A method for preparing and applying fermented Job's tears filtrate with anti-glycation and antioxidant effects.

Abstract

This invention relates to a method for preparing and applying a fermentation product filtrate of germinated Job's tears with anti-glycation and antioxidant effects, belonging to the field of microbial fermentation and cosmetic technology. This invention provides a brewer's yeast, specifically Saccharomyces cerevisiae A3, which was deposited on March 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37866. Using this fermentation strain to aerobically ferment the saccharified filtrate of germinated Job's tears not only efficiently converts the nutrients in Job's tears but also fully releases the bound active substances, resulting in multiple effects including whitening and brightening, anti-dullness, anti-glycation, yellowing removal, antioxidant, anti-inflammatory, and moisturizing. Furthermore, the product maintains a slightly acidic pH similar to that of the skin and is alcohol-free, avoiding the irritating effects of alcohol and excessively low pH on the skin.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation and cosmetics technology, and in particular to a method for preparing and applying a fermented product filtrate of sprouted Job's tears with anti-glycation and antioxidant effects. Background Technology

[0002] Job's tears, also known as coix seed or Job's tears kernel, is a common Chinese medicinal and edible herb with high nutritional value. Known as the "King of Gramineae," it contains various amino acids, polyphenols, flavonoids, triterpenoids, vitamins, and essential trace elements, among other active ingredients. In cosmetics, it possesses antioxidant, anti-glycation, whitening, brightening, anti-inflammatory, and moisturizing properties. In recent years, with the increasing demand for natural, green, and functional cosmetic ingredients, developing rice-based ingredients like Job's tears into skincare products has become a research hotspot. Especially abroad, in countries like Japan, rice-based ingredients are widely used in face creams, masks, and other skincare products, achieving positive market feedback and demonstrating its enormous potential in the cosmetics field.

[0003] To enhance the application value of Job's tears, it is usually fermented. The fermentation process promotes the degradation of starch in Job's tears, providing a carbon source for microbial growth, and releasing active substances bound to polysaccharides, thus giving the fermentation broth functional activity. Based on the theory of fermentation metabolic regulation, using yeast cell transformation can shorten the fermentation cycle and produce various active products during yeast growth, such as essential amino acids, sterols, phytic acid, and vitamins, thereby significantly improving the nutrient density and physiological activity of Job's tears. Therefore, fermented Job's tears have significantly improved nutritional and health benefits, and its application value far exceeds that of untreated Job's tears.

[0004] Gamma-aminobutyric acid (GABA), a naturally occurring non-protein amino acid, exhibits diverse bioactivities in skin care. Studies have shown that GABA can significantly promote the synthesis of type I collagen and elastin in human skin fibroblasts (HDFs), thereby enhancing skin firmness and elasticity. Simultaneously, it reduces collagen degradation and delays skin aging by inhibiting the expression of matrix metalloproteinase-1 (MMP-1). Furthermore, GABA possesses antioxidant properties, scavenging free radicals, maintaining skin barrier integrity, promoting hyaluronic acid production, improving skin's water retention capacity, and alleviating dryness. These properties make it an ideal active ingredient in anti-aging, moisturizing, and repairing skincare products.

[0005] Metallothionein is a special low-molecular-weight protein rich in cysteine, possessing a powerful ability to bind metal ions and scavenge free radicals. Compared to traditional single-target whitening agents, metallothionein's brightening effect has the following significant advantages: it does not directly interfere with melanocytes, but rather brightens skin tone by addressing the root causes of dullness—oxidation and inflammation—treating the underlying issues rather than just the symptoms. It simultaneously improves various skin tone problems such as yellowing, dullness, redness, acne scars, and roughness, resulting in an even, translucent, and radiant overall healthy complexion. It can protect and enhance the effects of other active ingredients, making it an excellent synergistic ingredient. While brightening, it also provides powerful antioxidant and anti-photoaging benefits, achieving multiple benefits of anti-aging and brightening in one step.

[0006] Glucocerebroside lipase is a key hydrolytic enzyme in the epidermis, participating in the lamellar body metabolism of intercellular lipids in the stratum corneum. Decreased or imbalanced β-glucocerebroside lipase activity disrupts the lipid layering structure of the stratum corneum, leading to barrier damage and rough skin. Therefore, enhancing or activating this enzyme helps promote ceramide production at its source, improving the skin barrier and texture. In cosmetics, glucocerebroside lipase can play a role in barrier repair and moisturizing, whitening and evening skin tone, and soothing and protecting the skin against photoaging.

[0007] Coixol, coixenolide, various amino acids, small molecule peptides, organic acids, phenolic compounds, vitamins, and enzymes also show good effects in whitening, anti-oxidation, anti-wrinkle, soothing, and moisturizing. For example, coixol can inhibit the release of inflammatory factors (such as TNF-α, IL-6, and PGE2), thus playing an anti-inflammatory and soothing role and alleviating sensitive skin problems. It can competitively inhibit tyrosinase activity (a key enzyme in melanin synthesis) and block the transport of melanosomes to keratinocytes, reducing epidermal pigmentation and inhibiting melanin production, thus whitening and fading spots. It can promote the orderly shedding of keratinocytes, reducing the accumulation of dead skin cells, and at the same time enhance the skin barrier function (by increasing the expression of filaggrin and ceramides), improving problems such as rough skin and keratosis pilaris, making the skin more delicate and smooth. It can regulate keratin metabolism, improve roughness, and brighten the skin. It can enhance the water-locking ability of the stratum corneum and relieve dryness. At the same time, it can scavenge free radicals (such as DPPH free radicals), reduce oxidative damage caused by ultraviolet rays or pollution, and delay photoaging, thus providing mild moisturizing and antioxidant effects. Coix seed oil can form a thin, breathable protective film on the skin surface, reducing transepidermal water loss while replenishing lipids for dry skin. It alleviates flaking and tightness caused by barrier damage, providing deep nourishment and improving dryness and flaking. It can be absorbed by the intercellular spaces of the stratum corneum, participating in the construction of the lipid barrier, promoting the production of barrier components such as ceramides and cholesterol, and enhancing the skin's tolerance to external stimuli (such as cold, heat, and cosmetic ingredients). It is suitable for repairing sensitive skin with a damaged barrier, helping to repair the skin barrier and enhance tolerance. It can reduce compensatory inflammation caused by dryness (such as redness caused by itching and scratching). When combined with coix seed oil, it can synergistically reduce skin sensitivity, providing auxiliary anti-inflammatory effects and relieving redness. Amino acids (such as glutamic acid, glycine, cysteine, and lysine) indirectly regulate melanin production by affecting tyrosinase activity and its signaling pathway, thus exerting a whitening effect. Phenolic compounds (such as flavonoids and phenolic acids) have strong antioxidant capabilities, neutralizing free radicals while inhibiting tyrosinase activity, achieving a dual whitening effect. Small molecule organic acids (such as kojic acid, citric acid, and succinic acid) not only have moisturizing, antibacterial, and exfoliating functions, but can also chelate metal ions and reduce oxidation reactions. Peptides can achieve anti-wrinkle effects through a dual mechanism: on the one hand, they promote collagen synthesis and skin structure repair; on the other hand, they inhibit muscle contraction through neurotransmitter regulation, reducing the formation of dynamic wrinkles. Enzymes (such as superoxide dismutase, SOD) have the ability to directly scavenge free radicals, enhancing antioxidant properties. Amylase, cellulase, and other enzymes can help decompose complex substances in the fermentation substrate, improving fermentation efficiency and product diversity. Proteases can promote the conversion and generation of proteins or peptides in the fermentation broth, for example, breaking down large protein molecules into smaller peptides and amino acids, thereby improving the whitening and antioxidant effects of the fermentation broth. Small molecule collagen peptides, vitamins, and other active factors in fermentation metabolites help repair, nourish, and reduce inflammation of the skin, lowering skin sensitivity.

