Fermented tea extract, method for preparing the same, and use thereof

Abstract

This invention discloses a fermented tea extract, its preparation method, and its applications. The strains used are specially selected *Aspergillus cristatus* PLT-PE and *Saccharomyces boulardii* PLT-HZ, which have higher polysaccharide or polypeptide content compared to other strains. This fermented extract meets the demand for effective natural oil-controlling skincare products, is rich in heteropolyphenols, polypeptides, and polysaccharides, and possesses strong anti-photoaging and oil-controlling activities.

Description

Technical Field

[0001] This invention relates to the field of fermentation, specifically to a fermented tea extract, its preparation method, and its application. In particular, it relates to a tea bifurcation fermented extract with anti-photoaging and oil-controlling effects, its preparation method, and its application, mainly used in skin care and hair care products. Background Technology

[0002] Skin aging is classified into two types: unavoidable intrinsic aging and preventable extrinsic aging (photoaging). Long-wave ultraviolet radiation (320-400nm, UVA) can reach the dermis. In photoaged skin, UV radiation increases reactive oxygen species (ROS) within skin cells, induces elevated levels of micronutrients (MMPs), and leads to the degradation of collagen and elastin, resulting in rough, loose, and wrinkled skin. In addition to significantly reducing UV-induced ROS levels, probiotics can directly regulate the expression levels of MMPs in skin cells through multiple signaling pathways, reducing the degradation of collagen and elastin after UV irradiation.

[0003] Normal sebum secretion is crucial for maintaining the normal physiological state of human skin. Excessive UV radiation produces a large amount of reactive oxygen species (ROS), which can react with unsaturated fatty acid chains in the body to undergo lipid peroxidation, forming lipid peroxides. Lipid peroxides disrupt the structure of the phospholipid bilayer, affecting cell membrane integrity. They also cause excessive sebum secretion, resulting in oily skin. Excessive oil can lead to clogged pores, hair loss, blackheads, and acne. Sebaceous glands are distributed throughout the body, especially on the face and scalp, producing sebum—a mixture of lipids. Lipids are produced by other glands in the body, but the two most unique lipids for sebaceous glands are squalene and wax esters. Sebum secretion is related to hormonal activity, and excessive sebum secretion is associated with the development of skin diseases such as acne vulgaris. Acne is not life-threatening, but it can leave permanent scars on the face and cause physical and psychological distress. Acne can be caused by a variety of mechanisms, including (a) increased sebum production by sebaceous glands; (b) changes in follicular keratinization; (c) proliferation of Propionibacterium acnes; and (d) inflammation of the hair follicles around the sebaceous glands.

[0004] With growing interest in anti-photoaging and oil-control products, finding effective solutions for these issues has become a key focus in the skincare industry. The most economical way to prevent photoaging is through sun protection, including sun umbrellas, sun-protective clothing and hats, sunglasses, and physical sunscreens containing ingredients like titanium dioxide and zinc oxide. However, these simple physical sunscreens cannot completely block UV rays and lack sufficient repair capabilities for damaged skin. Of course, retinoic acid has been a primary topical treatment for preventing and treating photoaging for many years, and there are also endogenous antioxidants like vitamin C and vitamin E that can act as antioxidants in the prevention of photoaging.

[0005] For oily skin care, most products on the market focus on thorough cleansing, controlling sebum secretion, and maintaining a balance of oil and moisture. These products often contain a large amount of surfactants. While many people believe surfactants are simply for removing dirt and oil, leaving skin feeling soft and clean, excessive surfactants can actually burden the skin. First, surfactants can damage the skin's natural protective barrier, leading to dryness, roughness, dullness, and a tendency to crack. Second, surfactants can irritate the skin, triggering allergic reactions such as itching, redness, and inflammation. Furthermore, some surfactants are harmful to the human body, such as sodium sulfate, sodium benzoate, and EDTA. Therefore, products with multiple oil-controlling capabilities and rich in natural active ingredients are increasingly popular with consumers and have a broader application prospect.

[0006] Natural ingredients such as tea extract have been reported to have anti-photoaging and oil-controlling effects. However, the content of active ingredients in natural plant raw materials is low, their structure is complex and unstable, and some may even have toxic side effects, seriously affecting the application of plant resources in the development of skincare product raw materials. We drew inspiration and theoretical basis from traditional Chinese fermented dark tea, utilizing the fermentation process of microorganisms to enrich the content of active ingredients in plant resources, enhance efficacy, and reduce toxic side effects, effectively improving the effectiveness and safety of plant-based active ingredients in skincare products. This invention solves the problems of low content and unstable, single-component nature of tea extract, while simultaneously improving its anti-photoaging and oil-controlling effects. Summary of the Invention

[0007] The purpose of this invention is to address the problems existing in the prior art by providing a fermented tea extract, its preparation method, and its application. This fermented extract can meet people's demand for effective natural oil-controlling skincare products. It is rich in polyphenols, polypeptides, and polysaccharides, and has strong anti-photoaging and oil-controlling activities.

[0008] This invention provides a method for preparing fermented tea extract, comprising the following steps: 1) Add tea leaves to water and heat to 90-100℃ for 1-10 hours to obtain tea extract; add glucose to the extract and sterilize to obtain tea fermentation culture medium; wherein, the mass ratio of tea leaves to water is 1:(5-20) and the mass ratio of glucose to tea extract is (1-5):100. 2) The concentration is (1~9)×10 7A suspension of *Aspergillus cristatus* PLT-PE bacteria per mL was inoculated into tea fermentation medium at an inoculation rate of 1–5% by volume. The fermentation temperature was 25–30°C, the shaking speed was 100–120 r / min, and the fermentation time was 4–7 days to obtain the first fermentation broth. 3) The concentration is (1~9)×10 8 A suspension of *Saccharomyces boulardii* PLT-HZ cells per mL was inoculated into the first fermentation broth at an inoculation rate of 1–5% by volume. The fermentation temperature was 25–30°C, the shaking speed was 100–120 r / min, and the fermentation time was 1–3 days to obtain the second fermentation broth. 4) Filter the second fermentation broth through a 200-mesh filter to obtain the mycelium of *Aspergillus cristatus* PLT-PE; then separate the filtrate using a tubular centrifuge to obtain *Saccharomyces boulardii* PLT-HZ cells and obtain the fermentation filtrate. 5) The mycelium of *Aspergillus cristatus* PLT-PE was placed in water at a mass ratio of 1:(1-20) and extracted at 30-90℃ for 0.5-10 h. After extraction, the extract was concentrated to 1 / 10 of its original volume. Then, 10%-100% ethanol was added at a volume ratio of 1:10 for alcohol precipitation for 0.5-10 h. The ethanol was then removed by vacuum distillation to obtain the polysaccharide extract of *Aspergillus cristatus* PLT-PE. 6) Place the Saccharomyces blazei PLT-HZ cells in water at a mass ratio of 1:(1-20), heat to 30-90℃, and homogenize under high pressure for 0.5-10 hours. After extraction, centrifuge and collect the supernatant to obtain the Saccharomyces blazei PLT-HZ extract. 7) Mix the fermentation filtrate, the polysaccharide extract of *Aspergillus cristatus* PLT-PE, and the extract of *Saccharomyces boulardii* PLT-HZ to obtain a mixed solution; add D101 resin at a mass ratio of 40%, adsorb for 0.5–10 h, filter, obtain the filtrate, add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol at a mass ratio to obtain the fermented tea extract; The *Eurotium cristatum* PLT-PE strain was deposited at the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences on June 30, 2023, with accession number CGMCC No. 40715. The *Saccharomyces boulardii* strain PLT-HZ was deposited on June 30, 2023, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 27760.

