Biological fermentation liquor with multiple efficacy, preparation method thereof and cosmetic

Abstract

The application provides a biological skin activity fermentation liquor with multiple effects, a preparation method thereof and a cosmetic, and relates to the cosmetic field.The method comprises the following steps: mixing camellia oleifera seed meal, protease, cellulase, lipase and water to obtain a first mixture; performing enzymolysis on the first mixture to obtain a fermentation substrate; performing second mixing on the fermentation substrate, glucose, peptone, yeast powder, disodium hydrogen phosphate and citric acid, and sterilizing to obtain a fermentation culture medium; activating bacillus velezensis through an LB culture medium to obtain a seed liquid; performing third mixing on the seed liquid and the fermentation culture medium, and performing fermentation to obtain a camellia oleifera seed meal fermentation liquor; performing centrifugation on the camellia oleifera seed meal fermentation liquor to obtain a centrifuged fermentation liquor; performing fourth mixing on the centrifuged fermentation liquor and polysaccharide-degrading enzyme, performing enzymolysis, and performing filtration to obtain the biological skin activity fermentation liquor with multiple effects.The method not only improves the high-value utilization of the camellia oleifera seed meal, but also obtains the biological skin activity fermentation liquor with high safety and outstanding activity.

Description

Technical Field

[0001] This application relates to the field of cosmetics, and more particularly to a bioactive fermentation broth with multiple functions, its preparation method, and cosmetics. Background Technology

[0002] Camellia seed cake, or tea seed meal, is a byproduct of oil extraction from camellia seeds. It is rich in various active ingredients such as tea saponins, polysaccharides, proteins, and polyphenols. Among them, tea saponins are a natural nonionic surfactant widely used in daily chemicals, pesticides, and animal feed. Traditional methods for extracting tea saponins often involve water extraction, organic solvent extraction, and enzymatic hydrolysis, but these methods suffer from problems such as low extraction rates, low purity, complex processes, and environmental pollution.

[0003] In recent years, microbial fermentation has attracted attention due to its green and environmentally friendly nature, simplified process, and high product activity. However, existing technologies mostly focus on improving the yield and purity of tea saponins or using extracts as cleansers and emulsifiers, but there is no evidence of directly using fermentation broth in high-end cosmetics (such as facial cleansers) or systematically evaluating its biosafety and cell activity. In addition, traditional water extracts or single-strain fermentation products have limitations in terms of cytotoxicity, antioxidant properties, skin repair, and anti-inflammation, such as high surface tension, insufficient cell safety, and limited content of active ingredients. Existing technologies also have problems such as complex extraction processes, heavy environmental burden: traditional water extraction or organic solvent extraction requires multiple steps, high energy consumption, and high risk of solvent residue; limited surfactant performance: water extracts or some fermentation products have high surface tension, limited emulsification and detergency effects, and strong skin irritation; poor cell safety: some extracts have high cytotoxicity and are not suitable for sensitive skin or infant products; insufficient bioactivity: the comprehensive functions of antioxidant, repair, anti-inflammation, and emulsification are not prominent, making it difficult to meet the needs of high-end cosmetics; and limited selection of fermentation strains.

[0004] Therefore, there is an urgent need to provide a method for preparing a bioactive fermentation broth with multiple functions to solve the above problems. Summary of the Invention

[0005] The purpose of this application is to provide a bioactive fermentation broth with multiple functions, its preparation method, and cosmetics, in order to solve the above-mentioned problems.

[0006] To achieve the above objectives, the first aspect of this application provides a method for preparing a bioactive fermentation broth with multiple functions, comprising: Camellia seed meal, protease, cellulase, lipase and water are first mixed to obtain a first mixture; the first mixture is then subjected to a first enzymatic hydrolysis to obtain a fermentation substrate. The fermentation substrate, glucose, peptone, yeast powder, disodium hydrogen phosphate, and citric acid are mixed a second time and sterilized to obtain the fermentation medium. Bacillus berberis was activated in LB medium to obtain seed liquid; the seed liquid and the fermentation medium were mixed and fermented to obtain camellia seed cake fermentation broth; the camellia seed cake fermentation broth was centrifuged to obtain centrifuged fermentation broth; the centrifuged fermentation broth and polysaccharide degrading enzyme were mixed, hydrolyzed, and filtered to obtain a bioactive fermentation broth with multiple functions.

[0007] Optionally, the camellia seed meal includes camellia seed cake and / or tea seed meal; And / or, the camellia seed meal is further pretreated before the first mixing; the pretreatment includes: drying the camellia seed meal and sieving it, wherein the sieve mesh size is ≥50; And / or, the solid-liquid ratio of the camellia seed meal to water is 1g:8-12mL; And / or, the mass ratio of the camellia seed meal, the protease, the cellulase, and the lipase is 100:0.1-0.3:0.1-0.3:0.1-0.3; And / or, the protease includes at least one of trypsin, neutral protease and pepsin.

[0008] Optionally, the temperature of the first enzymatic hydrolysis is 34-39℃, and the time is 3-6h.