[0008] Chinese patent CN110403881A discloses a fermentation product extract of Coix seed, a whitening herb, obtained by using 80-90 parts by weight of dried Coix seed powder as raw material and 1-20 parts by weight of a special yeast strain, after a series of fermentation cultures. This method does not employ a Coix seed germination process, thus failing to activate the active ingredients in Coix seed. Furthermore, the product only mentions coixol, without providing clear and detailed data on whether it contains other active ingredients. Moreover, the product's efficacy is only mentioned as reducing tyrosinase-induced melanin production, without specifying the exact tyrosinase inhibition rate or providing clear and detailed data on any other effects.

[0009] Chinese patent CN114990186A discloses a method for fermenting Job's tears using a mixture of three bacterial strains: Lactococcus lactis, Lactobacillus bulgaricus, and Streptococcus thermophilus. This method significantly increases the content of total phenols and total acids in the fermentation broth, improves the scavenging rate of hydroxyl radicals and the inhibitory effect on tyrosinase activity, and demonstrates superior antioxidant and whitening properties. However, this method does not utilize the Job's tears germination process, thus failing to activate the active ingredients in Job's tears. Furthermore, the final product from lactic acid bacteria fermentation tends to have a high acidity and may contain components that could adversely affect the skin. Moreover, the mixed-strain fermentation process is complex and difficult to control; different bacterial strains may produce antagonistic substances, affecting the efficacy of the final product. Finally, the product only explicitly states that it contains total sugars, total phenols, and total acids, without providing clear and detailed data on whether it contains other active ingredients.

[0010] In summary, the current research on coix seed fermentation products still has the following limitations: (1) The coix seed germination process is not used, so the active ingredients and enzyme system of coix seed cannot be activated, and the utilization rate of coix seed resources is not high; (2) The types of active ingredients in the fermentation product filtrate are relatively simple and the content is generally low. There are no reports of active ingredients such as metallothionein and glucocerebrosidase; (3) The fermentation product filtrate has the technical defect of single efficacy. When used alone, it is difficult to have multiple effects such as whitening, anti-wrinkle, brightening, anti-oxidation, anti-aging, soothing and moisturizing; (4) The existing fermentation process is relatively complicated and difficult to operate and control. Different strains of bacteria may produce antagonistic substances during fermentation, which will affect the efficacy and safety of the product; In addition, some fermentation processes produce irritating byproducts such as alcohol or the product acidity is too high, which will irritate the skin and limit its application in cosmetics and other fields with high safety requirements. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying a fermented coix seed product filtrate with anti-glycation and antioxidant effects.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a brewing yeast, wherein the brewing yeast is brewing yeast A3, which was deposited on March 6, 2026 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37866.

[0013] This invention has screened out a brewing yeast, which, when used as a fermentation strain, aerobically ferments germinated Job's tears. This process enriches the fermentation product filtrate with various active ingredients such as coixol, coixenolide, γ-aminobutyric acid, metallothionein, glucocerebrosidase, multiple amino acids, proteins, small molecule organic acids, and phenolic substances. Furthermore, the fermentation product filtrate is alcohol-free, avoiding the skin irritation caused by alcohol. It also possesses multiple skincare benefits, including whitening and brightening, anti-dullness, anti-glycation, yellowing removal, antioxidant, anti-inflammatory, and moisturizing effects.

[0014] Secondly, the present invention provides a Job's tears fermentation product, which is obtained by fermenting Job's tears with the brewing yeast.

[0015] Thirdly, the present invention provides a method for preparing the coix seed fermentation product, wherein coix seed is hydrolyzed with amylase to obtain a coix seed saccharification liquid; and the coix seed saccharification liquid is aerobically fermented with the brewing yeast to obtain the coix seed fermentation product.

[0016] Furthermore, the sprouted Job's tears were enzymatically hydrolyzed using amylase.

[0017] Furthermore, the sterilized Job's tears were soaked in a citric acid solution to germinate, resulting in sprouted Job's tears.

[0018] Furthermore, the Job's tears were sterilized using NaClO to obtain sterilized Job's tears.

[0019] Furthermore, the mass percentage concentration of the NaClO solution is preferably between 0.1% and 1%, for example, including but not limited to any point value or any range of two points from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1%.

[0020] The mass percentage concentration of citric acid solution is preferably 0.1% to 1%, for example, including but not limited to any point value or any range of two points from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%.

[0021] Furthermore, the germination treatment time should ideally be 6–24 hours, including but not limited to any point value or any range of two points from 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, and 24 h. A germination treatment time of 6–24 hours ensures sufficient enzyme activation and nutrient transformation while preventing nutrient loss due to excessive germination.

[0022] Furthermore, the amylase is α-amylase and / or glucoamylase.

