Composition based on lactobacillus fermented tea and application of composition

Abstract

The invention discloses a composition based on lactic acid bacteria fermented tea and application of the composition in preparation of skin care and beauty cosmetics, and belongs to the technical field of cosmetics. The lactic acid bacteria fermented tea is a fermentation liquid which is obtained by taking lactobacillus plantarum ZJU2303-RH17 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) 1.60149 as a fermentation strain and fresh tea leaves as a fermentation substrate and carrying out fermentation culture; separating the exosome from the fermentation liquor as a component I, removing the exosome from the fermentation liquor, and performing vacuum drying and superfine grinding to obtain a component II; the composition comprises a component I and a component II, and the mass ratio of the component I to the component II is 1: (10-20). The fermentation composition provided by the invention comprises exosome, polyphenol, a lactic acid bacteria fermentation product and lactic acid bacteria and tea leaf cell components, can give full play to the synergistic effect between the exosome and other active substances, and better gives play to the skin care and beauty efficacy.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, and in particular to a composition based on lactic acid bacteria fermented tea and application thereof in the preparation of skin care and beauty cosmetics. Background Art

[0002] Probiotics, mainly lactic acid bacteria, and their fermentation products are widely used in skin care and beauty. Studies have shown that lactic acid bacteria fermentation products have significant antioxidant activity, thus exerting skin care effects (Tang Liuying, Cao Xiaorui, Wu Wenyi, et al. Effects of prebiotics-lactic acid bacteria fermentation active ingredients on skin care effects. Medical Aesthetics and Cosmetology, 2024, 33(5):29-32.). Tea is also widely used in the cosmetics industry due to its skin care and beauty effects (Yang Huanyu, Qian Zhongying, Fu Xiuhua, et al. Synergistic skin care effects of black tea and green tea extracts. Fine Chemicals, 2020, 37(2):426-432; Liu Qian, Liu Yaxuan, Xiao Juan, et al. Research progress on the mechanism and application of tea in skin care and beauty. China Medical Herald, 2018, 15(33):40-42.). In recent years, it has also been found that vesicles secreted by cells to the outside of cells to exchange information between cells, namely exosomes, have a significant effect in promoting skin regeneration and renewal (Wu Shuxia, Shao Jiaqing, Yang Jian, et al. International status and trends of exosome cosmetic management. Chinese Journal of Pharmaceutical Biotechnology, 2024, 19(2): 104-107.). Exosomes have a particle size range of 30 to 150 nm and contain bioactive components such as lipids, proteins and nucleic acids for intercellular communication. Studies on exosomes in scar improvement, pigmentation correction, skin rejuvenation and hair loss recovery have shown that exosomes exert their antioxidant and cell regeneration properties to slow down skin aging, thereby improving skin firmness and radiance. Stem cell exosomes can accelerate wound healing and reduce inflammatory responses, especially in the care of sensitive skin and damaged skin, promoting skin barrier repair and regeneration. Exosomes also have a significant effect on promoting hair follicle repair and hair regeneration.

[0003] Many achievements have been made in the application of lactic acid bacteria fermentation products, active ingredients in tea, and exosomes from animals, plants and microorganisms in the field of cosmetics. For example, patent document CN114657107A discloses an acne-clearing essence cream prepared with plant lactobacillus (preservation number CCTCC NO: M2021683) lysate as the only effective ingredient. It has been confirmed by human trials that it can effectively improve mild to moderate acne, reduce the area of ​​acne and the oil content of the skin, reduce transepidermal water flow and increase the moisture content of the skin, make the skin color tend to normal, make the pores finer, and balance the pH of the skin. Patent document CN117815156A discloses a green tea polyphenol repair cream with good repair and firming effects on the skin. Patent document CN115025034A discloses a mesenchymal stem cell exosome composition, which has the effects of synergistically enhancing cell repair, whitening and anti-aging. Patent document CN114159373A discloses the application of exosome-like vesicles derived from dendrobium in skin care cosmetics, which can reduce the activity of β-galactosidase, inhibit the degradation of elastin, reduce skin wrinkles, enhance skin elasticity, and inhibit the synthesis of melanin in cells to achieve a whitening effect. Patent document CN116948921A discloses the application of Lactobacillus plantarum exosomes, which can promote the proliferation of epidermal cells, thereby increasing skin elasticity, reducing wrinkles, delaying skin aging, and accelerating the repair and healing of skin wounds.

