Catechin-lactobacillus acidophilus composite composition for improving skin

Abstract

The invention relates to the field of biotechnology and cosmetics, and particularly discloses a catechin-lactobacillus acidophilus composite composition for improving skin. The composition is in a microcapsule form, green tea catechin extract, lactobacillus acidophilus bacterial sludge and fermentation filtrate of the lactobacillus acidophilus bacterial sludge are taken as a core, and a pH responsive polymer and an enteric polymer form a double-layer shell layer; the problems that catechin is poor in stability and probiotics and metabolites thereof are poor in compatibility are effectively solved through a unique double-layer microcapsule structure, intelligent response release can be achieved based on the weak acid environment of the skin, and the bioavailability and the synergistic skin care effect of active ingredients are remarkably improved; the composition can be used for preparing anti-oxidation, anti-inflammatory, soothing, barrier repairing, moisturizing and anti-aging cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and cosmetics, in particular to a catechin-Lactobacillus acidophilus complex composition for improving skin. BACKGROUND

[0002] As the largest organ of the human body, skin is exposed to the external environment for a long time and is easily affected by various factors such as ultraviolet radiation, pollution, chemical stimulation and microbial invasion, thereby leading to a series of skin problems such as oxidative stress, inflammatory response, impaired barrier function and microecological imbalance, which are manifested as skin aging, sensitivity, dryness, acne and pigmentation, etc. In the prior art, antioxidant and anti-inflammatory are one of the core strategies for skin care. Although some natural active ingredients are widely studied due to their potential biological activities, their practical application still faces major challenges. Such ingredients generally have the problem of unstable chemical properties, are easily degraded by light, heat, oxygen and pH value, resulting in a significant decrease in their biological activities; in addition, their poor skin permeability and short residence time at the action site also seriously restrict the full play of their skin care efficacy.

[0003] In recent years, skin microecology has attracted great attention as a new emerging regulatory target for skin health. Although the application of microbial fermentation technology in cosmetics shows certain potential, its development still faces many bottlenecks. In the current related products, the active ingredients often lack effective protection and are easily inactivated in the formula system; there may be physical or chemical incompatibility between different components, leading to a decrease in the stability of the formula; more importantly, the conventional technology is difficult to achieve the controlled release of active ingredients at the target action site, which greatly reduces the bioavailability and expected efficacy of the product. The existing technology has not effectively solved the systematic problem of the stability, compatibility and targeted delivery of active ingredients.

[0004] It is worth noting that catechin substances and Lactobacillus acidophilus metabolites have significant complementary advantages at the theoretical level. Green tea catechin substances have strong antioxidant and antibacterial properties and can effectively resist skin damage caused by environmental stress; while Lactobacillus acidophilus metabolites are rich in various ingredients that are beneficial to the skin and can theoretically regulate the balance of skin microecology and enhance the skin barrier function. Both have synergistic potential in function and can provide a comprehensive solution to various skin problems. However, although both ingredients have their own advantages, the existing technology has not been able to effectively combine and complement the advantages of both, and has not been able to fully exert their theoretical synergistic effect.

[0005] Based on the above statements, the present application proposes a catechin-Lactobacillus acidophilus complex composition for improving skin. SUMMARY

[0006] In order to solve the problems of poor stability of active ingredients, poor compatibility between different components, lack of targeted delivery system and difficulty in achieving synergistic effect in the prior art, the application provides a catechin-lactobacillus acidophilus composite composition for improving skin.

[0007] In a first aspect, the application provides a catechin-lactobacillus acidophilus composite composition for improving skin, which is in the form of microcapsules, and has a core of catechin extract and lactobacillus acidophilus and its fermentation filtrate, and a double-layer shell of pH-responsive polymer and enteric polymer.

[0008] Preferably, the double-layer shell has an inner layer composed of pH-responsive polymer and an outer layer composed of enteric polymer, and the composite composition has the core, the inner layer and the outer layer in sequence from inside to outside.

[0009] Preferably, the pH-responsive polymer is chitosan, and the enteric polymer is hydroxypropyl methyl cellulose phthalate (HPMCP).

