Application of exosome from co-fermentation of lactic acid bacteria and yeast in preparation of skin care and beauty treatment composition

Abstract

The invention discloses an application of exosome from co-fermentation of lactic acid bacteria and yeast in preparation of a skin care and beauty treatment composition, and belongs to the technical field of cosmetics. Fresh tea leaves serve as a fermentation substrate, and the exosome is prepared through co-fermentation of lactic acid bacteria and yeast. Compared with exosomes prepared through single strain fermentation, the exosome prepared through co-fermentation has the advantages that the effects of promoting skin cell collagen generation, inhibiting skin cell melanin generation and the like are remarkably improved, a synergistic effect is displayed, the skin care and beauty treatment effects can be better exerted, and the exosome can be applied to preparation of skin care and beauty treatment products.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to an application of exosomes derived from co-fermentation of lactic acid bacteria and yeast in the preparation of a skin care beauty composition. Background Art

[0002] Exosomes are spherical lipid bilayer vesicles secreted by cells. They are a subpopulation of vesicles involved in intercellular communication. The particle size is roughly 30 to 150 nm. They contain bioactive components such as lipids, proteins and nucleic acids used for intercellular communication. They have significant effects in scar improvement, pigmentation correction, skin rejuvenation and hair loss recovery (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.).

[0003] Animal, plant and microbial cells can all produce exosomes. Considering safety and convenience, yeast and lactic acid bacteria have become ideal sources for preparing exosomes.

[0004] For example, patent document CN116042422A discloses a yeast (selected from brewer's yeast, Pichia pastoris, Candida) derived exosomes and a preparation method and application thereof, and the obtained exosomes have good anti-inflammatory and antioxidant effects.

[0005] Patent document CN116867479A discloses a skin-improving cosmetic composition containing exosomes derived from black yeast, which as an effective ingredient has the effects of improving skin elasticity, texture, skin tone and brightness, reducing skin wrinkles and whitening.

[0006] Patent document CN113876643A discloses that exosomes derived from Saccharomyces cerevisiae RD-03RI SC and their solubilized substances are used as effective ingredients, and have the functions of anti-aging, promoting skin regeneration, improving skin elasticity, and reducing skin wrinkles.

[0007] Patent document CN111225659A discloses an anti-aging composition with exosomes derived from lactic acid bacteria (selected from Bifidobacterium, Lactobacillus, Streptococcus, Leuconostoc, Pediococcus and Lactococcus) as an effective ingredient, which can promote collagen production by inhibiting MMP-1 gene expression, thereby reducing skin wrinkles, increasing skin elasticity, and preventing skin damage caused by ultraviolet rays.

[0008] The existing technology mainly obtains exosomes by culturing lactic acid bacteria or yeast alone and applies them to skin care and beauty. There are currently no reports on obtaining exosomes by co-fermenting with bacteria and fungi using tea as a fermentation substrate. Summary of the invention

[0009] The purpose of the present invention is to prepare exosomes with skin care and beauty effects by co-fermentation of lactic acid bacteria and yeast, and apply them to the development of cosmetics.

[0010] To achieve the above object, the present invention adopts the following technical solution:

[0011] The present invention provides an application of exosomes derived from co-fermentation of lactic acid bacteria and yeast in preparing a skin care and beauty composition. The exosomes are separated from a fermentation broth obtained by co-fermentation of lactic acid bacteria and yeast using tea leaves as a fermentation substrate.

[0012] Studies have shown that the exosomes prepared by co-fermentation with tea leaves as the fermentation substrate and lactic acid bacteria and yeast as the fermentation strains have the properties of both strains. Compared with the exosomes prepared by fermentation of a single strain, the exosomes prepared by co-fermentation of the present invention significantly improve the effects of promoting collagen production in skin cells and inhibiting melanin production in skin cells, showing a synergistic effect. Therefore, the present invention applies the exosomes prepared by co-fermentation to the preparation of skin care and beauty products.

[0013] The present invention uses fresh tea leaves as a co-fermentation substrate, which has a rich source of active substances. Furthermore, the co-fermentation culture medium is prepared by crushing fresh tea leaves, diluting them with water by 8-12 times, and then adding brown sugar, wherein the mass ratio of brown sugar to tea leaves is 1:5. Brown sugar is rich in sucrose for lactic acid bacteria fermentation, and because it is not crystallized, it retains a large amount of vitamins and mineral elements from sugar cane, which is beneficial to the growth of lactic acid bacteria.

