac glucomannan in ethanol, add sodium hydroxide and chloroacetic acid solution in sequence, stir continuously to react, collect the precipitate, wash and obtain carboxymethyl konjac glucomannan;

[0011] (4) Dissolve the carboxymethyl konjac glucomannan and prebiotics in water to obtain carboxymethyl konjac glucomannan solution and prebiotic solution respectively. Mix them after continuous stirring to obtain carboxymethyl konjac glucomannan-prebiotic composite solution.

[0012] (5) The carboxymethyl konjac glucomannan-prebiotic composite solution is added dropwise to the peanut protein nanocore suspension, the pH of the system is adjusted, and the mixture is stirred continuously. Through electrostatic interaction, the composite particles with a core-shell structure are formed through self-assembly.

[0013] Furthermore, in step (1), the mass concentration of peanut protein isolate is 1~10 mg / mL, and the heating conditions are: temperature 75~95℃, time 10~20 min.

[0014] Furthermore, the conditions for the ultrasound are: power 200~500 W, time 5~30 min.

[0015] Furthermore, the mass ratio of curcumin to resveratrol is 1:1, and the total mass concentration of curcumin and resveratrol in the ethanol aqueous solution is 0.5~2 mg / mL.

[0016] Furthermore, the volume ratio of the ethanol aqueous solution of curcumin and resveratrol to the peanut protein isolate dispersion is 1:7~10.

[0017] Furthermore, the dropping rate of the ethanol aqueous solution of curcumin and resveratrol is 1-3 drops / s.

[0018] Furthermore, the stirring conditions in step (2) include: a temperature of 25~35℃, a stirring rate of 300-500 r / min, and a time of 5-10 min.

[0019] Furthermore, the rotary evaporation conditions in step (2) include: a temperature of 30~45℃ and a time of 5~10 min.

[0020] Furthermore, the molar ratio of chloroacetic acid to sodium hydroxide is 1~3:1.

[0021] Furthermore, the reaction conditions in step (3) include: a temperature of 40~60℃ and a time of 2~4 h.

[0022] Furthermore, the washing method involves sequentially washing with 70% ethanol, 95% ethanol, and anhydrous ethanol by volume fraction.

[0023] Furthermore, in step (4), the prebiotics include fructooligosaccharides and / or resistant dextrin; the mass concentration of carboxymethyl konjac glucomannan in the solution is 1-5 mg / mL; the mass concentration of the prebiotics in the solution is 1-5 mg / mL; and the volume ratio of the prebiotic solution to the carboxymethyl konjac glucomannan solution during mixing is 1:1.

[0024] Furthermore, the stirring conditions in step (4) include: a temperature of 25°C, a stirring rate of 300-500 r / min, and a time of 30-60 min.

[0025] Furthermore, the volume ratio of the carboxymethyl konjac glucomannan-prebiotic composite solution to the peanut protein nanonucleus suspension is 1:2 to 2:1.

[0026] Furthermore, the dropping rate of the carboxymethyl konjac glucomannan-prebiotic composite solution is 1~3 drops / s.

[0027] Furthermore, the stirring conditions include: a temperature of 25~35℃, a stirring rate of 300~500 r / min, and a time of 5~10 min.

[0028] Furthermore, the pH of the system is 3 to 4.

[0029] The present invention provides composite particles prepared by the preparation method described above. The composite particles have a core-shell structure, with the core consisting of curcumin and resveratrol encapsulated by peanut protein isolate, and the shell consisting of a composite layer formed by carboxymethyl konjac glucomannan and prebiotics through intermolecular forces.

[0030] Furthermore, the composite particles are orange or yellow, relatively uniformly dispersed, and without obvious precipitation or aggregation.

[0031] Furthermore, the particle size of the composite particles is 100 ~ 400 nm.

[0032] Furthermore, the composite particles carry a negative charge.

[0033] The present invention also provides the application of the aforementioned composite particles in the preparation of functional foods or pharmaceuticals with colon-targeted delivery and intestinal microecological regulation functions.

[0034] The present invention also provides the use of the aforementioned composite particles in the preparation of formulations for the prevention or adjuvant treatment of colitis and / or colon cancer.

[0035] Compared with the prior art, the present invention has the following advantages and positive effects:

[0036] 1. The method for preparing composite particles provided by the present invention is simple, the proteins and polysaccharides involved are inexpensive and biodegradable, and the prepared composite particles have high safety and good biocompatibility.

[0037] 2. This invention involves heating and ultrasonically treating peanut protein isolate to enhance the encapsulation effect of the composite particles on fat-soluble curcumin and resveratrol.

[0038] 3. This invention modifies konjac glucomannan by carboxylation and further combines it with fructooligosaccharides to construct a composite shell that combines electronegativity, colon enzyme responsiveness, and prebiotic function. The polysaccharide is used to form a complex with the protein to further improve the stability of the protein-based colloidal particles encapsulating bioactive substances. The specific degradation characteristics of the selected polysaccharide in the gastrointestinal tract determine the delivery performance of the constructed delivery system, significantly improving the stability and targeting accuracy of the particles in the gastrointestinal environment.

[0039] 4. The composite particles prepared by this invention have a low release rate in simulated gastrointestinal fluid, but a high release rate in simulated colonic fluid containing β-mannanase. They have good colon-targeted co-delivery performance and can significantly improve the bioavailability of curcumin and resveratrol in the colon. This solves the problem that two active ingredients with synergistic effects, such as curcumin and resveratrol, have low oral bioavailability and are difficult to target the colon to exert synergistic effects.