[0010] Preferably, the catechin extract is green tea catechin extract, wherein the content of catechin is ≥40%, the content of EGCG is ≥18%, the content of EGC is ≥10%, the content of ECG is ≥8%, the content of DLC is ≥5%, the content of EC is ≥1.5%, and the content of GCG is ≥0.8%.

[0011] In a second aspect, the application provides a preparation method of a catechin-lactobacillus acidophilus composite composition for improving skin, which adopts the following technical scheme: The preparation method of the catechin-lactobacillus acidophilus composite composition for improving skin comprises the following steps: S1. After fermentation and centrifugation of lactobacillus acidophilus, lactobacillus acidophilus paste and centrifugal liquid are obtained, and the centrifugal liquid is concentrated by filtration and nanofiltration membrane to obtain lactobacillus acidophilus fermentation filtrate; S2. The lactobacillus acidophilus paste and the fermentation filtrate are mixed with green tea catechin extract to obtain a core composite solution; S3. The core composite solution is embedded with pH-responsive polymer and enteric polymer to obtain a microcapsule solution; S4. The microcapsule solution is subjected to solidification, washing and vacuum freeze-drying treatment to obtain dry microcapsule powder, i.e. the catechin-lactobacillus acidophilus composite composition.

[0012] Preferably, the specific operation method of step S1 is as follows: the third-generation activated lactobacillus acidophilus is inoculated into a fermentation medium to grow and ferment, and then centrifuged to obtain lactobacillus acidophilus paste and centrifugal liquid. Lactobacillus acidophilusThe seed liquid is inoculated into fresh MRS liquid culture medium at an inoculation amount of 1-5% (v / v) and fermented at 30-37°C for 36-48h; after fermentation, the fermentation liquid is centrifuged at 8000-12000rpm for 15-25min at 4-10°C to collect the bacterial slurry and supernatant; the supernatant is filtered through a filter membrane with a pore size of 0.1-0.45μm to remove bacteria and obtain sterile filtrate; the sterile filtrate is concentrated to 10-20% of the original volume through a nanofiltration membrane to obtain the L. acidophilus fermentation filtrate.

[0013] Preferably, the L. acidophilus bacterial slurry and the fermentation filtrate in step S2 are mixed with the green tea catechin extract at a mass ratio of 20-30:20-30:1.

[0014] Preferably, the specific operation method of step S3 is as follows: the core complex solution in S2 is mixed with a chitosan solution with a mass / volume concentration of 1-3% at a volume ratio of 1:3-5 to form a primary emulsion under the conditions of 4-10°C and 8000-12000rpm for 2-10min; then the primary emulsion is added dropwise into a hydroxypropyl methyl cellulose phthalate solution with a mass / volume concentration of 2-4% at a volume ratio of 1:4-6 and a speed of 10-20mL / min to form an outer layer to obtain a microcapsule solution.

[0015] Preferably, the specific operation method of step S4 is as follows: a sodium alginate solution with a mass / volume concentration of 1-3% is slowly added to the microcapsule solution in S3 at a volume ratio of 1:0.5-2 to form a complex coacervate; then the mixed solution is added dropwise into a calcium lactate solution with a mass / volume concentration of 1-5% at a volume ratio of 4-6:1, and solidified and crosslinked at room temperature for 20-40min; the solidified microcapsule particles are collected and washed with deionized water until the filtrate is neutral and no crosslinking agent is left; vacuum freeze-drying is then performed, which includes three stages of pre-freezing, sublimation and desorption, and the vacuum degree during the sublimation and desorption stages is maintained at 10-30Pa until the water content of the sample is ≤5% to obtain dry microcapsule powder, i.e. the catechin-L. acidophilus complex composition.

[0016] In a third aspect, the present application provides the catechin-L. acidophilus complex composition for improving skin in skin care products or cosmetics.

[0017] In summary, the present application has the following beneficial effects: (1) Excellent performance: By adopting the double-shell microcapsule structure composed of pH-responsive polymer and enteric polymer, and innovatively using calcium ions provided by food-grade calcium lactate for ion crosslinking solidification, the destruction of the core active ingredient by environmental factors such as light, oxygen and heat is effectively blocked, the chemical stability of green tea catechin extract is significantly improved, the technical bottleneck of easy oxidation and degradation is overcome, and the active period of the product is prolonged. The system uses calcium lactate, which is recognized as safe, as a crosslinking agent, avoiding the biological toxicity risk of synthetic crosslinking agents, and has extremely high biological safety and regulatory compatibility. The ion crosslinking process conditions are mild, which maximizes the protection of the activity of lactobacillus acidophilus and the structure of green tea catechin extract. The dense and high-mechanical-strength calcium alginate gel network structure can ensure the integrity of the core material through the gastric acid environment.