[0014] Furthermore, the tea leaves are fresh Anji white tea leaves. The fresh Anji white tea leaves can be used but not limited to fresh leaves in summer and autumn. Anji white tea is a local tea variety unique to Anji, planted in mountainous areas. Due to the large temperature difference between day and night in the mountainous areas, pests and diseases are not easy to breed, and pesticides do not need to be applied. It is safe to use it as a raw material. Compared with the spring Mingqian tea and the rain-pre-tea, the active substances such as tea polyphenols in the summer and autumn tea leaves are more abundant due to the long accumulation time.

[0015] The present invention adopts Lactobacillus rhamnosus and Saccharomyces cerevisiae as co-fermentation strains. Both Lactobacillus rhamnosus and Saccharomyces cerevisiae are food-grade microorganisms with wide distribution and application, good fermentation characteristics and sufficient safety, and the two have a mutually beneficial symbiotic relationship in terms of physiological and biochemical characteristics.

[0016] Furthermore, the lactic acid bacteria is Lactobacillus rhamnosus ZJU2303-RH11 with a preservation number of CGMCC 1.60148.

[0017] Furthermore, the yeast is Saccharomyces cerevisiae ZJU2014-RH05 with a preservation number of CGMCC 2.6673.

[0018] The Lactobacillus rhamnosus ZJU2303-RH11 and Saccharomyces cerevisiae ZJU2014-RH05 used in the present invention are both biological materials available to the public and are publicly deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee (CGMCC).

[0019] Furthermore, the method for preparing exosomes comprises the following steps:

[0020] (1) preparing fermentation medium: crush fresh Anji white tea leaves and mix with water, the ratio of tea leaves to water is 10 parts of tea leaves to 100 parts of water, and add 2 parts of brown sugar;

[0021] (2) Lactobacillus rhamnosus ZJU2303-RH11 with a preservation number of CGMCC 1.60148 and Saccharomyces cerevisiae ZJU2014-RH05 with a preservation number of CGMCC 2.6673 were activated and inoculated at a density of 0.5 to 1.5 × 10 7 cfu / mL was inoculated into the fermentation medium, and fermented at 25-30°C for 48-72 hours to obtain a fermentation liquid;

[0022] (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 with a 0.45 μm filter membrane and then centrifuged at 15000 g for 20 min at 4°C. The resulting precipitate was the exosome.

[0023] The exosomes prepared by the above method are spherical, have a double-layer membrane structure, and a particle size of 30 to 130 nm.

[0024] The skin care beauty composition provided by the present invention uses exosomes derived from co-fermentation of lactic acid bacteria and yeast as the main active substance, and any preparation or carrier medium that does not interfere with the biological activity of the active substance and has no toxic side effects on the host or the subject is added as an auxiliary material.

[0025] Furthermore, the skin care and beauty treatment includes at least one of reducing skin wrinkles, improving skin elasticity, and whitening.

[0026] Specifically, the exosomes prepared by co-fermentation in the present invention can promote the production of collagen in skin cells, reduce skin wrinkles and improve skin elasticity.

[0027] The exosomes can inhibit the production of melanin in skin cells and play a role in skin whitening.

[0028] Furthermore, the composition is a composition for external use on the skin.

[0029] Furthermore, the composition is a cosmetic composition.

[0030] The present invention has the following beneficial effects:

[0031] The present invention uses fresh tea leaves as a fermentation substrate and utilizes lactic acid bacteria and yeast to co-ferment and prepare exosomes. The fermentation raw materials are highly safe, contain rich active substances, and exhibit a synergistic effect. Compared with exosomes prepared by fermentation of a single strain, they can better exert skin care and beauty effects. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] In the following examples, the lactic acid bacteria used refer to Lactobacillus rhamnosus ZJU2303-RH11, which was deposited in the General Microbiology Center (CGMCC) of the China Microorganism 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 CGMCC1.60148.

[0035] Yeast refers to brewer's yeast (Saccharomyces cerevisiae ZJU2014-RH05), which was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on December 23, 2021, and was identified as alive. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and the deposit number is CGMCC 2.6673.

