Camellia oil nanoemulsion capsule for colon-targeted delivery, and preparation method and application thereof

Abstract

The application discloses a colon-targeted delivery camellia oil nanoemulsion capsule and a preparation method and application thereof, and belongs to the technical field of functional food. The colon-targeted delivery camellia oil nanoemulsion capsule comprises, in mass fraction, 30-40 parts of camellia oil nanoemulsion, 20-30 parts of probiotic microcapsules, 10-15 parts of PLGA-loaded traditional Chinese medicine active ingredient nanoparticles and 15-20 parts of tea stem ultrafine powder. The camellia oil nanoemulsion comprises camellia oil, quercetin and tea saponin. The probiotic microcapsules comprise a probiotic core, and the probiotic core is wrapped with sodium alginate, chitosan and pH-sensitive enteric material. The nanoemulsion microcapsule is embedded by multiple layers and colon-targeted technology, so that the traditional Chinese medicine active ingredients and probiotics can be efficiently delivered to the colon and released in coordination, and through multiple pathways such as anti-inflammatory, immune regulation and intestinal flora regulation, the nanoemulsion microcapsule plays a role in assisting in the intervention treatment of colitis.

Description

Technical Field

[0001] This application relates to a colon-targeted delivery of camellia oil nanoemulsion capsules, its preparation method and application, belonging to the field of functional food technology. Background Technology

[0002] The human gut is home to a vast array of microorganisms, approximately 100 trillion archaea and bacteria, forming a dynamic, complex, and individually diverse micro-ecosystem. These microorganisms participate in the body's metabolic processes, including nutrient metabolism, defense against invasion, and immune responses. The balance and stability of the gut micro-ecosystem are closely related to the body's nutrition, metabolism, immunity, and nervous system. Imbalance can lead to various diseases, such as ulcerative colitis, colorectal cancer, and functional gastrointestinal disorders. Colon-targeted delivery systems enable the specific release of drugs or active ingredients in the colon, which is of great significance for the treatment of localized diseases such as inflammatory bowel disease and colon cancer. However, products prepared using existing technologies face challenges such as degradation of active ingredients in the upper digestive tract, low bioavailability, and imprecise targeting.

[0003] Camellia oil (also known as wild camellia oil, tea seed oil, or oil-tea seed oil) is extracted from the seeds of the Camellia oleifera Able tree (family Camellia). It contains various nutrients such as camelliaside, tea polyphenols, squalene, and vitamin E. The polyphenols (such as squalene) and linoleic acid it contains have significant anti-inflammatory, gastric mucosa-repairing, and laxative effects. However, these fat-soluble active ingredients are easily degraded in the acidic environment of the stomach, resulting in low bioavailability. Based on these technical problems, those skilled in the art have used nanoemulsification technology (such as high-pressure homogenization to prepare nanoemulsions) to improve the stability of the active ingredients in camellia oil, thereby enhancing its targeting effect and therapeutic efficacy. However, these products are prone to stratification after long-term standing and lack targeted adhesion to the colonic mucosa, leading to reduced efficacy.

[0004] Probiotics need to colonize the colon to regulate gut microbiota. However, when medications are taken orally, they first pass through the stomach before entering the intestines. Stomach acid and bile salts can cause up to 90% mortality of live bacteria, preventing drugs with added probiotics from targeting the colon effectively. To address these issues, those skilled in the art have employed enteric microencapsulation technology to improve the drug's stomach acid tolerance. However, this process suffers from low mechanical strength, uneven coating thickness, and low utilization of lyophilized bacterial powder. Furthermore, existing technologies involve multi-layer coating of probiotics, which enhances protection but is complex and difficult to scale up for mass production.

[0005] Flavonoids, saponins, and other active ingredients in traditional Chinese medicine have anti-inflammatory and antioxidant properties, and are effective in treating diseases such as inflammatory bowel disease and colon cancer. However, these active ingredients have drawbacks such as poor water solubility and low colonic absorption rate, and are unstable in the gastrointestinal environment and are easily degraded.

[0006] During tea processing, waste products such as tea stems are generated. Tea stems specifically refer to the leaf stalks picked from finished tea, most commonly those of Tieguanyin tea. Tea stems are rich in tea polysaccharides (containing 3-5 times more than buds and leaves) and lignin, making them a potential prebiotic resource that can promote the proliferation of Bifidobacteria. However, traditional tea stem processing mainly involves discarding or composting, failing to fully utilize the prebiotic activity of their cellulose porous structure and fermentation derivatives (such as γ-aminobutyric acid). While ultrafine grinding technology can improve utilization, it has not yet formed a synergistic system with the probiotic microecological regulation.

[0007] The main shortcomings of the existing technology are as follows: (1) Camellia oil has a high rate of degradation by gastric acid, poor mucosal targeting, and insufficient concentration of effective components in the colon. (2) The multi-layer coating process for probiotic protection is complex, resulting in low survival rate of live bacteria and low colonization efficiency; (3) The encapsulation rate of PLGA in the delivery of traditional Chinese medicine components is low; (4) Tea stems do not activate prebiotic activity, and their ability to regulate the gut microbiota is limited.

[0008] Therefore, there is an urgent need for a camellia oil nanoemulsion capsule that is resistant to gastric acid, has colon-targeted drug release, and features multi-component synergistic effects.

[0009] Application content To address the aforementioned issues, a camellia oil nanoemulsion capsule for colon-targeted delivery is provided. This nanoemulsion microcapsule, through multi-layer encapsulation and colon-targeting technology, ensures that the active ingredients of traditional Chinese medicine and probiotics can be efficiently delivered to the colon and released synergistically. Through multiple pathways, including anti-inflammatory, immune regulation, and intestinal flora regulation, it plays an auxiliary role in the treatment of colitis.

[0010] One aspect of this application provides a colon-targeted delivery of camellia oil nanoemulsion capsule, comprising, by mass fraction: 30-40 parts of camellia oil nanoemulsion, 20-30 parts of probiotic microcapsules, 10-15 parts of PLGA-encapsulated Chinese herbal active ingredient nanoparticles, and 15-20 parts of tea stem ultrafine powder. The camellia oil nanoemulsion includes camellia oil, quercetin, and tea saponin; The probiotic microcapsules include a probiotic core, which is encapsulated with sodium alginate, chitosan, and a pH-sensitive enteric-coated material.

[0011] Camellia oil nanoemulsion, probiotic microcapsules, PLGA-encapsulated nanoparticles of active Chinese medicine ingredients, and ultrafine tea stem powder work synergistically to achieve targeted delivery of effective ingredients, effectively alleviating the symptoms of colitis and improving the bioavailability of multiple components.

[0012] Camellia oil nanoemulsions contain quercetin and tea saponins, which exhibit significant synergistic effects in antibacterial and anti-inflammatory properties. The combination of these two substances can depolarize bacterial cell membrane potential and damage membrane structure, thereby enhancing the antibacterial effect. Loading quercetin and tea saponins together into camellia oil to form camellia oil nanoemulsions can solve the problems of poor water and lipid solubility of quercetin and its low oral bioavailability. Furthermore, using camellia oil nanoemulsions as a carrier improves its gastrointestinal permeability.

[0013] Furthermore, camellia oil is rich in oleic acid, tea polyphenols, vitamin E, squalene, and other components, possessing effects such as lowering blood lipids, preventing hypertension and arteriosclerosis, anti-cancer properties, and regulating intestinal flora. As an oil-phase base, camellia oil can not only load quercetin and tea saponins, improving bioavailability, but also deliver quercetin and tea saponins to their target sites, enhancing product efficacy. It also has protective effects on the gastric mucosa and promotes bowel movements. Tea saponins, as a natural surfactant, can significantly improve the solubility and stability of the poorly soluble component quercetin, thereby increasing its bioavailability.

[0014] The probiotic microcapsules consist of a three-layer structure from the inside out, consisting of sodium alginate, chitosan, and a pH-sensitive enteric-coated material. This structure synergistically protects the probiotic core, improves its survival rate after passing through the gastrointestinal environment, and dissolves in the higher pH environment of the colon, thus achieving precise colon-targeted release. At the same time, it ensures that a sufficient amount of live bacteria reach the intestine, thereby more effectively regulating the intestinal flora and alleviating colitis.

[0015] Sodium alginate gels with calcium ions to form a primary capsule. Chitosan forms a polyelectrolyte composite membrane on the outer layer of the sodium alginate microcapsule through electrostatic interaction, which can effectively resist the damage of gastric acid and bile salts. The outermost enteric material ensures that the microcapsule remains intact in the acidic environment of the stomach, ensuring that probiotics can reach the colon in an active state and colonize to exert their effects to the maximum extent.

[0016] Specifically, the preparation method of the probiotic core is not specifically limited. Those skilled in the art can choose according to their needs. It can be obtained by freeze-drying or spray drying of high-concentration bacterial suspension, or by granulation after mixing high-density bacterial mud with a protectant, as long as the number of live bacteria in the probiotic core is ≥1.0×10¹¹ CFU / g.

[0017] Optionally, the water-soluble dietary fiber content in the ultrafine tea stem powder is ≥35%.