[0040] 5. By introducing fructooligosaccharides as a prebiotic component, this invention enables the delivery system to selectively promote the proliferation of beneficial bacteria in the colon and produce short-chain fatty acids while releasing polyphenols, thereby regulating the intestinal microecology and exerting a synergistic health effect with curcumin and resveratrol, forming an integrated "delivery-regulation" intervention strategy. Attached Figure Description

[0041] The accompanying drawings are provided to further understand the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof.

[0042] Figure 1 The image shows the appearance of the suspension of the composite particles co-encapsulated with curcumin and resveratrol prepared in Examples 1-4.

[0043] Figure 2 The particle size distribution, particle size and potential of the composite particles of co-encapsulated curcumin and resveratrol prepared in Examples 1-4 and Comparative Example 4 are shown.

[0044] Figure 3 The encapsulation efficiency of curcumin and resveratrol in the composite particles prepared in Examples 1-4 and Comparative Examples 2 and 4 is given.

[0045] Figure 4 The release rate of the co-encapsulated curcumin and resveratrol composite particles prepared in Examples 1-4 and Comparative Example 1 in simulated gastric juice is shown.

[0046] Figure 5 The release rate of the co-encapsulated curcumin and resveratrol composite particles prepared in Examples 1-4 and Comparative Example 1 in simulated colonic fluid.

[0047] Figure 6 This is a comparison chart of the short-chain fatty acid yields of the composite particles prepared in Example 1 and Comparative Example 3 in an in vitro colonic fermentation model.

[0048] Figure 7 The effects of the composite particles prepared in Example 1 and Comparative Example 3 on the changes in body weight, disease activity index, and colon length in mice with ulcerative colitis were investigated. Detailed Implementation

[0049] The specific implementation of the present invention will be further described below with reference to the accompanying drawings or embodiments. Experimental instruments or reagents whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0050] Example 1

[0051] The preparation method of the composite particles with prebiotic function and colon-targeted co-delivery performance in this embodiment includes the following steps:

[0052] 1. Heat a 1 mg / mL peanut protein isolate solution in an 80℃ water bath for 10 min, and then sonicate it at 200 W for 30 min to obtain the treated peanut protein dispersion.

[0053] 2. Curcumin and resveratrol were added to an ethanol solution in equal mass to prepare an ethanol aqueous solution with a final mass concentration of 1 mg / mL for the total content of curcumin and resveratrol. The ethanol aqueous solution containing curcumin and resveratrol was added dropwise to the peanut protein dispersion at a dropping rate of 3 drops / s. The volume ratio of the ethanol aqueous solution containing curcumin and resveratrol to the peanut protein dispersion was 1:9. Then, the mixture was stirred continuously at 500 rpm for 10 min at 25°C, followed by rotary evaporation at 40°C for 10 min to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol.

[0054] 3. Disperse 5 g of konjac glucomannan in 40 mL of 70% ethanol. Add sodium hydroxide and chloroacetic acid sequentially to the konjac glucomannan ethanol solution. The molar ratio of chloroacetic acid to sodium hydroxide is 1:1. Stir continuously at 50℃ for 2 h. Adjust the pH of the solution to 7.0. Collect the precipitate and wash it sequentially with 70% ethanol, 95% ethanol and anhydrous ethanol to obtain carboxymethyl konjac glucomannan.

[0055] 4. Dissolve carboxymethyl konjac glucomannan in water and stir continuously at 500 rpm for 60 min at 25℃ to obtain a carboxymethyl konjac glucomannan solution with a mass concentration of 2 mg / mL; dissolve oligofructose in water to prepare an oligofructose solution with a mass concentration of 2 mg / mL; mix the oligofructose solution and the carboxymethyl konjac glucomannan solution at a volume ratio of 1:1 and stir continuously at 500 rpm for 60 min at 25℃ to obtain an oligofructose-carboxymethyl konjac glucomannan composite solution.

[0056] 5. The fructooligosaccharide-carboxymethyl konjac glucomannan composite solution was added dropwise at a rate of 3 drops / s to the peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol. The volume ratio of the fructooligosaccharide-carboxymethyl konjac glucomannan composite solution to the peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol was 1:1. The pH of the system was adjusted to 3.5, and the mixture was stirred continuously at 500 rpm for 10 min at 25°C. Through electrostatic interaction, composite particles with a core-shell structure were formed through self-assembly.

[0057] Example 2

[0058] The preparation method of the composite particles with prebiotic function and colon-targeted co-delivery performance in this embodiment includes the following steps:

[0059] 1. Heat a 1 mg / mL peanut protein isolate solution in a 90℃ water bath for 10 min, and then sonicate it at 500 W for 15 min to obtain the treated peanut protein dispersion.

[0060] 2. Curcumin and resveratrol were added to an ethanol solution in equal mass to prepare an ethanol aqueous solution with a final mass concentration of 1 mg / mL for the total content of curcumin and resveratrol. The ethanol aqueous solution containing curcumin and resveratrol was added dropwise to the peanut protein dispersion at a dropping rate of 3 drops / s. The volume ratio of the ethanol aqueous solution containing curcumin and resveratrol to the peanut protein dispersion was 1:9. Then, the mixture was stirred continuously at 500 rpm for 10 min at 25°C, followed by rotary evaporation at 40°C for 10 min to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol.

