A sprouted brown rice fermentation product filtrate, method of making and use thereof

Abstract

The application discloses a germinated brown rice fermentation product filtrate, a preparation method thereof and application of the germinated brown rice fermentation product filtrate, wherein the steps of the preparation method comprise the following steps: S1, adjusting the pH of a CaCl2 solution by using glutamic acid to obtain a CaCl2 germination solution, then immersing sterilized brown rice in the CaCl2 germination solution for germination treatment, and obtaining a germinated brown rice slurry through polishing treatment; S2, adding liquefying enzymes and saccharifying enzymes into the germinated brown rice slurry, and performing enzymolysis until a terminal point to obtain a brown rice saccharification liquid; S3, inoculating a Saccharomyces cerevisiae CGMCC No. 33650 into a glucose culture medium to perform aerobic culture to obtain a seed liquid; S4, inoculating the seed liquid into a culture liquid containing the brown rice saccharification liquid, and performing aerobic fermentation until a terminal point to obtain a fermentation liquid; and S5, performing centrifugal treatment on the fermentation liquid to obtain the germinated brown rice fermentation product filtrate. The fermentation product filtrate is rich in GABA, amino acids, proteins, small-molecule organic acids and phenolic substances, retains a mellow wine aroma and does not contain alcohol, and has multiple functions such as whitening and brightening, anti-wrinkle repair, anti-aging, anti-oxidation, soothing and moisturizing.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and particularly relates to a filtrate of germinated brown rice fermentation product, its preparation method, and its application. Background Technology

[0002] my country is a major producer and consumer of rice, with an annual output exceeding 200 million tons. Brown rice, as a primary product of rice processing that retains the endosperm and bran, has higher nutritional value and bioactive component content compared to polished rice. In recent years, with the increasing demand for natural, green, and functional cosmetic ingredients, developing brown rice and other rice-based ingredients into skincare products has become a research hotspot. Especially abroad, such as in Japan, rice-based ingredients are widely used in face creams, masks, and other skincare products, achieving positive market feedback and demonstrating their enormous potential in the cosmetics field.

[0003] To enhance the application value of brown rice, it is typically fermented. The fermentation process promotes the degradation of polysaccharides in brown rice, providing a carbon source for microbial growth and releasing active substances bound to the polysaccharides, thus imbuing the fermentation broth with 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 increasing the nutrient density and physiological activity of brown rice. Therefore, fermented brown rice exhibits significantly improved nutritional and health benefits, making its application value far superior to untreated brown rice.

[0004] Gamma-aminobutyric acid (GABA), a naturally occurring non-protein amino acid, found in the saccharified brown rice solution obtained through liquefaction and saccharification, exhibits multifaceted bioactivity 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] Besides GABA, brown rice saccharification liquid is also rich in various amino acids, small molecule peptides, organic acids, phenolic compounds, vitamins, and enzymes. These substances also exhibit good effects in whitening, anti-oxidation, anti-wrinkle, soothing, and moisturizing. For example, 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 also chelate metal ions, reducing oxidation reactions. Peptides can achieve anti-wrinkle effects through a dual mechanism: on the one hand, promoting collagen synthesis and skin structure repair; on the other hand, inhibiting muscle contraction through neurotransmitter regulation mechanisms, reducing the formation of dynamic wrinkles. Enzymes (such as superoxide dismutase, SOD) have the ability to directly scavenge free radicals and enhance antioxidant properties. Amylases, cellulases, and other enzymes help break down 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 enhancing the whitening and antioxidant effects of the fermentation broth. Small-molecule collagen peptides, vitamins, and other active factors in fermentation metabolites contribute to skin repair, nourishment, and anti-inflammation, reducing skin sensitivity.

[0006] Although some patents have disclosed technical solutions for preparing active ingredients through fermentation of brown rice, they still have many shortcomings. For example, Chinese patents (CN114703074A, CN114703074B, CN117844582A) mainly focus on optimizing the fermentation stage. While a certain degree of biotransformation and nutrient enrichment can indeed be achieved during fermentation, they neglect the potential opportunity of the germination stage to enhance the nutritional value of the raw materials. Germination can not only activate the enzyme system in the seed and promote the decomposition of endogenous enzymes, thereby releasing more nutrients that can be utilized in subsequent fermentation processes, but it may also directly increase the content of target products such as GABA through biosynthetic pathways. Therefore, the failure to incorporate the germination step into the overall process design may limit the potential of these patented technologies in maximizing nutrient enrichment efficiency.

[0007] Chinese patents CN111184163A, CN118489817A, and CN108552526A disclose the preparation of fermentation products containing GABA, which have achieved significant application results in the food or health product fields. However, in the cosmetics field, which also has broad market prospects and high demand potential, the development and application of related patents are relatively weak, and the diversified value of fermentation products has not been fully explored and demonstrated. This restricts the application and promotion of fermentation products in a wider range of fields, especially in the field of cosmetic raw materials.

[0008] Chinese patent CN108324633A discloses a method for germinating brown rice, drying it at 60-80℃ for 5-10 hours, and then sieving it through an 80-200 mesh sieve to prepare germinated brown rice flour. Yeast cells are separated by centrifugation and discarding the supernatant as seed liquid. The concentration of germinated brown rice flour is 73-85 g / L. During fermentation, 55 g of glutamic acid powder is added to adjust the pH. The final fermentation filtrate contains 12 kinds of amino acids, with a total amino acid content of 85.3 g / L and a GABA concentration of 1.91-2.15 g / L. Although this patent claims to increase GABA content through a germination process, the GABA content of germinated brown rice is only 0.41~0.54g / L, which does not significantly increase the GABA content of brown rice. Furthermore, the process of preparing germinated brown rice flour is complex, time-consuming, and energy-intensive. In addition, the method of separating yeast cells by discarding the supernatant after centrifugation as seed liquid is complex and prone to infection by pathogenic bacteria, raising concerns about safety. Moreover, the product was only analyzed for amino acid content, and there is no clear and detailed data on whether it contains other active ingredients such as proteins, small molecule organic acids, or phenolic substances. Finally, the product's efficacy was only assessed for its antioxidant, whitening, and cell repair-promoting effects, and it is uncertain whether it has other effects such as anti-wrinkle, brightening, soothing, and moisturizing effects, nor is there any corresponding efficacy data.

