Method for preparing a cosmetic tea fermentation product having anti-aging efficacy

Abstract

The application discloses a preparation method of a tea fermentation product with anti-aging efficacy for cosmetics, and obtains a cosmetic yeast strain with cellulase, xylanase and lignin peroxidase production capacity through strain screening. The release of antioxidant and anti-aging substances in tea leaves is improved through fermentation and extraction of tea leaves by the preferred yeast strain, and the anti-aging performance of the fermentation product is further improved through the fermentation of gluconacetobacter kombuchia. Compared with conventional tea extracts, the tea fermentation product has more excellent performance in terms of antioxidant, elastase activity inhibition, collagenase activity inhibition, AGEs yield inhibition, human fibroblast protection and repair, and is suitable for use in skin care products such as essence, emulsion and cream with anti-aging efficacy.

Description

Technical Field

[0001] This invention belongs to the fields of plant fermentation and cosmetics, specifically relating to a method for preparing a tea fermentation product for cosmetics with anti-aging effects. Background Technology

[0002] Tea extract is a functional cosmetic ingredient rich in bioactive components, including tea polyphenols, theanine, alkaloids, tea polysaccharides, tea saponins, vitamins, and trace elements. Tea extract possesses strong antioxidant and free radical scavenging capabilities, exhibits significant inhibitory effects on matrix metalloproteinases and collagenases, and can also inhibit caspase, a key molecule in apoptosis, thereby prolonging cell lifespan. This indicates that tea extract is an ideal anti-aging cosmetic ingredient suitable for anti-aging cosmetic products.

[0003] However, the current mainstream extraction method for tea extracts involves heating and reflux with water or an alcohol-water mixture. This process is energy-intensive, and prolonged high temperatures can partially destroy the active ingredients. Furthermore, due to the robust cell wall structure protecting plant cells, conventional extraction methods struggle to fully release the active substances within the cells. Plant cell walls are primarily composed of cellulose, hemicellulose, and lignin. Microorganisms possess powerful enzyme systems that can degrade cellulose, hemicellulose, and lignin in plant cell walls, thereby breaking down the cell walls under gentle conditions and allowing for the complete release of active ingredients.

[0004] However, the microorganisms commonly used for the degradation of substances such as cellulose, hemicellulose, and lignin are mainly filamentous fungi—molds, which are rarely permitted in cosmetic raw material production. Therefore, screening for microorganisms with suitable enzyme systems and those permitted for use in cosmetic raw materials is a key factor in the gentle and thorough extraction of active ingredients from tea.

[0005] Yeast is one of the permitted microbial species in cosmetic raw materials and is also a microorganism closely related to people's production and daily life. Historically, it has been used in the food manufacturing industry, such as bread and beer, and has extremely high safety. Tan Jinlian et al. conducted a detailed analysis and screening of the extracellular enzyme activity of yeast from Fuxian Lake and Xingyun Lake in Yunnan Province. The results showed that lake yeast has the ability to synthesize and secrete multiple enzyme systems, including cellulase, xylanase, and lignin peroxidase. However, lake yeast, as a wild yeast, is not a common edible yeast, and its safety cannot be guaranteed. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a cosmetic tea fermentation product with anti-aging effects.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing a cosmetic tea fermentation product with anti-aging effects, comprising,

[0010] The tea leaves were crushed and sieved, mixed with glucose and yeast peptone, and distilled water was added. The mixture was stirred evenly, the pH was adjusted to 5.0, and sterilized to obtain the culture medium.

[0011] Seed cultures of yeast RY5, yeast RY16, and yeast RY17 were cultured to OD. 600nm ≥5, add to the above culture medium at a volume fraction of 1-5%, and incubate at 30℃ and 200r / min for 12-24h;

[0012] Seed culture of *Acetobacter xylinum* was cultured to OD0.05 600nm ≥4, add to the culture medium of the above-mentioned yeast cultured at a volume fraction of 1-6%, and culture at 30℃ and 50r / min for 8-12h;

[0013] The fermentation product is sterilized and then filtered through a water-based microporous membrane to obtain a cosmetic tea fermentation product with anti-aging effects.

[0014] The yeast strain RY5 is deposited at the China Center for Type Culture Collection (CCTCC) on July 9, 2021, with accession number CCTCC NO: M 2021849.

[0015] The yeast strain RY16 is deposited at the China General Microbiological Culture Collection Center on December 7, 2020, with accession number CGMCC NO.21316.

[0016] The yeast strain RY17 is deposited at the China Center for Type Culture Collection (CCTCC), classified and named Hanseniasporauvarum Q-8, with a deposit date of November 29, 2023, accession number CCTCC NO: M20232388, and deposit address at Wuhan University, Wuhan, China.

[0017] As a preferred embodiment of the preparation method described in this invention, the *Acetobacter xylinum* has the resource number ATCC 23767.

[0018] As a preferred embodiment of the preparation method described in this invention, the tea leaves are pulverized and sieved, including passing through a 100-mesh sieve.

[0019] As a preferred embodiment of the preparation method of the present invention, the culture medium comprises, by weight of raw materials, 1 to 10 parts of tea leaves, 1.5 parts of glucose, 1 part of yeast peptone, and distilled water to a total of 100 parts.

[0020] As a preferred embodiment of the preparation method described in this invention, the tea leaves include one or more of green tea, black tea, dark tea, white tea, oolong tea, and Pu-erh tea.

[0021] As a preferred embodiment of the preparation method described in this invention, the aqueous microporous filter membrane comprises a 0.22 μm aqueous microporous filter membrane.

[0022] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a tea fermentation product for cosmetic use with anti-aging effects.