[0018] (2) Targeted delivery and release: The complex composition uses the weakly acidic environment on the surface of the skin as a trigger mechanism to achieve intelligent responsive release of active substances. The double-shell structure can remain intact during transportation, and after precise arrival at the target site, it is dissolved in layers, thereby greatly improving the bioavailability and efficiency of the active ingredients.

[0019] (3) Excellent component compatibility and synergistic effect: By microencapsulation technology, green tea catechin extract and lactobacillus acidophilus fermentation filtrate are organically combined in the same system, not only solving the problem of component incompatibility caused by simple physical mixing, but also producing a synergistic effect between the two active ingredients, and simultaneously exerting multiple effects such as antioxidant, anti-inflammatory, regulation of skin microecology, and repair of skin barrier.

[0020] (4) Reliable process and suitable for large-scale production: The preparation method provided in the present application has clear parameters, mild and controllable process conditions, and can complete the entire production process using conventional equipment. In particular, the application of spray drying technology makes the final product have good flowability and stability, which is very suitable for industrial large-scale production.

[0021] (5) Safety and wide applicability: The catechin-lactobacillus acidophilus complex composition of the present application does not contain organic solvent residues and has good biocompatibility, and can be widely used in various skin care cosmetics, including the development of products for sensitive skin. DETAILED DESCRIPTION

[0022] The present application will be further described in conjunction with the following examples. It should be understood that the specific examples described herein are only for the purpose of explaining the present application and do not limit the scope of the present application.

[0023] If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0025] Biological material deposit: The *Lactobacillus acidophilus* strain LA11-Onlly involved in this application was provided by Shanghai Jiaotong University Angli Co., Ltd., and was deposited on July 13, 2007, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 2106. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0026] The green tea catechin extract was provided by Hunan Jinong Bioresources Co., Ltd. MRS liquid culture medium preparation: Weigh 10g peptone, 10g beef extract, 5g yeast extract, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1g Tween-80, 0.5g magnesium sulfate, and 0.25g manganese sulfate, dissolve them in 1L deionized water, and adjust the pH to 6.4.

[0027] Example 1 A method for preparing a catechin-Lactobacillus acidophilus complex composition for improving skin includes the following steps: S1, Lactobacillus acidophilus activated to the third generation ( Lactobacillus acidophilus LA11-Onlly seed culture was inoculated into fresh MRS liquid medium at an inoculum rate of 1% (v / v) and fermented at 30℃ for 36 h. After fermentation, the fermentation broth was centrifuged at 4℃ and 8000 rpm for 15 min, and the bacterial sludge and supernatant were collected separately. The supernatant was filtered through a PES microporous membrane with a pore size of 0.1 μm to remove bacteria and obtain sterile filtrate. The sterile filtrate was concentrated to 10% of its original volume through a nanofiltration membrane to obtain the Lactobacillus acidophilus fermentation filtrate. S2. Mix Lactobacillus acidophilus sludge, fermentation filtrate and green tea catechin extract in a mass ratio of 20:20:1 to obtain a core composite solution; S3. Mix the core composite solution from S2 with a 1% chitosan solution at a volume ratio of 1:3, and homogenize at 4℃ and 8000rpm for 2 minutes to form a primary emulsion. Then, add the primary emulsion dropwise to a 2% hydroxypropyl methylcellulose phthalate solution at a volume ratio of 1:4 and a rate of 10mL / min to form an outer layer, thus obtaining the microcapsule solution. S4. At a volume ratio of 1:0.5, slowly add a 1% sodium alginate solution to the microcapsule solution in S3, and mix evenly to form a composite aggregate. Then, at a volume ratio of 4:1, dropwise add the above mixture to a 1% calcium lactate solution and cure and crosslink at room temperature for 20 min. Collect the cured microcapsule particles, filter and wash with deionized water until the filtrate is neutral and there is no crosslinking agent residue. Then, perform vacuum freeze-drying, which includes three stages: pre-freezing, sublimation, and desorption. The vacuum degree is maintained at 10 Pa during the sublimation and desorption stages until the sample moisture content is 5%, and dry microcapsule powder is obtained, namely the catechin-Lactobacillus acidophilus composite composition.