[0009] Optionally, the mass ratio of the fermentation substrate, the glucose, the peptone, the yeast powder, the disodium hydrogen phosphate, and the citric acid is 50:1.5-2.5:0.4-0.6:0.4-0.6:0.25-0.3:0.1-0.12.

[0010] Optionally, the mass ratio of the seed liquid to the fermentation medium is 3-7:100.

[0011] Optionally, the fermentation temperature is 35-37°C and the time is 2-5 days.

[0012] Optionally, the polysaccharide-degrading enzyme includes at least one of glucanase, pectinase, cellobiase, and polygalacturonase; And / or, the mass ratio of the centrifuged fermentation broth to the polysaccharide degrading enzyme is 100:0.2-0.4.

[0013] Optionally, the pore size of the filter membrane is ≤0.2µm.

[0014] The second aspect of this application provides a bio-surfactant fermentation broth with multiple functions, which is prepared by the method for preparing the bio-surfactant fermentation broth with multiple functions.

[0015] A third aspect of this application provides a cosmetic product, including the aforementioned bioactive fermentation broth with multiple functions.

[0016] Compared with the prior art, the beneficial effects of this application include: The method for preparing a multi-functional bioactive fermentation broth provided in this application uses inexpensive and readily available camellia seed meal as raw material. First, the substrate is pretreated with digestive enzymes (protease, cellulase, lipase) to fully release active components such as saponins, oils, polysaccharides, proteins, polyphenols, and flavonoids, forming a culture medium suitable for the growth and metabolism of *Bacillus belyssioides* to produce highly active substances. Subsequently, fermentation is carried out using *Bacillus belyssioides*, where the bacteria utilize the nutrients released by the enzymes for growth and metabolism, moderately degrading or transforming irritating components such as tea saponins and tannins in the substrate, thereby effectively reducing the cytotoxicity of the final product. Simultaneously, during the fermentation process... During the process, Bacillus vesiculosus can efficiently synthesize lipopeptide metabolites (such as surfactants and subtilisin) and extracellular polysaccharides. These components not only endow the fermentation broth with excellent surface activity and anti-inflammatory capabilities, but also have multiple effects such as anti-oxidation, immunomodulation, and cell repair promotion. In addition, Bacillus vesiculosus fermentation also promotes the release and transformation of functional substances such as polyphenols, polypeptides, and polysaccharides in the substrate, further enhancing the antioxidant, anti-inflammatory, and repair activities of the fermentation broth. The bacterial-enzyme co-fermentation process provided in this application not only improves the high-value utilization of camellia seed meal resources, but also directly obtains a natural fermented surfactant stock solution with high safety, outstanding activity, and suitability for high-end cosmetics.

[0017] The bio-surfactant fermentation broth provided in this application has multiple functions and can be directly used in cosmetics. It is significantly superior to water extracts and traditional fermentation products, and has lower surface tension, higher cell safety, and stronger antioxidant, repair and anti-inflammatory activities.

[0018] The cosmetics provided in this application have good antioxidant, repair, and anti-inflammatory effects, and are safer. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0020] Figure 1 Comparison of surface tension properties of bioactive fermentation broths provided in Example 1 and Comparative Examples 1-5; Figure 2 Cytotoxicity test graphs of different concentrations of the bioactive fermentation broth with multiple functions provided in Example 1; Figure 3 Cytotoxicity test graphs of different concentrations of the bioactive fermentation broth provided for Comparative Example 1; Figure 4Cytotoxicity test graphs of different concentrations of the bioactive fermentation broth provided for Comparative Example 2; Figure 5 Cytotoxicity test graphs of different concentrations of bioactive fermentation broth provided for Comparative Example 3; Figure 6 Comparative graphs of scratch repair tests performed on the bio-surfactant fermentation broths provided in Example 1 and Comparative Examples 1-3; Figure 7 Comparative graphs showing the anti-inflammatory test results of the bioactive fermentation broths provided in Example 1 and Comparative Examples 1-3. Detailed Implementation

[0021] First, the solution provided in this application will be explained in more detail as follows: The first aspect of this application provides a method for preparing a bioactive fermentation broth with multiple functions, comprising: Camellia seed meal, protease, cellulase, lipase and water are first mixed to obtain a first mixture; the first mixture is then subjected to a first enzymatic hydrolysis to obtain a fermentation substrate. The fermentation substrate, glucose, peptone, yeast powder, disodium hydrogen phosphate, and citric acid are mixed a second time and sterilized to obtain the fermentation medium. Bacillus berberis was activated in LB medium to obtain seed liquid; the seed liquid and the fermentation medium were mixed and fermented to obtain camellia seed cake fermentation broth; the camellia seed cake fermentation broth was centrifuged to obtain centrifuged fermentation broth; the centrifuged fermentation broth and polysaccharide degrading enzyme were mixed, hydrolyzed, and filtered to obtain a bioactive fermentation broth with multiple functions.

