A whitening composition containing a fermented plant extract and use thereof

Abstract

The present application relates to the field of daily chemical technology, and more particularly to a whitening composition containing fermented plant extracts and application thereof.The composition comprises: (1) plant fermentation product; (2) nicotinamide; (3) trehalose complex extract; the plant extracts are grouped and then subjected to staged fermentation, and polysaccharide components are introduced during the fermentation process to obtain the plant fermentation product; wherein the plant extracts at least include: sasa albida smith extract, helichrysum angustifolium gaud extract, radix scutellariae extract, dictamnus dasycarpus turcz extract, lilium candidum extract, nymphaea alba l extract, panax ginseng c.a.meyer extract; the obtained composition shows good comprehensive performance in terms of melanin production inhibition, tyrosinase activity regulation and antioxidant performance.

Description

Technical Field

[0001] This invention relates to the field of daily chemical technology, and in particular to a whitening composition containing fermented plant extracts and its application. Background Technology

[0002] As research on skin pigmentation continues to deepen, whitening compositions derived from plant extracts are widely used in skin care products. Current technologies typically employ a combination of various plant extracts with niacinamide, vitamins, or antioxidants to regulate tyrosinase activity, melanin production, and oxidative stress processes.

[0003] However, there are still many problems with multi-plant bioactive systems in practical applications.

[0004] First, the chemical properties of active ingredients from different plant sources vary considerably. For example, flavonoids and phenolic compounds usually exist in glycoside form, and their activity is significantly affected by the degree of glycosylation; some sesquiterpene lactones, on the other hand, have high reactivity and are prone to degradation, polymerization, or increased irritation in complex systems. Furthermore, differences in redox reactions may exist between different plant extracts, leading to decreased system stability.

[0005] Secondly, most existing technologies for processing plant extracts employ a uniform extraction or fermentation method. While this approach can increase the content of some small-molecule active ingredients, the metabolic conditions for different plant active components vary, which can easily lead to the following problems: Some flavonoid glycosides are not fully converted, while other reactive active ingredients may be deactivated during long-term fermentation; some fat-soluble active components may also aggregate during continuous fermentation, thereby reducing system stability and uniformity of use.

[0006] In addition, some plant-derived components exhibit high activity in their free state. For example, sesquiterpene lactones in chrysanthemum and some active components in Dictamnus dasycarpus root may cause skin irritation at higher concentrations, thus limiting their application in whitening compositions.

[0007] In existing technologies, polysaccharides are typically used as thickeners, humectants, or rheology modifiers, primarily focusing on improving the feel of the system or enhancing viscosity stability. However, research on the stabilizing effects of polysaccharides on phenolic and flavonoid active ingredients during fermentation remains relatively limited.

[0008] The inventors discovered that some plant-derived polysaccharides can form intermolecular interactions with phenolic and flavonoid components, thereby reducing the tendency of small-molecule active substances to oxidize and aggregate during storage and improving their dispersion stability in aqueous systems. However, most existing technologies add polysaccharides after fermentation, making it difficult to regulate the state of active ingredients during the fermentation process.

[0009] On the other hand, trehalose has been used in some skincare systems to improve moisturizing properties and stability. However, trehalose in existing technologies is mostly a high-purity single ingredient, and its main function is concentrated on moisturizing and reducing water loss, with limited improvement on the oxidative stability of complex plant-based active systems.

[0010] In contrast, trehalose complex extracts derived from microalgae stress metabolism systems contain not only trehalose but also some low-molecular-weight polysaccharides and small-molecule metabolites. These components can improve the aqueous phase stability in complex plant systems and reduce the tendency of some phenolic active ingredients to oxidatively deactivate.

[0011] Therefore, how to process the metabolic characteristics of active components from different plant sources in stages and regulate the stability of active ingredients during fermentation to improve the stability of the composition, reduce irritation, and improve whitening activity remains a technical problem that needs to be solved in this field. Summary of the Invention

[0012] To address the problems of insufficient conversion of active components, poor oxidative stability, and high irritation of some active ingredients in existing multi-plant extract systems, this invention provides a whitening composition containing fermented plant extracts and its preparation method.

[0013] This invention does not involve a simple mixed fermentation of multiple plant extracts. Instead, it groups the plant extracts according to the chemical properties and metabolic characteristics of different plant active components and processes them separately using different fermentation stages to improve the conversion efficiency of flavonoid active components, while reducing the inactivation and increased irritation of some highly reactive active components during the fermentation process.

[0014] The present invention provides a whitening composition containing fermented plant extracts, the composition comprising: (1) plant fermentation products; (2) nicotinamide; and (3) microalgae-derived trehalose complex extract.

[0015] The plant fermentation products are obtained by the following method: First, the plant extracts are grouped according to the type of active components.

[0016] Among these processes, plant extracts rich in flavonoids and phenolic compounds are preferentially subjected to lactic acid bacteria fermentation. During this stage, β-glucosidase produced during lactic acid bacteria metabolism can promote the deglycosylation of some flavonoid glycosides, thereby increasing the content of flavonoid aglycones.

[0017] Subsequently, plant extracts containing sesquiterpene lactone active ingredients were added in the subsequent fermentation stage, and fermentation was continued using yeast. This stage can reduce the free exposure of some highly reactive active ingredients and improve the dispersion of lipid-soluble active components in the aqueous system.

[0018] Plant-derived polysaccharides and / or seaweed-derived polysaccharides are further added during subsequent fermentation to enable the polysaccharide components to participate in the stabilization process of active ingredients in the fermentation system.

[0019] The inventors discovered that when polysaccharide components are added during fermentation, polysaccharides can form intermolecular interactions with phenols, flavonoids, and some small molecule active ingredients, thereby reducing the tendency of active ingredients to oxidize and aggregate, and improving the stability of fermentation products during storage.

[0020] In this invention, the plant extracts include at least: *Saururus chinensis* extract, *Chrysanthemum indicum* extract, *Scutellaria baicalensis* extract, *Dictamnus dasycarpus* root extract, *Lilium lancifolium* extract, *Nymphaea rubra* extract, and ginseng extract.

[0021] Among them, extracts of Scutellaria baicalensis, Saururus chinensis and Nymphaea alba are preferred for the first stage of fermentation; extracts of Chrysanthemum indicum and Dictamnus dasycarpus are preferred for the second stage of fermentation; and extracts of ginseng and Lilium brownii are preferred for addition in subsequent stages.

[0022] This invention further incorporates a microalgae-derived trehalose complex extract.

[0023] The trehalose complex extract is derived from a microalgae system that has undergone stress-induced culture and is obtained through cell wall disruption, extraction, and selective separation.

[0024] Compared with single trehalose, the microalgae-derived trehalose complex extract contains not only trehalose but also retains some low-molecular-weight polysaccharides and microalgae-derived metabolites.

[0025] The inventors discovered that this type of composite component can reduce the water activity of the system and improve the oxidative stability in the fermentation plant activity system, thereby increasing the activity retention rate of the composition during storage.

[0026] Furthermore, the present invention further combines the plant fermentation product with nicotinamide.

[0027] Among them, plant fermentation products are mainly used to regulate oxidative stress processes and tyrosinase-related reactions, while nicotinamide is used to regulate skin pigment transport processes, thereby achieving combined regulation under different pathways of action.

[0028] To address the aforementioned technical problems, the present invention provides a whitening composition containing fermented plant extracts, the composition comprising: (1) Plant fermentation product; (2) Nicotinamide; (3) Trehalose complex extract from microalgae; wherein the plant fermentation product is prepared by the following method: Different plant extracts were grouped according to the type of active ingredients and processed using a staged fermentation method, so that the active ingredients from different plant sources would undergo selective metabolic transformation at different fermentation stages. in: In the first stage of fermentation, plant extracts rich in flavonoids and phenolic components are fermented with lactic acid bacteria. In the second stage of fermentation, plant extracts containing sesquiterpene lactones are introduced for yeast fermentation. In the third stage of fermentation, polysaccharide components are introduced to participate in the fermentation process; The plant extracts include at least: Saururus chinensis extract, Chrysanthemum indicum extract, Scutellaria baicalensis extract, Dictamnus dasycarpus root extract, Lilium brownii extract, Nymphaea alba extract, and ginseng extract; The polysaccharide components include polysaccharides derived from Bletilla striata and / or polysaccharides derived from seaweed; The microalgae-derived trehalose complex extract contains trehalose, low-molecular-weight polysaccharides, and small-molecule metabolites derived from microalgae.