[0018] Tea stem ultrafine powder provides energy to the probiotics in the probiotic core, promoting their proliferation, enhancing their probiotic effects, and further increasing their quantity so they can function better. The water-soluble dietary fiber in tea stem ultrafine powder has excellent water-holding capacity, swelling properties, and cholesterol adsorption characteristics. In the colon, its gel-like properties can delay drug release, achieving a sustained-release effect. Furthermore, its adsorption properties can adsorb harmful substances in the intestines, further enhancing the effectiveness of other active ingredients in the microcapsules and preventing harmful substances from affecting the product's therapeutic or alleviating effects.

[0019] Water-soluble dietary fiber itself helps regulate the gut microbiota, and the products obtained from the fermentation of water-soluble dietary fiber by Aspergillus niger have a nourishing effect on colonic epithelial cells. Together with the anti-inflammatory effects of probiotics and camellia oil nanoemulsion, they can synergistically treat colitis.

[0020] Optionally, the particle size D90 of the tea stem ultrafine powder is ≤20μm.

[0021] At this particle size, ultrafine pulverization ensures that the particle size of the tea stem ultrafine powder is similar to that of other components in the microcapsules, allowing for better and more uniform mixing and guaranteeing product quality consistency. Simultaneously, this particle size significantly increases the specific surface area of ​​the tea stem ultrafine powder, enhancing its adsorption, water and oil retention capacity, and synergistic effect with probiotic microcapsules and camellia oil nanoemulsions. This makes the entire microcapsule system more stable and synergistically enhances its therapeutic effect on colitis.

[0022] Optionally, the mass ratio of camellia oil, quercetin, and tea saponin in the camellia oil nanoemulsion is 1:(0.02-0.05):(0.1-0.3).

[0023] At this ratio, camellia oil can be loaded with quercetin and tea saponins, two effective active ingredients, to the maximum extent, thus improving its therapeutic effect on colitis. Furthermore, tea saponins can also act as a natural emulsifier, enhancing the emulsification process. At this ratio, tea saponins not only exert their therapeutic effect on colitis but also improve the stability of camellia oil nanoemulsions.

[0024] Optionally, the number of live bacteria in the probiotic core is ≥1.0×10⁻⁶. 11 CFU / g.

[0025] The camellia oil nanoemulsion capsules in this application exert their effects orally. Therefore, the camellia oil nanoemulsion capsules containing probiotics need to pass through the stomach, intestines, etc., and finally act on the colon. During this process, the number and content of probiotics will be greatly lost. At this level, the number of live bacteria in the probiotic core can ensure that even after the loss in the digestive tract, there are still enough live bacteria to reach the colon, which significantly improves the colonization efficiency.

[0026] If the number of live bacteria is low, the probiotics in the camellia oil nano-emulsion capsules will be lost during the delivery process, resulting in insufficient number of live bacteria reaching the colon. They will be unable to effectively colonize the colon and will be unable to competitively inhibit pathogens, thus greatly reducing the effect of regulating the balance of the gut microbiota.

[0027] Optionally, the probiotic core includes at least one of Lactobacillus plantarum, Bifidobacterium, and butyric acid bacteria.

[0028] Lactobacillus plantarum can inhibit harmful bacteria in the intestines and maintain the health of the intestinal mucosa. It also has good acid and bile salt resistance, allowing it to be effectively and stably transported to the colon through the upper digestive tract to exert its effects, thus improving the treatment efficacy of colitis. Bifidobacteria are dominant beneficial bacteria native to the intestines, crucial for restoring and maintaining a healthy intestinal microecological balance, and can effectively improve gastrointestinal dysfunction such as diarrhea and constipation. Butyric acid bacteria can metabolize and produce butyric acid, an important energy source for colonic epithelial cells, which has anti-inflammatory and intestinal mucosal repair effects, showing good therapeutic effects for colitis.

[0029] Preferably, the probiotic core includes Lactobacillus plantarum, Bifidobacterium, and butyric acid bacteria.

[0030] More preferably, the probiotic core comprises Lactobacillus plantarum, Bifidobacterium, and butyric acid bacteria in a mass ratio of 2:2:1.

[0031] Optionally, the fermentation temperature is 32-37℃ and the fermentation time is 72-96h.

[0032] Within this range, Aspergillus niger can be kept in its optimal growth state and metabolic activity, allowing a large amount of Aspergillus niger metabolites to accumulate in the ultrafine tea stem powder, thereby improving the therapeutic effect of the effective components in the Aspergillus niger metabolites on colitis.

[0033] If the fermentation temperature is too high, the Aspergillus niger fungus may die, preventing the tea stems from fermenting and potentially causing the degradation of metabolites, thus affecting the quality and efficacy of the tea stem ultrafine powder. If the fermentation temperature is too low, the Aspergillus niger fungus grows slowly, resulting in incomplete fermentation of the tea stems and a low conversion rate of active ingredients, which in turn affects the product's efficacy.

[0034] Optionally, the pH-sensitive enteric material accounts for 15-25% of the total weight of the probiotic microcapsules.

[0035] Within this range, a complete, dense, and appropriately thick coating membrane can be formed on the probiotic core, effectively protecting the probiotics as they safely pass through the stomach and small intestine, targeting the colon to dissolve and release their contents in a higher pH environment.

[0036] If the amount of pH-sensitive enteric-coated material used is too low, the coating layer will be too thin or unable to completely encapsulate the probiotic core, thus failing to effectively resist stomach acid and digestive enzymes. This causes the probiotics to be released prematurely and inactivated in large quantities before reaching the colon, preventing them from being delivered to the colon in a targeted manner. If the amount of pH-sensitive enteric-coated material used is too high, the coating layer will be too thick, causing it to dissolve slowly or not be completely released in the high pH environment of the colon, thus affecting the effectiveness of the probiotics.

[0037] Optionally, the enteric coating material is selected from one of the acrylic resins Eudragit® FS30D and Eudragit L30D-55.

[0038] Optionally, the drug loading of the PLGA-encapsulated nanoparticles of traditional Chinese medicine active ingredients is 5-15%.

[0039] Within this drug loading range, sufficient drug delivery is ensured for each administration while maintaining high stability of the PLGA-encapsulated Chinese medicine active ingredient nanoparticles. Excessive drug loading often leads to the precipitation or crystallization of the active ingredients within the nanoparticles, disrupting the particle structure and stability of the PLGA-encapsulated nanoparticles. It may also trigger burst release, failing to achieve sustained-release and targeted effects and potentially causing adverse effects on the human body.

[0040] Optionally, the encapsulation efficiency of the PLGA-encapsulated nanoparticles containing active ingredients of traditional Chinese medicine is ≥85%.

[0041] Another aspect of this application provides a method for preparing colon-targeted camellia oil nanoemulsion capsules, comprising the following steps: S1: Camellia oil, quercetin, and tea saponin are mixed in proportion to obtain the first oil phase. The first emulsifier is mixed with water to obtain the first aqueous phase. The first aqueous phase and the first oil phase are mixed and emulsified to obtain the first emulsion. After homogenization, camellia oil nano-emulsion is obtained. S2: Mix the probiotic core with sodium alginate solution to obtain a bacterial gel suspension. Then, drop the bacterial gel suspension into calcium chloride solution and mix to obtain a primary capsule. Then, immerse the primary capsule in chitosan solution to obtain chitosan-coated primary capsules. Then, spray the chitosan-coated primary capsules with a pH-sensitive enteric coating material to obtain probiotic microcapsules. S3: PLGA and the active ingredients of traditional Chinese medicine are dissolved in an organic solvent to obtain a second oil phase. Polyvinyl alcohol is mixed with water to obtain a second aqueous phase. The oil phase, aqueous phase and second emulsifier are mixed and emulsified to obtain a second emulsion. Then the solvent is evaporated, and the PLGA nanoparticles of the active ingredients of traditional Chinese medicine are collected after separation. S4: After sterilizing the tea stems, inoculate them with Aspergillus niger for fermentation, dry them, and then pulverize them into ultrafine tea stem powder. S5: The camellia oil nanoemulsion, probiotic microcapsules, PLGA herbal active ingredient nanoparticles and tea stem ultrafine powder obtained in steps S1-S4 are mixed in proportion and then aseptically packaged to obtain colon-targeted delivery camellia oil nanoemulsion capsules.

[0042] Optionally, the amount of organic solvent added in step S3 is 10-20 times the total mass of PLGA and the drug.

[0043] Optionally, the organic solvent in step S3 includes one of ethyl acetate, chloroform, and dichloromethane.

[0044] Optionally, high-pressure microfluidic technology is used for homogenization in step S1.

[0045] High-pressure microfluidic homogenization technology can provide huge energy in a very short time to obtain camellia oil nanoemulsions with small particle size and narrow distribution. Small particle size camellia oil nanoemulsions are beneficial to improving physical stability and bioavailability.

[0046] Optionally, the pressure value in the high-pressure microjet process is 100-180 MPa.

[0047] Under this high pressure, it can be ensured that the camellia oil nanoemulsion droplets are broken down to the nanoscale and uniformly distributed. If the pressure is insufficient, the particle size may be too large, the distribution may be too wide, and the stability may be poor. If the pressure is too high, excessive heat may be generated, which will affect the stability of the camellia oil nanoemulsion.

