An adelea longichia ferment and use thereof

Abstract

This invention discloses an *Adelie* longissimus ferment and its applications. The strain was isolated from a glacier in the Tibetan Plateau, with the preservation number CGMCC No. 39193. The fermentation product, prepared through low-temperature fermentation, homogenization, centrifugation, and sterilization by filtration, exhibits significant abilities to scavenge DPPH, ABTS, and ROS free radicals, and can significantly promote HaCaT cell proliferation. At a 5% addition level, the *Adelie* longissimus ferment product showed a DPPH free radical scavenging rate ≥60%; an ABTS free radical scavenging rate ≥90% at a 5% addition level; and in a ROS cell model, a scavenging rate ≥80% at a 1% addition level. At a 1% addition level and cultured for 48 h, the HaCaT cell proliferation rate was ≥50%, demonstrating promising application prospects.

Description

Technical Field

[0001] This application belongs to the field of microbial fermentation technology, specifically relating to an Adélie longissimus ferment and its application. Background Technology

[0002] Skin aging and barrier damage are closely related to the excessive accumulation of free radicals and decreased keratinocyte proliferation activity. Developing microbial-derived active ingredients that are naturally sourced, have clearly defined efficacy, and are highly safe has become an important direction in the research and development of high-end functional active ingredients.

[0003] High-altitude glacial microorganisms, living in extreme environments of low temperatures, strong ultraviolet radiation, and low nutrition, often possess unique antioxidant, stress-repairing, and cell-repairing activities in their metabolites. Yeast fermentation products, such as *Saccharomyces cerevisiae* and *Pichia pastoris*, are rich in components, have high safety, and good compatibility, making them a popular choice for skincare products. However, these yeasts typically have low levels of anti-inflammatory, antioxidant, repairing, and anti-wrinkle active ingredients, and the permitted levels often fail to achieve the expected significant efficacy. In other words, constrained by both the concentration of active ingredients and the permitted levels, relying solely on these traditional yeast ferments is insufficient to support the development of high-end raw materials that offer "high efficacy and low irritation."

[0004] The species *Lysimachia adélie* was first isolated in the Adélie region of Antarctica, and has since been isolated in other extreme environments around the world. It belongs to the kingdom Fungi, phylum Basidiomycota, class Tremella, order Ustilagoeales, family Ustilagoceae, genus *Lysimachia*, and species *Lysimachia adélie*. Current research uses it to produce low-temperature active xylanase and to enhance the aroma complexity of baijiu (Chinese liquor). Summary of the Invention

[0005] To address the shortcomings of existing technologies where microbial-derived active ingredients cannot simultaneously achieve high efficacy and low irritation, this invention creatively utilizes *Saccharomyces cerevisiae* for the development of active ingredients. *Saccharomyces cerevisiae* possesses free radical scavenging properties, reducing intracellular inflammatory factors IL-6, IL-8, and TNF-α, lowering intracellular ROS, promoting cell proliferation, and promoting the secretion of type I and type III collagen. The screened Adeli-1067 exhibits significantly improved performance compared to commonly used *Pichia pastoris* fermentation products, with a 48% increase in DPPH scavenging rate. The ROS intensity of HaCaT cells treated with *Saccharomyces cerevisiae* fermentation product extract was reduced by 37.56% compared to those treated with *Pichia pastoris* fermentation product extract.

[0006] Specifically, this invention provides the application of *Saccharomyces cerevisiae* ferment in anti-inflammatory, antioxidant, repairing, and anti-wrinkle effects. This includes the application of the *Saccharomyces cerevisiae* ferment in the preparation of anti-inflammatory drugs.

[0007] Furthermore, *Saccharomyces cerevisiae* fermentation products exhibit free radical scavenging effects, reduce intracellular inflammatory factors IL-6, IL-8, and TNF-α, reduce intracellular ROS, promote cell proliferation, and promote the secretion of type I and type III collagen. The *Saccharomyces cerevisiae* species include Adeli-1067, Adeli-267, Adeli-319, Adeli-388, Adeli-395, Adeli-2115, Adeli-2514, Adeli-2516, Adeli-2518, and Adeli-2519.

[0008] This invention provides a strain of *Naganishia adeliensis*, Adeli-1067, which was deposited on April 24, 2026, at the China General Microbiological Culture Collection Center (CGMCC). The depository code is CGMCC, and the address of the collection center is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The accession number is CGMCC No. 39193.

[0009] Compared with other fermentation products of *Saccharomyces cerevisiae* and *Pichia pastoris* fermentation products commonly used in the prior art, the fermentation product of this *Saccharomyces cerevisiae* exhibits significantly higher activity. At a 5% addition level, the DPPH free radical scavenging rate is ≥60%; at a 5% addition level, the ABTS free radical scavenging rate is ≥90%; in the ROS cell model, the scavenging rate is ≥80% at a 1% addition level; and at a 1% addition level and cultured for 48 h, the HaCaT cell proliferation rate is ≥50%.

[0010] The preparation method of the fermented product of *Saccharomyces cerevisiae* is as follows: after fermentation of *Saccharomyces cerevisiae*, the resulting fermentation broth is homogenized 1–3 times at 1200–1300 bar, centrifuged at 7000–8000 r / min for 10–15 min, and filtered through a 0.22 μm filter membrane to remove bacteria, thereby obtaining the fermentation product.

