Fermented vegetable oil with multiple effects of whitening, resisting ageing, resisting inflammation, relieving, repairing and inhibiting bacteria as well as preparation method and application of fermented vegetable oil

Abstract

The invention discloses fermented vegetable oil with multiple effects of whitening, resisting ageing, resisting inflammation, relieving, repairing and inhibiting bacteria as well as a preparation method and application of the fermented vegetable oil, and belongs to the field of microbial fermentation. The preparation method comprises the following steps: firstly, carrying out thallus fermentation on a basic culture medium for 24-48 hours to obtain basic fermentation liquor rich in active matters such as glycolipid and biological enzyme; then carrying out centrifugal filtration on the basic fermentation liquor to obtain cell-free basic fermentation liquor; then three factors and three levels with high sensory evaluation scores are screened out through a single factor to carry out an orthogonal test, optimal natural vegetable oil fermentation conditions are screened out to carry out vegetable oil fermentation, and after fermentation is finished, the fermented vegetable oil obtained by centrifuging, taking an oil phase and filtering is rich in various active matters such as glycolipid and biological enzyme; compared with natural vegetable oil, the natural vegetable oil has the multiple effects of better whitening, anti-aging, anti-inflammatory relieving, barrier repairing, good skin permeability, relatively strong acne bacillus inhibition capability and the like, is easier to absorb by skin and has skin feeling experience, and the economic value of the natural vegetable oil in the field of beauty and skin care is improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and in particular relates to a fermented plant oil with multiple effects such as whitening and anti-aging, anti-inflammatory and soothing, repair and antibacterial properties, as well as its preparation method and application. Technical Background

[0002] Natural plant oils are rich in fatty acids (such as oleic acid and linoleic acid), vitamins (such as vitamin E), antioxidants, and phytosterols. These components possess excellent moisturizing and repairing, antioxidant and anti-aging, anti-inflammatory and soothing, whitening, and spot-fading effects, making natural plant oils play multiple roles in skincare. "Oil-based skincare" has become a mainstream trend in the skincare market in recent years, and its scientific validity and efficacy have been widely recognized by consumers, thus gaining immense popularity. However, natural plant oils are triglycerides formed from unsaturated fatty acids and glycerol, which cannot be absorbed by human skin.

[0003] Fermented plant oils are produced by co-fermenting natural plant oils with specific microorganisms (such as yeast and lactic acid bacteria) under suitable conditions. This process generates beneficial metabolites such as polysaccharides, peptides, glycolipids, and free unsaturated fats. Simultaneously, the large molecular groups in the natural plant oils are broken down into smaller molecules. These smaller molecules can penetrate the stratum corneum, making the natural active skincare ingredients in the plant oils easier for the skin to absorb. Secondary metabolites produced during fermentation (such as antimicrobial peptides, glycolipids, and organic acids) can effectively inhibit Propionibacterium acnes, Escherichia coli, and Candida albicans, providing natural preservative and anti-acne benefits. The fermentation process typically improves the feel and odor of the oil, making it lighter and less greasy, and eliminating the "grassy smell" or "greasy heaviness" that crude oil may have, making it easier to use in formulations and enhancing the user experience. Furthermore, fermented oils have better stability and lower allergenicity because the microorganisms break down certain allergenic proteins in the crude oil.