[0023] Another objective of this invention is to overcome the shortcomings of the prior art by applying a tea fermentation product in the preparation of cosmetics with anti-aging effects.

[0024] Beneficial effects of this invention:

[0025] (1) The production process of the tea fermentation product of the present invention does not involve organic solvents, has low extraction energy consumption, high extraction efficiency, and the product filtrate is directly supplied as the product.

[0026] (2) The present invention screened out three yeast strains with the ability to synthesize cellulase, xylanase and lignin peroxidase; in addition to tea extract, the tea fermentation product also contains amino acids, nucleotides, vitamins and trace elements contained in yeast and xylo-glucosidase, which synergistically improve the anti-aging effect of the skin. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0028] Figure 1 A comparison chart of the antioxidant properties of various fermentation broths and extracts;

[0029] Figure 2 A comparison of the elastase inhibition capacity of various fermentation broths and extracts.

[0030] Figure 3 A comparison of the collagenase inhibition capacity of various fermentation broths and extracts;

[0031] Figure 4 A comparison of the AGEs inhibition capabilities of different fermentation broths and extracts;

[0032] Figure 5 A comparison chart of the cell protection capabilities of various fermentation broths and extracts;

[0033] Figure 6 A comparison chart of the cell repair capabilities of various fermentation broths and extracts;

[0034] Figure 7 This is a diagram showing the results of a chicken embryo chorioallantoic membrane vascular experiment. Detailed Implementation

[0035] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0036] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0037] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0038] The yeast strain RY5 in this embodiment of the invention is deposited at the China Center for Type Culture Collection on July 9, 2021, with accession number CCTCC NO: M 2021849, which is the same as the Saccharomyces cerevisiae in CN202211736240.9;

[0039] The yeast strain RY16 in this embodiment of the invention is deposited at the China General Microbiological Culture Collection Center on December 7, 2020, with accession number CGMCC NO.21316, the same as the yeast strain in CN202011604969.1.

[0040] The RY17 in this embodiment of the invention is deposited at the China Center for Type Culture Collection, classified and named as Hanseniasporauvarum Q-8, with a deposit date of November 29, 2023, accession number CCTCC NO: M20232388, and deposit address at Wuhan University, Wuhan, China.

[0041] The culture medium of this invention:

[0042] Yeast activation / proliferation medium (YPD medium) (g / L):

[0043] Add 10g yeast powder, 20g peptone, and 20g glucose to water to a final volume of 1000mL. Sterilize at pH 6.5 at 121℃ for 20min.

[0044] Cellulase activity screening medium (g / L):

[0045] Cellulose powder 2.0, peptone 10.0, yeast powder 5.0, glucose 2.0, potassium dihydrogen phosphate 1.0, magnesium sulfate heptahydrate 0.2, agar powder 15.0, distilled water 1.0L, sterilize at 121℃ for 20min. Do not add agar powder when preparing liquid culture medium.

[0046] Xylanase activity screening medium (g / L):

[0047] Xylan 2.0, peptone 10.0, yeast extract 5.0, glucose 0.5, xylose 5.0, potassium dihydrogen phosphate 1.0, magnesium sulfate heptahydrate 0.2, agar powder 15.0, distilled water 1.0L, sterilize at 121℃ for 20min. Do not add agar powder when preparing liquid culture medium.

[0048] Lignin peroxidase activity screening medium (g / L):

[0049] Sodium lignosulfonate 2.0g, peptone 10.0g, yeast extract 5.0g, glucose 2.0g, potassium dihydrogen phosphate 1.0g, magnesium sulfate heptahydrate 0.2g, agar powder 15.0g, distilled water 1.0L. Sterilize at 121℃ for 20min. Do not add agar powder when preparing liquid culture medium.

[0050] Activation / proliferation medium for *Acetobacter xylodis* (g / L):

[0051] Yeast extract 5.0g, glucose 20.0g, add water to a final volume of 1000mL, pH 6.5, sterilize at 121℃ for 20min.

[0052] Tea fermentation medium (g / L):

[0053] 10-100g of tea powder, 15g of glucose, 10g of yeast peptone, 1.0L of distilled water, pH 5.0, sterilized at 121℃ for 20min.

[0054] The methods for preparing yeast fermentation broth and yeast / Acetobacter xylitol fermentation broth used for comparison in this invention are as follows:

[0055] (1) Yeast fermentation broth

[0056] Seed cultures of yeast RY5, yeast RY16, and yeast RY17 were cultured until OD600nm≥5, and then added at 3% (v / L) to a tea-free fermentation medium (g / L) (15.0g glucose, 10.0g yeast peptone, 1.0L distilled water, pH5.0, sterilized at 121℃ for 20min). The cultures were then incubated at 30℃ and 200r / min for 24h.

[0057] The fermentation products were sterilized at 121°C for 20 minutes and collected after filtration through a 0.22μm aqueous microporous membrane.

[0058] (2) Yeast / Acetobacter xylitol fermentation broth

[0059] Seed cultures of yeast RY5, yeast RY16, and yeast RY17 were cultured until OD600nm≥5, and then added at 3% (v / L) to a tea-free fermentation medium (g / L) (15.0g glucose, 10.0g yeast peptone, 1.0L distilled water, pH5.0, sterilized at 121℃ for 20min). The cultures were then incubated at 30℃ and 200r / min for 24h.

[0060] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added to the culture medium of the above-mentioned yeast at a volume fraction of 5%, and cultured at 30℃ and 50r / min for 12h.

[0061] The fermentation products were sterilized at 121°C for 20 minutes and collected after filtration through a 0.22μm aqueous microporous membrane.