[0009] Chinese patent CN114703074A discloses a method of directly fermenting brown rice flour with Saccharomyces cerevisiae CF-37, then inoculating the fermentation broth into a culture medium containing enzymatic hydrolysate of brown rice flour for anaerobic fermentation. The fermentation broth is heated at 80-85℃ for 1 hour to break down the cells, and then the supernatant is collected by centrifugation to obtain the brown rice fermentation product filtrate. This method does not employ a brown rice germination process, thus failing to activate the active ingredients in brown rice. Furthermore, the high-temperature heating that lyses the bacterial cells easily leads to protein denaturation, denaturation and destruction of heat-sensitive substances such as phenols, and volatilization loss of small-molecule organic acids. Moreover, the anaerobic fermentation process produces alcohol, which can irritate the skin, limiting the product's application in the skincare field. Finally, the product analysis only covers total amino acids, GABA content, and ethanol content, without providing clear and detailed data on whether the product contains other active ingredients such as proteins, small-molecule organic acids, or phenolic substances.

[0010] Chinese patents (CN110592151A, CN112753914A, CN112890058A) disclose the use of lactic acid bacteria; Chinese patent CN114631613A discloses the use of *Lactobacillus plantarum* and *Lactobacillus acidophilus*; and Chinese patent CN104388514A discloses the use of *Lactobacillus plantarum* ZSM-002, *Castor yeast* ZSM-001, and *Rhizopus oryzae*. Chinese patents CN117099946A and CN1sm-005 disclose the use of a compound microbial agent comprising 1% acetic acid bacteria, 0.8% yeast, and 1.2% lactic acid bacteria. CN112931737A discloses the use of a compound microbial agent made from Lactobacillus acidophilus, Lactobacillus salivarius, and Saccharomyces cerevisiae for fermentation. While these methods can produce products with specific functions and are widely used in the food and health product industries, the fermentation process often results in high acidity in the final product or the presence of components that may have adverse effects on the skin. This problem is particularly prominent when fermentation products from the food and health product industries, such as brown rice fermentation liquid, are attempted to be used as cosmetic ingredients. Because the selected microbial strains and fermentation conditions are primarily optimized for the characteristics of food or health products without fully considering the physiological needs and tolerance of the skin, the high acidity components or specific metabolites in these products can easily damage the skin's natural barrier upon direct contact, causing adverse reactions such as irritation, sensitivity, and even inflammation.

[0011] Although Chinese patents (CN107541528A, CN115895809A, CN118440794A, CN116459203A, CN112971019A) have also been committed to increasing the GABA content in products, the GABA content in these products is still relatively low and insufficient in practical applications.

[0012] In summary, the current research on brown rice fermentation products still has the following limitations: (1) The content of γ-aminobutyric acid (GABA) in the fermentation product filtrate is low, the utilization rate of brown rice resources is not high, and the production efficiency of GABA per unit mass of brown rice is low; (2) The types and contents of active ingredients in the fermentation product filtrate are not yet clear, and there is a lack of systematic and comprehensive component analysis data, such as the composition and content information of functional components such as amino acids, peptides, organic acids, and phenolic substances; (3) When used alone, the fermentation product filtrate is difficult to have multiple effects such as whitening, anti-wrinkle, brightening, anti-oxidation, anti-aging, soothing, and moisturizing, and has the technical defect of single efficacy; (4) The existing fermentation process is relatively complex, the operation and control are difficult, and it is easy to cause contamination by miscellaneous bacteria, thereby affecting the quality and safety of the product; In addition, some fermentation processes produce irritating byproducts such as alcohol, which limits its application in fields with high safety requirements such as cosmetics. Summary of the Invention

[0013] To address the problems existing in the prior art, the purpose of this invention is to provide a germinated brown rice fermentation product filtrate, its preparation method, and its application. This filtrate is prepared using a specific strain of Saccharomyces cerevisiae CGMCC No. 33650 and germinated brown rice as raw material through an aerobic fermentation process. The filtrate is rich in γ-aminobutyric acid (GABA), amino acids, proteins, small molecule organic acids, and phenolic substances. It retains a rich, wine-like aroma but contains no alcohol, thus avoiding the skin irritation caused by alcohol. Furthermore, it possesses multiple skincare benefits, including whitening, brightening, anti-wrinkle, repairing, anti-aging, antioxidant, free radical scavenging, soothing, and moisturizing.

[0014] To achieve the above objectives, the first aspect of the present invention provides a method for preparing filtrate of germinated brown rice fermentation product, comprising the following steps:

[0015] S1. The pH of CaCl2 solution is adjusted by glutamic acid to obtain CaCl2 germination solution. Then, sterilized brown rice is soaked in CaCl2 germination solution for germination treatment. After germination, it is milled to obtain germinated brown rice paste.

[0016] S2. Mix the sprouted brown rice paste with water, heat and stir to obtain sprouted brown rice paste, then add liquefying enzyme and saccharifying enzyme to the sprouted brown rice paste, and enzymatically hydrolyze to the endpoint to obtain brown rice saccharification liquid.

[0017] S3. Inoculate Saccharomyces cerevisiae CGMCC No.33650 into glucose medium and culture aerobically to obtain seed culture;

[0018] S4. Inoculate the seed liquid into the culture medium containing brown rice saccharification liquid, and ferment aerobically until the endpoint to obtain the fermentation liquid;

[0019] S5. Centrifuge the fermentation broth, take the supernatant, and obtain the filtrate of germinated brown rice fermentation product.

[0020] Compared with the prior art, the beneficial effects of the method for preparing germinated brown rice fermentation product filtrate of the present invention are as follows:

[0021] (1) A CaCl2 solution containing glutamic acid was used as the germination culture medium. Calcium ions, as a key cofactor, effectively activated the activity of α-amylase, promoting the decomposition of starch into soluble sugars. This provided a sufficient energy source for germ growth, thus laying the material foundation for the subsequent germination process. Simultaneously, the calcium solution promoted water absorption by brown rice through osmosis, breaking the dormant state and initiating the germination process. Furthermore, calcium ions activated and released a large number of enzymes, such as glutamate decarboxylase and protease, converting bound enzymes into free enzymes, allowing proteins to hydrolyze under the action of protease to generate glutamic acid, which in turn is converted into GABA. In addition, the addition of glutamic acid, a GABA precursor, during the germination stage, generated GABA through the action of glutamate decarboxylase. This dual-effect strategy significantly increased the GABA content in the germinated brown rice slurry, laying the foundation for significantly increasing the GABA content in the filtrate of the germinated brown rice fermentation product.