[0004] Chinese patent applications CN116286414A, CN202311730896, CN202210941846, and CN201710046279 disclose methods for preparing fermented vegetable oils. These methods involve microorganisms throughout the entire process. During the processing of natural vegetable oils, microorganisms continuously hydrolyze and metabolize the resulting active substances and free fatty acids, producing a large number of undesirable byproducts. This significantly reduces the quality of the fermented vegetable oil, resulting in a darker color and a stronger fermented odor. Furthermore, it increases the difficulty and cost of downstream separation and purification. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for preparing fermented plant oil with multiple effects such as whitening, anti-aging, anti-inflammatory, soothing, repairing, and antibacterial properties, and its application. The method involves first culturing microorganisms under specific conditions to produce a large number of biological enzymes and active substances. Then, after centrifugation and filtration to remove the microorganisms, a cell-free basic fermentation broth rich in biological enzymes and fermentation active substances is obtained. Plant oil is then added in a specific ratio, and fermentation is carried out under specific conditions for a period of time. Samples are taken to measure the acid value and perform sensory evaluation. Fermentation is terminated when the acid value reaches the target value and the sensory evaluation meets the standard. After centrifugation and filtration, the fermented plant oil is obtained. In this method, microorganisms only participate in the first part of the fermentation process, i.e., producing biological enzymes and active substances, and do not participate in the latter part of the fermentation process. Therefore, the extraction and purification process of the fermented plant oil is simple, and the product has a pleasant aroma and excellent whitening, anti-aging, anti-inflammatory, soothing, repairing, and antibacterial effects, often providing consumers with a superior skin feel and skincare experience.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a method for preparing fermented plant oil with multiple effects such as whitening, anti-aging, anti-inflammatory, soothing, repairing, and antibacterial properties.

[0010] Includes the following steps:

[0011] 1) The bacterial cells are fermented in a basic culture medium to obtain a basic fermentation broth rich in bioactive substances such as enzymes and glycolipids;

[0012] 2) Centrifuge and filter the basic fermentation broth from step 1) to remove the bacterial cells and obtain cell-free basic fermentation broth:

[0013] 3) The cell-free basic fermentation broth from step 2) is fermented with natural plant oil or a mixture of natural plant oil under specific conditions for a period of time. Fermentation ends when the sensory score meets the requirements.

[0014] 4) Centrifuge, take the oil phase and filter to obtain fermented vegetable oil.

[0015] It is understood that the fermentation medium also contains conventional carbon sources, nitrogen sources, and inorganic salts. There are no strict limitations on its composition; preferably, the fermentation medium includes 0.6-1.8% carbon source, 0.1-0.5% nitrogen source, and 0.45-1.2% inorganic salts.

[0016] This invention first conducts single-factor experiments on three important fermentation process parameters: fermentation temperature, fermentation time, and the mass ratio of cell-free basic fermentation broth to natural vegetable oil or a mixture of natural and natural vegetable oils. The three levels with the highest sensory evaluation scores are then selected. Orthogonal experiments are then conducted to select the optimal fermentation conditions for a specific vegetable oil.

[0017] Specifically, the fermentation temperature is 20-60℃, the fermentation time is 4-48h, and the ratio of cell-free basic fermentation liquid to natural plant oil is 1:0.25-6; preferably, in step 3), the fermentation temperature is 26-32℃, the fermentation time is 24-34h, and the ratio of cell-free basic fermentation liquid to natural plant oil is 1:0.5-5.

[0018] For camellia seed oil, in step 3), the fermentation temperature is 27-29℃, the fermentation time is 25-27h, and the ratio of cell-free basic fermentation liquid to camellia seed oil is 1:3-5; more preferably, the fermentation temperature is 28℃, the fermentation time is 26h, and the ratio of cell-free basic fermentation liquid to camellia seed oil is 1:4.

[0019] For jojoba oil, in step 3), the fermentation temperature is 29-31℃, the fermentation time is 30-34h, and the ratio of cell-free basic fermentation broth to jojoba oil is 3:1-3; more preferably, in step 3), the fermentation temperature is 30℃, the fermentation time is 32h, and the ratio of cell-free basic fermentation broth to jojoba oil is 3:2.

[0020] Furthermore, prior to fermentation, the process may include: inoculating the strain into a seed culture medium for cultivation to obtain a seed liquid.

[0021] Furthermore, the composition of the seed culture medium is not strictly limited; preferably, the seed culture medium can be YPD medium.

[0022] Furthermore, seed culture conditions may include: a culture temperature of 25-30 degrees Celsius and a culture time of 30-48 hours.