[0048] Optionally, the concentration of the sodium alginate solution in step S2 is 1.5-2.0% (w / v).

[0049] Optionally, the concentration of the calcium chloride solution in step S2 is 1.5-2.0% (w / v).

[0050] Sodium alginate and calcium chloride solutions, within this concentration range, can form probiotic microcapsules with suitable mechanical strength and high encapsulation efficiency. If the concentration is too low, the capsule walls will be too soft and easily break; if the concentration is too high, the solution will be too viscous, which is not conducive to operation, and the quality uniformity of the capsule walls of the prepared probiotic microcapsules will be poor.

[0051] Optionally, the volume ratio of the bacterial gel mixture suspension to the calcium chloride solution is 1:(5-10).

[0052] Optionally, the concentration of the chitosan solution in step S2 is 0.5-1.5% (w / v).

[0053] Optionally, the volume ratio of the first aqueous phase to the first oil phase is 1:(3-5).

[0054] Optionally, the volume ratio of the second aqueous phase to the second oil phase is 1:(4-8).

[0055] Optionally, in step S2, the pH-sensitive enteric material is sprayed in a fluidized bed with an inlet air temperature of 35-40℃, an inlet air velocity of 8-10 m³ / h, and an atomization pressure of 0.8-1.2 bar.

[0056] At this temperature, high-temperature damage to probiotics can be avoided, while ensuring that the coating solution dries evenly to form a film, thus ensuring targeted performance.

[0057] Optionally, the spraying rate of the pH-sensitive enteric material is 3 g / min.

[0058] Optionally, step S3 also includes a post-processing step: washing the PLGA herbal active ingredient nanoparticles from step S3 with ultrapure water, adding a freeze-drying protectant, pre-freezing at -80℃ for 4 hours, and freeze-drying at -50℃ and 0.1mbar for 24 hours.

[0059] Adding a freeze-drying protectant and performing freeze-drying can transform PLGA nanoparticles into a solid powder form that is easy to store and transport, improving the long-term stability of PLGA nanoparticles containing active ingredients from traditional Chinese medicine and preventing aggregation, drug leakage, or degradation during storage.

[0060] Optionally, the preparation method of the Aspergillus niger spore suspension in step S4 includes the following steps: A1: Inoculate Aspergillus niger onto potato dextrose agar slant medium and incubate at 25-30℃ for 5-7 days; A2: Add sterile physiological saline containing Tween 80 to the culture medium obtained in step A1, elute the Aspergillus niger spores into the liquid, filter, and obtain the Aspergillus niger spore suspension.

[0061] Optionally, the inoculation amount of Aspergillus niger spore suspension in step S4 is 5%-8%.

[0062] At this inoculation level, Aspergillus niger grows rapidly and dominates, effectively converting substances in tea stems and increasing the content of active ingredients in the product. If the inoculation level is too low, Aspergillus niger will cause slow fermentation or contamination by other microorganisms; if the inoculation level is too high, excessive growth of the fungi or nutrient competition will lead to a decrease in the conversion rate of metabolites, affecting the therapeutic effect of the product.

[0063] In another aspect of this application, the use of the colon-targeted camellia oil nanoemulsion capsules described in this application, or the colon-targeted camellia oil nanoemulsion capsules prepared by the method for preparing colon-targeted camellia oil nanoemulsion capsules, in functional foods, health products, or drugs for the prevention and / or treatment of intestinal diseases and the regulation of intestinal flora.

[0064] The beneficial effects of this application include, but are not limited to: 1. The camellia oil nanoemulsion capsules for colon-targeted delivery according to this application are constructed by synergistic modification of quercetin and tea saponin to enhance the bioavailability of camellia oil and its adhesion to the colonic mucosa; probiotics are encapsulated in pH-sensitive enteric materials to achieve gastric juice environmental protection and colon-targeted release; active ingredients of traditional Chinese medicine are encapsulated in PLGA to provide sustained-release function; and ultrafine tea stem powder treated by microbial fermentation is used as a prebiotic carrier to promote probiotic colonization and synergistically regulate the intestinal microecology.

[0065] 2. The camellia oil nanoemulsion capsules delivered to the colon according to this application achieve a synergistic effect of bacteria, oil, and drug. The camellia oil nanoemulsion promotes the colonization of probiotics, and the active ingredients of traditional Chinese medicine stimulate the production of short-chain fatty acids, thereby improving the therapeutic effect of the drug and enhancing bioavailability.

[0066] 3. The camellia oil nanoemulsion capsules for colon-targeted delivery according to this application, which significantly improve the utilization rate of active ingredients by achieving precise release into the colon, while enhancing the stability of probiotics and reducing temperature and time limitations during storage and transportation. Detailed Implementation

[0067] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0068] Unless otherwise specified, the raw materials used in the embodiments and comparative examples of this application were all purchased commercially.

[0069] Unless otherwise specified, the methods used in the embodiments and comparative examples of this application are conventional methods in the prior art.

[0070] The camellia oil in this application's embodiments and comparative examples underwent low-temperature deodorization treatment at 60℃ and 0.1Pa before use, resulting in a polyphenol content of 50-300 mg / kg. This low-temperature deodorization technology is a conventional technique in the field, and those skilled in the art can choose it according to their needs, or directly purchase commercially available low-temperature deodorized camellia oil with a polyphenol content of 50-300 mg / kg. Quercetin underwent micronization treatment, with a particle size D90 ≤ 15 μm. The purity of tea saponin was ≥ 95%. The first emulsifier was a 1:2 mass ratio of soy protein isolate to tea saponin. The second emulsifier was polyvinyl alcohol (PVA17-88) with a molecular weight of 30,000-70,000.

[0071] The method for preparing the probiotic core is as follows: Inoculate the bacterial suspension into the MRS liquid culture medium at 1% of the medium volume, and culture at 37℃ and 100 rpm until the viable cell concentration reaches 10⁻⁶. 9The bacterial culture was then centrifuged at 6000 rpm for 15 min at 4 °C to collect the cells. The cells were washed twice with sterile physiological saline to remove the culture medium. The cells were then mixed with trehalose and skim milk in a 1:1 mass ratio, pre-frozen at -80 °C for 4 h, and then freeze-dried to obtain the probiotic core. The viable count of the probiotic core was ≥1.0 × 10⁻⁶. 11 CFU / g.

[0072] The PLGA in this application has a lactic acid hydroxyl group to acetic acid weight ratio of 75:25 and a molecular weight of 20 kDa; the chitosan has a degree of deacetylation ≥85% and a molecular weight of 100 kDa.

[0073] The *Lactobacillus plantarum* strain used in the embodiments and comparative examples of this application, with accession number ATCC 8014, was purchased from Shanghai Fuxiang Biotechnology Co., Ltd.; *Bifidobacterium bifidum* strain, with accession number ATCC 29521, was purchased from Shanghai Xuanke Biotechnology Co., Ltd.; *Clostridium butyricum* strain, with accession number ATCC 19398, was purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd.; *Lactobacillus rhamnosus* strain, with accession number ATCC 7469, was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.; and *Aspergillus oryzae* strain, with accession number ATCC 42149, was purchased from Shanghai Fuxiang Biotechnology Co., Ltd. All of the above strains are well-known type strains or standard strains in the art and can be obtained through publicly available commercial channels.

[0074] The Aspergillus niger spore suspensions prepared in the embodiments and comparative examples of this application can be stored for a short period at 4°C, and the viable count in the Aspergillus niger spore suspensions is 1.2 × 10⁻⁶. 9 CFU / mL; the viable count in the Aspergillus oryzae spore suspension was 1.2 × 10⁻⁶. 9 CFU / mL.

