Camellia oleifera seed fermented oil with multiple functions and preparation method and application thereof

Abstract

The application relates to the cosmetic technical field, and particularly discloses a Camellia nitidissima seed fermentation oil with multiple effects and a preparation method and application thereof. The preparation method of the Camellia nitidissima seed fermentation oil comprises the following steps: solid-state fermentation is carried out on Camellia nitidissima leaf blades by using Camellia nitidissima spore liquid, and the fermented Camellia nitidissima leaf blades are crushed into Camellia nitidissima leaf powder; the Camellia nitidissima seed oil and the Camellia nitidissima spore liquid are inoculated in a liquid culture medium, and a product is obtained by jointly carrying out liquid fermentation on the Camellia nitidissima spore liquid, Candida sp. and Lactobacillus plantarum bacterial liquid and the Camellia nitidissima leaf powder; the product is subjected to high-speed centrifugation, the supernatant oil phase is taken, and sterilization is carried out through a microporous filter membrane to obtain the Camellia nitidissima seed fermentation oil. The Camellia nitidissima seed fermentation oil has a fragrant smell, is not prone to deterioration and oxidation, has a better shelf life and stability, and has a better repair and anti-aging effect.

Description

Technical Field

[0001] This application relates to the field of cosmetic technology, and in particular to a fermented Camellia chrysantha seed oil with multiple effects, its preparation method and application. Background Technology

[0002] Golden camellia is rich in tea polyphenols, flavonoids, and saponins. Its seed oil is particularly high in unsaturated fatty acids, squalene, and vitamin E, giving it excellent moisturizing, antioxidant, and anti-inflammatory properties. In the skincare industry, golden camellia seed oil, obtained through pressing, can be applied directly or added as a base oil, quickly penetrating the skin to replenish lipids and repair the skin barrier. It slows down skin aging by locking in moisture, scavenging free radicals, and repairing UV damage and inflammatory skin problems. During tea fermentation, *Aspergillus cristatus* secretes extracellular enzymes such as amylase and oxidase, catalyzing the conversion of polyphenols, proteins, and glycosides in tea leaves into flavor compounds such as theaflavins and β-linalool, significantly enhancing the richness and unique aroma of the tea. Through fermentation combined with tea, not only are its active ingredients optimized, but its health benefits, such as lowering blood sugar, anti-oxidation, and anti-inflammation, are also enhanced.

[0003] Currently, plant oils face multiple technical bottlenecks in cosmetic applications. Traditional plant oils, such as peony seed oil and camellia seed oil, have large molecular structures like triglycerides that make it difficult to penetrate the skin barrier, resulting in low absorption rates of active ingredients and a heavy feel on the skin. The active ingredients in plants, such as polysaccharides and flavonoids, are encased in the cell walls, making them difficult to release effectively using conventional cold pressing or extraction processes. Although microbial fermentation can improve the texture of oils and generate small molecule active substances, existing processes have significant drawbacks: (1) Single-strain fermentation has limited efficiency, mixed-strain synergistic effects are insufficient, and there is a lack of precise control over the fermentation process. For example, solid-state fermentation is easily affected by humidity and uneven oxygen distribution, while in liquid fermentation, the contact between microorganisms and oils is insufficient, resulting in low conversion rates. (2) High-temperature sterilization easily damages heat-sensitive components and reduces product stability. (3) The special odor of plant oils and their tendency to oxidize and emit a sour smell limit their use as raw materials in skincare products. Summary of the Invention

[0004] The purpose of this application is to overcome the shortcomings of the prior art and provide a multi-functional fermented oil of Camellia chrysantha seeds, its preparation method, and its applications. The fermented oil of Camellia chrysantha seeds of this application has an aromatic odor, is not easily deteriorated or oxidized, has a better shelf life and stability, and also possesses superior repair and anti-aging effects.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0006] This application provides a method for preparing fermented Camellia chrysantha seed oil with multiple benefits, comprising the following steps:

[0007] S1. Solid-state fermentation of golden flower tea leaves using golden flower fungus spore liquid, and then crushing the fermented golden flower tea leaves into golden flower tea powder.

[0008] S2. Inoculate the golden camellia seed oil and golden camellia spore liquid into a liquid culture medium, add the bacterial liquid prepared by Candida albicans and Lactobacillus plantarum, and golden camellia tea powder together for liquid fermentation. During the fermentation period of 0 to 24 hours, the oxygen aeration rate is 0.8 to 2 vvm. After fermentation for 24 to 72 hours, allow the fermentation to stand to obtain the product.

[0009] S3. The product obtained in step S2 is centrifuged at high speed, the supernatant oil phase is collected, and sterilized by microporous filter membrane to obtain Camellia chrysantha seed fermented oil.

[0010] This application is the first to use *Tea chrysanthemum* to ferment tea leaves and co-ferment them with *Tea chrysanthemum* seed oil. This application of the unique flavoring technology of *Tea chrysanthemum* in tea leaves to skin care ingredients can improve the oily odor of *Tea chrysanthemum* seed oil, give it a fragrant aroma, slow down its deterioration and oxidation time, extend its shelf life and stability, and expand its application in skin care ingredients.

[0011] This application utilizes a combination of solid-liquid two-stage fermentation and multi-strain time-controlled oxygen technology to enrich the active ingredients in fermented Camellia chrysantha seed oil. Solid-state pre-fermentation with *Mycobacterium chrysantha* decomposes the cell walls of Camellia chrysantha tea leaves, releasing tea polyphenols, various flavonoids, and flavonol precursors. The synergistic fermentation of *Lactobacillus plantarum*, *Candida albicans*, and *Mycobacterium chrysantha* catalyzes the hydrolysis of oil molecules in Camellia chrysantha seed oil, generating small-molecule free fatty acids, theaflavins, and catechins. This enhances transdermal absorption, resulting in fermented Camellia chrysantha seed oil with superior repair and anti-aging effects.

[0012] In some specific embodiments, *Eurotium cristatum* can be any identified strain preserved in a fungal culture center or obtained by an individual through gene identification after purification from tea bricks. The identification method involves extracting the colony gene and performing PCR amplification of the strain using primers ITS1 and ITS4, which are universally used for fungal identification. The measured ITS gene sequence is then compared with the BLAST sequence in the NCBI microbial database to confirm that the obtained strain is *Eurotium cristatum*.

[0013] In some specific embodiments, Camellia chrysantha seed oil can be obtained by pressing Camellia chrysantha seeds, supercritical extraction, steam distillation, or ethanol extraction.

[0014] In some specific embodiments, the Candida species include at least one of Candida cruzia, Candida utilis, and Candida tropicalis, with Candida utilis being the preferred Candida species.

[0015] This application uses the aforementioned type of Candida, which can better cooperate with Camellia chrysanthemi and Lactobacillus plantarum. The synergistic fermentation of the three strains better catalyzes the hydrolysis of oil molecules such as Camellia chrysanthemi seed oil, generating small molecule free fatty acids and theaflavins and catechins, improving transdermal absorption rate, and thus enhancing the repair and anti-aging effects of Camellia chrysanthemi seed fermented oil.

[0016] In a preferred embodiment of the method for preparing the multi-functional fermented oil from Camellia chrysantha seeds described in this application, in step S1, the revived Camellia chrysantha fungus is removed using a sterile inoculation loop and evenly dispersed in sterile water. The spore concentration is measured, and the concentration of the Camellia chrysantha fungus spore solution is adjusted to 10% using sterile water. 6 ~10 8 The spore volume was measured at 1 / mL. The spore liquid and golden flower tea leaves were then mixed evenly and placed in a sealed fermentation environment at a temperature of 26–32℃ and a humidity of 80–85% for 8–20 days (stirring every 4–5 days to prevent local overheating) for solid-state fermentation culture.

[0017] This application employs the above-mentioned solid-state fermentation culture steps, using *Camellia sinensis* for solid-state pre-fermentation to decompose the cell walls of Camellia sinensis tea leaves, releasing tea polyphenols, various flavonoids, and flavonol precursors, thereby enhancing the repair and anti-aging effects of Camellia sinensis seed fermented oil.

