Cosmetic composition for improving skin condition or alopecia, comprising novel Saccharomyces cerevisiae strain and fermentation product thereof

Abstract

The present invention relates to a cosmetic composition for improving skin condition or hair loss, and more particularly, to a cosmetic composition for improving skin condition or hair loss, comprising a novel Saccharomyces cerevisiae strain and a fermented product thereof, the cosmetic composition according to one aspect exhibiting significantly excellent skin condition improving effect and hair loss improving effect.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to Korean Patent Application No. 10-2024-0147738, filed on October 25, 2024, which is incorporated herein by reference for all purposes as fully set forth herein. Technical Field

[0003] This invention relates to a cosmetic composition for improving skin condition or hair loss, comprising a novel brewer's yeast ( Saccharomyces cerevisiae ) strains and their ferments.

[0004] This invention was achieved with the support of the Ministry of Education (MOE) and Chungcheongbuk-do Province, specifically through the Regional Innovation System & Education (RISE) project of the Chungcheongbuk-do Regional Innovation System & Education Center. (2025-RISE-11-008-03) Background Technology

[0005] Kefir, also known as Tibetan mushroom, differs from regular yogurt in that it is a fermented milk produced by the co-fermentation of lactic acid bacteria and yeast. Its fermentation temperature is characterized by 22-25°C, rather than the 32-42°C suitable for conventional cultivation. Unlike the liquid or cell form of culture used for regular fermentation, kefir grains are used to cultivate a mixture of lactic acid bacteria and yeast. Lactic acid bacteria are primarily distributed on the surface of the kefir grain, while yeast is distributed inside. The most significant characteristic of kefir is that, unlike yogurt, it does not produce lactic acid; instead, it undergoes an alcoholic fermentation process, producing small amounts of alcohol, carbon dioxide, and kefir exopolysaccharides (Kefiran). These kefirs are classified as animal-based foods because they are typically produced using animal milk as a lactose substrate. However, unlike regular kefir, water kefir is classified as a plant-based food because it uses only sugar as a sucrose substrate for fermentation and, unlike milk kefir, has a transparent appearance. In addition, water kefir can also be used in vegan products.

[0006] Brewing yeast is typically derived from strains of brewer's yeast. While numerous studies have utilized strains derived from brewer's yeast, research on using brewing yeast derived from kefir to produce cosmetic compositions is scarce, and there are currently no inventions concerning brewing yeast derived from water kefir. Summary of the Invention

[0007] Technical issues

[0008] On the one hand, a brewing yeast ( Saccharomyces cerevisiae The HBWKY-1 strain, with accession number KCTC 15765BP.

[0009] On the other hand, a cosmetic composition for improving skin condition or hair loss is provided, comprising Saccharomyces cerevisiae with accession number KCTC 15765BP. Saccharomyces cerevisiae Fermentation product of strain HBWKY-1.

[0010] On the other hand, a ferment of Saccharomyces cerevisiae strain HBWKY-1 with accession number KCTC 15765BP is provided for use in improving skin condition or hair loss.

[0011] Another aspect provides a cosmetic composition for improving skin condition or hair loss, comprising Saccharomyces cerevisiae with accession number KCTC 15765BP. Saccharomyces cerevisiae HBWKY-1 strain and Lactobacillus pentosus ( Lactobacillus pentosus ) The complex fermentation product of strain .

[0012] On the other hand, a brewing yeast with accession number KCTC 15765BP is provided. Saccharomyces cerevisiae HBWKY-1 strain and Lactobacillus pentosus ( Lactobacillus pentosus The compound fermentation product of the strain is used to improve skin condition or for hair loss.

[0013] On the other hand, a brewing yeast is provided ( Saccharomyces cerevisiae A method for preparing fermentation products of strain HBWKY-1, comprising the following steps: (a) mixing plants with malt to prepare plant malt syrup; (b) mixing brewer's yeast ( Saccharomyces cerevisiae (c) Inoculating the HBWKY-1 strain into a culture medium containing plant malt syrup and culturing it; and (d) removing the bacterial cells from the cultured culture.

[0014] Technical solution

[0015] This relates to a cosmetic composition for improving skin condition or hair loss, comprising Saccharomyces cerevisiae with accession number KCTC15765BP. Saccharomyces cerevisiae The invention relates to the HBWKY-1 strain and the fermentation product obtained by fermenting plant malt syrup using the strain, and also to a method for preparing the plant malt syrup fermentation product using the strain.

[0016] The invention will be described in more detail below.

[0017] One aspect involves a strain of Saccharomyces cerevisiae, HBWKY-1, with accession number KCTC 15765BP.

[0018] The brewer's yeast strain HBWKY-1 was isolated from water kefir.

[0019] The *Saccharomyces cerevisiae* strain HBWKY-1 may have the 16S rRNA base sequence of SEQ ID NO:1.

[0020] The strain, strain culture medium, or strain fermentation product can have excellent effects on improving skin condition or hair loss.

[0021] In this specification, the term "culture medium" can be used interchangeably with "culture supernatant," "conditioned medium," or "adjustment medium," and can refer to the entire culture medium, including the strain, its metabolites, and additional nutrients obtained by culturing the strain in a nutrient-providing medium for a certain period of time to enable the strain to grow and survive in a test tube. Alternatively, the culture medium can refer to a culture medium from which bacterial cells have been removed during the culture of the strain. On the other hand, the liquid from which bacterial cells have been removed from the culture medium is called "supernatant," which can be obtained by: allowing the culture medium to stand for a certain period of time, removing the sediment at the bottom, and taking only the uppermost layer of liquid; filtering to remove bacterial cells; or centrifuging the culture medium to remove the sediment at the bottom and taking only the uppermost liquid. The term "bacterial cells" refers to the strain of the present invention itself, including strains isolated and selected from skin samples, etc., or strains isolated from the culture medium by culturing the strain. The bacterial cells can be obtained by centrifuging the culture medium and taking the portion that settles to the bottom, or by allowing the bacterial cells to stand for a certain period of time and removing the liquid at the top, since the bacterial cells settle to the bottom layer of the culture medium under the influence of gravity.

[0022] The culture medium may include the culture medium itself, its concentrate or freeze-dried form, obtained by culturing the strain, or the culture supernatant, its concentrate or freeze-dried form, obtained by removing the strain from the culture medium.