[0022] (2) A specific brewing yeast strain, CGMCC No. 33650, was selected as the fermentation strain. This strain has the characteristics of high specificity, high enzyme activity, good stability, and suitability for aerobic fermentation. Using this fermentation strain to carry out aerobic fermentation of brown rice saccharification liquid not only efficiently transforms the nutrients in brown rice, but also fully releases the bound active substances in brown rice, maximizing the organic combination of brown rice active substances and yeast metabolic active substances, producing a large number of active ingredients such as GABA, amino acids, proteins, small molecule organic acids, and phenolic substances. Moreover, it retains the mellow wine aroma, but does not contain alcohol, thus avoiding the irritating effect of alcohol on the skin. This significantly expands the application scope of germinated brown rice fermentation product filtrate in the daily chemical industry, thereby providing new ideas and innovative strategies for the widespread application of germinated brown rice fermentation product filtrate in skin care products.

[0023] (3) In this invention, liquid seed liquid is used as the inoculum and brown rice saccharification liquid is used as the 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 filtrate of germinated brown rice fermentation product. The whole process is simple to operate, no need to centrifuge to collect cells, no need to break cells, and no risk of contamination by pathogenic bacteria or other bacteria.

[0024] Furthermore, step S1 of the present invention includes sterilizing the brown rice with NaClO. Specifically, the NaClO concentration may be, but is not limited to, 0.1 mmol / L.

[0025] Further, step S1 of the present invention includes adjusting the pH of a 0.1-0.5 mmol / L CaCl2 solution to 4-7 using glutamic acid to obtain a CaCl2 germination solution; the germination treatment time is 6-24 h. Specifically, the concentration of the CaCl2 solution can be, but is not limited to, 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, or 0.5 mmol / L. The pH value can be, but is not limited to, 4.0, 4.4, 5.0, 5.3, 6.0, 6.6, 6.8, or 7.0. The germination treatment time can be, but is not limited to, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, or 24 h. Calcium ions, as cofactors of α-amylase and protease, have the most significant activation effect at pH 4-7. Therefore, an acidic environment with a pH of 4-7 can maintain the optimal activity of α-amylase and protease, promoting the complete hydrolysis of starch and protein. A 0.1–0.5 mmol / L CaCl2 solution exhibits optimal osmotic efficiency under these pH conditions, effectively promoting water absorption by brown rice. Controlling the germination time to 6–24 hours ensures sufficient enzyme activation and nutrient transformation while preventing nutrient loss due to excessive germination.

[0026] Further, in step S2 of the present invention, the total amount of liquefying enzyme and saccharifying enzyme added is 0.002~0.12% of the mass of the germinated brown rice milk; specifically, the total amount of liquefying enzyme and saccharifying enzyme added may be, but is not limited to, 0.002%, 0.016%, 0.028%, 0.030%, 0.048%, 0.055%, 0.068%, 0.075%, 0.082%, 0.085%, 0.094%, 0.099%, or 0.12% of the mass of the germinated brown rice milk.

[0027] Furthermore, in step S2 of the present invention, the mass ratio of liquefying enzyme to saccharifying enzyme is 1:20~60. The specific mass ratio of liquefying enzyme to saccharifying enzyme may be, but is not limited to, 1:20, 1:25, 1:30, 1:36, 1:42, 1:50, 1:56, or 1:60.

[0028] Furthermore, the liquefying enzyme of the present invention is α-amylase; the saccharifying enzyme is glucoamylase.

[0029] Further, in step S3 of the present invention, the glucose culture medium comprises the following components at the following mass concentrations: 5-20 g / L glucose, 1-20 g / L peptone, 1-20 g / L yeast extract, 0.1-5 g / L potassium dihydrogen phosphate, and 0.1-5 g / L magnesium sulfate. The specific concentrations of glucose can be, but are not limited to, 5 g / L, 8 g / L, 12 g / L, 15 g / L, 17 g / L, and 20 g / L. The specific concentrations of peptone can be, but are not limited to, 1 g / L, 2 g / L, 5 g / L, 8 g / L, 12 g / L, 15 g / L, 17 g / L, and 20 g / L. The specific concentrations of yeast extract can be, but are not limited to, 1 g / L, 2 g / L, 5 g / L, 8 g / L, 12 g / L, 15 g / L, 17 g / L, and 20 g / L. The specific concentrations of potassium dihydrogen phosphate can be, but are not limited to, 0.1 g / L, 0.6 g / L, 1.2 g / L, 1.8 g / L, 2.5 g / L, 3.2 g / L, 3.8 g / L, 4.4 g / L, and 5 g / L. The specific concentrations of magnesium sulfate can be, but are not limited to, 0.1 g / L, 0.6 g / L, 1.2 g / L, 1.8 g / L, 2.5 g / L, 3.2 g / L, 3.8 g / L, 4.4 g / L, and 5 g / L.

[0030] Further, the aerobic culture conditions in step S3 of the present invention are as follows: inoculum size of 0.5-5%, culture temperature of 20-40°C, shaking speed of 150-250 rpm, and culture time of 4-18 hours. Specifically, the inoculum size of *Saccharomyces cerevisiae* can be, but is not limited to, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5%. The culture temperature can be, but is not limited to, 20°C, 25°C, 30°C, 35°C, and 40°C. The shaking speed can be, but is not limited to, 150 rpm, 160 rpm, 180 rpm, 200 rpm, 210 rpm, 240 rpm, and 250 rpm. The culture time can be, but is not limited to, 4 hours, 6 hours, 8 hours, 10 hours, 14 hours, 16 hours, and 18 hours. Specifically, step S3 includes placing the Saccharomyces cerevisiae and glucose culture medium in a shake flask and placing the shake flask on a shaker for aerobic culture.

[0031] Furthermore, the Saccharomyces cerevisiae strain CGMCC NO.33650 selected in step S3 of the present invention was deposited at the China General Microbiological Culture Collection Center on February 25, 2025, with the accession number Saccharomyces cerevisiae CGMCC NO.33650 and the deposit address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0032] Furthermore, the selection and breeding of the Saccharomyces cerevisiae CGMCC NO.33650 strain of the present invention consists of the following process steps:

[0033] (1) Sampling and enrichment culture:

[0034] A small amount of soil was taken from the orchard and dissolved in sterilized physiological saline. The mixture was then plated and enriched. Colonies that were milky white, round and raised with neat and smooth edges were selected. The strain was then fermented, and the cells were sequenced using 16S gene sequencing. The sequence was compared with that of Saccharomyces cerevisiae to confirm that the starting strain was Saccharomyces cerevisiae.

