A tea polyphenol encapsulation and its preparation method and application

Abstract

The application discloses a kind of tea polyphenols and its preparation method and application, belong to tea polyphenols technical field, with hollow mesoporous silica (referred to as HMSN) as carrier, by loading tea polyphenols to improve the stability of tea polyphenols (referred to as TP).TP is loaded into HMSN by organic solvent impregnation, and HMSN@TP complex is obtained.The stability of HMSN@TP under the action of light, temperature, acid and alkali and gas and other factors is studied, and the free radical scavenging capacity of TP is detected by DPPH and ABTS method and its antibacterial performance is studied.HMSN's loading effectively improves the water dispersibility of TP;Compared with free TP, the TP retention rate of HMSN@TP is increased by 17% after 7d storage;Under different light, temperature, acid and alkali and gas environment, the wrapping of HMSN can significantly reduce the degradation rate of TP, and improve the antioxidant property of TP;The wrapping of HMSN does not affect the antibacterial performance of TP.HMSN loaded tea polyphenols effectively improve the stability of TP, so that its activity can be better maintained in different environments.

Description

Technical Field

[0001] This invention belongs to the field of encapsulated tea polyphenols technology, specifically relating to an encapsulated tea polyphenol, its preparation method, and its application. Background Technology

[0002] Tea has a long history in China and comes in many varieties, containing over 450 organic chemical components, making it a staple in the Chinese diet. Tea polyphenols (TP) are the main chemical components in tea that contribute to its health benefits. They are a general term for polyphenolic substances in tea and possess various functions such as anti-oxidation, anti-aging, antibacterial, anti-radiation, and preservative properties. They also have anti-tumor, lipid-lowering, antibacterial, antidiarrheal, and anti-inflammatory effects, and can help prevent and treat fatty liver and reduce fat deposition. Due to its significant antibacterial and antioxidant capabilities, it is widely used in the food and biopharmaceutical industries and has attracted considerable attention for its application in food preservation, becoming a research hotspot in these sectors. Despite its many health benefits, TP is inherently unstable. It is prone to oxidation and polymerization under light and alkaline conditions, and easily deteriorates and undergoes isomerization when exposed to strong acids, alkalis, light, and high heat. Therefore, it is easily affected by environmental factors during processing and storage, limiting its development and utilization. Thus, finding an effective way to protect the stability of TP has been an important direction for further developing its applications in food.

[0003] Numerous studies, both domestically and internationally, have focused on improving the stability of drug delivery systems (TPs), primarily employing methods such as liposome construction, nanoemulsions, and the preparation of nanosphere delivery systems. However, the use of hollow mesoporous silica nanoparticles (HMSNs) to encapsulate TPs and enhance their stability is yet to be reported. HMSNs are materials with a unique microstructure and high specific surface area. Their robust hollow structure and encapsulation capabilities have made them a research hotspot in drug delivery systems and sustained-release formulations, with significant applications in various fields, including as chromatographic packing materials, adsorbents, catalyst carriers, and drug carriers. Studies have shown that the stability of many active substances is improved after encapsulation in HMSNs. For example, using HMSNs to transport traditional antitumor drugs can address issues such as drug instability, poor solubility, and low recognition, and significantly improve antitumor efficacy. Zhao Yi loaded the photosensitive pesticide imazalil onto HMSNs, which then bound to ZnO quantum dots via electrostatic interactions to achieve a pH response, thereby controlling rice blast. He found that the encapsulated pesticide exhibited a 26.4% reduction in degradation rate, significantly improved photostability, and excellent pH-responsive characteristics. Hu Hongzhi used lycorine-encapsulated gold nanostars loaded onto HMSNs to combat osteosarcoma, finding that folic acid-modified HMSN-encapsulated gold nanostars possessed good photothermal stability and biocompatibility. Li Kun et al. encapsulated β-carboxyl in HMSNs, finding that encapsulation effectively improved stability and reduced the loss of antioxidant activity.

[0004] Given the instability of TP and the advantages of HMSN in encapsulating guest molecules, those skilled in the art urgently need to solve the technical problem of improving the storage stability of TP, thereby extending its shelf life and avoiding degradation. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing and applying tea polyphenols. HMSN was synthesized and characterized, and then TP was loaded into HMSN using a solvent impregnation method. Its stability under different light, temperature, acid-base, and gaseous conditions was investigated. The antioxidant activity of TP in HMSN was evaluated using the DPPH and ABTS methods, and its antibacterial properties were tested.

[0006] A method for encapsulating tea polyphenols, characterized in that the wall material of the encapsulated tea polyphenols is hollow mesoporous silica nanospheres, and the core material is tea polyphenols; the loading rate of the encapsulated tea polyphenols is 10.85% to 20.4%.

