A catechin-loaded pepper starch nanoparticle and a method for preparing the same

Abstract

The present application relates to the field of preparation of starch nanoparticles, and specifically provides a kind of epicatechin loaded pepper starch nanoparticles and its preparation method.The preparation method is to use pepper starch to load epicatechin, and through comparative experiment, it is proved that the specific process combination (debranching, stirring, homogenization) of the present application is crucial to realize high encapsulation rate.The finally prepared pepper starch nanoparticles can significantly improve the encapsulation rate of epicatechin.This means higher loading efficiency and lower waste of active ingredients, with significant industrial cost advantage.

Description

Technical Field

[0001] This invention relates to a method for preparing starch nanoparticles, specifically to a pepper starch nanoparticle loaded with epicatechin and its preparation method. Background Technology

[0002] Starch nanoparticles (SNPs) are considered a highly promising encapsulation carrier for active ingredients due to their good biocompatibility, biodegradability, and wide availability, and are widely used in functional foods, pharmaceuticals, and cosmetics. Currently, starch nanoparticles are commonly prepared from bulk raw materials such as corn starch and potato starch through physical, chemical, or enzymatic treatments for encapsulating fat-soluble vitamins, polyphenolic compounds, and other active substances.

[0003] Epicatechin, a natural polyphenol, possesses excellent antioxidant and anti-inflammatory activities, but it faces challenges such as easy degradation and low bioavailability in practical applications. Encapsulation using nanocarriers is an effective strategy to improve its application performance. However, highly efficient loading systems for epicatechin are still relatively rare, especially research on constructing nanocarriers using pepper starch as a matrix for epicatechin encapsulation and controlled release, which has not yet been systematically reported. The inventors discovered that existing starch-based nanocarriers have significant technical bottlenecks when loading epicatechin: the encapsulation efficiency (EE) is generally low. For example, nanoparticles prepared using common corn starch as a matrix often struggle to achieve an encapsulation rate of epicatechin exceeding 35% (see Example 6), resulting in a significant waste of active ingredients during the loading process, high production costs, and severely limiting its industrial application.

[0004] Studies have shown that the loading capacity of starch nanoparticles is closely related to their source (which directly affects the amylose / branched starch ratio, crystal structure, and other microscopic properties) and preparation process. Starches from different sources exhibit drastically different encapsulation properties under the same processing conditions. Therefore, finding a novel starch raw material with high natural affinity and high loading efficiency for epicatechin, and developing a matching preparation process, has become crucial to solving the problem of low encapsulation efficiency and is a direction that those skilled in the art continue to explore.

[0005] As a non-mainstream starch, pepper starch has not yet fully explored its potential as a nanocarrier, especially its performance in efficiently loading polyphenolic active ingredients (such as epicatechin) has not been reported.

[0006] Based on this, the present invention aims to provide nanoparticles made from pepper starch and a method for preparing the same, with the primary objective of significantly improving the encapsulation efficiency of epicatechin to overcome the aforementioned deficiencies in the prior art. Summary of the Invention

[0007] Based on the structural characteristics of piperine starch and previous research, this invention utilizes piperine starch nanoparticles to improve the encapsulation efficiency of epicatechin.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides pepper starch nanoparticles loaded with epicatechin, the preparation method of which includes the following steps:

[0010] Step 1: Preparation of pepper starch: Soak white peppercorns in a 0.5% w / v sodium metabisulfite solution at a ratio of 1g:6mL for 24 hours at 4℃ to obtain white pepper paste; grind the soaked white pepper paste for 1 minute and repeat the grinding process 3 times; filter the ground white pepper paste through a 270-mesh nylon cloth, wash with distilled water, centrifuge at 4000 r / min for 10 minutes, and collect the starch granules; wash the starch granules twice with anhydrous ethanol, then wash them three times with water, dry them at 40℃ for 48 hours, pulverize them, and pass them through a 100-mesh sieve to obtain pepper starch;