[0023] Furthermore, after fermentation, the process includes centrifuging the fermented broth, collecting the supernatant oily liquid, and then filtering it to obtain fermented vegetable oil. There are no strict limitations on the centrifugation and filtration conditions; for example, the centrifugation speed can be 2000-10000 r / min, and the centrifugation time can be 5-60 min.

[0024] This invention provides a fermented vegetable oil, prepared according to the above-described method. Specifically:

[0025] 1) The fermented camellia seed oil prepared by this invention has an acid value of <15mgKOH / g and a sugar and lipid content of >0.04%. It has multiple effects such as whitening, anti-aging, anti-inflammatory and soothing, barrier repair, easy skin penetration and absorption, and inhibition of acne bacteria.

[0026] 2) The fermented jojoba seed oil prepared by this invention has an acid value of <8mgKOH / g and a sugar and lipid content of >0.03%, and has multiple effects such as whitening, anti-aging, anti-inflammatory and soothing, barrier repair, easy skin penetration and absorption, and inhibition of acne bacteria.

[0027] Beneficial effects

[0028] This invention utilizes the enzymatic fermentation of natural plant oils using bio-enzymes in a cell-free basal fermentation broth. Simultaneously, some active substances such as glycolipids in the basal fermentation broth enter the oil phase, resulting in fermented plant oils containing several times more glycolipids and other components than natural plant oils. This results in multiple benefits compared to natural plant oils, including whitening, anti-aging, anti-inflammatory and soothing effects, barrier repair, easy skin penetration and absorption, and inhibition of acne bacteria.

[0029] Therefore, the method for preparing fermented vegetable oil according to the present invention can improve the quality of fermented vegetable oil and reduce the accumulation of microbial metabolic byproducts during fermentation. Secondly, it can reduce the complexity of downstream separation and extraction processes, thereby lowering production costs. The fermented vegetable oil prepared by the present invention has a high content of free fatty acids, which significantly improves its hydrophilicity and can be used to improve oil-water emulsion systems. Simultaneously, the fermented vegetable oil is rich in natural glycolipids and other active substances, possessing multiple effects such as whitening, anti-aging, anti-inflammatory and soothing, barrier repair, easy skin penetration and absorption, and inhibition of acne bacteria.

[0030] The fermented plant oil prepared by this invention is widely used in formulas such as lotions, creams, ointments, and serums due to its multiple effects, including whitening, anti-aging, anti-inflammatory and soothing, barrier repair, transdermal absorption, and inhibition of acne bacteria. Attached Figure Description

[0031] Figure 1 This is a bar chart comparing the tyrosinase inhibition rates of Examples 1 and 2 of the present invention and the corresponding crude oils.

[0032] Figure 2 These are Raman transdermal images of Examples 1 and 2 of the present invention and the corresponding crude oil.

[0033] Figure 3 The bar chart shows the cell healing rate of Examples 1 and 2 of the present invention and the corresponding crude oil.

[0034] Figure 4 These are cell scratch repair diagrams of Example 1, Comparative Example 1, and their crude oils of the present invention.

[0035] Figure 5 These are cell scratch repair diagrams of Example 2, Comparative Example 2, and their crude oils of the present invention.

[0036] Figure 6 This is a bar chart comparing the collagenase inhibition rates of Examples 1 and 2 of the present invention and their corresponding crude oils.

[0037] Figure 7 This is a comparison chart of the anti-inflammatory factor inhibition rates of Examples 1 and 2 of the present invention and their corresponding crude oils.

[0038] Information on the preservation of biological materials:

[0039] Starmerella bombicola BKSL02 is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​April 9, 2025, with accession number CGMCC No. 34152, and is classified as Starmerella bombicola. Detailed Implementation

[0040] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0041] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0042] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0043] The plant oils described in this invention include, but are not limited to, camellia seed oil, jojoba oil, perilla seed oil, peony seed oil, shea butter, coconut oil, Ganoderma lucidum spore oil, prickly ash fruit oil, almond oil, grape seed oil, sacha inchi oil, rice bran oil, sunflower seed oil, flaxseed oil, Xanthoceras sorbifolium oil, and safflower seed oil. This invention uses camellia seed oil, jojoba oil, perilla seed oil, and shea butter as examples for detailed description.