[0011] Homogenization is used to disrupt bacterial cells, typically performed 1–3 times at 1200–1300 bar, such as at 1200 bar, 1210 bar, 1220 bar, 1230 bar, 1240 bar, 1250 bar, 1260 bar, 1270 bar, 1280 bar, 1290 bar, and 1300 bar. Centrifugation is used to separate the supernatant and precipitate, typically performed at 7000–8000 r / min for 10–15 min, such as at 7000 r / min, 7100 r / min, 7200 r / min, 7300 r / min, 7400 r / min, 7500 r / min, 7600 r / min, 7700 r / min, 7800 r / min, 7900 r / min, and 8000 r / min. Sterilization is a common technique in this field, such as using a 0.22 μm filter membrane for sterilization.

[0012] Further, the fermentation method is as follows: *Saccharomyces cerevisiae* is inoculated into the fermentation medium at an inoculum size of 0.5% (v / v) to 5% (v / v); fermentation conditions: 16-24℃, aeration rate of 1.0–1.5 vvm, stirring at 200–300 r / min, fermentation for 30–60 h. The preferred inoculum size is 0.5–1.5% (v / v), for example, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%; fermentation conditions: 16-24℃, aeration rate of 1.0–1.5 vvm, stirring at 200–300 r / min, fermentation for 30–60 h.

[0013] Furthermore, the fermentation medium comprises the following components: 8-12 parts by weight of glucose, 2-4 parts by weight of yeast powder, 3-7 parts by weight of peptone, 2-4 parts by weight of malt extract, and a pH of 5.5-6.0.

[0014] In some embodiments of the present invention, the *Adeli* longissimus cerevisiae* Adeli-1067 is cultured in seed culture medium (YM liquid medium). The YM liquid medium, per 100 mL, has the following formulation: 0.3-0.7 g peptone, 0.2-0.4 g yeast extract, 0.2-0.4 g malt extract, and 0.8-1.2 g glucose, with ultrapure water as the solvent. The culture conditions are: 24°C, 180–200 rpm for 12–16 h until OD (dose retardation). 600 =1.0–3.0.

[0015] The beneficial effects of this invention are as follows: It creatively cultivates and purifies *Saccharomyces cerevisiae* to obtain highly bioactive active ingredients. At a 5% addition level, the *Saccharomyces cerevisiae* fermentation product exhibits a DPPH free radical scavenging rate ≥60%; an ABTS free radical scavenging rate ≥90% at a 5% addition level; in a ROS cell model, the scavenging rate at a 1% addition level is ≥80%; and at a 1% addition level and cultured for 48 h, the HaCaT cell proliferation rate is ≥50%. Attached Figure Description

[0016] Figure 1 Colony morphology of *Adelie* longissimus edulis; Figure 2 Comparison of DPPH free radical scavenging rate of Adeli-1067 fermentation products with that of Pichia pastoris; Figure 3 The ABTS free radical scavenging rate of 10 strains of *Saccharomyces cerevisiae* fermentation product extracts at a concentration of 5% was determined. Figure 4 Comparison of ABTS free radical scavenging rate of Adeli-1067 fermentation product with that of Pichia pastoris; Figure 5 Fluorescence representation of the scavenging effect of different concentrations of Adeli-1067 fermentation products on intracellular ROS; Figure 6 The inhibitory effect of HaCaT cell ROS on fermentation product extracts from 10 strains of *Saccharomyces cerevisiae*. Figure 7 A comparison of the scavenging effects of Adeli-1067 fermentation products and Pichia pastoris on intracellular ROS; Figure 8 To investigate the effects of different concentrations of Adeli-1067 fermentation product extracts on reducing intracellular ROS in HaCaT cells; Figure 9 The HaCaT cell proliferation rate of 10 strains of *Saccharomyces cerevisiae* fermentation product extracts; Figure 10 The cytotoxicity of different concentrations of Adeli-1067 fermentation products to HaCaT cells; Figure 11 The effects of different concentrations of Adeli-1067 fermentation products on the proliferation of HaCaT cells; Figure 12 A comparison of the effects of Adeli-1067 fermentation products and Pichia pastoris on HaCaT cell proliferation; Figure 13 HaCaT cell migration rate of 10 strains of *Saccharomyces cerevisiae* fermentation product extracts; Figure 14 Effects of Adeli-1067 fermentation products on the inflammatory cytokine IL-6 in HaCaT cells; Figure 15 Effects of Adeli-1067 fermentation products on the inflammatory cytokine IL-8 in HaCaT cells; Figure 16 Effects of Adeli-1067 fermentation products on the inflammatory factor TNF-α in HaCaT cells; Figure 17 The effect of Adeli-1067 fermentation products on the production of the inflammatory cytokine COLⅠ in HaCaT cells; Figure 18 The effect of Adeli-1067 fermentation products on the production of the inflammatory cytokine COLIII in HaCaT cells; Figure 19 The effect of Adeli-1067 fermentation products on cell p16 gene expression. Detailed Implementation

[0017] The following examples are provided to further illustrate the present invention and are intended to explain the invention, not to limit its scope. Unless otherwise specified, all terms are parts by weight and weight percentages.

[0018] Unless otherwise specified, all raw materials used in this invention are conventional commercially available products; all methods used in this invention are conventional methods in the art unless otherwise specified. Experimental methods in the following examples that do not specify specific conditions were performed according to conventional methods and conditions, or according to the product instructions.

[0019] The embodiments of the present invention will be further described below with reference to several examples.

[0020] It should be understood that the described embodiments are merely some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0021] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0022] In the following examples and comparative examples: LPS is endotoxin, and the CCK-8 kit was purchased from Beyotime.

[0023] In embodiments of the present invention, the amount of fermentation product extract added is calculated as the volume ratio of the fermentation product extract to the total reaction system.