[0004] The existing methods for obtaining exosomes are mainly based on simple cell culture. There are currently no research reports on using tea as a lactic acid bacteria fermentation substrate, isolating exosomes from the fermentation products, and combining them with active substances in the fermentation products for skin care and beauty. Summary of the invention

[0005] The purpose of the present invention is to provide a composition with skin care and beauty effects, which can reduce skin wrinkles, improve skin elasticity and whiten the skin, and develop cosmetics using the composition.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The invention provides a composition based on lactic acid bacteria fermented tea. The lactic acid bacteria fermented tea is prepared by fermenting Lactobacillus plantarum ZJU2303-RH17 with a preservation number of CGMCC 1.60149 as a fermentation strain and fresh tea leaves as a fermentation substrate to obtain a fermentation broth; exosomes are separated from the fermentation broth as component I, and the fermentation broth is vacuum dried and ultrafinely pulverized to obtain component II after the exosomes are removed; the composition comprises component I and component II, and the mass ratio of the two is 1:10-20.

[0008] The above-mentioned component II contains active substances such as polyphenols, lactic acid bacteria fermentation products, and lactic acid bacteria and tea cell components. Through the reasonable ratio of component I and component II, the synergistic effect between exosomes and other active substances is fully exerted to significantly improve skin wrinkles, melanin deposition and other problems.

[0009] The present invention adopts Lactobacillus plantarum as the fermentation strain. Lactobacillus plantarum is a lactic acid bacterium with wide distribution and application, good fermentation characteristics and sufficient safety.

[0010] The Lactobacillus plantarum ZJU2303-RH17 used in the present invention is a biological material available to the public and is publicly deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration with a deposit number of CGMCC 1.60149.

[0011] The present invention uses fresh leaves of Anji white tea as a lactic acid bacteria fermentation substrate, which has sufficient safety and a rich source of active substances. Anji white tea is a local tea species in Anji, and the tea leaves are rich in natural products represented by tea polyphenols. Anji white tea is planted in mountainous areas. Because the temperature difference between day and night in the mountainous areas is large, pests and diseases are not easy to breed, and pesticides do not need to be applied. Fresh leaves of Anji white tea can be used but are not limited to fresh leaves in summer and autumn. Compared with Mingqian tea and Yuqian tea in spring, the active substances in summer and autumn tea are richer in content due to the long accumulation time.

[0012] Preferably, the fresh tea leaves are crushed and diluted 8-12 times with water, and then MRS liquid medium is added to prepare the fermentation medium, and the mass ratio of MRS liquid medium to tea leaves is 1:2. MRS liquid medium is a medium suitable for lactic acid bacteria fermentation, which provides nutrients for the growth and expansion of lactic acid bacteria.

[0013] Further preferably, the preparation method of the lactic acid bacteria fermented tea comprises the following steps:

[0014] (1) Preparation of tea and water mixture: crush fresh Anji white tea leaves, add water and mix, and add 10 parts of tea leaves to every 100 parts of water by weight;

[0015] (2) Lactic acid bacteria inoculation and fermentation: Lactobacillus plantarum ZJU2303-RH17 was added to the tea and water mixture in step (1) at an inoculation density of 1 to 3 × 10 7 cfu / mL, by weight, add 5 parts of MRS liquid culture medium to every 100 parts of water, ferment at 25-30°C for 48-72 hours to obtain fermentation liquid.

[0016] The preparation method of the exosomes comprises: centrifuging the fermentation broth at 5000g for 10 min at 4°C, filtering the supernatant obtained after separating the bacteria with a 0.45 μm filter membrane, and then centrifuging at 15000g for 20 min at 4°C, and the obtained precipitate is the exosomes.

[0017] The exosomes prepared by the present invention are spherical, double-layer membrane structure, and particle size is 60 to 140 nm, which is consistent with the morphological characteristics of exosomes. Studies have shown that the exosomes provided by the present invention have the effect of promoting collagen synthesis in skin cells and inhibiting melanin production. The exosomes of the present invention are prepared by fermenting tea leaves with lactic acid bacteria, and have the advantages of exosomes, lactic acid bacteria fermentation products, and active substances in tea leaves.

[0018] In addition to exosomes, the fermentation broth also contains fermentation products, lactic acid bacteria and fermented tea leaves, which are rich in active substances. The present invention vacuum dries and concentrates the fermentation broth after removing the exosomes, and then grinds it using ultrafine grinding technology. The obtained component II also has the effect of promoting the synthesis of collagen in skin cells and inhibiting the production of melanin. It is compounded with component I to produce a synergistic effect.

[0019] Preferably, the ultrafine grinding conditions are: using a low-temperature ultrafine wall-breaking grinder, grinding at -10°C for 10 minutes and then storing at 4°C.