[0018] Preferably, in step S1, the spore liquid of *Gynostemma pentaphyllum* and the tea leaves of *Gynostemma pentaphyllum* are mixed evenly and placed in a sealed fermentation environment at a temperature of 28-30℃ and a humidity of 80-85% for 10-12 days (stirring once every 4-5 days to prevent local overheating) for solid-state fermentation culture.

[0019] Preferably, the revival step is as follows: the refrigerated *Aureobasidium aureum* strain is inoculated onto M40Y solid culture medium and cultured at 28°C for 7 days until the colonies are fully developed.

[0020] The M40Y culture medium formula is as follows: 20g of malt extract, 5g of yeast extract, 400g of sucrose, and 20g of agar are dissolved in 1L of water, and the pH is adjusted to 6.0.

[0021] Preferably, the golden flower tea leaves also include a pre-treatment step, which is to wash the fresh golden flower tea leaves and dry them at a low temperature of 60°C until the moisture content is 15%.

[0022] In a preferred embodiment of the method for preparing the multifunctional fermented oil from Camellia chrysantha seeds described in this application, in step S1, the ratio of Camellia chrysantha tea leaves to Camellia chrysantha spore liquid is 100g:(10-30ml).

[0023] Preferably, the ratio of the golden flower tea leaves to the golden flower fungus spore liquid is 100g:(20-30ml).

[0024] In the technical solution of this application, the golden chrysanthemum tea leaves and golden chrysanthemum spore liquid are in the above ratio. The golden chrysanthemum fungus can better decompose the cell wall of the golden chrysanthemum tea leaves through solid-state fermentation, and increase the release of tea polyphenols, various flavonoids, flavonol precursors and various trace elements in the golden chrysanthemum tea leaves and golden chrysanthemum seed oil, thereby improving the repair and anti-aging effects of the fermented golden chrysanthemum seed oil.

[0025] In a preferred embodiment of the method for preparing the multi-functional fermented Camellia chrysanthemi seed oil described in this application, in step S2, Candida albicans and Lactobacillus plantarum are respectively prepared with sterile water to a concentration of 10... 6 ~10 8 Candida albicans culture and Lactobacillus plantarum culture;

[0026] The ratio of the mixture of *Aureobasidium aureum* spore liquid, *Candida albicans* liquid, and *Lactobacillus plantarum* liquid to *Aureobasidium aureum* tea powder is (20-100g): (50-200g).

[0027] In the technical solution of this application, three strains of Camellia chrysanthemi, Lactobacillus plantarum, and Candida albicans are specifically used for synergistic fermentation in the above proportions. This can catalyze the hydrolysis of oil molecules in Camellia chrysanthemi seed oil, generating small molecule free fatty acids, theaflavins, and catechins, thereby improving transdermal absorption. The resulting fermented Camellia chrysanthemi seed oil has better repair and anti-aging effects.

[0028] Preferably, the ratio of the mixture of Lactobacillus plantarum liquid, Candida albicans liquid and Aureobasidium aureum spore liquid to Aureobasidium aureum tea powder is (40-60g): (80-130g).

[0029] Preferably, the ratio of the mixture of Lactobacillus plantarum liquid, Candida albicans liquid and Aureobasidium aureum spore liquid to Aureobasidium aureum tea powder is (40-60g):(100-110g).

[0030] The ratio of this mixture to Camellia chrysantha tea powder precisely balances the microbial community and substrate concentration, synergistically activating the complex enzyme system of Lactobacillus plantarum, Candida albicans, and Camellia chrysantha. This efficiently catalyzes the hydrolysis of Camellia chrysantha seed oil into small molecule free fatty acids, maximizing the conversion rate of flavonoid precursors in tea into active ingredients such as theaflavins and catechins, thereby significantly enhancing the various effects of the fermentation products.

[0031] Preferably, the mass ratio of the camellia seed oil to the camellia tea powder is 1 kg: (50-200 g).

[0032] This application involves fermenting Camellia chrysantha tea leaves with Mycorrhizal argentea and co-fermenting them with Camellia chrysantha seed oil. It was unexpectedly discovered that this method can improve the oily, characteristic odor of Camellia chrysantha seed oil, impart an aromatic scent to the fermented Camellia chrysantha seed oil, slow down its deterioration and oxidation time, extend its shelf life and stability, and expand the application of fermented Camellia chrysantha seed oil in skincare product ingredients.

[0033] Preferably, the preparation method further includes reviving Lactobacillus plantarum, inoculating Lactobacillus plantarum into MRS medium, and culturing at 37°C for 24 hours.

[0034] Preferably, the preparation method further includes reviving Candida albicans, inoculating Candida albicans into YM medium, and culturing at 28°C for 24 hours.

[0035] The formula for MRS medium is as follows: 10-15 g / L peptone, 5-10 g / L beef extract, 4-5 g / L yeast extract, 20-25 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.2-0.58 g / L magnesium sulfate, 0.05-0.25 g / L manganese sulfate, 10-20 g / L agar, and 1-2 mL Tween-80 dissolved in 1 L of water, adjusting the pH to approximately 6.0-6.5.

[0036] The YM medium formula is as follows: yeast extract 1.5-5 g / L, malt extract 2-3 g / L, glucose 10-20 g / L, peptone 5-10 g / L, and agar 10-20 g / L dissolved in 1 L of water, and the pH is adjusted to about 6.0-6.5.

[0037] As a preferred embodiment of the method for preparing the multifunctional Camellia chrysantha seed fermented oil described in this application, the mass ratio of the Lactobacillus plantarum liquid, Candida albicans liquid and Camellia chrysantha spore liquid is (1-3):(1-2):(2-5), preferably, the ratio of the Lactobacillus plantarum liquid, Candida albicans liquid and Camellia chrysantha spore liquid is 1:1:2.

[0038] In the technical solution of this application, three specific strains of bacteria—*Candida albicans*, *Candida*, and *Lactobacillus plantarum*—and their specific mass ratios are used for synergistic fermentation, which can better improve the repair and anti-aging effects of fermented camellia seed oil. However, when other strains are used, the repair and anti-aging effects of fermented camellia seed oil cannot be improved; furthermore, the mass ratios of *Candida albicans* spore liquid, *Candida* liquid, and *Lactobacillus plantarum* liquid are not within the aforementioned range, resulting in poor repair and anti-aging effects of the obtained fermented camellia seed oil.

[0039] In a preferred embodiment of the method for preparing fermented Camellia chrysantha seed oil with multiple effects as described in this application, the liquid fermentation in step S2 includes the following conditions:

[0040] Oxygen-controlled culture: during the first 0-24 hours of fermentation, the aeration rate of the fermenter is 0.8-2 vvm, and the stirring speed is 100-500 rpm; during the second 24-72 hours of fermentation, the system is closed and allowed to stand for fermentation.

[0041] This application employs multi-species time-sharing oxygen control technology to ensure the oxygen requirements of both anaerobic and aerobic bacteria, avoiding competitive inhibition or fermentation termination due to competition for oxygen resources among the microbial communities in the fermenter, thus shortening the fermentation time and improving fermentation efficiency.

[0042] This application utilizes a combination of solid-liquid two-stage fermentation and multi-strain time-controlled oxygen technology to achieve the enrichment of active ingredients in fermented Camellia chrysantha seed oil.

[0043] Preferably, during the fermentation period of 0 to 24 hours, the aeration rate of the fermenter is 0.8 to 1.2 vvm, the stirring speed is 150 to 200 rpm, and during the fermentation period of 24 to 72 hours, the closed system is allowed to stand for fermentation.

[0044] In the technical solution of this application, using the above parameters within 0 to 24 hours of fermentation can better activate the aerobic metabolism of Candida albicans and rapidly reduce dissolved oxygen.