[0024] Example 1: Strain Acquisition Water samples were collected from the Jiemayangzong Glacier in Tibet, also known as the source of the Yarlung Tsangpo River, at an altitude of 5300m. Acrylic glass samplers were used, and the samplers were thoroughly rinsed with river water before collection. At each sampling point, water samples were collected at a depth of approximately 0.5m using the acrylic glass sampler. Three water samples were collected from each sampling point and then homogenized into one sample, for a total of 6 L of water sample collected from each sampling point.

[0025] To isolate as many yeast species as possible, culture media with combinations of carbon and nitrogen sources were used for isolation and cultivation. Yeast strains were isolated using a membrane filtration upright culture method. After shaking the collected water samples, two gradients of 100 mL and 200 mL were used. The samples were then filtered through a sterile aquatic membrane with a pore size of 0.45 μm and a diameter of 50 mm using a sand filter. The filtered membranes were then placed upright on potato dextrose agar, Sabouraud dextrose agar, Bengal red agar, malt extract agar, and YM agar plates, with six replicates for each water sample gradient and each medium. The different media were incubated at 4 ℃ and 15 ℃. Colonies on the filter membranes were observed at 3, 7, 15, and 21 days of incubation. Different colonies were promptly picked and enriched on YM agar slants for further strain purification.

[0026] The purified strain was identified: DNA was extracted using the alkaline lysis method. Primers used were NL1 (5... -GCATATCAATAAGCGGAGGAAAAG-3 ), NL4 (5 -GGTCCGTGTTTCAAGACGG-3 The final amplified products were sequenced after 1% agarose gel electrophoresis. The sequencing results were manually proofread, and primers and bimodal sequences were removed. Homologous sequences were then searched in the GenBank nucleic acid sequence database. Those with a similarity of more than 99% to known sequences were identified as the same species. A total of 2145 strains were obtained.

[0027] Example 2: Fermentation Broth Acquisition Ten samples were randomly selected from 2145 strains: Adeli-1067, Adeli-267, Adeli-319, Adeli-388, Adeli-395, Adeli-2115, Adeli-2514, Adeli-2516, Adeli-2518, and Adeli-2519. Adeli-1067 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39193. Its colonies are shown in the figure below. Figure 1 As shown.

[0028] In contrast, a strain of Pichia-616 isolated from a glacier on the Tibetan Plateau was used for a comparative experiment.

[0029] Eleven bacterial strains were cultured according to the following steps to obtain fermentation broth: (1) Activation of strain: The strain was inoculated on YM solid medium plates and cultured at 24℃ for 72 h to obtain single colonies. The YM liquid medium was formulated as follows (per 100 mL): 0.5 g peptone, 0.3 g yeast powder, 0.3 g malt extract powder, 1 g glucose, 2 g agarose, and ultrapure water as solvent.

[0030] (2) Seed culture preparation: Pick the above single colonies and inoculate them into YM liquid medium, and culture at 24℃ and 200 r / min for 36 h with shaking, until OD 600 Once the viscosity reaches 2.0, the seed solution is obtained.

[0031] (3) Fermentation culture: The seed culture was inoculated into the fermentation medium at an inoculation rate of 3.0% (v / v). The composition of the fermentation medium per 1L was: 10 parts by weight of glucose, 3 parts by weight of yeast extract, 5 parts by weight of peptone, 3 parts by weight of malt extract, with ultrapure water as the solvent and pH 5.5-6.0. The fermentation conditions were: temperature 24℃, aeration rate 1.0 vvm, stirring speed 200 r / min, and fermentation endpoint OD. 600 =7-9, generally, the fermentation time is 60 h.

[0032] (4) Post-processing: The fermentation broth was subjected to high-pressure homogenization, centrifugation and filtration sterilization in sequence. The high-pressure homogenization conditions were 1100 bar pressure, 3 cycles; the centrifugation conditions were 8000 r / min, 4℃ centrifugation for 10 min, and the supernatant was collected; finally, it was filtered through a 0.22 μm microporous membrane for sterilization to obtain the fermentation product.

[0033] The fermentation products of Adélie have the following characteristics: (1) It is a pale yellow to brown transparent liquid with a pH of 5.0–6.5; (2) No bacterial colony growth was observed after filtration through a 0.22 μm filter membrane; (3) The active ingredients include yeast polysaccharides, small molecule polypeptides, free amino acids, SOD, etc.

[0034] Example 3: Fermentation Broth Test The 11 fermentation broth samples obtained in Example 2 above were used as samples and tested as follows: 1. DPPH free radical scavenging rate Weigh 12 mg of 1,1-diphenyl-2-trinitrophenylhydrazine into a 250 mL beaker, add 100 mL of 95% ethanol to prepare a 0.12 mg / mL DPPH ethanol solution, and stir with a glass rod until dissolved.

[0035] DPPH working solution: Dilute 0.12 mg / mL DPPH ethanol solution with 95% ethanol to A517 = 0.7 ± 0.02.

[0036]

[0037] Sample addition requirements: The reaction was carried out in the dark for 5 minutes, and the absorbance was measured at 517 nm.

[0038] Scavenging rate (%) = [1 - (sample group - sample background group) / (DPPH group - solvent background group)] × 100% like Figure 2 As shown, when the fermentation product extract was added at a concentration of 5%, the DPPH free radical scavenging rate of Adeli-1067 was 48% higher than that of Pichia pastoris, indicating that the Adeli-1067 fermentation product extract had a better DPPH free radical scavenging effect than the Pichia pastoris fermentation product extract. At a 5% addition level of fermentation product extract, the DPPH free radical scavenging rates of Adeli-1067, Adeli-267, Adeli-319, Adeli-388, Adeli-395, Adeli-2115, Adeli-2514, Adeli-2516, Adeli-2518, and Adeli-2519 were 61%, 38%, 50%, 44%, 61%, 47%, 48%, 52%, 53%, and 59%, respectively.