[0075] Example 1 This embodiment relates to a colon-targeted delivery of camellia oil nanoemulsion capsules, which, by mass fraction, include: 40 parts of camellia oil nanoemulsion, 25 parts of probiotic microcapsules, 15 parts of PLGA-encapsulated Chinese herbal active ingredient nanoparticles, and 15 parts of tea stem ultrafine powder. The camellia oil nanoemulsion comprises camellia oil, quercetin, and tea saponin, with a mass ratio of 35:1.5:1. The preparation method includes the following steps: S1: Camellia oil, quercetin, and tea saponin were mixed in a certain proportion to obtain the first oil phase. The first emulsifier was added to water and stirred at 50°C. The amount of the first emulsifier added was controlled to be 3% of the weight of the aqueous phase system. Then, xanthan gum (0.1% by weight of the aqueous phase system) and citrus fiber (0.5% by weight of the aqueous phase system) were added and stirred evenly to obtain the first aqueous phase. The first oil phase was heated to 50°C and then added to the first oil phase. The mixture was stirred and emulsified at 2000 rpm for 10 min to obtain the first emulsion. The volume ratio of the first oil phase to the first aqueous phase was 1:3. High-pressure micro-jet homogenization was carried out under 100 MPa pressure and the process was repeated 3 times. The temperature of the homogenization process was controlled to be ≤55°C to obtain camellia oil nanoemulsion. S2: Add the probiotic core to a 1.5% (w / v) sodium alginate solution and mix thoroughly using a vortex mixer to obtain a bacterial gel suspension. The probiotic core consists of lyophilized Lactobacillus plantarum and Bifidobacterium at a mass ratio of 1:1. The ratio of sodium alginate solution to probiotic core is 1:10 (w / v). Load the bacterial gel suspension into a syringe and push it through a syringe pump at a flow rate of 3 mL / min to form uniform droplets. Drop the droplets vertically into a solution containing 1.5% (w / v) calcium chloride. After being stirred and solidified in a coagulation bath for 20 minutes, the initial capsules were formed. The volume ratio of the bacterial gel suspension to the calcium chloride solution was 1:10. Then, the initial capsules were immersed in a 0.5% (w / v) chitosan solution and stirred for 10 minutes. After rinsing with a phosphate buffer solution with a pH of 7.0, chitosan-coated initial capsules were obtained. Then, a pH-sensitive enteric material was sprayed onto the chitosan-coated initial capsules in a fluidized bed. The inlet temperature of the fluidized bed was 40℃, the air velocity was 8m³ / h, and the spraying speed of the acrylic resin Eudragit® FS30D was 3g / min to obtain probiotic microcapsules. S3: PLGA and curcumin were dissolved in dichloromethane to obtain a second oil phase with a concentration of 10 mg / mL. The mass ratio of PLGA to curcumin was 10:1. Polyvinyl alcohol (PVA17-88) (molecular weight 30000) was mixed with water to obtain a second aqueous phase with a concentration of 2% (w / v). The second oil phase and the second aqueous phase were mixed at a volume ratio of 1:4 and ultrasonically emulsified under ice bath conditions to obtain a second emulsion. The ultrasonic power was 400 W, the working time was 3 s, and the interval was 5 s. The total emulsification time was 5 min. Then, the solvent was evaporated by treating the product in a rotary evaporator at 40℃ and 100 rpm for 12 h. After centrifugation at 10000 rpm for 15 min, the product was washed three times with ultrapure water. Trehalose and sucrose were added in a mass ratio of 1:1, and the total amount of trehalose and sucrose added was controlled to be in a mass ratio of 1:1 with PLGA. The product was pre-frozen at -80℃ for 4 h and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain PLGA nanoparticles containing active ingredients of traditional Chinese medicine. S4: The tea stems were crushed and passed through a 40-mesh sieve, then sterilized at 121℃ for 20 minutes, then inoculated with Aspergillus niger spore suspension and fermented at 32℃ for 72 hours, with stirring every 12 hours during the fermentation period. After that, the tea stems were dried under hot air at 60℃ until the moisture content was ≤8%, and then ultra-finely crushed by air jet mill under working fluid pressure of 0.7MPa and sorting frequency of 25Hz until the particle size D90≤20μm was obtained to obtain ultra-fine tea stem powder. The preparation method of the Aspergillus niger spore suspension in step S4 includes the following steps: A1: Inoculate Aspergillus niger onto potato dextrose agar slant medium and incubate at 25°C for 7 days; A2: Add sterile physiological saline containing 0.05% Tween 80 to the culture medium obtained in step A1. Gently scrape the surface with an inoculation loop and vortex to fully elute the Aspergillus niger spores into the liquid. Filter the resulting Aspergillus niger spore suspension through a 10μm filter membrane to remove mycelial fragments, obtaining the Aspergillus niger spore suspension. S5: The camellia oil nanoemulsion, probiotic microcapsules, PLGA herbal active ingredient nanoparticles and tea stem ultrafine powder obtained in steps S1-S4 are added to a three-dimensional motion mixer in proportion and mixed at 15 rpm for 30 min. Then, the mixture is aseptically packaged in a Class 10,000 clean environment to obtain camellia oil nanoemulsion capsules for colon-targeted delivery.

[0076] Example 2 This embodiment relates to a colon-targeted delivery of camellia oil nanoemulsion capsules, which, by mass fraction, include: 40 parts of camellia oil nanoemulsion, 30 parts of probiotic microcapsules, 10 parts of PLGA-encapsulated Chinese herbal active ingredient nanoparticles, and 20 parts of tea stem ultrafine powder. The camellia oil nanoemulsion comprises camellia oil, quercetin, and tea saponin, with a mass ratio of 35:4:1. The preparation method of camellia oil nanoemulsion capsules includes the following steps: S1: Camellia oil, quercetin, and tea saponin were mixed in a certain proportion to obtain the first oil phase. The first emulsifier was stirred and mixed with water at 60°C, and the amount of the first emulsifier added was controlled to be 3% of the weight of the aqueous phase system. Then, 0.1% xanthan gum and 0.5% citrus fiber of the aqueous phase system were added and stirred evenly to obtain the first aqueous phase. The first oil phase was heated to 60°C and then added to the first oil phase. The mixture was stirred and emulsified at 2000 rpm for 10 min to obtain the first emulsion. The volume ratio of the first oil phase to the first aqueous phase was 1:3. High-pressure micro-jet homogenization was carried out under 100 MPa pressure, and the process was repeated 3 times. The temperature of the homogenization process was controlled to be ≤55°C to obtain camellia oil nanoemulsion. S2: Add the probiotic core to a 2.0% (w / v) sodium alginate solution and mix thoroughly using a vortex mixer to obtain a bacterial gel suspension. The mass ratio of probiotic core to sodium alginate solution is 1:6. The probiotic core consists of lyophilized Lactobacillus plantarum, Bifidobacterium, and Butyric acid bacteria in a mass ratio of 2:2:1. The ratio of sodium alginate solution to probiotic core is 1:10 (w / v). Load the bacterial gel suspension into a syringe and pump it at a flow rate of 3-10 mL / min to form uniform droplets. Drop the mixture vertically into a container. After stirring and solidifying in a coagulation bath with 2.0% (w / v) calcium chloride solution for 20 min, primary capsules were formed. The volume ratio of the bacterial gel suspension to the calcium chloride solution was 1:7. Then, the primary capsules were immersed in a 1.5% (w / v) chitosan solution and stirred for 10 min. After rinsing with a phosphate buffer solution at pH 7.0, chitosan-coated primary capsules were obtained. Then, a pH-sensitive enteric coating material was sprayed onto the chitosan-coated primary capsules in a fluidized bed with an inlet temperature of 50℃, an air velocity of 10 m³ / h, and a spraying rate of Eudragit® FS30D acrylic resin of 3 g / min to obtain probiotic microcapsules. S3: PLGA and resveratrol were dissolved in dichloromethane to obtain a second oil phase with a concentration of 10 mg / mL. The mass ratio of PLGA to resveratrol was 10:1. Polyvinyl alcohol (molecular weight 30000) was mixed with water to obtain a second aqueous phase with a concentration of 2% (w / v). The second oil phase and the second aqueous phase were mixed at a volume ratio of 1:4 and ultrasonically emulsified in an ice bath to obtain a second emulsion. The ultrasonic power was 400W, the working time was 3s, and the interval was 5s. The total emulsification... After 5 minutes, the solvent was evaporated by treating the PLGA in a rotary evaporator at 40°C and 100 rpm for 12 hours. After centrifugation at 10,000 rpm for 15 minutes, the PLGA was washed three times with ultrapure water. Trehalose and sucrose were added in a mass ratio of 1:1, and the total amount of trehalose and sucrose added was controlled to be 1:1 with the mass ratio of PLGA. The PLGA was pre-frozen at -80°C for 4 hours and freeze-dried at -50°C and 0.1 mbar for 24 hours to obtain PLGA nanoparticles containing active ingredients of traditional Chinese medicine. S4: The tea stems were crushed and passed through a 40-mesh sieve, then sterilized at 121℃ for 20 minutes, then inoculated with Aspergillus niger spore suspension and fermented at 32℃ for 72 hours, with stirring every 12 hours during the fermentation period. After that, the tea stems were dried under hot air at 60℃ until the moisture content was ≤8%, and then ultra-finely crushed by air jet mill under working fluid pressure of 0.7MPa and sorting frequency of 25Hz until the particle size D90≤20μm was obtained to obtain ultra-fine tea stem powder. The preparation method of the Aspergillus niger spore suspension in step S4 includes the following steps: A1: Inoculate Aspergillus niger onto potato dextrose agar slant medium and incubate at 30°C for 5 days; A2: Add sterile physiological saline containing 0.05% Tween 80 to the culture medium obtained in step A1. Gently scrape the surface with an inoculation loop and vortex to fully elute the Aspergillus niger spores into the liquid. Filter the resulting Aspergillus niger spore suspension through a 10μm filter membrane to remove mycelial fragments, obtaining the Aspergillus niger spore suspension. S5: The camellia oil nanoemulsion, probiotic microcapsules, PLGA herbal active ingredient nanoparticles and tea stem ultrafine powder obtained in steps S1-S4 are added to a three-dimensional motion mixer in proportion and mixed at 15 rpm for 30 min. Then, the mixture is aseptically packaged in a Class 10,000 clean environment to obtain camellia oil nanoemulsion capsules for colon-targeted delivery.