[0011] Step 2: Preparation of debranched pepper starch: Using a pH 4.8 phosphoric acid / citric acid buffer solution as a solvent, dissolve the pepper starch prepared in Step 1 at a ratio of 100 g / L, gelatinize thoroughly in boiling water for 30 min, and cool to 58℃. The phosphoric acid / citric acid buffer solution consists of 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L sodium citrate. The starch dry weight is 3.09 × 10⁻⁶ g / L. -6 Pullulanase was added at kat / g to carry out debranching enzymatic hydrolysis for 8 h. After centrifugation at 2000 r / min for 5 min, the undebranched starch was removed and the supernatant was collected. The supernatant was then inactivated by enzyme at 100℃ for 10 min, followed by centrifugation at 2000 r / min for 2 min to remove the inactivated enzyme. After cooling to room temperature, the supernatant was collected as the debranched starch solution. The debranched starch was precipitated by mixing the debranched starch solution with anhydrous ethanol at a volume ratio of 1:3. The precipitate was washed three times with distilled water and then freeze-dried to obtain the debranched starch.

[0012] Step 3: Preparation of pepper starch nanoparticles: 10 g / L debranched starch was heated at 100℃ for 30 min, then 5 times the volume of anhydrous ethanol was added, and the mixture was magnetically stirred at 600 r / min for 1 h; homogenized by microjets at 11000 PSI pressure for 2 min; centrifuged at 8000 r / min for 10 min, the precipitate was collected, and the precipitate was washed 3 times with anhydrous ethanol; the mixture was freeze-dried to obtain pepper starch nanoparticles.

[0013] Step 4: Loading with epicatechin: Add 10 mg of pepper starch granules to 1.0 mL of 20 mmol / L catechin solution and stir magnetically at 600 r / min for 2 h; centrifuge at 12000 r / min for 20 min, remove the supernatant, collect the precipitate and wash it 3 times, and freeze dry under vacuum to obtain pepper starch granules loaded with catechin.

[0014] The present invention also provides the application of the aforementioned pepper starch nanoparticles loaded with epicatechin in the preparation of antioxidants.

[0015] The beneficial effects of this invention are:

[0016] (1) Significantly improved encapsulation efficiency and excellent load capacity:

[0017] This invention, through specific raw material selection and process combination, unexpectedly achieves extremely high encapsulation efficiency for epicatechin. As shown in Example 1, the encapsulation rate of epicatechin by the pepper starch nanoparticles reaches 84.67%, significantly better than the encapsulation effects of corn starch nanoparticles (Example 6, 34.45%) and banana starch nanoparticles (Example 7, 61.46%) in the comparative examples. This result demonstrates that pepper starch, as a nanocarrier material, exhibits unique high affinity and high loading capacity for epicatechin, effectively solving the technical problem of low encapsulation efficiency of existing starch carriers and significantly reducing the loss of active ingredients during the preparation process.

[0018] (2) Key to process synergy and good reproducibility:

[0019] This invention reveals the process synergy required to achieve high encapsulation efficiency. Comparative Examples 2-5 demonstrate that a complete process chain (including enzymatic debranching, magnetic stirring, and microfluidic homogenization) is crucial for achieving high encapsulation efficiency. The absence of any key step (such as debranching or stirring) or changes in parameters (such as shortening the gelatinization time) leads to a significant decrease in encapsulation efficiency and an increase in particle size. This proves that the method provided by this invention is not an obvious, conventional method, but rather a reproducible and efficient preparation scheme requiring a specific combination of steps.

[0020] (3) Raw material innovation has broadened the avenues for high-value applications:

[0021] This invention is the first to systematically utilize pepper starch to construct nanoparticles loaded with epicatechin, revealing its superior performance compared to traditional starches (such as corn starch). This not only provides a novel and efficient carrier option for the high-efficiency loading of epicatechin, but also opens up new avenues for the high-value utilization of pepper starch, demonstrating promising application development prospects and economic benefits.