[0044] The microorganisms described in this invention include, but are not limited to, yeasts, lactic acid bacteria, Bacillus, and Aspergillus. This invention uses *Candida aegypti* strain BKSL02 as an example for detailed description.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] The camellia seed oil, jojoba oil, perilla seed oil, and shea butter in the following examples are all commercially available products.

[0047] The *Starmerella Bombicola* BKSL02 in the following examples is deposited at the China General Microbiological Culture Collection Center, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, date of deposit: April 9, 2025, accession number: CGMCC 34152.

[0048] Sensory evaluation is a very important indicator in the field of cosmetics and skincare products. Therefore, the following examples use sensory evaluation to optimize the fermentation process parameters of fermented plant oils.

[0049] Thirty-five trained evaluators from the customer's product evaluation department conducted sensory evaluations of the vegetable oil before and after fermentation. The transparency, odor and taste were measured in accordance with the trial version of GB / T 5525-2005 "Identification Method of Transparency, Odor and Taste of Vegetable Oils". The average score of the 35 evaluators was the final sensory evaluation score.

[0050] Example 1

[0051] This embodiment takes fermented camellia seed oil as an example and optimizes the fermentation process parameters that affect the fermentation of camellia seed oil through sensory evaluation.

[0052] The specific operating steps for the comparative experiment of different fermentation process parameters are as follows:

[0053] 1) Yeast is fermented in a basic culture medium to obtain a basic fermentation broth rich in bioactive substances such as enzymes and glycolipids;

[0054] 2) Centrifuge the basic fermentation broth from step 1) at 4°C and 10,000 rpm for 10 min, take the upper phase, filter to remove the bacterial cells, and obtain cell-free basic fermentation broth;

[0055] 3) The cell-free basic fermentation broth from step 2) is mixed with the crude camellia seed oil in a certain proportion and fermented at an appropriate temperature for a certain period of time. Sensory evaluation is carried out according to Table 1, and fermentation ends when the requirements are met.

[0056] 4) Centrifuge at 4000 RPM for 5 minutes at room temperature, filter the oil phase, and obtain fermented camellia seed oil.

[0057] Table 1 Sensory rating table for fermented camellia seed oil

[0058]

[0059] 1. Single-factor experiment

[0060] A single-factor experiment was conducted on step 3 above, and the results are shown in Table 2.

[0061] 1) Fermentation temperature

[0062] Fermentation temperature is a crucial parameter in vegetable oil fermentation, directly affecting the activity of the enzyme system catalyzing the hydrolysis of camellia seed oil. Higher enzyme activity leads to more complete hydrolysis of the camellia seed oil; however, excessively high activity can result in over-hydrolysis and off-flavors. Conversely, insufficient activity leads to incomplete hydrolysis, resulting in a sticky texture or slow absorption. Single-factor experiments were conducted using nine temperatures from Table 2, with a 1:3 mass ratio of cell-free fermentation broth to camellia seed oil, and fermentation for 26 hours. Table 2 shows that a fermentation temperature of 28 degrees Celsius yielded the highest sensory score; temperatures below or above 28 degrees Celsius resulted in slightly lower final sensory evaluations.

[0063] 2) Fermentation time

[0064] Fermentation time is also an important process parameter in vegetable oil fermentation. Insufficient fermentation time results in a heavy texture, while excessive fermentation time can easily produce off-odors. A single-factor experiment was conducted at a fermentation temperature of 28 degrees Celsius, with a cell-free basal fermentation broth and camellia seed oil at a mass ratio of 1:3, according to the fermentation times listed in Table 2. Table 2 shows that the highest sensory score of 95.6 points was achieved after 26 hours of fermentation.