[0009] In some specific implementations, the *Eurotium cristatum* PLT-PE bacterial suspension is prepared by the following method: *Aspergillus cristatus* PLT-PE was inoculated onto PDA medium and incubated at 25–30°C for 5–7 days to activate it. Then, the mycelium was transferred to PDA solid medium and incubated for 5–7 days. Finally, the colony surface was rinsed with sterile water to obtain a *Aspergillus cristatus* PLT-PE spore suspension, and its concentration was adjusted to 1–9 × 10⁻⁶. 7 A suspension of *Aspergillus cristatus* PLT-PE bacteria was obtained by measuring cells / mL. In some specific implementations, the *Saccharomyces boulardii* PLT-HZ suspension is prepared by the following method: Saccharomyces boulardii PLT-HZ was inoculated onto YPD medium and incubated at 25–30°C for 1–2 days to activate it. Single colonies were then transferred to YPD liquid medium and incubated for 1–2 days. The cells were collected by centrifugation, and the colony surface was rinsed with sterile water to prepare a Saccharomyces boulardii PLT-HZ suspension, which was then adjusted to a concentration of 1–9 × 10⁻⁶. 8 A suspension of *Saccharomyces boulardii* PLT-HZ was obtained by measuring cells / mL.

[0010] Preferably, the tea leaves in step 1) are green tea, black tea, white tea, dark tea, Pu-erh tea, or oolong tea; the mass ratio of tea leaves to water is 1:20, the extraction time is 2 hours, and the mass ratio of glucose to tea extract is 2:100.

[0011] Preferably, in step 2), the concentration of the *Aspergillus cristatus* PLT-PE suspension is 1 × 10⁻⁶. 7 The inoculum size was 5%, the fermentation time was 4 days, and the inoculum size was 5%.

[0012] Preferably, in step 3), the concentration of the *Saccharomyces boulardii* PLT-HZ suspension is 1 × 10⁻⁶. 8 The inoculum size was 1% (1 / mL), and the fermentation time was 1 day.

[0013] Preferably, in step 5), the mycelium of *Aspergillus cristatus* PLT-PE is placed in water at a mass ratio of 1:20, the extraction temperature is 80℃, the extraction time is 1h, the ethanol concentration is 95%, and the ethanol precipitation time is 4h.

[0014] Preferably, in step 6), the *Saccharomyces boulardii* PLT-HZ cells are placed in water at a mass ratio of 1:20, the extraction temperature is 80℃, the high-pressure homogenization extraction time is 0.5h, and the high pressure for high-pressure homogenization is 170MPa.

[0015] In another aspect, the present invention provides a fermented tea extract prepared by the above method.

[0016] In another aspect, the present invention provides the application of the above-mentioned fermented tea extract in the preparation of anti-photoaging and oil-controlling products.

[0017] One of the strains used in this invention is *Eurotium cristatum* PLT-PE, isolated from Yunnan Pu'er tea. This strain has the highest polysaccharide content among the 20 common fermented tea screening strains. Another strain is *Saccharomyces boulardii* PLT-HZ, isolated from Anhua black brick tea. This strain has the highest polypeptide content among the 20 common fermented tea screening strains. These two strains are the most important beneficial bacteria in fermented tea, exhibiting strong adaptability to the tea system. *Eurotium cristatum* PLT-PE fermentation produces highly active extracellular polysaccharides, enhancing biological activity, and also generates various enzymes that promote methylation, sulfation, and glycosylation of tea polyphenols, improving stability. Yeast fermentation of tea significantly increases the biological activity and content of proteins, vitamin A, and other substances, enhancing the aroma, taste, flavor, and functionality of the tea.

[0018] Compared with the prior art, the present invention has the following advantages: 1) This invention utilizes the synergistic effect of tea extract and *Aspergillus cristatus* PLT-PE and *Saccharomyces boulardii* PLT-HZ dual-strain mimicry fermentation to simulate the natural fermentation process of tea. First, *Aspergillus cristatus* PLT-PE generates a large number of extracellular enzymes, causing methylation, sulfation, and glycosylation of tea polyphenols, thus improving the stability and functionality of the active ingredients, while simultaneously producing a large amount of bioactive fungal polysaccharides. Then, *Saccharomyces boulardii* PLT-HZ fully degrades a large amount of tea protein in the tea, resulting in a product rich in small molecule peptides. These small molecule peptides have strong functional activity and are easily absorbed by the skin. Furthermore, the small molecule peptides also promote yeast growth and fermentation, thereby enhancing the yeast's biotransformation capacity and generating more active ingredients.

[0019] 2) The tea fermentation extract obtained in this invention can regulate the sebum secretion of the skin. (a) The fermentation extract can reduce the sebum secreted by sebaceous gland cells SZ95; (b) The fermentation extract can increase the expression of GATA6 protein in keratinocytes and prevent excessive keratinization of hair follicles in the infundibulum; (c) The fermentation extract has a certain inhibitory effect on 5α reductase activity; (d) It regulates the inflamed hair follicles around the sebaceous glands and reduces the secretion of inflammatory factors and inflammatory mediators TNF-α, IL-6, and PGE2.

[0020] 3) The tea fermentation extract obtained by this invention can improve photoaging of the skin. (a) The fermentation extract can reduce the production of reactive oxygen species (ROS) in skin fibroblasts caused by ultraviolet radiation; (b) The fermentation extract can improve the damage of ultraviolet radiation to type I collagen in the skin; 4) This invention uses tea leaves as raw material, and the fermented tea leaves have anti-photoaging and oil-controlling effects. It has the characteristics of being natural and green, and is more likely to be recognized by modern consumers.

[0021] 5) After fermentation, the present invention selects different extraction methods according to the active ingredients of the species, and finally mixes and extracts them, which is targeted.

[0022] 6) This invention shows that tea extract, after being co-fermented by *Aspergillus cristatus* PLT-PE and *Saccharomyces boulardii* PLT-HZ, is significantly superior to non-fermented extract in terms of anti-photoaging and oil control effects, and is also superior to tea extract fermented by a single strain.

[0023] 7) This invention shows that the tea extract, after being co-fermented by *Aspergillus cristatus* PLT-PE and *Saccharomyces boulardii* PLT-HZ, is significantly superior to the commercially available fermented tea extracts fermented by *Aspergillus cristatus* PLT-PE and *Saccharomyces boulardii* PLT-HZ in terms of anti-photoaging and oil control effects.