[0022] (4) It provided a foundation for industrialized production:

[0023] High encapsulation efficiency directly translates to higher production efficiency and lower raw material costs. The method provided by this invention has a clear process route and well-defined parameters, resulting in products with high yield and stable performance, laying a solid technical foundation for their large-scale application in functional foods, health products, and other fields. Detailed Implementation

[0024] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0025] Example 1: A method for preparing pepper starch nanoparticles loaded with epicatechin

[0026] This embodiment provides a method for preparing pepper starch nanoparticles loaded with epicatechin, the method comprising the following steps:

[0027] Step 1: Preparation of pepper starch: Soak white peppercorns in a 0.5% w / v sodium metabisulfite solution at a ratio of 1g:6mL for 24 hours at 4℃ to obtain white pepper paste; grind the soaked white pepper paste for 1 minute and repeat the grinding process 3 times; filter the ground white pepper paste through a 270-mesh nylon cloth, wash with distilled water, centrifuge at 4000 r / min for 10 minutes, and collect the starch granules; wash the starch granules twice with anhydrous ethanol, then wash them three times with water, dry them at 40℃ for 48 hours, pulverize them, and pass them through a 100-mesh sieve to obtain pepper starch;

[0028] Step 2: Preparation of debranched pepper starch: Using a pH 4.8 phosphoric acid / citric acid buffer solution as a solvent, dissolve the pepper starch prepared in Step 1 at a ratio of 100 g / L, gelatinize thoroughly in boiling water for 30 min, and cool to 58℃. The phosphoric acid / citric acid buffer solution consists of 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L sodium citrate. The starch dry weight is 3.09 × 10⁻⁶ g / L. -6 Pullulanase was added at kat / g to carry out debranching enzymatic hydrolysis for 8 h. After centrifugation at 2000 r / min for 5 min, the undebranched starch was removed and the supernatant was collected. The supernatant was then inactivated by enzyme at 100℃ for 10 min, followed by centrifugation at 2000 r / min for 2 min to remove the inactivated enzyme. After cooling to room temperature, the supernatant was collected as the debranched starch solution. The debranched starch was precipitated by mixing the debranched starch solution with anhydrous ethanol at a volume ratio of 1:3. The precipitate was washed three times with distilled water and then freeze-dried to obtain the debranched starch.

[0029] Step 3: Preparation of pepper starch nanoparticles: 10 g / L debranched starch was heated at 100℃ for 30 min, then 5 times the volume of anhydrous ethanol was added, and the mixture was magnetically stirred at 600 r / min for 1 h; homogenized by microjets at 11000 PSI pressure for 2 min; centrifuged at 8000 r / min for 10 min, the precipitate was collected, and the precipitate was washed 3 times with anhydrous ethanol; the mixture was freeze-dried to obtain pepper starch nanoparticles.

[0030] Step 4: Loading with epicatechin: Add 10 mg of pepper starch granules to 1.0 mL of 20 mmol / L catechin solution and stir magnetically at 600 r / min for 2 h; centrifuge at 12000 r / min for 20 min, remove the supernatant, collect the precipitate and wash it 3 times, and freeze dry under vacuum to obtain pepper starch granules loaded with catechin.

[0031] Example 2: A method for preparing pepper starch nanoparticles loaded with epicatechin

[0032] This embodiment provides a method for preparing pepper starch nanoparticles loaded with epicatechin, the method comprising the following steps:

[0033] Step 1: Preparation of pepper starch: Soak white peppercorns in a 0.5% w / v sodium metabisulfite solution at a ratio of 1g:6mL for 24 hours at 4℃ to obtain white pepper paste; grind the soaked white pepper paste for 1 minute and repeat the grinding process 3 times; filter the ground white pepper paste through a 270-mesh nylon cloth, wash with distilled water, centrifuge at 4000 r / min for 10 minutes, and collect the starch granules; wash the starch granules twice with anhydrous ethanol, then wash them three times with water, dry them at 40℃ for 48 hours, pulverize them, and pass them through a 100-mesh sieve to obtain pepper starch;

[0034] Step 2: Preparation of debranched pepper starch: Using a pH 4.8 phosphate / citric acid buffer solution as a solvent, dissolve the pepper starch prepared in Step 1 at a ratio of 100 g / L, gelatinize thoroughly in boiling water for 10 min, and cool to 58℃. The phosphate / citric acid buffer solution consists of 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L sodium citrate. The starch dry weight is 3.09 × 10⁻⁶ g / L. -6 Pullulanase was added at kat / g to carry out debranching enzymatic hydrolysis for 8 h. After centrifugation at 2000 r / min for 5 min, the undebranched starch was removed and the supernatant was collected. The supernatant was then inactivated by enzyme at 100℃ for 10 min, followed by centrifugation at 2000 r / min for 2 min to remove the inactivated enzyme. After cooling to room temperature, the supernatant was collected as the debranched starch solution. The debranched starch was precipitated by mixing the debranched starch solution with anhydrous ethanol at a volume ratio of 1:3. The precipitate was washed three times with distilled water and then freeze-dried to obtain the debranched starch.