[0045] When the above parameters are used during fermentation of 24–72 hours, the facultative anaerobic bacteria *Lactobacillus plantarum* dominates acid production (pH drops to 4.5–5.0), inhibiting other bacteria and activating extracellular enzymes of *Aspergillus cristatus*.

[0046] In a preferred embodiment of the method for preparing the multi-functional fermented Camellia chrysantha seed oil described in this application, the liquid culture medium in step S2 comprises the following components:

[0047] Sucrose 50–80 g / L, fructooligosaccharides 10–15 g / L, yeast extract 5–8 g / L, peptone 3–5 g / L, KH₂PO₄ 1.5–2.0 g / L, MgSO₄·7H₂O 0.5–0.8 g / L, Tween-80 0.5–1.0 mL, L-cysteine ​​hydrochloride 0.3–0.5 g / L, dissolved in 1 L of water, with a liquid culture medium pH of 6–6.5.

[0048] Preferably, in step S3, the high-speed centrifugation conditions are 8000-12000 rpm for 5-10 minutes, and the microporous filter membrane diameter is 0.45 μm.

[0049] This application also provides the fermented Camellia chrysantha seed oil prepared by the above-mentioned method for preparing fermented Camellia chrysantha seed oil with multiple effects.

[0050] The fermented Camellia chrysantha seed oil prepared using the method described in this application has an aromatic odor, slows down the deterioration and oxidation time, and extends the shelf life and stability; furthermore, the fermented Camellia chrysantha seed oil of this application has a better repair and anti-aging effect.

[0051] This application also provides the application of the above-mentioned Camellia chrysantha seed fermented oil in the preparation of products with multiple effects such as moisturizing, soothing, repairing and anti-aging.

[0052] This application also provides a cosmetic with multiple effects of moisturizing, soothing, repairing and anti-aging, the cosmetic comprising a water-oil dual-phase essence;

[0053] The water-oil dual-phase essence comprises the following components by mass percentage:

[0054] The mixture contains 1-30% fermented Camellia chrysantha seed oil, 0.5-10% humectant, 0.5-3% preservative, and the remainder is deionized water.

[0055] Preferably, the moisturizer includes at least one of glycerin, D-panthenol, vitamin B5, 1,3-butanediol, 1,2-hexanediol, 1,3-propanediol, sodium hyaluronate, tremella polysaccharide, trehalose, betaine, allantoin, and low molecular weight sodium hyaluronate.

[0056] Preferably, the preservative includes at least one of p-hydroxyacetophenone and polyols.

[0057] The dosage forms of the cosmetics also include at least one of the following: lotion, cream, mask, serum, and spray.

[0058] Compared with the prior art, this application has the following beneficial effects:

[0059] This application provides a fermented oil from Camellia chrysantha seeds with multiple benefits, its preparation method, and its applications. This application utilizes a combination of solid-liquid two-stage fermentation and multi-strain time-controlled oxygen technology to enrich the active ingredients in the fermented oil. Specifically, *Lactobacillus plantarum* undergoes solid-state pre-fermentation to decompose the cell walls of Camellia chrysantha leaves, releasing tea polyphenols, various flavonoids, and flavonol precursors. The synergistic fermentation of *Lactobacillus plantarum*, *Candida albicans*, and *Lactobacillus plantarum* catalyzes the hydrolysis of oil molecules in the Camellia chrysantha seed oil, generating small-molecule free fatty acids, theaflavins, and catechins, thus improving transdermal absorption. Furthermore, the time-controlled oxygen technology ensures the oxygen requirements of both anaerobic and aerobic bacteria, preventing competitive inhibition or fermentation termination due to competition for oxygen resources in the fermenter, shortening fermentation time, and improving fermentation efficiency. The fermented oil from Camellia chrysantha seeds obtained using the method described in this application exhibits superior repair and anti-aging effects. Attached Figure Description

[0060] Figure 1 A photograph of the fermented Camellia chrysantha seed oil prepared in Example 1;

[0061] Figure 2 A photograph of the fermented Camellia chrysantha seed oil prepared in Comparative Example 1.

[0062] Figure 3 A photograph of the fermented Camellia chrysantha seed oil prepared in Comparative Example 2;

[0063] Figure 4 A photograph of the fermented Camellia chrysantha seed oil prepared in Comparative Example 13.

[0064] Figure 5 The image shows a before-and-after comparison of the two-way essential oil prepared in Application Example 1. Detailed Implementation

[0065] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0066] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the same raw materials were used in each parallel experiment. Where specific conditions are not specified in the examples, they were performed under conventional conditions or conditions recommended by the manufacturer.

[0067] In the following examples and comparative examples:

[0068] The *Eurotium cristatum* strain was purchased from the China Industrial Microbial Culture Collection Center (CSCEC), strain number: CICC 2099. It can also be obtained commercially.

[0069] Lactobacillus plantarum was purchased from the China General Microbiological Culture Collection Center (CGMCC 1.12935), but it can also be obtained commercially.

[0070] Candida utilis was purchased from the China General Microbiological Culture Collection Center (CGMCC 2.2876), but it can also be obtained commercially.

[0071] Candida krusei was purchased from the China General Microbiological Culture Collection Center (CGMCC 2.3196), but it can also be obtained commercially.

[0072] Lactobacillus casei was purchased from the China General Microbiological Culture Collection Center (CGMCC 1.8727) and can also be obtained commercially.

[0073] Sake yeast (Saccharomyces cerevisiae) was purchased from the China Industrial Microbial Culture Collection Center, strain number: CICC 30409, and can also be obtained commercially.

[0074] Camellia seed oil can be obtained by pressing, supercritical extraction, steam distillation, or ethanol extraction from Camellia seeds, or by conventional methods.

[0075] In the following embodiments:

[0076] The formula for MRS medium is as follows: 10.0 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 15 g / L agar, and 1.0 mL Tween 80 dissolved in 1 L of water, adjusting the pH to approximately 6.0.

[0077] The YM medium formula is as follows: 3 g / L yeast extract, 3 g / L malt extract, 10 g / L glucose, 5 g / L peptone, and 15 g / L agar are dissolved in 1 L of water, and the pH is adjusted to 6.0.

[0078] The liquid culture medium formula is as follows: 70 g / L sucrose, 15 g / L fructooligosaccharides (FOS), 5 g / L yeast extract, 5 g / L peptone, 1.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 1.0 mL Tween-80, and 0.5 g / L L-cysteine ​​hydrochloride, dissolved in 1 L of water, and the pH is adjusted to 6.

[0079] In the following embodiments and comparative examples:

[0080] The revival procedure for Camellia chrysantha is as follows: the refrigerated Camellia chrysantha strain is inoculated onto M40Y solid culture medium and cultured at 28°C for 7 days until the colonies are fully developed.

[0081] The resuscitation procedure for Candida albicans is as follows: the preserved Candida albicans is inoculated into YM medium and cultured at 28°C for 24 hours.

[0082] The resuscitation procedure for Lactobacillus plantarum is as follows: the preserved Lactobacillus plantarum is inoculated into MRS medium and cultured at 37°C for 24 hours.

[0083] Example 1: A method for preparing fermented Camellia chrysantha seed oil with multiple benefits

[0084] This application provides a method for preparing fermented Camellia chrysantha seed oil with multiple benefits, comprising the following steps:

[0085] S1. Pre-treatment of *Tea laurentii* tea leaves: Wash fresh leaves and dry at 60℃ until moisture content reaches 15%; use a sterile inoculation loop to remove the revived *Tea laurentii* fungus and evenly disperse it in sterile water; adjust the concentration of the *Tea laurentii* spore solution to 10% using sterile water. 8 The spores were mixed evenly with the golden flower tea leaves (20 mL of golden flower spores were added for every 100 g of tea leaves). The mixture was then placed in a sealed environment at a temperature of 28°C and a humidity of 80% for 10 days for solid-state fermentation. The mixture was stirred every 5 days to prevent local overheating. The fermented golden flower tea leaves were then crushed into golden flower tea powder.