[0039] 2. ABTS free radical scavenging rate (1) 7mmol / L ABTS stock solution: accurately weigh 0.1921g of ABTS (purity ≥98%), dissolve it in pure water and make up to 100mL, and store at 4℃ protected from light.

[0040] (2) 2.45 mmol / L potassium persulfate solution: Weigh 0.0614 g of potassium persulfate (K2S2O8), dissolve it in pure water and make up to 100 mL. Prepare and use immediately (potassium persulfate is easily decomposed and needs to be prepared again if left for more than 24 hours).

[0041] ABTS⁺・: Mix an equal volume of 7 mmol / L ABTS stock solution with 2.45 mmol / L potassium persulfate solution and place at room temperature (25±1℃) in the dark for 12-16 hours (to ensure complete oxidation to generate ABTS⁺・).

[0042] (3) ABTS⁺ working solution: dilute with pure water, measure absorbance at 734 nm and adjust to 0.7±0.02.

[0043] Sample addition requirements.

[0044]

[0045] The reaction was carried out in the dark for 5 minutes, and the absorbance was measured at 734 nm.

[0046] Scavenging rate (%) = [1 - (sample group - sample background group) / (ABTS group - solvent background group)] × 100% like Figure 3 As shown, the fermentation product extracts of 10 strains of *Saccharomyces cerevisiae*, numbered Adeli-1067, Adeli-267, Adeli-319, Adeli-388, Adeli-395, Adeli-2115, Adeli-2514, Adeli-2516, Adeli-2518, and Adeli-2519, all exhibited ABTS radical scavenging rates exceeding 60% at an addition level of 5%, with Adeli-1067 showing the highest ABTS radical scavenging rate.

[0047] like Figure 4As shown, at a 1% addition level of the fermentation product extract (i.e., 10 μL fermentation product extract + 40 μL water + 950 μL ABTS⁺ working solution), Adeli-1067 exhibited a 9% higher ABTS radical scavenging rate than Pichia pastoris; at a 5% addition level, Adeli-1067 showed a 5% higher ABTS radical scavenging rate than Pichia pastoris. The ABTS radical scavenging effect of the Adeli-1067 fermentation product extract was superior to that of Pichia pastoris. At a 5% addition level of the fermentation product extract, the ABTS radical scavenging rates of Adeli-1067, Adeli-267, Adeli-319, Adeli-388, Adeli-395, Adeli-2115, Adeli-2514, Adeli-2516, Adeli-2518, and Adeli-2519 were 98%, 70%, 80%, 85%, 80%, 70%, 68%, 89%, 81%, and 85%, respectively.

[0048] 3. ROS clearance rate of HaCaT cells 3.1 Cell Plating 3.1.1 Cell digestion. Take cells in the logarithmic growth phase, discard the old culture medium, and wash once with DPBS. HaCaT cells adhere relatively firmly, so trypsin digestion should take about 10 minutes. Closely monitor cell condition and adjust the digestion time accordingly. When the cells become rounded and slightly detach from the bottom of the culture flask, add serum-containing culture medium to stop the digestion.

[0049] 3.1.2 Centrifugation and resuspending. Centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant, add complete culture medium, and gently pipette to resuspend the cells.

[0050] 3.1.3 Counting.

[0051] Transfer 10 μL of cell suspension to an EP tube, add 10 μL of trypan blue dye, and gently pipette to mix. Trypan blue is a classic dye for cell viability testing. Its core function is to quickly distinguish between live and dead cells. Live cells have intact cell membranes, so the dye cannot enter (no color development); dead cells have damaged cell membranes, so the dye enters and turns the cell nucleus blue.

[0052] Use a hemocytometer to count cells. Wipe the counting chamber and coverslip with anhydrous ethanol, let them dry, and then smoothly place the coverslip over the counting chamber. Remove the deoxidized cell suspension and gently pipette a few times to ensure the cells are dispersed into single cells. If the cell concentration is too high, perform serial dilutions with culture medium to ensure 50-100 cells per large square for accurate counting. Add 10 μL of the diluted cell suspension slowly along the edge of the coverslip, allowing the suspension to flow naturally into the counting chamber through capillary action, avoiding air bubbles. After adding the sample, let it stand for 3 minutes to allow the cells to settle to the bottom of the counting chamber, preventing floating cells from affecting observation. Place the counting chamber on the stage of an inverted microscope and observe with a 10× objective lens, focusing on the grid of the counting chamber. Select the four corner squares in the counting chamber and count the cells in each square: for cells marked on the lines, follow the principle of "counting the top but not the bottom, counting the left but not the right" to avoid duplication or omission; only count intact cells, excluding cell debris and dead cells.

[0053] Calculation formula: Cell concentration (cells / mL) = Total number of cells in 4 large squares ÷ 4 × 10⁴ × dilution factor.

[0054] 3.1.4 Plate preparation. Adjust the cell concentration to the appropriate level using complete culture medium and add the cells to the wells.

[0055] 3.1.5 Observation. Observe the cell state under a microscope.

[0056] 3.2 Group Intervention Groups were set up: control group (complete culture medium. If the drug is diluted with DMSO, 0.1% DMSO should be added or the experimental group should use the maximum concentration of DMSO), oxidative damage group (with oxidation inducer added), and experimental group (complete culture medium containing different concentrations of samples and oxidation inducers).

[0057] Return to the incubator and continue culturing for 24 hours.

[0058] 3.3 ROS staining The most commonly used working concentration of the DCFH-DA probe is 10 μM, and staining for about 30 minutes is sufficient. The existing stock solution concentration is 10 mM; it should be diluted 1000-fold with serum-free medium before use. Fluorescent probes such as DCFH-DA must be strictly protected from light.