[0035] Step 3: Preparation of pepper starch nanoparticles: 10 g / L of debranched starch was heated at 100℃ for 30 min, followed by the addition of 5 times the volume of anhydrous ethanol. The mixture was magnetically stirred at 600 r / min for 1 h. The mixture was then homogenized by microjets at 11000 PSI pressure for 2 min. After centrifugation at 8000 r / min for 10 min, the precipitate was collected and washed three times with anhydrous ethanol. The precipitate was then freeze-dried to obtain pepper starch nanoparticles.

[0036] Step 4: Loading with epicatechin: Add 10 mg of pepper starch granules to 1.0 mL of 20 mmol / L catechin solution and stir magnetically at 600 r / min for 2 h; centrifuge at 12000 r / min for 20 min, remove the supernatant, collect the precipitate and wash it 3 times, and freeze dry under vacuum to obtain pepper starch granules loaded with catechin.

[0037] Example 3: A method for preparing pepper starch nanoparticles loaded with epicatechin

[0038] This embodiment provides a method for preparing pepper starch nanoparticles loaded with epicatechin, the method comprising the following steps:

[0039] Step 1: Preparation of pepper starch: Soak white peppercorns in a 0.5% w / v sodium metabisulfite solution at a ratio of 1g:6mL for 24 hours at 4℃ to obtain white pepper paste; grind the soaked white pepper paste for 1 minute and repeat the grinding process 3 times; filter the ground white pepper paste through a 270-mesh nylon cloth, wash with distilled water, centrifuge at 4000 r / min for 10 minutes, and collect the starch granules; wash the starch granules twice with anhydrous ethanol, then wash them three times with water, dry them at 40℃ for 48 hours, pulverize them, and pass them through a 100-mesh sieve to obtain pepper starch;

[0040] Step 2: Preparation of debranched pepper starch: Using a pH 4.8 phosphoric acid / citric acid buffer solution as a solvent, dissolve the pepper starch prepared in Step 1 at a ratio of 100 g / L, gelatinize thoroughly in boiling water for 30 min, and cool to 58℃. The phosphoric acid / citric acid buffer solution consists of 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L sodium citrate. The starch dry weight is 3.09 × 10⁻⁶ g / L. -6 Pullulanase was added at kat / g to carry out debranching enzymatic hydrolysis for 8 h. After centrifugation at 2000 r / min for 5 min, the undebranched starch was removed and the supernatant was collected. The supernatant was then inactivated by enzyme at 100℃ for 10 min, followed by centrifugation at 2000 r / min for 2 min to remove the inactivated enzyme. After cooling to room temperature, the supernatant was collected as the debranched starch solution. The debranched starch was precipitated by mixing the debranched starch solution with anhydrous ethanol at a volume ratio of 1:3. The precipitate was washed three times with distilled water and then freeze-dried to obtain the debranched starch.

[0041] Step 3: Preparation of pepper starch nanoparticles: 10 g / L debranched starch was heated at 100℃ for 30 min, and then 5 times the volume of anhydrous ethanol was added. The mixture was magnetically stirred at 600 r / min for 1 h. Then, it was centrifuged at 8000 r / min for 10 min, the precipitate was collected, and the precipitate was washed 3 times with anhydrous ethanol. The mixture was then freeze-dried to obtain pepper starch nanoparticles.

[0042] Step 4: Loading with epicatechin: Add 10 mg of pepper starch granules to 1.0 mL of 20 mmol / L catechin solution and stir magnetically at 600 r / min for 2 h; centrifuge at 12000 r / min for 20 min, remove the supernatant, collect the precipitate and wash it 3 times, and freeze dry under vacuum to obtain pepper starch granules loaded with catechin.