[0035] The enrichment culture medium consists of the following components at the following mass concentrations: glucose 10-30 g / L, peptone 1-20 g / L, yeast extract 1-20 g / L, potassium dihydrogen phosphate 0.1-5 g / L, magnesium sulfate 0.1-5 g / L, and agar 5-30 g / L. The enrichment culture conditions are: culture temperature 20-40℃ and culture time 4-24 hours.

[0036] (2) The enriched strains were subjected to compound mutagenesis:

[0037] The strain obtained in step (1) was inoculated into a test tube containing 5 ml of physiological saline. 0.5-2.0% (v / v) of diethyl sulfate mutagen was added to the test tube. The tube was shaken on a vortex mixer for 1-2 minutes. 0.5 ml of the bacterial suspension was spread onto the solid plate from step (1). The plate was irradiated with a UV lamp for 20-60 seconds and then incubated in the dark. The incubation conditions were: 20-40℃ and 4-24 hours.

[0038] (3) The mutagenized strains were screened by fermentation:

[0039] The screening medium consists of the following components at the following mass concentrations: 5-50 g / L brown rice saccharification liquid, 5-20 g / L peptone, 5-20 g / L yeast extract, 0.1-5 g / L potassium dihydrogen phosphate, 0.1-5 g / L magnesium sulfate, and 5-30 g / L agar;

[0040] The fermentation process conditions are as follows: the culture temperature is 25~40℃, the shaking speed is 100~200 rpm, and the fermentation time is 4~24 hours; strains with fast growth, high bacterial density, and high GABA content are screened.

[0041] (4) Perform subculture stability tests on the selected strains:

[0042] The selected strains 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.

[0043] Finally, the Saccharomyces cerevisiae strain CGMCC NO.33650, which has a fast growth rate, high bacterial density, and high content of metabolites, was obtained.

[0044] Further, in step S4 of the present invention, the culture medium comprises the following components at the following mass concentrations: 50-300 g / L brown rice saccharification solution, 1-20 g / L peptone, 1-20 g / L yeast extract, 0.1-5 g / L potassium dihydrogen phosphate, and 0.1-5 g / L magnesium sulfate. The concentration of the brown rice saccharification solution may be, but is not limited to, 50 g / L, 100 g / L, 150 g / L, 200 g / L, 250 g / L, or 300 g / L. The concentration of the peptone may be, but is not limited to, 1 g / L, 5 g / L, 8 g / L, 12 g / L, 15 g / L, or 20 g / L. The concentration of the yeast extract may be, but is not limited to, 1 g / L, 5 g / L, 8 g / L, 12 g / L, 15 g / L, or 20 g / L. The concentration of potassium dihydrogen phosphate can be, but is not limited to, 0.1 g / L, 0.5 g / L, 0.8 g / L, 1.5 g / L, 2.1 g / L, 2.9 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L, and 5 g / L. The concentration of magnesium sulfate can be, but is not limited to, 0.1 g / L, 0.5 g / L, 0.8 g / L, 1.5 g / L, 2.1 g / L, 2.9 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L, and 5 g / L.

[0045] Further, the aerobic fermentation conditions in step S4 of this invention are as follows: inoculum size of 1-15%, fermentation temperature of 20-40°C, ventilation rate of 1-2 vvm, fermentation tank stirring speed of 120-250 rpm, and fermentation time of 12-24 hours. Specifically, the inoculum size of the seed liquid can be, but is not limited to, 1%, 6%, 9%, 12%, or 15%. The fermentation temperature can be, but is not limited to, 20°C, 25°C, 30°C, 33°C, 36°C, 38°C, or 40°C. The ventilation rate can be, but is not limited to, 1 vvm, 1.2 vvm, 1.4 vvm, 1.6 vvm, 1.8 vvm, or 2 vvm. The fermentation tank stirring speed can be, but is not limited to, 120 rpm, 130 rpm, 150 rpm, 160 rpm, 190 rpm, 210 rpm, 230 rpm, or 250 rpm. The fermentation time can be, but is not limited to, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, or 24 hours.

[0046] Specifically, step S4 includes placing the seed liquid and the culture medium containing brown rice saccharification liquid in a fermenter for aerobic fermentation.

[0047] Accordingly, a second aspect of the present invention provides a germinated brown rice fermentation product filtrate, which is prepared by the aforementioned method for preparing germinated brown rice fermentation product filtrate.

[0048] Furthermore, the filtrate of the germinated brown rice fermentation product of the present invention contains 8.0~9.0 g / L GABA, 6.0~6.5 g / L free amino acids, 7.5~8.5 g / L total amino acids, 0.4~0.5 g / L protein, 4.5~5.0 g / L small molecule organic acids, 0 g / L ethanol, and 1.2~1.5 g / L total phenolic substances.

[0049] Furthermore, the free amino acids of the present invention include aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, tryptophan, arginine, and proline; small molecule organic acids include lactic acid, acetic acid, and succinic acid; phenolic substances include coumarins, 6-gingerol, β-carotene, methoxyphenylacetic acid, vanillic acid, epigallocatechin, eugenol, cinnamic acid, 6-gingerol, gentamicin, 2-hydroxybenzoic acid, p-coumaric acid, flavonoids, and apigenin.

[0050] Accordingly, a third aspect of the present invention provides the application of the aforementioned germinated brown rice fermentation product filtrate in cosmetics. This germinated brown rice fermentation product filtrate is rich in active ingredients such as GABA, amino acids, proteins, small molecule organic acids, and phenolic substances. It has been verified to have multiple skincare benefits, including whitening and brightening, anti-wrinkle repair, anti-aging, antioxidant and free radical scavenging, soothing and repairing, and long-lasting moisturizing effects. It can be widely used in various cosmetic products. Attached Figure Description

[0051] Figure 1 Neutrophil aggregation in zebrafish embryos of the model control group during the soothing efficacy test.

[0052] Figure 2 Neutrophil aggregation in zebrafish embryos of the positive control group in the soothing efficacy test.

[0053] Figure 3 Neutrophil aggregation in zebrafish embryos of the experimental group during the soothing efficacy test.

[0054] Figure 4 The image shows the tail area of ​​zebrafish embryos in the control group of the model during the moisturizing effect test.

[0055] Figure 5 The tail area of ​​zebrafish embryos in the blank control group during the moisturizing effect test.

[0056] Figure 6 The tail area of ​​zebrafish embryos in the experimental group during the moisturizing effect test. Detailed Implementation

[0057] To better illustrate the purpose, technical solution, and beneficial effects of this invention, the invention will be further described below in conjunction with specific embodiments and accompanying drawings. It should be noted that the methods described below are further explanations of this invention and should not be construed as limiting it.