[0028] Example 2 A method for preparing a catechin-Lactobacillus acidophilus complex composition for improving skin includes the following steps: S1, Lactobacillus acidophilus activated to the third generation ( Lactobacillus acidophilus LA11-Onlly seed culture was inoculated into fresh MRS liquid medium at an inoculum of 3% (v / v) and fermented at 35°C for 42 h. After fermentation, the fermentation broth was centrifuged at 8°C and 10,000 rpm for 20 min, and the bacterial sludge and supernatant were collected separately. The supernatant was filtered through a PES microporous membrane with a pore size of 0.22 μm to remove bacteria and obtain sterile filtrate. The sterile filtrate was concentrated to 15% of its original volume through a nanofiltration membrane to obtain the Lactobacillus acidophilus fermentation filtrate. S2. Mix Lactobacillus acidophilus sludge, fermentation filtrate and green tea catechin extract in a mass ratio of 25:25:1 to obtain a core composite solution; S3. Mix the core composite solution from S2 with a 2% (w / v) chitosan solution at a volume ratio of 1:4 until homogeneous. Homogenize the mixture at 8°C and 10,000 rpm for 6 min to form a primary emulsion. Then, add the primary emulsion dropwise to a 3% (w / v) hydroxypropyl methylcellulose phthalate solution at a volume ratio of 1:5 and a rate of 15 mL / min to form an outer layer, thus obtaining the microcapsule solution. S4. At a volume ratio of 1:1.5, slowly add a 2% sodium alginate solution to the microcapsule solution in S3, and mix evenly to form a composite aggregate. Then, at a volume ratio of 5:1, dropwise add the above mixture to a 1-5% calcium lactate solution, and cure and crosslink at room temperature for 30 minutes. Collect the cured microcapsule particles, filter and wash with deionized water until the filtrate is neutral and there is no crosslinking agent residue. Then, perform vacuum freeze-drying, which includes three stages: pre-freezing, sublimation, and desorption. The vacuum degree is maintained at 20 Pa during the sublimation and desorption stages until the sample moisture content is 5%, and dry microcapsule powder is obtained, namely the catechin-Lactobacillus acidophilus composite composition.

[0029] Example 3 A method for preparing a catechin-Lactobacillus acidophilus complex composition for improving skin includes the following steps: S1, Lactobacillus acidophilus activated to the third generation ( Lactobacillus acidophilus LA11-Onlly seed culture was inoculated into fresh MRS liquid medium at an inoculum of 5% (v / v) and fermented at 37℃ for 48 h. After fermentation, the fermentation broth was centrifuged at 10℃ and 12000 rpm for 15-25 min, and the bacterial sludge and supernatant were collected separately. The supernatant was filtered through a PES microporous membrane with a pore size of 0.45 μm to remove bacteria and obtain sterile filtrate. The sterile filtrate was concentrated to 20% of its original volume through a nanofiltration membrane to obtain the Lactobacillus acidophilus fermentation filtrate. S2. Mix Lactobacillus acidophilus sludge, fermentation filtrate and green tea catechin extract in a mass ratio of 30:30:1 to obtain a core composite solution; S3. Mix the core composite solution from S2 with a 3% chitosan solution at a volume ratio of 1:5 and homogenize at 10℃ and 12000rpm for 10min to form a primary emulsion. Then, add the primary emulsion dropwise to a 4% hydroxypropyl methylcellulose phthalate solution at a volume ratio of 1:6 and a rate of 20mL / min to form an outer layer, thus obtaining the microcapsule solution. S4. At a volume ratio of 1:2, slowly add a 3% sodium alginate solution to the microcapsule solution in S3, and mix evenly to form a composite aggregate. Then, at a volume ratio of 6:1, dropwise add the above mixture to a 5% calcium lactate solution and cure and crosslink at room temperature for 40 minutes. Collect the cured microcapsule particles, filter and wash with deionized water until the filtrate is neutral and there is no crosslinking agent residue. Then, perform vacuum freeze-drying, which includes three stages: pre-freezing, sublimation, and desorption. The vacuum degree is maintained at 30 Pa during the sublimation and desorption stages until the sample moisture content is 5%, and dry microcapsule powder is obtained, namely the catechin-Lactobacillus acidophilus composite composition.