[0020] Preferably, the mass ratio of component I to component II in the composition is 1:15.

[0021] The present invention also provides the use of the composition based on lactic acid bacteria fermented tea in the preparation of skin care and beauty cosmetics.

[0022] Furthermore, the skin care and beauty treatments include but are not limited to wrinkle removal and skin whitening.

[0023] The composition provided by the present invention can promote the production of collagen in skin cells, reduce skin wrinkles and improve skin elasticity.

[0024] The composition provided by the invention can inhibit the generation of melanin in skin cells and achieve the effect of skin whitening.

[0025] The present invention also provides a cosmetic having the efficacy of promoting collagen synthesis and / or inhibiting melanin production, comprising an effective dose of the lactic acid bacteria fermented tea-based composition and a cosmetic auxiliary material matrix.

[0026] The effective dose refers to the amount of the composition sufficient to improve skin problems. The auxiliary material matrix refers to any preparation or carrier medium that can deliver the effective dose of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or subject.

[0027] The present invention has the following beneficial effects:

[0028] The invention uses Lactobacillus plantarum CGMCC 1.60149 as a fermentation strain and fresh tea leaves as a fermentation substrate, has sufficient safety and a rich source of active substances, and the obtained fermentation composition of lactic acid bacteria fermented tea includes exosomes, polyphenols, lactic acid bacteria fermentation products, and lactic acid bacteria and tea cell components, which can give full play to the synergistic effect between exosomes and other active substances and better exert the skin care and beauty effects. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] In the following examples, the lactic acid bacteria used refer to Lactobacillus plantarum ZJU2303-RH17, which was publicly deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on October 26, 2023, and was identified as alive. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and the deposit number is CGMCC 1.60149. The plant lactobacillus was derived from a kimchi making experiment conducted by the College of Biosystems Engineering and Food Science, Zhejiang University, and was obtained by streaking single colony culture. The 16s rRNA sequencing results determined that it was classified as plant lactobacillus.

[0032] The tea used refers to the fresh leaves of Anji White Tea in summer and autumn, which are collected and provided by Zhejiang Anji Dashanwu White Tea Co., Ltd. Anji is a county under the jurisdiction of Huzhou City, China, and belongs to the mountainous area. Anji White Tea is a local unique tea variety. The temperature difference between day and night in the mountainous area is large, so pests and diseases are not easy to breed, and pesticides do not need to be used. Although the tenderness of summer and autumn tea is not as good as the Mingqian tea and Yuqian tea in spring, the active substances are richer because of the long accumulation time.

[0033] The experimental methods involved in the following examples are as follows:

[0034] 1. Exosome morphology analysis: After the exosomes were appropriately diluted with phosphate buffered saline (PBS), 10 μL was dropped onto a carbon-coated copper mesh. After drying, the morphology was observed and the particle size was measured using a transmission electron microscope, and the particle size was further calibrated using a nanoparticle tracking analyzer.

[0035] 2. Determination of collagen content in cells: Spectrophotometry was used, and the specific steps were referred to the literature (Zhu Ling et al. Effects of different negative pressure suction devices on the secretion of collagen, matrix metalloproteinase 1 and transforming growth factor-β by mouse skin. Chinese Journal of Medical Aesthetics and Cosmetology, 2021, 2: 145-148.).

[0036] The human skin fibroblasts (HFF-1) used in the experiment were purchased from the Experimental Animal Center of Zhejiang University School of Medicine.

[0037] 3. Determination of melanin content in cells: NaOH dissolution method was used. The specific steps were referred to the literature (Wang Zhaowei et al. Mechanism of tripeptide D-Trp-Arg-Leu-NH2 inhibiting melanin synthesis in B16 melanoma cells. Flavors and Fragrances Cosmetics, 2023, (1): 82-86.).

[0038] The mouse B16 melanoma cells used in the experiment were purchased from the Experimental Animal Center of Zhejiang University School of Medicine.

[0039] 4. Evaluation of synergistic effect: The evaluation of the synergistic effect between component I and component II in the composition is carried out by using the Jin (Zhengjun) formula in combination with the index Q value. First, the efficacy value E is calculated, E = (E 试验组 -E 阴性对照组 ) / E 试验组 × 100%, and then the Jin (Zhengjun) formula is used to calculate the combination index Q value, Q = E Ⅰ+Ⅱ / (E Ⅰ +E Ⅱ -E Ⅰ ·E Ⅱ );

[0040] In the above formula, E Ⅰ+Ⅱ is the effect of the combined application of component I and component II, E Ⅰ 、E Ⅱ The Q value is: 0.85≤Q≤1.15 is simple addition (+); Q>1.15 is enhancement (++); Q<0.85 is antagonism (-). The larger the Q value, the stronger the synergistic effect.