[0062] Example 1

[0063] Screening for the ability of yeast to synthesize cellulase:

[0064] (1) Twenty yeast strains were selected. The strain types and names are shown in Table 1. The strains were sourced from the strain bank built by Jiangsu Ruiting Biotechnology Co., Ltd.

[0065] (2) The frozen yeast cryopreservation tubes were thawed by shaking in a 30°C water bath, and then transferred to YPD medium in a clean bench and cultured in a shake flask at 30°C for 48 hours at 200 r / min.

[0066] (3) Let the yeast culture in the shake flask stand for 5 minutes, obtain the upper bacterial suspension by decantation, centrifuge at 2000 r / min for 10 minutes, and collect the precipitated bacterial cells.

[0067] (4) The precipitated bacterial cells were redispersed with liquid cellulase activity screening medium and cultured in shake flasks at 200 r / min and 30 ℃ for 48 h.

[0068] (5) Let the cultured yeast liquid stand for 5 minutes, obtain the upper bacterial suspension by decantation, centrifuge at 2000 r / min for 10 minutes, and collect the precipitated bacterial cells.

[0069] (6) The precipitated bacterial cells were redispersed with a small amount of liquid cellulase activity screening medium and adjusted to OD600nm=0.5.

[0070] (7) Pick up the bacterial resuspension from (6) with an inoculation needle and inoculate the bacterial cells into the center of an 11cm culture dish pre-filled with solid cellulase activity screening medium using the puncture inoculation method. Inoculate three culture dishes for each type of bacteria.

[0071] (8) Place the inoculated petri dishes in a 30℃ constant temperature incubator for 48 hours. After removing them, measure the thickness of the transparent zone around the colonies using calipers. The results are shown in Table 1.

[0072] (9) Through comparison, it was found that yeast RY5 has the largest transparent zone thickness and the strongest ability to synthesize cellulase.

[0073] Table 1

[0074]

[0075]

[0076] Example 2

[0077] Screening for the ability of yeast to synthesize xylanase:

[0078] (1) Twenty yeast strains were selected. The strain types and names are shown in Table 1. The strains were sourced from the strain bank built by Jiangsu Ruiting Biotechnology Co., Ltd.

[0079] (2) The frozen yeast cryopreservation tubes were thawed by shaking in a 30°C water bath, and then transferred to YPD medium in a clean bench and cultured in a shake flask at 30°C for 48 hours at 200 r / min.

[0080] (3) Let the yeast culture in the shake flask stand for 5 minutes, obtain the upper bacterial suspension by decantation, centrifuge at 2000 r / min for 10 minutes, and collect the precipitated bacterial cells.

[0081] (4) The precipitated bacterial cells were redispersed in liquid xylanase activity screening medium and cultured in shake flasks at 200 r / min and 30 ℃ for 48 h.

[0082] (5) Let the cultured yeast liquid stand for 5 minutes, obtain the upper bacterial suspension by decantation, centrifuge at 2000 r / min for 10 minutes, and collect the precipitated bacterial cells.

[0083] (6) The precipitated bacterial cells were redispersed with a small amount of liquid xylanase activity screening medium and adjusted to OD. 600nm =0.5.

[0084] (7) Pick up the bacterial resuspension from (6) with an inoculation needle and inoculate the bacterial cells into the center of an 11cm culture dish pre-filled with solid xylanase activity screening medium using the puncture inoculation method. Inoculate three culture dishes for each type of bacteria.

[0085] (8) Place the inoculated petri dishes in a 30℃ constant temperature incubator for 48 hours. After removing them, measure the thickness of the transparent zone around the colonies using calipers. The results are shown in Table 1.

[0086] (9) Through comparison, it was found that yeast RY17 has the largest transparent zone thickness and its ability to synthesize xylanase is the strongest.

[0087] Example 3

[0088] Screening of yeast's ability to synthesize lignin peroxidase:

[0089] (1) Twenty yeast strains were selected. The strain types and names are shown in Table 1. The strains were sourced from the strain bank built by Jiangsu Ruiting Biotechnology Co., Ltd.

[0090] (2) The frozen yeast cryopreservation tubes were thawed by shaking in a 30°C water bath, and then transferred to YPD medium in a clean bench and cultured in a shake flask at 30°C for 48 hours at 200 r / min.

[0091] (3) Let the yeast culture in the shake flask stand for 5 minutes, obtain the upper bacterial suspension by decantation, centrifuge at 2000 r / min for 10 minutes, and collect the precipitated bacterial cells.

[0092] (4) The precipitated bacterial cells were redispersed in liquid lignin peroxidase activity screening medium and cultured in shake flasks at 200 r / min and 30 ℃ for 48 h.

[0093] (5) Let the cultured yeast liquid stand for 5 minutes, obtain the upper bacterial suspension by decantation, centrifuge at 2000 r / min for 10 minutes, and collect the precipitated bacterial cells.

[0094] (6) The precipitated bacterial cells were redispersed with a small amount of liquid lignin peroxidase activity screening medium and adjusted to OD600nm=0.5.

[0095] (7) Pick up the bacterial resuspension from (6) with an inoculation needle and inoculate the bacterial cells into the center of an 11cm culture dish pre-filled with solid lignin peroxidase activity screening medium using the puncture inoculation method. Inoculate three culture dishes for each type of bacteria.

[0096] (8) Place the inoculated petri dishes in a 30℃ constant temperature incubator for 48 hours. After removing them, measure the thickness of the transparent zone around the colonies using calipers. The results are shown in Table 1.