[0029] Preferably, the method for preparing the plant fermentation product includes the following steps: (1) The plant extract was divided into the following components: Component A: Contains Scutellaria baicalensis extract, Saururus chinensis extract and Nymphaea alba extract; Component B: Contains chrysanthemum extract and dictamnus root extract; Component C: Contains ginseng extract and white lily extract; (2) Component A undergoes the first stage of lactic acid bacteria fermentation; (3) Add component B to the first stage fermentation system and carry out the second stage yeast fermentation; (4) Add component C and polysaccharide components to the second-stage fermentation system and carry out the third-stage fermentation; (5) The fermentation system is inactivated and separated to obtain plant fermentation products; (6) Add nicotinamide and microalgae-derived trehalose complex extract to the obtained plant fermentation product to obtain the whitening composition.

[0030] Preferably, the microalgae-derived trehalose complex extract is derived from a microalgae system cultured under stress-induced conditions; wherein the stress-induced conditions include one or more of high-salt stress, osmotic stress, and temperature stress.

[0031] Preferably, the trehalose complex extract is selectively separated using an ultrafiltration membrane with a molecular weight cutoff of 3 to 10 kDa.

[0032] Preferably, the polysaccharide derived from Bletilla striata is obtained by enzyme-assisted extraction and alcohol precipitation.

[0033] Preferably, the fermentation time for the first stage is 24–48 hours; the fermentation time for the second stage is 12–36 hours; and the fermentation time for the third stage is 12–36 hours.

[0034] Preferably, the first stage of lactic acid bacteria includes Lactobacillus plantarum; the second stage of yeast includes Saccharomyces cerevisiae.

[0035] A second aspect of the present invention provides the use of the whitening composition in the preparation of products for regulating skin pigmentation, reducing melanin production, improving dull skin, improving skin oxidation state, and enhancing skin barrier stability.

[0036] By adopting the above technical solution, the present invention has the following beneficial effects: (1) By performing staged fermentation treatment on different plant active components, the conversion efficiency of flavonoid active components is improved and the inactivation tendency of some highly reactive active components is reduced. (2) By adding polysaccharide components during fermentation, the stability of phenolic and flavonoid active ingredients is improved and oxidative aggregation is reduced; (3) By using trehalose complex extracts derived from microalgae, the stability and activity retention of complex plant systems during storage can be improved; (4) The resulting composition exhibits good comprehensive performance in terms of melanin production inhibition, tyrosinase activity regulation and antioxidant properties. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0038] This invention provides a whitening composition containing fermented plant extracts and its preparation method.

[0039] Unlike existing technologies that involve mixing multiple plant extracts and then directly fermenting or simply compounding them, this invention processes plant extracts in stages based on the chemical properties, polarity characteristics, and metabolic stability of active ingredients from different plant sources.

[0040] The inventors discovered that the metabolic direction of active ingredients from different plant sources varies significantly during the fermentation process.

[0041] For example, flavonoids and phenolic compounds usually exist in the form of glycosides, and their activity is significantly affected by the degree of glycosylation. Some flavonoid glycosides can undergo deglycosylation under the action of β-glucosidase during lactic acid bacteria metabolism, thereby generating the corresponding flavonoid aglycones.

[0042] Compared to glycosides, flavonoid aglycones typically have a higher degree of phenolic hydroxyl exposure, thus exhibiting greater activity in terms of antioxidant capacity and tyrosinase interactions.

[0043] However, some sesquiterpene lactones or highly reactive small molecule active ingredients are prone to structural changes, aggregation, or decreased activity under prolonged acidic fermentation conditions.

[0044] Therefore, this invention does not employ a uniform fermentation method, but instead prioritizes the first-stage fermentation treatment of plant extracts rich in flavonoids and phenolic components.

[0045] In this invention, Scutellaria baicalensis extract, Saururus chinensis extract and Nymphaea alba extract are preferably used as the first-stage fermentation components.

[0046] in: Scutellaria baicalensis extract contains flavonoid glycosides such as baicalin and wogonin; The extract of Saururus chinensis contains polyphenols and flavonoids as active ingredients; White water lily extract contains some phenols and aromatic active components.

[0047] The above-mentioned components can undergo partial glycosylation and small molecule transformation in the metabolic environment of lactic acid bacteria, thereby increasing the degree of free release of some active ingredients.

[0048] Preferably, the first stage uses Lactobacillus plantarum (CICC 25125, China Industrial Microbial Culture Collection Center) for fermentation.

[0049] The inventors discovered that Lactobacillus plantarum can produce a certain amount of β-glucosidase and related metabolic enzymes during metabolism, thereby promoting the conversion of flavonoid glycosides into flavonoid aglycones.

[0050] Meanwhile, the organic acids produced during the metabolism of lactic acid bacteria can lower the pH of the system, keeping some phenolic components in a relatively stable state.

[0051] In this invention, the first stage of fermentation is preferably carried out under relatively mild conditions to avoid the aggregation or inactivation of highly reactive active ingredients during long-term fermentation.

[0052] Furthermore, this invention introduces a second-stage fermentation component after the first-stage fermentation is completed. Preferably, the second stage involves the addition of chrysanthemum extract and dictamnus root extract. The inventors have discovered that chrysanthemum contains sesquiterpene lactone active ingredients with high reactivity; dictamnus root also contains some fat-soluble active components.

[0053] If these active ingredients are continuously exposed to acidic fermentation conditions for an extended period, they may polymerize, degrade, or become more irritating.

[0054] Therefore, in this invention, it is preferable to introduce the above-mentioned plant extracts after the flavonoid components have completed their initial conversion.

[0055] Preferably, the second stage involves continued fermentation using Saccharomyces cerevisiae (CICC®1392, China Industrial Microbial Culture Collection Center).

[0056] The inventors discovered that metabolites produced during yeast metabolism can improve the dispersion of some lipid-soluble active components in aqueous systems, while reducing the free exposure of some highly reactive active components.

[0057] In addition, yeast metabolism can also generate some amino acids, small molecule peptides and metabolic byproducts, which can improve the system's ability to stabilize some phenolic active ingredients.

[0058] In this invention, a third stage further incorporates plant-derived polysaccharides and / or seaweed-derived polysaccharides. Unlike existing technologies that add polysaccharides after fermentation, this invention preferably involves the polysaccharide components in the fermentation process. The inventors have discovered that when polysaccharides are present during fermentation, they can interact with phenols, flavonoids, and other small-molecule active ingredients through intermolecular interactions. For example, the phenolic hydroxyl groups in polyphenolic components can form hydrogen bonds or other intermolecular interactions with polysaccharide segments, thereby reducing the tendency of small-molecule active ingredients to oxidatively aggregate during storage.

[0059] Meanwhile, some polysaccharide segments can also reduce the local aggregation of fat-soluble small molecules in the aqueous phase, thereby improving the dispersion stability of fermentation products in the system.

[0060] In this invention, the polysaccharide components preferably include polysaccharides derived from Bletilla striata and polysaccharides derived from brown algae. Bletilla striata polysaccharides have a high hydroxyl density and good hydration capacity, which can improve the stability of some phenolic active ingredients in an aqueous system; while polysaccharides derived from brown algae have good hydrophilicity and ion stability, thus helping to improve the stability of complex plant active systems during storage.

[0061] Furthermore, this invention also incorporates a complex extract of trehalose derived from microalgae. While most existing trehalose formulations utilize single, purified trehalose, this invention employs a complex extract of trehalose derived from the stress-induced metabolic system of microalgae.

[0062] The inventors discovered that under high-salt or osmotic stress conditions, the trehalose content in microalgae systems significantly increases, while also generating some low-molecular-weight metabolites and low-molecular-weight polysaccharides. This invention does not completely remove these accompanying metabolic components, but rather retains some low-molecular-weight polysaccharides and metabolites.

[0063] The inventors discovered that this type of composite component can reduce the water activity of the system and improve the stability of phenolic and flavonoid active ingredients during storage.

[0064] Furthermore, the present invention further utilizes the above-mentioned plant fermentation products together with nicotinamide.

[0065] Among them, plant fermentation products are mainly used to regulate oxidative stress-related processes and tyrosinase activity, while nicotinamide is mainly used to regulate melanin transport-related processes.

[0066] Therefore, the present invention can act on different pigment generation-related pathways simultaneously, thereby improving the overall whitening performance of the composition.

[0067] In some preferred embodiments of the present invention, the whitening composition containing fermented plant extracts is made from the following components: (1) Plant fermentation products 20.0-60.0%; (2) Nicotinamide 1.0-6.0%; (3) Trehalose complex extract from microalgae 0.5-5.0%; (4) Deionized water and cosmetic acceptable excipients balance.

[0068] The plant fermentation product is preferably prepared by the following method.