[0022] It should be noted that this application provides a method for preparing biosurfactants from agricultural product processing waste such as camellia seed meal. The substrate is inexpensive and readily available, the preparation process is simple, and it can obtain fermentation products with better performance. The purpose of existing camellia seed meal extraction and fermentation is generally to obtain tea saponins and improve the purity of tea saponins. The method provided in this application ultimately obtains a fermentation filtrate containing a variety of surfactants such as tea saponins and lipopeptides, which has a stronger surface tension than tea saponins. The fermentation product obtained by this application is rich in saponins, lipopeptides, extracellular polysaccharides, and active peptides, and has lower cytotoxicity, better antioxidant properties, scratch repair, hydration, and anti-inflammatory effects than the aqueous extract of camellia seed meal.

[0023] It should also be noted that this application uses Bacillus vesiculosus for fermentation of camellia seed meal mainly because it can have a beneficial effect on the composition and properties of active substances in the fermentation system during the fermentation process, thereby obtaining better overall efficacy (the corresponding effects are described later). Figure 1 Table 1 and Figure 2-7(As embodied in the text). Without being constrained by any theoretical framework, the applicant hypothesizes that its mechanism of action includes, but is not limited to: Bacillus belyssus can secrete extracellular enzyme systems and carry out metabolic transformation during fermentation, promoting the release and transformation of functional substances such as saponins, polyphenols, polypeptides, lipids, and polysaccharides in the substrate, enhancing the antioxidant, anti-inflammatory, and repair activities of the fermentation broth, and reducing cytotoxicity; at the same time, Bacillus belyssus can synthesize lipopeptide biosurfactants (such as surfactants) and secrete extracellular polysaccharides. These components synergistically form with the surface-active components derived from the substrate, improving the interfacial properties of the system and jointly contributing to the enhancement of antioxidant, anti-inflammatory, and repair activities.

[0024] In some embodiments, the camellia seed meal includes camellia seed cake and / or tea seed meal; And / or, the camellia seed meal is further pretreated before the first mixing; the pretreatment includes: drying the camellia seed meal and sieving it, wherein the sieve mesh size is ≥50; And / or, the solid-liquid ratio of the camellia seed meal to water is 1g:8-12mL; Optionally, the solid-liquid ratio of camellia seed meal to water can be any value between 1g:8mL, 1g:9mL, 1g:10mL, 1g:11mL, 1g:12mL or 1g:8mL-12mL; And / or, the mass ratio of the camellia seed meal, the protease, the cellulase, and the lipase is 100:0.1-0.3:0.1-0.3:0.1-0.3; Optionally, the mass ratio of camellia seed meal, protease, cellulase, and lipase can be any value between (100:0.1:0.1:0.1), (100:0.2:0.1:0.1), (100:0.3:0.1:0.1), (100:0.1:0.2:0.1), (100:0.1:0.3:0.1), (100:0.1:0.1:0.2), (100:0.1:0.1:0.3), or 100:0.1-0.3:0.1-0.3:0.1-0.3. And / or, the protease includes at least one of trypsin, neutral protease and pepsin.

[0025] In some embodiments, the temperature of the first enzymatic hydrolysis is 34-39°C, and the time is 3-6 hours.

[0026] Optionally, the temperature of the first enzymatic hydrolysis can be any value between 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 34-39℃, and the time can be any value between 3h, 4h, 5h, 6h or 3-6h.

[0027] In some embodiments, the mass ratio of the fermentation substrate, the glucose, the peptone, the yeast extract, the disodium hydrogen phosphate, and the citric acid is 50:1.5-2.5:0.4-0.6:0.4-0.6:0.25-0.3:0.1-0.12.

[0028] Optionally, the mass ratio of fermentation substrate, glucose, peptone, yeast extract, disodium hydrogen phosphate, and citric acid can be (50:1.5:0.4:0.4:0.25:0.1), (50:2:0.4:0.4:0.25:0.1), (50:2.5:0.4:0.4:0.25:0.1), (50:1.5:0.5:0.4:0.25:0.1), (50:1.5:0.6:0.4:0.25:0.1), (50:1.5:0.4:0.5:0.25:0.1), or (50:1.5:0.4:0.5:0.25:0.1). .1), (50:1.5:0.4:0.6:0.25:0.1), (50:1.5:0.4:0.4:0.27:0.1), (50:1.5:0.4:0.4:0.3:0.1), (50:1.5:0.4:0.4:0.25:0.11), (50:1.5:0.4:0.4:0.25:0.12) or any value between 50:1.5-2.5:0.4-0.6:0.4-0.6:0.25-0.3:0.1-0.12.

[0029] In some embodiments, the mass ratio of the seed culture to the fermentation medium is 3-7:100.

[0030] Optionally, the mass ratio of seed culture to fermentation medium can be any value between 3:100, 4:100, 5:100, 6:100, 7:100 or 3:7:100.

[0031] In some embodiments, the fermentation temperature is 35-37°C and the time is 2-5 days.

[0032] Optionally, the fermentation temperature can be any value between 35°C, 36°C, 37°C or 35-37°C, and the time can be any value between 2 days, 3 days, 4 days, 5 days or 2-5 days.