[0097] (9) Through comparison, it was found that yeast RY16 has the largest transparent zone thickness and the strongest ability to synthesize lignin peroxidase.

[0098] Example 4

[0099] The production process of black tea extract:

[0100] Take commercially available black tea leaves, pulverize them, pass them through a 100-mesh sieve, weigh out 50g, mix them with 1L of distilled water, stir well, and then extract using the reflux extraction method for 2 hours;

[0101] The product was centrifuged at 8000 r / min for 10 min, filtered through a 0.22 μm aqueous microporous membrane and collected as black tea extract for later use.

[0102] Example 5

[0103] Production process of black tea / yeast fermentation liquid:

[0104] Take commercially available black tea leaves, crush them, pass them through a 100-mesh sieve, weigh out 50g, mix them with 15g glucose, 10g yeast peptone, and 1L distilled water, stir well, adjust the pH to 5.0, and then sterilize at 121℃ for 20 minutes to prepare a culture medium for later use.

[0105] Seed cultures of yeast RY5, yeast RY16, and yeast RY17 were cultured until OD600nm≥5, and then added to tea fermentation medium at a volume fraction of 3% and cultured at 30℃ and 200r / min for 24h.

[0106] The fermentation products were sterilized at 121°C for 20 minutes and collected after filtration through a 0.22μm aqueous microporous membrane.

[0107] Example 6

[0108] Production process of black tea / yeast / Acetobacter xyloside fermentation broth:

[0109] Take commercially available black tea leaves, crush them, pass them through a 100-mesh sieve, weigh out 50g, mix them with 15g glucose, 10g yeast peptone, and 1L distilled water, stir well, adjust the pH to 5.0, and then sterilize at 121℃ for 20 minutes to prepare a culture medium for later use.

[0110] Seed cultures of yeast RY5, yeast RY16, and yeast RY17 were cultured until OD600nm≥5, and then added to tea fermentation medium at a volume fraction of 3% and cultured at 30℃ and 200r / min for 24h.

[0111] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added to the culture medium of the above-mentioned yeast at a volume fraction of 5%, and cultured at 30℃ and 50r / min for 12h.

[0112] The fermentation products were sterilized at 121°C for 20 minutes and collected after filtration through a 0.22μm aqueous microporous membrane.

[0113] To compare the performance differences between products obtained by inoculating multiple yeast strains together and those obtained by inoculating a single yeast strain, in addition to the above experiments, only one yeast strain and xylosinophil were used to inoculate the culture medium, resulting in three samples of black tea fermentation liquid obtained by co-fermentation using only one yeast strain and xylosinophil.

[0114] The specific method is as follows: the seed liquid of yeast RY5, yeast RY16 and yeast RY17 is cultured until OD600nm≥5, and one of them is selected and added to the tea fermentation medium at a volume fraction of 9% and cultured at 30℃ and 200r / min for 24h.

[0115] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added to the culture medium of the previously cultured yeast at a volume fraction of 5%. The culture was carried out at 30℃ and 50r / min for 12h. The fermentation product was sterilized at 121℃ for 20min and collected after filtration through a 0.22μm aqueous microporous membrane.

[0116] Preparation of fermentation broth from yeast RY5, yeast RY16, and *Acetobacter xylinum*:

[0117] Seed cultures of yeast RY5 and yeast RY16 were cultured until OD600nm≥5, and then added to tea fermentation medium at a volume fraction of 4.5% and cultured at 30℃ and 200r / min for 24h.

[0118] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added at 5% (v / v) to the culture medium previously used for yeast culture. The culture was incubated at 30℃ and 50 rpm for 12 h. The fermentation product was sterilized at 121℃ for 20 min and collected after filtration through a 0.22 μm aqueous microporous membrane.

[0119] Preparation of fermentation broth from yeast RY5, yeast RY17, and *Acetobacter xylosiderin*:

[0120] Seed cultures of yeast RY5 and yeast RY17 were cultured until OD600nm≥5, and then added to tea fermentation medium at a volume fraction of 4.5% and cultured at 30℃ and 200r / min for 24h.

[0121] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added to the culture medium of the previously cultured yeast at a volume fraction of 5%. The culture was carried out at 30℃ and 50r / min for 12h. The fermentation product was sterilized at 121℃ for 20min and collected after filtration through a 0.22μm aqueous microporous membrane.

[0122] Preparation of fermentation broth from yeast RY16, yeast RY17, and *Acetobacter xylinum*:

[0123] Seed cultures of yeast RY5 and yeast RY17 were cultured until OD600nm≥5, and then added to tea fermentation medium at a volume fraction of 4.5% and cultured at 30℃ and 200r / min for 24h.

[0124] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added to the culture medium of the previously cultured yeast at a volume fraction of 5%. The culture was carried out at 30℃ and 50r / min for 12h. The fermentation product was sterilized at 121℃ for 20min and collected after filtration through a 0.22μm aqueous microporous membrane.

[0125] Example 7

[0126] Production process of black tea / Acetobacter xylobiobacterium fermentation broth:

[0127] Take commercially available black tea leaves, crush them, pass them through a 100-mesh sieve, weigh out 50g, mix them with 15g glucose, 10g yeast peptone, and 1L distilled water, stir well, adjust the pH to 5.0, and then sterilize at 121℃ for 20 minutes to prepare a culture medium for later use.

[0128] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added to the culture medium of the previously cultured yeast at a volume fraction of 5%. The culture was carried out at 30℃ and 50r / min for 12h. The fermentation product was sterilized at 121℃ for 20min and collected after filtration through a 0.22μm aqueous microporous membrane.