[0077] Example 3 This embodiment relates to a colon-targeted delivery of camellia oil nanoemulsion capsules, which, by mass fraction, include: 30 parts of camellia oil nanoemulsion, 20 parts of probiotic microcapsules, 15 parts of PLGA-encapsulated Chinese herbal active ingredient nanoparticles, and 15 parts of tea stem ultrafine powder. The camellia oil nanoemulsion comprises camellia oil, quercetin, and tea saponin, with a mass ratio of 25:2:3. The preparation method of camellia oil nanoemulsion capsules includes the following steps: S1: Camellia oil, quercetin, and tea saponin were mixed in a mass ratio of 25:2:3 to obtain the first oil phase. The first emulsifier was stirred and mixed with water at 50°C, and the amount of the first emulsifier added was controlled to be 3% of the weight of the aqueous phase system. Then, 0.1% xanthan gum and 0.5% citrus fiber of the aqueous phase system were added and stirred evenly to obtain the first aqueous phase. The first oil phase was heated to 50°C and then added to the first oil phase. The mixture was stirred and emulsified at 2000 rpm for 10 min to obtain the first emulsion. The volume ratio of the first oil phase to the first aqueous phase was 1:3. High-pressure micro-jet homogenization was carried out under a pressure of 100 MPa and the process was repeated 3 times. The temperature of the homogenization process was controlled to be ≤55°C to obtain camellia oil nanoemulsion. S2: Add the probiotic core to a 1.5% (w / v) sodium alginate solution and mix thoroughly using a vortex mixer to obtain a bacterial gel suspension. The mass ratio of the probiotic core to the sodium alginate solution is 1:8. The probiotic core is lyophilized Lactobacillus plantarum powder, and the ratio of the sodium alginate solution to the probiotic core is 1:10 (w / v). Load the bacterial gel suspension into a syringe and push it through a syringe pump at a flow rate of 10 mL / min to form uniform droplets. Drop the droplets vertically into a coagulation bath containing a 1.5% (w / v) calcium chloride solution. After stirring and solidifying for 20 minutes, the initial capsules were formed. Then, the initial capsules were immersed in a 0.5% (w / v) chitosan solution and stirred for 10 minutes. After rinsing with a phosphate buffer solution with a pH of 7.0, chitosan-coated initial capsules were obtained. The volume ratio of the bacterial gel suspension to the calcium chloride solution was 1:5. Then, a pH-sensitive enteric coating material was sprayed onto the chitosan-coated initial capsules in a fluidized bed. The inlet temperature of the fluidized bed was 45℃, the air velocity was 9m³ / h, and the spraying speed of UTEC L30D-55 was 3g / min to obtain probiotic microcapsules. S3: PLGA and resveratrol were dissolved in dichloromethane to obtain a second oil phase with a concentration of 10 mg / mL. The mass ratio of PLGA to resveratrol was 10:1. Polyvinyl alcohol (PVA17-88) (molecular weight 30000) was mixed with water to obtain a second aqueous phase with a concentration of 2% (w / v). The second oil phase and the second aqueous phase were mixed at a volume ratio of 1:4 and ultrasonically emulsified under ice bath conditions to obtain a second emulsion. The ultrasonic power was 400W, the working time was 3s, and the interval was 5s. The total emulsification time was 5 min. Then, the solvent was evaporated by treating the product in a rotary evaporator at 40℃ and 100 rpm for 12 h. After centrifugation at 10000 rpm for 15 min, the product was washed three times with ultrapure water. Trehalose and sucrose were added in a mass ratio of 1:1, and the total amount of trehalose and sucrose added was controlled to be 1:1 with the mass ratio of PLGA. The product was pre-frozen at -80℃ for 4 h and freeze-dried at -50℃ and 0.1 mbar for 24 h to obtain PLGA nanoparticles containing active ingredients of traditional Chinese medicine. S4: The tea stems were crushed and passed through a 40-mesh sieve, then sterilized at 121℃ for 20 minutes, then inoculated with Aspergillus niger spore suspension and fermented at 37℃ for 96 hours, turning them over every 12 hours. After that, they were dried under hot air at 60℃ until the moisture content was ≤8%, and then ultra-finely crushed by air jet mill under working pressure of 0.7MPa and sorting frequency of 25Hz until the particle size D90≤20μm was obtained to obtain ultra-fine tea stem powder. The preparation method of the Aspergillus niger spore suspension in step S4 includes the following steps: A1: Inoculate Aspergillus niger onto potato dextrose agar slant medium and incubate at 30°C for 5 days; A2: Add sterile physiological saline containing 0.05% Tween 80 to the culture medium obtained in step A1. Gently scrape the surface with an inoculation loop and vortex to fully elute the Aspergillus niger spores into the liquid. Filter the resulting Aspergillus niger spore suspension through a 10μm filter membrane to remove mycelial fragments, obtaining the Aspergillus niger spore suspension. S5: The camellia oil nanoemulsion, probiotic microcapsules, PLGA herbal active ingredient nanoparticles and tea stem ultrafine powder obtained in steps S1-S4 are added to a three-dimensional motion mixer in proportion and mixed at 15 rpm for 30 min. Then, the mixture is aseptically packaged in a Class 10,000 clean environment to obtain camellia oil nanoemulsion capsules for colon-targeted delivery.

[0078] Example 4 The difference between this embodiment and Embodiment 2 is that in step S2, the probiotic microcapsules are replaced with primary capsules, and the chitosan coating and the acrylic resin Eudragit® FS30D are not applied. The rest is the same as in Embodiment 2.

[0079] Example 5 The difference between this embodiment and embodiment 2 is that the homogenization method in step S1 is to homogenize the first emulsion at a speed of 5000 rpm for 4 minutes, while the rest is the same as in embodiment 2.

[0080] Example 6 The difference between this embodiment and embodiment 2 is that in step S4, the tea stems are directly subjected to ultrafine grinding in an airflow pulverizer under the conditions of working fluid pressure of 0.7MPa and sorting frequency of 25Hz without fermentation by Aspergillus niger, and the particle size D90 is ≤20μm. The rest is the same as in embodiment 2.

[0081] Example 7 The difference between this embodiment and embodiment 2 is that in step S2, the pH-sensitive enteric material accounts for 10% of the total weight of the probiotic microcapsules, and the rest is the same as in embodiment 2.

[0082] Example 8 The difference between this embodiment and embodiment 2 is that in step S1, the mass ratio of camellia oil, quercetin, and tea saponin in the camellia oil nanoemulsion is 35:0.5:1, while the rest is the same as in embodiment 2.

[0083] Example 9 The difference between this embodiment and embodiment 2 is that in step S2, the pH-sensitive enteric material is sprayed without a fluidized bed. Instead, the precapsule is directly immersed in the enteric material solution, stirred and mixed, and then taken out and dried. The rest is the same as in embodiment 2.

[0084] Example 10 The difference between this embodiment and embodiment 2 is that the inoculation amount of Aspergillus niger spore suspension in step S4 is 2%, while the rest is the same as in embodiment 2.

[0085] Example 11 The difference between this embodiment and embodiment 2 is that in step S4, the Aspergillus niger spore suspension is replaced with Aspergillus oryzae spore suspension, and the rest is the same as in embodiment 2.

[0086] Comparative Example 1 The difference between this comparative example and Example 2 is that the amount of camellia oil nanoemulsion added is 10 parts, while the rest is the same as in Example 2.

[0087] Comparative Example 2 The difference between this comparative example and Example 2 is that the probiotic microcapsules are replaced with probiotic freeze-dried powder, while the rest is the same as in Example 2.

[0088] Comparative Example 3 The difference between this comparative example and Example 2 is that the PLGA-encapsulated nanoparticles of active Chinese medicine ingredients are replaced with resveratrol, while the rest is the same as in Example 2.

[0089] Comparative Example 4 The difference between this comparative example and Example 2 is that camellia oil nanoemulsion is replaced with camellia oil, while the rest is the same as in Example 2.

[0090] Test Example 1 The water-soluble dietary fiber content, particle size D90, viable bacteria count in the probiotic core, encapsulation efficiency, and drug loading of PLGA-encapsulated active ingredient nanoparticles of traditional Chinese medicine in Examples 1-11 and Comparative Examples 1-4 were tested using the following methods: Test method for water-soluble dietary fiber content in ultrafine tea stem powder: refer to the enzyme gravimetric method in the national standard GB5009.88 Determination of Dietary Fiber in Food.

[0091] Test method for the particle size D90 of tea stem ultrafine powder: The particle size is determined using a laser particle size analyzer. D90 indicates that 90% of the particles in the sample have a diameter smaller than this value.

[0092] Methods for testing the viable count of probiotic core cells: Traditional plate counting method: After serially diluting the sample, spread it on MRS agar plates, incubate under suitable conditions, and then count the colony-forming units.

[0093] Methods for testing the encapsulation efficiency and drug loading of PLGA-encapsulated nanoparticles containing active ingredients from traditional Chinese medicine: A1. Separation: The free drug and PLGA-encapsulated active ingredient nanoparticles of traditional Chinese medicine were separated by ultrafiltration centrifugation or dialysis. A2. Determination: The concentration of free drug was determined using HPLC.