[0036] Both originated from a kimchi-making experiment conducted by the College of Biosystems Engineering and Food Science of Zhejiang University. They were obtained by streaking single colony culture, and rRNA sequencing results confirmed that they belonged to Lactobacillus rhamnosus and Saccharomyces cerevisiae in terms of classification.

[0037] Anji white tea fresh leaves are summer and autumn fresh leaves, collected and provided by Zhejiang Anji Dashanwu White Tea Co., Ltd. Anji white tea summer and autumn fresh leaves have the advantages of large temperature difference between day and night in mountainous areas, pests and diseases are not easy to breed, no pesticides are used, and rich in active substances such as tea polyphenols.

[0038] MRS liquid culture medium, which can be purchased commercially or prepared by yourself, includes the following components per liter: peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g, glucose 20g, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, manganese sulfate 0.2g, Tween 80 1.5mL, dilute to 1L, sterilize at 121℃ for 15min;

[0039] YPD liquid culture medium, commercially available or self-prepared, includes the following components per 1 L: 20 g peptone, 10 g yeast extract, 20 g glucose, fixed to 1 L, sterilized at 121°C for 15 min.

[0040] Brown sugar refers to any commercially available product that meets the standard “T / CNFIA 110-2018 Brown Sugar”.

[0041] The human skin fibroblasts (HFF-1) used in the experiment were purchased from the Experimental Animal Center of Zhejiang University School of Medicine.

[0042] The mouse B16 melanoma cells used in the experiment were purchased from the Experimental Animal Center of Zhejiang University School of Medicine.

[0043] The experimental methods involved in the following examples are as follows:

[0044] 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.

[0045] 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.).

[0046] 3. Determination of melanin content in cells: NaOH dissolution method was used. The specific steps were referred to the literature (Wang Zhaowei et al. Tripeptide D-Trp-Arg-Leu-NH 2 Mechanism of inhibiting melanin synthesis in B16 melanoma cells. Flavors and Fragrances Cosmetics, 2023, (1): 82-86.).

[0047] 4. Synergistic effect evaluation: The synergistic effect evaluation uses the Jin (Zhengjun) formula combined with the index Q value. First, the efficacy value E is calculated, E = (E 试验 -E 空白对照 ) / E 试验 × 100%, and then the Jin (Zhengjun) formula is used to calculate the combination index Q value, Q = EⅠ+Ⅱ / (E Ⅰ +E Ⅱ -E Ⅰ ·E Ⅱ ).

[0048] In the above formula, E Ⅰ+Ⅱ E is the effect of two factors with expected synergistic effects when used together. Ⅰ 、E Ⅱ The Q value is the effect of two factors that are expected to have synergistic effects when applied separately. The meaning of Q value: 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.

[0049] 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.).

[0050] Example 1

[0051] 1. Preparation of co-fermented exosomes

[0052] (1) Obtaining bacterial suspension: Inoculate the slant strain into liquid culture medium, inoculate Lactobacillus rhamnosus CGMCC 1.60148 into MRS liquid culture medium, and inoculate Saccharomyces cerevisiae CGMCC 2.6673 into YPD liquid culture medium, and culture them at 33°C to 39°C with shaking for 20 to 36 h until the bacterial suspension density reaches 10 8 ~10 9 cfu / mL to obtain bacterial suspension.

[0053] (2) Preparation of fermentation medium: Crush the fresh leaves of Anji white tea in summer and autumn, add water and mix, the ratio of tea leaves to water is 10 parts of tea leaves to 100 parts of water, and add 2 parts of brown sugar.

[0054] (3) Fermentation: The density of the two bacterial suspensions obtained above was adjusted to 5×10 8 cfu / mL, added into the fermentation medium at a 1% (v / v) inoculation amount, and fermented at 25-30°C for 48 hours to obtain a fermentation broth.

[0055] (4) 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 with a 0.45 μm filter membrane and then centrifuged at 15000 g for 20 min at 4°C. The resulting precipitate was the exosomes produced by co-fermentation of Saccharomyces cerevisiae CGMCC 2.6673 and Lactobacillus rhamnosus CGMCC 1.60148.

[0056] 2. Exosome morphology analysis

[0057] Transmission electron microscopy showed that the exosomes produced after co-fermentation were spherical in shape, with a double-layer membrane structure and a particle size of 30 to 130 nm.