[0024] 8) The main raw material of this invention is tea leaves, which are abundant and inexpensive. It does not require the use of large amounts of organic solvents, has high bioavailability, is environmentally friendly and does not put any environmental pressure on the environment. It also does not require special treatment of the raw materials and has a very good prospect for industrialization. Attached Figure Description

[0025] Figure 1 This is a diagram showing the sebum secretion of SZ95 sebaceous gland cells in test example 1.

[0026] Figure 2 This figure shows the expression of GATA6 protein in HaCaT cells in test example 2.

[0027] Figure 3 The graph shows the activity of 5α-reductase in test example 3.

[0028] Figure 4 This is a diagram showing the amount of TNF-α produced in HaCaT cells in test example 4.

[0029] Figure 5 This is a diagram showing the amount of IL-6 produced in HaCaT cells in test example 4.

[0030] Figure 6 The figure shows the amount of PGE2 produced in HaCaT cells in test example 4.

[0031] Figure 7 This is a diagram showing the amount of ROS generated in keratinocytes in test example 5.

[0032] Figure 8 This is a diagram showing the amount of type I collagen produced in fibroblasts in test example 6. Detailed Implementation

[0033] The following examples will illustrate the implementation of this application in detail, so that the process of how this application uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0034] Unless otherwise specified, all raw materials and equipment used in this application are commonly used in the field and are derived from commercially available products. Unless otherwise specified, all methods used in this application are conventional methods in the field.

[0035] There are many other feasible technical solutions in this application, which will not be listed here. All technical solutions claimed in the claims of this application are feasible.

[0036] The terms "comprising" or "including" are intended to indicate that a composition (e.g., a medium) and a method include the listed elements, but do not exclude other elements. When used to define compositions and methods, "consisting substantially of" means excluding other elements that are of any significance to the combination for the stated purpose. Therefore, a composition consisting substantially of the elements defined herein does not exclude other materials or steps that do not materially affect the essential and novel features of the claimed application. "Constitutes" means excluding trace elements and substantial method steps that are other components. Embodiments defined by each of these transitional terms are within the scope of this application.

[0037] By analyzing the intracellular polysaccharide content of 20 strains of *Aureobasidium triflorum* selected from 20 well-known Chinese fermented tea varieties and purchased *Aureobasidium triflorum* strains, a strain with the highest intracellular polysaccharide content, GT-10, was obtained and named *Aureobasidium triflorum* PLT-PE.

[0038] By analyzing the protein content of 20 yeast strains screened from 20 well-known Chinese fermented tea varieties and a purchased *Saccharomyces bladderwracki* strain after cell wall disruption, a yeast strain JY-8 with the highest protein content was obtained and named *Saccharomyces bladderwracki* PLT-HZ. The results are shown in Tables 7 and 8. *Eurotium cristatum* PLT-PE: deposited on June 30, 2023, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40715.

[0039] Purchased *Eurotium cristatum*: Purchased from Beina Biotechnology, product number BNCC46563. Saccharomyces boulardii PLT-HZ: deposited on June 30, 2023, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27760.

[0040] Purchased Saccharomyces boulardii: Commercially available Saccharomyces boulardii was isolated from purchased Saccharomyces boulardii powder.

[0041] Example 1: Tea extract PL01 (without added fermentation bacteria) 1) After washing the tea leaves, add them to water at a ratio of 1:20 (tea leaves:water). Heat to 90-100℃ and extract for 2 hours. Filter and dilute with water to obtain a tea extract. Add glucose to the extract to obtain a tea fermentation medium, then sterilize it. The glucose:tea extract ratio in the tea fermentation medium is 2:100.

[0042] 2) Under aseptic conditions, inoculate 10 ml of sterile water without bacterial solution and incubate in an incubator at 25-30°C for 4 days.

[0043] 4) After the fermentation is completed, add sterile water to the tea fermentation medium at a volume ratio of 1%. The fermentation conditions are: temperature 25-30℃, shaking speed 100-120r / min, and fermentation time 1 day.

[0044] 5) After completion, centrifuge and filter, add 40% D101 resin, adsorb for 1 hour, filter, and obtain filtrate. Add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol to the filtrate as preservatives to obtain tea extract.

[0045] Example 2: Tea fermentation extract PL02 (with only *Aspergillus cristatus* PLT-PE added) 1) After washing the tea leaves, add them to water at a ratio of 1:20 (tea leaves:water). Heat to 90-100℃ and extract for 2 hours. Filter and dilute with water to obtain a tea extract. Add glucose to the extract to obtain a tea fermentation medium, then sterilize it. The glucose:tea extract ratio in the tea fermentation medium is 2:100.

[0046] 2) Under aseptic conditions, the frozen *Aspergillus cristatus* PLT-PE was inoculated onto PDA medium for activation and incubated at 25–30°C for 5–7 days. Then, the mycelia were transferred to PDA solid medium and incubated for another 5–7 days. The colony surface was then rinsed with sterile water to prepare a *Aspergillus cristatus* PLT-PE spore suspension, and its concentration was adjusted to 1 × 10⁻⁶. 7 The bacterial suspension was obtained by dividing the bacterial count by 100 cells / mL.

[0047] 3) By volume, inoculate the bacterial suspension of *Aspergillus cristatus* PLT-PE into sterilized tea fermentation medium at a 5% inoculation rate for fermentation. The fermentation conditions are: temperature 25-30℃, shaking speed 100-120 r / min, and fermentation time 4 days.

[0048] 4) After fermentation is complete, inoculate with sterile water at a volume ratio of 1%. Fermentation conditions are: temperature 25-30℃, shaking speed 100-120r / min, and fermentation time 1 day.

[0049] 5) After fermentation is complete, use a 200-mesh filter to collect the mycelium of *Aspergillus cristatus* PLT-PE, and use a tubular centrifuge to obtain the fermentation filtrate.

[0050] 6) The collected mycelia of *Aspergillus cristatus* PLT-PE were added to deionized water at a mass ratio of 1:20 and extracted at 80℃ for 1 hour. After extraction, the extract was concentrated to 1 / 10 of its original volume. Precipitation was then performed using 95% ethanol at a ratio of 1:10 for 4 hours. The ethanol was removed by vacuum distillation to obtain the polysaccharide extract of *Aspergillus cristatus* PLT-PE.

[0051] 7) Mix the fermentation filtrate and the polysaccharide extract of *Aspergillus cristatus* PLT-PE, add 40% D101 resin, adsorb for 0.5-10 hours, filter, and obtain the filtrate. Add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol to the filtrate as preservatives to obtain tea fermentation filtrate PL02.

[0052] Example 3: Tea fermentation extract PL03 (with only *Bladic yeast bradycamii* PLT-HZ added) 1) After washing the tea leaves, add them to water at a ratio of 1:20 (tea leaves:water). Heat to 90-100℃ and extract for 2 hours. Filter and dilute with water to obtain a tea extract. Add glucose to the extract to obtain a tea fermentation medium, then sterilize it. The glucose:tea extract ratio in the tea fermentation medium is 2:100.

[0053] 2) Under aseptic conditions, the frozen *Saccharomyces boulardii* PLT-HZ was inoculated onto YPD medium for activation and incubated at 25–30°C for 1–2 days. Single colonies were then transferred to YPD liquid medium and incubated for 1–2 days. The cells were collected by centrifugation, and the colony surface was rinsed with sterile water to prepare a *Saccharomyces boulardii* PLT-HZ suspension, which was adjusted to a concentration of 1 × 10⁻⁶. 8 The bacterial suspension was obtained by dividing the bacterial count by 100 cells / mL.