[0023] Furthermore, the mass ratio of α-amylase to glucoamylase is preferably α-amylase:glucoamylase = 1:(20~60), for example, including but not limited to any point value or any range of two points from 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55 and 1:60.

[0024] Furthermore, α-amylase and glucoamylase are mixed to obtain a mixed enzyme. The total amount of the mixed enzyme added is preferably 0.002% to 0.12% of the mass of the sprouted Job's tears slurry, for example, including but not limited to any point value or any range of any two points from 0.002%, 0.016%, 0.028%, 0.030%, 0.048%, 0.055%, 0.068%, 0.07%, 0.075%, 0.082%, 0.085%, 0.094%, 0.099%, 0.12%. The sprouted Job's tears are then milled to obtain sprouted Job's tears slurry.

[0025] Furthermore, the enzymatic hydrolysis temperature is preferably 50~70℃, for example, including but not limited to any point value or any two points of 50℃, 55℃, 60℃, 65℃ and 70℃; the enzymatic hydrolysis time is preferably 0.3~0.7 h, for example, including but not limited to any point value or any two points of 0.3 h, 0.35 h, 0.4 h, 0.45 h, 0.5 h, 0.55 h, 0.6 h, 0.65 h and 0.7 h.

[0026] Furthermore, the concentration of the coix seed saccharification solution is preferably 50~300 g / L, for example, including but not limited to any point value or any range of two points from 50 g / L, 100 g / L, 150 g / L, 200 g / L, 250 g / L and 300 g / L.

[0027] Further, the *Saccharomyces cerevisiae* was inoculated into a glucose medium and cultured aerobically to obtain a fermentation seed liquid. The mass concentrations of each component in the glucose medium were: 5-20 g / L glucose, 1-20 g / L peptone, 1-20 g / L yeast extract, and 0.1-5 g / L potassium dihydrogen phosphate. The aerobic culture conditions were: inoculum size of 0.5%-5% (volume concentration), culture temperature of 20-40℃, shaking speed of 150-250 r / min, and culture time of 4-18 h.

[0028] Furthermore, the glucose concentration is preferably 5-20 g / L, for example, including but not limited to any point value or any range of two points such as 5 g / L, 8 g / L, 12 g / L, 15 g / L, 17 g / L and 20 g / L; The peptone concentration is preferably 1~20 g / L, for example, including but not limited to any point value or any range of two points from 1 g / L, 2 g / L, 5 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 17 g / L and 20 g / L; The concentration of yeast powder should be 1~20 g / L, including but not limited to any point value or any range of two points such as 1 g / L, 2 g / L, 5 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 17 g / L and 20 g / L. The concentration of potassium dihydrogen phosphate is preferably 0.1~5 g / L, including but not limited to any point value or any range of two points from 0.1 g / L, 0.6 g / L, 1.2 g / L, 1.8 g / L, 2 g / L, 2.5 g / L, 3.2 g / L, 3.8 g / L, 4.4 g / L and 5 g / L. The inoculation amount should be between 0.5% and 5%, including but not limited to any point value or any range of two points from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5%. The OD value of the initial inoculation of Saccharomyces cerevisiae should preferably be 0.1 to 0.3, such as any point value or any range of any two points including but not limited to 0.1, 0.15, 0.2, 0.25 and 0.3; The optimal culture temperature is 20-40℃, including but not limited to 20℃, 25℃, 30℃, 35℃ and 40℃.

[0029] The shaking speed is preferably 150~250 r / min, for example, including but not limited to any point value or any range of any two points from 150 r / min, 160 r / min, 180 r / min, 200 r / min, 210 r / min, 240 r / min and 250 r / min; The incubation time should be 4 to 18 hours, including but not limited to any point value or any range of two points such as 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h and 18 h.

[0030] Further, the fermentation seed liquid is inoculated into a culture medium containing Job's tears saccharification liquid, and aerobic fermentation is carried out until the endpoint to obtain fermentation liquid. The mass concentration of each component in the culture medium containing Job's tears saccharification liquid is: 50~300 g / L Job's tears saccharification liquid, 1~20 g / L peptone, 1~20 g / L yeast powder, and 0.1~5 g / L potassium dihydrogen phosphate.

[0031] Furthermore, the inoculation amount of the fermentation seed liquid is preferably 1% to 15%, for example, including but not limited to any point value or any range of two points such as 1%, 3%, 5%, 8%, 10%, 12% and 15%.

[0032] Furthermore, the concentration of the coix seed saccharification solution should preferably be 50~300 g / L, for example, including but not limited to any point value or any range of two points such as 50 g / L, 100 g / L, 150 g / L, 200 g / L, 250 g / L and 300 g / L; The peptone concentration is preferably 1~20 g / L, for example, including but not limited to any point value or any range of two points from 1 g / L, 2 g / L, 5 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 17 g / L and 20 g / L; The concentration of yeast powder should be 1~20 g / L, including but not limited to any point value or any range of two points such as 1 g / L, 2 g / L, 5 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 17 g / L and 20 g / L. The concentration of potassium dihydrogen phosphate is preferably 0.1~5 g / L, including but not limited to any point value or any range of two points from 0.1 g / L, 0.5 g / L, 0.8 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L and 5 g / L.

[0033] Furthermore, the fermentation temperature is 20~40℃, the ventilation rate is 1~2 vvm, the stirring speed of the fermentation tank is 120~250 r / min, and the fermentation time is 12~24 h.

[0034] Furthermore, the fermentation temperature is preferably 20~40℃, such as any point value or any range of any two points including but not limited to 20℃, 25℃, 30℃, 33℃, 36℃, 38℃ and 40℃. The ventilation volume should be 1~2 VVM, such as any point value or any range of any two points including but not limited to 1 VVM, 1.2 VVM, 1.4 VVM, 1.5 VVM, 1.6 VVM, 1.8 VVM and 2 VVM. The stirring speed of the fermenter should be 120~250 r / min, including but not limited to any point value or any range of any two points from 120 r / min, 130 r / min, 150 r / min, 160 r / min, 190 r / min, 210 r / min, 230 r / min and 250 r / min. The fermentation time should be 12 to 24 hours, including but not limited to any point value or any range of two points such as 12 h, 14 h, 16 h, 18 h, 20 h and 24 h.