[0061] 3. Disperse 5 g of konjac glucomannan in 40 mL of 70% ethanol. Add sodium hydroxide and chloroacetic acid sequentially to the konjac glucomannan ethanol solution. The molar ratio of chloroacetic acid to sodium hydroxide is 1:1. Stir continuously at 50℃ for 2 h. Adjust the pH of the solution to 7.0. Collect the precipitate and wash it sequentially with 70% ethanol, 95% ethanol and anhydrous ethanol to obtain carboxymethyl konjac glucomannan.

[0062] 4. Dissolve carboxymethyl konjac glucomannan in water and stir continuously at 500 rpm for 60 min at 25℃ to obtain a carboxymethyl konjac glucomannan solution with a mass concentration of 2 mg / mL; dissolve oligofructose in water to prepare an oligofructose solution with a mass concentration of 2 mg / mL; mix the oligofructose solution and the carboxymethyl konjac glucomannan solution at a volume ratio of 1:1 and stir continuously at 500 rpm for 60 min at 25℃ to obtain an oligofructose-carboxymethyl konjac glucomannan composite solution.

[0063] 5. The fructooligosaccharide-carboxymethyl konjac glucomannan composite solution was added dropwise at a rate of 3 drops / s to the peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol. The volume ratio of the fructooligosaccharide-carboxymethyl konjac glucomannan composite solution to the peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol was 1:1. The pH of the system was adjusted to 3.5, and the mixture was stirred continuously at 500 rpm for 10 min at 25°C. Through electrostatic interaction, composite particles with a core-shell structure were formed through self-assembly.

[0064] Example 3

[0065] The preparation method of the composite particles with prebiotic function and colon-targeted co-delivery performance in this embodiment includes the following steps:

[0066] 1. Heat a 1 mg / mL peanut protein isolate solution in an 80℃ water bath for 10 min, and then sonicate it at 200 W for 30 min to obtain the treated peanut protein dispersion.

[0067] 2. Curcumin and resveratrol were added to an ethanol solution in equal mass to prepare an ethanol aqueous solution with a final mass concentration of 1 mg / mL for the total content of curcumin and resveratrol. The ethanol aqueous solution containing curcumin and resveratrol was added dropwise to the peanut protein dispersion at a dropping rate of 3 drops / s. The volume ratio of the ethanol aqueous solution containing curcumin and resveratrol to the peanut protein dispersion was 1:9. Then, the mixture was stirred continuously at 500 rpm for 10 min at 25°C, followed by rotary evaporation at 40°C for 10 min to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol.

[0068] 3. Disperse 5 g of konjac glucomannan in 40 mL of 70% ethanol. Add sodium hydroxide and chloroacetic acid sequentially to the konjac glucomannan ethanol solution. The molar ratio of chloroacetic acid to sodium hydroxide is 1:1. Stir continuously at 50℃ for 2 h. Adjust the pH of the solution to 7.0. Collect the precipitate and wash it sequentially with 70% ethanol, 95% ethanol and anhydrous ethanol to obtain carboxymethyl konjac glucomannan.

[0069] 4. Dissolve carboxymethyl konjac glucomannan in water and stir continuously at 500 rpm for 60 min at 25℃ to obtain a carboxymethyl konjac glucomannan solution with a mass concentration of 2 mg / mL; dissolve oligofructose in water to prepare an oligofructose solution with a mass concentration of 4 mg / mL; mix the oligofructose solution and the carboxymethyl konjac glucomannan solution at a volume ratio of 1:1 and stir continuously at 500 rpm for 60 min at 25℃ to obtain an oligofructose-carboxymethyl konjac glucomannan composite solution.

[0070] 5. The fructooligosaccharide-carboxymethyl konjac glucomannan composite solution was added dropwise at a rate of 3 drops / s to the peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol. The volume ratio of the fructooligosaccharide-carboxymethyl konjac glucomannan composite solution to the peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol was 1:1. The pH of the system was adjusted to 3.5, and the mixture was stirred continuously at 500 rpm for 10 min at 25°C. Through electrostatic interaction, composite particles with a core-shell structure were formed through self-assembly.

[0071] Example 4

[0072] The preparation method of the composite particles with prebiotic function and colon-targeted co-delivery performance in this embodiment includes the following steps:

[0073] 1. Heat a 1 mg / mL peanut protein isolate solution in an 80℃ water bath for 10 min, and then sonicate it at 200 W for 30 min to obtain the treated peanut protein dispersion.

[0074] 2. Add equal amounts of curcumin and resveratrol to an ethanol solution to prepare an ethanol aqueous solution with a final mass concentration of 1 mg / mL for the total content of curcumin and resveratrol. Add the ethanol aqueous solution containing curcumin and resveratrol dropwise to the peanut protein dispersion at a dropping rate of 3 drops / s. The volume ratio of the ethanol aqueous solution containing curcumin and resveratrol to the peanut protein dispersion is 1:9. Then, stir continuously at 500 rpm for 10 min at 25°C, followed by rotary evaporation at 40°C for 10 min to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol.

[0075] 3. Disperse 5 g of konjac glucomannan in 40 mL of 70% ethanol. Add sodium hydroxide and chloroacetic acid sequentially to the konjac glucomannan ethanol solution. The molar ratio of chloroacetic acid to sodium hydroxide is 1:1. Stir continuously at 50℃ for 2 h. Adjust the pH of the solution to 7.0. Collect the precipitate and wash it sequentially with 70% ethanol, 95% ethanol and anhydrous ethanol to obtain carboxymethyl konjac glucomannan.