[0054] 3) By volume, inoculate with 5% sterile water. Fermentation conditions: temperature 25-30℃, shaking speed 100-120 r / min, fermentation time 4 days.

[0055] 4) After fermentation is complete, inoculate the Saccharomyces boulardii PLT-HZ suspension into the sterilized fermentation medium at a volume ratio of 1% for fermentation. The fermentation conditions are: temperature 25-30℃, shaking speed 100-120r / min, and fermentation time 1 day.

[0056] 5) After fermentation is complete, the filtrate is centrifuged using a tubular centrifuge to collect the PLT-HZ cells of Saccharomyces boulardii and obtain the fermentation filtrate.

[0057] 6) The collected Saccharomyces bladderwracki PLT-HZ bodies were added to deionized water at a mass ratio of 1:20, and homogenized at 170 MPa for 0.5 h at 80 °C. After extraction, the mixture was centrifuged and the supernatant was collected to obtain the Saccharomyces bladderwracki PLT-HZ extract.

[0058] 7) Mix the fermentation filtrate and the Saccharomyces boulardii PLT-HZ extract, add 40% D101 resin, adsorb for 0.5-10 hours, filter, and obtain the filtrate. Add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol to the filtrate as preservatives to obtain tea fermentation filtrate PL03.

[0059] Example 4: Tea fermentation extract PL04 ​​(with added *Aspergillus cristatus* PLT-PE and *Saccharomyces boulardii* PLT-HZ) 1) After washing the tea leaves, add them to water at a ratio of 1:20 (tea leaves: water). Heat to 90-100℃ and extract for 2 hours. Filter and dilute with water to obtain a tea extract. Add glucose to the extract to obtain a tea fermentation medium, then sterilize it. The glucose:tea extract ratio in the tea fermentation medium is 2:100.

[0060] 2) Under aseptic conditions, the frozen *Aspergillus cristatus* PLT-PE was inoculated onto PDA medium for activation and incubated at 25–30°C for 5–7 days. Then, the mycelia were transferred to PDA solid medium and incubated for another 5–7 days. The colony surface was then rinsed with sterile water to prepare a *Aspergillus cristatus* PLT-PE spore suspension, and its concentration was adjusted to 1 × 10⁻⁶. 7A *Aureobasidium cristatum* PLT-PE suspension was obtained by inoculating *Saccharomyces boulardii* PLT-HZ onto YPD medium for activation. The culture was then incubated at 25–30°C for 1–2 days. Single colonies were then transferred to YPD liquid medium and incubated for 1–2 days. The cells were collected by centrifugation, and the colony surface was rinsed with sterile water to prepare a *Saccharomyces boulardii* PLT-HZ suspension, which was adjusted to a concentration of 1 × 10⁻⁶. 8 A suspension of *Saccharomyces boulardii* PLT-HZ was obtained by measuring cells / mL.

[0061] 3) By volume, inoculate the *Aspergillus cristatus* PLT-PE suspension into the sterilized tea fermentation medium at a 5% inoculation rate for fermentation. The fermentation conditions are: temperature 25-30℃, shaking speed 100-120 r / min, and fermentation time 4 days.

[0062] 4) After fermentation is complete, inoculate with a 1% (by volume) suspension of Saccharomyces boulardii PLT-HZ for fermentation. The fermentation conditions are: temperature 25-30℃, shaking speed 100-120 r / min, and fermentation time 1 day.

[0063] 5) After fermentation is complete, first use a 200-mesh filter to collect the mycelium of *Aspergillus cristatus* PLT-PE, then use a tubular centrifuge to filter it a second time to collect *Saccharomyces boulardii* PLT-HZ cells and obtain the fermentation filtrate.

[0064] 6) The collected mycelia of *Aspergillus cristatus* PLT-PE were added to deionized water at a mass ratio of 1:20 and extracted at 80℃ for 1 hour. After extraction, the extract was concentrated to 1 / 10 of its original volume. Precipitation was then performed using 95% ethanol at a ratio of 1:10 for 4 hours. The ethanol was removed by vacuum distillation to obtain the polysaccharide extract of *Aspergillus cristatus* PLT-PE.

[0065] 7) The collected Saccharomyces bladderwracki PLT-HZ bodies were added to deionized water at a mass ratio of 1:20, and homogenized at 170 MPa for 0.5 h at 80 °C. After extraction, the mixture was centrifuged and the supernatant was collected to obtain the Saccharomyces bladderwracki PLT-HZ extract.

[0066] 8) Mix the fermentation filtrate, the polysaccharide extract of *Aspergillus cristatus* PLT-PE, and the extract of *Saccharomyces boulardii* PLT-HZ, add 40% D101 resin, adsorb for 0.5–10 h, filter, and obtain the filtrate. Add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol to the filtrate as preservatives to obtain tea fermentation filtrate PL04.

[0067] Example 5: Tea fermentation extract PL05 (purchased *Aurotriarcha cristatum* and *Saccharomyces boulardii*) 1) After washing the tea leaves, add them to water at a ratio of 1:20 (tea leaves: water). Heat to 90-100℃ and extract for 2 hours. Filter and dilute with water to obtain a tea extract. Add glucose to the extract to obtain a tea fermentation medium, then sterilize it. The glucose:tea extract ratio in the tea fermentation medium is 2:100.

[0068] 2) Under aseptic conditions, the purchased *Aspergillus cristatus* was inoculated onto PDA medium for activation and cultured at 25–30°C for 5–7 days. Then, the mycelium was transferred to PDA solid medium and cultured for another 5–7 days. The colony surface was then rinsed with sterile water to prepare a *Aspergillus cristatus* spore suspension, and its concentration was adjusted to 1 × 10⁻⁶. 7 The concentration of the yeast cells was increased to 1 × 10⁶ cells / mL to obtain a bacterial suspension. Commercially available *Saccharomyces boulardii* was inoculated onto YPD medium for activation and incubated at 25–30°C for 1–2 days. Single colonies were then transferred to YPD liquid medium and incubated for 1–2 days. The cells were collected by centrifugation, and the colony surface was rinsed with sterile water to prepare a yeast suspension, which was then adjusted to a concentration of 1 × 10⁶ cells / mL. 8 The bacterial suspension was obtained by dividing the bacterial count by 100 cells / mL.

[0069] 3) By volume, inoculate a suspension of *Aspergillus cristatus* spores into a sterile fermentation medium at a 5% inoculation rate. Fermentation conditions are: temperature 25–30°C, shaking speed 100–120 r / min, and fermentation time 4 days.

[0070] 4) After fermentation is complete, inoculate the yeast suspension into the sterilized fermentation medium at a volume ratio of 5% for fermentation. The fermentation conditions are: temperature 25-30℃, shaking speed 100-120r / min, and fermentation time 1 day.

[0071] 5) After fermentation is complete, use a 200-mesh filter to collect the mycelium of *Aspergillus cristatus*, and use a tubular centrifuge to collect the yeast cells and obtain the fermentation filtrate.