[0086] S2. First, revive *Lactobacillus plantarum* and *Candida pruriens*. Then, prepare 10 solutions of *Lactobacillus plantarum*, *Candida pruriens*, and *Aureobasidium aureum* spores from solid-state fermentation using sterile water. 8 A mixture of *Lactobacillus plantarum* culture (1 / mL) and *Candida oryzae* culture was added to a fermenter. 1 kg of *Camellia chrysantha* seed oil was added, along with a mixture of 60 g of *Lactobacillus plantarum* culture, *Candida oryzae* culture, and *Camellia chrysantha* spores (mass ratio 1:1:2), and 100 g of *Camellia chrysantha* tea powder was added as substrate. Co-fermentation was carried out in a liquid culture medium. During fermentation from 0 to 24 hours, the oxygen aeration rate was 1.5 vvm, and the stirring speed was 300 rpm. After 24 to 72 hours of static fermentation, the product was obtained.

[0087] S3. The product obtained in step S2 is centrifuged at 10,000 rpm for 10 minutes. The supernatant oil phase is collected and sterilized by microporous filtration membrane with a diameter of 0.45 μm to obtain Camellia chrysantha seed fermented oil.

[0088] Example 2: A method for preparing fermented Camellia chrysantha seed oil with multiple benefits

[0089] This application provides a method for preparing fermented Camellia chrysantha seed oil with multiple benefits, comprising the following steps:

[0090] S1. Pre-treatment of *Tea laurentii* tea leaves: Wash fresh leaves and dry at 60℃ until moisture content reaches 15%; use a sterile inoculation loop to remove the revived *Tea laurentii* fungus and evenly disperse it in sterile water; adjust the concentration of the *Tea laurentii* spore solution to 10% using sterile water. 6 The concentration of spores / mL was increased, and then the spore liquid and golden flower tea leaves were mixed evenly (10mL of golden flower spore liquid was added for every 100g of tea leaves). The mixture was placed in a sealed fermentation environment at a temperature of 26℃ and a humidity of 80% for 8 days for solid-state fermentation. The mixture was stirred every 4-5 days to prevent local overheating. The fermented golden flower tea leaves were then crushed into golden flower tea powder.

[0091] S2. First, revive *Lactobacillus plantarum* and *Candida pruriens*. Then, prepare 10 solutions of *Lactobacillus plantarum*, *Candida pruriens*, and *Aureobasidium aureum* spores from solid-state fermentation using sterile water. 6 A mixture of *Lactobacillus plantarum* culture (1 spores / mL) and *Candida przewalskii* culture was added to a fermenter. 1 kg of *Camellia chrysantha* seed oil was added, along with a mixture of 20 g of *Lactobacillus plantarum* culture, *Candida przewalskii* culture, and *Camellia chrysantha* spores (mass ratio 1:2:5), and 50 g of *Camellia chrysantha* tea powder as substrate. Co-fermentation was carried out in a liquid culture medium. During fermentation from 0 to 24 hours, the oxygen aeration rate was 0.8 vvm, and the stirring speed was 100 rpm. After 24 hours of static fermentation, the product was obtained.

[0092] S3. The product obtained in step S2 is centrifuged at 8000 rpm for 5 minutes. The supernatant oil phase is collected and sterilized by microporous filtration membrane with a diameter of 0.45 μm to obtain Camellia chrysantha seed fermented oil.

[0093] Example 3: A method for preparing fermented Camellia chrysantha seed oil with multiple benefits

[0094] This application provides a method for preparing fermented Camellia chrysantha seed oil with multiple benefits, comprising the following steps:

[0095] S1. Pre-treatment of *Tea laurentii* tea leaves: Wash fresh leaves and dry at 60℃ until moisture content reaches 15%; use a sterile inoculation loop to remove the revived *Tea laurentii* fungus and evenly disperse it in sterile water; adjust the concentration of the *Tea laurentii* spore solution to 10% using sterile water. 8 The concentration of spores / mL was increased, and then the spore liquid and golden flower tea leaves were mixed evenly (30mL of golden flower spore liquid was added for every 100g of tea leaves). The mixture was placed in a sealed fermentation environment at a temperature of 32℃ and a humidity of 85% for 20 days for solid-state fermentation. The mixture was stirred every 4-5 days to prevent local overheating. The fermented golden flower tea leaves were then crushed into golden flower tea powder.

[0096] S2. First, revive *Lactobacillus plantarum* and *Candida pruriens*. Then, prepare 10 solutions of *Lactobacillus plantarum*, *Candida pruriens*, and *Aureobasidium aureum* spores from solid-state fermentation using sterile water. 8 A mixture of *Lactobacillus plantarum* culture (3 cells / mL) and *Candida przewalskii* culture was added to a fermenter. 1 kg of *Camellia chrysantha* seed oil was added, along with a mixture of 100 g of *Lactobacillus plantarum* culture, *Candida przewalskii* culture, and *Camellia chrysantha* spores (mass ratio 3:2:5). 200 g of *Camellia chrysantha* tea powder was added as a substrate. Co-fermentation was carried out in a liquid culture medium. During fermentation from 0 to 24 hours, the oxygen flow rate was 2 vvm, and the stirring speed was 500 rpm. After 24 to 72 hours of static fermentation, the product was obtained.

[0097] S3. The product obtained in step S2 is centrifuged at 12,000 rpm for 10 minutes. The supernatant oil phase is collected and sterilized by microporous filtration membrane with a diameter of 0.45 μm to obtain Camellia chrysantha seed fermented oil.

[0098] Example 4: A method for preparing fermented Camellia chrysantha seed oil with multiple benefits

[0099] This application provides a method for preparing fermented Camellia chrysantha seed oil with multiple benefits, comprising the following steps:

[0100] S1. Pre-treatment of *Tea laurentii* tea leaves: Wash fresh leaves and dry at 60℃ until moisture content reaches 15%; use a sterile inoculation loop to remove the revived *Tea laurentii* fungus and evenly disperse it in sterile water; adjust the concentration of the *Tea laurentii* spore solution to 10% using sterile water. 8 The spores were mixed evenly with the golden flower tea leaves (20 mL of golden flower spores were added for every 100 g of tea leaves). The mixture was then placed in a sealed environment at a temperature of 28°C and a humidity of 80% for 10 days for solid-state fermentation. The mixture was stirred every 5 days to prevent local overheating. The fermented golden flower tea leaves were then crushed into golden flower tea powder.

[0101] S2. First, revive *Lactobacillus plantarum* and *Candida pruriens*. Then, prepare 10 solutions of *Lactobacillus plantarum*, *Candida pruriens*, and *Aureobasidium aureum* spores from solid-state fermentation using sterile water. 8 A mixture of *Lactobacillus plantarum* culture (1 spores / mL) and *Candida przewalskii* culture was added to a fermenter. 1 kg of *Camellia chrysantha* seed oil was added, along with a mixture of 50 g of *Lactobacillus plantarum* culture, *Candida przewalskii* culture, and *Camellia chrysantha* spores (mass ratio 1:1:2), and 80 g of *Camellia chrysantha* tea powder as substrate. Co-fermentation was carried out in a liquid culture medium. During fermentation from 0 to 24 hours, the oxygen aeration rate was 0.8 vvm, and the stirring speed was 150 rpm. After 24 to 72 hours of static fermentation, the product was obtained.

[0102] S3. The product obtained in step S2 is centrifuged at 8000 rpm for 5 minutes. The supernatant oil phase is collected and sterilized by microporous filtration membrane with a diameter of 0.45 μm to obtain Camellia chrysantha seed fermented oil.

[0103] Example 5: A method for preparing fermented Camellia chrysantha seed oil with multiple benefits

[0104] This application provides a method for preparing fermented Camellia chrysantha seed oil with multiple benefits, comprising the following steps:

[0105] S1. Pre-treatment of *Tea laurentii* tea leaves: Wash fresh leaves and dry at 60℃ until moisture content reaches 15%; use a sterile inoculation loop to remove the revived *Tea laurentii* fungus and evenly disperse it in sterile water; adjust the concentration of the *Tea laurentii* spore solution to 10% using sterile water.6 The spores were mixed evenly with the golden flower tea leaves (30 mL of golden flower spores were added for every 100 g of tea leaves). The mixture was then placed in a sealed environment at 30°C and 85% humidity for 12 days for solid-state fermentation. The mixture was stirred every 5 days to prevent local overheating. The fermented golden flower tea leaves were then crushed into golden flower tea powder.