[0043] Example 4: A method for preparing pepper starch nanoparticles loaded with epicatechin

[0044] This embodiment provides a method for preparing pepper starch nanoparticles loaded with epicatechin, the method comprising the following steps:

[0045] Step 1: Preparation of pepper starch: Soak white peppercorns in a 0.5% w / v sodium metabisulfite solution at a ratio of 1g:6mL for 24 hours at 4℃ to obtain white pepper paste; grind the soaked white pepper paste for 1 minute and repeat the grinding process 3 times; filter the ground white pepper paste through a 270-mesh nylon cloth, wash with distilled water, centrifuge at 4000 r / min for 10 minutes, and collect the starch granules; wash the starch granules twice with anhydrous ethanol, then wash them three times with water, dry them at 40℃ for 48 hours, pulverize them, and pass them through a 100-mesh sieve to obtain pepper starch;

[0046] Step 2: Preparation of debranched pepper starch: Using a pH 4.8 phosphoric acid / citric acid buffer solution as a solvent, dissolve the pepper starch prepared in Step 1 at a ratio of 100 g / L, gelatinize thoroughly in boiling water for 30 min, and cool to 58℃. The phosphoric acid / citric acid buffer solution consists of 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L sodium citrate. The starch dry weight is 3.09 × 10⁻⁶ g / L. -6 Pullulanase was added at kat / g to carry out debranching enzymatic hydrolysis for 8 h. After centrifugation at 2000 r / min for 5 min, the undebranched starch was removed and the supernatant was collected. The supernatant was then inactivated by enzyme at 100℃ for 10 min, followed by centrifugation at 2000 r / min for 2 min to remove the inactivated enzyme. After cooling to room temperature, the supernatant was collected as the debranched starch solution. The debranched starch was precipitated by mixing the debranched starch solution with anhydrous ethanol at a volume ratio of 1:3. The precipitate was washed three times with distilled water and then freeze-dried to obtain the debranched starch.

[0047] Step 3: Preparation of pepper starch nanoparticles: 10 g / L debranched starch was heated at 100℃ for 30 min, then 5 times the volume of anhydrous ethanol was added, and the mixture was homogenized by microfluidic jet for 2 min at 11000 PSI pressure; the mixture was centrifuged at 8000 r / min for 10 min, the precipitate was collected, and the precipitate was washed 3 times with anhydrous ethanol; the mixture was freeze-dried to obtain pepper starch nanoparticles.

[0048] Step 4: Loading with epicatechin: Add 10 mg of pepper starch granules to 1.0 mL of 20 mmol / L catechin solution and stir magnetically at 600 r / min for 2 h; centrifuge at 12000 r / min for 20 min, remove the supernatant, collect the precipitate and wash it 3 times, and freeze dry under vacuum to obtain pepper starch granules loaded with catechin.

[0049] Example 5: A method for preparing pepper starch nanoparticles loaded with epicatechin

[0050] Step 1: Preparation of pepper starch: Soak white peppercorns in a 0.5% w / v sodium metabisulfite solution at a ratio of 1g:6mL for 24 hours at 4℃ to obtain white pepper paste; grind the soaked white pepper paste for 1 minute and repeat the grinding process 3 times; filter the ground white pepper paste through a 270-mesh nylon cloth, wash with distilled water, centrifuge at 4000 r / min for 10 minutes, and collect the starch granules; wash the starch granules twice with anhydrous ethanol, then wash them three times with water, dry them at 40℃ for 48 hours, pulverize them, and pass them through a 100-mesh sieve to obtain pepper starch;

[0051] Step 2: Preparation of pepper starch nanoparticles: 100 g / L pepper starch was heated at 100℃ for 30 min, then 5 times the volume of anhydrous ethanol was added, and the mixture was magnetically stirred at 600 r / min for 1 h; homogenized by microjets at 11000 PSI pressure for 2 min; centrifuged at 8000 r / min for 10 min, the precipitate was collected, and the precipitate was washed 3 times with anhydrous ethanol; the mixture was freeze-dried to obtain pepper starch nanoparticles.