[0007] Furthermore, the hollow mesoporous silica nanospheres are prepared by mixing 0.64 g of CTAB, 60 mL of ethanol, and 2 mL of mixed silicon source at room temperature, adding 100 mL of water, stirring for 60 min, adding 1 mL of concentrated ammonia, stirring for 2.5 h, centrifuging at 8000 r / min for 10 min after the reaction is complete, washing the precipitate three times with ethanol, then resuspending it in a 1:9 hydrochloric acid-ethanol solution and refluxing for 12 h, centrifuging at 8000 r / min for 10 min after the reaction is complete, washing the precipitate twice with ethanol and twice with water, and finally resuspending it in water and freeze-drying to obtain the hollow mesoporous silica nanospheres. The mixed silicon source is composed of 0.6 mL of tetraethyl silicate, 0.6 mL of 1,2-bis(triethoxysilyl)ethane, and 0.8 mL of cyclohexane.

[0008] The above-mentioned method for preparing encapsulated tea polyphenols includes the following steps: weigh 200 mg of tea polyphenols and 6 mL of ethanol, sonicate them evenly, add 300 mg of hollow mesoporous silica nanospheres, the mass ratio of tea polyphenols to hollow mesoporous silica nanospheres is 1:1.5, place them in a shaker at 1000 r / min and shake at room temperature in the dark for 24 h, after which centrifuge at 8000 rpm for 5 min to remove the supernatant, freeze dry in the dark for 4 h, weigh the difference in mass before and after to obtain the precipitate mass, resuspend the precipitate in PBS solution, blow evenly and store it in a 4℃ refrigerator in a sealed container.

[0009] The application of the above-mentioned encapsulated tea polyphenols in anti-degradation and antioxidant antibacterial agents is characterized in that the bacteria inhibited by the antibacterial agent are Gram-negative bacteria, Escherichia coli.

[0010] The beneficial effects of this invention are:

[0011] This invention discloses a method for preparing encapsulated tea polyphenols and its applications. Hollow mesoporous silica (HMSN) is used as a carrier, and the stability of tea polyphenols (TP) is improved by encapsulating TP. HMSN is prepared using an emulsion template method, and TP is loaded into HMSN through impregnation with an organic solvent to obtain an HMSN@TP complex. The stability of HMSN@TP under the influence of light, temperature, acidity / alkalinity, and gas is studied. The free radical scavenging ability of TP is detected by DPPH and ABTS methods, and its antibacterial properties are investigated. Results: Hollow microspheres were successfully prepared. The encapsulation with HMSN effectively improved the water dispersibility of TP. Compared with free TP, the TP retention rate of HMSN@TP increased by 17% after 7 days of storage. Under different light, temperature, acidity / alkalinity, and gas environments, HMSN encapsulation significantly reduced the degradation rate of TP while improving its antioxidant properties. Furthermore, the antibacterial experiment with *E. coli* showed that HMSN encapsulation did not affect the antibacterial properties of TP. Conclusion: HMSN@TP effectively improves the stability of TP, allowing its activity to be better maintained under different environments. Therefore, HMSN can be used as a candidate carrier for TP to improve its stability during various processing steps. Attached Figure Description

[0012] The present invention will now be described in detail with reference to the accompanying drawings. Figure 1 yes Figure 1 In Example 1, SEM and TEM images of HMSN were obtained, where (A) are SEM images magnified 100,000 times and 20,000 times, and (B) is a TEM image; Figure 2 The diagram shows the adsorption of TP by HMSN in the example, where 2-A is a comparison photograph of TP water dispersibility (a is free TP, b is HMSN@TP), 2-B is the UV spectrum of TP, and C is the standard curve of TP. Figure 3 This is a graph showing the TP retention rate, where 3-A represents the change in the UV spectrum of TP samples after different storage days; 3-B represents the TP sample retention rate after different storage days. Figure 4 The graph shows the photostability of TP under HMSN, where 4-A is a comparison of the UV spectra of TP after 6 hours of different light exposures; 4-B is a comparison of the TP retention rate in the light-protected group after 6 hours of different light exposures; and 4-C is the color of the TP sample solution after 6 hours of different light exposures. Figure 5 The results show the thermal stability of TP under HMSN support. 5-A shows the changes in the UV spectrum of TP after 6 hours at different temperatures; 5-B shows the change rate of TP at 0℃ after 6 hours at different temperatures compared to TP at different temperatures; and 5-C shows the color comparison of the sample solution after TP was heated at 100℃ for 6 hours, where X represents the untreated control solution. Figure 6The results show the acid-base stability of TP under HMSN, including the changes in the UV spectrum of TP after adding different acid and base solutions to sample 6-A for 30 min; the TP retention rate of sample 6-B after adding different acid and base solutions for 30 min compared with the neutral group; and the color comparison of sample solution after adding different acid and base solutions to sample 6-C for 30 min, where a is the initial sample solution before adding acid and base, and b is the black precipitate after centrifugation of the sample solution after the reaction. Figure 7 The results show the oxygen stability of TP under HMSN support, including the TP spectrum change of sample 7-A after 6 hours of reaction with different gases and the rate of change of sample 7-B after 6 hours of reaction with different gases. Figure 8 HMSN-loaded TP antioxidant properties, including spectral changes of reaction solution 8-A (a is DPPH spectrum, b is ABTS spectrum); free radical scavenging rate after reaction 8-B; and uncentrifuged sample solution after reaction 8-C. Figure 9 These are photos of E. coli inhibition zones in HMSN-PBS, TP-PBS, and HMSN@TP-PBS from Example 3. Detailed Implementation