[0106] S2. First, revive *Lactobacillus plantarum* and *Candida pruriens*. Then, prepare 10 solutions of *Lactobacillus plantarum*, *Candida pruriens*, and *Aureobasidium aureum* spores from solid-state fermentation using sterile water. 6 A mixture of *Lactobacillus plantarum* culture (1 spores / mL) and *Candida pruriens* culture was added to a fermenter. 1 kg of *Camellia chrysantha* seed oil was added, along with a mixture of 40 g of *Lactobacillus plantarum* culture, *Candida pruriens* culture, and *Camellia chrysantha* spores (mass ratio 1:1:2), and 130 g of *Camellia chrysantha* tea powder was added as substrate. Co-fermentation was carried out in a liquid culture medium. During fermentation from 0 to 24 hours, the oxygen aeration rate was 1.2 vvm, and the stirring speed was 200 rpm. After 24 to 72 hours of static fermentation, the product was obtained.

[0107] S3. The product obtained in step S2 is centrifuged at 10,000 rpm for 10 minutes. The supernatant oil phase is collected and sterilized by microporous filtration membrane with a diameter of 0.45 μm to obtain Camellia chrysantha seed fermented oil.

[0108] Example 6: A method for preparing fermented Camellia chrysantha seed oil with multiple benefits

[0109] This application provides a method for preparing fermented Camellia chrysantha seed oil with multiple benefits, comprising the following steps:

[0110] S1. Pre-treatment of *Tea laurentii* tea leaves: Wash fresh leaves and dry at 60℃ until moisture content reaches 15%; use a sterile inoculation loop to remove the revived *Tea laurentii* fungus and evenly disperse it in sterile water; adjust the concentration of the *Tea laurentii* spore solution to 10% using sterile water. 8 The spores were mixed evenly with the golden flower tea leaves (20 mL of golden flower spores were added for every 100 g of tea leaves). The mixture was then placed in a sealed environment at a temperature of 28°C and a humidity of 80% for 10 days for solid-state fermentation. The mixture was stirred every 5 days to prevent local overheating. The fermented golden flower tea leaves were then crushed into golden flower tea powder.

[0111] S2. First, revive *Lactobacillus plantarum* and *Candida cruzi*. Then, prepare 10 solutions of *Lactobacillus plantarum*, *Candida cruzi*, and *Aureobasidium aureum* spores extracted during solid-state fermentation using sterile water. 8A mixture of *Lactobacillus plantarum* culture and *Candida croceta* culture was prepared. 1 kg of *Camellia chrysantha* seed oil was added to a fermentation tank, along with a mixture of 60 g of *Lactobacillus plantarum* culture, *Candida croceta* culture, and *Camellia chrysantha* spores (mass ratio 1:1:2). 100 g of *Camellia chrysantha* tea powder was added as a substrate. Co-fermentation was carried out in a liquid culture medium. During fermentation from 0 to 24 hours, the oxygen aeration rate was 1.5 vvm, and the stirring speed was 300 rpm. After 24 to 72 hours of static fermentation, the product was obtained.

[0112] S3. The product obtained in step S2 is centrifuged at 10,000 rpm for 10 minutes. The supernatant oil phase is collected and sterilized by microporous filtration membrane with a diameter of 0.45 μm to obtain Camellia chrysantha seed fermented oil.

[0113] Example 7: A method for preparing fermented Camellia chrysantha seed oil with multiple benefits

[0114] Compared with Example 1, in Example 7, the mass ratio of Lactobacillus plantarum culture, Candida pruriens culture, and Aureobasidium aureum spore culture was 0.5:0.5:6, and the amount of the mixture of Lactobacillus plantarum culture, Candida pruriens culture, and Aureobasidium aureum spore culture added was 60g. The remaining steps were the same as in Example 1.

[0115] Example 8: A method for preparing fermented Camellia chrysantha seed oil with multiple benefits

[0116] Compared with Example 1, in Example 8, the mass ratio of Lactobacillus plantarum culture, Candida pruriens culture, and Aureobasidium aureum spore liquid was 5:4:1, and the amount of the mixture of Lactobacillus plantarum culture, Candida pruriens culture, and Aureobasidium aureum spore liquid added was 60g. The remaining steps were the same as in Example 1.

[0117] Comparative Example 1

[0118] Compared with Example 1, Comparative Example 1 does not include step S1, the solid-state fermentation culture step. The specific steps are as follows:

[0119] S1: First, revive *Lactobacillus plantarum* and *Candida pruriens*. Then, prepare 10 solutions of *Lactobacillus plantarum*, *Candida pruriens*, and *Aureobasidium aureum* spores extracted during solid-state fermentation using sterile water. 8 A mixture of *Lactobacillus plantarum* culture (1 spores / mL) and *Candida przewalskii* culture was added to a fermenter. 1 kg of *Camellia chrysantha* seed oil was added, along with a mixture of 60 g of *Lactobacillus plantarum* culture, *Candida przewalskii* culture, and *Camellia chrysantha* spores (mass ratio 1:1:2). 100 g of unfermented *Camellia chrysantha* tea powder was added as substrate. Co-fermentation was carried out in a liquid culture medium. During fermentation from 0 to 24 hours, the oxygen flow rate was 1.5 vvm, and the stirring speed was 300 rpm. After 24 to 72 hours of static fermentation, the product was obtained.

[0120] S2. The product obtained in step S2 is centrifuged at 10,000 rpm for 10 minutes. The supernatant oil phase is collected and sterilized by microporous filtration membrane with a diameter of 0.45 μm to obtain Camellia chrysantha seed fermented oil.

[0121] Comparative Example 2

[0122] Compared with Example 1, this example does not include step S2, the liquid fermentation culture step. The specific steps are as follows:

[0123] S1. Pre-treatment of *Tea laurentii* tea leaves: Wash fresh leaves and dry at 60℃ until moisture content reaches 15%; use a sterile inoculation loop to remove the revived *Tea laurentii* fungus and evenly disperse it in sterile water; adjust the concentration of the *Tea laurentii* spore solution to 10% using sterile water. 8 The spores were mixed evenly with the golden flower tea leaves (20 mL of golden flower spores were added for every 100 g of tea leaves). The mixture was then placed in a sealed environment at a temperature of 28°C and a humidity of 80% for 10 days for solid-state fermentation. The mixture was stirred every 5 days to prevent local overheating. The fermented golden flower tea leaves were then crushed into golden flower tea powder.

[0124] S2. Add 1kg of Camellia chrysantha seed oil to a fermentation tank, add 60g of sterile water, and add 100g of Camellia chrysantha tea powder as a substrate. Maintain a constant temperature of 28℃, mechanically stir at 300rpm for 24h, and let stand for 24-72h.

[0125] S3. The product obtained in step S2 is centrifuged at 10,000 rpm for 10 minutes. The supernatant oil phase is collected and sterilized by microporous filtration membrane with a diameter of 0.45 μm to obtain Camellia chrysantha seed oil.

[0126] Comparative Example 3

[0127] Compared with Example 1, Comparative Example 3 did not contain Lactobacillus plantarum culture, the mass ratio of Candida pruriens culture and Aureobasidium spore culture was 1:1, the amount of the mixture of Candida pruriens culture and Aureobasidium spore culture added was 60g, and the remaining steps were the same as in Example 1.

[0128] Comparative Example 4

[0129] Compared with Example 1, Comparative Example 4 did not contain Candida pruriens culture, the mass ratio of Lactobacillus plantarum culture and Aureobasidium spore culture was 1:1, the amount of the mixture of Lactobacillus plantarum culture and Aureobasidium spore culture added was 60g, and the remaining steps were the same as in Example 1.