[0072] 6) The collected mycelia of *Aspergillus cristatus* were added to deionized water at a mass ratio of 1:20 and extracted at 80℃ for 1 hour. After extraction, the extract was concentrated to 1 / 10 of its original volume. Precipitation was then performed using 95% ethanol at a ratio of 1:10 for 4 hours. The ethanol was removed by vacuum distillation to obtain the polysaccharide extract of *Aspergillus cristatus*.

[0073] 7) The collected yeast cells were added to deionized water at a mass ratio of 1:20 and homogenized at 170 MPa for 0.5 h at 80℃. After extraction, the cells were centrifuged and the supernatant was collected to obtain the yeast extract.

[0074] 8) Mix the fermentation filtrate, the polysaccharide extract of *Aspergillus cristatus* and the yeast extract, add D101 resin, adsorb for 1 hour, filter, and obtain the filtrate. Add a certain amount of preservative to the filtrate to obtain tea fermentation filtrate PL05.

[0075] The following tests were performed in all of the above Examples 1, 2, 3, 4, and 5: Test Example 1: Effect of tea fermentation extract on lipid content in LA-induced SZ95 cells.

[0076] 1.1 Cell Culture Take SZ95 cells from liquid nitrogen, quickly place the cells in a 37°C water bath to dissolve, then add 5 ml of preheated culture medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, add preheated complete culture medium, gently pipette to resuspend the cells, and culture.

[0077] 1.2 Cell Seeding and Culture Collect cells in the logarithmic growth phase. After culturing for 24 hours, collect and count SZ95 cells that have grown to 80-90% confluence. Based on the counting results, distribute 0.5 ml of cell suspension (3.0 × 10⁻⁶ cells) per well. 5 -6.0×10 5 (cells / ml) were seeded into 24-well plates and incubated at 37°C in a 5.0% CO2 incubator for 24 hours.

[0078] 1.3 Solution preparation Prepare the working solution of the test substance according to Table 1, with 500 μl of culture medium per well.

[0079] Table 1. Preparation of tea fermentation broth for LA-induced lipid content in SZ95 cells. Note: BC represents the blank group, and NC represents the model group; 1.4 Administration According to the experimental design, the drug was administered in groups, with 500 μl per well. Each group had three replicates, and the drugs were incubated in an incubator (37℃, 5% CO2, 95% RH) for 24 h.

[0080] 1.5LA induces SZ95 cells After 24 hours of cell culture, the culture medium in the wells was discarded, and the experimental group samples were added to each well in sequence. Except for the blank group, the other groups were induced with LA at a final concentration of 0.25 mg / ml. The blank group was added to each well with complete culture medium, and the sample groups were added to complete culture medium containing different concentrations of samples in sequence. The 24-well plate was placed in a 37℃, 5.0% CO2 incubator and cultured for 24 hours.

[0081] 1.6 Oil Red O Staining After culturing cells for 24 hours, the culture medium in the wells was discarded, and 300 μl of Oil Red O staining solution was added to each well. After incubation for 30 min, the staining solution was discarded, and the cells were washed twice with 500 μl of PBS. The cells were then photographed under a microscope.

[0082] 1.7 Lipid content detection After taking photos under a microscope, discard the liquid in the well plate, add 500 μl of isopropanol to each well, and measure the absorbance at 495 nm using a microplate reader.

[0083] 1.8 Results from Figure 1 The results showed that, based on the LA (linoleic acid)-induced SZ95 cell detection, compared with the blank group BC, the negative group NC significantly increased lipid content (p<0.001); compared with NC, the positive group PC significantly decreased lipid content (p<0.001); and compared with NC, 10 mg / ml tea fermentation broth significantly decreased lipid content (p<0.001). Among them, the tea fermentation filtrate PL04 ​​obtained in Example 4 showed the highest reduction rate of lipid content, reaching 54.25%. The reduction of sebum content in other examples was lower than that of the tea fermentation filtrate PL04 ​​obtained in Example 4. This indicates that fermented tea extract is more effective than non-fermented extract in inhibiting the sebum secretion function of sebaceous gland cells, and the effect of dual-strain fermentation is better than that of single-strain fermentation.

[0084] Test Example 2: Test on the effect of tea fermentation extract on the expression of GATA6 protein in HaCaT cells.

[0085] 2.1 Cell Culture Take keratinocytes from liquid nitrogen, quickly place the cells in a 37°C water bath to dissolve them, then add 5 ml of preheated culture medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, add preheated complete culture medium, gently pipette to resuspend the cells, and culture them.

[0086] 2.2 Cell Seeding and Culture Collect cells in the logarithmic growth phase. After culturing for 24 hours, collect and count the keratinocytes that have grown to 80-90%. Based on the counting results, distribute 0.5 ml of cell suspension (3.0 × 10⁻⁶ cells / well) per well. 5 -6.0×10 5(cells / ml) were seeded into 24-well plates and incubated at 37°C in a 5.0% CO2 incubator for 24 hours.

[0087] 2.3 Prepare the working solution of the test substance according to Table 2, with 500 μl of culture medium per well.

[0088] Note: BC represents the blank group, and NC represents the model group; 2.4 UV-induced keratinocyte model After 24 hours of cell culture, the culture medium in the wells was discarded, and the experimental group samples were added to each well in sequence for 2 hours of cell pretreatment. The culture medium in the wells was then discarded, and 500 μl of PBS was added to each well. Except for the blank group, the other groups were induced with 90 mJUVB + 0.8 JUVA. 500 μl of complete culture medium was added to each well of the blank group, and 500 μl of complete culture medium solution of different doses of tea fermentation broth was added to each well of the sample group. The 24-well plates were placed in a 37℃, 5.0% CO2 incubator for 24 hours.

[0089] 2.5GATA6 test Follow the kit's instructions.

[0090] 2.6 Results from Figure 2 It was found that, based on the UV (ultraviolet light) induced keratinocyte detection results, compared with the blank group BC, the negative group NC significantly reduced GATA6 content (p<0.001); compared with NC, both 10 mg / ml tea fermentation extract and tea extract increased GATA6 content (p<0.001). Among them, the tea fermentation filtrate PL04 ​​obtained in Example 4 showed the highest increase in GATA6 content, reaching 26%. The increases in GATA6 in other examples were all lower than those of the tea fermentation filtrate PL04 ​​obtained in Example 4. This indicates that the tea fermentation filtrate PL04 ​​obtained in Example 4 prevents excessive keratinization of the infundibulum by negatively regulating the proliferation of ORS and IFE keratinocytes and sebaceous gland cells through GATA6.

[0091] Test Example 3: Test on the inhibitory effect of tea fermentation extract on 5α reductase activity.