[0023] The culture medium can be obtained by culturing the strain in a culture medium (e.g., a culture medium containing magnesium sulfate and manganese sulfate).

[0024] In one specific embodiment, the culture supernatant of the strain can be obtained by centrifuging or filtering the strain culture medium to remove the strain.

[0025] In another specific embodiment, the obtained supernatant can be concentrated by centrifugation or by filtering the culture medium itself or the culture medium to obtain a concentrate.

[0026] Those skilled in the art can appropriately select or modify the culture medium and culture conditions used to culture the strain.

[0027] The improvement in skin condition can be achieved by improving skin damage caused by reactive oxygen species, reducing inflammation, moisturizing the skin, improving wrinkles, improving acne, improving sebum production, improving skin keratinization, or improving skin elasticity.

[0028] In this specification, the term "improvement of skin damage caused by reactive oxygen species" can refer to all effects of inhibiting or restoring the oxidative damage to skin cells caused by reactive oxygen species (ROS) generated by environmental factors or internal metabolic processes, thereby including the effect of preventing skin cell aging or restoring damaged skin tissue.

[0029] In this specification, the term "anti-inflammatory" can refer to all actions that inhibit or modulate an inflammatory response that is overactivated by inflammatory factors, thereby preventing tissue damage or alleviating the inflammatory state.

[0030] In this specification, the term "skin moisturizing" can refer to all functions that retain skin moisture or prevent moisture loss.

[0031] In this specification, the term "wrinkle" refers to a state of looseness caused by loss of skin elasticity, such as when the skin may fold. "Improving skin wrinkles" can refer to any action that prevents or improves wrinkles by inhibiting the expression of wrinkle-related factors, or by increasing the total amount of collagen.

[0032] In this specification, the term "improvement of acne" can refer to all effects of relieving or preventing acne symptoms caused by excessive sebum secretion, inflammation within hair follicles, or bacterial proliferation of the skin, and reducing acne scars.

[0033] In this specification, the term "improved sebum" can refer to all the effects of regulating excessive sebum secretion or inhibiting sebum production, thereby maintaining the oil balance on the skin surface or reducing excess sebum.

[0034] In this specification, the term "improvement of hair loss" can refer to the state of preventing hair from falling out due to abnormal hair growth, or all effects that promote hair growth and thus inhibit or reduce the progression of hair loss.

[0035] In this specification, the term "improved skin keratinization" can refer to the removal of the layer of dead cells accumulated on the skin surface—keratin—or to promoting the normal shedding process of keratin, thereby smoothing the skin texture and helping the skin regenerate.

[0036] In this specification, the term "improved skin elasticity" can refer to all the effects of increasing structural proteins such as collagen and elastin in the skin or repairing damaged skin tissue, thereby maintaining skin elasticity and firmness.

[0037] Another aspect relates to a cosmetic composition for improving skin condition or hair loss, comprising Saccharomyces cerevisiae with accession number KCTC 15765BP ( Saccharomyces cerevisiae Fermentation product of strain HBWKY-1.

[0038] The fermentation product can be obtained by inoculating the Saccharomyces HBWKY-1 strain into a culture medium containing plant malt syrup and then culturing it.

[0039] The culture medium may also include magnesium sulfate, manganese sulfate, or a combination thereof.

[0040] In this specification, the term "malt syrup" may refer to a liquid obtained by breaking down the starch of grains into sugars using enzymes contained in malt.

[0041] In this specification, the term "malt" can refer to grains that have been soaked in water to germinate and then dried to stop germination, and in which enzymes (especially amylases) generated during the process can break down the starch in the grains into sugars.

[0042] In one specific embodiment, the plant may be one or more selected from the group consisting of Polygonum cuspidatum root, plum, fig, arborvitae, sprouted black rice, dragon's blood, black bean, trumpet creeper, snow lotus, rowan, pollen, duckweed, juniper, fern, bleeding heart, mistletoe, sedum, golden camellia, mulberry fungus, tomato, broccoli, cabbage, houttuynia cordata, dried tangerine peel, peony, chestnut flower, burdock, kudzu root, walnut, corn, water celery, mugwort, toon, Holly wheat, and Aralia elata.

[0043] The improvement in skin condition can be achieved by improving skin damage caused by reactive oxygen species, reducing inflammation, moisturizing the skin, improving wrinkles, improving acne, improving sebum production, improving skin keratinization, or improving skin elasticity.

[0044] The cosmetic composition can be formulated into dosage forms such as toner, lotion, conditioning water, serum, essence, gel, ampoule, or cream, but is not limited thereto.

[0045] In addition to the composition as an active ingredient, the ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions, such as conventional adjuvants and carriers such as stabilizers, solvents, vitamins, pigments, and fragrances.

[0046] Another aspect involves a brewing yeast with accession number KCTC 15765BP ( Saccharomycescerevisiae Fermentation products of strain HBWKY-1 are used to improve skin condition or treat hair loss.

[0047] The same applies as described above to the uses of improving skin condition or hair loss.

[0048] Another aspect relates to a cosmetic composition for improving skin condition or hair loss, comprising Saccharomyces cerevisiae with accession number KCTC 15765BP ( Saccharomyces cerevisiae HBWKY-1 strain and Lactobacillus pentosus ( Lactobacillus pentosus ) The complex fermentation product of strain .

[0049] The compound ferment can be prepared by inoculating the Saccharomyces HBWKY-1 strain and the Lactobacillus pentosus strain into a culture medium containing plant malt syrup and then culturing them.

[0050] The culture medium may also include magnesium sulfate, manganese sulfate, or a combination thereof.

[0051] The *Lactobacillus pentosaccharide* strain can be *Lactobacillus pentosaccharide* strain HB-8023 with accession number KCTC 13322BP. This strain, *Lactobacillus pentosaccharide* strain HB-8023 with accession number KCTC 13322BP, has been disclosed in Korean Patent Registration No. 10-2152878. This *Lactobacillus pentosaccharide* strain HB-8023 was deposited at the Korean Collection for Type Cultures (KCTC) on August 30, 2017, and is classified and named *Lactobacillus pentosaccharide* HB-8023 (…). Lactobacillus pentosus HB-8023), with accession number KCTC 13322BP.