[0076] 4. Dissolve carboxymethyl konjac glucomannan in water and stir continuously at 500 rpm for 60 min at 25℃ to obtain a carboxymethyl konjac glucomannan solution with a mass concentration of 2 mg / mL; dissolve oligofructose in water to prepare an oligofructose solution with a mass concentration of 2 mg / mL; mix the oligofructose solution and the carboxymethyl konjac glucomannan solution at a volume ratio of 1:1 and stir continuously at 500 rpm for 60 min at 25℃ to obtain an oligofructose-carboxymethyl konjac glucomannan composite solution.

[0077] 5. The fructooligosaccharide-carboxymethyl konjac glucomannan composite solution was added dropwise at a rate of 3 drops / s to the peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol. The volume ratio of the fructooligosaccharide-carboxymethyl konjac glucomannan composite solution to the peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol was 2:1. The pH of the system was adjusted to 3.5, and the mixture was stirred continuously at 400 rpm for 10 min at 25°C. Through electrostatic interaction, composite particles with a core-shell structure were formed through self-assembly.

[0078] Comparative Example 1

[0079] The preparation method of the composite particles with prebiotic function and colon-targeted co-delivery performance in this embodiment includes the following steps:

[0080] 1. Heat a 1 mg / mL peanut protein isolate solution in an 80℃ water bath for 10 min, and then sonicate it at 200 W for 30 min to obtain the treated peanut protein dispersion.

[0081] 2. Curcumin and resveratrol were added to an ethanol solution in equal mass to prepare an ethanol aqueous solution with a final mass concentration of 1 mg / mL for the total content of curcumin and resveratrol. The ethanol aqueous solution containing curcumin and resveratrol was added dropwise to the peanut protein dispersion at a dropping rate of 3 drops / s, with a volume ratio of 1:9 between the ethanol aqueous solution containing curcumin and resveratrol and the peanut protein dispersion. The mixture was stirred continuously at 500 rpm for 10 min at 25°C, followed by rotary evaporation at 40°C for 10 min to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol.

[0082] 3. Dissolve konjac glucomannan in water and stir continuously at 500 rpm for 60 min at 25℃ to obtain a konjac glucomannan solution with a mass concentration of 2 mg / mL.

[0083] 4. Dissolve fructooligosaccharides in water to prepare a fructooligosaccharide solution with a mass concentration of 2 mg / mL; mix the fructooligosaccharide solution with the konjac glucomannan solution at a volume ratio of 1:1, and stir continuously at 500 rpm for 60 min at 25℃ to obtain a fructooligosaccharide-konjac glucomannan composite solution.

[0084] 5. The fructooligosaccharide-konjac glucomannan composite solution was added dropwise to the peanut protein nanonucleus suspension co-encapsulated with curcumin and resveratrol at a dropping rate of 3 drops / s. The volume ratio of the fructooligosaccharide-konjac glucomannan composite solution to the peanut protein nanonucleus suspension co-encapsulated with curcumin and resveratrol was 1:1. The pH of the system was adjusted to 3.5, and the mixture was stirred continuously at 500 rpm for 10 min at 25°C to obtain composite particles.

[0085] Comparative Example 2

[0086] The preparation method of the composite particles with prebiotic function and colon-targeted co-delivery performance in this embodiment includes the following steps:

[0087] 1. Prepare a peanut protein isolate solution with a mass concentration of 1 mg / mL.

[0088] 2. Curcumin and resveratrol were added to an ethanol solution in equal mass to prepare an ethanol aqueous solution with a final mass concentration of 1 mg / mL for the total content of curcumin and resveratrol. The ethanol aqueous solution containing curcumin and resveratrol was added dropwise to the peanut protein dispersion at a dropping rate of 3 drops / s. The volume ratio of the ethanol aqueous solution containing curcumin and resveratrol to the peanut protein dispersion was 1:9. Then, the mixture was stirred continuously at 500 rpm for 10 min at 25°C, followed by rotary evaporation at 40°C for 10 min to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol.

[0089] 3. Disperse konjac glucomannan in ethanol, add sodium hydroxide and chloroacetic acid sequentially to the konjac glucomannan ethanol solution, with a molar ratio of chloroacetic acid to sodium hydroxide of 1:1, and stir continuously at 50°C for 4 h. Collect the precipitate and wash it sequentially with 70% ethanol, 95% ethanol and anhydrous ethanol to obtain carboxymethyl konjac glucomannan.

[0090] 4. Dissolve carboxymethyl konjac glucomannan in water and stir continuously at 500 rpm for 60 min at 25℃ to obtain a carboxymethyl konjac glucomannan solution with a mass concentration of 2 mg / mL; dissolve oligofructose in water to prepare an oligofructose solution with a mass concentration of 2 mg / mL; mix the oligofructose solution and the carboxymethyl konjac glucomannan solution at a volume ratio of 1:1 and stir continuously at 500 rpm for 60 min at 25℃ to obtain an oligofructose-carboxymethyl konjac glucomannan composite solution.