[0041] For specific steps, please refer to the literature (Liu Lijuan et al. Effects of Smilax glabra, Prunus mume and their combination on inflammatory factors and Notch signaling pathway in skin lesions of mice with psoriasis-like model. Journal of Traditional Chinese Medicine, 2022, 63(19): 1880-1887.).

[0042] Example 1

[0043] 1. Preparation of fermentation composition

[0044] (1) Preparation of tea and water mixture: crush the fresh leaves of Anji white tea in summer and autumn, add water and mix, the ratio of tea to water is 10 parts of tea to 100 parts of water.

[0045] (2) Lactic acid bacteria inoculation and fermentation: a density of 1×10 9 cfu / mL Lactobacillus plantarum CGMCC 1.60149 bacterial liquid is added to the tea and water mixture in step (1), the added amount is 1 part of bacterial liquid in 100 parts of water, and 5 parts of MRS liquid culture medium are added at the same time, and fermented at 25-30°C for 48 hours to obtain a fermentation liquid.

[0046] (3) Exosome preparation: The fermentation broth was centrifuged at 5000 g for 10 min at 4°C. The supernatant obtained after separating the bacteria was first filtered through 0.45 μm and then centrifuged at 15000 g for 20 min at 4°C. The resulting precipitate was the exosomes produced by Anji white tea fermented by Lactobacillus plantarum CGMCC 1.60149, i.e., component I.

[0047] (4) Preparation of fermentation composition: The components other than exosomes obtained during the preparation of exosomes are vacuum dried and ultrafinely ground to become component II. The ultrafine grinding conditions are to use a low-temperature ultrafine wall-breaking grinder, grind at -10°C for 10 minutes and then store at 4°C. The ultrafinely ground component II includes bacteria, ultrafine fragments of tea leaves, fermentation products and culture medium components. Component II is compounded with component I in a certain ratio to obtain a fermentation composition (component I + component II).

[0048] 2. Exosome morphology analysis

[0049] Transmission electron microscopy showed that the exosomes prepared in step (3) were spherical in shape, with a double-layer membrane structure and a particle size of 60 to 140 nm.

[0050] 3. Analysis of the efficacy of the fermented composition of lactic acid bacteria fermented tea in promoting collagen production

[0051] Human skin fibroblasts were dispersed in DMEM medium containing fetal bovine serum at a rate of 5 × 10 cells per well. 4 The strains were divided into 24-well plates at a concentration of 80 μL per well and cultured for 24 hours.

[0052] Component I was added to DMEM medium and diluted to a final concentration of 0.1% (w / v), component II was added to DMEM medium and diluted to a final concentration of 2% (w / v), component I and component II were added to DMEM medium to form "component I + component II" (final concentrations of 0.1% and 2%, w / v), respectively, and used to treat human skin fibroblasts that had been cultured in 24-well plates for 24 hours, and the grouping treatment was as follows:

[0053] Control group (Control): treated with DMEM medium only, with 20 μL added to each well, and 6 wells were set up for replication.

[0054] Experimental group I (T1): Component I was added, with 20 μL added to each well, and 6 wells were repeated.

[0055] Experimental group II (T2): Component II was added, with 20 μL added to each well, and 6 wells were repeated.

[0056] Experimental group III (T3): Add "component I + component II" treatment, the amount of liquid added to each well is 20 μL, and 6 wells are repeated.

[0057] After 24 hours of continuous culture, the total cell number was determined using an MTT detection kit (purchased from Sigma Aldrich), centrifuged, and the absorbance was measured at a specific wavelength using an EIA kit for procollagen type IC peptide (PIP) (purchased from Takara), which was converted into the amount of collagen synthesized by human skin fibroblasts and accumulated in the culture medium. The measured amount of collagen was normalized by dividing it by the total number of cells, and the relative amount of collagen was calculated based on the control group (Control): absorbance of different groups / absorbance of control group × 100%. The results are shown in Table 1.

[0058] Table 1. Relative collagen content in cells

[0059]

[0060] Note: a, b, c, and d are significantly different from each other (p<0.01).

[0061] As can be seen from the above table, compared with the control group, both the T1 group treated with component I and the T2 group treated with component II showed a significant increase in collagen synthesis, among which the increase in the T1 group was significantly higher than that in the T2 group, and the T3 group treated with "component I + component II" showed a significant increase in collagen synthesis and the increase was significantly higher than that in the T1 group. The results showed that both component I and component II in the fermentation composition of lactic acid bacteria fermented tea can promote collagen synthesis, component I is more effective than component II, and the effect of component I and component II combined (ie, T3) is significantly better than component I or component II alone (ie, T1 and T2).