[0129] Example 8

[0130] Production process of green tea / yeast / Acetobacter xylitol fermentation broth:

[0131] Take commercially available green tea leaves, crush them, pass them through a 100-mesh sieve, weigh out 35g, mix them with 15g glucose, 10g yeast peptone, and 1L distilled water, stir well, adjust the pH to 5.0, and then sterilize at 121℃ for 20 minutes to prepare a culture medium for later use.

[0132] Seed cultures of yeast RY5, yeast RY16, and yeast RY17 were cultured until OD600nm≥5, and then added to tea fermentation medium at a volume fraction of 3% and cultured at 30℃ and 200r / min for 24h.

[0133] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added to the culture medium of the previously cultured yeast at a volume fraction of 5%. The culture was carried out at 30℃ and 50r / min for 12h. The fermentation product was sterilized at 121℃ for 20min and collected after filtration through a 0.22μm aqueous microporous membrane.

[0134] Example 9

[0135] Production process of white tea / yeast / Acetobacter xylitol fermentation broth:

[0136] Take commercially available white tea leaves, pulverize them, pass them through a 100-mesh sieve, weigh out 85g, mix them with 15g glucose, 10g yeast peptone, and 1L distilled water, stir well, adjust the pH to 5.0, and then sterilize at 121℃ for 20 minutes to prepare a culture medium for later use.

[0137] Seed cultures of yeast RY5, yeast RY16, and yeast RY17 were cultured until OD600nm≥5, and then added to tea fermentation medium at a volume fraction of 3% and cultured at 30℃ and 200r / min for 24h.

[0138] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added to the culture medium of the previously cultured yeast at a volume fraction of 5%. The culture was carried out at 30℃ and 50r / min for 12h. The fermentation product was sterilized at 121℃ for 20min and collected after filtration through a 0.22μm aqueous microporous membrane.

[0139] Example 10

[0140] Production process of Pu-erh tea / yeast / Acetobacter xylan fermentation broth:

[0141] Take commercially available Pu-erh tea cakes, crush them, pass them through a 100-mesh sieve, weigh out 50g, mix them with 15g glucose, 10g yeast peptone, and 1L distilled water, stir well, adjust the pH to 5.0, and then sterilize at 121℃ for 20 minutes to prepare a culture medium for later use.

[0142] Seed cultures of yeast RY5, yeast RY16, and yeast RY17 were cultured until OD600nm≥5, and then added to tea fermentation medium at a volume fraction of 3% and cultured at 30℃ and 200r / min for 24h.

[0143] The seed culture of *Acetobacter xylose* was cultured until OD600nm≥4, and then added to the culture medium of the previously cultured yeast at a volume fraction of 5%. The culture was carried out at 30℃ and 50r / min for 12h. The fermentation product was sterilized at 121℃ for 20min and collected after filtration through a 0.22μm aqueous microporous membrane.

[0144] The antioxidant properties of the above-mentioned black tea extract, black tea / yeast fermentation broth, black tea / Acetobacter xyloside fermentation broth, black tea / yeast / Acetobacter xyloside fermentation broth, and fermentation broth without black tea were compared.

[0145] Because China has a wide variety of tea varieties and products, this invention selects black tea, which is popular both domestically and internationally, as a representative target for evaluating the efficacy of fermentation products.

[0146] This invention uses DPPH free radical scavenging rate to evaluate the antioxidant properties of tea extract and tea fermentation broth before and after fermentation. The specific steps are as follows:

[0147] The tea extract or tea fermentation liquid was diluted with distilled water to concentrations of 1%, 0.5%, 0.1%, 0.05%, and 0.01%, respectively, to prepare sample solutions.

[0148] Accurately pipette 1 mL of sample solution, add 5 mL of DPPH solution, mix well, and let stand at room temperature (25℃) in the dark for 30 min. Measure the absorbance at 517 nm and calculate the DPPH free radical scavenging rate using the following formula:

[0149]

[0150] y—Clearance rate, %;

[0151] A0—The absorbance of 1 mL distilled water + 5 mL DPPH solution;

[0152] A1—Absorbance of 1 mL sample solution + 5 mL DPPH solution;

[0153] A2—Absorbance of 1 mL sample solution + 5 mL ethanol solution (to eliminate errors between different samples).

[0154] See [link / reference] for DPPH radical scavenging ability. Figure 1 Both 1% black tea / yeast fermentation broth and black tea / yeast / Acetobacter xyloside fermentation broth can achieve a DPPH free radical scavenging capacity of over 90%.

[0155] The scavenging abilities of 1% black tea extract, black tea / Acetobacter xylinum fermentation broth, and black tea / single yeast / Acetobacter xylinum fermentation broth were all less than 90%. Clearly, when the three yeasts act together on black tea, its antioxidant active ingredients are released more fully, and the combined action of Acetobacter xylinum and yeasts has a synergistic effect. Meanwhile, compared to other fermentation broths, the scavenging ability of simple microbial fermentation broths is weak. This indicates that the fermentation process can effectively improve the release and acquisition of antioxidants in black tea.

[0156] Example 11

[0157] Comparison of elastase inhibition capacity of black tea extract, black tea / yeast fermentation broth, black tea / Acetobacter xyloside fermentation broth, black tea / yeast / Acetobacter xyloside fermentation broth, and no black tea fermentation broth.

[0158] Elastase has the ability to degrade elastin in human skin, thereby causing skin tissue aging. Inhibiting elastase activity is of great significance for anti-aging effects.