[0091] 5. The fructooligosaccharide-carboxymethyl konjac glucomannan composite solution was added dropwise at a rate of 3 drops / s to the peanut protein nanonucleus suspension co-encapsulated with curcumin and resveratrol. The volume ratio of the fructooligosaccharide-carboxymethyl konjac glucomannan composite solution to the peanut protein nanonucleus suspension co-encapsulated with curcumin and resveratrol was 1:1. The pH of the system was adjusted to 3.5, and the mixture was stirred continuously at 500 rpm for 10 min at 25°C to obtain composite particles.

[0092] Comparative Example 3

[0093] The preparation method of the composite particles with prebiotic function and colon-targeted co-delivery performance in this embodiment includes the following steps:

[0094] 1. Heat a 1 mg / mL peanut protein isolate solution in an 80℃ water bath for 10 min, and then sonicate it at 200 W for 30 min to obtain the treated peanut protein dispersion.

[0095] 2. Add equal amounts of curcumin and resveratrol to an ethanol solution to prepare an ethanol aqueous solution with a final mass concentration of 1 mg / mL for the total content of curcumin and resveratrol. Add the ethanol aqueous solution containing curcumin and resveratrol dropwise to the peanut protein dispersion at a dropping rate of 3 drops / s. The volume ratio of the ethanol aqueous solution containing curcumin and resveratrol to the peanut protein dispersion is 1:9. Stir continuously at 500 rpm for 10 min at 25°C, and then rotary evaporate at 40°C for 10 min to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol.

[0096] 3. Disperse konjac glucomannan in ethanol, add sodium hydroxide and chloroacetic acid sequentially to the konjac glucomannan ethanol solution, with a molar ratio of chloroacetic acid to sodium hydroxide of 1:1, and stir continuously at 50°C for 4 h. Collect the precipitate and wash it sequentially with 70% ethanol, 95% ethanol and anhydrous ethanol to obtain carboxymethyl konjac glucomannan.

[0097] 4. Dissolve carboxymethyl konjac glucomannan in water and stir continuously at 500 rpm for 60 min at 25℃ to prepare a carboxymethyl konjac glucomannan solution with a mass concentration of 2 mg / mL.

[0098] 5. The carboxymethyl konjac glucomannan solution was added dropwise to the peanut protein nanonucleus suspension co-encapsulated with curcumin and resveratrol at a dropping rate of 3 drops / s. The volume ratio of the carboxymethyl konjac glucomannan solution to the peanut protein nanonucleus suspension co-encapsulated with curcumin and resveratrol was 1:1. The pH of the system was adjusted to 3.5, and the mixture was stirred continuously at 500 rpm for 10 min at 25°C to obtain composite particles.

[0099] Comparative Example 4

[0100] The preparation method of the composite particles with prebiotic function and colon-targeted co-delivery performance in this embodiment includes the following steps:

[0101] 1. Heat a 1 mg / mL peanut protein isolate solution in an 80℃ water bath for 10 min, and then sonicate it at 200 W for 30 min to obtain the treated peanut protein dispersion.

[0102] 2. Curcumin and resveratrol were added to an ethanol solution in equal mass to prepare an ethanol aqueous solution with a final mass concentration of 1 mg / mL for the total content of curcumin and resveratrol. The ethanol aqueous solution containing curcumin and resveratrol was added dropwise to the peanut protein dispersion at a dropping rate of 3 drops / s, with a volume ratio of 1:9 between the ethanol aqueous solution containing curcumin and resveratrol and the peanut protein dispersion. The mixture was stirred continuously at 500 rpm for 10 min at 25°C, followed by rotary evaporation at 40°C for 10 min to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol.

[0103] 3. Disperse konjac glucomannan in ethanol, add sodium hydroxide and chloroacetic acid sequentially to the konjac glucomannan ethanol solution, with a molar ratio of chloroacetic acid to sodium hydroxide of 1:1, and stir continuously at 50°C for 4 h. Collect the precipitate and wash it sequentially with 70% ethanol, 95% ethanol and anhydrous ethanol to obtain carboxymethyl konjac glucomannan.

[0104] 4. Dissolve carboxymethyl konjac glucomannan in water and stir continuously at 500 rpm for 60 min at 25℃ to obtain a carboxymethyl konjac glucomannan solution with a mass concentration of 2 mg / mL; dissolve oligofructose in water to prepare an oligofructose solution with a mass concentration of 2 mg / mL; mix the oligofructose solution and the carboxymethyl konjac glucomannan solution at a volume ratio of 1:1 and stir continuously at 500 rpm for 60 min at 25℃ to obtain an oligofructose-carboxymethyl konjac glucomannan composite solution.

[0105] 5. The fructooligosaccharide-carboxymethyl konjac glucomannan composite solution was added dropwise at a rate of 3 drops / s to the peanut protein nanonucleus suspension co-encapsulated with curcumin and resveratrol. The volume ratio of the fructooligosaccharide-carboxymethyl konjac glucomannan composite solution to the peanut protein nanonucleus suspension co-encapsulated with curcumin and resveratrol was 1:1. The pH of the system was adjusted to 6.0, and the mixture was stirred continuously at 500 rpm for 10 min at 25°C to obtain composite particles.

[0106] Example 5

[0107] This embodiment provides a method for observing the apparent turbidity of composite particles, including the following steps: taking freshly prepared composite particles into a transparent glass bottle and recording the apparent turbidity of the suspension using a digital camera.

[0108] The appearance of the suspensions of the co-encapsulated curcumin and resveratrol composite particles prepared in Examples 1-4 above was tested, and the results are as follows: Figure 1 As shown.