[0035] Further, the saccharified Job's tears liquid is aerobically fermented with the aforementioned brewing yeast to obtain a fermentation broth, which is then centrifuged to obtain the Job's tears fermentation product. Fourthly, the present invention provides a cosmetic product containing the aforementioned Job's tears fermentation product.

[0036] Fifthly, the present invention provides a method for increasing the content of active ingredients in coix seed fermentation products, wherein the coix seed is fermented with the brewing yeast; the active ingredients include at least one selected from coixol, coixenolide, γ-aminobutyric acid, metallothionein, glucocerebrosidase, amino acids, proteins, succinic acid, lactic acid, and phenolic substances.

[0037] Furthermore, the phenolic substances include at least one of coumarin, 6-gingerol, β-carotene, methoxyphenylacetic acid, vanillic acid, epigallocatechin, eugenol, cinnamic acid, 6-gingerol, gentamicin, 2-hydroxybenzoic acid, p-coumaric acid, flavonoids, and apigenin.

[0038] Furthermore, the active ingredients include 200-500 μg / mL coixol, 500-1000 μg / mL coixenolide, 50-100 μg / mL γ-aminobutyric acid, 2000-3000 pd / mL metallothionein, 100-200 pg / mL glucocerebrosidase, 4500-10000 mg / L total free amino acids, 6.5-10 g / L total amino acids, 0.15-0.2 g / L protein, 0.045-0.1 g / L succinic acid, 0.2-0.3 g / L lactic acid, and 3.5-5 g / L total phenolic substances.

[0039] More preferably, the active ingredients include 314-351 μg / mL coixol, 798-825 μg / mL coixenolide, 82-86 μg / mL γ-aminobutyric acid, 2832-2879 pd / mL metallothionein, 143-152 pg / mL glucocerebrosidase, 5013-5667 mg / L total free amino acids, 6.987-7.096 g / L total amino acids, 0.165-0.171 g / L protein, 0.048-0.058 g / L succinic acid, 0.218-0.242 g / L lactic acid, and 3.64-3.97 g / L total phenolic substances.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) After germination and saccharification treatment, Job's tears help activate the enzymes and initiate metabolism within the seeds, inducing the synthesis of various new secondary metabolites, such as an increase in γ-aminobutyric acid (GABA) content. The vitamin content also increases significantly during germination, and the content and activity of antioxidants such as polyphenols and flavonoids are also enhanced. Citric acid is an effective color protectant. Soaking Job's tears in citric acid water before germination can adjust the pH value of the water (creating a slightly acidic environment), protect the color (preventing browning), inhibit bacteria and decay, and promote water absorption and activation of internal enzymes in the seeds. These effects work together to increase the accumulation of beneficial active ingredients during the germination process of Job's tears, laying a good foundation for significantly improving the efficacy of Job's tears fermentation filtrate. At the same time, it can also improve the sensory quality of the final germinated Job's tears, such as color, flavor, and nutritional value.

[0041] (2) Saccharomyces cerevisiae A3 (CGMCC No. 37866) was specifically selected as the fermentation strain. This strain has strong specificity, high enzyme activity, good stability, and is suitable for aerobic fermentation. Using this fermentation strain to carry out aerobic fermentation of the saccharified liquid of germinated Job's tears not only efficiently transforms the nutrients in Job's tears, but also fully releases the bound active substances in Job's tears, maximizing the organic combination of Job's tears active substances and yeast metabolic active substances, producing a large amount of coixol, coixenolide, γ-aminobutyric acid, metallothionein, glucocerebrosidase, various amino acids, proteins, and small amounts of coixol. The active ingredients, such as molecular organic acids and phenolic substances, can simultaneously provide multiple benefits including whitening and brightening, anti-dullness, anti-glycation, yellowing removal, anti-oxidation, anti-inflammation, and moisturizing. Moreover, the product maintains a slightly acidic pH similar to that of the skin and is alcohol-free, avoiding the irritating effects of alcohol and excessively low pH on the skin. This significantly expands the application scope of germinated Job's tears fermentation product filtrate in the daily chemical industry, thus providing new ideas and innovative strategies for the widespread application of germinated Job's tears fermentation product filtrate in skin care products.

[0042] (3) This invention uses liquid seed liquid as inoculum and coix seed saccharification liquid as fermentation raw material for aerobic fermentation. There is no need to adjust the pH value during the fermentation process. After the fermentation is completed, the supernatant is directly centrifuged to obtain the fermentation product filtrate of germinated coix seed. The whole process is simple to operate, does not require multi-strain fermentation, collects cells by centrifugation, does not require cell disruption, and has no risk of contamination by pathogenic bacteria or other bacteria. It has the advantages of simple process, high product quality and low production cost. Attached Figure Description

[0043] Figure 1 This is a colony diagram of Saccharomyces cerevisiae A3.

[0044] Figure 2 This is a summary of the apparent colorimetric results.

[0045] Figure 3 This is a compilation of images showing the distribution of melanin. The scale bar is 50 μm.

[0046] Figure 4 This is a summary of images showing the results of AGEs (CML) detection. The scale bar is 50 μm.

[0047] Figure 5 This is a summary of ROS detection results images. The scale bar is 50 μm. Detailed Implementation

[0048] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0049] Example 1 1. Sampling and enrichment culture A small amount of soil was taken from the coix seed planting area in Xingren, Qianxinan Buyi and Miao Autonomous Prefecture, Guizhou Province, and dissolved in sterilized physiological saline. The mixture was then plated and enriched. Colonies that were milky white, round, raised, with neat and smooth edges were selected. The strain was fermented, and ITS gene sequencing was performed. The results were compared with the gene sequence of Saccharomyces cerevisiae to confirm that the starting strain was Saccharomyces cerevisiae.

[0050] The enrichment culture medium consisted of the following components at the specified mass concentrations: 20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract (Beijing Aoboxing Biotechnology Co., Ltd., catalog number 01-013), 1 g / L potassium dihydrogen phosphate, and 15 g / L agar. The enrichment culture conditions were: a culture temperature of 30℃ and a culture time of 18 h.