[0059] 3.4 Taking photos Discard the culture medium from the wells and gently wash the cells twice with sterile DPBS.

[0060] Add DPBS or phenol red-free medium, and photograph under an inverted fluorescence microscope using the GFP channel (488nm). Phenol red-containing medium will increase the fluorescence background signal; DPBS does not contain the core essential components for cell survival, and if used, photograph immediately.

[0061] After washing off the DCFH-DA probe, the imaging process must be completed within 30 minutes; if more than 1 hour passes, the probe will gradually quench.

[0062] The DCFH-DA probe can only stain viable cells. Cells undergo oxidative damage under fluorescent stimulation, and the fluorescence directly catalyzes the rapid hydrolysis of unhydrolyzed DCFH-DA within HaCaT cells into DCFH. DCFH is then immediately oxidized by intracellular ROS into the fluorescent substance DCF. The longer the imaging time and the more light exposure, the more newly generated DCF is produced, resulting in a sustained and irreversible brightening of the fluorescence. All samples should be photographed using the same settings, and the time the fluorescence is exposed to the sample should be minimized to avoid abnormal enhancement of the fluorescence signal, which could affect the experimental results.

[0063] 3.5 Data Statistical Analysis Open the captured images using ImageJ software and calculate the average fluorescence intensity for each group: The data were analyzed using GraphPad Prism software, and bar charts comparing the fluorescence intensity of each group were plotted.

[0064] like Figure 5 As shown, the addition of Adeli-1067 fermentation product extract can significantly reduce fluorescence intensity, i.e., reduce intracellular ROS in HaCaT cells.

[0065] like Figure 6 As shown, the fermentation product extracts of 10 strains of *Saccharomyces cerevisiae*, numbered Adeli-1067, Adeli-267, Adeli-319, Adeli-388, Adeli-395, Adeli-2115, Adeli-2514, Adeli-2516, Adeli-2518, and Adeli-2519, all exhibited inhibitory effects on ROS in HaCaT cells, with Adeli-1067 and Adeli-2519 showing the best effects.

[0066] like Figure 7 As shown, at a concentration of 0.5%, the Adeli-1067 fermentation product extract significantly reduced intracellular ROS in HaCaT cells, while the Pichia pastoris fermentation product extract had almost no effect. The ROS intensity of HaCaT cells treated with the Adeli-1067 fermentation product extract was reduced by 37.56% compared to those treated with the Pichia pastoris fermentation product extract. The effects of different concentrations of Adeli-1067 fermentation product extract on reducing intracellular ROS in HaCaT cells, such as Figure 8 As shown, the ROS intensity of the experimental groups with added amounts of 0.25-1% was significantly lower than that of the LPS group without added Adeli-1067 fermentation product extract.

[0067] 4. HaCaT cell proliferation 4.1 Cell Plating 4.1.1 Cell digestion. Take cells in the logarithmic growth phase, discard the old culture medium, and wash once with DPBS. HaCaT cells adhere relatively firmly, so trypsin digestion should take about 10 minutes. Closely monitor cell condition and adjust the digestion time accordingly. When the cells become rounded and slightly detach from the bottom of the culture flask, add serum-containing culture medium to stop the digestion.

[0068] 4.1.2 Centrifugation and resuspending. Centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant, add complete culture medium, and gently pipette to resuspend the cells.

[0069] 4.1.3 Counting.

[0070] Transfer 10 μL of cell suspension to an EP tube, add 10 μL of trypan blue dye, and gently pipette to mix. Trypan blue is a classic dye for cell viability testing. Its core function is to quickly distinguish between live and dead cells. Live cells have intact cell membranes, so the dye cannot enter (no color development); dead cells have damaged cell membranes, so the dye enters and turns the cell nucleus blue.

[0071] Use a hemocytometer to count cells. Wipe the counting chamber and coverslip with anhydrous ethanol, let them dry, and then smoothly place the coverslip over the counting chamber. Remove the deoxidized cell suspension and gently pipette a few times to ensure the cells are dispersed into single cells. If the cell concentration is too high, perform serial dilutions with culture medium to ensure 50-100 cells per large square for accurate counting. Add 10 μL of the diluted cell suspension slowly along the edge of the coverslip, allowing the suspension to flow naturally into the counting chamber through capillary action, avoiding air bubbles. After adding the sample, let it stand for 3 minutes to allow the cells to settle to the bottom of the counting chamber, preventing floating cells from affecting observation. Place the counting chamber on the stage of an inverted microscope and observe with a 10× objective lens, focusing on the grid of the counting chamber. Select the four corner squares in the counting chamber and count the cells in each square: for cells marked on the lines, follow the principle of "counting the top but not the bottom, counting the left but not the right" to avoid duplication or omission; only count intact cells, excluding cell debris and dead cells.

[0072] Calculation formula: Cell concentration (cells / mL) = Total number of cells in 4 large squares ÷ 4 × 10⁴ × dilution factor.

[0073] 4.1.4 Plating. Adjust the cell concentration to the appropriate level using complete culture medium. Add 100 μL of cell suspension to each well of a 96-well plate, and add sterile DPBS to the edge wells to prevent errors caused by evaporation of the culture medium in the edge wells during culture.

[0074] 4.1.5 Observation. Observe the cell quantity and dispersion under a microscope. If the cells are unevenly distributed, gently tap the walls of the 96-well plate to disperse the cells.

[0075] 4.2 Cell Culture Incubate overnight in a 37°C, 5% CO2 incubator to allow cells to adhere and grow.

[0076] 4.3 Group Intervention Discard the old culture medium from the well and wash gently once with DPBS.