[0092] 3.15α-Reductase Solution Preparation Four clean-grade male rats were used, fasted but allowed free access to water, and euthanized overnight by dislocation. Liver samples were rapidly collected to obtain 5α-reductase extract. The protein content of the 5α-reductase extract was quantified using a Bradford protein quantification kit, and the total protein content was expressed as the 5α-reductase content. The kit was operated according to the instructions. 3.2 Preparation of the testosterone standard curve Testosterone stock solution was diluted with methanol to obtain a series of concentrations: 5 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L, 400 μmol / L, 800 μmol / L, 1000 μmol / L, 1500 μmol / L, and 2000 μmol / L. 1 mL of each graded dilution of testosterone was precisely pipetted and filtered through a 0.22 μm microporous membrane into a sample vial. The injection volume was set to 20 μL. The peak area of ​​testosterone was detected by HPLC. The HPLC conditions were as follows: column: Luna C18(2) column (4.6 × 250 mm, 5 μm) stainless steel column; column temperature: 30℃; mobile phase: methanol / water 70 / 30 (v / v); flow rate: 1.0 mL / min; detection wavelength: 242 nm; injection volume: 20 μL. A standard curve was plotted with peak area A (AU*min) on the ordinate and testosterone concentration T (μmol / L) on the abscissa.

[0093] 3.35α-reductase inhibition rate determination In a 1000 μL bioassay system, 300 μL of PBS buffer (pH 7.4), 500 μL of 0.76 mg / mL 5α-reductase extract dilution, 50 μL of 2 mmol / L testosterone solution, 50 μL of the test sample, and 100 μL of 2 mmol / L NADPH solution were added sequentially to a centrifuge tube. The mixture was rapidly mixed in a vortex mixer for 3 seconds and reacted in a 37°C water bath for 30 minutes. At 0 min (t0) and 30 min (t30), 400 μL of the reaction solution was quickly added to 800 μL of methanol, and the reaction was terminated by vortexing for 5 seconds. The mixture was then centrifuged at 10000 rpm for 15 minutes to remove protein precipitate. The supernatant was filtered through a 0.22 μm microporous membrane into a liquid chromatography bottle for HPLC analysis under the same conditions as in section 3.2. The change in peak area (T) at the start of the reaction (t0) and at t30 indicates the change in reaction concentration.

[0094] Set up BC (enzyme inactivated with methanol), NC, PC (dutasteride), and sample tubes (tea fermentation broth), with 3 replicates per tube. See Table 3 for details. Table 3. Substances and their contents in different reaction tubes The relevant calculation formulas are as follows.

[0095] △T(μmol / L)=Ct0-Ct 30 C t0 C represents the testosterone concentration at 0 min. t30 The value represents the testosterone concentration at 30 minutes, and ΔT represents the difference in testosterone concentration before and after the reaction.

[0096] 5α-reductase activity (μmolT / gprot·30min) = 5α-reductase activity (μmolT / gprot·30min) is defined as the amount of testosterone converted from one gram of 5α-reductase extract per 30 minutes, and C5α-reductase (g / L) represents the concentration of 5α-reductase extract, which is the concentration of 5α-reductase extract protein.

[0097] Sample / Dutaxiongan 5α-reductase inhibition rate (%) = 3.4 Results from Figure 3 It was found that the positive group PC significantly reduced 5α-reductase activity (p<0.001); compared with NC, both 100 mg / ml tea fermentation extract and tea extract reduced 5α-reductase activity (p<0.001). Among them, the tea fermentation filtrate PL04 ​​obtained in Example 4 showed the highest reduction in 5α-reductase activity, reaching 19.28%. The reduction rates of 5α-reductase activity in other examples were all lower than those of the tea fermentation filtrate PL04 ​​obtained in Example 4. This indicates that the tea fermentation filtrate PL04 ​​obtained in Example 4 achieves the oil control effect by reducing 5α-reductase activity.

[0098] Test Example 4: Test of the effect of tea fermentation extract on UVB-induced secretion of TNF-α, IL-6 and PGE2 by keratinocytes.

[0099] 4.1 Cell resuscitation and passage Keratinocytes were taken from liquid nitrogen and quickly placed in a 37°C water bath to dissolve. Then, 5 ml of preheated culture medium was added, and the cells were centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended by gently pipetting with preheated complete culture medium for further culture. 4.2 Cell Seeding and Culture Collect and count the keratinocytes that have grown to 80-90%. Based on the count results, distribute 0.5 ml of cell suspension (3.0 × 10⁶ cells / well) into each well. 5 After inoculation (cells / ml), the 24-well plates were placed in a 37°C, 5.0% CO2 incubator and incubated for 24 hours.

[0100] 4.3 Solution preparation: Prepare the working solution of the test substance according to Table 4 below, with 500 μl of culture medium per well.

[0101] Table 4. Preparation of solutions for detecting inflammatory factors in the UVB-induced keratinocyte model. Note: BC is the blank group, NC is the model group, and PC is the positive group.

[0102] 4.4 UVB induces keratinocytes After culturing cells for 24 hours, discard the culture medium in each well, add the experimental group samples to each well sequentially, and pretreat the cells for 2 hours; discard the culture medium in each well, add 500 μl of PBS to each well, and except for group BC, use 50 mJ / cm² PBS for the other groups. 2 After UVB induction, 500 μl of complete culture medium was added to each well of the BC group, 20 ng / ml of dexamethasone was added to the PC group, and 500 μl of complete culture medium solution containing tea fermentation broth was added to each of the sample groups. The 24-well plates were placed in a 37℃, 5.0% CO2 incubator for 24 h.

[0103] 4.5 Detection of TNF-α, IL-6, and PGE2 Collect the supernatant and detect the levels of TNF-α, IL-6, and PGE2 according to the Elisα kit instructions.

[0104] 4.6 Results from Figure 4 As observed in Figures 5 and 6, based on the UV (ultraviolet light) induced keratinocyte detection results, compared with the blank group BC, the negative group NC significantly increased the levels of TNF-α, IL-6, and PGE2 (p<0.001); compared with NC, the positive group PC significantly decreased the levels of TNF-α, IL-6, and PGE2 (p<0.001); compared with NC, both 10 mg / ml tea fermentation extract and tea extract decreased the levels of TNF-α, IL-6, and PGE2 (p<0.001). Among them, the tea fermentation filtrate PL04 ​​obtained in Example 4 showed the highest reduction rate of TNF-α, IL-6, and PGE2, reaching 25.8%, 25.2%, and 39.1%, respectively. The reduction rates of TNF-α, IL-6, and PGE2 in other examples were all lower than those of the tea fermentation filtrate PL04 ​​obtained in Example 4. This indicates that the tea fermentation filtrate PL04 ​​obtained in Example 4 can reduce skin inflammation, thereby preventing inflammation of hair follicles around the sebaceous glands.

[0105] Test Example 5: Test of the effect of tea fermentation extract on UVB-induced ROS secretion in keratinocytes.

[0106] 5.1 Cell resuscitation and passage Keratinocytes were collected from liquid nitrogen; the cells were quickly dissolved in a 37°C water bath, then 5 ml of preheated complete culture medium was added, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended by gently pipetting with preheated complete culture medium, and cultured. Keratinocytes that had grown to 80-90% were washed twice with PBS, digested with 0.25% trypsin, incubated at 37°C for 5 min, digestion was stopped by adding complete culture medium, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended by gently pipetting with preheated complete culture medium, and passaged at a ratio of 1:3 to 1:5. 5.2 Cell Seeding and Culture Collect and count keratinocytes that have grown to 80-90%. Based on the count results, distribute 0.5 ml of cell suspension (3.0 x 10⁻⁶ cells / well) into each well. 5 (cells / ml) were seeded into 24-well plates, and the seeded 24-well plates were placed in a 37℃, 5.0% CO2 incubator for 24 h.