[0069] (a) Grouping and pretreatment of plant extracts In this invention, plant extracts are preferably grouped according to the polarity, metabolic stability and fermentation adaptability of active ingredients from different plant sources.

[0070] The inventors discovered that if different plant materials are extracted uniformly and continuously fermented directly, the conversion efficiency of some flavonoid components is low, and some highly reactive active components are prone to oxidative aggregation or decreased activity during the fermentation process.

[0071] Therefore, in this invention, the plant extract is preferably divided into the following components: Component A: Scutellaria baicalensis extract, Saururus chinensis extract and Nymphoides pubescens extract; Component B: Chrysanthemum indicum extract and Dictamnus dasycarpus root extract; Component C: Ginseng extract and Lilium lancifolium extract.

[0072] in: Component A has a high content of flavonoids and phenolic active ingredients, making it suitable for preferential lactic acid bacteria fermentation; Component B contains some sesquiterpene lactones and lipophilic active components with high reactivity, therefore it is preferred to add them in a later stage; Component C mainly contains polysaccharides, saponins, and nutritional active ingredients. It has good stability under long-term fermentation conditions, so it is preferred to introduce it in subsequent stages.

[0073] (II) Preparation of Component A Extract In this invention, component A is preferably obtained by extracting Scutellaria baicalensis, Saururus chinensis, and Nymphaea alba.

[0074] Among them, Scutellaria baicalensis mainly contains flavonoid glycosides, including baicalin and wogonin; Saururus chinensis contains phenols and flavonoid active substances; and Nymphaea alba contains some aromatic phenolic active ingredients.

[0075] The inventors discovered that the above-mentioned active ingredients have good extraction efficiency in a moderately polar extraction system.

[0076] Therefore, the extraction is preferably performed using an alcohol-water mixture system in this invention.

[0077] Preferably, the raw materials of Scutellaria baicalensis, Saururus chinensis, and Nymphoides pubescens are dried and pulverized to 60-80 mesh, and then mixed in a mass ratio of 2-4:2-4:1-2. Subsequently, an aqueous ethanol solution with a volume fraction of 50-70% is added, preferably with a material-to-liquid ratio of 1:8-1:15, and the mixture is extracted at 50-70°C for 1.5-3 hours.

[0078] The inventors discovered that the above-mentioned ethanol concentration can balance the extraction efficiency of flavonoid glycosides and some phenolic components, while reducing the content of highly polar impurities. After extraction, the mixture is filtered and concentrated under reduced pressure at 40–50°C to prevent oxidation or aggregation of flavonoid active ingredients under high-temperature conditions. The resulting extract A is preferably used for the first stage of fermentation.

[0079] (III) Preparation of Component B Extract In this invention, component B is preferably obtained from the extraction of Chrysanthemum indicum and Dictamnus dasycarpus root.

[0080] The inventors discovered that some sesquiterpene lactone active ingredients in chrysanthemum are prone to structural changes under high temperature or strong acid conditions. Therefore, milder extraction conditions are preferred in this invention.

[0081] Preferably, the raw materials of white chrysanthemum and white dictamnus root are dried and pulverized to 60-80 mesh, and then mixed in a mass ratio of 1:1 to 2:1.

[0082] Subsequently, extraction was performed using an ethanol solution with a volume fraction of 30-50%, with a preferred material-to-liquid ratio of 1:10-1:15, and extraction was carried out at 35-50°C for 1-2 hours.

[0083] The inventors discovered that lower extraction temperatures help reduce the degradation tendency of some highly reactive active ingredients during the extraction process.

[0084] After extraction, the sample was filtered and concentrated under reduced pressure to obtain component B extract.

[0085] The resulting component B extract is preferably added after the first stage of fermentation.

[0086] (iv) Preparation of component C extract In this invention, component C is preferably obtained from ginseng and white lily extracts.

[0087] Ginseng mainly contains ginsenosides, while white lily contains some polysaccharides and hydrophilic active ingredients.

[0088] The inventors discovered that this type of active ingredient has a better extraction efficiency in systems with high water content.

[0089] Therefore, in this invention, a low-concentration alcohol-water system or a water extraction system is preferably used for extraction.

[0090] Preferably, ginseng and white lily raw materials are dried and pulverized, then mixed in a mass ratio of 1:1 to 2:1, and 30% ethanol aqueous solution or deionized water is added. The preferred material-to-liquid ratio is 1:10 to 1:15.

[0091] Further optimization involves adding cellulase and pectinase for auxiliary enzymatic hydrolysis.

[0092] The inventors discovered that enzymatic hydrolysis can improve the release efficiency of some bound saponins and polysaccharides.

[0093] Preferably, the amount of cellulase added is 0.1-1.0 wt%, the amount of pectinase added is 0.05-0.5 wt%, the enzymatic hydrolysis temperature is preferably 40-50℃, and the enzymatic hydrolysis time is preferably 1-3 hours.

[0094] After enzymatic hydrolysis, enzyme inactivation treatment was performed, followed by further extraction, filtration, and vacuum concentration to obtain component C extract.

[0095] (v) Extract Pretreatment Preferably, to improve the stability of the subsequent fermentation process, the extracts of each component are further pretreated in this invention.

[0096] Preferably, the pH of each extract is adjusted to 5.0–6.0.

[0097] The inventors discovered that this pH range can improve the metabolic stability of lactic acid bacteria and yeast during subsequent fermentation, while reducing the oxidation tendency of some phenolic active ingredients during storage.

[0098] In addition, it is preferable to further filter the extract to remove insoluble impurities and some large particles, thereby improving the uniformity of the subsequent fermentation process.

[0099] (vi) First stage fermentation In this invention, the first stage of fermentation is preferably used to promote the initial metabolic transformation of flavonoids and phenolic active ingredients.

[0100] The inventors discovered that some of the flavonoid active ingredients in Scutellaria baicalensis, Saururus chinensis, and Nymphaea alba exist in the form of glycosides, which have high hydrophilicity, but some glycosylation structures can affect the degree of exposure of active sites.

[0101] Therefore, the present invention preferably uses lactic acid bacteria for fermentation in the first stage to promote the deglycosylation conversion of some flavonoid glycosides.

[0102] Preferably, the extract of component A is added to deionized water to adjust the solid content of the system to 5-15 wt%, preferably 8-12 wt%.

[0103] The inventors discovered that when the solid content is too low, the concentration of active ingredients is insufficient, affecting the efficiency of subsequent metabolic transformation; while when the solid content is too high, it can easily lead to an increase in the viscosity of the system, thereby affecting the uniformity of microbial metabolism.

[0104] Preferably, the pH of the system is adjusted to 5.0–6.0, more preferably 5.3–5.7.

[0105] The inventors discovered that this pH range is beneficial for maintaining the metabolic activity of Lactobacillus plantarum, while also reducing the oxidation rate of some phenolic components during fermentation.

[0106] Fermentation is preferably carried out by inoculating with Lactobacillus plantarum.

[0107] The preferred inoculation amount is 2.0–5.0 wt%, and more preferably 2.5–3.5 wt%.

[0108] The inventors discovered that Lactobacillus plantarum can produce β-glucosidase and some esterases during metabolism, thereby promoting the conversion of flavonoid glycosides to flavonoid aglycones.

[0109] For example, baicalin in Scutellaria baicalensis can be partially converted into baicalein during fermentation, thereby increasing the exposure of phenolic hydroxyl groups.

[0110] Some flavonoid aglycones typically exhibit higher activity in free radical scavenging and tyrosinase activity.

[0111] In addition, lactic acid and other organic acids produced during the metabolism of lactic acid bacteria can reduce the oxidation rate of the system and improve the stability of some phenolic active ingredients.

[0112] Preferably, the fermentation temperature in the first stage is controlled at 30–35°C, more preferably at 31–33°C.

[0113] The inventors discovered that while higher temperatures can increase the metabolic rate, some phenolic components are prone to oxidative aggregation at higher temperatures. Therefore, the present invention preferably uses a relatively mild fermentation temperature.

[0114] Preferably, the fermentation time for the first stage is 24 to 48 hours, more preferably 30 to 40 hours.

[0115] The inventors discovered that when the fermentation time is insufficient, some flavonoid glycosides are not fully converted; while when the fermentation time is too long, some active ingredients may be further degraded.

[0116] Further optimization was performed to ensure the control system was in a slightly aerobic state.

[0117] The inventors discovered that microaerobic conditions can reduce the oxidation rate of some phenolic active ingredients while maintaining the normal metabolism of lactic acid bacteria.

[0118] The stirring speed is preferably controlled at 50–150 rpm, more preferably 80–120 rpm, to improve the uniformity of the system and reduce local concentration differences.