[0051] 2. The enriched strains were subjected to compound mutagenesis. The bacterial strain obtained in step 1 was inoculated into a test tube containing 5 mL of physiological saline. 1% (v / v) of diethyl sulfate mutagen was added to the test tube, and the mixture was vortexed for 1 min to obtain a bacterial suspension. 0.5 mL of the bacterial suspension was spread onto the solid plate from step 1, irradiated with UV light for 30 s, and then incubated in the dark. The incubation conditions were: 30℃ and 24 h.

[0052] 3. The mutagenized strains were then screened through fermentation. The screening medium consisted of the following components at the following concentrations: 200 g / L coix seed saccharification solution, 10 g / L peptone, 5 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, and 15 g / L agar.

[0053] The fermentation process conditions were as follows: the culture temperature was 30℃, the shaking speed was 200 r / min, and the fermentation time was 16 h; strains with fast growth, high bacterial density, and high coixol content were screened.

[0054] The preparation method of coix seed saccharification liquid is as follows: (1) Coix seed is disinfected with NaClO at a mass percentage concentration of 0.1% to obtain disinfected coix seed; the disinfected coix seed is soaked in citric acid solution at a mass percentage concentration of 1% for germination treatment for 18 h to obtain germinated coix seed; the germinated coix seed is ground to obtain germinated coix seed syrup.

[0055] (2) Mix α-amylase and glucoamylase to obtain a mixed enzyme with a mass ratio of 1:20. Add the mixed enzyme to the sprouted coix seed slurry. The total amount of the mixed enzyme added is 0.12% of the mass of the sprouted coix seed slurry. Enzymatic hydrolysis is carried out at 60℃ for 0.5 h until the endpoint to obtain the coix seed saccharification solution.

[0056] 4. Perform subculture stability experiments on the selected strains. The strains selected in step 3 were cultured for more than 8 generations, and each generation was fermented to confirm that the growth rate, cell density and metabolite content of the strains were stable across 8 generations.

[0057] Finally, a strain of Saccharomyces Cerevisiae with rapid growth, high cell density, and high content of metabolites was obtained. The ITS gene sequence of this strain is as follows: (SEQ ID NO: 1).

[0058] The colonies of this strain are milky white, with a smooth, moist, and glossy surface and neat edges. Figure 1 The bacterial cells are single-celled, oval-shaped, with a cell width of 3-7 μm and a length of 5-10 μm. The optimal growth temperature is 20-40℃. It is adapted to aerobic fermentation and grows and metabolizes in a neutral to slightly acidic environment with a pH of 3-7.

[0059] The obtained brewing yeast strain was named *Saccharomyces cerevisiae* and deposited on March 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37866. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0060] Example 2 This embodiment provides a method for preparing the filtrate of germinated Job's tears fermentation product, the steps of which are as follows: 1. Sterilize Job's tears with 0.1% NaClO to obtain sterilized Job's tears; soak the sterilized Job's tears in 1% citric acid solution for germination treatment for 18 h to obtain germinated Job's tears; grind the germinated Job's tears to obtain germinated Job's tears slurry.

[0061] 2. Mix α-amylase and glucoamylase to obtain a mixed enzyme with a mass ratio of 1:20. Add the mixed enzyme to the germinated Job's tears slurry, with the total amount of mixed enzyme added being 0.12% of the mass of the germinated Job's tears slurry. Enzymatically hydrolyze at 60℃ for 0.5 h until the endpoint to obtain Job's tears saccharification solution.

[0062] 3. The A3 seed culture of Saccharomyces cerevisiae was inoculated into glucose medium and cultured aerobically to obtain the fermentation seed culture. The mass concentrations of each component of the glucose medium were: 20 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, and 2 g / L potassium dihydrogen phosphate. The aerobic culture conditions were: seed culture OD value of 0.2, inoculation amount of 5% (volume concentration), culture temperature of 30℃, shaking speed of 250 r / min, and culture time of 18 h.

[0063] 4. Inoculate the fermentation seed liquid into the culture medium containing Job's tears saccharification solution, and aerobic fermentation to the endpoint to obtain the fermentation broth. The mass concentrations of each component in the culture broth are: 150 g / L Job's tears saccharification solution, 10 g / L peptone, 10 g / L yeast powder, and 2 g / L potassium dihydrogen phosphate. The aerobic fermentation conditions are: inoculum amount of 10% (volume concentration), fermentation temperature of 30℃, aeration rate of 1.5 vvm, fermentation tank stirring speed of 200 r / min, and fermentation time of 20 h.

[0064] 5. Centrifuge the fermentation broth using a disc centrifuge, collect the supernatant, and obtain the filtrate of germinated Job's tears fermentation product.

[0065] Example 3 This embodiment provides a method for preparing the filtrate of germinated Job's tears fermentation product, the steps of which are as follows: 1. Sterilize Job's tears with 0.5% NaClO to obtain sterilized Job's tears; soak the sterilized Job's tears in 0.1% citric acid solution for germination treatment for 24 h to obtain germinated Job's tears; grind the germinated Job's tears to obtain germinated Job's tears slurry.

[0066] 2. Mix α-amylase and glucoamylase to obtain a mixed enzyme with a mass ratio of 1:40. Add the mixed enzyme to the germinated Job's tears slurry, with the total amount of mixed enzyme added being 0.07% of the mass of the germinated Job's tears slurry. Enzymatically hydrolyze at 60℃ for 0.5 h until the endpoint to obtain Job's tears saccharification solution.

[0067] 3. The A3 seed culture of Saccharomyces cerevisiae was inoculated into glucose medium and cultured aerobically to obtain the fermentation seed culture. The mass concentrations of each component of the glucose medium were: 5 g / L glucose, 1 g / L peptone, 20 g / L yeast extract, and 5 g / L potassium dihydrogen phosphate. The aerobic culture conditions were: seed culture OD value of 0.2, inoculation amount of 2% (volume concentration), culture temperature of 20℃, shaking speed of 200 r / min, and culture time of 12 h.

[0068] 4. Inoculate the fermentation seed liquid from step 3 into a culture medium containing Job's tears saccharification liquid, and aerobic fermentation to the endpoint to obtain the fermentation broth. The mass concentrations of each component in the culture medium are: 50 g / L Job's tears saccharification liquid, 1 g / L peptone, 20 g / L yeast powder, and 5 g / L potassium dihydrogen phosphate. The aerobic fermentation conditions are: inoculum amount of 1% (volume concentration), fermentation temperature of 20℃, aeration rate of 1 vvm, fermentation tank stirring speed of 250 r / min, and fermentation time of 24 h.