[0058] Example 1

[0059] This embodiment provides a method for preparing filtrate of germinated brown rice fermentation product, the steps of which include:

[0060] S1. The brown rice was disinfected with 0.1 mmol / L NaClO. The pH of the 0.5 mmol / L CaCl2 solution was adjusted to 4.5 with glutamic acid to obtain a CaCl2 germination solution. The disinfected brown rice was then soaked in the CaCl2 germination solution for 18 hours. After germination, the rice was milled to obtain germinated brown rice paste.

[0061] S2. Add α-amylase and glucoamylase at a mass ratio of 1:20 to the germinated brown rice slurry, and enzymatically hydrolyze to the endpoint to obtain brown rice saccharified liquid. The total amount of α-amylase and glucoamylase added is 0.1% of the mass of the germinated brown rice slurry.

[0062] S3. Inoculate Saccharomyces cerevisiae CGMCC No.33650 into glucose medium and conduct aerobic culture to obtain seed culture. The glucose medium contains the following components at the following mass concentrations: 20 g / L glucose, 5 g / L peptone, 10 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, and 1 g / L magnesium sulfate. The aerobic culture conditions are: inoculum size of 4%, culture temperature of 30℃, shaker speed of 250 rpm, and culture time of 16 hours.

[0063] S4. Inoculate the seed culture into a culture medium containing brown rice saccharification liquid, and aerobic fermentation to the endpoint to obtain the fermentation broth, wherein the culture medium includes the following components at the following mass concentrations: 100 g / L brown rice saccharification liquid, 10 g / L peptone, 10 g / L yeast powder, 1 g / L potassium dihydrogen phosphate, and 1 g / L magnesium sulfate; the aerobic fermentation conditions are: inoculum size of 10%, fermentation temperature of 30℃, aeration rate of 1.5 vvm, fermentation tank stirring speed of 200 rpm, and fermentation time of 20 hours;

[0064] S5. Centrifuge the fermentation broth using a disc centrifuge, and take the supernatant to obtain the filtrate of germinated brown rice fermentation product.

[0065] Example 2

[0066] This embodiment provides a method for preparing filtrate of germinated brown rice fermentation product, the steps of which include:

[0067] S1. The brown rice was disinfected with 0.1 mmol / L NaClO. The pH of the 0.35 mmol / L CaCl2 solution was adjusted to 5 with glutamic acid to obtain a CaCl2 germination solution. The disinfected brown rice was then soaked in the CaCl2 germination solution for 24 hours. After germination, the rice was milled to obtain germinated brown rice paste.

[0068] S2. Add α-amylase and glucoamylase at a mass ratio of 1:40 to the germinated brown rice slurry, and enzymatically hydrolyze to the endpoint to obtain brown rice saccharified liquid. The total amount of α-amylase and glucoamylase added is 0.07% of the mass of the germinated brown rice slurry.

[0069] S3. Inoculate Saccharomyces cerevisiae CGMCC No.33650 into glucose medium and conduct aerobic culture to obtain seed culture. The glucose medium contains the following components at the following mass concentrations: 15 g / L glucose, 10 g / L peptone, 15 g / L yeast extract, 5 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate. The aerobic culture conditions are: inoculum size of 3%, culture temperature of 30℃, shaker speed of 220 rpm, and culture time of 8 hours.

[0070] S4. Inoculate the seed liquid into a culture medium containing brown rice saccharification liquid, and ferment aerobically until the endpoint to obtain the fermentation broth. The culture medium includes the following components at the following mass concentrations: 200 g / L brown rice saccharification liquid, 10 g / L peptone, 15 g / L yeast powder, 1 g / L potassium dihydrogen phosphate, and 5 g / L magnesium sulfate. The aerobic fermentation conditions are: inoculum size of 10%, fermentation temperature of 25℃, aeration rate of 1.5 vvm, fermentation tank stirring speed of 220 rpm, and fermentation time of 16 hours.

[0071] S5. Centrifuge the fermentation broth using a disc centrifuge, and take the supernatant to obtain the filtrate of germinated brown rice fermentation product.

[0072] Example 3

[0073] This embodiment provides a method for preparing filtrate of germinated brown rice fermentation product, the steps of which include:

[0074] S1. The brown rice was disinfected with 0.1 mmol / L NaClO. The pH of the 0.15 mmol / L CaCl2 solution was adjusted to 6.5 with glutamic acid to obtain a CaCl2 germination solution. The disinfected brown rice was then soaked in the CaCl2 germination solution for 8 hours. After germination, the rice was milled to obtain germinated brown rice paste.

[0075] S2. Add α-amylase and glucoamylase at a mass ratio of 1:60 to the germinated brown rice slurry, and enzymatically hydrolyze to the endpoint to obtain brown rice saccharified liquid. The total amount of α-amylase and glucoamylase added is 0.04% of the mass of the germinated brown rice slurry.

[0076] S3. Inoculate *Saccharomyces cerevisiae* CGMCC No. 33650 into glucose medium and culture aerobically to obtain seed culture. The glucose medium contains the following components at the following mass concentrations: 20 g / L glucose, 15 g / L peptone, 20 g / L yeast extract, 0.1 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate. The aerobic culture conditions are: inoculum size 1%, culture temperature 28℃, shaker speed 180 rpm, and culture time 12 hours.

[0077] S4. Inoculate the seed liquid into a culture medium containing brown rice saccharification liquid, and aerobic fermentation to the endpoint to obtain the fermentation broth, wherein the culture medium includes the following components at the following mass concentrations: 300 g / L brown rice saccharification liquid, 5 g / L peptone, 15 g / L yeast powder, 5 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate; the aerobic fermentation conditions are: inoculum size of 5%, fermentation temperature of 37℃, aeration rate of 2 vvm, fermentation tank stirring speed of 250 rpm, and fermentation time of 24 hours;

[0078] S5. Centrifuge the fermentation broth using a disc centrifuge, and take the supernatant to obtain the filtrate of germinated brown rice fermentation product.

[0079] Comparative Example 1

[0080] Comparative Example 1 and Example 2 are basically the same, except that the only difference is in step S1. Step S1 of Comparative Example 1 is as follows: the brown rice is disinfected with 0.1 mmol / L NaClO, and then the pH of the deionized water is adjusted to 5 with glutamic acid to obtain a weak acid solution. The disinfected brown rice is then soaked in the weak acid solution for 24 hours and then milled to obtain brown rice slurry.