[0013] Raw materials and equipment: 98% tea polyphenols (analytical grade): Shanghai Titan Technology Co., Ltd.; 95% ethanol: Xilong Scientific Co., Ltd.; >99.8% dimethyl sulfoxide and 99% triethylamine (analytical grade): Shanghai Maclean Biochemical Technology Co., Ltd.; chloroform (analytical grade), ethyl acetate (analytical grade), and acetone (analytical grade): Sinopharm Chemical Reagent Co., Ltd.; methanol (analytical grade) and sodium hydroxide (analytical grade): Tianjin Yongda Chemical Reagent Co., Ltd.; hydrofluoric acid, 99% hexadecyltrimethylammonium bromide (CTAB), 98% tetraethyl silicate, 96% 1,2-bis(triethoxysilyl)ethane, 99.5% cyclohexane, and concentrated ammonia (analytical grade): Shanghai Aladdin Biochemical Technology Co., Ltd.; 20×PBS buffer, LB broth medium, and agar powder: Sangon Biotech (Shanghai) Co., Ltd.; Escherichia coli culture - prepared in the laboratory; all water used in this study was ultrapure water. DTC-15J* Ultrasonic Cleaner: Dingtai (Hubei) Biochemical Technology Equipment Manufacturing Co., Ltd.; Thermo Multiskan Go* Microplate Reader: Shanghai Danding International Trade Co., Ltd.; Catcher H3-18K Benchtop High-Speed ​​Centrifuge: Catcher Technology; UV Lamp Box; Nitrogen Cylinder: Nanjing Airport Special Gases Co., Ltd.; Oxygen Cylinder: Nanjing Gas Plant; BCD-649* Refrigerator: Qingdao Haier Co., Ltd.; DKT200-4* Metal Bath: Hangzhou Mio Instrument Co., Ltd.; Milli-Q Advantage A10* Ultrapure Water System (Imported): Merck Millipore; MTC-100* Thermostatic Mixer: Changzhou Maike Nuo Instrument Co., Ltd.; alphal-2LDplus* Freeze Dryer: CHRIST GmbH, Germany; ZQPW-70 Benchtop Full-Temperature Shaking Incubator: Tianjin Laiboteri Instrument Equipment Co., Ltd.; HVE-50* Autoclave: Shanghai Lailan Biotechnology Co., Ltd.; BHC-1300 II A2 Biosafety Cabinet: Altai Laboratory Equipment (Beijing) Co., Ltd.; PWT-P150B Electric Thermostatic Incubator: Sichuan Zhonglang Technology Co., Ltd.; (0~150)mm / 0.01mm Digital Calibrator: Dongguan Sanliang Measuring Tools Co., Ltd. Example 1

[0014] I. Synthesis and Characterization of HMSN:

[0015] (1) Synthesis of HMSN: 0.64 g of CTAB, 60 mL of ethanol and 2 mL of mixed silicon source were mixed evenly at room temperature, 100 mL of water was added, and after stirring for 60 min, 1 mL of concentrated ammonia was added and the reaction was stirred for 2.5 h. After the reaction was completed, the mixture was centrifuged at 8000 r / min for 10 min, and the precipitate was washed three times with ethanol. Then, it was resuspended in a hydrochloric acid-ethanol solution with a volume ratio of 1:9 and refluxed for 12 h. After the reaction was completed, the mixture was centrifuged at 8000 r / min for 10 min, and the precipitate was washed twice with ethanol and twice with water. Finally, it was resuspended in water and freeze-dried to obtain HMSN. The 2 mL of mixed silicon source in this part was composed of 0.6 mL of tetraethyl silicate, 0.6 mL of 1,2-bis(triethoxysilyl)ethane and 0.8 mL of cyclohexane.

[0016] (2) Characterization of HMSN nanoparticles: The surface morphology, size and dispersibility of the prepared HMSN were characterized by TEM and SEM. 10 mg of HMSN sample was taken and sonicated for 10 min to be uniformly dispersed in 5 mL of deionized water. The sample was dipped into a copper mesh and dried using carbon conductive adhesive. The sample was then placed on the sample stage of a vacuum chamber for adjustment and the accelerating voltage was tested at 10.0 kV.