[0159] The elastase activity inhibition assay used in this invention comprises the following specific steps:

[0160] Accurately weigh 5 mg of Congo red elastin, add 1 mL of 0.05 mol / L Tris-HCl buffer (pH 8.8), vortex for 10 s, and incubate at 37°C for 15 min to obtain phase A.

[0161] A 2 mg / mL elastase solution was prepared using 0.05 mol / L Tris-HCl buffer (pH 8.8). 1 mL of the enzyme solution was added to 200 μL of the sample solution, vortexed for 10 s, and incubated at 37°C for 15 min as phase B. In the blank control group, 1 mL of Tris-HCl buffer was used instead of the enzyme solution, and 200 μL of water was used instead of the sample solution. In the negative control group, 200 μL of water was used instead of the sample solution.

[0162] Phase B was transferred into Phase A and then shaken at 37°C and 200 rpm for 3 hours.

[0163] After shaking, add 2 mL of 0.5 mol / L phosphate buffer solution to each group, vortex for 10 s, and let stand at room temperature for 15 min.

[0164] The above test products were centrifuged for 10 minutes under a gravitational acceleration of 10000g.

[0165] Transfer 200 μL of the centrifuged supernatant to a 96-well plate, measure the absorbance at 495 nm using a microplate reader, and calculate the elastase inhibition rate using the following formula:

[0166]

[0167] Where As is the absorbance value of the sample group; Ab is the absorbance value of the blank group; and Ac is the absorbance value of the negative control group.

[0168] The inhibitory effect on elastase is shown in [reference needed]. Figure 2 When the concentration of the black tea / yeast / Acetobacter xylinum fermentation broth was 0.1%, its inhibitory effect on elastase activity reached over 90%, while the inhibitory effect of 1% black tea extract did not reach 90%. When the concentration of the black tea / yeast fermentation broth was 0.5%, its inhibitory effect on elastase activity reached over 90%, while the inhibitory effects of the black tea / Acetobacter xylinum fermentation broth and the black tea / single yeast / Acetobacter xylinum fermentation broth at a concentration of 0.5% were close to 90% or even lower than 90%. Clearly, the combined effect of the three yeasts is better than that of a single yeast, and the three yeasts have the best synergistic effect with Acetobacter xylinum. Meanwhile, compared to each other, the inhibitory effect of simple microbial fermentation broth on elastase activity is weak. This indicates that the fermentation process can effectively improve the release and extraction of anti-aging related active substances in black tea.

[0169] Example 12

[0170] Comparison of collagenase inhibition capabilities of black tea extract, black tea / yeast fermentation broth, black tea / Acetobacter xyloside fermentation broth, black tea / yeast / Acetobacter xyloside fermentation broth, and non-black tea fermentation broth:

[0171] Similar to elastase, collagenase has the ability to degrade collagen in human skin, thereby causing skin tissue aging. Inhibiting collagenase activity is of great significance for anti-aging effects. The specific steps of the collagenase activity inhibition test used in this invention are as follows:

[0172] Collagenase solution was prepared using 0.05 mol / L Tris-HCl buffer (containing 400 mmol / L sodium chloride and 10 mmol / L calcium chloride, pH 7.5), with an enzyme activity of 0.9 U / mL.

[0173] Accurately weigh 1 mg of collagen substrate—N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA)—and dissolve it in 1 mL of Tris-HCl buffer.

[0174] Transfer 50 μL of sample solution, 100 μL of Tris-HCl buffer, and 50 μL of collagenase solution to a container. Vortex for 10 s and incubate at 37°C for 15 min. For the sample control group, use 50 μL of buffer instead of the enzyme solution; for the negative control group, use 50 μL of water instead of the sample solution; for the blank group, use 50 μL of water instead of the sample solution and 50 μL of buffer instead of the enzyme solution.

[0175] Add 50 μL of collagen substrate solution to each of the above reaction systems, vortex for 10 s, and incubate at 37°C for 20 min. For the sample control group and blank group, use 50 μL of buffer solution instead of the substrate solution.

[0176] Transfer 200 μL of the reaction solution into a 96-well plate, measure the absorbance at 340 nm using a microplate reader, and calculate the elastase inhibition rate using the following formula:

[0177]

[0178] Where As is the absorbance value of the sample group; Asb is the absorbance value of the sample control group; Ac is the absorbance value of the negative control group; and Acb is the absorbance value of the blank group.

[0179] The inhibitory effect on collagenase is shown in the figure. Figure 3 When the concentration of black tea / yeast / Acetobacter xyloside fermentation broth is 1%, its inhibitory capacity on collagenase activity can reach more than 80%, and when the concentration of black tea / yeast fermentation broth is 1%, its inhibitory capacity on collagenase activity can reach more than 75%, while the inhibitory capacity of 1% black tea extract is less than 60%.

[0180] A single yeast strain exhibits a synergistic effect with *Acetobacter xylinum*, significantly enhancing its inhibitory capacity on collagenase activity compared to the fermentation broth of black tea / *Acetobacter xylinum*. Conversely, the inhibitory capacity of a single microbial fermentation broth on collagenase activity is weaker. This indicates that the fermentation process effectively enhances the release and extraction of anti-aging-related active substances in black tea.

[0181] Example 13

[0182] Comparison of the inhibitory effects of black tea extract, black tea / yeast fermentation broth, black tea / Acetobacter xyloside fermentation broth, black tea / yeast / Acetobacter xyloside fermentation broth, and no black tea fermentation broth on the inhibition of late-stage protein glycosylation products.

[0183] Excess sugar in the body reacts with proteins (including collagen and elastin in the skin), lipids, and nucleic acids through a series of complex non-enzymatic reactions to produce advanced glycation end products (AGEs). Components such as proteins bound to sugar lose their original functions. Severe glycation in the skin can lead to aging-related symptoms such as dullness and loss of elasticity.