[0107] 5.3 Solution preparation: Prepare the working solution of the test substance according to the table below, with 500 μl of culture medium per well.

[0108] Table 5. Preparation of solutions for detecting ROS in UVB-induced keratinocytes using tea fermentation extract. Note: BC is the blank group, NC is the negative group, and PC is the positive group.

[0109] 5.4 UVB induces keratinocytes After culturing cells for 24 hours, discard the culture medium in each well, add the experimental group samples to each well sequentially, and pretreat the cells for 2 hours; discard the culture medium in each well, add 500 μl of PBS to each well, and except for BC, use 50 mJ / cm² water for the other groups. 2 After UVB induction, 500 μl of complete culture medium was added to each well of NC, and 500 μl of complete culture medium solution containing 0.1% ascorbic acid was added to each well of PC. 500 μl of complete culture medium solution containing tea extract or tea fermentation filtrate was added to each sample group in turn. The 24-well plates were placed in an incubator at 37℃ and 5.0% CO2 for 24 h.

[0110] 5.5 Reactive Oxygen Detection Wash cells twice with PBS. Add 500 μl of DCFH-DA probe diluted with FluoroBrite™ complete medium according to the ROS detection kit instructions. Incubate at 37°C and 5.0% CO2 for 20 min. Detect fluorescence intensity with a microplate reader. Wash three times with FluoroBrite™ complete medium.

[0111] 5.6 Results from Figure 7 It was found that the positive group PC significantly reduced ROS (p<0.001); compared with NC, the tea fermentation filtrate PL04 ​​obtained in Example 4 showed the highest ROS reduction, reaching 16.28%, while the ROS reduction rates of other examples were all lower than those of the tea fermentation filtrate PL04 ​​obtained in Example 4. This indicates that the tea fermentation filtrate PL04 ​​obtained in Example 4 achieves its anti-photoaging effect by reducing ROS.

[0112] Test Example 6: Test of the effect of tea fermentation extract on UVB-induced secretion of type I collagen in fibroblasts 6.1 Cell resuscitation and passage Fibroblasts were taken from liquid nitrogen and quickly placed in a 37°C water bath to thaw (the 37°C water bath was turned on in advance), thawing within 2 minutes. Then, they were added to preheated culture medium (at a ratio of 1 mL frozen cells + 5 mL preheated complete culture medium), centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended by gently pipetting with preheated complete culture medium. The cells were then cultured. Fibroblasts that had grown to 80-90% were washed twice with PBS, digested with 0.25% trypsin, incubated at 37°C for 1 minute, the digestion was stopped by adding culture medium, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended by gently pipetting with preheated complete culture medium. The cells were then passaged at a ratio of 1:2 to 1:3. 6.2 Cell Seeding and Culture Cell culture was performed using high-glucose DMEM medium (containing 10% FBS and 1% penicillin antibiotics). HSF cells that had grown to 80-90% confluence were collected and counted. Based on the count results, cells were divided into groups of 2-8 × 10⁶ cells per cell line. 4 Cells / wells were seeded into 24-well cell culture plates.

[0113] 6.3 Solution preparation: Prepare the working solution of the test substance according to the table below, with 500 μl of culture medium per well.

[0114] Table 6. Preparation of solutions for type I collagen detection in UVB-induced fibroblast model. Note: BC represents the blank group, and NC represents the model group. 6.4 Administration Drug administration was initiated when cell deposition in the 24-well plates reached 40%–60%. The control group received 500 μl of PBS, while the PC and sample groups each received 500 μl of PBS containing the corresponding concentration of the sample. Except for the control group, the other groups received 50 mJ / cm² PBS. 2 After UVB irradiation, PBS was discarded. 500 μl of culture medium was added to each well of the Control group, and 500 μl of culture medium containing the corresponding concentration of the sample was added to each well of the PC and sample groups. After drug administration, the 96-well plate was placed in an incubator (37℃, 5% CO2) and incubated for 24 h.

[0115] 6.5 Collagen I Detection: Collect the supernatant and detect the content of type I collagen using ELISA according to the kit instructions.

[0116] 6.6 Results from Figure 8It was found that NC significantly reduced the secretion of type I collagen (p<0.001). Compared with NC, the tea fermentation filtrate PL04 ​​obtained in Example 4 had the highest rate of promoting type I collagen secretion, reaching 49.29%. The rate of promoting type I collagen secretion in other examples was lower than that of the tea fermentation filtrate PL04 ​​obtained in Example 4. This indicates that the tea fermentation filtrate PL04 ​​obtained in Example 4 achieves its anti-photoaging effect by promoting type I collagen.

[0117] Screening of *Eurotium cristatum* strains: The content of intracellular polysaccharides was determined by screening 20 strains of *Aurotriarcha* from 20 well-known Chinese fermented tea varieties and purchasing *Aurotriarcha* from external sources.

[0118] Under aseptic conditions, *Aspergillus cristatus* was inoculated onto PDA medium for activation and cultured at 25–30°C for 5–7 days. Then, the mycelia were transferred to PDA solid medium and cultured for another 5–7 days. The colony surface was then rinsed with sterile water to prepare a *Aspergillus cristatus* PLT-PE spore suspension, and its concentration was adjusted to 1 × 10⁻⁶. 7 The spore suspension was obtained by extracting spores per mL. 3 mL of the spore suspension was inoculated into 300 mL of PDA-containing liquid medium and cultured at 28°C and 150 rpm for 5 days using a shaker. The mixture was then filtered through a 200-mesh filter and squeezed. Deionized water was added, and the mixture was washed and squeezed again. This process was repeated twice to obtain wet mycelia, which were then weighed. Deionized water was added to a final volume of 50 g, and the mixture was boiled at 90°C for 1 hour. Then, 95% ethanol was added at a ratio of 1:4. The mixture was allowed to stand overnight, allowing polysaccharides to precipitate. The precipitated polysaccharides were then filtered to obtain crude polysaccharide. The results are shown in Table 7, where GT-21 was a purchased *Aspergillus cristatus* strain, and GT-10 was *Aspergillus cristatus* PLT-PE.

[0119] Screening of *Saccharomyces blazei* strains: The content of intracellular proteins (peptides) was determined by screening 20 strains of Saccharomyces boulardii from 20 well-known Chinese fermented teas and purchasing Saccharomyces boulardii.