[0062] Calculate the Q value between component I and component II:

[0063] E Ⅰ =(E Ⅰ -E 阴性对照组 ) / E Ⅰ ×100%=0.200;

[0064] E Ⅱ =(E Ⅱ -E 阴性对照组 ) / E Ⅱ ×100%=0.115;

[0065] E Ⅰ+Ⅱ =(E Ⅰ+Ⅱ -E 阴性对照组 ) / E Ⅰ+Ⅱ ×100%=0.346;

[0066] Q Ⅰ+Ⅱ =E Ⅰ+Ⅱ / (E Ⅰ +E Ⅱ -E Ⅰ ·E Ⅱ )=1.186;

[0067] Q Ⅰ+Ⅱ >1.15, component I and component II show a synergistic effect, that is, the fermentation composition of lactic acid bacteria fermented tea composed of the two has the best effect in promoting collagen synthesis, which can reduce skin wrinkles and improve skin elasticity.

[0068] 4. Analysis of the efficacy of the fermented composition of lactic acid bacteria fermented tea in inhibiting melanin production

[0069] Mouse melanoma cells were dispersed in DMEM medium containing fetal bovine serum at a rate of 1×10 per well. 4 The strains were divided into 24-well plates at a concentration of 80 μL per well and cultured for 24 hours.

[0070] Afterwards, melanin synthesis stimulator αMSH (α melanocyte stimulating hormone, α melanocyte stimulating hormone) (0.10%, v / v) was added to each group, and the component I, component II and "component I + component II" previously diluted in DMEM medium were used to treat mouse melanoma cells that had been cultured in a 24-well plate for 24 hours. The grouping treatment was as follows:

[0071] Negative control group (C1): treated with DMEM medium only, 20 μL of liquid was added to each well, and 6 wells were set up for replication.

[0072] Positive control group (C2): Arbutin was added to a final concentration of 1 mM, and 6 wells were set up for replication.

[0073] Experimental group I (T1): Component I was added, with 20 μL added to each well, and 6 wells were repeated.

[0074] Experimental group II (T2): Component II was added, with 20 μL added to each well, and 6 wells were repeated.

[0075] Experimental group III (T3): Add "component I + component II" treatment, the amount of liquid added to each well is 20 μL, and 6 wells are set to replicate.

[0076] The culture was continued for 48 hours, the supernatant was discarded after centrifugation, and the mouse melanoma cells were treated with 1N NaOH (purchased from Merck Millipore) mixed with 10% DMSO. The plate was sealed and heated at 85°C for 20 minutes to extract intracellular melanin. The absorbance was measured at 405 nm, and the relative melanin amount was calculated: absorbance of different groups / absorbance of C1 group × 100%. The results are shown in Table 2.

[0077] Table 2. Relative melanin content in cells

[0078]

[0079] Note: a, b, c, and d are significantly different from each other (p<0.01).

[0080] As can be seen from the above table, compared with the negative control group (C1), the T1 group treated with component I and the T2 group treated with component II both showed significant inhibition of melanin production, among which the inhibition amplitude of the T1 group was significantly stronger than that of the T2 group, which was equivalent to the positive control group (C2), while the T3 group treated with "component I + component II" showed significant inhibition of melanin production and the inhibition amplitude was significantly stronger than that of the T1 group and the positive control group (C2). The results showed that both component I and component II in the fermentation composition of lactic acid bacteria fermented tea can inhibit melanin production, component I is more effective than component II, and the effect of component I and component II combined (ie, T3) is significantly better than component I or component II alone (ie, T1 and T2).

[0081] Calculate the Q value between component I and component II (the reciprocal of the melanin content represents the whitening effect):

[0082] E Ⅰ =(E Ⅰ -E 阴性对照组 ) / E Ⅰ ×100%=0.150;

[0083] E Ⅱ =(E Ⅱ -E 阴性对照组 ) / E Ⅱ ×100%=0.110;

[0084] E Ⅰ+Ⅱ =(E Ⅰ+Ⅱ -E 阴性对照组 ) / E Ⅰ+Ⅱ×100%=0.300;

[0085] Q Ⅰ+Ⅱ =E Ⅰ+Ⅱ / (E Ⅰ +E Ⅱ -E Ⅰ ·E Ⅱ )=1.232;

[0086] Q Ⅰ+Ⅱ >1.15, component I and component II show a synergistic effect, that is, the fermentation composition of lactic acid bacteria fermented tea composed of the two has the best effect of inhibiting melanin production, and the efficacy is higher than arbutin (positive control), which can play a role in skin whitening.