[0065] 3) The mixing ratio of cell-free basic fermentation broth to crude camellia seed oil

[0066] The ratio of cell-free basal fermentation broth to crude camellia seed oil is also an important parameter in vegetable oil fermentation. Cell-free basal fermentation broth is rich in biological enzymes; the higher its proportion, the more thoroughly the camellia seed oil is hydrolyzed. However, if its proportion is too high, on the one hand, oil-soluble pigments in the cell-free basal fermentation broth may dissolve into the fermented oil, causing the oil to darken in color; on the other hand, it may lead to excessive hydrolysis of the camellia seed oil, producing an off-flavor. Using 28 degrees Celsius as the fermentation temperature, and following the mixing ratio of cell-free basal fermentation broth to crude camellia seed oil as shown in Table 2, a single-factor experiment was conducted for 26 hours. The results are shown in Table 2. Table 2 shows that when the mixing ratio of cell-free basal fermentation broth to crude camellia seed oil is 1:4, the fermented camellia seed oil achieves the highest comprehensive sensory score of 96.8 points.

[0067] Table 2 Summary of Single-Factor Experiment Results for Fermented Camellia Seed Oil

[0068]

[0069] 2. Orthogonal experiment

[0070] Orthogonal experiments were conducted on three factors with high sensory evaluation scores. Fermentation temperatures were selected at 25℃, 28℃, and 30℃; fermentation times were selected at 21h, 26h, and 32h; and the ratio of cell-free basal fermentation broth to camellia seed oil was selected at 1:3, 1:4, and 1:5. Range analysis was performed using orthogonal experimental design to optimize the process parameters. The results of the orthogonal experiments are detailed in Table 3.

[0071] Table 3 shows that, under the selected factor levels, the order of influence on the sensory evaluation of fermented camellia seed oil from largest to smallest is: fermentation time > ratio of cell-free basal fermentation broth to camellia seed oil > fermentation temperature. The optimal fermentation level is A2B2C2, i.e., fermentation temperature of 28 degrees Celsius, fermentation time of 26 hours, and a ratio of cell-free basal fermentation broth to camellia seed oil of 1:4. Under these fermentation conditions, the fermented camellia seed oil achieved a sensory evaluation score of 96.8, an acid value of 11.2 mgKOH / g, and a sugar and lipid content of 405 mg / kg.

[0072] Table 3. Results of the orthogonal experiment on fermented camellia seed oil

[0073] .

[0074] Example 2

[0075] This embodiment takes fermented jojoba oil as an example and optimizes the fermentation process parameters that affect the fermentation of jojoba oil through sensory evaluation.

[0076] The specific operating steps for the comparative experiment of different fermentation process parameters are as follows:

[0077] 1) Yeast is fermented in a basic culture medium to obtain a basic fermentation broth rich in bioactive substances such as enzymes and glycolipids;

[0078] 2) Centrifuge the basic fermentation broth from step 1) at 4°C and 10,000 rpm for 10 min, take the upper phase, filter to remove the bacterial cells, and obtain cell-free basic fermentation broth;

[0079] 3) The cell-free basic fermentation broth from step 2) is mixed with jojoba oil crude oil in a certain proportion and fermented at an appropriate temperature for a certain period of time. Sensory evaluation is carried out according to Table 4. Fermentation is ended after the requirements are met.

[0080] 4) Centrifuge at 4000 RPM for 5 minutes at room temperature, take the oil phase and filter to obtain fermented jojoba oil.

[0081] Table 4 Sensory rating table for fermented jojoba oil

[0082]

[0083] 1. Single-factor experiment

[0084] A single-factor experiment was conducted on step 3 above, and the results are shown in Table 5.

[0085] 1) Fermentation temperature

[0086] Fermentation temperature directly affects the activity of the enzyme system catalyzing the hydrolysis of jojoba oil. Higher enzyme activity leads to more complete hydrolysis of the jojoba oil; however, excessively high activity results in over-hydrolysis, which can cause off-odors. Conversely, insufficient activity leads to incomplete hydrolysis, resulting in a sticky texture or slow absorption. Using a cell-free fermentation broth to crude jojoba oil ratio of 1:1, and with fermentation temperature as a single factor in Table 5, the sensory evaluation scores after 32 hours of fermentation are detailed in Table 5. Table 5 shows that the highest sensory score of 95.2 was obtained at a fermentation temperature of 30 degrees Celsius.