[0184] Bovine serum albumin (BSA) solution was prepared using 0.05 mol / L phosphate buffer (PBS, pH 7.4) to a concentration of 8 mg / mL, and sodium azide was added to bring the concentration to 0.04%.

[0185] Take 200 μL of BSA solution, 200 μL of sample solution, and 200 μL of 0.2 mol / L glucose solution, respectively, vortex for 10 s, and let stand at room temperature for 15 min. For the negative control group and blank group, use 200 μL of water instead of the sample solution; for the blank group and sample control group, use 200 μL of PBS solution instead of the glucose solution.

[0186] The above reaction system was incubated in a 55°C water bath for 4 days and then cooled to room temperature.

[0187] Add 200 μL of 5% trichloroacetic acid aqueous solution to the reaction system, vortex for 10 s, then centrifuge at 12000 rpm for 5 min and discard the supernatant.

[0188] Add 600 μL of phosphate buffer (pH 10) to the precipitate, let stand for 10 min, vortex for 20 s, and filter the solution through a 0.22 μm filter membrane.

[0189] Take 200 μL of the above filtrate and place it in a 96-well plate. Measure the fluorescence intensity using a microplate reader at an excitation wavelength of 370 nm and an emission wavelength of 440 nm, and calculate the AGEs inhibition rate using the following formula:

[0190]

[0191] Where As is the absorbance value of the sample group; Asb is the absorbance value of the sample control group; Ac is the absorbance value of the negative control group; and Acb is the absorbance value of the blank group.

[0192] The inhibitory effect on AGEs synthesis is shown in the figure. Figure 4 When the concentration of black tea / yeast fermentation broth and black tea / yeast / Acetobacter xyloside fermentation broth was 1%, both showed an inhibition rate of over 50% against AGEs, while the inhibition ability of 1% black tea extract was close to 40%. The inhibition ability of 1% black tea / single yeast / Acetobacter xyloside fermentation broth was between 45% and 50%, which was superior to that of 1% black tea / Acetobacter xyloside fermentation broth, demonstrating the synergistic effect between yeast and Acetobacter xyloside.

[0193] Meanwhile, compared to other substances, the simple microbial fermentation broth has a weak inhibitory effect on AGEs, which indicates that the fermentation process can effectively improve the release and acquisition of anti-aging-related active substances in black tea.

[0194] Example 14

[0195] Skin cell protection and repair performance tests of black tea extract, black tea / yeast fermentation broth, black tea / Acetobacter xyloside fermentation broth, black tea / yeast / Acetobacter xyloside fermentation broth and no black tea fermentation broth.

[0196] Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT (thiazolyl blue) to water-insoluble blue-purple formazan crystals, which are then deposited in the cells. The lower the cell viability, the worse this function. Therefore, the MTT assay can be used to determine the cell viability of human fibroblasts in different environments, thereby characterizing the protective and repairing effects of environmental factors on cells.

[0197] Each group of samples was diluted with distilled water to obtain a series of sample solutions of different concentrations;

[0198] The concentration of human fibroblasts in the logarithmic growth phase was adjusted to 20,000 cells / mL using culture medium, and 100 μL of cell suspension was added to 96-well plates and cultured for 12 hours.

[0199] Add a certain amount of sample solution to each well to make the final concentration 1%, continue culturing for 12 hours, completely discard the culture medium, then add culture medium containing 0.5 mg / mL MTT to each well, and continue culturing the cells for 3-5 hours until crystals are formed;

[0200] Discard the culture medium completely, add 150 μL LDMSO to each well, and gently shake at room temperature for 5 minutes to completely dissolve the crystals;

[0201] Quickly remove the 96-well plate and measure the absorbance of each well at a wavelength of 490 nm using an ELISA reader.

[0202] Cell protection experimental conditions:

[0203] Positive control: serum-free culture medium + fibroblasts, without ultraviolet irradiation treatment;

[0204] Negative control: serum-free culture medium + fibroblasts, treated with ultraviolet light irradiation;

[0205] Experimental group: serum-free culture medium + fibroblasts, after adding sample solution, were treated with ultraviolet light irradiation.

[0206] Ultraviolet irradiation conditions: UVA lamp (irradiation wavelength: 365nm), light intensity 20J / cm² 2 .

[0207] Experimental results: such as Figure 5 As shown, both the 1% concentration of black tea / yeast fermentation broth and the black tea / yeast / Acetobacter xyloside fermentation broth exhibit good cell protection capabilities.

[0208] Cell repair experimental conditions:

[0209] Positive control: serum-free culture medium + fibroblasts, without ultraviolet irradiation treatment;

[0210] Negative control: serum-free culture medium + fibroblasts, treated with ultraviolet light irradiation;

[0211] Experimental group: serum-free culture medium + fibroblasts, treated with ultraviolet light and then added to each sample solution;

[0212] Ultraviolet irradiation conditions: UVA lamp (irradiation wavelength: 365nm), light intensity 20J / cm² 2 .

[0213] Experimental results: such as Figure 6 As shown, both the 1% concentration of black tea / yeast fermentation broth and the black tea / yeast / Acetobacter xyloside fermentation broth exhibit good cell repair capabilities.

[0214] Example 15

[0215] Safety evaluation of black tea / yeast fermentation broth and black tea / yeast / Acetobacter xyloside fermentation broth

[0216] (1) Human skin patch test

[0217] The procedure shall be carried out in accordance with the methods specified in the 2015 Cosmetic Technical Specifications.