[0058] Example 2

[0059] 1. Preparation of co-fermented exosomes

[0060] (1) Obtaining bacterial suspension: Same as Example 1.

[0061] (2) Preparation of fermentation medium: same as in Example 1.

[0062] (3) Fermentation: The density of the two bacterial suspensions obtained above was adjusted to 5×10 8 cfu / mL, added into the fermentation medium at an inoculum rate of 3% (v / v), fermented at 25-30°C for 72 hours to obtain a fermentation broth.

[0063] (4) Exosome preparation: same as Example 1.

[0064] 2. Exosome morphology analysis

[0065] Transmission electron microscopy showed that the exosomes produced after co-fermentation were spherical in shape, with a double-layer membrane structure and a particle size of 30 to 130 nm.

[0066] Example 3

[0067] 1. Preparation of co-fermented exosomes

[0068] (1) Obtaining bacterial suspension: Same as Example 1.

[0069] (2) Preparation of fermentation medium: same as in Example 1.

[0070] (3) Fermentation: The density of the two bacterial suspensions obtained above was adjusted to 5×10 8 cfu / mL, added into the fermentation medium at an inoculum rate of 2% (v / v), fermented at 25-30°C for 60 hours to obtain a fermentation broth.

[0071] (4) Exosome preparation: same as Example 1.

[0072] 2. Exosome morphology analysis

[0073] Transmission electron microscopy showed that the exosomes produced after co-fermentation were spherical in shape, with a double-layer membrane structure and a particle size of 30 to 130 nm.

[0074] Comparative Example 1

[0075] 1. Lactobacillus rhamnosus is cultured alone to obtain exosomes

[0076] (1) Obtain bacterial suspension: inoculate the Lactobacillus rhamnosus CGMCC 1.60148 slant strain into MRS liquid culture medium and culture at 33°C to 39°C with shaking for 20 to 36 hours until the bacterial suspension density reaches 10 8 ~10 9 cfu / mL to obtain bacterial suspension.

[0077] (2) Preparation of fermentation medium: same as in Example 1.

[0078] (3) Fermentation: The density of the bacterial suspension obtained above was adjusted to 5×10 8 cfu / mL, added into the fermentation medium at an inoculum rate of 2% (v / v), fermented at 25-30°C for 60 hours to obtain a fermentation broth.

[0079] (4) Exosome preparation: same as Example 1.

[0080] 2. Exosome morphology analysis

[0081] Transmission electron microscopy showed that the exosomes produced after co-fermentation were spherical in shape, with a double-layer membrane structure and a particle size of 30 to 100 nm.

[0082] Comparative Example 2

[0083] 1. Cultivate Saccharomyces cerevisiae alone to obtain exosomes

[0084] (1) Obtain bacterial suspension: Inoculate the slant strain of Saccharomyces cerevisiae CGMCC 2.6673 into YPD liquid medium and culture at 33°C to 39°C with shaking for 20 to 36 h until the bacterial suspension density reaches 10 8 ~10 9 cfu / mL to obtain bacterial suspension.

[0085] (2) Preparation of fermentation medium: same as in Example 1.

[0086] (3) Fermentation: The density of the bacterial suspension obtained above was adjusted to 5×10 8 cfu / mL, added into the fermentation medium at an inoculum rate of 2% (v / v), fermented at 25-30°C for 60 hours to obtain a fermentation broth.

[0087] (4) Exosome preparation: same as Example 1.

[0088] 2. Exosome morphology analysis

[0089] Transmission electron microscopy showed that the exosomes produced after co-fermentation were spherical in shape, with a double-layer membrane structure and a particle size of 40 to 130 nm.

[0090] Comparative Example 3

[0091] 1. Preparation of exosomes obtained by culturing Lactobacillus rhamnosus alone: ​​the same as comparative example 1.

[0092] 2. Preparation of exosomes obtained by culturing Saccharomyces cerevisiae alone: ​​the same as in Comparative Example 2.

[0093] 3. The exosomes obtained in step "1" and step "2" of this comparative example were mixed at a ratio of 1:1 (v / v). Test Example 1: Effect of Promoting Collagen Production

[0094] The exosomes obtained in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3 were named LS-1, LS-2, LS-3 and L, S, L+S, respectively, and DMEM medium was used as a blank control and named C.