[0119] (vii) Second stage fermentation After the first stage of fermentation is completed, the present invention preferably adds component B extract and carries out a second stage of fermentation.

[0120] The inventors discovered that some active ingredients in chrysanthemum and dictamnus root have high reactivity.

[0121] For example, sesquiterpene lactones in chrysanthemum tend to undergo structural changes in a prolonged acidic environment; while some fat-soluble active ingredients in Dictamnus dasycarpus root tend to aggregate locally during continuous fermentation.

[0122] Therefore, in this invention, the plant extracts were not added simultaneously in the first stage, but were introduced after the flavonoids had completed their initial conversion.

[0123] Preferably, glucose or other fermentable carbon sources are added during the second stage of fermentation.

[0124] The inventors discovered that an appropriate amount of carbon source can enhance the metabolic activity of yeast and improve the stability of subsequent fermentation.

[0125] The preferred amount of glucose added is 0.5–1.5 wt%.

[0126] Further selection was made to inoculate with Saccharomyces cerevisiae for the second stage of fermentation.

[0127] The preferred inoculation amount is 1.0–3.0 wt%, more preferably 1.5–2.5 wt%.

[0128] The inventors discovered that yeast metabolism can generate some amino acids, small peptides, and metabolic byproducts.

[0129] These metabolites can improve the dispersion stability of some lipid-soluble active components in aqueous systems and reduce the free exposure of highly reactive active ingredients.

[0130] In addition, some metabolic components produced during yeast metabolism can improve the fermentation system's tolerance to oxidative stress.

[0131] Preferably, the fermentation temperature in the second stage is controlled at 28–32°C.

[0132] The inventors discovered that this temperature range can balance the metabolic efficiency of yeast with the stability of plant active ingredients.

[0133] Further preferred fermentation time is 12–36 hours, more preferably 18–30 hours.

[0134] The inventors discovered that when the second stage is too short, some fat-soluble active components are not sufficiently dispersed; while when the stage is too long, some sesquiterpene active components may be further degraded.

[0135] Preferably, the pH of the system is maintained at 5.0 to 5.5 during the second stage of fermentation.

[0136] The inventors discovered that this pH range can reduce the tendency of some highly reactive active ingredients to become inactive during fermentation.

[0137] (viii) Third stage fermentation After the second stage of fermentation is completed, the present invention further adds component C extract and polysaccharide components, and carries out the third stage of fermentation.

[0138] In this invention, the ginsenosides and polysaccharides in component C have good stability under relatively mild fermentation conditions, so they are preferably introduced in subsequent stages.

[0139] Meanwhile, in this invention, it is preferable to involve the polysaccharide component in the third stage of fermentation, rather than adding it directly after fermentation.

[0140] The inventors discovered that when polysaccharides are present during fermentation, they can interact with some phenolic and flavonoid active ingredients through intermolecular interactions.

[0141] For example, phenolic hydroxyl groups can form hydrogen bonds or other intermolecular interactions with polysaccharide segments, thereby reducing the tendency of small molecule active ingredients to oxidize and aggregate during storage.

[0142] In addition, polysaccharide segments can also improve the dispersion of some fat-soluble small molecules in the aqueous phase.

[0143] Compared to adding polysaccharides after fermentation, the fermentation products obtained when polysaccharides are involved in the fermentation process in this invention typically exhibit higher stability and lower precipitation tendency.

[0144] Preferably, the polysaccharide component includes polysaccharides derived from Bletilla striata and polysaccharides derived from brown algae.

[0145] in: Polysaccharides derived from Bletilla striata have a high hydroxyl density and good hydration capacity; Polysaccharides derived from brown algae have good ionic stability and hydrophilicity.

[0146] The inventors discovered that when the two types of polysaccharides mentioned above coexist, they have a good stabilizing effect on phenolic and flavonoid active ingredients in complex plant active systems.

[0147] Preferably, the amount of polysaccharide from Bletilla striata is 1.0 to 8.0 wt%, more preferably 3.0 to 6.0 wt%.

[0148] The preferred amount of brown algae-derived polysaccharide is 0.1–2.0 wt%, more preferably 0.3–1.2 wt%.

[0149] Preferably, the fermentation temperature in the third stage is controlled at 28–32°C, more preferably at 29–31°C.

[0150] The preferred fermentation time is 12 to 36 hours, and more preferably 18 to 30 hours.

[0151] Further optimization was performed to control the pH of the system to be 5.0–5.5.

[0152] The inventors discovered that within this pH range, polysaccharides and some phenolic active ingredients can maintain a good dispersion stability.

[0153] The stirring speed is preferably controlled at 80–150 rpm to improve the uniformity of the system and reduce local aggregation.

[0154] Example 1 This embodiment provides a whitening composition containing fermented plant extracts. The whitening composition comprises the following components by weight percentage: Phase A: Plant fermentation products 40.00%; microalgae-derived trehalose complex extract 3.00%; 1,3-propanediol 5.00%; glycerol 3.00%; β-glucan 0.20%; sodium hyaluronate (multi-molecular weight compound) 0.15%; xanthan gum 0.15%.

[0155] Phase B: Nicotinamide 4.00%; Panthenol 0.50%; Disodium EDTA 0.05%.

[0156] Phase C: Hydrogenated lecithin 0.30%; polyglycerol-10 laurate 0.50%.

[0157] Phase D: 1,2-Hexanediol 0.80%; p-hydroxyacetophenone 0.30%; appropriate amount of citric acid / sodium citrate buffer system; deionized water to 100%.

[0158] Sodium hyaluronate (multi-molecular weight compound) includes: 1500 kDa (30%), 500 kDa (40%), and 30 kDa (30%).

[0159] The raw materials for preparing the plant fermentation product include: Component A: Scutellaria baicalensis extract, Saururus chinensis extract, and Nymphaea alba extract; Component B: Chrysanthemum indicum extract and Dictamnus dasycarpus root extract; Component C: Ginseng extract and white lily extract.

[0160] The mass ratio of component A, component B and component C is 9:5:1.

[0161] (a) Preparation of component A Scutellaria baicalensis, Saururus chinensis, and Nymphoides pubescens were dried separately and then pulverized to 60 mesh. They were then mixed at a mass ratio of 3:3:1. A 60% (v / v) ethanol aqueous solution was added, maintaining a material-to-liquid ratio of 1:10. Extraction was carried out at 60℃ with stirring for 2 hours.

[0162] After extraction, the solution was filtered and concentrated under reduced pressure at 45°C to a solid content of 80%. The resulting extract was designated as component A.

[0163] (II) Preparation of Component B The raw materials of white chrysanthemum and Dictamnus dasycarpus were dried separately and then pulverized to 60 mesh. They were mixed at a mass ratio of 1:1. A 40% (v / v) ethanol aqueous solution was added, with a material-to-liquid ratio of 1:12. Extraction was carried out at 40℃ for 1.5 hours. After extraction, the mixture was filtered and concentrated under reduced pressure to obtain component B, with a solid content of 80%.

[0164] (III) Preparation of component C Ginseng and white lily raw materials were mixed at a mass ratio of 1:1 and then pulverized to 60 mesh. Deionized water was added, maintaining a material-to-liquid ratio of 1:12. Subsequently, 0.2 wt% cellulase and 0.1 wt% pectinase were added. Enzymatic hydrolysis was carried out at 45℃ for 1.5 hours. After enzymatic hydrolysis, the temperature was raised to 90℃ and held for 10 minutes to inactivate the enzymes. Extraction was then carried out at 60℃ for 2 hours. The mixture was filtered and concentrated under reduced pressure to obtain component C, with a solid content of 80%.

[0165] Preparation of plant fermentation products (iv) First stage fermentation Component A was added to deionized water to adjust the solid content of the system to 10 wt%. The pH was adjusted to 5.5 using a citric acid buffer system. Lactobacillus plantarum was then inoculated at a rate of 3 wt% of the total system. The first stage of fermentation was carried out at 32°C for 36 hours. During fermentation, the stirring speed was controlled at 100 rpm, and microaerophilic conditions were maintained.

[0166] (v) Second stage fermentation After the first stage of fermentation, component B was added to the system. Then, 1.0 wt% glucose (based on the total system weight) was added, followed by inoculation with 2 wt% Saccharomyces cerevisiae (based on the total system weight). Fermentation continued at 30°C for 24 hours.

[0167] During the second stage of fermentation, the pH of the system was maintained at 5.2–5.5.

[0168] (vi) Third stage fermentation After the second stage of fermentation is completed, component C is added to the system. This includes: 5.0 wt% of polysaccharides from Bletilla striata (based on the entire system); and 0.8 wt% of polysaccharides from brown algae (based on the entire system).