[0077] Grouping was set up as follows: blank group (cell-free), control group (complete culture medium. If the drug is diluted with DMSO, 0.1% DMSO should be added or the experimental group should use the minimum concentration of DMSO), and experimental group (complete culture medium containing samples of different concentrations).

[0078] Each group was set up with 6 replicates, and the cells were returned to the incubator for 24 hours of further incubation (the drug treatment time was adjusted according to the specific situation).

[0079] 4.4 Incubation with CCK-8 reagent After the set incubation time is reached, remove the 96-well plate, discard the old culture medium, and add 100 μL of DMEM / F12 containing 10% CCK8 reagent.

[0080] Place the 96-well plate back into the incubator and incubate in the dark for 1-4 hours (the incubation time can be adjusted according to the cell density, with the OD value of the control group being around 1.0).

[0081] 4.5 Absorbance Detection The absorbance (OD value) of each well was measured sequentially at a wavelength of 450 nm using an ELISA reader, and the data were recorded.

[0082] 4.6 Data Statistical Analysis Calculate the OD value for each group and subtract the OD value of the blank group to eliminate background interference.

[0083] Cell viability (%) = (experimental group - blank group) / (control group - blank group) × 100%.

[0084] Use GraphPad Prism for data analysis.

[0085] Depend on Figure 9 It can be seen that the fermentation product extracts of all 10 strains of *Adeli* longissimus edulis* have HaCaT cell proliferation effects, and except for Adeli-319 and Adeli-388, the rest all have significant proliferation effects.

[0086] Cytotoxicity tests were conducted on different concentrations of Adeli-319 fermentation product extracts. The results showed no significant toxicity to HaCaT cells within the concentration range of 1%-25%, and the cell proliferation rate remained at no less than 90%. Figure 10 Within the concentration range of 0.1-1.6%, its concentration showed a linear relationship with the proliferative effect of HaCaT cells. Figure 11 ).

[0087] Depend on Figure 12 It can be seen that at addition levels of 0.4%, 0.8%, and 1.6%, the cell proliferation of Adeli-1067 fermentation product extract was significantly higher than that of Pichia pastoris, indicating that Adeli-1067 fermentation product extract has a better effect on promoting cell proliferation than Pichia pastoris fermentation product extract.

[0088] 5. HaCaT cell migration 5.1 Orifice Plate Marking Beforehand, use a ruler to draw even straight lines on the bottom of the 6-hole plate for later photo positioning.

[0089] 5.2 Cell Plating 5.2.1 Cell digestion. Take cells in the logarithmic growth phase, discard the old culture medium, and wash once with DPBS. HaCaT cells adhere relatively firmly, so trypsin digestion should take about 10 minutes. Closely monitor cell condition and adjust the digestion time accordingly. When the cells become rounded and slightly detach from the bottom of the culture flask, add serum-containing culture medium to stop the digestion.

[0090] 5.2.2 Centrifugation and resuspending. Centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant, add complete culture medium, and gently pipette to resuspend the cells.

[0091] 5.2.3 Counting.

[0092] Transfer 10 μL of cell suspension to an EP tube, add 10 μL of trypan blue dye, and gently pipette to mix. Trypan blue is a classic dye for cell viability testing. Its core function is to quickly distinguish between live and dead cells. Live cells have intact cell membranes, so the dye cannot enter (no color development); dead cells have damaged cell membranes, so the dye enters and turns the cell nucleus blue.

[0093] Use a hemocytometer to count cells. Wipe the counting chamber and coverslip with anhydrous ethanol, let them dry, and then smoothly place the coverslip over the counting chamber. Remove the deoxidized cell suspension and gently pipette a few times to ensure the cells are dispersed into single cells. If the cell concentration is too high, perform serial dilutions with culture medium to ensure 50-100 cells per large square for accurate counting. Add 10 μL of the diluted cell suspension slowly along the edge of the coverslip, allowing the suspension to flow naturally into the counting chamber through capillary action, avoiding air bubbles. After adding the sample, let it stand for 3 minutes to allow the cells to settle to the bottom of the counting chamber, preventing floating cells from affecting observation. Place the counting chamber on the stage of an inverted microscope and observe with a 10× objective lens, focusing on the grid of the counting chamber. Select the four corner squares in the counting chamber and count the cells in each square: for cells marked on the lines, follow the principle of "counting the top but not the bottom, counting the left but not the right" to avoid duplication or omission; only count intact cells, excluding cell debris and dead cells.

[0094] Calculation formula: Cell concentration (cells / mL) = Total number of cells in 4 large squares ÷ 4 × 10⁴ × dilution factor.

[0095] 5.2.4 Plating. Adjust the cell concentration to the appropriate level using complete culture medium, and add 2 mL of cell suspension to each well of a 6-well plate. The cells need to be densely packed; after overnight culture, the cells should reach 100% confluence (forming a dense monolayer).

[0096] 5.2.5 Observation. Observe the cell state under a microscope.

[0097] 5.3 Cell Culture Incubate overnight in a 37°C, 5% CO2 incubator to allow cells to adhere and grow.

[0098] 5.4 Cell scratches Discard the culture medium in the wells and gently wash the cells twice with sterile PBS to remove residual serum and floating cells.

[0099] Using a 10μL pipette tip perpendicular to the bottom of the well plate, make three parallel scratches evenly on the cell monolayer in each well, avoiding tilting to prevent uneven scratch width.

[0100] Wash again with PBS 2-3 times to completely remove cell debris caused by the scratches.

[0101] 5.5 Taking photos The scratches were photographed under an inverted microscope, and their location was determined based on the scratch lines.