[0052] Example 6: A method for preparing corn starch nanoparticles loaded with epicatechin

[0053] This embodiment provides a method for preparing corn starch nanoparticles loaded with epicatechin, the method comprising the following steps:

[0054] Step 1: Purchase corn starch (purity above 95%, amylose content 10%), food grade, purchased from Guangxi Cenxi Triangle Starch Co., Ltd.

[0055] Step 2: Preparation of debranched corn starch: Using a pH 4.8 phosphate / citric acid buffer solution as a solvent, dissolve the corn starch purchased in Step 1 at a ratio of 100 g / L, gelatinize thoroughly in boiling water for 30 min, and cool to 58℃. The phosphate / citric acid buffer solution consists of 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L sodium citrate. The starch dry weight is 3.09 × 10⁻⁶ g / L. -6 Pullulanase was added at kat / g to carry out debranching enzymatic hydrolysis for 8 h. After centrifugation at 2000 r / min for 5 min, the undebranched starch was removed and the supernatant was collected. The supernatant was then inactivated by enzyme at 100℃ for 10 min, followed by centrifugation at 2000 r / min for 2 min to remove the inactivated enzyme. After cooling to room temperature, the supernatant was collected as the debranched starch solution. The debranched starch was precipitated by mixing the debranched starch solution with anhydrous ethanol at a volume ratio of 1:3. The precipitate was washed three times with distilled water and then freeze-dried to obtain the debranched starch.

[0056] Step 3: Preparation of corn starch nanoparticles: 10 g / L debranched starch was heated at 100℃ for 30 min, followed by the addition of 5 times the volume of anhydrous ethanol. The mixture was magnetically stirred at 600 r / min for 1 h. The mixture was then homogenized by microfluidic jet for 2 min at 11000 PSI pressure. After centrifugation at 8000 r / min for 10 min, the precipitate was collected and washed three times with anhydrous ethanol. The precipitate was then freeze-dried to obtain corn starch nanoparticles.

[0057] Step 4: Loading epicatechin: Add 10 mg of corn starch granules to 1.0 mL of 20 mmol / L catechin solution and stir magnetically at 600 r / min for 2 h; centrifuge at 12000 r / min for 20 min, remove the supernatant, collect the precipitate and wash it 3 times, and freeze dry under vacuum to obtain corn starch granules loaded with catechin.

[0058] Example 7: A method for preparing banana starch nanoparticles loaded with epicatechin.

[0059] This embodiment provides a method for preparing banana starch nanoparticles loaded with epicatechin, the method comprising the following steps:

[0060] Step 1: Prepare banana starch. Wash, peel, and cut green bananas into small pieces. Soak the banana pieces in a color-protecting solution (20 g / L citric acid + 2 g / L ascorbic acid) for 15 min. After color protection, grind the banana pieces in deionized water using a pulper, and sieve through a 200-mesh sieve. Treat the resulting starch with 0.25% NaOH solution to remove surface proteins and plant pigments until the starch is free of impurities. Finally, wash the starch precipitate with deionized water, freeze-dry under vacuum at -40 ℃ for 48 h, grind into a fine powder, sieve, and store in a sealed container.

[0061] Step 2: Preparation of debranched banana starch: Using a pH 4.8 phosphate / citric acid buffer solution as a solvent, dissolve the banana starch prepared in Step 1 at a ratio of 100 g / L, gelatinize thoroughly in boiling water for 30 min, and cool to 58℃. The phosphate / citric acid buffer solution consists of 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L sodium citrate. The starch dry weight is 3.09 × 10⁻⁶ g / L. -6 Pullulanase was added at kat / g to carry out debranching enzymatic hydrolysis for 8 h. After centrifugation at 2000 r / min for 5 min, the undebranched starch was removed and the supernatant was collected. The supernatant was then inactivated by enzyme at 100℃ for 10 min, followed by centrifugation at 2000 r / min for 2 min to remove the inactivated enzyme. After cooling to room temperature, the supernatant was collected as the debranched starch solution. The debranched starch was precipitated by mixing the debranched starch solution with anhydrous ethanol at a volume ratio of 1:3. The precipitate was washed three times with distilled water and then freeze-dried to obtain the debranched starch.