[0120] Under aseptic conditions, *Saccharomyces boulardii* was inoculated onto YPD medium for activation and incubated at 25–30°C for 1–2 days. Single colonies were then transferred to YPD liquid medium and incubated for 1–2 days. The cells were collected by centrifugation, and the colony surface was rinsed with sterile water to prepare a *Saccharomyces boulardii* suspension, which was then adjusted to a concentration of 1 × 10⁻⁶. 8A *Saccharomyces boulardii* suspension was obtained by inoculating 3 ml of the yeast suspension into an Erlenmeyer flask containing YPD liquid medium and incubating at 30°C with a shaker at 150 rpm for 2 days. After shaking, the bacterial cells were obtained, centrifuged at 4000 rpm for 20 min, the supernatant was discarded, deionized water was added, and the cells were washed and centrifuged again at 4000 rpm for 20 min. This process was repeated twice to obtain wet bacterial cells. Deionized water was added to the wet bacterial cells to a final volume of 50 g, and the cells were sonicated for 15 min with a 7-s interval at 400 W. The cells were then disrupted in an ice bath. The protein content was determined using the Folin-phenol reagent method after disruption, and the results are shown in Table 8. JY-21 was a commercially available *Saccharomyces boulardii* strain, and JY-8 was *Saccharomyces boulardii* PLT-HZ.

[0121] Table 7. Polysaccharide content (mg) of different *Coronavirus cristatum* strains Table 8. Protein content (mg) of different yeast strains The contents not described in detail in this application specification are common knowledge to those skilled in the art.

[0122] As used throughout the specification and claims, the term "comprising" is an open-ended term and should therefore be interpreted as "comprising but not limited to". "Approximately" means that within an acceptable margin of error, those skilled in the art can solve the technical problem and substantially achieve the technical effect within a certain margin of error.

[0123] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a product or system comprising a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a product or system. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the product or system that includes said element.

[0124] The foregoing description illustrates and describes several preferred embodiments of this application. However, as previously stated, it should be understood that this application is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the foregoing teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of this application should be protected within the scope of the appended claims.

Claims

1. A method for preparing a fermented tea extract, characterized in that, Includes the following steps: 1) Add tea leaves to water and heat to 90-100℃ for 1-10 hours to obtain tea extract; add glucose to the extract and sterilize to obtain tea fermentation culture medium; wherein, the mass ratio of tea leaves to water is 1:(5-20) and the mass ratio of glucose to tea extract is (1-5):

100. 2) The concentration is (1~9)×10 7 *Eurotium cristatum* per mL Eurotium cristatum PLT-PE bacterial suspension was inoculated into tea fermentation medium at a volume ratio of 1-5% for fermentation at a fermentation temperature of 25-30℃, a shaking speed of 100-120 r / min, and a fermentation time of 4-7 days to obtain the first fermentation broth. 3) The concentration is (1~9)×10 8 Saccharomyces boulardii CFU / mL Saccharomyces boulardii PLT-HZ bacterial suspension was inoculated into the first fermentation broth at an inoculation rate of 1-5% by volume. The fermentation temperature was 25-30℃, the shaking speed was 100-120 r / min, and the fermentation time was 1-3 days to finally obtain the second fermentation broth. 4) Filter the second fermentation broth through a 200-mesh filter to obtain the mycelium of *Aspergillus cristatus* PLT-PE; then separate the filtrate using a tubular centrifuge to obtain *Saccharomyces boulardii* PLT-HZ cells and obtain the fermentation filtrate. 5) The mycelium of *Aspergillus cristatus* PLT-PE was placed in water at a mass ratio of 1:(1-20) and extracted at 30-90℃ for 0.5-10 h. After extraction, the extract was concentrated to 1 / 10 of its original volume. Then, 95%-100% ethanol was added at a volume ratio of 1:10 for alcohol precipitation for 0.5-10 h. The ethanol was then removed by vacuum distillation to obtain the polysaccharide extract of *Aspergillus cristatus* PLT-PE. 6) Place the Saccharomyces blazei PLT-HZ cells in water at a mass ratio of 1:(1-20), heat to 30-90℃, and homogenize under high pressure for 0.5-10 hours. After extraction, centrifuge and collect the supernatant to obtain the Saccharomyces blazei PLT-HZ extract. 7) Mix the fermentation filtrate, the polysaccharide extract of *Aspergillus cristatus* PLT-PE, and the extract of *Saccharomyces boulardii* PLT-HZ to obtain a mixed solution; add D101 resin at a mass ratio of 40%, adsorb for 0.5–10 h, filter, obtain the filtrate, add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol at a mass ratio to obtain the fermented tea extract; The *Eurotium cristatum* PLT-PE strain was deposited on June 30, 2023, at the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 40715. The *Saccharomyces boulardii* strain PLT-HZ was deposited on June 30, 2023, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 27760.

2. The production method according to claim 1, characterized by, The *Eurotium cristatum* PLT-PE bacterial suspension was prepared by the following method: *Aspergillus cristatus* PLT-PE was inoculated onto PDA medium and incubated at 25–30°C for 5–7 days to activate it. Then, the mycelium was transferred to PDA solid medium and incubated for 5–7 days. Finally, the colony surface was rinsed with sterile water to obtain a *Aspergillus cristatus* PLT-PE spore suspension, and its concentration was adjusted to (1–9) × 10⁻⁶. 7 A suspension of *Aspergillus cristatus* PLT-PE was obtained by measuring 100 cells / mL.

3. The preparation method according to claim 1, characterized in that, The *Saccharomyces bladderwyn* PLT-HZ suspension was prepared by the following method: Saccharomyces boulardii PLT-HZ was inoculated onto YPD medium and incubated at 25–30°C for 1–2 days to activate it. Single colonies were then transferred to YPD liquid medium and incubated for 1–2 days. The cells were collected by centrifugation, and the colony surface was rinsed with sterile water to prepare a Saccharomyces boulardii PLT-HZ suspension. The concentration was adjusted to (1–9) × 10⁻⁶. 8 A suspension of *Saccharomyces boulardii* PLT-HZ was obtained by measuring cells / mL.

4. The preparation method according to claim 1, characterized in that, In step 1), the tea leaves are green tea, black tea, white tea, dark tea, Pu-erh tea, or oolong tea; the mass ratio of tea leaves to water is 1:20, the extraction time is 2 hours, and the mass ratio of glucose to tea extract is 2:

100.

5. The preparation method according to claim 1, characterized in that, The concentration of the Eurotium amstelodami PLT-PE bacterial suspension in the step 2) is 1×10 7 individuals / mL, the inoculation amount is 5%, and the fermentation time is 4 days.

6. The method of claim 1, wherein, In step 3), the concentration of the *Saccharomyces boulardii* PLT-HZ suspension is 1 × 10⁻⁶. 8 The inoculum size was 1% (1 / mL), and the fermentation time was 1 day.

7. The preparation method according to claim 1, characterized in that, In step 5), the mycelium of *Aspergillus cristatus* PLT-PE was placed in water at a mass ratio of 1:20, the extraction temperature was 80℃, and the extraction time was 1 hour; the ethanol concentration was 95%, and the ethanol precipitation time was 4 hours.

8. The method of claim 1, wherein, In step 6), the PLT-HZ cells of Saccharomyces boulardii were placed in water at a mass ratio of 1:20, the extraction temperature was 80℃, the high-pressure homogenization extraction time was 0.5h, and the high pressure for high-pressure homogenization was 170MPa.

9. A fermented tea extract prepared by the method according to any one of claims 1-8.

10. The application of the fermented tea extract according to claim 9 in the preparation of anti-photoaging and oil-controlling products.

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