[0130] Comparative Example 5

[0131] Compared with Example 1, Comparative Example 5 did not contain *Lactobacillus plantarum* spore liquid, the mass ratio of *Lactobacillus plantarum* liquid and *Candida pruriens* liquid was 1:1, the amount of the mixture of *Lactobacillus plantarum* liquid and *Candida pruriens* liquid added was 60g, and the remaining steps were the same as in Example 1.

[0132] Comparative Example 6

[0133] Compared with Example 1, Comparative Example 6 did not contain Lactobacillus plantarum culture, the mass ratio of Candida pruriens culture and Aureobasidium spore culture was 1:2, the amount of the mixture of Candida pruriens culture and Aureobasidium spore culture added was 45g, and the remaining steps were the same as in Example 1.

[0134] Comparative Example 7

[0135] Compared with Example 1, Comparative Example 7 did not contain Candida pruriens culture, the mass ratio of Lactobacillus plantarum culture and Aureobasidium brevicornu spore culture was 1:2, the amount of the mixture of Lactobacillus plantarum culture and Aureobasidium brevicornu spore culture added was 45g, and the remaining steps were the same as in Example 1.

[0136] Comparative Example 8

[0137] Compared with Example 1, Comparative Example 8 did not contain *Lactobacillus plantarum* spore liquid, the mass ratio of *Lactobacillus plantarum* liquid and *Candida pruriens* liquid was 1:1, the amount of the mixture of *Lactobacillus plantarum* liquid and *Candida pruriens* liquid added was 30g, and the remaining steps were the same as in Example 1.

[0138] Comparative Example 9

[0139] Compared with Example 1, Comparative Example 9 used Lactobacillus casei instead of Lactobacillus plantarum. Lactobacillus casei was revived and cultured in MRS medium at 37°C for 24 hours. The bacterial cells were washed off with sterile water, resuspended, and the spore concentration was adjusted to 10. 8 The number of Lactobacillus casei spores per mL was used to prepare a Lactobacillus casei spore solution; the remaining steps were the same as in Example 1.

[0140] The formula for MRS medium is as follows: 10.0 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 15.0 g / L agar, and 1.0 ml Tween 80 dissolved in 1 L of water, adjusting the pH to approximately 6.0.

[0141] Comparative Example 10

[0142] Compared with Example 1, Comparative Example 10 used sake yeast instead of Candida albicans. The sake yeast was revived and cultured in YM medium at 28°C for 24 hours. The cells were removed with a sterile inoculation loop and evenly dispersed in sterile water, resuspended, and the spore concentration was adjusted to 10. 8 The number of yeast cells per mL was used to prepare a sake yeast culture; the remaining steps were the same as in Example 1.

[0143] The YM medium formula is as follows: 3 g / L yeast extract, 3 g / L malt extract, 10 g / L glucose, 5 g / L peptone, and 15 g / L agar are dissolved in 1 L of water, and the pH is adjusted to 6.0.

[0144] Comparative Example 11

[0145] Compared with Example 1, Comparative Example 11 used Monascus purpureus instead of Aureobasidium aureum. Monascus purpureus was revived and cultured in PDA medium at 28°C for 24 hours. The bacterial cells were removed with a sterile inoculation loop and evenly dispersed in sterile water, resuspended, and the spore concentration was adjusted to 10. 8 The concentration of Monascus purpureus spores was increased to 1 / mL to prepare a solution of Monascus purpureus spores; the remaining steps were the same as in Example 1.

[0146] The PDA culture medium formula is as follows: 200 g / L potato filtrate, 20 g / L glucose, and 15 g / L agar are dissolved in 1 L of water, and the pH is adjusted to 6.0.

[0147] Comparative Example 12

[0148] Compared with Example 1, in step S2 of Comparative Example 12, time-sharing oxygen control is not used. The specific steps are as follows:

[0149] S2. First, revive *Lactobacillus plantarum* and *Candida pruriens*. Then, prepare 10 solutions of *Lactobacillus plantarum*, *Candida pruriens*, and *Aureobasidium aureum* spores from solid-state fermentation using sterile water. 8 A mixture of *Lactobacillus plantarum* culture (60g / mL), *Candida pruriens* culture, and *Camellia chrysantha* spores (mass ratio 1:1:2) was added to a fermentation tank along with 1kg of Camellia chrysantha seed oil. 100g of Camellia chrysantha tea powder was also added as a substrate. Co-fermentation was carried out in a liquid culture medium. The fermentation was allowed to proceed statically for 0–72 hours to obtain the product.

[0150] The remaining steps are the same as in Example 1.

[0151] Experiment Example 1: In vitro antioxidant verification experiment

[0152] This application provides in vitro antioxidant verification experiments to demonstrate that fermented Camellia japonica seed oil has antioxidant properties.

[0153] The fermented Camellia chrysantha seed oils prepared in Examples 1-8 and Comparative Examples 1-12 were subjected to antioxidant experiments, and the DPPH free radical scavenging experiment was used to investigate the process. The specific steps included:

[0154] (1) Dissolve 0.001972g of DPPH in anhydrous ethanol solution, and make up to 25mL with anhydrous ethanol solution. Measure the absorbance at 517nm and record the absorbance value between 200-800 to obtain the DPPH test solution.

[0155] (2) Using Examples 1-8 and Comparative Examples 1-12 as test samples, 0.1 mL of the sample solution was pipetted into anhydrous ethanol to prepare a 1% sample test solution. The solutions were prepared according to the following A, B, and C tubes, and incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm and the absorbance value was recorded. Each sample was repeated 3 times.

[0156] Tube A: 2 mL sample + 2 mL DPPH test solution;

[0157] Tube B: 2 mL DPPH test solution + 2 mL PBS;

[0158] Tube C: 2 mL PBS + 2 mL sample;

[0159] Zeroing: Anhydrous ethanol solution;

[0160] The calculation method is as follows: DPPH free radical scavenging rate = 1 - (AC) / B × 100%. The experiment was repeated 3 times and the average value was taken. The test results are shown in Table 1.

[0161] Table 1

[0162]

[0163]

[0164] Conclusion: The scavenging rate of Camellia chrysantha seed fermented oil prepared in Examples 1-6 of this application was 90.2-99.1%, which showed good antioxidant effect. Among them, Comparative Example 1 did not contain a solid-state fermentation culture step, and the efficiency of the Camellia chrysantha powder substrate was reduced. Comparative Example 2 did not contain a liquid fermentation culture step, and the scavenging rate of the obtained Camellia chrysantha seed fermented oil was lower, and the antioxidant effect was not as good as that of Examples 1-6.

[0165] In Examples 7-8, the bacterial solutions of Lactobacillus plantarum, Candida albicans, and Camellia chrysantha spores were not within the range of (1-3):(1-2):(2-5), and the antioxidant effect of the fermented Camellia chrysantha seed oil prepared was not as good as that of Examples 1-6.

[0166] In the preparation methods of Comparative Examples 3-5, when the total amount of each ingredient was kept constant, removing one of *Candida albicans*, *Candida*, and *Lactobacillus plantarum*, or in the preparation methods of Comparative Examples 6-8, removing only one of *Candida albicans*, *Candida*, and *Lactobacillus plantarum*, the scavenging rate of the fermented Camellia chrysantha seed oil obtained was lower, and the antioxidant effect was not as good as that of Examples 1-6.

[0167] When the specific combination of Camellia chrysanthemi, Candida albicans, and Lactobacillus plantarum was not used in Comparative Examples 9–11, the scavenging rate of Camellia chrysanthemi seed fermented oil was low and the antioxidant effect was poor.

[0168] Comparative Example 12 did not employ time-sharing oxygen control, and the added Candida pruriens, as an aerobic bacterium, could not ferment normally, resulting in a weakened efficiency in the liquid fermentation stage of Camellia chrysantha seed fermentation oil and poor antioxidant effect.