[0033] In some embodiments, the polysaccharide-degrading enzyme includes at least one of glucanase, pectinase, cellobiase, and polygalacturonase; And / or, the mass ratio of the centrifuged fermentation broth to the polysaccharide degrading enzyme is 100:0.2-0.4.

[0034] Optionally, the mass ratio of the fermentation broth to the polysaccharide degrading enzyme after centrifugation can be any value between 100:0.2, 100:0.3, 100:0.4, or 100:0.2-0.4.

[0035] In some embodiments, the pore size of the filter membrane is ≤0.2µm.

[0036] Optionally, the pore size of the filter membrane can be any value of 0.01µm, 0.1µm, 0.2µm or ≤0.2µm.

[0037] The second aspect of this application provides a bio-surfactant fermentation broth with multiple functions, which is prepared by the method for preparing the bio-surfactant fermentation broth with multiple functions.

[0038] A third aspect of this application provides a cosmetic product, including the aforementioned bioactive fermentation broth with multiple functions.

[0039] The implementation schemes of this application will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustrating this application and should not be regarded as limiting the scope of this application. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0040] Example 1 This embodiment provides a bio-surfactant fermentation broth with multiple functions and its preparation method. The specific preparation steps are as follows: S1: Pretreatment of Camellia oleifera seed cake: The dried Camellia oleifera seed cake is crushed into powder with uniform particle size and passed through a 50-mesh sieve. Water is added at a solid-liquid ratio of 1g:10mL. In addition, trypsin, cellulase and lipase, accounting for 0.1% of the total mass of the sieved Camellia oleifera seed cake powder, are added. The resulting material is placed in a 37℃ water bath and stirred for 6 hours for enzymatic hydrolysis to obtain the fermentation substrate. S2: Preparation of fermentation medium: Weigh the fermentation substrate, glucose, peptone, yeast powder, disodium hydrogen phosphate and citric acid according to the mass ratio of 50:1.5:0.6:0.4:0.3:0.12. Mix the weighed components well and then heat to 121℃ for sterilization. S3: Fermentation culture: After activating Bacillus belye with LB medium, the activated seed liquid was inoculated into the fermentation medium prepared in step S2 at a mass ratio of 7%, and fermented in a shaker at 37°C for 2 days to obtain Camellia oleifera fermentation liquid. S4: Remove bacteria and residue from the fermentation broth of Camellia oleifera by centrifugation. Add 0.1% glucanase and 0.1% pectinase by mass to the fermentation broth after centrifugation, and enzymatically hydrolyze at 37℃ for 1 hour. After enzymatic hydrolysis, rapidly raise the temperature to 80℃ for 10 minutes to inactivate the enzyme. S5: After sterilizing the liquid obtained in step S4 by passing it through a 0.2-micron filter membrane, a bioactive fermentation broth with multiple functions is obtained.

[0041] Example 2 This embodiment provides a bio-surfactant fermentation broth with multiple functions and its preparation method. The specific preparation steps are as follows: S1: Pretreatment of Camellia oleifera seed cake: The dried Camellia oleifera seed cake is crushed into powder with uniform particle size and passed through a 50-mesh sieve. Water is added at a solid-liquid ratio of 1g:8mL. In addition, 0.3% of neutral protease, 0.2% of cellulase and 0.3% of lipase are added according to the total mass of the sieved Camellia oleifera seed cake powder. The resulting material is placed in a 34℃ water bath and stirred for 3 hours to obtain the fermentation substrate. S2: Preparation of fermentation medium: Weigh the fermentation substrate, glucose, peptone, yeast powder, disodium hydrogen phosphate and citric acid according to the mass ratio of 50:2:0.5:0.5:0.25:0.1. Mix the weighed components well and then heat to 121℃ for sterilization. S3: Fermentation culture: After activating Bacillus belye with LB medium, the activated seed liquid was inoculated into the fermentation medium prepared in step S2 at a mass ratio of 5%, and fermented in a shaker at 35℃ for 4 days to obtain Camellia oleifera fermentation liquid. S4: Remove bacteria and residue from the fermentation broth of Camellia oleifera by centrifugation. Add 0.15% by mass of glucanase and 0.15% by mass of polygalacturonase to the centrifuged fermentation broth. Enzymatically hydrolyze at 37°C for 1 hour. After enzymatic hydrolysis, rapidly raise the temperature to 80°C for 10 minutes to inactivate the enzyme. S5: After sterilizing the liquid obtained in step S4 by passing it through a 0.2-micron filter membrane, a bioactive fermentation broth with multiple functions is obtained.