[0030] Comparative Example 1 A method for preparing a catechin-Lactobacillus acidophilus complex composition for improving skin includes the following steps: S1, Lactobacillus acidophilus activated to the third generation ( Lactobacillus acidophilusLA11-Onlly seed culture was inoculated into fresh MRS liquid medium at an inoculum rate of 1% (v / v) and fermented at 30℃ for 36 h. After fermentation, the fermentation broth was centrifuged at 4℃ and 8000 rpm for 15 min, and the bacterial sludge and supernatant were collected separately. The supernatant was filtered through a PES microporous membrane with a pore size of 0.1 μm to remove bacteria and obtain sterile filtrate. The sterile filtrate was concentrated to 10% of its original volume through a nanofiltration membrane to obtain the Lactobacillus acidophilus fermentation filtrate. S2. Mix Lactobacillus acidophilus sludge, fermentation filtrate and green tea catechin extract in a mass ratio of 20:20:1 to obtain a composite solution; S3. The composite solution is subjected to vacuum freeze-drying, which includes three stages: pre-freezing, sublimation and desorption. The vacuum degree is maintained at 10 Pa during the sublimation and desorption stages until the water content of the sample is 5%, and the dried microcapsule powder, namely the catechin-Lactobacillus acidophilus composite composition, is obtained.

[0031] Comparative Example 2 A method for preparing a catechin-Lactobacillus acidophilus complex composition for improving skin includes the following steps: S1, Lactobacillus acidophilus activated to the third generation ( Lactobacillus acidophilus LA11-Onlly seed culture was inoculated into fresh MRS liquid medium at an inoculum rate of 1% (v / v) and fermented at 30℃ for 36 h. After fermentation, the fermentation broth was centrifuged at 4℃ and 8000 rpm for 15 min, and the bacterial sludge and supernatant were collected separately. The supernatant was filtered through a PES microporous membrane with a pore size of 0.1 μm to remove bacteria and obtain sterile filtrate. The sterile filtrate was concentrated to 10% of its original volume through a nanofiltration membrane to obtain the Lactobacillus acidophilus fermentation filtrate. S2. Mix Lactobacillus acidophilus sludge, fermentation filtrate and green tea catechin extract in a mass ratio of 20:20:1 to obtain a core composite solution; S3. Mix the core composite solution in S2 with a chitosan solution with a mass-volume concentration of 1% at a volume ratio of 1:3, and homogenize at 4℃ and 8000rpm for 2 minutes to form a primary emulsion. S4. At a volume ratio of 1:0.5, slowly add a 1% sodium alginate solution to the microcapsule solution in S3, and mix evenly to form a composite aggregate. Then, at a volume ratio of 4:1, dropwise add the above mixture to a 1% calcium lactate solution and cure and crosslink at room temperature for 20 min. Collect the cured microcapsule particles, filter and wash with deionized water until the filtrate is neutral and there is no crosslinking agent residue. Then, perform vacuum freeze-drying, which includes three stages: pre-freezing, sublimation, and desorption. The vacuum degree is maintained at 10 Pa during the sublimation and desorption stages until the sample moisture content is 5%, and dry microcapsule powder is obtained, namely the catechin-Lactobacillus acidophilus composite composition.