[0169] Experiment Example 2: Verification Experiment on Inhibiting UV-Induced Inflammation

[0170] Experiment 2 verifies the anti-UV inflammation effect of fermented Camellia chrysantha seed oil.

[0171] The specific steps are as follows:

[0172] Zebrafish species: Spawning tests were conducted using wild-type AB strain zebrafish (Danio rerio) from reliable sources (such as the China National Zebrafish Resource Center);

[0173] Zebrafish embryo preparation: At 26°C, fertilized fish embryos complete their first division in approximately 45 minutes, followed by division into 4, 8, 16, and 32 cells. At this stage, the fertilized fish embryos are clearly identifiable. Healthy fish embryos in the 4–32 cell stage are selected for use. Exposure to the test substance should begin within the 4–128 cell stage.

[0174] Test sample preparation: Dissolve Camellia chrysantha seed fermented oil (Examples 1-8, Comparative Examples 1-12) in 10 times dimethyl sulfoxide, dilute with zebrafish culture medium to a concentration of 1% by mass, record as sample solution, and refrigerate;

[0175] (3) UVB fin irradiation procedure: Zebrafish were placed in a 6-well plate and irradiated with UVB for 30 seconds (20 mJ / cm²). 2 );

[0176] (4) Group setup: The fish were divided into a model control group (UVB irradiation), a blank control group (normal culture without irradiation) and a test group (solution of each example / comparative example + UVB irradiation). Six fish embryos were selected for each group and placed in a constant temperature incubator at 28±1℃ for 24h.

[0177] (5) Data analysis: Zebrafish were anesthetized with tricaine and fluorescent images of the dorsal region of the zebrafish were taken under a fluorescence microscope. The data were analyzed and collected using the advanced image processing software ImageJ. The target area was precisely delineated using the rectangle tool, and the average fluorescence intensity of the selected area, i.e. the number of neutrophils in the zebrafish, was measured using the gray intensity method.

[0178] Neutrophil quantification: Data was analyzed and collected using the advanced image processing software ImageJ. The target area was precisely delineated using the rectangle tool, and the average fluorescence intensity of the selected area was measured using the grayscale intensity method, which is the number of neutrophils in zebrafish.

[0179] Calculation of the inhibition rate of zebrafish neutrophils

[0180]

[0181] Specific data are shown in Table 2 (neutrophil inhibition rate of zebrafish in each group of samples).

[0182] Table 2

[0183]

[0184]

[0185] Conclusion: The neutrophil inhibition rate of the fermented Camellia chrysantha seed oil prepared in Examples 1-6 of this application was 62.3-78.2%, which showed good anti-UV inflammation effect. Among them, Comparative Example 1 did not contain a solid-state fermentation culture step, and Comparative Example 2 did not contain a liquid fermentation culture step. The neutrophil inhibition rate of the fermented Camellia chrysantha seed oil obtained was lower, and the anti-UV inflammation effect was not as good as that of Examples 1-6.

[0186] In Examples 7-8, the bacterial suspensions of Lactobacillus plantarum, Candida albicans, and Camellia chrysantha spores were not within the range of (1-3):(1-2):(2-5), and the anti-UV inflammation effect of the fermented Camellia chrysantha seed oil prepared was not as good as that in Examples 1-6.

[0187] In the preparation methods of Comparative Examples 3-5, when the total amount of each ingredient was kept constant, removing one of *Candida albicans*, *Candida*, and *Lactobacillus plantarum*, or in the preparation methods of Comparative Examples 6-8, removing only one of *Candida albicans*, *Candida*, and *Lactobacillus plantarum*, the neutrophil inhibition rate of the fermented Camellia chrysantha seed oil obtained was lower, and the anti-UV inflammation effect was not as good as that of Examples 1-6.

[0188] When the specific combination of Camellia chrysanthemi, Candida albicans, and Lactobacillus plantarum was not used in Comparative Examples 9–11, the neutrophil inhibition rate of Camellia chrysanthemi seed fermentation oil was lower and the anti-UV inflammation effect was poor.

[0189] Comparative Example 12 did not employ time-sharing oxygen control, and the added Candida pruriens, as an aerobic bacterium, could not ferment normally, resulting in a weakening of the efficiency of the liquid fermentation stage of Camellia chrysantha seed oil, a reduction in the production of polyphenols and flavonoids, and a poorer anti-inflammatory and anti-UV inflammation effect.

[0190] Experiment Example 3: Stability Test of Fermented Oil from Camellia chrysantha Seeds

[0191] The stability of the Camellia chrysantha seed fermented oils obtained in Examples 1-8 and Comparative Examples 1-12 was tested, and the test results are shown in Table 3.

[0192] Test method: The samples were placed in constant temperature equipment at 50℃, 25℃, 5℃, and -8℃, as well as in a UV light aging chamber for stability testing. The samples were removed at 0 and 30 days to observe whether there were any obvious abnormal changes in appearance, such as discoloration, layering, precipitation, or exudation. The color change results after 30 days are shown in Table 3.

[0193] Table 3

[0194]

[0195]

[0196] Conclusion: Based on the fermented Camellia chrysantha seed oil at 25℃, the fermented Camellia chrysantha seed oils prepared in Examples 1-8 of this application did not undergo any changes in color or properties under high temperature (50℃), low temperature (5℃), ultra-low temperature (-8℃), or UV irradiation, and still maintained a stable bright golden color. The fermented Camellia chrysantha seed oil prepared in Example 1 of this application... Figure 1 As shown.

[0197] like Figure 2 As shown, Comparative Example 1 did not undergo a solid-state fermentation step, resulting in insufficient cell wall disruption and dissolution of the active ingredients from the Camellia chrysantha seed fermented oil. Consequently, material exchange could not occur during the liquid fermentation stage, leading to a reduction in colored active ingredients such as flavonoids and a paler color compared to Example 1. However, no changes in color or properties occurred under high temperature (50°C), low temperature (5°C), ultra-low temperature (-8°C), or UV irradiation, indicating relatively stable color.

[0198] The color and properties of the fermented Camellia chrysantha seed oil prepared in Comparative Example 2 were as follows: Figure 3 As shown, since no liquid fermentation step was performed, the three bacteria, Lactobacillus plantarum, Candida albicans, and Aureobasidium aureum, could not work synergistically to catalyze the hydrolysis of oils and generate small molecule free fatty acids (such as oleic acid and linoleic acid). At the same time, the conversion of flavonoid precursors in tea into highly active polyphenols such as theaflavins and catechins, which are powerful natural antioxidants, was also reduced. Therefore, the color was lighter than in Example 1, and the oils were more prone to discoloration, resulting in poor stability.

[0199] Comparative examples 5, 9, 10, and 12 involved replacing or reducing the microbial strains used in the liquid fermentation stage of Camellia chrysantha seed fermented oil, or failing to use the optimal ratio of microbial solution and optimal fermentation conditions. This resulted in a reduction in colored active ingredients such as flavonoids in the fermented oil, leading to a lighter color. However, no changes in color or properties occurred under high temperature (50℃), low temperature (5℃), ultra-low temperature (-8℃), or UV irradiation; the color remained relatively stable.

[0200] Comparative Example 11 used *Russula rubra* instead of *Mycorrhiza glabra* to complete the solid-state fermentation of the strain and the liquid fermentation of *Camellia chrysantha* seed oil. This resulted in the production of a large amount of red pigment, affecting the color of the *Camellia chrysantha* seed oil and producing a color similar to... Figure 4 The orange-yellow fermented oil shown.

[0201] Experimental Example 4, Application Example

[0202] The fermented Camellia chrysantha seed oils prepared in Examples 1-8 and Comparative Examples 1-12 were formulated into water-oil dual-phase essences, as shown in Table 4. The water-oil dual-phase essences in Application Examples 1-8 used the fermented Camellia chrysantha seed oils prepared in Examples 1-8, respectively; the water-oil dual-phase essences in Comparative Application Examples 1-12 used the fermented Camellia chrysantha seed oils prepared in Comparative Examples 1-12, respectively; and Application Examples 9-10 used the fermented Camellia chrysantha seed oil prepared in Example 1.