[0052] In one specific embodiment, the plant may be selected from the group consisting of Polygonum cuspidatum root, plum, fig, arborvitae, sprouted black rice, dragon's blood, black bean, trumpet creeper, snow lotus, rowan, pollen, duckweed, juniper, fern, bleeding heart, mistletoe, sedum, golden camellia, mulberry fungus, tomato, broccoli, cabbage, houttuynia cordata, dried tangerine peel, peony, chestnut flower, burdock, kudzu root, walnut, corn, water celery, mugwort, toon, Holly wheat, and Aralia elata.

[0053] The improvement in skin condition can be achieved by improving skin damage caused by reactive oxygen species, reducing inflammation, moisturizing the skin, improving wrinkles, improving acne, improving sebum production, improving skin keratinization, or improving skin elasticity.

[0054] The cosmetic composition can be formulated into dosage forms such as toner, lotion, conditioning water, serum, essence, gel, ampoule, or cream, but is not limited thereto.

[0055] In addition to the composition as an active ingredient, the ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions, such as conventional adjuvants and carriers such as stabilizers, solvents, vitamins, pigments, and fragrances.

[0056] Another aspect involves a brewing yeast with accession number KCTC 15765BP ( Saccharomyces cerevisiae HBWKY-1 strain and Lactobacillus pentosus ( Lactobacillus pentosus The compound fermentation product of the strain is used to improve skin condition or for hair loss.

[0057] The same applies as described above to the uses of improving skin condition or hair loss.

[0058] Another aspect involves a type of brewing yeast ( Saccharomyces cerevisiae A method for preparing fermentation products of strain HBWKY-1, comprising the following steps: (a) mixing plants with malt to prepare plant malt syrup; (b) Add brewing yeast ( Saccharomyces cerevisiae HBWKY-1 strain was inoculated into a medium containing plant malt syrup and cultured; and (c) Remove bacterial cells from the culture after it has been cultured.

[0059] In one specific embodiment, in step (a), the plant and the malt may be mixed in a weight ratio of 1:0.1 to 10, 1:0.1 to 5, 1:0.1 to 3, 1:1 to 10, 1:1 to 5, 1:1 to 3, 1:2 to 10, 1:2 to 5 or 1:2 to 3.

[0060] In one specific embodiment, step (a) can be performed at 0 to 100°C, 10 to 100°C, or 20 to 100°C.

[0061] In one specific embodiment, step (a) can be performed for 0.5 to 48 hours, 0.5 to 36 hours, 1 to 48 hours, 1 to 36 hours, 6 to 48 hours, or 6 to 36 hours.

[0062] In one specific embodiment, the plant may be one or more selected from the group consisting of Polygonum cuspidatum root, plum, fig, arborvitae, sprouted black rice, dragon's blood, black bean, trumpet creeper, snow lotus, rowan, pollen, duckweed, juniper, fern, bleeding heart, mistletoe, sedum, golden camellia, mulberry fungus, tomato, broccoli, cabbage, houttuynia cordata, dried tangerine peel, peony, chestnut flower, burdock, kudzu root, walnut, corn, water celery, mugwort, toon, Holly wheat, and Aralia elata.

[0063] Step (b) may also include additional inoculation and culture of Lactobacillus pentosus (Lactobacillus pentosus ) strains.

[0064] The Lactobacillus pentosus strain can be Lactobacillus pentosus strain HB-8023 with accession number KCTC 13322BP.

[0065] Step (b) can be performed at 0 to 50°C, 0 to 40°C, 10 to 50°C, 10 to 40°C, 20 to 50°C, or 20 to 40°C.

[0066] Step (b) can be performed at 0 to 300 rpm, 0 to 200 rpm, 10 to 300 rpm, 10 to 200 rpm, 100 to 300 rpm, or 100 to 200 rpm.

[0067] The plant malt syrup fermentation product prepared by the above preparation method can exhibit excellent skin condition or hair loss improvement effects.

[0068] The same applies to the preparation method as described above.

[0069] Beneficial effects

[0070] This relates to a cosmetic composition for improving skin condition or hair loss, comprising a novel strain of Saccharomyces cerevisiae and its ferments, which, according to one aspect of the cosmetic composition, exhibits a significant and superior effect in improving skin condition.

[0071] Brief description of the attached diagram

[0072] Figure 1 Figure showing the isolation, culture, and identification of the Saccharomyces HBWKY-1 strain derived from fig water kefir.

[0073] Figure 2 A diagram illustrating the classification of the Saccharomyces cerevisiae HBWKY-1 strain.

[0074] Figure 3 This is a graph showing the growth curve of the Saccharomyces cerevisiae HBWKY-1 strain over time.

[0075] Figure 4 The image shows the process of plating colonies of the HBWKY-1 strain of Saccharomyces cerevisiae onto a culture medium to confirm whether they secrete organic acids. Detailed Implementation

[0076] The present invention will be described in more detail below with reference to the following embodiments. However, these embodiments are for illustrative purposes only, and the scope of the invention is not limited to these embodiments.

[0077] Preparation Example 1: Isolation, culture, and identification of a *Saccharomyces cerevisiae* strain derived from fig water kefir.

[0078] In fig water kefir, the mycelium is separated from the fermentation liquid. Figure 1 After homogenization, the bacterial particles were spread onto SDA, TSA, and MRS agar to pick colonies. Morphologically identified yeast strains were then confirmed from the picked colonies, and the confirmed strains were analyzed by PCR using 18S primers.

[0079] The 18S rRNA sequence of the finally selected HBWKY-1 was identical to that of SEQ ID NO:1. The isolated strain is genetically related to *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae The sequences are similar, meaning they share 99% sequence homology, therefore final classification is performed through biochemical experiments. Figure 2 (and Table 1).

[0080] Specifically, prepare the inoculum for *Saccharomyces cerevisiae* HBWKY-1. Suspend the solid-cultured colonies on suspension medium, adjust the turbidity to 2 McFarlane, and then aliquot the culture into test strips to avoid air bubbles. Cover the trays and incubate at 30°C for 48 to 72 hours, then assess the turbidity. The results are shown in Table 1 below. Input the assessment results into the program and classify it as *Saccharomyces cerevisiae*.