[0069] 5. Centrifuge the fermentation broth using a disc centrifuge, collect the supernatant, and obtain the filtrate of germinated Job's tears fermentation product.

[0070] Example 4 This embodiment provides a method for preparing the filtrate of germinated Job's tears fermentation product, the steps of which are as follows: 1. Sterilize the Job's tears with 1% NaClO to obtain sterilized Job's tears; soak the sterilized Job's tears in 0.5% citric acid solution for germination treatment for 6 hours, and grind the germinated Job's tears to obtain germinated Job's tears slurry.

[0071] 2. Mix α-amylase and glucoamylase to obtain a mixed enzyme with a mass ratio of 1:60. Add the mixed enzyme to the germinated Job's tears slurry, with the total amount of mixed enzyme added being 0.002% of the mass of the germinated Job's tears slurry. Enzymatically hydrolyze at 60℃ for 0.5 h until the endpoint to obtain Job's tears saccharification solution.

[0072] 3. The A3 seed culture of Saccharomyces cerevisiae was inoculated into glucose medium and cultured aerobically to obtain the fermentation seed culture. The mass concentrations of each component of the glucose medium were: 12 g / L glucose, 20 g / L peptone, 1 g / L yeast extract, and 0.1 g / L potassium dihydrogen phosphate. The aerobic culture conditions were: OD value of seed culture of 0.2, inoculation amount of 0.5% (volume concentration), culture temperature of 40℃, shaking speed of 150 r / min, and culture time of 4 h.

[0073] 4. Inoculate the fermentation seed liquid from step 3 into a culture medium containing Job's tears saccharification liquid, and aerobic fermentation to the endpoint to obtain the fermentation broth. The mass concentrations of each component in the culture medium are: 300 g / L Job's tears saccharification liquid, 20 g / L peptone, 1 g / L yeast powder, and 0.1 g / L potassium dihydrogen phosphate. The aerobic fermentation conditions are: inoculum size of 15% (volume concentration), fermentation temperature of 40℃, aeration rate of 2 vvm, fermentation tank stirring speed of 120 r / min, and fermentation time of 12 h.

[0074] 5. Centrifuge the fermentation broth using a disc centrifuge, collect the supernatant, and obtain the filtrate of germinated Job's tears fermentation product.

[0075] Comparative Example 1 The only difference between Comparative Example 1 and Example 2 is step 1. In Comparative Example 1, step 1 is: the sterilized Job's tears are directly ground to make Job's tears paste without germination treatment.

[0076] Comparative Example 2 The only difference between Comparative Example 2 and Example 2 is the use of different brewing yeasts. Comparative Example 2 used brewing yeast CGMCC 2.101 purchased from the China General Microbiological Culture Collection Center.

[0077] Comparative Example 3 Comparative Example 3 used commercially available Japanese coix seed cosmetics (brand name: TECHNOLONG, product number: ORGANIC ADLAY-FF organic coix seed fermentation liquid).

[0078] Test Example 1 The components of the germinated coix seed fermentation product filtrate of Examples 2-4, the coix seed fermentation product filtrate of Comparative Example 1, the germinated coix seed fermentation product filtrate of Comparative Example 2, and the commercially available Japanese coix seed cosmetic of Comparative Example 3 were analyzed.

[0079] γ-aminobutyric acid (GABA) content analysis: High-performance liquid chromatography (HPLC) was used. HPLC conditions were as follows: column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm); mobile phase: water: acetonitrile (v:v) = 80:20; flow rate: 1.0 mL / min; column temperature: 25℃; detector: UV detector, wavelength 228 nm. The GABA standard curve was calculated as y = 1493x + 275933 (R²). 2 =0.9991).

[0080] Content analysis of coixol and coixenolide: High-performance liquid chromatography (HPLC) was used. HPLC conditions were as follows: column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm); mobile phase: water: acetonitrile (v:v) = 80:20; flow rate: 1.0 mL / min; column temperature: 25℃; detector: UV detector, 235 nm. The standard curve for coixol was y = 32568x - 162.48 (R²). 2 =0.9997), the standard curve of coixol is y = 25018x + 22035 (R = 0.9997). 2 =0.9996).

[0081] Metallothionein content detection: Microorganism metallothionein (MT) ELISA kit (Shanghai Yanjin Biotechnology Co., Ltd., catalog number F05011) was used for detection.

[0082] Detection of glucocerebrosidase content: Microorganism glucocerebrosidase (GBA) ELISA kit (Shanghai Yanjin Biotechnology Co., Ltd., catalog number ybs-11779R).

[0083] The total amino acid content was determined using the ninhydrin method, and the standard curve equation for the amino acid was plotted as y = 0.0371x - 0.0081 (R²). 2 =0.9910).

[0084] Protein content was detected using the Coomassie Brilliant Blue method, and the protein standard curve equation was plotted as y = 5.8671x + 0.0325 (R²). 2 =0.9909).

[0085] The content of free amino acids was detected using an automated amino acid analyzer.

[0086] Analysis of small molecule organic acid content: High performance liquid chromatography (HPLC) was used. The HPLC conditions were as follows: column: Aminex HPX-87H (300 × 7.8 mm); mobile phase: 5 mmol / L H₂SO₄; flow rate: 0.5 mL / min; column temperature: 40°C; detector: differential detector. The lactic acid standard curve was: y = 712891x + 7596 (R² + π / 4)². 2 =0.9958); the succinic acid standard curve is: y=1119366x+4590 (R = 0.9958). 2 =0.9976).

[0087] The alcohol content was determined using the gas chromatography method specified in GB 5009.225-2023, "National Food Safety Standard - Determination of Ethanol Concentration in Wine and Edible Alcohol".

[0088] Analysis of phenolic compounds: Following the People's Republic of China agricultural industry standard "Determination of Major Phenolic Compounds in Apples by High Performance Liquid Chromatography," high performance liquid chromatography (HPLC) was used to analyze the phenolic compounds in the samples. Simultaneously, liquid chromatography-mass spectrometry (LC-MS) was used to identify total phenolic compounds. The results showed that the fermentation product filtrate contained phenolic compounds such as coumarin, 6-gingerol, β-carotene, methoxyphenylacetic acid, vanillic acid, epigallocatechin, eugenol, cinnamic acid, 6-gingerol, amitidine, 2-hydroxybenzoic acid, p-coumaric acid, flavonoids, and apigenin.