[0062] Step 3: Preparation of banana starch nanoparticles: 10 g / L debranched starch was heated at 100℃ for 30 min, then 5 times the volume of anhydrous ethanol was added, and the mixture was magnetically stirred at 600 r / min for 1 h; homogenized by microjets at 11000 PSI pressure for 2 min; centrifuged at 8000 r / min for 10 min, the precipitate was collected, and the precipitate was washed 3 times with anhydrous ethanol; the precipitate was freeze-dried to obtain banana starch nanoparticles.

[0063] Step 4: Loading with epicatechin: Add 10 mg of banana starch granules to 1.0 mL of 20 mmol / L catechin solution and stir magnetically at 600 r / min for 2 h; centrifuge at 12000 r / min for 20 min, remove the supernatant, collect the precipitate and wash it 3 times, and freeze-dry under vacuum to obtain banana starch granules loaded with catechin.

[0064] Experiment 1: Particle size determination. Using a disposable dropper, the original starch was added drop by drop into the inlet of the laser particle size analyzer. When the PIDS detector value reached 40%, the particle size of the starch sample was measured. Each sample was measured in parallel three times.

[0065] Experiment 2: Encapsulation efficiency determination. The prepared epicatechin-loaded pepper starch solution was ultracentrifuged at 15,000 rpm for 20 min at 25℃ to obtain free epicatechin. HPLC determination conditions: Column: Agilent XDB-C18 (4.6 mm × 250 mm, 5 μm); Column temperature: 35℃; Injection volume: 10 μL;

[0066] Flow rate: 1 mL / min. Measurement wavelength: 278 nm; Mobile phase A: 90 mL acetonitrile, 20 mL acetic acid, 2 mL LEDTA-2Na solution, diluted to 1000 mL with water, filtered through a 0.45 μm organic phase membrane; Mobile phase B: 800 mL acetonitrile, 20 mL acetic acid, 2 mL LEDTA-2Na solution, diluted to 1000 mL with water, filtered through a 0.45 μm organic phase membrane. Gradient elution: 0–10 min, 100% A (volume ratio, same below); 10–25 min, 100% A–68% A, 32% B; 25–35 min, maintain 68% A, 32% B; 35–50 min, elute with 100% A. Quantification was performed according to the epicatechin standard curve. All measurements were performed in triplicate. EE% = (A − B) ÷ A × 100%. In the formula, A is the initial amount of epicatechin, and B is the amount of free epicatechin.

[0067] Table 1. Particle size of different starch granules and their encapsulation efficiency of epicatechin.

[0068] Group Particle size (nm) Encapsulation rate (%) Example 1 <![CDATA[80.79±4.57 g ]]> <![CDATA[84.67±2.72 a ]]> Example 2 <![CDATA[165.91±12.46 c ]]> <![CDATA[47.82±3.96 e ]]> Example 3 <![CDATA[115.75±3.25 f ]]> <![CDATA[72.47±3.83 b ]]> Example 4 <![CDATA[150.69±11.75 d ]]> <![CDATA[55.62±2.77 d ]]> Example 5 <![CDATA[275.47±15.87 a ]]> <![CDATA[31.49±2.15 g ]]> Example 6 <![CDATA[245.27±12.46 b ]]> <![CDATA[34.45±1.27 f ]]> Example 7 <![CDATA[135.72±6.75 e ]]> <![CDATA[61.46±2.42 c ]]>

[0069] Note: Different letters indicate significant differences between different groups. p< 0.05)

[0070] As can be seen from Table 1:

[0071] (1) The advantages of pepper starch as a carrier are obvious: Compared with Examples 6-7, the epicatechin loaded with pepper starch in Example 1 has the smallest nanoparticle size (80.79 nm) and the highest encapsulation efficiency (84.67%), which is significantly better than other starch types (such as corn starch and banana starch). The encapsulation efficiencies of Examples 6 and 7 are 34.45% and 61.46%, respectively, and the particle size is also larger, indicating that pepper starch has structural advantages and higher affinity in loading epicatechin.