[0218] Volunteer requirements: 30 healthy subjects (aged 20-40) who do not have clinically unhealed inflammatory skin diseases, whose skin test sites have no scars, pigmentation, atrophy, port-wine stains or other blemishes, who have not participated in other clinical trials, who do not have a highly sensitive constitution, and who have not undergone patch testing in the past month.

[0219] The black tea / yeast fermentation broth and the black tea / yeast / xylactone fermentation broth were successively diluted with distilled water to obtain sample solutions with concentrations of 20%, 10%, 5%, 1%, and 0.5%, respectively.

[0220] Transfer 0.020 to 0.025 mL of sample solution onto the filter paper attached to the spot tester, and place the filter paper inside the spot tester. At the same time, use the same volume of distilled water as a blank control. Apply the spot tester containing the test substance to the flexor side of the forearm with non-irritating adhesive tape, and gently press it with your palm to make it evenly adhered to the skin. Maintain for 24 hours.

[0221] After removing the spot tester, wait 30 minutes until the indentation disappears and observe the skin reaction. If the skin reaction is negative, observe the skin reaction again at 24 hours and 48 hours.

[0222] The experimental results are shown in Table 2.

[0223] Table 2 Results of human skin patch test

[0224]

[0225] Note: -(0): negative reaction; ±(1): suspicious reaction (weak erythema); +(2): weak positive reaction (erythema reaction); erythema, infiltration, edema

[0226] In the human patch experiment, all 30 subjects showed a grade 0 reaction. According to the relevant provisions of the Cosmetic Safety Technical Specifications, neither the black tea / yeast ferment broth nor the black tea / yeast / Acetobacter xyloside ferment broth caused adverse reactions on human skin at the above concentrations. Therefore, the product has a high degree of safety for human use.

[0227] (2) Chicken embryo chorioallantoic membrane vascular assay (HET-CAM assay)

[0228] The chicken embryo chorioallantoic membrane vascular test evaluates irritation by detecting damage to the chorioallantoic membrane caused by chemical substances.

[0229] Experimental Design:

[0230] 1. Positive control: 0.1 mol / L NaOH (sodium hydroxide) solution;

[0231] 1% SDS (sodium dodecyl sulfate) solution;

[0232] 2. Negative control: 0.9% sterile physiological saline (NaCl) solution;

[0233] 3. Test sample: 20% black tea / yeast fermentation broth;

[0234] 20% Black Tea / Yeast / Acetobacter xylinum fermentation broth;

[0235] Experimental results are as follows Figure 7 As shown:

[0236] In the HET-CAM assay, the addition of 0.9% sterile saline showed no significant change.

[0237] After adding 1% SDS solution, the capillaries in the chicken embryo gradually disappeared;

[0238] After adding 0.1 mol / L NaOH solution, the edges of some capillaries gradually became blurred after 1 minute, the capillaries began to disappear after 5 minutes, and the main vessels also gradually became blurred after 10 minutes.

[0239] Adding 20% ​​black tea / yeast fermentation broth and black tea / yeast / Acetobacter xyloside fermentation broth did not cause significant changes.

[0240] Therefore, black tea / yeast fermentation broth and black tea / yeast / Acetobacter xyloside fermentation broth with a concentration of 20% or less have no obvious irritation or corrosiveness and are highly safe.

[0241] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing a cosmetic tea fermentation product with anti-aging effects, characterized in that: include, The tea leaves were crushed and sieved, mixed with glucose and yeast peptone, and distilled water was added. The mixture was stirred evenly, the pH was adjusted to 5.0, and sterilized to obtain the culture medium. Seed cultures of yeast RY5, yeast RY16, and yeast RY17 were cultured to OD. 600nm ≥5, add to the above culture medium at a volume fraction of 1~5%, and incubate at 30℃ and 200 r / min for 12~24 h; Seed culture of *Acetobacter xylinum* was cultured to OD0.05 600nm ≥4, add 1~6% by volume to the culture medium that has been cultured with yeast above, and culture at 30℃ and 50 r / min for 8~12 h; The fermentation product is sterilized and then filtered through a water-based microporous membrane to obtain a cosmetic tea fermentation product with anti-aging effects. The yeast strain RY5 is deposited at the China Center for Type Culture Collection (CCTCC) on July 9, 2021, with accession number CCTCC NO: M 2021849. The yeast strain RY16 is deposited at the China General Microbiological Culture Collection Center on December 7, 2020, with accession number CGMCC NO.21316. The yeast strain RY17 is deposited at the China Center for Type Culture Collection and is classified as *Hansenula polymorpha*. Hanseniaspora uvarum Q-8, deposited on November 29, 2023, with accession number CCTCC NO: M20232388; The *Acetobacter xylinum* species described is resource number ATCC 23767.

2. The preparation method according to claim 1, characterized in that: The tea leaves are pulverized and sieved, including through a 100-mesh sieve.

3. The preparation method according to claim 1, characterized in that: The culture medium, by the mass fraction of the raw materials, comprises 1-10 parts tea leaves, 1.5 parts glucose, 1 part yeast peptone, and distilled water to a total of 100 parts.

4. The preparation method according to claim 1, characterized in that: The tea mentioned includes one or more of the following: green tea, black tea, dark tea, white tea, oolong tea, and pu-erh tea.

5. The preparation method according to claim 1, characterized in that: The aqueous microporous filter membrane includes a 0.22 mm aqueous microporous filter membrane.

6. The tea fermentation product obtained by any one of the preparation methods described in claims 1 to 5.

7. The use of the tea fermentation product according to claim 6 in the preparation of cosmetics with anti-aging effects.

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