[0081] Therefore, this strain was named *Saccharomyces cerevisiae* (Saccharomyces cerevisiae Saccharomyces cerevisiae HBWKY-1 was deposited at the Korean Collection for Type Cultures (KCTC) on January 10, 2024, with accession number KCTC 15765BP.

[0082]

SEQ ID NO: 1

[0083]

[0084] Table 1

[0085] Preparation Example 2: Preparation of Polygonum cuspidatum root extract

[0086] Preparation of Polygonum cuspidatum root ( Polygonum Cuspidatum Extraction. Specifically, purified water was extracted for 6 hours, yielding 10% by weight of dried and pulverized Polygonum cuspidatum root extract. The extract was then filtered using Whatman No. 5 filter paper to prepare the Polygonum cuspidatum root extract.

[0087] Preparation Example 3: Preparation of Polygonum cuspidatum root malt syrup

[0088] Preparation of Polygonum cuspidatum root malt syrup. Specifically, an enzymatic reaction was slowly carried out for 24 hours at 25 to 90°C in purified water containing 10% by weight of dried and pulverized Polygonum cuspidatum root extract and 25% by weight of malt. The syrup was then filtered using Whatman No. 5 filter paper.

[0089] Preparation Example 4: Preparation of Prune Malt Syrup

[0090] In addition to using plums ( Prunus Domestica In addition to the above, prune malt syrup was prepared using the same method as in Preparation Example 3.

[0091] Preparation Example 5: Preparation of Fig Malt Syrup

[0092] Besides using figs ( Ficus Carica; Fig In addition to the above, fig malt syrup was prepared using the same method as in Preparation Example 3.

[0093] Example 1: Preparation of Polygonum cuspidatum root malt syrup using HBWKY-1 brewer's yeast ferment.

[0094] In Preparation Example 3, a culture medium containing 2% by weight magnesium sulfate and 2% by weight manganese sulfate was prepared by inoculating *Saccharomyces cerevisiae* HBWKY-1 to produce a ferment. The ferment was then prepared by centrifugation and syringe filtration.

[0095] Comparative Example 1: Saccharomyces cerevisiae HBWKY-1 ferment for preparing Polygonum cuspidatum root extract

[0096] In Preparation Example 2, the Polygonum cuspidatum root extract was mixed with a culture medium containing 2% by weight magnesium sulfate and 2% by weight manganese sulfate. Saccharomyces cerevisiae HBWKY-1 was inoculated and cultured to prepare a ferment. The ferment was then prepared by centrifugation and syringe filtration.

[0097] Example 2: A combined fermentation product of *Saccharomyces cerevisiae* HBWKY-1 and *Lactobacillus pentosus* HB-8023 for preparing *Polygonum cuspidatum* root malt syrup.

[0098] In a culture medium containing 1 to 30% by weight of Polygonum cuspidatum root malt syrup prepared in Preparation Example 3, 2% by weight of magnesium sulfate and 2% by weight of manganese sulfate were mixed, and then HBWKY-1, a brewer's yeast derived from fig water kefir, was mixed in the same amount. Saccharomyces cerevisiae ; KCTC 15765 BP) and Lactobacillus pentosaccharide HB-8023 derived from Jeju citrus kimchi ( Lactobacillus pentosus (KCTC 13322 BP), and then fermented under culture conditions of 25 to 37°C and 150 rpm to prepare a compound ferment of yeast and lactic acid bacteria.

[0099] Comparative Example 2: The combined fermentation product of *Saccharomyces cerevisiae* ATCC 9763 and *Lactobacillus pentosus* HB-8023 used to prepare *Polygonum cuspidatum* root malt syrup.

[0100] In addition to using Saccharomyces cerevisiae ATCC 9763 and Lactobacillus pentosus HB-8023 ( Lactobacillus pentosus In addition to KCTC 13322 BP as the fermentation strain, a compound ferment of yeast and lactic acid bacteria was prepared using the same method as in Example 2.

[0101] Example 3: A combined fermentation product of *Saccharomyces cerevisiae* HBWKY-1 and *Lactobacillus pentosus* HB-8023 for preparing prune malt syrup.

[0102] Except for using the prune malt syrup from Preparation Example 4, a compound ferment of prune malt syrup yeast and lactic acid bacteria was prepared using the same method as in Example 2.

[0103] Example 4: A combined fermentation product of *Saccharomyces cerevisiae* HBWKY-1 and *Lactobacillus pentosus* HB-8023 for preparing fig malt syrup.

[0104] Except for using the fig malt syrup from Preparation Example 5, a compound ferment of fig malt syrup yeast and lactic acid bacteria was prepared using the same method as in Example 2.

[0105] Example 1: Characteristic Analysis of Saccharomyces HBWKY-1 Derived from Fig Water Kefir

[0106] 1-1 Growth Curve

[0107] The isolated *Saccharomyces cerevisiae* HBWKY-1 was inoculated into YPD liquid medium and cultured at 30°C. After incubation, bacterial culture was collected according to the incubation time and spread onto YPD agar at the appropriate dilution ratio, and incubated at 30°C for 2 days. After incubation, the resulting colonies were counted to confirm the bacterial count. The measured bacterial counts were converted to logarithmic values ​​to plot a growth curve. Figure 3 ).

[0108] 1-2. Enzyme activity

[0109] The API ZYM kit was used to confirm enzyme activity. Suspensions were prepared by centrifuging the liquid culture. The suspensions were aliquoted and reacted at 37°C for 4 hours, and the color changes were observed [Table 2]. The *Saccharomyces cerevisiae* derived from water kefir and *Lactobacillus pentosus* strains used for co-fermentation were reacted separately. The results confirmed that the yeast derived from water kefir exhibited higher activity for both alkaline phosphatase and acid phosphatase.

[0110] Table 2

[0111] 1-3. Acid secretion

[0112] After culturing, the generated colonies were picked and plated using BCP plate counting agar (EIKEN) solid medium to confirm the presence of organic acid secretion. Because the medium contains bromocresol purple reagent, the organic acids secreted by the strain cause a decrease in pH, resulting in a yellow border around the colony, thus confirming that the strain is secreting organic acids. Figure 4 ).