[0081] Comparative Example 2

[0082] Comparative Example 2 used commercially available Japanese women's sake lees cosmetics (brand name: TECHNOLONG, product number: LADY'S SAKELEES).

[0083] The components of the germinated brown rice fermentation product filtrate of Examples 1-3, the ungerminated brown rice fermentation product filtrate of Comparative Example 1, and the commercially available Japanese sake lees cosmetic of Comparative Example 2 were tested according to the following test methods. The results of the GABA content test are shown in Table 1; the results of the free amino acid content test are shown in Table 2; the results of the total amino acid content test are shown in Table 3; the results of the small molecule organic acid content test are shown in Table 4; the results of the alcohol content test are shown in Table 5; the results of the total phenolic substance content test are shown in Table 6; and the results of the protein content test are shown in Table 7.

[0084] GABA content analysis: High performance liquid chromatography (HPLC) was used. The HPLC conditions were as follows: column, mobile phase: 20 mmol / L sodium acetate aqueous solution: acetonitrile = 80:20, flow rate: 1.0 mL / min, column temperature: 25℃, detector: UV detector; the GABA standard curve was y = 1517.2x + 399971 (R²). 2 =0.9911).

[0085] 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).

[0086] 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).

[0087] The content of free amino acids was detected using an automated amino acid analyzer.

[0088] High performance liquid chromatography (HPLC) was used to detect the content of small molecule organic acids.

[0089] The alcohol content was detected using the gas chromatography method specified in GB 5009.225—2023, "National Food Safety Standard - Determination of Ethanol Concentration in Wine and Edible Alcohol".

[0090] The total phenolic content was detected by liquid chromatography-mass spectrometry (LC-MS), and the fermentation product filtrate was identified to contain phenolic substances including 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.

[0091] Table 1. Results of GABA content detection

[0092]

[0093] Table 2 Results of Free Amino Acid Content Detection

[0094]

[0095] Table 3. Results of Total Amino Acid Content Detection

[0096]

[0097] Table 4 Results of small molecule organic acid content

[0098]

[0099] Table 5. Results of alcohol content detection

[0100]

[0101] Table 6. Detection of total phenolic content

[0102]

[0103] Table 7 Protein content detection results

[0104]

[0105] As can be seen from Tables 1 to 5 and Tables 5 to 7, the content of γ-aminobutyric acid (GABA), free amino acid, total amino acid, protein, total phenolic substances, and small molecule organic acids in the filtrate of germinated brown rice fermented product prepared by the method of the present invention is significantly higher than that of the ungerminated brown rice treatment group (Comparative Example 1) and significantly higher than that of commercially available Japanese women's sake lees cosmetics (Comparative Example 2). This is because germination pretreatment activates the endogenous enzyme system of brown rice, effectively promoting the natural accumulation of GABA and providing a high-quality substrate rich in GABA for subsequent fermentation, thereby significantly increasing the final yield of GABA in the fermentation product. Secondly, the combination of the selected CGMCC No.33650 fermentation strain with the saccharified liquid of germinated brown rice not only efficiently transforms the nutrients in brown rice, but also fully releases the bound active substances in brown rice, maximizing the organic combination of the active substances in brown rice with the metabolic active substances of yeast, producing a large number of active ingredients such as GABA, amino acids, proteins, small molecule organic acids, and phenolic substances. This significantly expands the application scope of germinated brown rice 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.

[0106] Furthermore, according to the data in Table 4, the germinated brown rice fermentation product filtrate prepared using the method of this invention contained three small-molecule organic acids: lactic acid (approximately 2.4 g / L), acetic acid (approximately 1.5 g / L), and succinic acid (approximately 0.8 g / L). The contents of these three acids were significantly higher than those in the ungerminated brown rice treatment group (Comparative Example 1). Notably, no small-molecule organic acids were detected in the Japanese female sake lees cosmetic sample (Comparative Example 2). Based on this, the germinated brown rice fermentation product filtrate of this invention, due to its unique organic acid composition, exhibits multiple beneficial effects in skin repair applications, including pH regulation, antibacterial properties, exfoliation, and whitening.

[0107] Furthermore, as shown in Table 5, the germinated brown rice fermentation product filtrate prepared by the method of the present invention contains no alcohol at all, while the alcohol content of commercially available Japanese sake lees cosmetics (Comparative Example 2) is as high as 55.14 g / L. This indicates that traditional sake lees products inevitably contain alcohol due to the limitations of the fermentation process, while the germinated brown rice fermentation product filtrate obtained by the method of the present invention contains no alcohol at all. This avoids the skin irritation, dryness and other problems that alcohol may cause, and is especially suitable for sensitive skin, children and people with alcohol intolerance.

[0108] Furthermore, as shown in Table 6, the total phenolic content in the filtrate of germinated brown rice fermented product prepared by the method of the present invention is significantly higher than that of the ungerminated brown rice treatment group (Comparative Example 1) and significantly higher than that of commercially available Japanese women's sake lees cosmetics (Comparative Example 2). In addition, liquid chromatography-mass spectrometry analysis identified 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 in the product. These phenolic substances work synergistically to enable the germinated brown rice fermented product filtrate to exhibit a unique mellow and rich sake aroma even without alcohol.

[0109] Whitening and brightening efficacy test:

[0110] The inhibition of tyrosinase activity in the germinated brown rice fermentation product filtrate of Examples 1-3, the ungerminated brown rice fermentation product filtrate of Comparative Example 1, and the commercially available Japanese sake lees cosmetic of Comparative Example 2 were tested according to the group standard T / SHRH 015-2018 "Cosmetics - Test Method for Inhibition of Tyrosinase Activity" of Shanghai Daily Chemical Industry Association. The test results are shown in Table 8.

[0111] The germinated brown rice fermentation product filtrate from Example 1 was applied to female sake lees extract (labeled as sample S1). The formulation (by mass percentage) was: 6% butylene glycol, 0.8% polyacrylate crosspolymer-6, 70.17% water, 0.03% disodium ethylenediaminetetraacetate, 2% 1,2-pentanediol, 0.5% p-hydroxyacetophenone, 0.5% 1,2-hexanediol, and 20% germinated brown rice fermentation product filtrate from Example 1. Sample S1 was tested by a third-party testing agency, Guangdong Microspection Testing Technology Co., Ltd., according to T / GDCDC 019-2021 standards, to evaluate its skin whitening and brightening effects on 33 subjects with dull and lackluster skin. The results are shown in Table 9.