[0017] Figure 1 The images are SEM and TEM images of HMSN obtained in Example 1, where (A) are SEM images magnified 100,000 times and 20,000 times, and (B) is a TEM image. The SEM image shows the mesoporous structure of HMSN with a particle size of about 1 μm. The TEM image shows that HMSN microspheres with obvious hollow structure were successfully prepared.

[0018] II. Preparation and Adsorption Study of HMSN@TP:

[0019] (1) Establishment of the TP standard curve

[0020] Weigh 10 mg of TP powder, add 10 mL of ethanol, sonicate to homogenize, and then dilute to prepare 10 equal-concentration TP-ethanol mixtures ranging from 10 to 100 μg / mL, using ethanol as a blank control. Detect the absorbance at 200-800 nm using a microplate reader, identify the characteristic peaks, and construct a standard curve.

[0021] (2) Preparation and drug loading determination of HMSN@TP

[0022] Weigh 200 mg TP and 6 mL ethanol, sonicate until homogeneous, then add 300 mg HMSN (TP:HMSN mass ratio 1:1.5). Place in a shaker at 1000 rpm and shake at room temperature in the dark for 24 h. After shaking, centrifuge at 8000 rpm for 5 min to remove the supernatant. Freeze-dry in the dark for 4 h, and weigh the difference between the initial and final mass to obtain the precipitate mass. Resuspend the precipitate in 8 mL 1×PBS, mix well, and store in a sealed container at 4℃. Take 100 μL of this drug loading, dilute 50 times with ethanol, and extract by sonication. Centrifuge at 8000 rpm for 5 min, collect the supernatant, and measure its absorbance. Substitute the absorbance into the standard curve to determine the concentration and drug loading. The formula is:

[0023] (1)

[0024] In the formula: LE represents the drug loading of TP in HMSN, %; W e Indicates the amount of TP encapsulated within the HMSN, in mg; W m This indicates the total weight of HMSN@TP, in mg.

[0025] Water dispersibility study

[0026] Based on the TP concentration in HMSN@TP, a free TP control group was prepared. After the two groups of sample solutions were thoroughly mixed by blowing, they were placed in the dark for 6 hours, and the morphological changes of the two solutions were compared.

[0027] HMSN adsorption of TP

[0028] Figure 2 This is a graph showing the adsorption of TP by HMSN, where A is a comparison of TP water dispersibility (a is free TP, b is HMSN@TP); B is the UV spectrum of TP; and C is the TP standard curve.

[0029] TP is soluble in water, but its solubility in water at room temperature is not high. Figure 2 In the -A group, after standing in the dark for 6 hours at the same concentration of TP, the TP control group showed obvious brown precipitate at the bottom, while the HMSN@TP experimental group showed a more uniform distribution due to the good water dispersibility of HMSN, indicating that HMSN@TP improved the water dispersibility of TP.

[0030] Studies have shown that the absorption peaks of catechins often appear at wavelengths of 270–280 nm. Figure 2 -B indicates that TP exhibits a characteristic absorption peak at 278 nm. This is likely due to the presence of the basic structure 2-phenylphenylpyran, and therefore the peak position does not change with mass concentration. Using this characteristic peak as a benchmark, a linear regression equation is derived: y = 0.026x - 0.0329, where RB... 2=0.9995, indicating a linear relationship. The average absorbance of the diluted sample at 276 nm was 2.2557. Substituting this into the standard curve, the mass of TP loaded in the drug was found to be 35.2097 mg, with a drug loading of 10.85% at 24 h and 66.012 mg at 48 h. Example 2

[0031] HMSN@TP stability study:

[0032] Take 80 μL of HMSN@TP from the examples, dilute it 40 times with ethanol, sonicate for 3 min, centrifuge at 8000 rpm for 5 min, collect the supernatant and measure its absorbance at 276 nm, substitute it into the standard curve to calculate the TP concentration. Accurately weigh TP powder according to this concentration, dissolve it in a small amount of ethanol by sonication, and resuspend it in 1×PBS to prepare a free TP control group with the same TP concentration as HMSN@TP. Each experiment was conducted independently, and the control sample solution was re-measured and prepared before each experiment.

[0033] (1) Retention rate determination: The two groups of stock solutions were diluted 8 times with 1×PBS to prepare experimental samples, and their initial absorbance values ​​were recorded. 1.5 mL of each sample was transferred into EP tubes of the same diameter and stored at room temperature in the dark for 7 days, with measurements taken every 24 hours. After the experiment, 160 μL of the post-experiment sample solution was added to 1440 μL of ethanol, sonicated for 5 min, and centrifuged at 12000 rpm for 5 min. The supernatant was then used to measure the absorbance values ​​of each group.