[0032] Comparative Example 3 A method for preparing a catechin-Lactobacillus acidophilus complex composition for improving skin includes the following steps: S1, Lactobacillus acidophilus activated to the third generation ( Lactobacillus acidophilus LA11-Onlly seed culture was inoculated into fresh MRS liquid medium at an inoculum rate of 1% (v / v) and fermented at 30℃ for 36 h. After fermentation, the fermentation broth was centrifuged at 4℃ and 8000 rpm for 15 min, and the bacterial sludge and supernatant were collected separately. The supernatant was filtered through a PES microporous membrane with a pore size of 0.1 μm to remove bacteria and obtain sterile filtrate. The sterile filtrate was concentrated to 10% of its original volume through a nanofiltration membrane to obtain the Lactobacillus acidophilus fermentation filtrate. S2. Mix Lactobacillus acidophilus sludge, fermentation filtrate and green tea catechin extract in a mass ratio of 20:20:1 to obtain a core composite solution; S3. Mix the core composite solution from S2 with a 1% chitosan solution at a volume ratio of 1:3, and homogenize at 4℃ and 8000rpm for 2 minutes to form a primary emulsion. Then, add the primary emulsion dropwise to a 2% hydroxypropyl methylcellulose phthalate solution at a volume ratio of 1:4 and a rate of 10mL / min to form an outer layer, thus obtaining the microcapsule solution. S4. At a volume ratio of 1:0.5-2, slowly add a sodium alginate solution with a mass-volume concentration of 1-3% to the microcapsule solution in S3, and mix evenly to form a composite aggregate; then, at a volume ratio of 4-6:1, dropwise add the above mixture to a glutaraldehyde solution with a mass-volume concentration of 1-5%, and cure and crosslink at room temperature for 20-40 minutes; collect the cured microcapsule particles, filter and wash with deionized water until the filtrate is neutral and there is no crosslinking agent residue; then perform vacuum freeze-drying, which includes three stages: pre-freezing, sublimation and desorption, wherein the vacuum degree is maintained at 10-30 Pa during the sublimation and desorption stages, until the sample water content is ≤5%, and dry microcapsule powder is obtained, namely the catechin-Lactobacillus acidophilus composite composition.

[0033] Product performance comparison test 1. Determination of microcapsule encapsulation efficiency and drug loading Sample groups: Examples 1-3 and Comparative Examples 1-3.

[0034] Methods: Ultrafiltration-centrifugation-HPLC was used. 5g of each sample was accurately weighed, dissolved in PBS buffer (pH 7.4), and placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kDa. The tubes were centrifuged at 4000 rpm for 15 min, and the filtrate was collected. The content of free green tea catechin extract was determined by HPLC. Encapsulation efficiency (EE%) and drug loading (DL%) were calculated using the following formulas: EE% = (Total W - Free W) / Total W × 100% DL% = (W total - W free) / W microcapsules × 100% Wherein, Wtotal represents the total amount of green tea catechin extract added (1g), Wfree represents the amount of free green tea catechin extract measured in the filtrate, and Wmicrocapsule represents the sample weight (5g). The results are shown in Table 1.

[0035] Table 1 Encapsulation efficiency and drug loading of microcapsules in each group

[0036] As shown in Table 1, the embodiments of this application employ a double-layer encapsulation technique, achieving a high encapsulation rate (>90%) and high drug loading. Comparative Example 1 (physical mixing) showed no encapsulation effect and was not a microcapsule; Comparative Example 2 (single-layer chitosan encapsulation) lacked an outer protective layer, resulting in significant loss of active ingredients during washing and drying, leading to a significantly reduced encapsulation rate; Comparative Example 3 (glutaraldehyde crosslinking) had an encapsulation rate and drug loading comparable to Example 1, but the glutaraldehyde crosslinking product was pale yellow to brown, which is crucial for the appearance of cosmetics.

[0037] 2. Accelerated stability test Sample groups: Examples 1-3 and Comparative Examples 1-3.

[0038] Methods: Each group of samples was placed in a constant temperature and humidity chamber at 40℃ and 75% relative humidity. Samples were taken at 0, 10, 20 and 30 days to determine the retention rate of green tea catechin extract.

[0039] Retention rate % = (Ct / C0) × 100%, where C0 is the content of green tea catechin extract at day 0, Ct is the content of green tea catechin extract at day t, and t is 10, 20, or 30. The results are shown in Table 2.

[0040] Table 2 Retention rate of green tea catechin extract in each group of samples

[0041] As shown in Table 2, after 30 days of accelerated testing, the retention rate of green tea catechin extracts in Examples 1-3 of this application remained above 90%, demonstrating extremely high stability. Particularly at the 10-day and 20-day testing points, the retention rate of each example exceeded 94%, indicating that the bilayer microcapsule structure of this application provided continuous and effective protection for the internal green tea catechin extracts throughout the entire testing period, significantly delaying their oxidative degradation process.