[0113] 1-4. Acid resistance

[0114] In this embodiment, an experiment was conducted to test whether the isolated and identified *Saccharomyces cerevisiae* strain HBWKY-1 possessed acid resistance. The strain was cultured at 35–37°C for 2 days, and the pH was adjusted to pH 2 using citric acid or HCl. It was then mixed with 1 mg / ml pepsin. Under these conditions, the reaction was carried out for 1 hour, followed by plating according to the dilution factor. After culturing, the resulting white colonies were counted as *Saccharomyces cerevisiae* HBWKY-1 colonies, and the results are shown in Table 3 below.

[0115] Table 3

[0116] Example 2: Composition of the culture medium used to culture a strain of Saccharomyces cerevisiae derived from fig water kefir

[0117] The minimum culture conditions for the isolated *Saccharomyces cerevisiae* HBWKY-1 were determined by measuring the bacterial count based on the composition of the culture medium used for culturing. Fermentations from Example 1 and Comparative Example 1 were inoculated onto YPD liquid medium and cultured at 30°C. The bacterial suspensions were collected, spread onto YPD agar at the appropriate dilution, and cultured at 30°C for 2 days. After culturing, the resulting colonies were counted, and the results are shown in Table 4 below.

[0118] Table 4

[0119] As a result, it was confirmed that the culture medium composition of Example 1, which contained Polygonum cuspidatum root malt syrup, was suitable for the culture of the fig water kefir strain.

[0120] Experiment Example 3: Confirming the content of active ingredients

[0121] The contents of polyphenols and flavonoids in ferments containing natural maltose syrup as a culture medium composition according to a specific embodiment, as well as in complex ferments of yeast and lactic acid bacteria, were confirmed. Polyphenol content was determined using the Folin-Denis method, and total flavonoid content was determined using a modified method of Nieva Moreno et al. (2000). First, for polyphenol content, the sample was dissolved in distilled water at a concentration of 10 mg / ml. 0.2 ml of the sample was placed in a test tube, diluted to 2 ml with distilled water, and then 0.2 ml of Foiln-Ciocalteu phenol reagent was added. After mixing, the mixture was reacted at room temperature for 3 minutes. Then, 0.4 ml of saturated Na2CO3 solution was added and mixed, followed by 1.4 ml of distilled water. The mixture was reacted at room temperature for 1 hour, and the absorbance was measured at 725 nm using a UV / Vis spectrophotometer. At this point, for the total polyphenolic compounds, gallic acid (Sigma Co, USA) was dissolved in distilled water at a concentration of 10 mg / ml to prepare solutions with final concentrations of 0, 37.5, 75, 150, and 300 μg / ml. Standard curves were plotted according to the method described above, and the total polyphenolic compound content in the extract was calculated using the standard curves. For the flavonoid content, the method of Nieva Moreno et al. (2000) was modified. The solution was diluted to a concentration of 10 mg / ml using 80% ethanol as a solvent. 0.1 ml of 10% aluminum nitrate, 0.1 ml of 1M potassium acetate, and 4.7 ml of 80% ethanol were added to 0.1 ml of the sample, and the mixture was reacted at 25°C for 40 minutes. The absorbance was then measured at 415 nm using a UV / Vis spectrophotometer. For the quantification of total flavonoids, quercetin (Sigma Chemical Co.) was used to make final concentrations of 0, 10, 25, 50, 100, 250 and 500 μg / ml. The content of flavonoid compounds for each extract was calculated using the same method as described above, based on the measured standard curves, and the contents are shown in Table 5.

[0122] Table 5

[0123] As shown in Table 5, in Examples 2 to 4, which were compound ferments using the Saccharomyces cerevisiae HBWKY-1 strain, the contents of total polyphenols and total flavonoids were significantly higher than those in Comparative Example 2, which was a compound ferment using the Saccharomyces cerevisiae ATCC 9763 strain. Furthermore, compared to Example 1, which was a fermentation using a single yeast strain, the contents of total polyphenols and total flavonoids increased in Examples 2 to 4, which were compound ferments of yeast and lactic acid bacteria.

[0124] In Examples 2 to 4, the total polyphenol and total flavonoid content in the yeast and lactic acid bacteria complex fermentation product, which included Polygonum cuspidatum root malt syrup as the culture medium in Example 2, was significantly higher than in the other examples. This confirms that the active ingredients can be maximized by the fermentation product manufacturing process according to a specific example.

[0125] Experiment Example 4: Confirmation of Cytotoxicity

[0126] The cytotoxicity of yeast ferments and a combination of yeast and lactic acid bacteria ferments to skin cells using a natural maltose syrup culture medium composition according to a specific embodiment was confirmed. Specifically, cytotoxicity was measured using the EZ-Cytox kit. Each cell line was cultured at 2 × 10⁻⁶. 5 Cells / mL were aliquoted into 24-well plates and incubated at 37°C for 24 hours. Samples were then diluted in DMEM medium at each concentration and incubated for 24 hours. The original medium was removed, and phenol red-free DMEM and CCK-8 reagent diluted 10:1 were added. The plates were incubated for 30 minutes, and the absorbance at 450 nm was measured using a microplate reader. Cell viability (%) was calculated using Equation 1 below, and the sample concentration that did not affect cell viability was determined. The results are shown in Table 6 below.

[0127]

Mathematical Formula 1

[0128] Cell viability (%) = [(St-Bo) / (Bt-Bo)] × 100

[0129] Bo: Absorbance at 450 nm of wells containing only cell culture medium and used for colorimetric reactions.

[0130] Bt: Absorbance at 450 nm of the well before sample treatment and colorimetric reaction.

[0131] St: Absorbance at 450 nm of the well after sample processing and colorimetric reaction.

[0132] Table 6

[0133] As shown in Table 6, compared with Example 1, which was a fermentation product of a single strain, Examples 2 to 4 and Comparative Example 2, which were fermentations of a combination of yeast and lactic acid bacteria, showed a higher 100% cell viability concentration.

[0134] Therefore, the compound ferment according to a specific embodiment can be used as a safe and effective composition because it is less likely to cause skin irritation and has a wide range of useful concentrations.