[0034] (2)

[0035] In the formula: OD0 is the initial absorbance of the sample solution, and OD1 is the absorbance of the sample solution after the experiment.

[0036] (2) Photostability determination: The two groups of stock solutions were diluted 6 times with 1×PBS to prepare the experimental samples, and their initial absorbance values ​​were recorded. 1 mL of each sample was taken into an EP tube, sealed, and placed in sunlight, indoor light, ultraviolet light, and dark environments for 6 h. After the experiment, 160 μL of the post-experiment sample solution was added to 1440 μL of ethanol, sonicated for 5 min, and centrifuged at 12000 rpm for 5 min. The supernatant was then used to detect the absorbance values ​​of each group.

[0037] (3) Thermal stability determination: Both groups of stock solutions were diluted 6 times with 1×PBS to prepare experimental samples, and their initial absorbance values ​​were recorded. 1.5 mL of each sample was transferred to EP tubes and placed in refrigerators at temperatures of 0, 30, 60, 80, and 100℃, respectively, and in preheated metal baths. The tubes were sealed and protected from light for 2 hours. After the experiment, 160 μL of the HMSN@TP group sample was added to 1440 μL of ethanol, sonicated for 5 min, and centrifuged at 12000 rpm for 5 min. The supernatant was collected for later use. The TP-PBS control group underwent the same procedure, with homogenization by sonication. The absorbance values ​​of each group were detected using an ELISA reader.

[0038] (3)

[0039] In the formula: OD0 is the initial absorbance of the sample solution, and OD1 is the absorbance of the sample solution after the experiment.

[0040] (4) Acid-base stability determination: The two groups of stock solutions were diluted 5 times with 1×PBS to prepare experimental samples, and their initial absorbance values ​​were recorded. 1.5 mL of HMSN@TP and the free TP control group were each placed in an EP tube, and 100 μL of hydrofluoric acid, triethylamine, and ethanol were added respectively to simulate acid, alkali, and neutral environments, and the samples were allowed to stand for 30 min. After the experiment, 160 μL of the post-experiment sample solution was added to 1440 μL of ethanol, sonicated for 5 min, and centrifuged at 12000 rpm for 5 min. The supernatant was then used to detect the absorbance values ​​of each group.

[0041] (5) Gas stability determination: The two groups of stock solutions were diluted 6 times with 1×PBS to prepare experimental samples, and their initial absorbance values ​​were recorded. 1.5 mL of HMSN@TP and the free TP control group were each placed in EP tubes of the same diameter and left open in air and oxygen environments for 6 h, respectively. The initial detection concentration was used as the control group. After the experiment, 160 μL of the experimental sample solution was added to 1440 μL of ethanol, sonicated for 5 min, centrifuged at 12000 rpm for 5 min, and the supernatant was collected to detect the absorbance values ​​of each group.

[0042] (6) Antioxidant performance determination: Refer to Liang Jie's method (Liang Jie, Zhao Xiaoxu, Liu Tao, et al. Preparation and preservation effect of tea polyphenol-chitosan composite film [J]. Journal of Tropical Crops, 2022, 43(06): 1267-1279.). Weigh 1.97 mg of 1,1-diphenyl-2-picrylhydrazyl free radical, add 50 mL of ethanol, sonicate for 1 min, and let stand for 30 min to prepare a 0.1 mmol / L DDPH ethanol mixture. Use an enzyme-linked immunosorbent assay reader, zero with ethanol, adjust the sample solution to 519 nm and detect the absorbance value to 1.2-1.3, and store in the dark. Prepare two control group stock solutions according to the HMSN and TP concentrations in HMSN@TP, and dilute the three stock solutions 30 times with 1×PBS to prepare experimental sample solutions. Take 1335 μL of LPPH-ethanol mixture and add 665 μL of ethanol. After thorough mixing, measure the A0 value at 519 nm to 0.7-0.9. Use this mixture to mix with the experimental sample solutions of each group at a ratio of 2:1, let stand in the dark for 10 min, centrifuge at 12000 rpm, collect the supernatant, and measure the absorbance value at 519 nm for each group. Record this value as the A value. The calculation formula is as follows:

[0043] (4)

[0044] In the formula: A0 is the absorbance value of the blank control group, and A1 is the absorbance value of the sample group.