[0042] The retention rate of green tea catechin extract in Comparative Group 1 (physical mixing) showed a precipitous decline, reaching only 42.5% after 30 days. This directly confirms that the unencapsulated physical mixing method cannot solve the inherent defect of the unstable chemical properties of catechins, and the active ingredients are rapidly degraded under high temperature and high humidity conditions.

[0043] Although the stability of Comparative Example 2 (single-layer chitosan encapsulation) was better than that of Comparative Example 1, it was significantly lower than that of all examples. This strongly suggests that single-layer encapsulation alone cannot provide sufficient protection. The lack of an outer enteric material (HPMCP) makes its barrier function incomplete and unable to effectively isolate moisture and oxygen, resulting in the core material stability being significantly lower than that of the double-layer structure of the present invention.

[0044] 3. Safety evaluation Cytotoxicity testing is a key indicator for assessing the biocompatibility of cosmetic raw materials. To investigate the biocompatibility of the catechin-Lactobacillus acidophilus composite prepared in this invention, the CCK-8 assay was used to detect the cytotoxicity of each sample to human immortalized keratinocytes (HaCaT).

[0045] Sample groups: Examples 1-3 and Comparative Examples 1-3.

[0046] Methods: HaCaT cells in logarithmic growth phase were introduced into each well at a density of 1 × 10⁶ cells / well. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured at 37°C with 5% CO2 for 24 hours to allow complete cell adhesion. After discarding the original culture medium, 100 μL of sample solutions from Examples 1-3 and Comparative Examples 1-3, serially diluted with culture medium to different concentrations (1000 μg / mL, 500 μg / mL, 100 μg / mL), were added to each well. The culture medium-only group served as a blank control (100% cell viability). Each group was configured with 6 replicates. After incubation for another 24 hours, 10 μL of CCK-8 solution was added to each well, and the reaction was carried out for 2 hours. The absorbance (OD value) of each well was then measured using a microplate reader at 450 nm. Cell viability was calculated using the following formula: Cell viability (%) = (OD sample group - OD blank well) / (OD control group - OD blank well) × 100%. The results are shown in Table 3.

[0047] Table 3. Cytotoxicity of each sample group to HaCaT cells

[0048] Note: Data are expressed as mean ± standard deviation (n=6); one-way ANOVA was used for inter-group comparisons, followed by Tukey post-hoc tests. Compared with the control group, *p<0.05, **p<0.01.

[0049] As shown in Table 3, Comparative Example 3 (crosslinked with glutaraldehyde) exhibited concentration-dependent cytotoxicity. At a high concentration of 1000 μg / mL, cell viability dropped sharply to 78.3 ± 5.6%, a significant difference from the control group, indicating significant cytotoxicity. Even when the concentration was reduced to 500 μg / mL, cell viability remained significantly lower than in the Example group. This suggests that due to the high reactivity and cytotoxicity of glutaraldehyde, even after multiple washes, trace amounts of residues or incompletely removed free aldehyde groups in the crosslinking bonds may remain, causing damage to cell membranes and proteins, and inducing apoptosis or necrosis.

[0050] This safety evaluation experiment confirms that the catechin-Lactobacillus acidophilus composite composition prepared using calcium lactate as a cross-linking agent in this application exhibits significantly better biosafety than the process using glutaraldehyde as a traditional chemical cross-linking agent. This not only solves the technical problems of microencapsulation but also ensures the high safety of the final product for use in cosmetics.

[0051] The above specific embodiments are merely explanations of this application and are not intended to limit this application. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A catechin-Lactobacillus acidophilus complex composition for improving skin, characterized in that, The catechin-Lactobacillus acidophilus complex is in the form of microcapsules, with catechin extract and Lactobacillus acidophilus and its fermentation filtrate as the core, and pH-responsive polymer and enteric polymer as the double shell.

2. The catechin-Lactobacillus acidophilus complex composition for improving skin according to claim 1, characterized in that, The double shell consists of an inner layer made of a pH-responsive polymer and an outer layer made of an enteric polymer. The composite composition consists of a core, an inner layer, and an outer layer from the inside out.