[0087] Example 2

[0088] 1. Preparation of fermentation composition

[0089] (1) Preparation of tea and water mixture: same as in Example 1.

[0090] (2) Lactic acid bacteria inoculation and fermentation: a density of 1×10 9 cfu / mL Lactobacillus plantarum CGMCC 1.60149 bacterial liquid is added to the tea and water mixture of step (1), the added amount is 3 parts of bacterial liquid in 100 parts of water, and 5 parts of MRS liquid culture medium are added at the same time, and fermented at 25-30°C for 72 hours to obtain a fermentation liquid.

[0091] (3) Exosome preparation: same as Example 1.

[0092] (4) Preparation of fermentation composition: same as Example 1.

[0093] 2. Exosome morphology

[0094] Transmission electron microscopy showed that the exosomes prepared in step (3) were spherical in shape, with a double-layer membrane structure and a particle size of 60 to 140 nm.

[0095] 3. Analysis of the efficacy of the fermented composition of lactic acid bacteria fermented tea in promoting collagen production

[0096] The specific experimental method is the same as that of Example 1. Component I was added to DMEM medium and diluted to a final concentration of 0.1% (w / v), and component II was added to DMEM medium and diluted to a final concentration of 1% (w / v). Component I and component II were added to DMEM medium to form "component I + component II" (final concentrations of 0.1% and 1%, w / v, respectively), and used to treat human skin fibroblasts cultured in 24-well plates for 24 hours, respectively, and the grouping treatment was as follows:

[0097] Control group (Control): treated with DMEM medium only, with 20 μL added to each well, and 6 wells were set up for replication.

[0098] Experimental group I (T1): Add component I, add 20 μL to each well, and set 6 wells for replication.

[0099] Experimental group II (T2): Add component II, add 20 μL to each well, and set 6 wells for replication.

[0100] Experimental group III (T3): Add "component I + component II", add 20 μL of liquid to each well, and set 6 wells for replication.

[0101] The results are shown in Table 3. Although the specific data are different from those in Example 1, the effects of component I, component II and "component I + component II" in promoting collagen production are similar to those in Example 1.

[0102] Table 3. Relative collagen content in cells

[0103]

[0104] Note: a, b, c, and d are significantly different from each other (p<0.01).

[0105] Calculate the Q value between component I and component II:

[0106] E Ⅰ =(E Ⅰ -E 阴性对照组 ) / E Ⅰ ×100%=0.180;

[0107] E Ⅱ =(E Ⅱ -E 阴性对照组 ) / E Ⅱ ×100%=0.123;

[0108] E Ⅰ+Ⅱ =(E Ⅰ+Ⅱ -E 阴性对照组 ) / E Ⅰ+Ⅱ ×100%=0.324;

[0109] Q Ⅰ+Ⅱ =E Ⅰ+Ⅱ / (E Ⅰ +E Ⅱ -E Ⅰ ·E Ⅱ )=1.153;

[0110] Q Ⅰ+Ⅱ >1.15, component I and component II show synergistic effect.

[0111] 4. Analysis of the efficacy of the fermented composition of lactic acid bacteria fermented tea in inhibiting melanin production

[0112] The specific experimental method is the same as that of Example 1. The dilution of component I, component II and "component I + component II" is the same as that of "3. Analysis of the efficacy of the fermented composition of lactic acid bacteria fermented tea in promoting collagen production" in this example. The grouping process is as follows:

[0113] Negative control group (C1): treated with DMEM medium only, 20 μL of liquid was added to each well, and 6 wells were set up for replication.

[0114] Positive control group (C2): Arbutin was added to a final concentration of 1 mM, and 6 wells were set up for replication.

[0115] Experimental group I (T1): Component I was added, with 20 μL added to each well, and 6 wells were repeated.

[0116] Experimental group II (T2): Add component II, add 20 μL to each well, and set 6 wells for replication.

[0117] Experimental group III (T3): Add "component I + component II", add 20 μL of liquid to each well, and set 6 wells for replication.

[0118] The results are shown in Table 4. Although the specific data are different from those in Example 1, the efficacy of component I, component II and "component I + component II" in inhibiting melanin production is similar to that in Example 1.

[0119] Table 4. Relative melanin content in cells

[0120]

[0121] Note: a, b, c, and d are significantly different from each other (p<0.01).