[0045] Weigh 38.4076 mg ABTS and 6.6418 mg potassium persulfate, and dilute to 10 mL with deionized water to prepare an ABTS reaction solution with a molar concentration of 7 mmol / L. Store at room temperature in the dark for 12 h. Take 1 mL of the reaction solution and dilute it 50 times with deionized water, then adjust its absorbance at 734 nm to 0.70 ± 0.02. Prepare two control group stock solutions based on the concentrations of HMSN and TP in HMSN@TP. Simultaneously dilute all three stock solutions 30 times with 1×PBS to prepare experimental sample solutions. Mix 2 mL of the reaction solution with 1 mL of each group's experimental sample solution, let stand in the dark for 5 min, centrifuge, and measure the absorbance at 734 nm for each group. The ABTS scavenging rate formula is:

[0046] (5)

[0047] In the formula: A0 is the absorbance value of the blank control group, and A1 is the absorbance value of the sample group.

[0048] The impact of HMSN's integration with TP on its retention rate:

[0049] The retention rate of the sample solution during storage is a direct reflection of its stability. TP is hygroscopic and easily undergoes oxidation in air, just as... Figure 3As shown in Figure A, during open storage at room temperature and away from light, the absorbance of TP in both groups of samples showed a gradual decreasing trend. The free TP group showed the most significant change in the first 4 days of the experiment, while the HMSN@TP group remained relatively stable. This may be because the HMSN coating protected TP from contact with oxygen, thereby improving the TP retention rate. By day 7 of storage, the retention rate of the HMSN@TP group was on average 17% higher than that of the TP group, indicating that HMSN coating has a certain effect on improving the TP retention rate and effectively extending the shelf life of TP.

[0050] Figure 3 This is a TP retention rate graph, where A represents the change in TP UV spectrum after different storage days; B represents the TP sample retention rate after different storage days.

[0051] The impact of HMSN with TP on its optical stability:

[0052] Tea polyphenols are highly sensitive to light; therefore, the photosensitivity of tea polyphenols (TP) can be measured by comparing the absorbance values ​​of TP under different light conditions. For example... Figure 4 As shown, light exposure causes a decrease in the absorbance of TP, and the degradation rate of free TP after 6 hours of light exposure is significantly higher than that of HMSN@TP. The photosensitivity of TP, from highest to lowest, is: ultraviolet light, sunlight, room light, and darkness. It is relatively stable under normal temperature and light-protected conditions, while ultraviolet light and sunlight exposure easily cause its oxidation and polymerization, leading to a darker color (e.g., ...). Figure 4 -C), this result is consistent with Chen's conclusion (CHEN X, CHEN T, LIU J, et al. Physicochemical stability and antibacterial mechanism of theabrownins prepared from tea polyphenols catalyzed by polyphenol oxidase and peroxidase.[J]. Food science and biotechnology,2024,33(1).). According to the photosensitivity order, with light avoidance as the control, HMSN@TP has a higher retention rate than TP by 15.83%, 10.61%, and 7.86%, respectively. After 6 hours of UV irradiation, the loss rate of HMSN@TP is only 2.36% ( Figure 4 -B). TP is highly sensitive to light, and its molecular oxidative degradation is accelerated under light. HMSN@TP has a significant improvement effect, indicating that HMSN encapsulation of TP can improve its photostability.

[0053] Figure 4This is a graph showing the photostability of TP under HMSN, where A is a comparison of the UV spectra of TP after 6 hours of different light exposures; B is a comparison of the TP retention rate in the light-protected group after 6 hours of different light exposures; and C is the color of the TP sample solution after 6 hours of different light exposures.

[0054] The impact of HMSN's TP (Transistor) on its thermal stability:

[0055] Most chemical reactions are related to temperature; therefore, temperature is also an important factor affecting the stability of substances. Studies on the stability of TP in samples at different temperatures revealed a positive correlation between absorbance and temperature; the solution color gradually deepened, eventually turning dark brown. This indicates TP degradation and instability. The darker the sample solution, the higher the degree of TP oxidation and the lower its antioxidant capacity. This is one reason why cold-brewed tea is gaining increasing attention as an alternative to traditional hot-brewed tea. Figure 5 -A, When heated to 60~100℃, the degradation rate of TP accelerated, and the absorbance values ​​all showed an upward trend, indicating that temperature can significantly affect the degradation rate of TP, and the degradation rate accelerates with increasing temperature. This is consistent with the experimental conclusions of Guo Ziyu (Guo Ziyu, Feng Cuimin, Wang Changzheng, et al. Study on the chemical stability of tea polyphenols and their degradation kinetics during disinfection [J]. Chinese Science and Technology Papers, 2018, 13(03):304-309.). In particular, when the test solution was heated to 100℃, the absorbance value of the TP group sample solution changed by 71%, and the color of the test solution also changed from colorless to reddish-brown. However, under the same conditions, the color change of the HMSN@TP group was not significant. With 0℃ as the initial control, it was found that the HMSN@TP group had a lower degradation rate than the TP group at 60℃ and 100℃, respectively, by 20.57% and 71%. This indicates that HMSN coating can reduce the oxidation of TP caused by high temperature environment during heat treatment, effectively reducing the degradation of TP.