[0087] 2) Fermentation time

[0088] A single-factor experiment was conducted with a fermentation temperature of 30 degrees Celsius, a cell-free basic fermentation broth, and jojoba oil crude oil in a 1:1 ratio, using the fermentation times listed in Table 5. Table 5 shows that the highest sensory evaluation score (95.2 points) was achieved at 32 hours of fermentation. Fermentation times <32 hours resulted in a thicker, heavier texture, while fermentation times >32 hours produced off-odors.

[0089] 3) The mixing ratio of cell-free basic fermentation broth to jojoba crude oil

[0090] The higher the ratio of cell-free fermentation broth to crude jojoba oil, the more free fatty acids are produced during jojoba oil fermentation. However, an excessively high ratio can cause oil-soluble pigments in the cell-free fermentation broth to dissolve in the fermented oil, resulting in a pale yellow or even yellow color. A single-factor experiment was conducted at a fermentation temperature of 30 degrees Celsius, using the cell-free fermentation broth to crude jojoba oil ratios shown in Table 5. Fermentation lasted 32 hours, and sensory evaluation scores are detailed in Table 5. Table 5 shows that when the ratio of cell-free fermentation broth to crude jojoba oil is 3:2, the highest overall sensory score of the fermented jojoba oil is 96.4 points.

[0091] Table 5 Summary of Single-Factor Experiment Results for Fermented Jojoba Oil

[0092]

[0093] 2. Orthogonal experiment

[0094] Orthogonal experiments were conducted on three factors with high sensory evaluation scores. Fermentation temperatures were selected at 30℃, 32℃, and 35℃; fermentation times were selected at 26h, 32h, and 37h; and the ratios of cell-free basal fermentation broth to jojoba oil were selected at 2:1, 3:2, and 1:1. Range analysis was performed using orthogonal experimental design to optimize the process parameters. The results of the orthogonal experiments are detailed in Table 6.

[0095] Table 6 shows that, under the selected factor levels, the order of influence on the sensory evaluation of fermented jojoba oil from largest to smallest is: fermentation temperature > ratio of cell-free basal fermentation broth to jojoba oil > fermentation time. The optimal fermentation level is A1B2C2, i.e., fermentation temperature of 30 degrees Celsius, fermentation time of 32 hours, and a ratio of cell-free basal fermentation broth to jojoba oil of 3:2. Under these fermentation conditions, the fermented jojoba oil achieved a sensory evaluation score of 96.4, an acid value of 4.6 mg KOH / g, and a sugar and lipid content of 350 mg / kg.

[0096] Table 6 Results of the orthogonal experiment on fermented jojoba oil

[0097]

[0098] Comparative Example 1

[0099] The steps for fermenting camellia seed oil are as follows:

[0100] 1) Yeast is fermented in a basic culture medium to obtain a basic fermentation broth rich in bioactive substances such as enzymes and glycolipids;

[0101] 2) The basic fermentation liquid in step 1) is mixed with the crude camellia seed oil at a ratio of 1:4, fermented at 28℃ for 26 hours, and then the fermentation is completed.

[0102] 3) Centrifuge at 4000 RPM for 5 minutes at room temperature, filter the oil phase, and obtain fermented camellia seed oil.

[0103] Comparative Example 2

[0104] The steps for fermenting jojoba oil are as follows:

[0105] 1) Yeast is fermented in a basic culture medium to obtain a basic fermentation broth rich in bioactive substances such as enzymes and glycolipids;

[0106] 2) The basic fermentation broth in step 1) is mixed with jojoba crude oil in a 3:2 ratio and fermented at 30°C for 32 hours to complete the fermentation.