[0089] Table 1. Results of Coixol Content Detection Table 2 Results of Coix Seed Extract Content Detection Table 3. Detection results of γ-aminobutyric acid content Table 4 Results of Metallothionein Content Detection Table 5 Results of glucocerebrosidase content detection Table 6 Results of Free Amino Acid Content Detection Table 7 Results of Total Amino Acid Content Detection Table 8. Results of Protein Content Detection Table 9 Results of small molecule organic acid content Table 10 Detection of Total Phenolic Content Table 11 Results of alcohol content detection Table 12 pH test results of fermentation filtrate The results of the coixol content detection are shown in Table 1, the coixenolide content detection results are shown in Table 2, the γ-aminobutyric acid content detection results are shown in Table 3, the metallothionein content detection results are shown in Table 4, the glucocerebrosidase content detection results are shown in Table 5, the free amino acid content detection results are shown in Table 6, the total amino acid content detection results are shown in Table 7, the protein content detection results are shown in Table 8, the succinic acid and lactic acid content detection results are shown in Table 9, the total phenolic substances content detection results are shown in Table 10, the alcohol content detection results are shown in Table 11, and the pH detection results are shown in Table 12.

[0090] As can be seen from Tables 1 to 10, the contents of coixol, coixenolide, γ-aminobutyric acid, metallothionein, glucocerebrosidase, free amino acids, total amino acids, protein, succinic acid, lactic acid and total phenolic substances in the fermented coix seed product filtrate prepared by the preparation method of the present invention are significantly higher than those in the unsprouted coix seed treatment group (Comparative Example 1), and significantly higher than those in commercially available Japanese coix seed cosmetics (Comparative Example 3). This is because germination pretreatment can activate the endogenous enzyme system of Job's tears, effectively promoting the natural accumulation of these active substances and providing a high-quality substrate for subsequent fermentation, thereby significantly increasing the final yield of various active substances in the fermentation product. Secondly, the combination of selected brewing yeast A3 with the saccharification liquid of germinated Job's tears not only efficiently transforms the nutrients in Job's tears, but also fully releases the bound active substances in Job's tears, maximizing the organic combination of Job's tears active substances and yeast metabolic active substances, producing a large number of active ingredients such as coixol, coixenolide, γ-aminobutyric acid, metallothionein, glucocerebrosidase, various amino acids, proteins, succinic acid, lactic acid and phenolic substances. This significantly expands the application scope of germinated Job's tears fermentation product filtrate in the daily chemical industry, providing new ideas and innovative strategies for the widespread application of the product in skin care products.

[0091] Furthermore, according to the data in Tables 3, 6, and 9, the germinated coix seed fermentation product filtrate prepared using the preparation method of Example 2 of this invention contains 86 μg / mL of γ-aminobutyric acid (GABA), 45 mg / L of free tyrosine, 0.058 g / L of succinic acid, and 0.235 g / L of lactic acid. In contrast, other Saccharomyces cerevisiae samples (Comparative Example 2) and Japanese coix seed cosmetic samples (Comparative Example 3) did not contain free tyrosine, succinic acid, or lactic acid; γ-aminobutyric acid was not detected in Comparative Example 3. Based on this, the germinated coix seed fermentation product filtrate of this invention, due to its unique organic acid composition, exhibits multiple advantages in skin repair applications, including pH regulation, antibacterial, exfoliating, and whitening effects.

[0092] Furthermore, as shown in Table 11, the germinated coix seed fermentation product filtrate prepared using the preparation method of this invention contains no alcohol at all, while the alcohol content of other brewing yeast fermentation (Comparative Example 2) and commercially available Japanese coix seed cosmetics (Comparative Example 3) is as high as 35.5 g / L and 31.4 g / L, respectively. This indicates that traditional coix seed products inevitably contain alcohol due to limitations in fermentation strains and processes, while the germinated coix seed fermentation product filtrate obtained using the strains and preparation method of this invention contains no alcohol at all. This avoids the skin irritation, dryness, and other problems that alcohol may cause, making it particularly suitable for sensitive skin, children, and people with alcohol intolerance.

[0093] Furthermore, as shown in Table 10, the total phenolic content in the germinated coix seed fermentation product filtrate prepared by the method of this embodiment of the invention is significantly higher than that in the ungerminated coix seed treatment group (Comparative Example 1), and significantly higher than that in other brewing yeast fermentation products (Comparative Example 2) and commercially available Japanese coix seed cosmetics (Comparative Example 3). In addition, liquid chromatography-mass spectrometry analysis identified that the germinated coix seed fermentation product filtrate prepared by this embodiment of the invention contains phenolic substances such as coumarin, 6-gingerol, β-carotene, methoxyphenylacetic acid, vanillic acid, epigallocatechin, eugenol, cinnamic acid, 6-gingerol, amitidine, 2-hydroxybenzoic acid, p-coumaric acid, flavonoids, and apigenin. These phenolic substances work synergistically to enable the germinated coix seed fermentation product filtrate to exhibit a unique mellow wine aroma even without alcohol.

[0094] Test Example 2 The tyrosinase activity inhibition rate of the germinated coix seed fermentation product filtrate of Examples 2-4, the coix seed fermentation product filtrate of Comparative Example 1, the germinated coix seed fermentation product filtrate of Comparative Example 2, and the commercially available Japanese coix seed cosmetic of Comparative Example 3 were tested according to the test method of Shanghai Daily Chemical Industry Association group standard T / SHRH 015-2018 "Cosmetics - Test Method for Tyrosinase Activity Inhibition". The test results are shown in Table 13.

[0095] Table 13 Tyrosinase activity inhibition rate As shown in Table 13, the tyrosinase inhibition rate of the germinated coix seed fermentation product filtrate prepared by the preparation method of the present invention can reach up to 98.9%, which is significantly better than that of comparative examples 1 to 3. This indicates that the germinated coix seed fermentation product filtrate of the present invention has excellent skin whitening and brightening effects.