[0169] The fermentation temperature for the third stage was controlled at 30℃. The fermentation time was 24 hours. The pH of the system was controlled at 5.2. The stirring speed was controlled at 100 rpm.

[0170] (vii) Termination of fermentation After fermentation, the system was heated to 80°C and maintained for 20 minutes. Then, it was centrifuged at 8000 rpm for 15 minutes. The bacterial cells and insoluble substances were removed, and the supernatant was collected as the plant fermentation product.

[0171] The preparation method of the whitening composition is as follows: S10 First, add xanthan gum to glycerol and part of 1,3-propanediol, and stir to form a xanthan gum pre-dispersion without obvious clumping.

[0172] β-glucan and sodium hyaluronate were added to the remaining 1,3-propanediol for pre-dispersion to obtain an active polysaccharide pre-dispersion.

[0173] Add some deionized water to the main ingredient tank, control the temperature at 35℃, and add xanthan gum predispersant and active polysaccharide predispersant in sequence while stirring at 400 rpm. Continue stirring for 20 minutes to fully hydrate xanthan gum, β-glucan and sodium hyaluronate.

[0174] Lower the system temperature to no higher than 35°C, add the plant fermentation product and the microalgae-derived trehalose complex extract in sequence, and continue stirring for 15 minutes to obtain phase A.

[0175] S11 Take an appropriate amount of deionized water, control the temperature at 35-40℃, and add disodium EDTA, nicotinamide and panthenol in sequence. Stir until all components are completely dissolved to obtain a clear or homogeneous phase B.

[0176] Slowly add phase B to phase A, keeping the system temperature below 40°C and the stirring speed at 400 rpm during the addition process; continue stirring for 15 minutes after the addition is complete.

[0177] S12 Hydrogenated lecithin is mixed with polyglycerol-10 laurate and stirred at 55-65°C until the hydrogenated lecithin is uniformly dispersed to obtain phase C.

[0178] Under continuous stirring, phase C was slowly added to the mixture formed by phases A and B. During the addition process, the temperature of the mixing system was controlled at 40°C and the stirring speed was 500 rpm. After the addition was completed, stirring was continued for 15 minutes to ensure that the hydrogenated lecithin and polyglycerol-10 laurate were evenly dispersed.

[0179] The mixture was then cooled to no higher than 35°C.

[0180] S13 Add p-hydroxyacetophenone to 1,2-hexanediol and stir at 50°C until dissolved to form a premixed solution of preservative auxiliary components; cool the premixed solution to no higher than 35°C and add it to the main ingredient tank, and continue stirring for 10 minutes.

[0181] The pH of the composition was adjusted to 5.5 using a buffer system consisting of citric acid and sodium citrate.

[0182] Finally, deionized water was added to bring the volume to 100%, and the mixture was stirred at 400 rpm for 20–30 minutes. Vacuum degassing was then performed for 10 minutes at a vacuum degree of -0.09 MPa, followed by filtration through a 100 μm pore size filter to obtain the whitening composition containing fermented plant extracts.

[0183] Example 2 This embodiment provides a whitening composition containing fermented plant extracts.

[0184] Compared with Example 1, this example reduces the amount of polysaccharide components added during the third stage of fermentation and reduces the proportion of trehalose complex extract from microalgae.

[0185] In this embodiment, the whitening composition comprises the following components by weight percentage: Phase A: Plant fermentation products 35.00%; microalgae-derived trehalose complex extract 2.00%; 1,3-propanediol 5.00%; glycerol 3.50%; β-glucan 0.20%; sodium hyaluronate (multi-molecular weight compound) 0.12%; xanthan gum 0.12%.

[0186] Phase B: Nicotinamide 3.00%; Panthenol 0.50%; Disodium EDTA 0.05%.

[0187] Phase C: Hydrogenated lecithin 0.25%; Polyglycerol-10 laurate 0.40%.

[0188] Phase D: 1,2-Hexanediol 0.80%; p-hydroxyacetophenone 0.30%; appropriate amount of citric acid / sodium citrate buffer system; deionized water to 100%.

[0189] in: The extraction methods for components A, B, and C are the same as in Example 1.

[0190] The conditions for the first and second stages of fermentation are the same as in Example 1.

[0191] During the third stage of fermentation: The amount of polysaccharide from Bletilla striata was adjusted to 3.0 wt%; the amount of polysaccharide from brown algae was adjusted to 0.5 wt%; and the fermentation time for the third stage was adjusted to 18 hours.

[0192] In addition, the stress conditions for the microalgae-derived trehalose complex extract in this embodiment were adjusted as follows: The final NaCl concentration was adjusted to 1.5 wt%; the stress time was adjusted to 18 hours. The trehalose content in the obtained trehalose complex extract was approximately 35 wt%.

[0193] The remaining steps are the same as in Example 1.

[0194] Example 3 This embodiment provides a whitening composition containing fermented plant extracts and its preparation method.

[0195] In this embodiment, the whitening composition comprises the following components by weight percentage: Phase A: Plant fermentation products 50.00%; microalgae-derived trehalose complex extract 5.00%; 1,3-propanediol 4.00%; glycerol 2.50%; β-glucan 0.30%; sodium hyaluronate (multi-molecular weight compound) 0.20%; xanthan gum 0.18%.

[0196] Phase B: Nicotinamide 5.00%; Panthenol 0.50%; Disodium EDTA 0.05%.

[0197] Phase C: Hydrogenated lecithin 0.40%; Polyglycerol-10 laurate 0.60%.

[0198] Phase D: 1,2-Hexanediol 0.80%; p-hydroxyacetophenone 0.30%; appropriate amount of citric acid / sodium citrate buffer system; deionized water to 100%.

[0199] in: The fermentation conditions for the first and second stages are the same as in Example 1.

[0200] During the third stage of fermentation: the amount of polysaccharides from Bletilla striata was increased to 6.0 wt%; the amount of polysaccharides from brown algae was increased to 1.2 wt%; and the fermentation time for the third stage was extended to 30 hours.

[0201] In addition, the stress conditions for the microalgae-derived trehalose complex extract in this embodiment were adjusted as follows: The final NaCl concentration was increased to 2.5 wt%; the stress time was increased to 30 hours. The obtained trehalose complex extract contained approximately 55 wt% trehalose.

[0202] The remaining steps are the same as in Example 1.

[0203] Comparative Example 1 In this comparative example, the other components are the same as in Example 1.

[0204] The only difference is that this comparative example does not involve staged processing of the plant extracts. Specifically, the plant materials corresponding to components A, B, and C are extracted uniformly and then directly mixed.

[0205] Specifically, all plant extracts were mixed, and deionized water was added to adjust the solid content to 10% and the pH to 5.5. Simultaneously, 3 wt% *Lactobacillus plantarum* and 2 wt% *Saccharomyces cerevisiae* were inoculated, and fermentation was carried out continuously at 30°C for 48 hours. After fermentation, the mixture was heated to 80°C and held for 20 minutes to inactivate the bacteria. The mixture was then centrifuged (8000 rpm, 15 min), and the supernatant was collected as the plant fermentation product.

[0206] After fermentation, the mixture was inactivated and centrifuged. The resulting supernatant was used as the plant fermentation product.

[0207] Comparative Example 2 In this comparative example, all other components and fermentation conditions are the same as in Example 1.

[0208] The only difference is that no polysaccharides from Bletilla striata or brown algae are added during the third stage of fermentation.

[0209] The remaining steps are the same as in Example 1.

[0210] Comparative Example 3 In this comparative example, the other components are the same as in Example 1.

[0211] The only difference is that no microalgae-derived trehalose complex extract is added to the system.

[0212] The remaining steps are the same as in Example 1.

[0213] Comparative Example 4 In this comparative example, the other components are the same as in Example 1.

[0214] The only difference is that brewer's yeast is no longer inoculated during the second and third stages of fermentation. The entire fermentation process uses only Lactobacillus plantarum for continuous fermentation.

[0215] The preparation of plant fermentation products involves using only a single strain of microorganisms in the staged fermentation process.

[0216] The first stage of fermentation was the same as in Example 1: Lactobacillus plantarum 3wt%, 32℃ × 36 h.

[0217] During the second and third stages of fermentation, yeast is no longer inoculated; fermentation continues using only *Lactobacillus plantarum*. Temperature: 30℃; Time: 24 hours for the second stage and 24 hours for the third stage. All other conditions are the same as in Example 1.

[0218] The remaining steps are the same as in Example 1.

[0219] Comparative Example 5 In this comparative example, the other components are the same as in Example 1.

[0220] The only difference is that no high-salt stress treatment is performed during the microalgae cultivation process. Preparation of the microalgae-derived trehalose complex extract (difference from Example 1): After the microalgae are cultured to the logarithmic growth phase, no salt stress treatment is performed.