[0135] Experiment 5: Confirmation of free radical scavenging activity

[0136] To determine the free radical scavenging activity of ferments using a natural maltose syrup culture medium composition according to a specific embodiment, as well as a complex ferment of yeast and lactic acid bacteria, free radical scavenging activity was determined under laboratory conditions using an extract with excellent antioxidant activity (e.g., green tea extract) as a comparative sample, employing the DPPH method. Free radical scavenging activity by reducing power was determined using DPPH (2,2-bis(4-tert-octylphenyl)-1-picrylhydrazine radical). The degree of absorbance decrease after reduction of the DPPH test sample was compared with the absorbance of the control solution, and the free radical scavenging rate was determined at a wavelength of 560 nm. To measure the DPPH free radical scavenging activity, extracts of different concentrations were prepared. The extracts of these concentrations were placed in 96-well plates, and DPPH prepared as a 100 μM methanol solution was added to bring the total solution volume to 200 μL. The plates were incubated at 37°C for 30 minutes, and then the absorbance was measured at 560 nm. The free radical scavenging activity (%) was calculated using the following mathematical formula 2, and the results are shown in Table 7 below. Butylated hydroxytoluene (BHT) was used as a positive control.

[0137]

Mathematical Formula 2

[0138] Free radical scavenging activity (%) = 100 - (BA) × 100

[0139] A: Absorbance of the untreated control group wells

[0140] B: Absorbance of the test group wells

[0141] Table 7

[0142] As shown in Table 7, in Examples 2 to 4 and Comparative Example 2, which are combined ferments of yeast and lactic acid bacteria, the free radical scavenging rate was significantly higher than that of Example 1, which is a ferment of yeast alone. In particular, in the combined ferment of yeast and lactic acid bacteria containing Polygonum cuspidatum root malt syrup from Example 2 as the culture medium, the free radical scavenging rate was higher than that of the other examples. This confirms that antioxidant activity can be maximized by the fermentation preparation process according to a specific embodiment.

[0143] Because the compound ferment according to a specific embodiment has excellent free radical scavenging activity, it can be effectively used to prevent or improve aging.

[0144] Experimental Example 6: Inhibition of Inflammatory Cytokine Expression Caused by Ultraviolet Radiation Irradiation

[0145] To confirm whether the expression of inflammatory cytokines induced by ultraviolet radiation in Examples 2 to 4 and Comparative Example 2, which were selected as the compound ferments in Experimental Example 5, could be suppressed, fibroblasts isolated from human epidermal tissue were seeded into 24-well test plates, 5 × 10⁶ cells per well. 4 Cells were cultured and adhered for 24 hours. Then, each well was washed once with PBS, and 500 μL of PBS was added to each well. The fibroblasts were irradiated with UVB lamp (F15T8, UVB 15W, Sankyo Electric Co., Ltd., Japan) at 10 mJ / cm², the PBS was removed, and 350 μL of cell culture medium (DMEM medium without FBS) was added. The samples to be evaluated were treated in this medium and then cultured at 37°C for 5 hours. Then, 150 μL of the culture supernatant was taken, and the inhibitory effect of the culture medium on the expression of inflammatory cytokines was assessed by quantifying IL-1α. The IL-1α content was quantified using an enzyme-linked immunosorbent assay (ELISA), and the IL-1α production rate was calculated according to the following mathematical formula (Table 8).

[0146]

Mathematical Expression 3

[0147] Inhibition rate of inflammatory cytokine expression (%) = [1 - (St-Bo) / (Bt-Bo)] × 100

[0148] Bo: IL-1α formation in wells that were not exposed to UV light and were not sample-treated.

[0149] Bt: IL-1α formation in wells exposed to ultraviolet light without sample treatment

[0150] St: IL-1α formation in wells treated with UV light and sample.

[0151] Table 8

[0152] As a result, in Examples 2 to 4, the inhibition rate of inflammatory cytokine expression was confirmed to be higher than that in Comparative Example 2. Among them, Example 2 showed the highest inhibition rate.

[0153] Experiment Example 7: Confirmation of gene expression related to promoting moisturization

[0154] To confirm the expression of genes related to moisturizing in the samples selected in Experimental Example 5, the following experiments were conducted. To evaluate the moisturizing effect, RT-PCR was used to determine the expression of filaggrin, a precursor of the skin's natural moisturizing factor (NMF); aquaporin 3, which transports water and glycerol to various parts of the skin to maintain moisture; and hyaluronan synthase 2 (HAS 2), an enzyme that synthesizes hyaluronic acid in cells. Keratinocyte cell lines were cultured at 3 × 10⁻⁶ cells / year. Dilute to 1 / ml, dispense 3ml into each 35µm culture dish, and incubate for 24 hours in medium supplemented with 10% FBS. Then, dilute the sample in medium containing 2% FBS and incubate for another 48 hours. After incubation, total RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany). Using 1 μg of total RNA as a template, cDNA was synthesized by reacting with oligo(dT)15 primers, 0.5 μM dNTPs, 1 unit RNase inhibitor, and 4 units Omniscript reverse transcriptase (Qiagen, Hilden, Germany) at 37°C for 60 minutes, followed by heating at 93°C for 5 minutes to terminate the reaction. For PCR, to amplify AQP3, HAS2, and GAPDH using the cDNA as a template, mix 2 μL cDNA, 0.5 μM forward and reverse primers, 10× buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 200 μM dNTPs, and 25 mM MgCl2. 2.5 units of Taq polymerase (Qiagen, Hilden, Germany) were added to a final volume of 25 μL with distilled water for PCR. The generated PCR products were separated by electrophoresis on a 2% agarose gel. Gene expression was confirmed using an image analyzer (Davinch K, South Korea), and the density of each band was quantified using ImageJ software (NIH Image software, Maryland, USA).

[0155] The results, as shown in Table 9, showed that compared with 1% hyaluronic acid as a positive control group, the expression of moisturizing-related genes was increased in the examples, especially in Examples 2 and 3, where the expression of filaggrin, which is important for maintaining skin hydration, was twice that of the control group.