[0042] Example 3 This embodiment provides a bio-surfactant fermentation broth with multiple functions and its preparation method. The specific preparation steps are as follows: S1: Pretreatment of Camellia oleifera seed cake: The dried Camellia oleifera seed cake is crushed into powder with uniform particle size and passed through a 50-mesh sieve. Water is added at a solid-liquid ratio of 1g:12mL. In addition, 0.2% of pepsin, 0.3% of cellulase and 0.2% of lipase are added according to the total mass of the sieved Camellia oleifera seed cake powder. The resulting material is placed in a 39℃ water bath and stirred for 4 hours to obtain the fermentation substrate. S2: Preparation of fermentation medium: Weigh the fermentation substrate, glucose, peptone, yeast powder, disodium hydrogen phosphate and citric acid according to the mass ratio of 50:2.5:0.4:0.6:0.27:0.11, mix the weighed components, and then heat to 121℃ for sterilization. S3: Fermentation culture: After activating Bacillus belye with LB medium, the activated seed liquid was inoculated into the fermentation medium prepared in step S2 at a mass ratio of 3%, and fermented in a shaker at 35°C for 5 days to obtain Camellia oleifera fermentation liquid. S4: Remove bacteria and residue from the fermentation broth of Camellia oleifera by centrifugation. Add 0.2% cellobiase and 0.2% pectinase by mass to the centrifuged fermentation broth and enzymatically hydrolyze at 35℃ for 1 hour. After enzymatic hydrolysis, rapidly raise the temperature to 80℃ to inactivate the enzyme for 10 minutes. S5: After sterilizing the liquid obtained in step S4 by passing it through a 0.2-micron filter membrane, a bioactive fermentation broth with multiple functions is obtained.

[0043] Comparative Example 1 This comparative example provides a bio-active fermentation broth and its preparation method, which involves direct fermentation without enzymatic hydrolysis. The specific preparation method is as follows: S1: Pretreatment of Camellia oleifera seed cake: The dried Camellia oleifera seed cake is crushed into powder with uniform particle size, passed through a 50-mesh sieve, and water is added according to a solid-liquid ratio of 1g:10mL. The resulting material is placed in a 37℃ water bath and stirred for 6 hours to obtain the fermentation substrate. S2: Preparation of fermentation medium: Weigh the fermentation substrate, glucose, peptone, yeast powder, disodium hydrogen phosphate and citric acid according to the mass ratio of 50:1.5:0.6:0.4:0.3:0.12. Mix the weighed components well and then heat to 121℃ for sterilization. S3: Fermentation culture: After activating Bacillus belye with LB medium, the activated seed liquid was inoculated into the fermentation medium prepared in step S2 at a mass ratio of 7%, and fermented in a shaker at 37°C for 2 days to obtain Camellia oleifera fermentation liquid. S4: Remove bacteria and residue from the fermentation broth of Camellia oleifera by centrifugation. Add 0.1% glucanase and 0.1% pectinase by mass to the fermentation broth after centrifugation, and enzymatically hydrolyze at 35℃ for 1 hour. After enzymatic hydrolysis, rapidly raise the temperature to 80℃ for 10 minutes to inactivate the enzyme. S5: After sterilizing the liquid obtained in step S4 by passing it through a 0.2-micron filter membrane, a bioactive fermentation broth with multiple functions is obtained.

[0044] Comparative Example 2 This comparative example provides a bio-active fermentation broth and its preparation method, the specific preparation method of which is as follows: S1: Pretreatment of Camellia oleifera seed cake: The dried Camellia oleifera seed cake is crushed into powder with uniform particle size and passed through a 50-mesh sieve. Water is added at a solid-liquid ratio of 1g:10mL. In addition, trypsin, cellulase and lipase, accounting for 0.1% of the total mass of the sieved Camellia oleifera seed cake powder, are added. The resulting material is placed in a 37℃ water bath and stirred for 6 hours for enzymatic hydrolysis to obtain the fermentation substrate. S2: Mix the fermentation substrate and water at a mass ratio of 1:1, and then sterilize at 121°C; S3: Centrifuge to remove the residue from the matrix obtained in step S2. Add 0.1% glucanase and 0.1% pectinase to the centrifuged liquid, and enzymatically hydrolyze at 35°C for 1 hour. After enzymatic hydrolysis, rapidly heat to 80°C to inactivate the enzyme for 10 minutes. S4: After sterilizing the liquid obtained in step S3 by passing it through a 0.2-micron filter membrane, a bioactive fermentation broth with multiple functions is obtained.

[0045] Comparative Example 3 This comparative example provides a bio-active fermentation broth and its preparation method, which involves only water extraction. The specific preparation method is as follows: S1: Pretreatment of Camellia oleifera seed cake: Crush dried Camellia oleifera seed cake into a uniform powder, pass it through a 50-mesh sieve, and add water at a material-to-liquid ratio of 1:10. Place the mixture in a 37℃ water bath and stir for 6 hours to obtain the fermentation substrate; S2: Mix the fermentation substrate and water in a 1:1 ratio, sterilize at 121℃ to obtain the water extract; S3: Centrifuge to remove residue, add 0.1% glucanase and 0.1% pectinase to the centrifuged liquid, enzymatically hydrolyze at 35℃ for 1 hour, and then rapidly heat to 80℃ to inactivate the enzyme for 10 minutes after enzymatic hydrolysis. S4: The feed solution is sterilized by passing it through a 0.2-micron filter membrane to obtain the enzymatic hydrolysate.