[0109] Figure 1 The appearance of the suspension of the composite particles co-encapsulated with curcumin and resveratrol is shown by... Figure 1 It can be seen that the composite particles prepared in Examples 1-4 are orange or yellow, relatively uniformly dispersed, and without obvious precipitation or aggregation, indicating that the system has good stability.

[0110] Example 6

[0111] This embodiment provides a method for measuring the particle size distribution, particle size, and potential of composite particles, including the following steps: measuring the particle size and zeta potential of the composite particles using a nanoparticle size analyzer and a zeta potential analyzer, appropriately diluting with pure water to avoid multiple scattering effects, testing at 25°C, and performing three parallel measurements.

[0112] The particle size distribution, particle size, and potential of the composite particles co-encapsulated with curcumin and resveratrol prepared in Examples 1-4 above were tested, and the results are as follows: Figure 2 As shown.

[0113] Figure 2 To determine the particle size distribution, particle size, and potential of the composite particles co-encapsulated with curcumin and resveratrol, the following analysis was conducted. Figure 2 It can be seen that the composite particles co-encapsulated with curcumin and resveratrol prepared in Examples 1-4 have a uniform particle size distribution, and the particle size is all in the nanometer range. Among them, the composite particles prepared in Example 1 have the smallest particle size, about 145.4 nm, and the composite particles prepared in Example 4 have the largest particle size, about 322.8 nm. The composite particles co-encapsulated with curcumin and resveratrol prepared in Examples 1-4 all have a negative charge, indicating that the fructooligosaccharide-carboxymethyl konjac glucomannan complex was successfully coated onto the surface of peanut protein particles.

[0114] Compared to the pH setting of 3.5 in Example 1, the electrostatic interaction between peanut protein and the fructooligosaccharide-carboxymethyl konjac glucomannan complex was weaker under the pH setting of 6 in Comparative Example 4, resulting in poor composite particle assembly, large particle size, and wide particle distribution.

[0115] Example 7

[0116] This embodiment provides a method for determining the encapsulation efficiency of composite particles, including the following steps: adding ethanol to the composite particles and sonicating them at 200 W power and 40 kHz frequency for 30 min; determining the total amount of curcumin and resveratrol using high performance liquid chromatography; placing the composite particles in the inner tube of an ultrafiltration centrifuge tube (Amicon Ultra-10 K) and centrifuging at 4000 g for 20 min; collecting the filtrate from the outer tube; and determining the content of free curcumin and resveratrol using high performance liquid chromatography.

[0117] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method for curcumin determination were as follows: detection wavelength: 425 nm; mobile phase: A: 4% acetic acid, B: acetonitrile; elution program: gradient elution, 0-10 min, 30-100% B; 10-15 min, 100-30% B; flow rate: 1.0 mL / min; column temperature: 25 ℃; injection volume: 20 μL. The HPLC conditions for the chromatographic method for resveratrol determination were as follows: detection wavelength: 306 nm; mobile phase: methanol:water (50%:50%, v / v); elution program: isocratic elution; flow rate: 0.9 mL / min; column temperature: 35 ℃; injection volume: 20 μL.

[0118] The encapsulation efficiency of curcumin and resveratrol in the composite particles was calculated using the following formula:

[0119] ;

[0120] The encapsulation efficiency of the co-encapsulated curcumin and resveratrol composite particles prepared in Examples 1-4 above was tested, and the results are as follows: Figure 3 As shown.

[0121] Figure 3 The encapsulation efficiency of the composite particles co-encapsulating curcumin and resveratrol was determined by... Figure 3 It can be seen that the composite particles prepared in Examples 1-4 have an encapsulation effect of more than 85% on curcumin and resveratrol. The encapsulation rate of resveratrol is slightly higher than that of curcumin, which is related to the poorer water solubility of curcumin compared to resveratrol.

[0122] The encapsulation rates of curcumin and resveratrol in the composite particles prepared in Comparative Examples 2 and 4 were significantly lower than those in Example 1, indicating that heating and ultrasonic treatment plays a key role in exposing the hydrophobic sites of proteins and improving their loading capacity. Similarly, in Comparative Example 4, under the condition of pH 6, the weak interaction between peanut protein and the fructooligosaccharide-carboxymethyl konjac glucomannan complex prevented it from achieving the effect of encapsulating curcumin and resveratrol.

[0123] Example 8

[0124] This embodiment provides a method for determining the release rate of composite particles in simulated gastrointestinal fluid, comprising the following steps: dispersing the composite particles in simulated gastric fluid, incubating in a 37°C water bath shaker, periodically sampling, centrifuging to collect the supernatant, and determining the release amount of active substances using high-performance liquid chromatography; after each centrifugation, resuspending the composite particles in fresh simulated fluid until the next sampling; after incubation for 2 hours, suspending the composite particles in simulated small intestinal fluid, incubating in a water bath shaker under the same conditions for 3 hours, and periodically sampling and measuring; finally, resuspending the composite particles in simulated colonic fluid, and repeating the above operations until the experiment is completed. The release rate of the composite particles in simulated gastrointestinal fluid is expressed as the percentage of the cumulative release of active substances to the total amount of active substances.

[0125] The simulated gastric juice was prepared as follows: 6 mL of concentrated hydrochloric acid was added to 1 L of pure water, the pH was adjusted to 1.2 with 0.1 mol / L HCl, and pepsin (9600 U / L) was added under stirring to dissolve it completely. The mixture was then filtered and set aside.