[0156] Table 9

[0157] Experiment Example 8: Confirming the effects of skin wrinkles and moisturizing

[0158] For the samples selected in Experimental Example 5, the skin wrinkle and moisturizing improvement activities were confirmed. Specifically, human dermal fibroblasts (Modern Tissue Technology, Korea) were used at a rate of 1×1 Cells were seeded at a density of cells / cm² in DMEM / F-12 (3:1) medium supplemented with 10% fetal bovine serum (FBS) and cultured. When the cells grew to approximately 70-80% confluence, they were aliquoted and passaged at a ratio of 1:3, and cells from passages 3-4 were used for experiments. After culturing the fibroblasts to over 90% confluence in 48-well plates, Examples 2-4 and Comparative Example 2 were treated with non-cytotoxic concentrations. After 24 hours of culture, the amount of procollagen released in the culture medium was measured using a procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan), and the results are shown in Table 10 below. Ascorbic acid was used as a positive control.

[0159] Table 10

[0160] As shown in Table 10, the pre-collagen production rates of Examples 2 to 4 were significantly higher than those of Comparative Example 2. This confirms that the fermentation product of the strain according to a specific embodiment can be effectively used to prevent or improve skin wrinkles and for moisturizing.

[0161] Experiment 9: Confirming the effects of improving acne and sebum, as well as preventing or improving hair loss.

[0162] For the samples selected in Experimental Example 5, the effects of preventing or improving acne and sebum were confirmed. Specifically, 0.1 μCi of tritium (3H) radiolabeled testosterone, along with samples from Examples 2 to 4 and Comparative Example 2, were added to each well of a microplate, and then 10,000 fibroblasts were seeded and cultured. After 24 hours of culture, the supernatant was collected, and steroids were extracted using 1 ml of ethyl acetate-cyclohexane (1:1) as the extraction solvent. The steroids were placed on a thin-film chromatography plate and developed with a chloroform / methanol mixture (98 / 2, v / v). The radioactivity corresponding to the spots of testosterone and dihydrotestosterone (DHT) was measured using a densitometer, and the conversion rate was calculated accordingly. The 5α-reductase inhibition rate was evaluated by comparing it with the negative control group according to the following mathematical formula 4, and the results are shown in Table 11 below.

[0163] DHT plays a role in promoting sebum secretion in sebaceous glands and is considered one of the main causes of hair loss. Therefore, when the inhibition rate of 5α-reductase is high, the production of DHT is reduced, thereby decreasing the activity of sebaceous glands, which can alleviate acne and other skin problems, inhibit hair loss, and improve the hair growth cycle.

[0164]

Mathematical Expression 4

[0165] 5α-Reductase Inhibition Rate (%) = (AB) / A × 100

[0166] A: Conversion rate of testosterone to dihydrotestosterone when no sample was added.

[0167] B: Conversion rate of testosterone to dihydrotestosterone when the sample is added.

[0168] Table 11

[0169] As shown in Table 11, the 5α-reductase inhibition rates of Examples 2 to 4 were significantly higher than those of Comparative Example 2. This confirms that the fermentation product of the strain according to a specific embodiment inhibits the conversion of dihydrotestosterone induced by 5α-reductase, and thus can be used not only to prevent or improve acne and sebum, but also to prevent, improve, or treat hair loss, or to promote hair growth or nourish hair.

[0170] Experimental Example 10: Analysis of Lactic Acid Content in Compound Fermented Products

[0171] The content of lactic acid, one of the AHA components in the samples selected in Experimental Example 5, was analyzed. Specifically, high performance liquid chromatography (HPLC) was used to analyze the lactic acid production of the complex strains. The results showed that lactic acid was detected in all the complex ferments, and in particular, the highest lactic acid content was found in Example 3 (Table 12).

[0172] Lactic acid is known to loosen the intercellular connections in the stratum corneum of the skin, thereby promoting the natural shedding of dead skin cells. Therefore, it is confirmed that the fermentation product of the strain according to a specific embodiment can be effectively used to improve skin keratinization.

[0173] Table 12

[0174] Experiment 11: Confirming the improvement effect of exfoliation

[0175] The exfoliation-improving effect of a cosmetic composition containing a yeast and lactic acid bacteria complex fermentation product according to a specific embodiment was confirmed. First, a cream-form cosmetic composition containing the ingredients and amounts listed in Table 13 below was prepared. Specifically, phase B of Table 13 was heated and stored at 70°C, and then phase A was added for pre-emulsification. Then, the mixture was homogenized using a homogenizer and subsequently slowly cooled to prepare the cosmetic composition.

[0176] Both formulation 1 and comparative formulation 1 were administered to 30 women aged 20 to 35 years, twice daily for 30 consecutive days. The degree of exfoliation was then assessed using a questionnaire, and the results are shown in Table 14 below.

[0177] Table 13

[0178] Table 14

[0179] (Assessment levels: -3 = very inadequate, -2 = inadequate, -1 = slightly inadequate, 0 = no change, 1 = slightly improved, 2 = improved, 3 = very improved)

[0180] As shown in Table 14, compared with Comparative Formulation Example 1, Formulation Example 1 has a superior effect in improving keratin removal.

[0181] Experiment 12: Confirming the skin moisturizing effect

[0182] The clinical efficacy of a cosmetic composition containing a yeast and lactic acid bacteria complex ferment according to a specific embodiment in improving skin moisturizing was confirmed. Specifically, two skin sites were selected for testing, and skin moisture content was measured three times at each site. The dosage form of Table 13 of Experimental Example 11 was then administered to 20 women aged 20 to 35 years in the same manner. The moisture content of the epidermis beneath the stratum corneum at the application site was then measured using a Corneometer (CM 825, C+K, Germany) at different time points (immediately after application, 30 minutes after application, 60 minutes after application, 90 minutes after application, and 120 minutes after application). The results are shown in Table 15 below.

[0183] Table 15

[0184] As shown in Table 15, compared with Comparative Formulation Example 1, Formulation Example 1 exhibited superior water content and long-lasting moisturizing effect after 90 minutes of application. This confirms that the fermentation product of the strain according to a specific embodiment can be effectively used for skin moisturizing.

[0185] Experiment 13: Confirming the effect of improving skin elasticity

[0186] The skin elasticity-improving effect of the cosmetic composition comprising an embodiment according to a specific example was confirmed. First, the dosage form of Experimental Example 11 (Table 13) was administered to 20 women aged 20 to 35 years twice daily for two consecutive months. Then, the elasticity before and after using dosage form 1 and the comparative dosage form 1 was measured using a skin elasticity meter (Cutometer SEM 575, C+K Electric Co., Germany), and the results are shown in Table 16 below. The measured values ​​are expressed as the R7 value of the Cutometer SEM 575, which reflects the viscoelasticity of the skin.