[0112] Table 8 Inhibition of tyrosinase activity

[0113]

[0114] As shown in Table 8, the tyrosinase inhibition rate of the germinated brown rice fermentation product filtrate prepared by the method of the present invention can reach 99.0%, which is significantly better than that of Comparative Examples 1 and 2. This indicates that the germinated brown rice fermentation product filtrate of the present invention has excellent skin whitening and brightening effects.

[0115] Table 9. Human test results of the whitening efficacy of sample S1

[0116]

[0117] According to the human test data in Table 9, sample S1 has a significant effect on improving the skin melanin index (MI), and this effect continues to increase with prolonged use. Specifically, after 7 days of use, the MI value showed a significant decrease (5.78%, p<0.001); after 14 days, the decrease reached 10.22%; and by the 28-day test endpoint, the decrease rate further increased to 16.90%, with all time points showing statistical significance (p<0.001).

[0118] According to the human test data in Table 9, sample S1 has a significant effect on improving skin radiance, and this effect continues to increase with prolonged use. Specifically, a significant improvement of 6.09% was observed after 7 days of use (0.001≤p<0.01); the improvement reached 14.91% after 14 days; and the maximum improvement (20.24% improvement) was achieved at 28 days. The significance level at all later time points was p<0.001.

[0119] According to the human test data in Table 9, sample S1 can significantly improve skin color parameters, and this improvement continues to increase with prolonged use. Specifically, L* value (brightness): shows a significant increase from day 7 (1.15%, 0.01≤p<0.05), with an increase rate of 3.20% (p<0.001) at day 28; a* value (redness): decreases by 2.25% (0.01≤p<0.05) at day 7, and by 7.19% (p<0.001) at day 28; b* value (yellowness): decreases by 1.83% (0.01≤p<0.05) at day 7, and by 3.27% (0.001≤p<0.01) at day 28.

[0120] The above results indicate that the female sake lees extract containing the germinated brown rice fermentation product filtrate of this invention can effectively inhibit melanin production, improve skin radiance, and significantly improve skin color parameters, and these improvement effects continue to enhance with prolonged use. All test indicators reached their optimal improvement effect at 28 days, confirming that cosmetics containing the germinated brown rice fermentation product filtrate of this invention have significant whitening and brightening effects.

[0121] Anti-wrinkle and repair efficacy test

[0122] The female sake lees extract, labeled as sample S1, was tested for its anti-wrinkle and repair effects on 33 healthy Chinese male and female subjects aged 20-55 years, according to the T / GDCDC 019-2021 standard by Guangdong Microspection Testing Technology Co., Ltd. The subjects exhibited dull, lackluster skin, sagging and inelastic facial skin, and visible crow's feet wrinkles meeting the crow's feet wrinkle registration grade 1-5. The experimental results are shown in Table 10.

[0123] Table 10. Results of anti-wrinkle and repair human body tests

[0124]

[0125] Table 10 shows that sample S1 has a significant effect on improving wrinkles around the eyes and facial wrinkles, and this effect continues to increase with prolonged use. Specifically, the depth of crow's feet wrinkles decreased significantly by 5.23% after 14 days (0.01≤p<0.05) and reached 8.71% after 28 days (0.001≤p<0.01); the number of crow's feet wrinkles decreased by 11.86% after 7 days (0.001≤p<0.01) and improved by 24.90% after 28 days (p<0.001); the area of ​​crow's feet wrinkles decreased by 13.69% after 7 days (p<0.001) and reached 21.28% after 28 days (p<0.001); the depth of under-eye wrinkles decreased significantly by 5.99% after 14 days (0.01≤p<0.05) and reached 7.89% after 28 days (0.01≤p<0.05). .001≤p<0.01); Number of under-eye wrinkles: decreased by 14.98% in 7 days (p<0.001), and improved by 28.06% in 28 days (p<0.001); Area of ​​under-eye wrinkles: decreased by 11.88% in 7 days (p<0.001), and reached 21.35% in 28 days (p<0.001); Depth of nasolabial folds: significantly decreased by 4.34% in 14 days (p<0.001), and reached 6.28% in 28 days (p<0.001); Area of ​​nasolabial folds: decreased by 10.91% in 7 days (0.001≤p<0.01), and improved by 18.02% in 28 days (p<0.001). In summary, the depth of crow's feet, under-eye wrinkles, and nasolabial folds did not show significant improvement at 7 days (p≥0.05), but all showed significant improvement after 14 days. This indicates that the anti-wrinkle effect of the female sake lees essence containing the germinated brown rice fermentation product filtrate of this invention requires a certain amount of time to work. However, the improvement in the number and area of ​​wrinkles reached a significant level at 7 days (p<0.001), indicating that the female sake lees essence containing the germinated brown rice fermentation product filtrate of this invention has a rapid repair ability for superficial wrinkles.

[0126] Table 10 shows that the VISIA test results indicate that sample S1 has a significant and lasting effect on improving facial contours. Regarding skin tone brightness (ITA° value), it increased by 2.07% after 7 days (0.01≤p<0.05) and reached an improvement of 5.94% (p<0.001) after 28 days, indicating that the product effectively brightens skin tone. For lower facial contour firmness, the width of the lower face decreased by 1.19% after 7 days (0.01≤p<0.05) and the improvement rate reached 3.33% (p<0.001) after 28 days. Simultaneously, the lifting effect on the corners of the eyes was significant, with an angle improvement of 3.28% (0.01≤p<0.05) after 7 days and an improvement of 9.85% (p<0.001) after 28 days. Furthermore, significant improvements were observed in facial contour and jawline: the improvement angle of the facial contour increased by 1.33% (0.01≤p<0.05) at 7 days and reached 4.24% (p<0.001) at 28 days; the jawline angle increased by 1.93% (0.01≤p<0.05) at 7 days and significantly improved by 6.27% (p<0.001) at 28 days. These data fully demonstrate that sample S1 can comprehensively improve facial contour, and the effect continues to enhance with prolonged use.

[0127] Anti-aging, antioxidant, and free radical scavenging effects

[0128] The anti-aging, antioxidant, and free radical scavenging effects of the germinated brown rice fermentation product filtrate of Examples 1-3, the ungerminated brown rice fermentation product filtrate of Comparative Example 1, and the commercially available Japanese sake lees cosmetic of Comparative Example 2 were tested according to the following test methods. The test results are shown in Table 11.

[0129] 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).