[0102] 5.6 Group Intervention Groups were set up: control group (serum-free culture medium. If the drug is diluted with DMSO, 0.1% DMSO should be added or the experimental group should use the minimum concentration of DMSO), and experimental group (serum-free culture medium containing samples of different concentrations).

[0103] Return to the incubator and continue incubation for 24 hours (adjust the drug treatment time according to the specific situation).

[0104] 5.7 Taking photos Discard the culture medium in the wells and gently wash the cells twice with sterile DPBS to remove residual serum and floating cells.

[0105] Based on the marked location, the healing process of the scratch was photographed under an inverted microscope at the same field of view.

[0106] 5.8 Data Statistical Analysis Open the photographed image using ImageJ software and measure the width / area of ​​the scratches at various time points.

[0107] Healing rate (%) = (0h scratch width - 24h scratch width) / 0h scratch width × 100%.

[0108] Depend on Figure 13 It can be seen that all 10 strains of *Adeli* longissimus fermentation product extracts have HaCaT cell migration effects, with Adeli-1067 and Adeli-2516 showing the best migration effects.

[0109] 6. Detection of IL6 levels in HaCaT cells 6.1 Cell seeding: Seed cells into 24-well plates at an appropriate seeding density (2×10⁴ / well) and incubate overnight in an incubator (37°C, 5% CO₂).

[0110] 6.2 Experimental grouping: The experiment was set up with a blank control group, a model group (LPS), a positive control group (dexamethasone), and a sample group.

[0111] 6.3 Solution preparation: Prepare working solutions of different concentrations of the test substance according to the test concentration setting table.

[0112] 6.4 Drug administration: Drug administration was performed when the cell deposition rate in the 24-well plate reached 40%–60%. 1 mL of cell culture medium was added to each well in the blank control group; 1 mL of culture medium containing the corresponding concentration of the test substance was added to each well in the sample group.

[0113] 6.5 Collect cell supernatant: After incubation for 48 h, collect the cell culture supernatant in EP tubes and freeze at -80℃.

[0114] 6.6 IL6 Detection: The IL6 content was detected and analyzed according to the operating instructions of the ELISA detection kit.

[0115] from Figure 14 It can be seen that the Adeli-1067 fermentation product extract, at an addition level of 4%, exhibits an excellent effect in reducing IL-6, indicating that the Adeli-1067 fermentation product extract has anti-inflammatory effects.

[0116] 7. Detection of IL8 content in HaCaT cells The ELISA kit for detecting IL6 levels in HaCaT cells is replaced with IL8, similar to the method used for detecting IL6 levels in HaCaT cells.

[0117] like Figure 15 The Adeli-1067 fermentation product extract, at a concentration of 4%, showed excellent IL-8 reduction, indicating that the Adeli-1067 fermentation product extract has anti-inflammatory effects.

[0118] 8. Detection of TNFα content in HaCaT cells The ELISA kit for detecting IL6 levels in HaCaT cells was replaced with TNFα, similar to the method used for detecting IL6 levels in HaCaT cells.

[0119] Depend on Figure 16 It can be seen that the Adeli-1067 fermentation product extract, at an addition level of 4%, exhibits an excellent effect in reducing TNFα, indicating that the Adeli-1067 fermentation product extract has anti-inflammatory effects.

[0120] 9. Detection of COLOI content in HaCaT cells 9.1 Cell seeding: Seed cells into 24-well plates at an appropriate seeding density (2×10⁴ / well) and incubate overnight in an incubator (37°C, 5% CO₂).

[0121] 9.2 Experimental grouping: The experiment included a blank control group, a positive control group (TGFβ), and a sample group.

[0122] 9.3 Solution preparation: Prepare working solutions of different concentrations of the test substance according to the test concentration setting table.

[0123] 9.4 Drug administration: Drug administration was performed when the cell deposition rate in the 24-well plate reached 40%–60%. 1 mL of cell culture medium was added to each well in the blank control group; 1 mL of culture medium containing the corresponding concentration of the test substance was added to each well in the sample group.

[0124] 9.5 Collection of cell supernatant: After incubation for 48 h, collect the cell culture supernatant in EP tubes and store them frozen at -80℃.

[0125] 9.6 Collagen I Detection: The Collagen I content was detected and analyzed according to the operating instructions of the ELISA kit.

[0126] Depend on Figure 17 It can be seen that when the Adeli-1067 fermentation product extract is added at a concentration of 1%, it can significantly increase the COLI content of HaCaT cells, indicating that the Adeli-1067 fermentation product extract has anti-wrinkle and firming effects.

[0127] 10. Detection of COLⅢ content in HaCaT cells The same method for detecting COLⅠ content in HaCaT cells was used, but the ELISA kit was replaced with Collagen Ⅲ.

[0128] Depend on Figure 18 It can be seen that when the Adeli-1067 fermentation product extract is added at a concentration of 1%, it can significantly increase the COLⅢ content of HaCaT cells, indicating that the Adeli-1067 fermentation product extract has anti-wrinkle and firming effects.

[0129] 11. Detection of relative expression levels of the HaCaT-p16 gene 11.1 Cell Culture 11.1.1 Cell seeding: Seed cells into 24-well plates at an appropriate seeding density and incubate overnight in an incubator (37°C, 5% CO2).

[0130] 11.1.2 Experimental grouping: The experiment was set up with a blank control group, a model group (D-galactose) and a sample group.

[0131] 11.1.3 Solution preparation: Prepare working solutions of different concentrations of the test substance according to the test concentration setting table.

[0132] 11.1.4 Drug administration: 1 mL of cell culture medium was added to each well of the blank control group; 1 mL of culture medium containing the corresponding concentration of the test substance was added to each well of the sample group.