[0095] 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 96-well plates at a concentration of 80 μL per well and cultured for 24 hours.

[0096] LS-1, LS-2, LS-3 and L, S, L+S were diluted in DMEM medium to a final concentration of 0.2% (w / v) and used to treat human skin fibroblasts that had been cultured in 96-well plates for 24 hours. 20 μL was added to each well, and 20 μL DMEM medium was added to the control group, with 6 replicates each.

[0097] 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 blank control (C): absorbance of different groups / absorbance of group C × 100%. The results are shown in Table 1.

[0098] Table 1. Relative collagen content in cells

[0099]

[0100] Note: a, b, and c are significantly different from each other (p<0.01).

[0101] As can be seen from the above table, compared with C, LS-1, LS-2, LS-3 and L, S, L+S all showed a significant increase in collagen synthesis, there was no significant difference between LS-1, LS-2, LS-3, there was no significant difference between L, S, L+S, and there were significant differences between LS-1, LS-2, LS-3 and L, S, L+S, the former had a significant effect on promoting collagen synthesis than the latter. The results showed that in the present invention, the exosomes prepared by fermentation of Lactobacillus rhamnosus CGMCC 1.60148 and Saccharomyces cerevisiae CGMCC 2.6673 can promote collagen synthesis. The Q value was calculated to evaluate whether there is a synergistic effect:

[0102] E L =(E L -E 空白对照 ) / E Ⅰ ×100%=0.145;

[0103] E S =(E S -E 空白对照 ) / E Ⅱ ×100%=0.123;

[0104] E L+S =(E L+S -E 空白对照 ) / E L+S ×100%=0.219;

[0105] E LS-1 =(E LS-1 -E 空白对照 ) / E LS-1 ×100%=0.306;

[0106] E LS-2 =(E LS-2 -E 空白对照 ) / E LS-2 ×100%=0.301;

[0107] E LS-3 =(E LS-3 -E 空白对照 ) / E LS-3 ×100%=0.315;

[0108] Q L+S =E L+S / (E L +E S -E L ·E S )=0.875;

[0109] Q LS-1 =E LS-1 / (EL +E S -E L ·E S )=1.224;

[0110] Q LS-2 =E LS-2 / (E L +E S -E L ·E S )=1.204;

[0111] Q LS-3 =E LS-3 / (E L +E S -E L ·E S )=1.260;

[0112] 0.85≤Q L+S ≤1.15, pure additive effect, Q LS-1 , Q LS-2 and Q LS-3 All of them were >1.15, indicating that when Lactobacillus rhamnosus CGMCC 1.60148 and Saccharomyces cerevisiae CGMCC 2.6673 were co-fermented, the prepared exosomes showed a synergistic effect, had the best effect in promoting collagen synthesis, and could play a role in reducing skin wrinkles and improving skin elasticity.

[0113] Test Example 2: Efficacy of inhibiting melanin production

[0114] The exosomes obtained in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3 were named LS-1, LS-2, LS-3 and L, S, L+S, respectively, and DMEM medium was used as a blank control and named C.

[0115] 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.

[0116] Then, melanin synthesis stimulator αMSH (α melanocyte stimulating hormone, α melanocyte stimulating hormone) (0.10%, v / v) was added to each group, and LS-1, LS-2, LS-3 and L, S, L+S were diluted in DMEM medium to a final concentration of 0.2% (w / v) for treating mouse melanoma cells that had been cultured in 96-well plates for 24 hours. 20 μL was added to each well, and 20 μL DMEM medium was added to the control group, with 6 replicates each.

[0117] 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 content was calculated: absorbance of different groups / absorbance of group C×100%. The results are shown in Table 2.

[0118] Table 2. Relative melanin content in cells

[0119]

[0120] Note: a, b, and c are significantly different from each other (p<0.01).