[0130] Table 11 Results of Antioxidant Capacity and Free Radical Scavenging Rate Tests

[0131]

[0132] As shown in Table 11, the antioxidant capacity of the germinated brown rice fermentation product filtrate of Examples 1 to 3 reached 1.81 mM, 1.79 mM and 1.80 mM, respectively, which were significantly higher than those of Comparative Example 1 (0.81 mM) and commercially available Japanese sake lees cosmetic (0.01 mM). In terms of free radical scavenging rate, Examples 1 to 3 all maintained a high level of over 86%, while Comparative Example 1 and Comparative Example 2 were only 42.55% and 2.36%, 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.

[0133] Soothing efficacy test

[0134] The female fermented grains extract labeled S1 was subjected to a zebrafish embryo neutrophil aggregation inhibition test by Water Silver (International) Biotechnology Co., Ltd. Specifically, a neutrophil aggregation model induced by copper sulfate-induced damage to neurothalamic cells in the lateral line region of zebrafish embryos was used for testing. In the experiment, 24 zebrafish embryos were exposed to sample S1 containing 10 μM anhydrous copper sulfate and 20% germinated brown rice fermentation product filtrate (experimental group). A model control group (10 μM anhydrous copper sulfate) and a positive control group (10 μM anhydrous copper sulfate + indomethacin) were also set up. After 40 min of exposure, the embryos were fixed and stained with Sudan Black. The number of neutrophils in the lateral line region was counted and statistically analyzed. The experimental results are as follows: Figures 1-3 As shown.

[0135] Statistical analysis showed that the experimental group had an inhibition rate of 88% on neutrophil aggregation (p<0.05), which was significantly better than the model control group (0% inhibition rate on neutrophil aggregation) and the positive control group (30% inhibition rate on neutrophil aggregation). Figures 1-3 This further demonstrates the significant effect of the S1 sample in inhibiting neutrophil aggregation. This confirms that the S1 sample containing 20% ​​germinated brown rice fermentation product filtrate has excellent soothing effects and can effectively suppress inflammatory responses.

[0136] Moisturizing effect test

[0137] The above-mentioned female sake lees extract, labeled as sample S1, was tested by Water Silver (International) Biotechnology Co., Ltd. to assess its moisturizing effect in inhibiting water loss in zebrafish embryos using a water loss model induced by increasing the osmotic pressure of the fish embryo culture medium. In the experiment, 24 three-day-old zebrafish embryos were exposed to 15 g / L NaCl and sample S1 solution respectively (experimental group). A model control group (15 g / L NaCl) and a blank control group (fish embryo culture medium) were also set up. After 3 hours of exposure, the fish embryos were photographed under a microscope to measure the tail area and perform statistical analysis.

[0138] Statistical analysis showed that the experimental group inhibited the shrinkage of the tail area of ​​zebrafish embryos by 86% (p<0.05), which was significantly better than the blank control group (tail area shrinkage inhibition rate of 30%) and the model control group (tail area shrinkage inhibition rate of 0%). Figures 4-6 The study further and visually demonstrated the significant effect of sample S1 in inhibiting the shrinkage of zebrafish embryo tail area. This indicates that sample S1, containing 20% ​​germinated brown rice fermentation product filtrate, can significantly reduce the water loss and shrinkage of zebrafish embryos, exhibiting a significant moisturizing effect.

[0139] 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 filtrate of germinated brown rice fermentation product, characterized in that, It contains 8.0~9.0g / L GABA, 6.0~6.5g / L free amino acids, 7.5~8.5g / L total amino acids, 0.4~0.5g / L protein, 4.5~5.0g / L small molecule organic acids, 0g / L ethanol, and 1.2~1.5g / L total phenolic substances; The preparation steps of the germinated brown rice fermentation product filtrate are as follows: S1. The pH of a 0.1-0.5 mmol / L CaCl2 solution is adjusted to 4-7 using glutamic acid to obtain a CaCl2 germination solution. The sterilized brown rice is then soaked in the CaCl2 germination solution for germination treatment. After germination, the rice is milled to obtain germinated brown rice paste. S2. Add liquefying enzyme and saccharifying enzyme to the germinated brown rice slurry, and enzymatically hydrolyze to the endpoint to obtain brown rice saccharified liquid. The mass ratio of liquefying enzyme to saccharifying enzyme is 1:20~60. The liquefying enzyme is α-amylase and the saccharifying enzyme is glucoamylase. S3. Inoculate Saccharomyces Cerevisiae CGMCC No. 33650 into glucose medium and culture aerobically to obtain seed culture; S4. The seed liquid is inoculated into a culture medium containing the brown rice saccharification liquid, and aerobic fermentation is carried out until the endpoint to obtain the fermentation liquid; S5. The fermentation broth is centrifuged, and the supernatant is collected to obtain the filtrate of germinated brown rice fermentation product.

2. The filtrate of germinated brown rice fermentation product as described in claim 1, characterized in that, The germination treatment time is 6~24 hours.

3. The filtrate of germinated brown rice fermentation product as described in claim 1, characterized in that, The total amount of the liquefying enzyme and the saccharifying enzyme added is 0.002~0.12% of the mass of the germinated brown rice slurry.

4. The filtrate of germinated brown rice fermentation product as described in claim 1, characterized in that, The glucose culture medium described in step S3 comprises the following components at the following mass concentrations: 5-20 g / L glucose, 1-20 g / L peptone, 1-20 g / L yeast extract, 0.1-5 g / L potassium dihydrogen phosphate, and 0.1-5 g / L magnesium sulfate.

5. The filtrate of germinated brown rice fermentation product as described in claim 1, characterized in that, The conditions for aerobic culture in step S3 are as follows: inoculum size of 0.5-5%, culture temperature of 20-40℃, shaking speed of 150-250 rpm, and culture time of 4-18 hours.

6. The filtrate of germinated brown rice fermentation product as described in claim 1, characterized in that, The culture medium in step S4 comprises the following components in mass concentrations: 50-300 g / L brown rice saccharification liquid, 1-20 g / L peptone, 1-20 g / L yeast powder, 0.1-5 g / L potassium dihydrogen phosphate, and 0.1-5 g / L magnesium sulfate.

7. The filtrate of germinated brown rice fermentation product as described in claim 1, characterized in that, The conditions for aerobic fermentation in step S4 are as follows: inoculum size of 1-15%, fermentation temperature of 20-40℃, ventilation rate of 1-2 vvm, stirring speed of fermentation tank of 120-250 rpm, and fermentation time of 12-24 hours.

8. The application of the germinated brown rice fermentation product filtrate as described in any one of claims 1 to 7 in cosmetics.

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