[0056] Figure 5 This section describes the thermal stability of TP under HMSN support. A shows the change in the UV spectrum of TP after 6 hours at different temperatures; B shows the change rate of TP at different temperatures compared to 0℃ after 6 hours; C shows the color comparison of the sample solution after heating TP at 100℃ for 6 hours, with the untreated control solution on the far right.

[0057] The effect of HMSN carrying TP on its acid-base stability:

[0058] Studies have shown that pH has a significant impact on the stability of TP. Therefore, this invention simulates an extreme pH environment using hydrofluoric acid and triethylamine to investigate the effect of pH on the stability of different TP forms. It was found that TP is highly stable in the ethanol system, and the absorbance hardly changes under the experimental conditions. In the acidic system simulated by hydrofluoric acid, the retention rates of TP and TP@HMSN are close, at 100.92% and 101.67%, respectively, indicating that TP is relatively stable under acidic conditions, consistent with the conclusion of Su (SU YL, LAI KL, HY, et al. Stability of tea theaflavins and catechins. Food Chemistry, 2003, 83(2): 189-195.). In the presence of triethylamine, the absorbance spectrum of free TP changed significantly, with a retention rate of only 51.27%, while the retention rate of TP@HMSN reached 100.15%. This indicates that the alkaline environment simulated by triethylamine can rapidly induce the deterioration of free TP. The alkaline environment directly destroys the structure of TP, leading to TP molecule fusion and the formation of a dark brown precipitate. Due to the encapsulation of HMSN, the stability of TP@HMSN is significantly improved.

[0059] Figure 6 The results show the acid-base stability of TP under HMSN, including the changes in the UV spectrum of TP after adding different acid and base solutions to sample 6-A for 30 min; the TP retention rate of sample 6-B after adding different acid and base solutions for 30 min compared with the neutral group; and the color comparison of sample solution after adding different acid and base solutions to sample 6-C for 30 min, where a is the initial sample solution before adding acid and base, and b is the black precipitate after centrifugation of the sample solution after the reaction.

[0060] The impact of HMSN equipped with TP on its gas stability:

[0061] As oxygen concentration increases, the characteristic absorption of TP shows an increasing trend. Figure 7 A). It is possible that oxygen accelerates the oxidative degradation of TP, resulting in an isomerization reaction and causing an increase in absorbance. Figure 7 Compared to the control group, in group B, the TP content in the TP group increased by 3.92% and 6.68% in air and oxygen, respectively, while the HMSN@TP group only increased by 2.06% and 3.24%. This indicates that under oxygen conditions, HMSN encapsulation can reduce the oxidation rate of TP and improve its stability.

[0062] Figure 7 The oxygen stability of TP under HMSN was measured, including the TP spectrum change of sample 7-A after 6 hours of reaction under different gases and the rate of change of sample 7-B after 6 hours of reaction under different gases.

[0063] The effect of HMSN carrying TP on its antioxidant properties:

[0064] Figure 8 The results show the antioxidant properties of TP under HMSN, including the spectral changes of the 8-A reaction solution (a is the DPPH spectrum, b is the ABTS spectrum); the free radical scavenging rate after the 8-B reaction; and the uncentrifuged sample solution after the 8-C reaction.

[0065] TP possesses extremely strong antioxidant capabilities; therefore, it is necessary to investigate whether the antioxidant capacity of TP changes after encapsulation. This invention employs the most commonly used DPPH and ABTS methods to evaluate its antioxidant capacity. Figure 8 As shown in Figure -A, HMSN exhibited virtually no scavenging ability in DPPH and ABTS detection, while TP itself achieved DPPH and ABTS scavenging rates of 91.57% and 95.06%, respectively. Studies have indicated that when TP concentration reaches 20.4 μg / mL or higher, 90% of DPPH radicals can be scavenged within 1.5 minutes, which is largely consistent with these findings. Comparison of the two indicator detection results revealed that after HMSN encapsulation, the DPPH and ABTS radical scavenging rates of TP increased by 1.78% and 3.5%, respectively. The post-reaction sample solution showed... Figure 8 As shown in -C. Therefore, the antioxidant capacity test results indicate that the antioxidant capacity of TP was not inhibited after encapsulation, but rather improved to a certain extent. This may be due to the good water dispersibility of HMSN, which allows TP to be more uniformly distributed in the detection system, thereby enhancing its free radical scavenging ability. Example 3

[0066] HMSN@TP antibacterial properties:

[0067] Antibacterial performance test

[0068] Escherichia coli is the most common bacterium in clinical practice and one of the most studied bacteria in modern biology. Excessive levels of E. coli in food can destroy its nutritional components, accelerate spoilage, and reduce its nutritional value. Therefore, this invention uses E. coli as the experimental strain to investigate the effect of nanomaterials encapsulating TP on its antibacterial properties. The experiment combines the method of Luo and Zhang (Luo Aiguo, Qing Yifan, Hu Bianfang, et al. Performance study of nano-silica modified algal polysaccharide and chitosan composite membrane [J]. Food Industry Technology, 2022, 43(09):243-250. Zhang Tingting. Preparation and performance study of antibacterial micro-nanofibers based on catechin / chitosan microcapsules [D]. Donghua University, 2023.) with slight modifications. Under aseptic conditions, sterilized LB broth medium was poured in, cooled, and then 150 μL of E. coli bacterial suspension with a concentration of approximately 5 × 10⁷ CFU / mL was coated onto the medium. Two control group sample solutions were prepared based on the HMSN and TP concentrations in HMSN@TP. Sterilized filter paper discs with a diameter of 10 mm were soaked in the three sample solutions for 30 min and then attached to the plates. The plates were inverted and placed in a 37℃ constant temperature incubator for 24 h. The size of the inhibition zone of each group was measured using calipers. The measurement results of the Escherichia coli inhibition zone are shown in Table 1.

[0069] Comparison of inhibition zones revealed that HMSN itself does not exhibit antibacterial activity against *E. coli*, while TP showed significant inhibitory effects and strong antibacterial activity. Its main mechanism is through disrupting cell membrane permeability, causing metabolic imbalances, and ultimately leading to bacterial death. The antibacterial activity of the HMSN@TP experimental group against *E. coli* was second only to the TP group, with an average inhibition zone difference of only 0.93 mm. This indicates that the antibacterial activity of TP after encapsulation remains, although not enhanced, but essentially consistent with that of free TP, and the antibacterial activity was not significantly inhibited by encapsulation.

[0070] Table 1 Results of Escherichia coli inhibition zone measurement

[0071]

[0072] In summary, this invention successfully prepared and characterized a hollow-structured HMSN, successfully encapsulated TP into HMSN using a solvent impregnation method, and investigated the stability of the encapsulated TP. The results show that, compared with free TP, TP@HMSN not only improves the aqueous dispersibility of TP but also enhances its stability to light, temperature, oxygen, and pH, thereby improving its antioxidant capacity to a certain extent, while maintaining its antibacterial activity largely unaffected. This paper provides a valuable reference for addressing the problem of poor TP stability. Therefore, in practical applications of TP, HMSN can be selected as a candidate carrier system, offering a new approach to improving TP stability.

Claims

1. A coating material containing tea polyphenols, characterized in that, The wall material for the encapsulated tea polyphenols is hollow mesoporous silica nanospheres, and the core material is tea polyphenols; the loading rate of the encapsulated tea polyphenols is 10.85%–20.4%; the preparation method of the hollow mesoporous silica nanospheres is as follows: 0.64 g of hexadecyltrimethylammonium bromide, 60 mL of ethanol and 2 mL of mixed silicon source are mixed evenly at room temperature, 100 mL of water is added, and after stirring for 60 min, 1 mL of concentrated ammonia is added, and the reaction is stirred for 2.5 h. After the reaction is completed, the mixture is centrifuged at 8000 r / min for 10 m. The precipitate was washed three times with ethanol, then resuspended in a 1:9 hydrochloric acid-ethanol solution and refluxed for 12 hours. After resuspending, it was centrifuged at 8000 rpm for 10 minutes. The precipitate was washed twice with ethanol and twice with water, and finally resuspended in water and freeze-dried to obtain hollow mesoporous silica nanospheres. The mixed silicon source was composed of 0.6 mL of tetraethyl silicate, 0.6 mL of 1,2-bis(triethoxysilyl)ethane, and 0.8 mL of cyclohexane. [The text then abruptly shifts to a different topic:] Preparation method for encapsulating tea polyphenols... The process includes the following steps: Weigh 200 mg of tea polyphenols and 6 mL of ethanol, sonicate until homogeneous, add 300 mg of hollow mesoporous silica nanospheres (the mass ratio of tea polyphenols to hollow mesoporous silica nanospheres is 1:1.5), place in a shaker at 1000 r / min, shake at room temperature in the dark for 24-48 h, centrifuge at 8000 rpm for 5 min to remove the supernatant, freeze-dry in the dark for 4 h, weigh the difference between the mass before and after to obtain the precipitate mass, resuspend the precipitate in PBS solution, mix well, and store in a sealed container at 4 °C.

2. The application of the tea polyphenol-encapsulated agent as described in claim 1 in the preparation of an antibacterial agent with anti-degradation and anti-oxidation properties, characterized in that, The antibacterial agent inhibits Gram-negative bacteria, specifically Escherichia coli.

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