3. The catechin-Lactobacillus acidophilus complex composition for improving skin according to claim 2, characterized in that, The pH-responsive polymer is chitosan, and the enteric polymer is hydroxypropyl methylcellulose phthalate.

4. The catechin-Lactobacillus acidophilus complex composition for improving skin according to claim 1, characterized in that, The catechin extract is a green tea catechin extract, wherein the catechin content is ≥40%, the EGCG content is ≥18%, the EGC content is ≥10%, the ECG content is ≥8%, the DL-C content is ≥5%, the EC content is ≥1.5%, and the GCG content is ≥0.8%.

5. A method for preparing the catechin-Lactobacillus acidophilus complex composition for improving skin as described in claims 1-4, characterized in that, Includes the following steps: S1. After fermenting and centrifuging Lactobacillus acidophilus, Lactobacillus acidophilus sludge and centrifuged liquid are obtained. After filtration and concentration by nanofiltration membrane, Lactobacillus acidophilus fermentation filtrate is obtained. S2. Mix Lactobacillus acidophilus sludge and fermentation filtrate with green tea catechin extract to obtain a core composite solution; S3. The core composite solution is encapsulated using pH-responsive polymers and enteric polymers to obtain microcapsule liquid; S4. The microcapsule liquid is solidified, washed, and vacuum freeze-dried to obtain dried microcapsule powder, namely the catechin-Lactobacillus acidophilus composite composition.

6. The method for preparing the catechin-Lactobacillus acidophilus complex composition for improving skin according to claim 5, characterized in that, The specific operation method of step S1 is as follows: the seed culture of Lactobacillus acidophilus activated to the third generation is inoculated into fresh MRS liquid culture medium at an inoculation amount of 1-5% (v / v) and fermented at 30-37℃ for 36-48h; after fermentation, the fermentation broth is centrifuged at 4-10℃ and 8000-12000rpm for 15-25min, and the bacterial sludge and supernatant are collected separately; the supernatant is filtered through a filter membrane with a pore size of 0.1-0.45μm to remove bacteria and obtain sterile filtrate; the sterile filtrate is concentrated to 10-20% of the original volume through a nanofiltration membrane to obtain Lactobacillus acidophilus fermentation filtrate.

7. The method for preparing the catechin-Lactobacillus acidophilus complex composition for improving skin according to claim 5, characterized in that, In step S2, Lactobacillus acidophilus sludge, fermentation filtrate, and green tea catechin extract are mixed at a mass ratio of 20-30:20-30:

1.

8. The method for preparing the catechin-Lactobacillus acidophilus complex composition for improving skin according to claim 5, characterized in that, The specific operation method of step S3 is as follows: the core composite solution in S2 is mixed with a chitosan solution with a mass-volume concentration of 1-3% at a volume ratio of 1:3-5, and homogenized at 4-10℃ and 8000-12000rpm for 2-10min to form a primary emulsion; then the primary emulsion is added dropwise to a hydroxypropyl methylcellulose phthalate solution with a mass-volume concentration of 2-4% at a volume ratio of 1:4-6 at a rate of 10-20mL / min to form an outer layer, thereby obtaining the microcapsule solution.

9. The method for preparing the catechin-Lactobacillus acidophilus complex composition for improving skin according to claim 5, characterized in that, The specific operation method of step S4 is as follows: Sodium alginate solution with a mass-volume concentration of 1-3% is slowly added to the microcapsule liquid in S3 at a volume ratio of 1:0.5-2, and mixed evenly to form a composite aggregate; then, the above mixture is added dropwise to a calcium lactate solution with a mass-volume concentration of 1-5% at a volume ratio of 4-6:1, and cured and crosslinked at room temperature for 20-40 minutes; the cured microcapsule particles are collected and washed with deionized water until the filtrate is neutral and free of crosslinking agent residue; then, vacuum freeze-drying is performed, which includes three stages: pre-freezing, sublimation, and desorption. The vacuum degree is maintained at 10-30 Pa during the sublimation and desorption stages until the sample water content is ≤5%, resulting in dried microcapsule powder, i.e., the catechin-Lactobacillus acidophilus composite composition.

10. The use of the catechin-Lactobacillus acidophilus complex composition for improving skin according to any one of claims 1-4 in skin care products or cosmetics.

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