[0221] Directly perform: centrifugation (4000 rpm, 12 min), washing, and ultrasonic disruption (300 W, 15 min).

[0222] Subsequently, extraction was performed at a material-to-liquid ratio of 1:10 at 40°C for 1.5 hours, followed by centrifugation to collect the supernatant. The supernatant was then purified using a 5 kDa ultrafiltration membrane, with ethanol added to bring the concentration to 40%. The final product was a trehalose extract with a trehalose content of approximately 15–20%.

[0223] The remaining steps are the same as in Example 1.

[0224] Comparative Example 6 In this comparative example, the other components are the same as in Example 1. The only difference is that the multi-molecular weight sodium hyaluronate compound system uses only 1500 kDa sodium hyaluronate.

[0225] The remaining steps are the same as in Example 1.

[0226] Comparative Example 7 In this comparative example, the other components are the same as in Example 1.

[0227] The only difference is that the microalgae extract is not subjected to ultrafiltration separation. The remaining steps are the same as in Example 1.

[0228] Comparative Example 8 In this comparative example, the other components are the same as in Example 1.

[0229] The only difference is: Polysaccharides derived from Bletilla striata and brown algae do not participate in the third stage of fermentation. Instead, they are added directly after all fermentation is complete.

[0230] The remaining steps are the same as in Example 1.

[0231] Comparative Example 9 In this comparative example, the other components are the same as in Example 1.

[0232] The only difference is that nicotinamide is added directly to the fermentation system before the first stage of fermentation begins. All fermentation steps are then carried out.

[0233] Preparation of plant fermentation products (differences from Example 1) Nicotinamide (4.00%) was added directly to the fermentation system before the start of the first stage of fermentation.

[0234] Then, fermentation was carried out in stages as in Example 1: Stage 1: 3 wt% Lactobacillus plantarum, 32℃, 36 hours; Stage 2: 2 wt% yeast, 30℃, 24 hours; Stage 3: Polysaccharide system added, 30℃, 24 hours; After fermentation, inactivation and centrifugation are performed. No additional nicotinamide is added during subsequent component preparation.

[0235] The remaining steps are the same as in Example 1.

[0236] Comparative Example 10 In this comparative example, the other components are the same as in Example 1.

[0237] The only difference is that all the ingredients—Scutellaria baicalensis, Saururus chinensis, Nymphaea alba, Chrysanthemum indicum, Dictamnus dasycarpus, ginseng, and Lilium brownii—are mixed together and extracted in a single step. Specifically, the raw materials are pulverized to 60 mesh, and a 50% (by volume) ethanol solution is added to the total mixture at a ratio of 1:10. Extraction is carried out at 60°C for 2 hours, followed by filtration and vacuum concentration to obtain the mixed plant extract. Subsequent fermentation steps are then performed.

[0238] The remaining steps are the same as in Example 1.

[0239] Performance testing I. Flavonoid Glycoside Conversion Rate Test 1. Test Objective This test was used to evaluate the effect of the first stage of lactic acid bacteria fermentation on the conversion of flavonoid glycosides.

[0240] In this invention, extracts of Scutellaria baicalensis, Saururus chinensis, and Nymphoides pubescens are preferentially subjected to lactic acid bacteria fermentation. The purpose is to utilize β-glucosidase produced during lactic acid bacteria metabolism to promote the deglycosylation conversion of some flavonoid glycosides. Baicalin and baicalein are used as representative detection indicators, where baicalin represents glycosidic flavonoids and baicalein represents aglycone flavonoids.

[0241] 2. Sample processing Take 1.0 g of the plant fermentation products obtained in Examples 1-3 and Comparative Examples 1, 4, and 10, respectively, add methanol to a final volume of 10 mL, extract ultrasonically for 20 min, and centrifuge at 8000 rpm for 10 min. Filter the supernatant through a 0.22 μm organic filter membrane as the test solution. Separately, take the extract of component A before fermentation as the initial control sample and process it using the same method.

[0242] 3. HPLC detection conditions High performance liquid chromatography (HPLC) was used for detection.

[0243] Chromatographic column: C18 reversed-phase column, 250 mm × 4.6 mm, 5 μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; gradient elution program: 0–10 min, 20% B; 10–30 min, 20%–45% B; 30–35 min, 45% B; flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 280 nm; injection volume: 10 μL.

[0244] 4. Calculation Formula The conversion rate of baicalin is calculated using the following formula: Baicalin conversion rate (%) = (C0 - Ct) / C0 × 100% Where: C0 is the baicalin content in the sample before fermentation; Ct is the baicalin content in the sample after fermentation.

[0245] The increase rate of baicalin is calculated using the following formula: Baicalein increase rate (%) = (Bt - B0) / B0 × 100% Where: B0 is the baicalin content in the sample before fermentation; Bt is the baicalin content in the sample after fermentation.

[0246] II. Test for Free Residue Rate of Sesquiterpene Lactones 1. Test Objective This test was used to evaluate the effect of the second-stage yeast fermentation on the free exposure of highly reactive sesquiterpene lactones in chrysanthemum.

[0247] Parthenolide, a component of Chrysanthemum indicum, was used as a representative detection indicator. Parthenolide belongs to the sesquiterpene lactone class of compounds and has high reactivity; in its free state, it may increase the risk of system irritation.

[0248] 2. Sample processing Take 2.0 mL of the plant fermentation products obtained in Examples 1-3 and Comparative Examples 1, 4, and 10, respectively, add 5.0 mL of ethyl acetate, and extract by shaking for 10 min. Centrifuge at 6000 rpm for 10 min. Take the upper organic phase, dry it under nitrogen, and redissolve it in 1.0 mL of methanol. Filter through a 0.22 μm filter membrane to obtain the test solution.

[0249] 3. HPLC detection conditions Chromatographic column: C18 reversed-phase column, 250 mm × 4.6 mm, 5 μm; mobile phase: acetonitrile / water = 55 / 45 v / v; flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 210 nm; injection volume: 10 μL.

[0250] 4. Calculation Formula The free residual rate of parthenolide was calculated using the following formula: Free residue rate (%) = Ct / C0 × 100% Where: C0 is the content of parthenolide before component B is added to the fermentation system; Ct is the content of free parthenolide in the sample after fermentation.

[0251] III. Intermolecular Interaction Tests of Polysaccharides and Active Substances (FTIR) 1. Test Objective This test is used to evaluate whether there are intermolecular interactions between polysaccharides and phenolic and flavonoid active ingredients after polysaccharides participate in fermentation.

[0252] 2. Sample processing The plant fermentation products obtained in Examples 1-3, Comparative Example 2 and Comparative Example 8 were taken respectively.

[0253] The sample was freeze-dried in a freeze dryer at -50℃ for 24 h to obtain a lyophilized powder. Approximately 2 mg of the lyophilized powder was taken and thoroughly ground and mixed with approximately 200 mg of dried KBr, then compressed into tablets for later use.

[0254] 3. FTIR test conditions Scanning range: 4000–400 cm -1 Resolution: 4 cm -1 Number of scans: 32.

[0255] Test method: Transmission method.

[0256] 4. Result Determination Focus on the 3200–3400 cm range. -1 The regional hydroxyl stretching vibration peak, and the 1600–1700 cm⁻¹ peak. -1 Absorption peaks related to regional aromatic rings and carbonyl groups.

[0257] IV. Particle size and zeta potential testing 1. Test Objective This test is used to evaluate the dispersion stability of plant fermentation products in an aqueous system. If the active ingredients in the fermentation products are unevenly dispersed or locally aggregated, phenomena such as increased particle size, increased PDI, and storage turbidity are likely to occur.

[0258] 2. Sample processing Take the compositions obtained in Examples 1-3 and Comparative Examples 1-10, respectively, dilute them 50 times with deionized water, gently shake them evenly, and let them stand for 10 minutes. Take the supernatant for testing.

[0259] 3. Test Conditions Dynamic light scattering was used for testing. Instrument: Malvern Zetasizer; Test temperature: 25℃; Equilibrium time: 120 s; Each sample was tested in triplicate, and the average value was taken.

[0260] 4. Test Indicators The following indicators were measured: average particle size, nm; PDI (polydispersity index); and Zeta potential, mV.

[0261] V. Melanin Production Inhibition Experiment 1. Test Objective This test is used to evaluate the ability of the composition to inhibit melanin production.

[0262] In this invention, plant fermentation products reduce melanin production through flavonoid conversion, antioxidant regulation, and tyrosinase-related effects; nicotinamide is mainly used to regulate the melanin transport process.