[0096] The filtrate of the germinated Job's tears fermentation product from Example 2 was tested at a concentration of 5% (v / v) by a third-party testing agency, Shaanxi Boxi General Testing Technology Co., Ltd., according to the "Appearance Color and Apparent Brightness (L) Based on 3D Melanocyte Skin Model (MelaKutis)". The whitening efficacy, anti-glycation effect, and antioxidant effect were evaluated using the methods for detecting the distribution of melanin and reactive oxygen species (ROS) and the content of glycation end products (carboxymethyl lysine) based on the isolated skin model.

[0097] through Figure 2 The statistical analysis results of the apparent colorimetric results (Table 14) show that, based on the 3D melanin skin model (MelaKutis), compared with the control group, the sample at a concentration of 5% (vNv) showed a significantly whiter apparent color and a significantly lower apparent brightness (L). The apparent color value increased significantly, with an improvement rate of 11.06% (Table 14). This indicates that the sample can improve the apparent color and increase the apparent brightness (L). (Value), with significant skin whitening and brightening effects.

[0098] Table 14. Apparent brightness of human body samples (L) (Value) Test Results Note: When performing statistical analysis using the t-test method, significance compared to the BC group (blank control) is indicated by #, with P-value < 0.05 indicated by # and P-value < 0.01 indicated by ##; significance compared to the NC group (negative control) is indicated by #. This means that a p-value < 0.05 indicates that... A p-value < 0.01 indicates that... .

[0099] through Figure 3 The statistical analysis of the melanin distribution images (Table 15) shows that, compared with the control group, the sample at a concentration of 5% (v / v) showed a significant reduction in melanin granules, with an inhibition rate of 55.38%. This indicates that the sample can reduce melanin granules and has a significant skin whitening and brightening effect.

[0100] Table 15. Melanin distribution data in human samples. Note: When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by #, P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##, and significance compared to the NC group is indicated by ##. This means that a p-value < 0.05 indicates that... A p-value < 0.01 indicates that... .

[0101] Test Example 3 through Figure 4 Statistical analysis of the human anti-glycation effect test results (Table 16) shows that, based on the ex vivo skin model, compared with the control group, the sample at a concentration of 5% (v / v) showed a significant decrease in the content of advanced glycation end products (carboxymethyl lysine), with an inhibition rate of 77.01%, indicating that the sample can reduce advanced glycation end products (carboxymethyl lysine) and has a significant anti-glycation effect.

[0102] Table 16 Results of human samples' anti-glycation effects Note: When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by #, P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##, and significance compared to the NC group is indicated by ##. This means that a p-value < 0.05 indicates that... A p-value < 0.01 indicates that... .

[0103] Test Example 4 The antioxidant and free radical scavenging effects of the germinated coix seed fermentation product filtrate of Examples 2-4, the coix seed fermentation product filtrate of Comparative Example 1, the germinated coix seed fermentation product filtrate of Comparative Example 2, and the commercially available Japanese coix seed cosmetic of Comparative Example 3 were tested according to the following test methods.

[0104] Using a 96-well plate, the absorbance of Trolox standard solutions of different concentrations at 734 nm was measured, and the Trolox standard curve equation was plotted as: y = -0.2995x + 0.6457 (R²). 2 =0.9991).

[0105] As shown in Table 17, the antioxidant capacity of the germinated Job's tears fermentation product filtrate of Examples 2-4 reached 1.78 mM, 1.69 mM, and 1.71 mM, respectively, which were significantly higher than those of Comparative Example 1 (0.91 mM), Comparative Example 2 (0.15 mM), and commercially available Japanese Job's tears cosmetic (0.03 mM). In terms of free radical scavenging rate, Examples 2-4 all maintained a high level of over 76%, while Comparative Examples 1-3 were only 43.55%, 9.66%, and 2.78%, respectively. These data indicate that the preparation method of the present invention can significantly improve the antioxidant and free radical scavenging performance of the product, thus having important application value in the development of anti-aging cosmetics.

[0106] Table 17 Results of Antioxidant Capacity and Free Radical Scavenging Rate Tests through Figure 5 The statistical analysis of human ROS data (Table 18) shows that, based on the ex vivo skin model, compared with the control group, the ROS content of the active liquid in the sample was significantly reduced at a concentration of 5% (v / v), with an inhibition rate of 57.91%, indicating that the sample can reduce the ROS content and has a significant antioxidant effect.

[0107] Table 18 Results of the human body antioxidant (ROS) test for samples Note: When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by #, P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##, and significance compared to the NC group is indicated by ##. This means that a p-value < 0.05 indicates that... A p-value < 0.01 indicates that... .

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A brewing yeast, characterized in that, The brewing yeast is brewing yeast A3, which was deposited on March 6, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37866.

2. A fermented Job's tears product, characterized in that, It is obtained by fermenting Job's tears with the brewing yeast described in claim 1.

3. The method for preparing the coix seed fermentation product according to claim 2, characterized in that, Job's tears are hydrolyzed with amylase to obtain a Job's tears saccharified liquid; the Job's tears saccharified liquid is aerobically fermented with the brewing yeast of claim 1 to obtain the Job's tears fermentation product.

4. The preparation method according to claim 3, characterized in that, Germinated Job's tears were hydrolyzed using amylase.

5. The preparation method according to claim 3, characterized in that, The amylase is α-amylase and / or glucoamylase.

6. The preparation method according to claim 3, characterized in that, The concentration of the coix seed saccharification solution is 50~300 g / L.

7. The preparation method according to claim 3, characterized in that, The fermentation temperature is 20~40℃, the ventilation rate is 1~2 vvm, the stirring speed of the fermentation tank is 120~250 r / min, and the fermentation time is 12~24 h.

8. A cosmetic product, characterized in that, The cosmetic contains the coix seed fermentation product as described in claim 2.

9. A method for increasing the content of active ingredients in coix seed fermentation products, characterized in that, The yeast of claim 1 is used to ferment Job's tears; the active ingredients include at least one of coixol, coixenolide, γ-aminobutyric acid, metallothionein, glucocerebrosidase, amino acids, proteins, succinic acid, lactic acid and phenolic substances.

10. The preparation method according to claim 9, characterized in that, The phenolic substances include at least one of the following: coumarin, 6-gingerol, β-carotene, methoxyphenylacetic acid, vanillic acid, epigallocatechin, eugenol, cinnamic acid, 6-gingerol, amitine, 2-hydroxybenzoic acid, p-coumaric acid, flavonoids, and apigenin.

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