[0046] Comparative Example 4 The difference from Example 1 is that Bacillus berberis is replaced with Saccharomyces cerevisiae.

[0047] Comparative Example 5 The difference from Example 1 is that Bacillus belye is replaced with Lactobacillus rhamnosus.

[0048] Test Example 1 The surface tension properties of the bioactive fermentation broths obtained in Example 1 and Comparative Examples 1-5 were tested. Surfactants can significantly reduce the surface tension of liquids. The surface tension change of the fermentation broth was measured to determine its surfactant activity. In this test, the platinum ring method was used. The test results are as follows: Figure 1 As shown.

[0049] from Figure 1The results show that all samples exhibited a significant reduction in surface tension relative to water, indicating that the camellia seed extract possesses good surface activity. Comparative Examples 2 and 3 showed similar surface tensions, suggesting that enzymatic hydrolysis alone had limited effect on enhancing the leaching of saponins and other surfactants. In contrast, Comparative Example 1 (fermentation treatment) showed even lower surface tension, indicating that fermentation not only helps increase the release of saponins but also generates new surfactants through microbial metabolism. Conversely, Comparative Examples 4 and 5 showed surface tensions similar to Comparative Example 3, indicating that yeast and lactic acid bacteria fermentation did not enhance the leaching of saponins and other surfactants, nor did it generate new surfactants to further reduce surface tension like *Bacillus belyssima*. Notably, Example 1 (enzymatic hydrolysis combined with fermentation treatment) showed a further reduction in surface tension, suggesting that enzymatic pretreatment not only effectively released saponins from the substrate but also provided more nutrients such as oils, proteins, and polysaccharides, providing a richer substrate for the growth and metabolic activities of *Bacillus belyssima*, promoting the synthesis of novel surfactants such as lipopeptides, and thus significantly enhancing the surface activity of the final product.

[0050] Test Example 2 The total antioxidant capacity of the bioactive fermentation broths provided in Examples 1-3 and Comparative Examples 1-5 was tested. The total antioxidant capacity was detected using a BIOSS kit, the principle of which is to reduce Fe under acidic conditions. 3+ - Tripyridine triazine (Fe 3+ -TPTZ) produces blue Fe 2+ The ability of -TPTZ reflects the total antioxidant capacity. The reaction system was prepared according to the kit instructions. After reacting for 10 minutes, the absorbance was measured at 593 nm using a microplate reader. A standard curve was calculated, which was y = 10.641x + 0.0006, R0. 2 =1. x is Fe 2+ Concentration, y is the difference in absorbance between the sample and the control.

[0051] The sample tube contained 180 μL of test reagent + 6 μL of sample + 18 μL of distilled water; the control tube contained 180 μL of test reagent + 24 μL of distilled water. Based on the absorbance difference, x (μ mol / mL) was calculated, and the total antioxidant capacity T-AOC = x·34. The final total antioxidant capacity test results are shown in Table 1.

[0052] Table 1 Total Antioxidant Capacity

[0053] As shown in Table 1, the total antioxidant capacity (T-AOC) of the fermentation filtrates from Examples 1-3 was significantly higher than that of Comparative Examples 1-3. Specifically, the T-AOC values ​​of the samples treated with bacterial-enzyme co-fermentation (Examples 1-3) were all between 4.21 and 4.26 μmol / mL, which was significantly higher than that of samples treated with fermentation alone (Comparative Examples 1, 4, and 5, 1.24–2.85 μmol / mL), enzymatic hydrolysis alone (Comparative Example 2, 1.94 μmol / mL), and water extraction (Comparative Example 3, 1.72 μmol / mL). This indicates that bacterial-enzyme co-fermentation can not only effectively enhance the release and transformation of active substances in Camellia oleifera seed cake, but also significantly enhance the antioxidant capacity of the final product. Further comparison of Examples 1-3 with Comparative Example 1 shows that enzymatic pretreatment helps release and transform nutrients in the substrate, optimizes the nutrient composition ratio of the substrate, provides a richer substrate for the growth and metabolism of *Bacillus belyssioides*, and promotes the generation of antioxidant active substances (active peptides, organic acids, extracellular polysaccharides, and polyphenols, etc.), thereby significantly improving the total antioxidant capacity of the fermentation filtrate. The results of Comparative Examples 1, 4, and 5 indicate that, compared with *Bacillus belyssioides*, yeast and lactic acid bacteria have a poorer effect on improving the total antioxidant capacity of the fermentation broth in the fermentation of *Camellia oleifera* seed cake. It is speculated that the tea saponins in *Camellia oleifera* seed cake have a certain inhibitory effect on the growth of yeast and lactic acid bacteria. At the same time, the growth and metabolism of yeast and lactic acid bacteria consume reducing components such as polyphenols in the substrate, therefore the total reducing power of the fermentation broth will not be significantly improved.