[0126] The simulated intestinal fluid was prepared as follows: 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate were mixed in a volume ratio of 49:51, and then pure water was added to bring the volume to 1 L. The pH was adjusted to 6.8 with 0.1 mol / L NaOH, and trypsin (25000 U / L) was added under stirring to dissolve it completely. The mixture was then filtered and set aside for later use.

[0127] The simulated colonic fluid was prepared as follows: 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate were mixed at a volume ratio of 81:19, and then pure water was added to bring the volume to 1 L. The pH was adjusted to 7.4 with 0.1 mol / L NaOH, and β-mannanase (6000 U / L) was added under stirring to dissolve the mixture completely. The solution was then filtered and set aside for later use.

[0128] The release rate of the co-encapsulated curcumin and resveratrol composite particles prepared in Examples 1-4 above was tested in simulated gastric juice, and the results are as follows: Figure 4 As shown.

[0129] Figure 4 The release rate of the co-encapsulated curcumin and resveratrol composite particles in simulated gastric juice was determined by... Figure 4It can be seen that, compared with commercially available peanut protein particles, the release rates of the co-encapsulated curcumin and resveratrol composite particles prepared in Examples 1-4 in simulated gastric juice were all less than 25%. Among them, the co-encapsulated curcumin and resveratrol composite particles prepared in Example 1 had the lowest release rate in simulated gastric juice. After incubation in simulated gastric juice for 2 hours, the release rates of curcumin and resveratrol were 15.5% and 17.3%, respectively, which were lower than the release rates of curcumin (72.6%) and resveratrol (79.5%) in single peanut protein particles, and also significantly lower than the release rates of curcumin and resveratrol in the composite particles in Comparative Example 1. This indicates that the coating of fructooligosaccharide-carboxymethyl konjac glucomannan can improve the stability of peanut protein particles in the gastric environment and inhibit the release of the encapsulated active substances in simulated gastric juice.

[0130] The release rate of the co-encapsulated curcumin and resveratrol composite particles prepared in Examples 1-4 above was tested in simulated colonic fluid, and the results are as follows: Figure 5 As shown.

[0131] Figure 5 The release rate of the co-encapsulated curcumin and resveratrol composite particles in simulated colonic fluid was determined by... Figure 5 It can be seen that, compared with commercially available peanut protein particles, the composite particles co-encapsulated with curcumin and resveratrol prepared in Examples 1-4 all showed higher release rates in simulated colonic fluid. Among them, the peanut protein isolate / carboxymethyl konjac glucomannan composite particles co-encapsulated with curcumin and resveratrol prepared in Example 1 showed the highest release rate in simulated colonic fluid, after incubation in simulated colonic fluid for 7 days. After h, the release rates of curcumin and resveratrol were 61.5% and 60.3%, respectively, which were much higher than the release rates of curcumin (10.5%) and resveratrol (8.6%) in single peanut protein particles. This indicates that the encapsulation of fructooligosaccharide-carboxymethyl konjac glucomannan can achieve colon-targeted co-delivery of the active substances encapsulated in the composite particles. This is related to the characteristics of carboxymethyl konjac glucomannan. Carboxymethyl konjac glucomannan can remain relatively stable in the gastric environment, but it can be specifically degraded by β-mannanase in simulated colonic fluid. Therefore, the composite particles prepared in this invention can be degraded in simulated colonic fluid, thereby promoting the large-scale release of the encapsulated active substances.

[0132] In addition, the composite particles prepared in Comparative Example 1 showed poor stability in simulated gastrointestinal fluid and low release rate in simulated colonic fluid, failing to achieve colon-targeted co-delivery of curcumin and resveratrol. This indicates that the negatively charged carboxymethyl konjac glucomannan is beneficial to the electrostatic stability of the particles, and that carboxymethylation modification is the key to achieving enzyme responsiveness.

[0133] Example 9

[0134] This embodiment provides the determination of short-chain fatty acid yield in an in vitro fermentation model of composite particles, including the following steps:

[0135] Under anaerobic conditions, a fermentation medium was prepared using fecal microbiota from healthy individuals as inoculum and composite particles as a carbon source. Fermentation was carried out in an anaerobic incubator at 37°C for 24 h. Samples were taken at the end of fermentation, and the supernatant was obtained by centrifugation. The concentration of short-chain fatty acids in the supernatant was determined by gas chromatography. By comparing the differences in the concentration of short-chain fatty acids produced by the composite particles, their activity in regulating intestinal microbiota metabolism was evaluated.

[0136] The short-chain fatty acid yield of the co-encapsulated curcumin and resveratrol composite particles prepared in Example 1 and Comparative Example 3 was tested in an in vitro colonic fermentation model. The results are as follows: Figure 6 As shown.

[0137] Figure 6 The results of short-chain fatty acid yield in an in vitro fermentation model were obtained from the co-encapsulated curcumin and resveratrol composite particles. Figure 6 It can be seen that the composite particles co-encapsulated with curcumin and resveratrol prepared in Example 1 significantly promoted probiotic metabolism during in vitro fermentation, and the total short-chain fatty acid and butyric acid yields in its fermentation broth were significantly higher than those of the composite particles in Comparative Example 3. Although the composite particles prepared in Comparative Example 3 had colon-targeted co-delivery performance, they lacked prebiotic function. Combined with in vitro fermentation experiments, it was shown that its ability to promote the production of short-chain fatty acids by probiotics was significantly lower than that of the composite particles in Example 1. This directly proves that the composite particles prepared in this invention, while achieving colon-targeted delivery, also have a prebiotic effect that regulates the intestinal microecology, and can produce synergistic health benefits with curcumin and resveratrol.