[0094] A3. Calculation: a. Encapsulation rate (%) = (1 - Free drug amount / Total drug amount) × 100%; where, the total drug amount refers to the total mass of curcumin or resveratrol added in step S3; the free drug amount refers to the mass of curcumin or resveratrol not encapsulated in PLGA nanoparticles obtained after separation in step A1.

[0095] b. Drug loading = (Mass amount of active ingredient added to PLGA-encapsulated Chinese medicine nanoparticles / PLGA-encapsulated Chinese medicine nanoparticles) × 100%; Test results are shown in Table 1.

[0096] Table 1

[0097] Note: In Comparative Example 3, the PLGA-encapsulated Chinese medicine active ingredient nanoparticles were replaced with resveratrol. Since the Chinese medicine active ingredient was not encapsulated, the encapsulation efficiency and drug loading of the PLGA-encapsulated Chinese medicine active ingredient nanoparticles could not be measured, so it is indicated as "not applicable".

[0098] Based on the data from Examples 1-3 in Table 1, it can be seen that the technical solution using camellia oil nanoemulsion, probiotic microcapsules, PLGA drug-loaded nanoparticles, and Aspergillus niger fermented tea stem powder is reliable and has good reproducibility.

[0099] Example 4 used uncoated calcium alginate pre-capsules. Because chitosan and pH-sensitive enteric-coating materials were not used, the probiotics were not well protected, leading to a sharp decrease in the viable bacteria count in the prepared camellia oil nanoemulsion capsules. Furthermore, Example 4 used calcium alginate pre-capsules with a porous gel structure, which affected the physical state of the PLGA nanoparticles during the preparation of the camellia oil nanoemulsion capsules, thus reducing the encapsulation efficiency and drug loading of the PLGA nanoparticles. In addition, Example 4 directly demonstrates that the complete coating of the three-layer protective structure formed by sodium alginate, chitosan, and pH-sensitive enteric-coating material effectively protects probiotics from gastric acid erosion and achieves colon-targeting. This complete coating also forms a smooth, dense interface, reducing interference from other components in the camellia oil nanoemulsion capsules and improving the stability of the camellia oil nanoemulsion system.

[0100] In Example 5, the rotational speed during the high-speed shear homogenization process was 5000 rpm. At this speed, more heat is generated, which affects the overall microenvironment stability of the camellia oil nanoemulsion. This, in turn, interferes with the physical stability of the PLGA nanoparticles in the final product, leading to aggregation and leakage during storage or testing, resulting in a decrease in encapsulation efficiency and drug loading. This indicates that the preparation process of the camellia oil nanoemulsion not only affects the preparation of the camellia oil nanoemulsion itself but also indirectly affects the stability of other functional components.

[0101] In Example 6, the tea stem powder was not fermented, which ultimately led to a decrease in the release rate of prebiotics and active ingredients in the tea stem powder. Consequently, it could not provide a good storage microenvironment for the probiotics in the probiotic microcapsules, thus indirectly but significantly affecting the number of live probiotics in the probiotic microcapsules. In Examples 1-3, the tea stems were fermented with Aspergillus niger, which effectively converted insoluble dietary fiber into water-soluble dietary fiber, and its content was ≥30%. The number of live probiotics in Examples 1-3 was higher than that in Example 6.

[0102] Example 7: Insufficient addition of enteric material resulted in an excessively thin, non-dense, and uneven enteric coating layer of the probiotic microcapsules. This not only reduced the protective effect on the probiotics but also increased the particle size of the probiotic microcapsules, causing them to adhere to other functional components in the camellia oil nanoemulsion capsules. This fully demonstrates that sufficient and well-film-forming enteric material is crucial for maintaining the structural stability and functional realization of the entire colon-targeted delivery system.

[0103] In Example 8, the excessive amount of camellia oil added to the camellia oil nanoemulsion led to a decrease in its self-stability, resulting in phase separation, flocculation, and Austronesian ripening, thus causing it to lose its uniform and stable nanoscale carrier characteristics. Simultaneously, the unstable camellia oil nanoemulsion could not provide a uniform and stable dispersion environment for the PLGA nanoparticles. At this point, the PLGA nanoparticles would aggregate and settle. During the determination of the encapsulation efficiency of PLGA-encapsulated active ingredient nanoparticles, steps such as washing and centrifugation were involved. These aggregated and settled PLGA-encapsulated active ingredient nanoparticles were easily eluted and lost during the determination process, thereby reducing the encapsulation efficiency and drug loading of the PLGA-encapsulated active ingredient nanoparticles. Due to the instability of the camellia oil nanoemulsion carrier, a large number of PLGA-encapsulated active ingredient nanoparticles were lost during microencapsulation, ultimately significantly reducing the total amount of PLGA-encapsulated active ingredient nanoparticles that could be successfully encapsulated and retained in the product.

[0104] Example 9 uses an immersion stirring method instead of fluidized bed spraying, which results in uneven thickness and non-dense enteric coating of probiotic microcapsules, reducing the integrity of the microcapsule structure and making the probiotics more susceptible to damage during processing. Consequently, the number of viable probiotics decreases. Due to the uneven surface of the probiotic microcapsules, the dispersibility of the tea stem ultrafine powder deteriorates during the mixing process with PLGA-encapsulated Chinese medicine active ingredient nanoparticles and tea stem ultrafine powder, leading to a decrease in the encapsulation rate and drug loading of PLGA-encapsulated Chinese medicine active ingredient nanoparticles.

[0105] In Example 10, the amount of Aspergillus niger inoculated was reduced, which led to insufficient fermentation of the tea stem ultrafine powder and failed to maximize the conversion of insoluble fiber in the tea stem into water-soluble dietary fiber, resulting in a decrease in the water-soluble dietary fiber content in the camellia oil nanoemulsion capsules.

[0106] Example 11, compared to Examples 1, 2, and 3, demonstrates that *Aspergillus niger* fermentation effectively increases the water-soluble dietary fiber content in tea stems. Different fermentation strains produce different enzyme systems, resulting in varying degrees and methods of degradation of tea stem fiber. This alters the porosity, specific surface area, and surface roughness of the tea stem powder. The fermentation process also produces different metabolites, which may change the surface charge, hydrophilicity / hydrophobicity, and pH microenvironment of the tea stem powder. Although *Aspergillus niger* and *Aspergillus oryzae* belong to the same genus, their secreted enzyme systems differ in composition and activity. *Aspergillus niger* is known for producing potent cellulase and hemicellulase, which can more thoroughly degrade insoluble fiber in tea stems, converting it into water-soluble dietary fiber and making its structure more porous and easier to ultrafine grind. The enzyme system of *Aspergillus oryzae* may be more inclined towards the degradation of starch and protein, with relatively lower efficiency in fiber degradation. Therefore, its water-soluble dietary fiber content and grinding fineness are slightly inferior to those of *Aspergillus niger* fermentation products. This demonstrates that not all fermentation strains can achieve the optimal results of this application. Aspergillus niger is the key parameter for achieving high water-soluble dietary fiber content and suitable particle size in this application.

[0107] Comparative Example 1 showed a low addition of camellia oil nanoemulsion, resulting in a reduction in the amount of nanocarriers. This led to uneven dispersion and incomplete encapsulation of the camellia stem ultrafine powder. Simultaneously, due to insufficient oil phase, the ultrafine camellia stem powder, probiotic microcapsules, and PLGA-encapsulated active ingredient nanoparticles could not be adequately encapsulated and separated, causing severe adhesion and aggregation between the components, resulting in a larger overall particle size. Firstly, the probiotic microcapsules were partially exposed or embedded in the incomplete camellia oil nanoemulsion, making them more susceptible to damage from oxygen and moisture during storage, significantly reducing the survival rate of the probiotics. Secondly, the incomplete encapsulation of the PLGA-encapsulated active ingredient nanoparticles led to significant leakage in the later stages of preparation, resulting in a decrease in the encapsulation rate of the PLGA-encapsulated active ingredient nanoparticles. This indicates that a sufficient amount of camellia oil nanoemulsion is indispensable for forming structurally stable and functionally complete camellia oil nanoemulsion capsules.

[0108] Comparative Example 2 used probiotic freeze-dried powder. During the product preparation process, the number of live probiotics will decrease. However, the probiotics in the freeze-dried powder are in a dry, dormant, and stable state. Although there is no encapsulation protection, the metabolism of the bacteria basically stops during product mixing and short-term storage, and the decay rate is slow. Therefore, the number of live bacteria will decrease, but not by much.

[0109] In Comparative Example 3, the absence of PLGA nanoparticles as hydrophobic solid particles may make the system more prone to phase separation or localized concentration unevenness. Probiotic microcapsules may be more directly exposed at the interface or aggregate, making them more susceptible to damage in subsequent processing steps. The absence of PLGA nanoparticles alters the physical properties of the system, indirectly affecting the survival environment of the probiotics. This demonstrates that the tea stem ultrafine powder, probiotic microcapsules, PLGA-encapsulated traditional Chinese medicine active ingredient nanoparticles, and camellia oil nanoemulsion in this application have a synergistic effect; the absence or alteration of any component will affect the performance of the entire system.