[0122] Calculate the Q value between component I and component II (the inverse of the melanin content represents the whitening effect):

[0123] E Ⅰ =(E Ⅰ -E 阴性对照组 ) / E Ⅰ ×100%=0.150;

[0124] E Ⅱ =(E Ⅱ -E 阴性对照组 ) / E Ⅱ ×100%=0.100;

[0125] E Ⅰ+Ⅱ =(E Ⅰ+Ⅱ -E 阴性对照组 ) / EⅠ+Ⅱ ×100%=0.290;

[0126] Q Ⅰ+Ⅱ =E Ⅰ+Ⅱ / (E Ⅰ +E Ⅱ -E Ⅰ ·E Ⅱ )=1.184;

[0127] Q Ⅰ+Ⅱ >1.15, component I and component II show synergistic effect.

[0128] Example 3

[0129] 1. Preparation of fermentation composition

[0130] (1) Preparation of tea and water mixture: same as in Example 1.

[0131] (2) Lactic acid bacteria inoculation and fermentation: a density of 1×10 9 cfu / mL Lactobacillus plantarum CGMCC 1.60149 bacterial liquid is added to the tea and water mixture of step (1), the added amount is 2 parts of bacterial liquid in 100 parts of water, and 5 parts of MRS liquid culture medium are added at the same time, and fermented at 25-30°C for 60 hours to obtain a fermentation liquid.

[0132] (3) Exosome preparation: same as Example 1.

[0133] (4) Preparation of fermentation composition: same as Example 1.

[0134] 2. Exosome morphology

[0135] Transmission electron microscopy showed that the exosomes prepared in step (3) were spherical in shape, with a double-layer membrane structure and a particle size of 60 to 140 nm.

[0136] 3. Analysis of the efficacy of the fermented composition of lactic acid bacteria fermented tea in promoting collagen production

[0137] The specific experimental method is the same as that of Example 1. Component I was added to DMEM medium and diluted to a final concentration of 0.1% (w / v), and component II was added to DMEM medium and diluted to a final concentration of 1.5% (w / v). Component I and component II were added to DMEM medium to form "component I + component II" (final concentrations of 0.1% and 1.5%, w / v, respectively), and used to treat human skin fibroblasts that had been cultured in a 24-well plate for 24 hours. The grouping treatments were as follows:

[0138] Control group (Control): treated with DMEM medium only, with 20 μL added to each well, and 6 wells were set up for replication.

[0139] Experimental group I (T1): Add component I, add 20 μL to each well, and set 6 wells for replication.

[0140] Experimental group II (T2): Add component II, add 20 μL to each well, and set 6 wells for replication.

[0141] Experimental group III (T3): Add "component I + component II", add 20 μL of liquid to each well, and set 6 wells for replication.

[0142] The results are shown in Table 5. Although the specific data are different, the effects of component I, component II and "component I + component II" in promoting collagen synthesis are similar to those of Example 1 and Example 2.

[0143] Table 5. Relative collagen content in cells

[0144]

[0145] Note: a, b, c, and d are significantly different from each other (p<0.01).

[0146] Calculate the Q value between component I and component II:

[0147] E Ⅰ =(E Ⅰ -E 阴性对照组 ) / E Ⅰ ×100%=0.194;

[0148] E Ⅱ =(E Ⅱ -E 阴性对照组 ) / E Ⅱ ×100%=0.138;

[0149] E Ⅰ+Ⅱ =(E Ⅰ+Ⅱ -E 阴性对照组 ) / E Ⅰ+Ⅱ ×100%=0.363;

[0150] Q Ⅰ+Ⅱ =E Ⅰ+Ⅱ / (E Ⅰ +E Ⅱ -E Ⅰ ·E Ⅱ )=1.190;

[0151] Q Ⅰ+Ⅱ >1.15, component I and component II show synergistic effect.

[0152] 4. Analysis of the efficacy of the fermented composition of lactic acid bacteria fermented tea in inhibiting melanin production

[0153] The specific experimental method is the same as that of Example 1. The dilution of component I, component II and "component I + component II" is the same as that of "3. Analysis of the efficacy of the fermented composition of lactic acid bacteria fermented tea in promoting collagen production" in this example. The grouping process is as follows:

[0154] Negative control group (C1): treated with DMEM medium only, 20 μL of liquid was added to each well, and 6 wells were set up for replication.

[0155] Positive control group (C2): Arbutin was added to a final concentration of 1 mM, and 6 wells were set up for replication.

[0156] Experimental group I (T1): Add component I, add 20 μL to each well, and set 6 wells for replication.

[0157] Experimental group II (T2): Add component II, add 20 μL to each well, and set 6 wells for replication.