[0138] Example 10

[0139] This embodiment provides an evaluation of the efficacy of compound granules in relieving ulcerative colitis, including the following steps:

[0140] Male C57BL / 6 mice, approximately 6 weeks old and weighing between 20 ± 2 g, were purchased and housed separately in an SPF-grade animal facility. The room temperature was maintained at 20-22 ℃, the relative humidity at 60%, and the daily light exposure was 12 h. After one week of acclimatization, they were used for experiments, during which time they were allowed to eat and drink. A mouse model of ulcerative colitis was established using DSS (distilled water without DSS). The normal control group was given distilled water without DSS, while the model group (DSS) and experimental groups were given 3% DSS solution for 7 consecutive days. From the second day after modeling, mice in all groups were administered saline by gavage. Mice in the normal control group and model group were given an equal volume of physiological saline by gavage once a day for 7 consecutive days. On the 8th day, the mice were sacrificed and their tissues were collected.

[0141] During the experiment, the mice's weight changes were measured and recorded daily. Their activity level, fur luster, fecal characteristics, and the degree of occult blood were observed, and mortality was recorded. The mouse disease activity index score was used to assess the clinical severity of colitis. After sacrifice, colon tissue was harvested from the mice to measure the length of the colon from the anterior end of the cecum to the posterior end of the rectum in a naturally extended state, used to assess macroscopic damage to the colon.

[0142] The efficacy of the co-encapsulated curcumin and resveratrol composite particles prepared in Example 1 and Comparative Example 3 in relieving ulcerative colitis was evaluated, and the results are as follows: Figure 7 As shown.

[0143] Figure 7 This study investigated the effects of co-encapsulated curcumin and resveratrol compound particles on body weight changes, disease activity index, and colon length in mice with ulcerative colitis. Figure 7 It can be seen that the composite particles co-encapsulated with curcumin and resveratrol prepared in Example 1 can significantly alleviate clinical symptoms such as weight loss, diarrhea and bloody stools in mice with DSS-induced colitis, and can also inhibit the shortening of the colon in mice caused by DSS. Its improvement effect is better than that of the composite particles in Comparative Example 3, indicating that the prebiotic components can enhance the synergistic effect of curcumin and resveratrol in relieving colitis.

[0144] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A method for preparing composite particles with prebiotic function and colon-targeted co-delivery properties, characterized in that, The method includes the following steps: (1) Dissolve peanut protein isolate in water and heat and sonicate to obtain the treated peanut protein dispersion; (2) Add the ethanol aqueous solution containing curcumin and resveratrol dropwise to the peanut protein dispersion, stir continuously and then remove the ethanol by rotary evaporation to obtain a peanut protein nanocore suspension co-encapsulated with curcumin and resveratrol. (3) Disperse konjac glucomannan in ethanol, add sodium hydroxide and chloroacetic acid solution in sequence, stir continuously to react, collect the precipitate, wash and obtain carboxymethyl konjac glucomannan; (4) Dissolve the carboxymethyl konjac glucomannan and prebiotics in water to obtain carboxymethyl konjac glucomannan solution and prebiotic solution respectively. Mix them after continuous stirring to obtain carboxymethyl konjac glucomannan-prebiotic composite solution. (5) The carboxymethyl konjac glucomannan-prebiotic composite solution is added dropwise to the peanut protein nanocore suspension, the pH of the system is adjusted, and the mixture is stirred continuously. Through electrostatic interaction, the composite particles with a core-shell structure are formed through self-assembly.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of peanut protein isolate is 1-10 mg / mL, and the heating conditions are: temperature 75-95℃, time 10-20 min.

3. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of curcumin to resveratrol is 1:1, and the total mass concentration of curcumin and resveratrol in the ethanol aqueous solution is 0.5~2 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step (3), the molar ratio of chloroacetic acid to sodium hydroxide is 1~3:

1.

5. The preparation method according to claim 1, characterized in that, In step (4), the prebiotics include fructooligosaccharides and / or resistant dextrin; the mass concentration of carboxymethyl konjac glucomannan in the solution is 1~5 mg / mL; the mass concentration of the prebiotics in the solution is 1~5 mg / mL; and the volume ratio of the prebiotic solution to the carboxymethyl konjac glucomannan solution during mixing is 1:

1.

6. The preparation method according to claim 1, characterized in that, In step (5), the volume ratio of carboxymethyl konjac glucomannan-prebiotic composite solution to peanut protein nanonucleus suspension is 1:2 to 2:1; the pH of the system is 3 to 4.

7. The composite particles prepared by the preparation method according to any one of claims 1 to 6, characterized in that, The composite particles have a core-shell structure, with the core consisting of curcumin and resveratrol encapsulated by peanut protein isolate, and the shell consisting of a composite layer formed by carboxymethyl konjac glucomannan and prebiotics through intermolecular forces.

8. The use of the composite particles according to claim 7 in the preparation of functional foods or pharmaceuticals with colon-targeted delivery and intestinal microecological regulation functions.

9. The use of the composite particles according to claim 7 in the preparation of formulations for the prevention or adjuvant treatment of colitis and / or colon cancer.

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