[0187] Table 16

[0188] As shown in Table 16, the skin viscoelasticity of dosage form 1 increased compared to comparative dosage form 1. This confirms that the bacterial ferment according to a specific embodiment can be effectively used to improve skin elasticity.

[0189] Experiment 14: Confirming the effect of improving skin wrinkles

[0190] The skin wrinkle-improving effect of the cosmetic composition comprising an embodiment according to a specific example was confirmed. Specifically, the same subjects used the dosage form of Experimental Example 11 as described in Table 13 using the same method. Then, a silicone material replica was created, and the skin wrinkle state after using the sample of dosage form 1 and the comparative dosage form 1 was measured using an image analyzer—a wrinkle measuring instrument (Visiometer: SV60, C+K Electronic, Germany). The results are shown in Table 17 below. The measured values ​​are expressed as the average of the parameter values ​​after 2 months of use minus the parameter values ​​before use, where a more negative measured value indicates a more significant wrinkle-improving effect.

[0191] Table 17

[0192] R1: The difference between the maximum and minimum values ​​of the wrinkle contour lines.

[0193] R2: The average R1 value of each segment after arbitrarily dividing the wrinkle contour line into 5 segments.

[0194] R3: The maximum value of R1 among the five segments.

[0195] R4: The average value obtained by subtracting each peak and valley value from the baseline of the wrinkle contour lines.

[0196] R5: The average value after subtracting each wrinkle contour from the baseline of the wrinkle contour lines.

[0197] As shown in Table 17, the parameter values ​​representing the state of skin wrinkles in dosage form 1 were significantly lower than those in comparative dosage form 1. This confirms that the fermentation product of the strain according to a specific embodiment can be effectively used to improve skin wrinkles.

[0198] Experiment 15: Confirming the effect of improving sebum production

[0199] The sebum-improving effect of the cosmetic composition comprising an embodiment according to a specific example was confirmed. Specifically, subjects used the dosage forms listed in Table 18 below. Then, objective visual observation was performed by multiple skilled personnel, and subjective visual observation was performed by the subjects, and the degree of sebum-improving efficacy was evaluated according to the following levels, the results of which are shown in Table 19 below.

[0200] Table 18

[0201] Table 19

[0202] (Assessment levels: -3 = Very poor, -2 = Poor, -1 = Slightly poor, 0 = No change, 1 = Slight improvement, 2 = Improvement, 3 = Very improved)

[0203] As shown in Table 19, dosage form 2 exhibited a better sebum-improving effect than comparative dosage form 2. This confirms that the fermentation product of the strain according to a specific embodiment can be effectively used to improve sebum production.

Claims

1. A type of brewing yeast ( Saccharomyces cerevisiae The HBWKY-1 strain, with accession number KCTC15765BP, is a strain of HBWKY-1.

2. The strain according to claim 1, wherein, The strain has the 16S rRNA base sequence of SEQ ID NO:

1.

3. A cosmetic composition for improving skin condition or hair loss, comprising a ferment of Saccharomyces cerevisiae strain HBWKY-1 with accession number KCTC 15765BP.

4. The cosmetic composition according to claim 3, wherein, The fermentation product is prepared by inoculating the Saccharomyces HBWKY-1 strain into a culture medium containing plant malt syrup and then culturing it.

5. The cosmetic composition according to claim 4, wherein, The plants mentioned are selected from one or more of the following groups: Japanese knotweed root, plum, fig, arborvitae, sprouted black rice, dragon's blood, black bean, trumpet creeper, snow lotus, rowan, pollen, duckweed, juniper, fern, bleeding heart, mistletoe, sedum, golden camellia, mulberry fungus, tomato, broccoli, cabbage, houttuynia cordata, dried tangerine peel, peony, chestnut flower, burdock, kudzu root, walnut, corn, water celery, mugwort, toon, Holly wheat, and Aralia elata.

6. The cosmetic composition according to claim 3, wherein, The improvement in skin condition refers to the improvement of skin damage caused by reactive oxygen species, anti-inflammation, skin moisturization, improvement of skin wrinkles, improvement of acne, improvement of sebum, improvement of skin keratinization, or improvement of skin elasticity.

7. A cosmetic composition for improving skin condition or hair loss, comprising a compound ferment of Saccharomyces cerevisiae strain HBWKY-1 and Lactobacillus pentosus strain KCTC 15765BP.

8. The cosmetic composition according to claim 7, wherein, The compound ferment is prepared by inoculating the Saccharomyces HBWKY-1 strain and the Lactobacillus pentosus strain into a culture medium containing plant malt syrup and then culturing them.

9. The cosmetic composition according to claim 7, wherein, The Lactobacillus pentosus strain is Lactobacillus pentosus strain HB-8023 with accession number KCTC13322BP.

10. The cosmetic composition according to claim 8, wherein, The plants mentioned are selected from one or more of the following groups: Japanese knotweed root, plum, fig, arborvitae, sprouted black rice, dragon's blood, black bean, trumpet creeper, snow lotus, rowan, pollen, duckweed, juniper, fern, bleeding heart, mistletoe, sedum, golden camellia, mulberry fungus, tomato, broccoli, cabbage, houttuynia cordata, dried tangerine peel, peony, chestnut flower, burdock, kudzu root, walnut, corn, water celery, mugwort, toon, Holly wheat, and Aralia elata.

11. The cosmetic composition according to claim 7, wherein, The improvement in skin condition refers to the improvement of skin damage caused by reactive oxygen species, anti-inflammation, skin moisturization, improvement of skin wrinkles, improvement of acne, improvement of sebum, improvement of skin keratinization, or improvement of skin elasticity.

12. A method for preparing a ferment of Saccharomyces cerevisiae strain HBWKY-1, comprising the following steps: (a) Mixing plants with malt to prepare plant malt syrup; (b) Inoculating the *Saccharomyces cerevisiae* strain HBWKY-1 into a medium containing plant malt syrup and culturing it; and (c) Remove bacterial cells from the culture after it has been cultured.

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