[0121] As can be seen from the above table, compared with C, LS-1, LS-2, LS-3 and L, S, L+S all showed significant inhibition of melanin production, there was no significant difference between LS-1, LS-2, LS-3, there was no significant difference between L, S, L+S, and there was a significant difference between LS-1, LS-2, LS-3 and L, S, L+S, the former had a significant inhibitory effect on melanin production than the latter. The results showed that in the present invention, the exosomes prepared by fermentation of Lactobacillus rhamnosus CGMCC 1.60148 and Saccharomyces cerevisiae CGMCC 2.6673 can inhibit melanin production, and the Q value is calculated to evaluate whether there is a synergistic effect (the reciprocal of the melanin content represents the whitening effect):

[0122] E L =(E L -E 空白对照 ) / E Ⅰ ×100%=0.130;

[0123] E S =(E S -E 空白对照 ) / E Ⅱ ×100%=0.120;

[0124] E L+S =(E L+S -E 空白对照 ) / E L+S ×100%=0.250;

[0125] E LS-1 =(E LS-1 -E 空白对照 ) / E LS-1 ×100%=0.320;

[0126] E LS-2 =(E LS-2 -E 空白对照 ) / E LS-2×100%=0.330;

[0127] E LS-3 =(E LS-3 -E 空白对照 ) / E LS-3 ×100%=0.310;

[0128] Q L+S =E L+S / (E L +E S -E L ·E S )=1.067;

[0129] Q LS-1 =E LS-1 / (E L +E S -E L ·E S )=1.365;

[0130] Q LS-2 =E LS-2 / (E L +E S -E L ·E S )=1.408;

[0131] Q LS-3 =E LS-3 / (E L +E S -E L ·E S )=1.323;

[0132] 0.85≤Q L+S ≤1.15, pure additive effect, Q LS-1 , Q LS-2 and Q LS-3 All of them were >1.15, indicating that when Lactobacillus rhamnosus CGMCC 1.60148 and Saccharomyces cerevisiae CGMCC 2.6673 were co-fermented, the prepared exosomes showed a synergistic effect, had the best effect in promoting collagen synthesis, and could play a role in reducing skin wrinkles and improving skin elasticity.

[0133] Summarize:

[0134] The exosomes prepared by fermentation of Lactobacillus rhamnosus CGMCC 1.60148 and Saccharomyces cerevisiae CGMCC 2.6673 can promote collagen synthesis and inhibit melanin production. There is a synergistic effect when the two are co-fermented. The prepared exosomes have the best effect of promoting collagen synthesis and inhibiting melanin production, and can play a role in skin care and beauty.

Claims

1. Application of exosomes derived from co-fermentation of lactic acid bacteria and yeast in the preparation of a skin care and beauty composition, characterized in that: The exosomes are separated from a fermentation broth obtained by co-fermentation of lactic acid bacteria and yeast using tea leaves as a fermentation substrate.

2. The use according to claim 1, characterized in that The co-fermentation culture medium is prepared by grinding fresh tea leaves, diluting the mixture with water by 8-12 times, and then adding brown sugar, wherein the mass ratio of brown sugar to tea leaves is 1:

5.

3. The use according to claim 2, characterized in that The tea leaves used are fresh leaves of Anji white tea from summer and autumn.

4. The use according to claim 1, characterized in that The lactic acid bacteria is Lactobacillus rhamnosus ZJU2303-RH11 with a preservation number of CGMCC 1.60148.

5. The use according to claim 1, characterized in that The yeast used is Saccharomyces cerevisiae ZJU2014-RH05 with a preservation number of CGMCC 2.6673.

6. The use according to claim 1, characterized in that The method for preparing exosomes comprises the following steps: (1) preparing fermentation medium: crush fresh Anji white tea leaves and mix with water, the ratio of tea leaves to water is 10 parts of tea leaves to 100 parts of water, and add 2 parts of brown sugar; (2) Lactobacillus rhamnosus ZJU2303-RH11 with a preservation number of CGMCC 1.60148 and Saccharomyces cerevisiae ZJU2014-RH05 with a preservation number of CGMCC 2.6673 were activated and inoculated at a density of 0.5 to 1.5 × 10 7 cfu / mL was inoculated into the fermentation medium, and fermented at 25-30°C for 48-72 hours to obtain a fermentation liquid; (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 with a 0.45 μm filter membrane and then centrifuged at 15000 g for 20 min at 4°C. The resulting precipitate was the exosome.

7. The use according to claim 1, characterized in that The particle size of the exosomes is 30 to 130 nm.

8. The use according to claim 1, characterized in that The skin care and beauty treatment includes at least one of reducing skin wrinkles, improving skin elasticity, and whitening.

9. The use according to claim 1, characterized in that The composition is a composition for external use on skin.

10. The use according to claim 1, characterized in that The composition is a cosmetic composition.

Images