[0263] 2. Cell Culture B16-F10 mouse melanoma cells were used. Cells were seeded in 96-well plates at a density of 1 × 10⁶ cells / well. 4 Cells / well. DMEM medium containing 10% fetal bovine serum was used, and the cells were cultured at 37°C and 5% CO2 for 24 h to allow the cells to adhere.

[0264] 3. Model building and sample processing The culture medium was discarded, and fresh culture medium containing 100 nM α-MSH was added to induce melanin production. Subsequently, samples from Examples 1-3 and Comparative Examples 1-10 were added to bring the final sample concentration to 0.1 wt%. Six parallel wells were set up for each group. A blank group, a model group, and a positive control group were also included; culture was continued for 48 h.

[0265] 4. Melanin extraction After culture, discard the culture medium. Wash cells twice with PBS. Add 1 mol / L NaOH solution containing 10% DMSO; incubate at 80°C for 30 min to fully dissolve the melanin in the cells.

[0266] 5. Testing conditions The absorbance was measured at 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0267] 6. Calculation Formula The melanin production inhibition rate is calculated using the following formula: Melanin production inhibition rate (%) = (Model group A - Sample group A) / (Model group A - Blank group A) × 100% in: Model group A is the absorbance of the group induced by α-MSH without sample addition; Sample group A is the absorbance of the group treated with sample addition; Blank group A is the absorbance of the uninduced group.

[0268] VI. Tyrosinase Activity Inhibition Experiment 1. Test Objective This test was used to evaluate the inhibitory effect of the composition on tyrosinase activity. Increased flavonoid aglycone content leads to increased exposure of phenolic hydroxyl groups, which generally enhances the interaction with the active site of tyrosinase.

[0269] 2. Reagent preparation PBS buffer: pH 6.8; L-tyrosine solution: 2 mmol / L; tyrosinase solution: 200 U / mL; sample solution: 0.1 wt%.

[0270] 3. Reaction System Add the following to a 96-well plate in sequence: 100 μL PBS buffer; 50 μL L-tyrosine solution; and 50 μL sample solution. Pre-incubate at 37°C for 10 min. Then add 50 μL tyrosinase solution and continue the reaction for 30 min.

[0271] 4. Testing conditions The absorbance was measured at 475 nm.

[0272] 5. Calculation Formula The tyrosinase inhibition rate is calculated using the following formula: Tyrosinase inhibition rate (%) = (1 - Sample A / Control A) × 100% in: Control A represents the absorbance without the addition of the sample; Sample A represents the absorbance after the addition of the sample.

[0273] VII. Antioxidant Capacity Test (DPPH Method) 1. Test Objective This test is used to evaluate the composition's ability to scavenge free radicals.

[0274] In this invention, the antioxidant capacity is mainly related to the content of flavonoid aglycones, the stable state of polyphenols, and the stabilizing effect of the microalgae-derived trehalose complex extract.

[0275] 2. Reagent preparation Prepare a 0.1 mmol / L DPPH ethanol solution and store it protected from light.

[0276] The samples from Examples 1-3 and Comparative Examples 1-10 were diluted with deionized water to 0.10 wt% to obtain the sample solutions.

[0277] 3. Reaction steps Take 1.0 mL of the sample solution and mix it with 1.0 mL of DPPH ethanol solution. Incubate in the dark for 30 min at a controlled temperature of 25℃. A blank sample group and a DPPH control group were also set up.

[0278] 4. Testing conditions The absorbance was measured at 517 nm.

[0279] 5. Calculation Formula The DPPH free radical scavenging rate is calculated using the following formula: Clearance rate (%) = [1 - (Sample A - Blank A) / Control A] × 100% in: Sample A is the absorbance of the sample mixed with DPPH; Blank A is the background absorbance of the sample; Control A is the absorbance of the DPPH solution.

[0280] 8. Oxidative Stability Test 1. Test Objective This test is used to evaluate the color stability and activity retention of the composition under accelerated oxidation conditions.

[0281] This test is mainly used to verify the role of polysaccharides in fermentation and the inhibitory effect of microalgae-derived trehalose complex extract on the oxidative inactivation of phenolic and flavonoid components.

[0282] 2. Test Conditions Take samples from Examples 1-3 and Comparative Examples 1-10 respectively, place them in transparent glass bottles, and seal them.

[0283] The following storage conditions were set: Condition A: Store at 45℃ in the dark for 14 days; Condition B: Irradiate with 365 nm ultraviolet light at 25℃ for 8 hours a day for 14 consecutive days.

[0284] 3. Detection Indicators Before and after the test, the following were measured: appearance; color change; flavonoid retention rate; and DPPH scavenging rate.

[0285] 4. Color difference detection The L, a, and b values ​​were measured using a colorimeter.

[0286] The color difference ΔE is calculated using the following formula: ΔE = [(L-L0)] 2 +(a-a0) 2 +(b-b0) 2 ] 1 / 2 in: L0, a0, and b0 are the color values ​​of the sample before storage; L, a, and b are the color values ​​of the sample after storage.

[0287] 5. Calculation of flavonoid retention rate The total flavonoid retention rate is calculated using the following formula: Total flavonoid retention rate (%) = Ct / C0 × 100% Where: C0 is the total flavonoid content before storage; Ct is the total flavonoid content after storage.

[0288] IX. Long-term storage stability test 1. Test Objective This test is used to evaluate the appearance, pH, viscosity, and activity retention of the composition under different storage conditions.

[0289] 2. Storage conditions Samples from Examples 1-3 and Comparative Examples 1-10 were placed in sealed containers under the following conditions: Store at 4℃; store at 25℃; store at 40℃; storage time is 90 days.

[0290] 3. Detection time point 0 d, 30 d, 60 d, 90 d.

[0291] 4. Detection Indicators The following items were tested: appearance (whether there was layering, precipitation, or turbidity); pH value; viscosity; baicalin retention rate; nicotinamide retention rate; and total flavonoid retention rate.

[0292] 5. Formula for Activity Retention Rate Activity retention rate (%) = Ct / C0 × 100% Where: C0 represents the content of active ingredients before storage; Ct represents the content of active ingredients after storage.

[0293] 10. Centrifugal stability test 1. Test Objective Used to evaluate the phase stability of a composition under external force.

[0294] 2. Testing Methods Place 50 mL of sample into a centrifuge tube; centrifuge at 4000 rpm for 30 min. After centrifugation, observe whether the sample shows stratification, precipitation, flocculation, or turbidity.

[0295] 3. Judgment Criteria No stratification, no sedimentation, and no obvious turbidity are recorded as stable; slight sedimentation or slight turbidity are recorded as slightly unstable; obvious stratification or sedimentation are recorded as unstable.

[0296] XI. Freeze-thaw stability test 1. Test Objective Used to evaluate the stability of a composition under temperature cycling conditions.

[0297] 2. Testing Methods Take 50 mL of sample and place it in a sealed container. Perform freeze-thaw cycles under the following conditions: -5℃ for 12 h; 40℃ for 12 h; the above process constitutes one cycle, and a total of 5 cycles are performed.

[0298] 3. Detection Indicators Observe the appearance, layering, precipitation, color changes, and viscosity changes of the sample.

[0299] 12. pH stability test 1. Test Objective Used to evaluate the stability of the composition under different pH conditions.

[0300] 2. Sample processing Using a citrate / disodium hydrogen phosphate buffer system, the samples were adjusted to pH 4.5, pH 5.5, pH 6.5, and pH 7.0, respectively.

[0301] 3. Storage conditions The conditioned sample was stored at 40°C for 30 days.

[0302] 4. Detection Indicators Before and after storage: appearance; pH change; viscosity change; presence or absence of precipitation; total flavonoid retention rate.

[0303] XIII. Viscosity and Rheological Properties Testing 1. Test Objective Used to evaluate the rheological stability of a composition system.

[0304] 2. Test Conditions Instrument: Rotational rheometer; Temperature: 25℃; Shear rate range: 0.1~100 s⁻¹ -1 Sample volume: approximately 10 mL.

[0305] 3. Testing Procedures Place the sample on the test platform and allow it to equilibrate for 2 minutes. Gradually increase the shear rate and record the viscosity change. Test the sample before storage and the sample after storage at 40℃ for 30 days.

[0306] 4. Formula for the rate of change of viscosity Viscosity change rate (%) = (ηt - η0) / η0 × 100% Where: η0 is the viscosity before storage; ηt is the viscosity after storage.

[0307] XIV. Moisturizing Performance Test 1. Test Objective Used to evaluate the in vitro moisturizing ability of the composition.