[0203] Table 4

[0204]

[0205] This application provides a method for preparing a water-oil dual-action essence, the method comprising the following steps:

[0206] (1) Mix the humectant and all the water thoroughly to obtain pre-prepared component A;

[0207] (2) Mix the preservatives and heat them to 60°C to melt them, to obtain pre-prepared component B;

[0208] (3) Add pre-prepared component B to pre-prepared component A and stir until evenly mixed. Then slowly add fermented Camellia chrysantha seed oil to obtain the final product.

[0209] Experimental Example 5: The Repairing and Anti-aging Effects of a Water-Oil Dual-Purpose Serum on Sensitive Skin

[0210] Experimental Methods: Sixty-nine volunteers aged 35-50 with sensitive skin (self-assessed as sensitive, with a lactic acid stinging score ≥3) were enrolled in the test and divided into 23 groups of 3 participants each. All volunteers signed informed consent forms. Enrolled volunteers were instructed to use the serum twice daily, morning and evening, for 4 weeks. Follow-up visits were conducted on day 1 (initial values) and day 28, and skin moisture was measured using a skin moisture measurement probe. The CM 825 was used to detect skin hydration, the Tewameter™ Hex transdermal water loss probe was used to detect skin TEWL values, the Colorimeter CL400 skin color probe was used to detect skin color content, and Visia was used to detect the improvement of fine lines under the eyes. All tests were conducted after cleansing the face with a uniform cleanser and sitting still for 30 minutes in the experimental environment (temperature 20±1℃, humidity 50±10%). The efficacy of the serum was expressed as the improvement rate, calculated as |(data after use - data before use) / data before use| × 100%. Data processing and graphing were performed using GraphPad Prism 8.0 software, and one-way ANOVA was conducted using SPSS 20.0 software (P<0.5). All experiments were repeated three times. The test results are shown in Table 5.

[0211] Table 5

[0212]

[0213] The fermented Camellia chrysanthemum seed oil prepared in Examples 1-6 of this application, when applied to a water-oil dual-action essence, exhibits superior repair and anti-aging effects. However, Comparative Application Example 1, which does not contain a solid-state fermentation step, and Comparative Application Example 2, which does not contain a liquid-state fermentation step, show inferior repair and anti-aging effects to the water-oil dual-action essence obtained in Comparative Application Example 1 compared to Examples 1-6. The improvement in skin achieved by Comparative Application Example 1 is as follows: Figure 5 As shown.

[0214] In Application Examples 7-8, the liquids of Lactobacillus plantarum, Candida albicans, and Aureobasidium aureum spores in the water-oil dual-action essences were not within the range of (1-3):(1-2):(2-5), and the repair and anti-aging effects of the prepared water-oil dual-action essences were not as good as those in Application Examples 1-6.

[0215] Compared with the preparation methods of Application Examples 3-5, when the total amount is kept constant, removing one of the following: *Candida albicans*, *Candida*, and *Lactobacillus plantarum*, or compared with the preparation methods of Application Examples 6-8, when only one of the following is removed, the repair and anti-aging effects of the obtained water-oil dual-action essence are not as good as those of Application Examples 1-6.

[0216] Compared with the application examples 9-11, when the specific combination of Aureobasidium aureum, Candida albicans and Lactobacillus plantarum was not used, the water-oil dual-action essence prepared had reduced moisturizing, soothing, repairing and anti-aging effects.

[0217] In contrast, application example 12 did not employ time-based oxygen control, so the added Candida albicans, as an aerobic bacterium, could not ferment normally. This resulted in a weakening of the liquid fermentation stage of Camellia chrysantha seed oil, a reduction in the production of polyphenols and flavonoids, and an impact on the efficacy of active ingredients. Consequently, the moisturizing, soothing, repairing, and anti-aging effects of the water-oil dual-action essence were weakened.

[0218] 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 the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for preparing fermented Camellia chrysantha seed oil with multiple benefits, characterized in that, Includes the following steps: S1. Solid-state fermentation of golden flower tea leaves using golden flower fungus spore liquid, and then crushing the fermented golden flower tea leaves into golden flower tea powder. S2. Inoculate the golden camellia seed oil and golden camellia spore liquid into a liquid culture medium, add the bacterial liquid prepared by Candida albicans and Lactobacillus plantarum, and golden camellia tea powder together for liquid fermentation. During the fermentation period of 0 to 24 hours, the oxygen aeration rate is 0.8 to 2 vvm. After fermentation for 24 to 72 hours, allow the fermentation to stand to obtain the product. S3. The product obtained in step S2 is centrifuged at high speed, the supernatant oil phase is collected, and sterilized by microporous filter membrane to obtain Camellia chrysantha seed fermented oil. The mass ratio of Lactobacillus plantarum bacterial solution, Candida albicans bacterial solution and Aureobacillus spore solution was (1~3):(1~2):(2~5).

2. The method for preparing fermented Camellia chrysantha seed oil with multiple effects as described in claim 1, characterized in that, In step S1, the revived *Aureobasidium aureum* is washed off with sterile water, evenly dispersed, and the spore concentration is measured. The concentration of the *Aureobasidium aureum* spore solution is then adjusted to 10% with sterile water. 6 ~10 8 The spore volume was measured at 1 / mL. The spore liquid and golden flower tea leaves were then mixed evenly and placed in a sealed environment at a temperature of 26-32℃ and a humidity of 80-85% for 8-20 days for solid-state fermentation.

3. The method for preparing fermented Camellia chrysantha seed oil with multiple effects as described in claim 1, characterized in that, In step S1, the ratio of golden flower tea leaves to golden flower spore liquid is 100g:(10~30ml).

4. The method for preparing fermented Camellia chrysantha seed oil with multiple effects as described in claim 1, characterized in that, In step S2, *Candida albicans* and *Lactobacillus plantarum* are respectively prepared with sterile water to a concentration of 10... 6 ~10 8 Candida albicans culture and Lactobacillus plantarum culture; The ratio of a mixture of Lactobacillus plantarum bacterial solution, Candida albicans bacterial solution and Aureobasidium aureum spore solution to Aureobasidium aureum tea powder is (20~100g): (50~200g).

5. The method for preparing fermented Camellia chrysantha seed oil with multiple effects as described in claim 1, characterized in that, In step S2, liquid fermentation includes the following conditions: Oxygen-controlled culture: during the first 0-24 hours of fermentation, the aeration rate of the fermenter is 0.8-2 vvm, and the stirring speed is 100-500 rpm; during the second 24-72 hours of fermentation, the system is closed and allowed to stand for fermentation.

6. The method for preparing fermented Camellia chrysantha seed oil with multiple effects as described in claim 1, characterized in that, In step S2, the liquid culture medium comprises the following components: Sucrose 50-80 g / L, fructooligosaccharides 10-15 g / L, yeast extract 5-8 g / L, peptone 3-5 g / L, KH2PO4 1.5-2.0 g / L, MgSO4·7H2O 0.5-0.8 g / L, Tween-80 0.5-1.0 mL, L-cysteine ​​hydrochloride 0.3-0.5 g / L, dissolved in 1 L of water, with a liquid culture medium pH of 6-6.

5.

7. The fermented Camellia chrysantha seed oil prepared by the method for preparing fermented Camellia chrysantha seed oil with multiple effects as described in any one of claims 1 to 6.

8. The application of the fermented Camellia chrysantha seed oil as described in claim 7 in the preparation of products with multiple effects of moisturizing, soothing, repairing and anti-aging.

9. A cosmetic product with multiple functions including moisturizing, soothing, repairing, and anti-aging, characterized in that, The cosmetics include a water-oil dual-phase serum; The water-oil dual-phase essence comprises the following components by mass percentage: The tea plant seed fermentation oil as described in claim 7 contains 1-30% humectant, 0.5-10% humectant, 0.5-3% preservative, and the balance is deionized water.

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