[0110] Comparative Example 4 used camellia oil. First, during the preparation of camellia oil nanoemulsion capsules with other ingredients, the surface properties of the camellia oil may not be compatible with those of the probiotic microcapsules. During processing and storage, lipid peroxides generated by camellia oil can directly attack and damage the cell membranes of probiotics, leading to the death of live bacteria and resulting in uneven dispersion and easy aggregation of the probiotic microcapsules. Second, PLGA nanoparticles are hydrophobic particles, and their stable dispersion depends on the emulsification strength of the oil emulsion. Using camellia oil, a strong interfacial film cannot be formed, which can lead to leakage and aggregation of the PLGA-encapsulated active ingredient nanoparticles during homogenization and drying, ultimately resulting in a decrease in the encapsulation efficiency and drug loading of the PLGA-encapsulated active ingredient nanoparticles.

[0111] Test Example 2 The following tests were performed on Examples 1-11 and Comparative Examples 1-4, and the test methods were as follows: (1) Method for probiotic gastric acid survival rate: Referring to the "Group Standard for Gastric Juice Tolerance of Probiotic Agents", an in vitro static simulated digestion model was used. The sample was placed in simulated gastric juice (containing pepsin) at pH 3.0 (simulating fasting gastric juice) for 2 hours, and then the number of viable bacteria was measured; (2) Method for colonic release rate of camellia oil: An in vitro simulated digestion model was used, and the camellia oil was incubated in simulated gastric juice (2 hours), simulated small intestinal juice (2 hours) and simulated colonic juice (pH 7.2 phosphate buffer containing colonic contents enzymes, 24 hours) in sequence. After the completion of the entire simulated digestion (stomach 2h + small intestine 2h + colon 24h), the cumulative release rate of oleic acid in camellia oil in the colon stage was measured. (3) Accelerated stability test: The products were placed in a constant temperature and humidity chamber at 40±2℃ and 75±5% relative humidity, and samples were taken for testing after one month.

[0112] a. Viable bacteria retention rate test method: The viable bacteria count is determined as described above, and the data results are tested after 1 month of treatment; b. Peroxide value test method: The test shall be conducted in accordance with GB5009.227 Determination of peroxide value in food, and the degree of oxidation of camellia oil after 1 month shall be evaluated. c: The retention rate test method of active ingredients in traditional Chinese medicine was to determine the content of curcumin and resveratrol after 1 month using HPLC; the test results are shown in Table 2.

[0113] Table 2

[0114] According to the data in Table 2, Examples 1-3, using camellia oil nanoemulsions, probiotic microcapsules, PLGA-encapsulated nanoparticles of active Chinese medicine ingredients, and Aspergillus niger fermented tea stem ultrafine powder in specific ratios, exhibited a synergistic effect. Firstly, the products from these examples showed significant probiotic gastric acid survival rates (≥84.7%) and camellia oil colonic release rates (≥81.5%), demonstrating good upper gastrointestinal tolerance and colonic targeting. Secondly, the accelerated stability results in Table 2 showed a viable bacteria retention rate >90% and a component retention rate >95%, indicating excellent shelf-life stability. Finally, peroxide value is mainly related to the oxidative stability of oils in the product, directly reflecting the integrity and protective effect of the formulation system. The low peroxide values ​​in Examples 1-3 indicate low oxidation levels and product stability.

[0115] Example 4: The probiotic microcapsules lacked chitosan and enteric coating. After treatment with a simulated gastric acid solution, a large number of probiotics in the capsules died in the gastric acid. The incomplete microcapsule structure accelerated component degradation. Furthermore, due to the lack of chitosan and enteric coating, the surface of the probiotic microcapsules was calcium alginate, exhibiting a porous gel structure. This affected the stability of the camellia oil nanoemulsion during the preparation of the finished capsules, thus reducing the colonic release rate of camellia oil. In Example 7, insufficient enteric material resulted in a non-dense coating layer, leading to a decreased gastric acid survival rate and premature release, demonstrating that sufficient enteric material is crucial for forming an effective barrier. Example 9: Uneven stirring and impregnation coating led to decreased protective performance and a comprehensive decline in all indicators, indicating the importance of fluidized bed spraying technology in forming a uniform and dense enteric coating.

[0116] Example 5: High-speed polar shear homogenization at high rotation speeds led to instability and increased particle size of the camellia oil nanoemulsion, affecting the integrity of the camellia oil nanoemulsion carrier and resulting in irregular release and accelerated oxidation. Example 8: Excessive camellia oil in the camellia oil nanoemulsion caused the product capsules to rapidly demulsify and flocculate in simulated gastric juice, failing to effectively protect the probiotics and active ingredients in the probiotic microcapsules. The precise proportions of each component in the camellia oil nanoemulsion are crucial for the stability of the product capsules. In Comparative Example 1: Insufficient camellia oil nanoemulsion resulted in the inability to form a continuous carrier, resulting in an uneven, loose mixture that rapidly collapsed during simulated digestion, making it impossible to determine the camellia oil colon release rate. Comparative Example 4: Using ordinary camellia oil, it failed to form a structured dispersion and protective network. Its antioxidant capacity was far inferior to that of the nanoemulsion system stabilized by emulsifiers, and the oil was more prone to oxidative rancidity. The resulting lipid peroxides and free radicals directly attacked and damaged the cell membranes and DNA of probiotics, leading to a sharp decline in viable bacteria count and extremely high peroxide values.

[0117] Example 6: The tea stems were not fermented, resulting in a significantly lower content of water-soluble dietary fiber compared to Examples 1-3. However, the probiotic microcapsules and camellia oil nanoemulsion remained intact, so the gastric acid survival rate and release rate were not directly affected. The viable bacteria retention rate decreased due to the lack of a microenvironmental support from water-soluble dietary fiber. Example 10: Changes in the fermentation inoculum amount resulted in lower water-soluble dietary fiber content and viable bacteria retention rate compared to Example 2. This may indirectly affect the long-term survival of probiotics by influencing the yield of water-soluble dietary fiber, but has limited impact on the immediate survival rate of probiotics through the gastric acid stage. Example 11: The content and quality of water-soluble dietary fiber produced by Aspergillus oryzae fermentation may be lower than that of Aspergillus niger, leading to weakened nutritional support for probiotics during product storage, resulting in a slight decrease in viable bacteria retention rate in accelerated stability testing. Simultaneously, insufficient water-soluble dietary fiber available for probiotic utilization after simulated colon release may also affect their colonization and proliferation effects.

[0118] The probiotics in Comparative Example 2 showed an extremely low survival rate in gastric acid (0.8%), demonstrating that unprotected probiotics cannot withstand gastric acid. Due to structural changes, free probiotic freeze-dried powder particles compete with camellia oil nanoemulsions for the interface, disrupting the uniformity and stability of the nanoemulsions and causing premature demulsification. Without the protection of the emulsification system, the camellia oil nanoemulsions are released, dispersed, or degraded in large quantities in the stomach and small intestine, failing to reach the colon intact. Its colonic release behavior could not be effectively assessed (marked as 'unmeasurable' in Table 2). During preparation, free probiotic particles interfere with the uniform encapsulation and embedding of PLGA-encapsulated active ingredient nanoparticles by the camellia oil nanoemulsion. The lack of effective protection may lead to premature drug leakage, aggregation, or degradation of the PLGA-encapsulated active ingredient nanoparticles, resulting in a significant reduction in the "effective drug amount" that ultimately reaches and acts on the colon. In Comparative Example 3, the drug was not encapsulated, and the free resveratrol was rapidly released in the gastrointestinal stage, with a simulated colon release rate of only 28.7%, a stark contrast to Example 2 (84.1%). This demonstrates that PLGA nanoparticles, as hydrophobic solid microparticles, when uniformly dispersed in camellia oil nanoemulsions, help increase the viscosity and steric hindrance of the system, thereby improving its stability.

[0119] Test Example 3 Experimental animal grouping: 70 male C57BL / 6 mice aged 6-8 weeks were randomly divided into 7 groups of 10 mice each.

[0120] Modeling methods: Blank control: Drinking sterile water and then receiving physiological saline by gavage; Model control: Drinking 2.5% DSS solution and then receiving physiological saline by gavage; Example 2: Drinking 2.5% DSS solution and then receiving the sample from Example 2 by gavage (500 mg / kg); Comparative example 2: Drinking 2.5% DSS solution and then receiving the sample from Comparative example 2 by gavage (equal dose); Comparative example 3: Drinking 2.5% DSS and then receiving the sample from Comparative example 3 by gavage (equal dose); Comparative example 4: Drinking 2.5% DSS and then receiving the sample from Comparative example 4 by gavage (equal dose); Positive control: Drinking 2.5% DSS and then receiving a commonly used clinical drug (e.g., mesalazine, 50 mg / kg) by gavage; Except for the blank group, all other groups freely drank 2.5% DSS solution for 7 consecutive days to induce acute colitis. Gavage administration began 1 week before modeling, once daily, and continued until the end of the experiment (14 days in total). The samples from both examples and comparative examples were suspended in sterile physiological saline, and the gavage dose was 500 mg / kg. The blank control group and the model control group were administered an equal volume of physiological saline by gavage; the evaluation indicators and detection methods are shown in Table 3. Table 3

[0121] The Disease Activity Index (DAI) is the gold standard for assessing the severity of experimental colitis. It quantifies disease activity by comprehensively considering three key clinical indicators. The scoring formula is: DAI = (Weight Loss Score + Stool Characteristics Score + Rectal Blood Score) / 3. The specific procedure is as follows: (1) Daily monitoring: From the start of modeling, all experimental animals are weighed and observed at a fixed time every day (e.g., 9-11 am).