[0308] 2. Testing Procedures Take a dry filter paper and weigh its initial mass W0. Evenly coat 0.5 g of the sample onto the filter paper surface and weigh it, recording the weight as W1. Place the sample in a constant temperature and humidity environment of 25℃ and 50% RH for 24 h.

[0309] Weigh the baby 24 hours later and record the weight as W2.

[0310] 3. Calculation Formula Moisturizing rate (%) = [1 - (W1 - W2) / (W1 - W0)] × 100% Where: W0 is the initial mass of the filter paper; W1 is the mass after coating the sample; W2 is the mass after standing for 24 hours.

[0311] Table 1. Flavonoid glycoside conversion rate test

[0312] Table 2 Results of test on free residue of sesquiterpene lactones

[0313] Table 3 Results of intermolecular interactions between polysaccharides and active ingredients

[0314] Table 4. Results of particle size and zeta potential tests

[0315] As shown in Table 4, the fermentation compositions obtained in the embodiments of the present invention have an average particle size of only 116.8–156.2 nm, a particle size distribution index (PDI) of 0.148–0.211, and an absolute Zeta potential of 28.6–35.1 mV, which are significantly better than those of the comparative examples. The smaller and more uniform particle size indicates more complete dispersion of the system, the lower PDI indicates a more uniform particle size distribution, and the larger absolute Zeta potential indicates better electrostatic stability of the system, making it less prone to aggregation, sedimentation, or stratification. This demonstrates that the staged fermentation of plants, the participation of polysaccharides in fermentation, and the combined effect of the microalgae-derived trehalose complex extract improve the dispersion of plant active components in the aqueous system, resulting in a more stable colloidal structure in the obtained compositions, thus ensuring subsequent storage stability and efficacy stability.

[0316] Table 5. Results of Whitening and Antioxidant Performance Tests

[0317] As shown in Table 5, the embodiments of the present invention are significantly superior to the comparative examples in terms of melanin production inhibition rate, tyrosinase inhibition rate, and DPPH free radical scavenging rate. Example 3, in particular, achieved 74.6%, 71.2%, and 85.8% respectively, demonstrating the best overall performance. This indicates that the present invention improves the bioactivity of flavonoid active ingredients by promoting the conversion of flavonoid glycosides to flavonoid aglycones through lactic acid bacteria in the first stage; reduces the adverse effects of highly reactive active ingredients through yeast fermentation in the second stage; and further enhances the stability of active substances through polysaccharide fermentation in the third stage. Combined with the trehalose complex extract from microalgae, it enhances the antioxidant capacity of the system. Therefore, it can simultaneously enhance the regulation of melanin production, tyrosinase inhibition, and free radical scavenging capabilities, thereby significantly improving the overall whitening effect of the composition.

[0318] Table 6 Stability Test Results

[0319] As shown in Table 6, the embodiments of the present invention exhibit excellent performance in various tests, including oxidative stability, long-term storage stability, centrifugal stability, freeze-thaw stability, and pH stability. Overall color difference changes are minimal, and the retention rates of total flavonoids, baicalin, and nicotinamide remain at high levels. Viscosity changes are low, and no stratification, precipitation, or significant appearance changes occur after long-term storage, centrifugation, and multiple freeze-thaw cycles. In contrast, the comparative examples generally show decreased retention of active ingredients, increased color changes, precipitation, stratification, or decreased system stability. These results demonstrate that the present invention, through staged fermentation, polysaccharide-involved fermentation, and the synergistic effect of microalgae-derived trehalose complex extract, can effectively inhibit the oxidative degradation of active ingredients, improve the physical and chemical stability of the entire composition, and thus ensure the stable quality of the product after long-term storage.

[0320] Table 7. Viscosity and Rheological Properties Test Results

[0321] As shown in Table 7, the initial viscosity of the embodiments of the present invention is relatively high, and the viscosity change rate after 30 days of storage is only 2.6% to 5.8%, which is much lower than that of the comparative examples. Simultaneously, the shear thinning index is low, indicating that the system has more stable and suitable rheological properties under external forces. The low viscosity change rate suggests that the product is less prone to network structure damage during storage, while the better shear thinning properties facilitate uniform spreading of the product during use, improving skin feel and application uniformity. These results further demonstrate that the fermented plant active system obtained by the present invention can form a stable dispersion structure, ensuring good performance while improving the rheological stability of the product during long-term storage.

[0322] Table 8. Results of Moisturizing Performance Tests

[0323] As shown in Table 8, the 24-hour moisturizing rate of the present invention in Examples 1-2 of this invention reached 78.5%–87.3%, significantly higher than that of the comparative examples, with Example 3 reaching 87.3%, indicating that the composition of the present invention has excellent long-lasting moisturizing ability. Analysis suggests that, on the one hand, the polysaccharides participate in fermentation to form a stable hydration structure, improving the system's water-holding capacity; on the other hand, the trehalose complex extract from microalgae, in addition to providing trehalose, also retains low-molecular-weight polysaccharides and microalgae metabolites, which can further reduce water loss and improve the water-retention performance of the stratum corneum. Simultaneously, the small-molecule active ingredients produced by staged fermentation are more easily and evenly dispersed in the system, forming a synergistic effect with the moisturizing components. Therefore, it can achieve whitening effects while also maintaining skin moisturizing performance, resulting in a more superior comprehensive skincare effect.

[0324] As shown in Tables 1 to 8, the embodiments of the present invention exhibit superior overall performance compared to the comparative examples in terms of flavonoid glycoside conversion rate, free residual rate of sesquiterpene lactones, intermolecular interaction between polysaccharides and active substances, dispersion stability, melanin production inhibition ability, tyrosinase activity inhibition ability, antioxidant capacity, and oxidative stability. The phased fermentation of different plant extracts significantly improves the biotransformation efficiency of flavonoid active ingredients and reduces the free exposure of highly reactive sesquiterpene lactones. The participation of polysaccharides in the fermentation process enhances the intermolecular interactions between polysaccharides and phenolic and flavonoid active ingredients, improving the dispersion and storage stability of the fermentation products in an aqueous system. Simultaneously, the microalgae-derived trehalose complex extract further improves the antioxidant stability and activity retention of the active ingredients, resulting in a more superior overall whitening performance in terms of melanin production inhibition, tyrosinase activity regulation, and free radical scavenging. This demonstrates that the phased fermentation of plant extracts, the participation of polysaccharides in fermentation, and the synergistic application of the microalgae-derived trehalose complex extract employed in this invention have a good synergistic effect, comprehensively improving the stability, safety, and whitening efficacy of the composition.

[0325] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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; and these 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 the present invention.

Claims

1. A whitening composition containing fermented plant extracts, characterized in that, The composition comprises: (1) Plant fermentation products; (2) Nicotinamide; (3) Trehalose complex extract from microalgae; The method for preparing the plant fermentation product includes the following steps: (1) The plant extract was divided into the following components: Component A: Contains Scutellaria baicalensis extract, Saururus chinensis extract and Nymphaea alba extract; Component B: Contains chrysanthemum extract and dictamnus root extract; Component C: Contains ginseng extract and white lily extract; (2) Component A undergoes the first stage of lactic acid bacteria fermentation; (3) Add component B to the first stage fermentation system and carry out the second stage yeast fermentation; (4) Add component C and polysaccharide components to the second-stage fermentation system and carry out the third-stage fermentation; (5) The fermentation system is inactivated and separated to obtain plant fermentation products; (6) Add nicotinamide and microalgae-derived trehalose complex extract to the obtained plant fermentation product to obtain the whitening composition; The polysaccharide components include polysaccharides derived from Bletilla striata and / or polysaccharides derived from seaweed; The microalgae-derived trehalose complex extract is derived from a microalgae system cultured under stress-induced conditions; wherein, the stress-induced conditions include one or more of high-salt stress, osmotic stress, and temperature stress.

2. The whitening composition according to claim 1, characterized in that: The microalgae-derived trehalose complex extract was selectively separated using an ultrafiltration membrane with a molecular weight cutoff of 3–10 kDa.

3. The whitening composition according to claim 1, characterized in that: The polysaccharide derived from Bletilla striata was prepared by enzyme-assisted extraction and alcohol precipitation.

4. The whitening composition according to claim 1, characterized in that: The first stage of fermentation takes 24–48 hours; the second stage takes 12–36 hours; and the third stage takes 12–36 hours.

5. The whitening composition according to claim 1, characterized in that: The first stage of lactic acid bacteria includes Lactobacillus plantarum; the second stage of yeast includes Saccharomyces cerevisiae.

6. The use of the whitening composition according to any one of claims 1 to 5 in the preparation of products for regulating skin pigmentation, reducing melanin production, improving dull skin, improving skin oxidation state, and enhancing skin barrier stability.

Images