[0054] Test Example 3 By observing the effects of the bioactive fermentation broths provided in Example 1 and Comparative Examples 1-3 on Hacat cell viability, it was found that enzymes in the mitochondria of living cells reduced yellow MTT to purple crystalline formazan, a function absent in dead cells. The amount of crystalline formazan was directly proportional to the number of living cells, and the absorbance was measured using a microplate reader to indirectly reflect cytotoxicity. The specific steps were as follows: 1. Hacat cells were seeded in 96-well plates and cultured until adherent; 2. Add samples of different concentrations and intervene for 24-72 hours; 3. Add MTT solution and incubate for 4 hours; 4. Remove the supernatant and add DMSO to dissolve and crystallize; 5. Measure the absorbance at 490 / 570 nm using an ELISA reader and calculate the cell viability. Viability = (Experimental group OD - Blank OD) / (Control group OD - Blank OD) × 100%.

[0055] The cytotoxicity test results of different concentrations of the multifunctional bioactive fermentation broth provided in Example 1 are as follows: Figure 2 As shown.

[0056] The cytotoxicity test results of different concentrations of the multifunctional bioactive fermentation broth provided in Comparative Example 1 are as follows: Figure 3 As shown.

[0057] The cytotoxicity test results of different concentrations of the multifunctional bioactive fermentation broth provided in Comparative Example 2 are as follows: Figure 4 As shown.

[0058] The cytotoxicity test results of different concentrations of the multifunctional bioactive fermentation broth provided in Comparative Example 3 are as follows: Figure 5 As shown.

[0059] Depend on Figure 2-5 The results showed that the cytotoxicity of the sample from Example 1 (co-fermentation treatment) was significantly lower than that of Comparative Examples 1-3 (fermentation alone, enzymatic hydrolysis, and water extraction, respectively), and the cell viability remained above 80% at a concentration of 1%. Specifically, the sample treated only by fermentation maintained cell viability above 80% at concentrations of 0.2% and below, indicating that the fermentation process can effectively reduce the cytotoxicity of Camellia oleifera seed meal extract. This is presumably related to the biotransformation of some unfavorable components during Bacillus belyssus fermentation and the optimization of the fermentation product composition, thereby improving cell compatibility. In contrast, the samples treated only by enzymatic hydrolysis and water extraction could barely maintain cell viability above 80% at concentrations of 0.04% and below, indicating that these two treatment methods had limited improvement on cytotoxicity.

[0060] Comprehensive analysis shows that the co-fermentation of bacteria and enzymes not only more fully degrades and transforms irritating components in the substrate (such as tea saponins and tannins), but also promotes the synthesis of safer bioactive substances such as extracellular polysaccharides and bioactive peptides, thereby significantly reducing the cytotoxicity of the final product and enhancing its biosafety and application potential.

[0061] Test Example 4 The bioactive fermentation broths provided in Example 1 and Comparative Examples 1-3 were subjected to scratch repair tests. The scratch repair experiment simulates the in vitro wound healing process, evaluating the scratch repair capability of the samples by observing cell migration and proliferation on both sides of an artificially created monolayer cell gap. The specific steps are as follows: 1. Fix the sterile insert to the bottom of a 6-well plate, inoculate equal amounts of Hacat cell suspension into the wells on both sides of the insert, and incubate at 37°C and 5% CO2 for 24 hours until the cells are completely adhered. 2. Remove the plugin to create a scratch test area and take an initial scratch image; 3. Add the test sample and culture medium to the cell wells. The negative control group does not add the sample, but only adds an equal amount of culture medium. Place the 6-well plate back into the 37℃, 5% CO2 incubator and continue to incubate for 24 hours. 4. Take 24-hour microscopic images of the scratched area to record cell migration status; 5. Quantitative analysis using ImageJ software: The area of ​​the scratch blank region at 0h (A0) and 24h (A24) was measured respectively; the healing rate (%) was calculated according to the formula [(A0-A24) / A0]×100 to calculate the scratch healing percentage.

[0062] The scratch repair test results of the bio-surfactant fermentation broths provided in Example 1 and Comparative Examples 1-3 are as follows: Figure 6 As shown.

[0063] Depend on Figure 6 It is evident that Example 1 (co-fermentation treatment with bacteria and enzymes) demonstrated the most outstanding performance in promoting cell migration and proliferation, and scratch repair. Compared with Comparative Examples 1-3 (fermentation, enzymatic hydrolysis, or water extraction treatment only), the samples treated with Example 1 significantly improved the healing rate of cell scratch areas. The water extract group showed almost no scratch repair effect, while the co-fermentation group significantly promoted cell migration and filling of the scratch area, exhibiting a higher repair capacity. The results indicate that, corresponding to the cytotoxicity results, co-fermentation with bacteria and enzymes can not only effectively release and transform functional components in the substrate, but also promote the generation of extracellular polysaccharides, bioactive peptides, and other cell repair-related active substances, reduce cytotoxicity, enhance cell viability and migration ability, thereby significantly enhancing the scratch healing effect of the fermentation broth.