[0122] (2) Indicator Recording: ① Weight: Using an electronic balance with an accuracy of 0.1 g, calculate the percentage decrease compared to the previous day; ② Stool Characteristics: Directly observe the stool in the mouse cage, or gently stimulate the anus to induce defecation before making a judgment. It is important to distinguish between loose stool (semi-formed) and watery stool (watery and possibly sticking to the anus); ③ Blood in Stool: Use guaiac ester method fecal occult blood test strips for detection. Avoid interference caused by food pigments (such as red feed). Visible blood in stool is scored directly according to the severity; The scoring criteria are shown in Table 4: Table 4

[0123] (3) Experimental results and data The following tables show the key indicator data of each group of mice after the experiment (expressed as mean ± standard deviation, n=6). The test results are shown in Table 5 and Table 6.

[0124] Table 5

[0125] Table 6

[0126] In Test Example 3, the model control used DSS to disrupt the colonic mucosal barrier, triggering a strong immune inflammatory response. The DAI score reached as high as 3.5, with a sharp increase in pro-inflammatory factor levels and a decrease in the anti-inflammatory factor IL-10, presenting a typical state of severe acute colitis. Example 2 used the complete colon-targeted delivery system of this application, which significantly reduced the DAI score to near normal levels (1.2), with a substantial decrease in pro-inflammatory factors (TNF-α, IL-6, IL-1β) and an increase in the anti-inflammatory factor IL-10. Example 2 used camellia oil nanoemulsion as a stable carrier. The three-layer encapsulated probiotic microcapsules ensured that a large number of live bacteria reached the colon and played a role in regulating the flora and repairing the barrier. PLGA-encapsulated nanoparticles of active Chinese medicine ingredients achieved colon-targeted sustained release of anti-inflammatory components. Tea stem ultrafine powder provided soluble dietary fiber, thus forming a multi-target, multi-component synergistic effect in the colon, with strong anti-inflammatory and immunomodulatory effects. The positive control used mesalazine, a classic anti-inflammatory drug, which mainly works through local anti-inflammatory action. In test example 3, the positive control showed a clear therapeutic effect, but the DAI score and pro-inflammatory factor levels were higher than in example 2, while the level of the anti-inflammatory factor IL-10 was close to that in example 2. This indicates that the proposed treatment has multiple effects, including anti-inflammatory, immunomodulatory, and barrier repair, and possesses more comprehensive therapeutic advantages.

[0127] In Comparative Example 2, the probiotics were added in the form of lyophilized powder, which significantly reduced the therapeutic effect, but was still better than the model group. The pro-inflammatory factor levels were higher, and the increase in IL-10 was limited. The vast majority of probiotics were killed in gastric acid, failing to reach the colon in sufficient quantities to exert their beneficial effects, resulting in a near-complete loss of their functions in regulating gut microbiota, anti-inflammation, and barrier repair.

[0128] In Comparative Example 3, the active ingredient of the traditional Chinese medicine (resveratrol) was added in free form, without being formulated into PLGA-encapsulated nanoparticles. Its efficacy was significantly reduced, and the level of pro-inflammatory factors was higher. Free drug is partially absorbed or destroyed in the upper gastrointestinal tract, failing to achieve an effective therapeutic concentration in the colon. Encapsulating the drug in PLGA nanoparticles is a traditional Chinese medicine approach to achieve targeted drug release in the colon, increase local drug concentration and efficacy; simply mixing drugs cannot achieve targeted therapy.

[0129] Comparative Example 4, which used ordinary camellia oil instead of camellia oil nanoemulsion, showed the worst efficacy, with only slightly better results than the model group in all comparisons. Ordinary camellia oil cannot form a stable nanoemulsion carrier, resulting in uneven dispersion and poor physical stability of probiotic microcapsules and PLGA-encapsulated Chinese medicine active ingredient nanoparticles in the formulation. After oral administration, the system rapidly disintegrates, and the components cannot be synergistically delivered to the colon.

[0130] The DAI score of Example 2 was significantly lower than that of Comparative Examples 2-4, and close to that of the blank control and positive control, indicating that it can effectively alleviate the clinical symptoms of colitis. The levels of pro-inflammatory factors in the colonic tissue of Example 2 were significantly reduced, indicating that it has a strong anti-inflammatory effect. The camellia oil nanoemulsion capsules prepared in this application can effectively protect the contents as they pass through the upper digestive tract, allowing for precise release in the colon, facilitating storage and transportation, and effectively alleviating colitis in animal models.

[0131] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A colon-targeted camellia oil nanoemulsion capsule, characterized in that, By mass fraction, it includes: 30-40 parts of camellia oil nanoemulsion, 20-30 parts of probiotic microcapsules, 10-15 parts of PLGA-encapsulated Chinese herbal active ingredient nanoparticles, and 15-20 parts of tea stem ultrafine powder. The camellia oil nanoemulsion includes camellia oil, quercetin, and tea saponin; The probiotic microcapsules include a probiotic core, which is encapsulated with sodium alginate, chitosan, and a pH-sensitive enteric-coated material.

2. The camellia oil nanoemulsion capsule for colon-targeted delivery according to claim 1, characterized in that, The mass ratio of camellia oil, quercetin, and tea saponin in the camellia oil nanoemulsion is 1:(0.02-0.05):(0.1-0.3).

3. The camellia oil nanoemulsion capsule for colon-targeted delivery according to claim 2, characterized in that, The viable count of the probiotic core is ≥1.0×10⁻⁶. 11 CFU / g; and / or The probiotic core includes at least one of Lactobacillus plantarum, Bifidobacterium, and butyric acid bacteria.

4. The camellia oil nanoemulsion capsule for colon-targeted delivery according to claim 2, characterized in that, The pH-sensitive enteric-coated material accounts for 15-25% of the total weight of the probiotic microcapsules.

5. The camellia oil nanoemulsion capsule for colon-targeted delivery according to claim 1, characterized in that, The drug loading of the PLGA-encapsulated nanoparticles containing active ingredients of traditional Chinese medicine is 5-15%.

6. The method for preparing colon-targeted camellia oil nanoemulsion capsules according to any one of claims 1-5, characterized in that, Includes the following steps: S1: Camellia oil, quercetin, and tea saponin are mixed in proportion to obtain the first oil phase. The first emulsifier is mixed with water to obtain the first aqueous phase. The first aqueous phase and the first oil phase are mixed and emulsified to obtain the first emulsion. After homogenization, camellia oil nano-emulsion is obtained. S2: Mix the probiotic core with sodium alginate solution to obtain a bacterial gel suspension. Then, drop the bacterial gel suspension into calcium chloride solution and mix to obtain a primary capsule. Then, immerse the primary capsule in chitosan solution to obtain chitosan-coated primary capsules. Then, spray the chitosan-coated primary capsules with a pH-sensitive enteric coating material to obtain probiotic microcapsules. S3: PLGA and the active ingredients of traditional Chinese medicine are dissolved in an organic solvent to obtain a second oil phase. Polyvinyl alcohol is mixed with water to obtain a second aqueous phase. The oil phase, aqueous phase and second emulsifier are mixed and emulsified to obtain a second emulsion. Then the solvent is evaporated, and the PLGA nanoparticles of the active ingredients of traditional Chinese medicine are collected after separation. S4: After sterilizing the tea stems, inoculate them with Aspergillus niger for fermentation, dry them, and then pulverize them into ultrafine tea stem powder. S5: The camellia oil nanoemulsion, probiotic microcapsules, PLGA herbal active ingredient nanoparticles and tea stem ultrafine powder obtained in steps S1-S4 are mixed in proportion and then aseptically packaged to obtain colon-targeted delivery camellia oil nanoemulsion capsules.

7. The preparation method according to claim 6, characterized in that, In step S2, the pH-sensitive enteric-coated material is sprayed in a fluidized bed with an inlet air temperature of 35-40℃, an inlet air velocity of 8-10 m³ / h, and an atomization pressure of 0.8-1.2 bar; and / or The spraying rate of the pH-sensitive enteric material is 3 g / min.

8. The preparation method according to claim 6, characterized in that, The preparation method of the Aspergillus niger spore suspension in step S4 includes the following steps: A1: Inoculate Aspergillus niger onto potato dextrose agar slant medium and incubate at 25-30℃ for 5-7 days; A2: Add sterile physiological saline containing Tween 80 to the culture medium obtained in step A1, elute the Aspergillus niger spores into the liquid, filter, and obtain the Aspergillus niger spore suspension.

9. The preparation method according to claim 6, characterized in that, In step S4, the inoculum size of the Aspergillus niger spore suspension is 5-8%.

10. The use of camellia oil nanoemulsion capsules delivered to the colon according to any one of claims 1-9 in functional foods, health products or drugs for the prevention and / or treatment of intestinal diseases and the regulation of intestinal flora.

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