[0133] 11.1.5 Collection of cell samples: After incubation for 72 h, cells were collected in EP tubes.

[0134] 11.2 Total RNA Extraction 11.2.1 Sample preparation. Take the sample and add 1 mL of TRIzol. ® Reagent, shake vigorously, then add 0.2 mL of chloroform.

[0135] 11.2.2 Let stand at room temperature for 2-3 min, then centrifuge (12,000g, 4℃, 15 min).

[0136] 11.2.3 Transfer the supernatant to a new 1.5 mL centrifuge tube, then add an equal volume of 70% ethanol and vortex to mix.

[0137] 11.2.4 Transfer the supernatant to a Spin Cartridge (including the cannula), centrifuge (12,000g, room temperature, 15 sec), and discard the waste liquid.

[0138] 11.2.5 Add 50 μL of RNase-Free DNase Buffer and incubate at room temperature for 10 min to completely remove residual genomic DNA.

[0139] 11.2.6 Add 700 μL Wash Buffer I to Spin Cartridge (including the tube), centrifuge (12,000g, room temperature, 15 sec), and discard the waste liquid.

[0140] 11.2.7 Add 500 μL Wash Buffer II to Spin Cartridge (including the tube), centrifuge (12,000g, room temperature, 15 sec), and discard the waste liquid; repeat step 4.1.7.

[0141] 11.2.8 Emptying: Centrifugation (12,000g, room temperature, 1 min).

[0142] 11.2.9 Elution: Add 40-100 μL of RNase-Free Water to the center of the Spin Cartridge (with a new 1.5 ml centrifuge tube), incubate at room temperature for 1 min; then centrifuge (12,000 g, room temperature, 2 min).

[0143] 11.2.10 The content, purity and quality of the sample were determined by ultraviolet spectrophotometer and electrophoresis, and then stored at -80℃ for later use.

[0144] 11.3 Reverse Transcription Experiment 1st-Strand cDNA Synthesis Reaction System and Conditions

[0145] 11.4 Real-Time PCR Detection 11.4.1 Primer Design and Synthesis for Quantitative Real-Time PCR Primers for quantitative PCR were designed using Primer Premier 6.0 and Beacon Designer 7.8 software, and then synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are as follows: Real-Time PCR Primers and Conditions

[0146] 11.4.2 Real-Time PCR Amplification System and Reaction Conditions Quantitative PCR reaction system and conditions

[0147] 11.4.3 Statistical analysis of differential gene expression by real-time PCR Each sample was repeated three times, and the relative expression levels of each gene were calculated in increments of 2. (Ct内参基因－Ct目的基因） Perform statistical analysis.

[0148] The stimulation conditions were D-galactose (100 mM), the concentration of the fermentation product extract of the sample Adeli-1067 was 4%, the detection model was HaCaT human immortalized keratinocytes, and the results were characterized by qRT-PCR.

[0149] Compared with the model group (without sample), the sample at a concentration of 4% showed a significant inhibitory effect on the expression of p16 in HaCaT immortalized keratinocytes (p<0.05), with an inhibition rate of 23.66%, indicating that it has anti-aging effects.

[0150] The above embodiments describe in detail the structure, features, and effects of the present invention. The above description is only a preferred embodiment of the present invention. Any changes made in accordance with the concept of the present invention, or equivalent embodiments modified to have equivalent changes, shall still fall within the scope of protection of the present invention if they do not exceed the scope covered by the specification.

Claims

1. Application of Adelie Longsi yeast ferment in anti-inflammatory, antioxidant, repair, and anti-wrinkle effects.

2. The application according to claim 1, characterized in that, The application of the *Adelie* longissimus ferment broth in the preparation of anti-inflammatory drugs.

3. The application according to claim 1, characterized in that, Adelie's long-lived yeast ferment has the effect of scavenging free radicals and reducing intracellular inflammatory factors IL-6, IL-8, and TNF-α.

4. The application according to claim 1, characterized in that, Adélie longissimus ferment has the effect of reducing intracellular ROS.

5. The application according to claim 1, characterized in that, Adélie Longsi yeast ferment has the effect of promoting cell proliferation.

6. The application according to claim 1, characterized in that, Adélie Longi yeast ferment has the effect of promoting the secretion of type I and type III collagen.

7. The application according to any one of claims 1-6, wherein the *Adeli* longissimus yeast comprises Adeli-1067, Adeli-267, Adeli-319, Adeli-388, Adeli-395, Adeli-2115, Adeli-2514, Adeli-2516, Adeli-2518, and Adeli-2519.

8. The application according to claim 7, characterized in that, The accession number of Adeli-1067 is CGMCC No. 39193.

9. The application according to any one of claims 1-8, characterized in that, The preparation method of the fermented product of *Saccharomyces cerevisiae* is as follows: after fermentation of *Saccharomyces cerevisiae*, the resulting fermentation broth is homogenized 1–3 times at 1200–1300 bar, centrifuged at 7000–8000 r / min for 10–15 min, and filtered through a 0.22 μm filter membrane to remove bacteria, thereby obtaining the fermentation product.

10. The application according to claim 9, characterized in that, The fermentation method is as follows: *Saccharomyces cerevisiae* is inoculated into the fermentation medium at an inoculation rate of 0.5% (v / v) to 5% (v / v); fermentation conditions are: 16-24℃, aeration rate of 1.0–1.5 vvm, stirring at 200–300 r / min, and fermentation time of 30–60 h; the fermentation medium contains the following components: 8-12 parts by weight of glucose, 2-4 parts by weight of yeast extract, 3-7 parts by weight of peptone, 2-4 parts by weight of malt extract, and a pH of 5.5-6.0.

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