[0064] Test Example 5 The bioactive fermentation broths provided in Example 1 and Comparative Examples 1-3 were subjected to anti-inflammatory tests. ANA-1 macrophages were stimulated with LPS (lipopolysaccharide) to produce and release the inflammatory factor TNFα. The inhibitory effect of the samples on the inflammatory response, i.e., the anti-inflammatory effect, was evaluated by measuring the amount of TNFα released. The specific steps are as follows: 1. The ANA-1 mouse macrophage cell line was cultured at 37°C and 5% CO2 until it reached the logarithmic growth phase; 2. Divide the cells into groups: blank control group, negative (LPS model group), positive group, and sample group. Treat the cells with the sample to be tested for 1 hour. 3. LPS was added to all groups except the control group at a final concentration of 100 ng / mL, and the cells were incubated for another 24 hours to induce an inflammatory response and release TNFα. 4. Collect cell supernatant and use an ELISA kit to detect the TNFα content in each group.

[0065] The anti-inflammatory test results of the bioactive fermentation broths provided in Example 1 and Comparative Examples 1-3 are as follows: Figure 7 As shown.

[0066] Depend on Figure 7The results showed that, at the tested concentration, the tea seed meal sample treated with bacterial-enzyme co-fermentation (Example 1) exhibited the most significant inhibitory effect on inflammatory factors, with a significantly better anti-inflammatory effect than other treatments. Although the samples in Comparative Examples 1-3 (fermentation, enzymatic hydrolysis, or water extraction alone) also reduced inflammatory factor levels to some extent, their anti-inflammatory activity was inferior to that of the bacterial-enzyme co-fermentation group. These results indicate that the synergistic effect of enzymatic hydrolysis and Bacillus belye fermentation not only promotes the release and transformation of anti-inflammatory active ingredients in the substrate but also synthesizes metabolites with anti-inflammatory effects, such as lipopeptides, extracellular polysaccharides, and bioactive peptides, thereby synergistically enhancing the anti-inflammatory effect of the final product.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0068] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of this application and form different embodiments. For example, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for preparing a bioactive fermentation broth with multiple functions, characterized in that, include: Camellia seed meal, protease, cellulase, lipase and water are mixed to obtain a first mixture; The first mixture is subjected to a first enzymatic hydrolysis to obtain a fermentation substrate; The fermentation substrate, glucose, peptone, yeast powder, disodium hydrogen phosphate, and citric acid are mixed a second time and sterilized to obtain the fermentation medium. Bacillus belye was activated in LB medium to obtain seed culture; The seed liquid and the fermentation medium are mixed and fermented in a third manner to obtain camellia seed cake fermentation liquid. The camellia seed cake fermentation liquid is centrifuged to obtain centrifuged fermentation liquid. The centrifuged fermentation liquid and polysaccharide degrading enzyme are mixed in a fourth manner, subjected to a second enzymatic hydrolysis, and filtered to obtain a bioactive fermentation liquid with multiple functions.

2. The method for preparing the multi-functional bio-surfactant fermentation broth according to claim 1, characterized in that, The camellia seed meal includes camellia seed cake and / or tea seed meal; And / or, the camellia seed meal is further pretreated before the first mixing; the pretreatment includes: drying the camellia seed meal and sieving it, wherein the sieve mesh size is ≥50; And / or, the solid-liquid ratio of the camellia seed meal to water is 1g:8-12mL; And / or, the mass ratio of the camellia seed meal, the protease, the cellulase, and the lipase is 100:0.1-0.3:0.1-0.3:0.1-0.3; And / or, the protease includes at least one of trypsin, neutral protease and pepsin.

3. The method for preparing the multi-functional bio-surfactant fermentation broth according to claim 1, characterized in that, The first enzymatic hydrolysis was performed at a temperature of 34-39℃ for 3-6 hours.

4. The method for preparing the multi-functional bio-surfactant fermentation broth according to claim 1, characterized in that, The mass ratio of the fermentation substrate, the glucose, the peptone, the yeast powder, the disodium hydrogen phosphate, and the citric acid is 50:1.5-2.5:0.4-0.6:0.4-0.6:0.25-0.3:0.1-0.

12.

5. The method for preparing the bio-surfactant fermentation broth with multiple functions according to claim 1, characterized in that, The mass ratio of the seed liquid to the fermentation medium is 3-7:

100.

6. The method for preparing the bio-surfactant fermentation broth with multiple functions according to claim 1, characterized in that, The fermentation temperature is 35-37℃, and the time is 2-5 days.

7. The method for preparing the multi-functional bio-surfactant fermentation broth according to claim 1, characterized in that, The polysaccharide-degrading enzyme includes at least one of glucanase, pectinase, cellobiase, and polygalacturonase; And / or, the mass ratio of the centrifuged fermentation broth to the polysaccharide degrading enzyme is 100:0.2-0.

4.

8. The method for preparing the multifunctional bioactive fermentation broth according to any one of claims 1-7, characterized in that, The pore size of the filter membrane used for filtration is ≤0.2µm.

9. A bio-surfactant fermentation broth with multiple functions, characterized in that, It is prepared by the method for preparing the bioactive fermentation broth with multiple functions as described in any one of claims 1-8.

10. A cosmetic product, characterized in that, Including the bioactive fermentation broth with multiple functions as described in claim 9.

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