[0072] (2) The complete process is crucial to the performance of nanoparticles: Compared with Example 1, Examples 2-5 show that Example 2 (shortening the gelatinization time to 10 minutes) resulted in increased particle size and decreased encapsulation efficiency, indicating that sufficient gelatinization is necessary. Examples 3 (lacking the microfluidic homogenization step) and Example 4 (lacking the magnetic stirring step) both resulted in increased particle size and decreased encapsulation efficiency, indicating that magnetic stirring and microfluidic homogenization both contribute significantly to the formation of nanoparticles. Example 5 (without enzymatic debranching) showed the worst results, with the largest particle size (275.47 nm) and the lowest encapsulation efficiency (31.49%), indicating that enzymatic debranching is a key step in the formation of nanostructures. In summary, changes in the conditions for preparing catechin-piper starch granules are all detrimental to the preparation of nanoparticles. Among them, the absence of a pH 4.8 phosphate / citric acid buffer solution is the least conducive to the formation of catechin-piper starch granules; microfluidic homogenization has the weakest effect on nanoparticle formation.

[0073] (3) Process parameters have a significant impact on performance: The integrity of steps such as gelatinization time, stirring, homogenization, and debranching directly affects the particle size distribution and encapsulation efficiency of the final nanoparticles. Microfluidic homogenization has a weak effect on particle size control, but it is still indispensable; magnetic stirring is more critical for particle dispersion and loading. The integrity of the process steps and their order have a decisive impact on the performance of the final product.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing pepper starch nanoparticles loaded with epicatechin, comprising the following steps: Step 1: Preparation of pepper starch: Soak white peppercorns in a 0.5% w / v sodium metabisulfite solution at a ratio of 1g:6mL for 24 hours at 4℃ to obtain white pepper paste; grind the soaked white pepper paste for 1 minute, and repeat the grinding process 3 times; filter the ground white pepper paste through a 270-mesh nylon cloth, wash with distilled water, centrifuge at 4000 r / min for 10 minutes, and collect the starch granules; wash the starch granules twice with anhydrous ethanol, then wash them three times with water, dry them at 40℃ for 48 hours, pulverize them, and pass them through a 100-mesh sieve to obtain pepper starch; Step 2: Preparation of debranched pepper starch: Using a pH 4.8 phosphate / citric acid buffer solution as the solvent, wherein the phosphate / citric acid buffer solution consists of 0.2 mol / L sodium hydrogen phosphate and 0.1 mol / L sodium citrate, dissolve the pepper starch obtained in Step 1 at a ratio of 100 g / L, gelatinize thoroughly in boiling water for 30 min, and cool to 58℃; based on a starch dry weight of 3.09 × 10⁻⁶... -6 Pullulanase was added at kat / g to carry out debranching enzymatic hydrolysis for 8 h. After centrifugation at 2000 r / min for 5 min, the undebranched starch was removed and the supernatant was collected. The supernatant was then inactivated by enzyme at 100℃ for 10 min, followed by centrifugation at 2000 r / min for 2 min to remove the inactivated enzyme. After cooling to room temperature, the supernatant was collected as the debranched starch solution. The debranched starch was precipitated by mixing the debranched starch solution with anhydrous ethanol at a volume ratio of 1:

3. The precipitate was washed three times with distilled water and then freeze-dried to obtain the debranched starch. Step 3: Preparation of pepper starch nanoparticles: 10 g / L debranched starch was heated at 100℃ for 30 min, followed by the addition of 5 times the volume of anhydrous ethanol. The mixture was then magnetically stirred at 600 r / min for 1 h. The mixture was then homogenized by microjets at 11000 PSI pressure for 2 min. The mixture was then centrifuged at 8000 r / min for 10 min and the precipitate was collected. The precipitate was washed three times with anhydrous ethanol and then freeze-dried to obtain pepper starch nanoparticles. Step 4: Loading with epicatechin: Add 10 mg of pepper starch granules to 1.0 mL of 20 mmol / L catechin solution and stir magnetically at 600 r / min for 2 h; centrifuge at 12000 r / min for 20 min and remove the supernatant, collect the precipitate and wash it 3 times, and freeze dry under vacuum to obtain pepper starch granules loaded with catechin.

2. A pepper starch nanoparticle loaded with epicatechin prepared by the preparation method according to claim 1.