[0158] Experimental group III (T3): Add "component I + component II", add 20 μL of liquid to each well, and set 6 wells for replication.

[0159] The results are shown in Table 6. Although the specific data are different, the efficacy of component I, component II and "component I + component II" in inhibiting melanin production is similar to that of Example 1 and Example 2.

[0160] Table 6. Relative melanin content in cells

[0161]

[0162] Note: a, b, c, and d are significantly different from each other (p<0.01).

[0163] Calculate the Q value between component I and component II (the reciprocal of the melanin content represents the whitening effect):

[0164] E Ⅰ =(E Ⅰ -E 阴性对照组 ) / E Ⅰ ×100%=0.170,

[0165] E Ⅱ =(E Ⅱ -E 阴性对照组 ) / E Ⅱ ×100%=0.120,

[0166] E Ⅰ+Ⅱ =(E Ⅰ+Ⅱ -E 阴性对照组 ) / E Ⅰ+Ⅱ ×100%=0.320,

[0167] QⅠ+Ⅱ =E Ⅰ+Ⅱ / (E Ⅰ +E Ⅱ -E Ⅰ ·E Ⅱ )=1.185,

[0168] Q Ⅰ+Ⅱ >1.15, component I and component II show synergistic effect.

[0169] Summarize:

[0170] Both component I (exosomes) and component II in the fermentation composition of lactic acid bacteria fermented tea can promote collagen synthesis and inhibit melanin production. The effect of component I (exosomes) is stronger than that of component II, and component I (exosomes) and component II have a synergistic effect, that is, the fermentation composition of lactic acid bacteria fermented tea composed of the two has the best effect of promoting collagen synthesis and inhibiting melanin production, and can play a role in skin care and beauty.

Claims

1. A composition based on lactic acid bacteria fermented tea, characterized in that: The lactic acid bacteria fermented tea is a fermentation broth obtained by fermenting and culturing Lactobacillus plantarum ZJU2303-RH17 with a preservation number of CGMCC 1.60149 and fresh tea leaves as a fermentation substrate; exosomes are separated from the fermentation broth as component I, and after removing the exosomes from the fermentation broth, component II is obtained by vacuum drying and ultrafine grinding; the composition comprises component I and component II, and the mass ratio of the two is 1:10-20.

2. The composition based on lactic acid bacteria fermented tea according to claim 1, characterized in that Fresh tea leaves are crushed and diluted with water for 8-12 times, and then MRS liquid culture medium is added to prepare a fermentation medium, wherein the mass ratio of MRS liquid culture medium to tea leaves is 1:

2.

3. The composition based on lactic acid bacteria fermented tea according to claim 1, characterized in that The preparation method of the lactic acid bacteria fermented tea comprises the following steps: (1) Preparation of tea and water mixture: crush fresh Anji white tea leaves, add water and mix, and add 10 parts of tea leaves to every 100 parts of water by weight; (2) Lactic acid bacteria inoculation and fermentation: Lactobacillus plantarum ZJU2303-RH17 was added to the tea and water mixture in step (1) at an inoculation density of 1 to 3 × 10 7 cfu / mL, by weight, add 5 parts of MRS liquid culture medium to every 100 parts of water, ferment at 25-30°C for 48-72 hours to obtain fermentation liquid.

4. The composition based on lactic acid bacteria fermented tea according to claim 1 or 3, characterized in that The preparation method of the exosomes comprises: centrifuging the fermentation broth at 5000g for 10 min at 4°C, filtering the supernatant obtained after separating the bacteria with a 0.45 μm filter membrane, and then centrifuging at 15000g for 20 min at 4°C, and the obtained precipitate is the exosomes.

5. The composition based on lactic acid bacteria fermented tea according to claim 1, characterized in that The ultrafine grinding conditions are: using a low-temperature ultrafine wall-breaking grinder, grinding at -10°C for 10 minutes and then storing at 4°C.

6. The composition based on lactic acid bacteria fermented tea according to claim 1, characterized in that The mass ratio of component I to component II in the composition is 1:

15.

7. Use of the composition based on lactic acid bacteria fermented tea according to any one of claims 1 to 5 in the preparation of cosmetics for skin care and beauty.

8. The use according to claim 7, characterized in that The skin care and beauty treatment includes wrinkle removal and skin whitening.

9. A cosmetic having the effect of promoting collagen synthesis and / or inhibiting melanin production, characterized in that: The invention comprises an effective dose of the composition based on lactic acid bacteria fermented tea as described in any one of claims 1 to 5 and a cosmetic auxiliary material matrix.

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