[0107] 3) Centrifuge at 4000 RPM for 5 minutes at room temperature, filter the oil phase, and obtain fermented jojoba oil.

[0108] The sensory evaluation scores, active ingredient content, and acid value of Example 1, Camellia seed oil crude oil, Comparative Example 1, Example 2, Jojoba oil crude oil, and Comparative Example 2 are detailed in Table 7. Specific analysis is as follows:

[0109] 1. From the perspective of sensory evaluation scores: After fermentation, the scores for color, odor, and transparency of Examples 1 and 2 were slightly lower than those of the corresponding crude oil, but higher than those of the corresponding comparative examples; the skin feel and absorption effect of Examples 1 and 2 were significantly higher than those of the corresponding crude oil, and slightly higher than those of the corresponding comparative examples. The final sensory evaluation score of Example 1 was 96.8 points, and the final sensory evaluation score of Example 2 was 96.4 points, both significantly higher than the total sensory evaluation score of 90.5 points for the corresponding crude oil, and slightly higher than the total sensory evaluation scores of 92.9 points and 92.7 points for the corresponding comparative examples;

[0110] 2. Regarding glycolipid content: Natural vegetable oils themselves do not contain glycolipids. Glycolipids are metabolically active substances produced by microorganisms during fermentation in the basal culture medium. As can be seen from Table 7, the glycolipid content of Examples 1 and 2 is higher than that of Comparative Examples 1 and 2, respectively.

[0111] 3. From the perspective of acid value: Acid value is an assessment method that can indirectly reflect the amount of biological enzymes. The higher the amount of biological enzymes, the faster the hydrolysis of vegetable oil is catalyzed, and the higher the acid value of the vegetable oil per unit time. As can be seen from Table 7, the amount of biological enzymes in Example 1 and Comparative Example 1, and Example 2 and Comparative Example 2 are not significantly different.

[0112] Table 7 Comparison of Sensory Evaluation Scores and Active Ingredients

[0113]

[0114] Test case

[0115] The following test cases respectively tested the whitening, transdermal, cell barrier repair, anti-aging, anti-inflammatory and soothing, and acne-causing effects of Example 1, Example 2 and the corresponding crude oil.

[0116] Test Example 1: Whitening Efficacy Test

[0117] 1. Test samples: Example 1 and its crude oil (1.25% concentration), Example 2 and its crude oil (7.5% concentration);

[0118] 2. Positive control: kojic acid;

[0119] 3. Main reagents: PBS buffer solution, tyrosine solution, tyrosinase solution;

[0120] 4. Instruments: Analytical balance, microplate reader, constant temperature reactor;

[0121] 5. Experimental methods: Using a biochemical method - tyrosinase inhibition test model, the whitening efficacy of cosmetic raw materials and finished products was evaluated by detecting the inhibition of tyrosinase activity in the samples.

[0122] Add the components as per Table 8: solvent background group, solvent reaction group, sample / positive control background group, and sample / positive control reaction group. At the end of the experiment, transfer each reaction solution to the corresponding 96-well plate, with three replicates per group. Read the OD value at 475 nm using a microplate reader after the reaction.

[0123] Table 8 Component Addition Table

[0124]

[0125] "-" indicates that it has not been added, and "+" indicates that it has been added.

[0126] 6. Experimental results are as follows Figure 1 As shown.

[0127] Test Example 2: Skin Penetration Efficacy

[0128] 1. Test samples: Example 1, Example 2, and the corresponding crude oil;

[0129] 2. Test method: The drug was applied to isolated pig skin at a dosage of 2 mg / cm³. 2

[0130] 3. Experimental Instruments: LabRAM Soleil ultra-high resolution confocal Raman microscope;

[0131] 4. Experimental results: see Figure 2 ;

[0132] 5. Experimental results show that Examples 1 and 2 have more significant skin penetration effects than the corresponding crude oil.

[0133] Test Example 3: Barrier Repair - Cell Scratch Assay

[0134] 1. Test samples: Example 1, Comparative Example 1 and camellia seed oil crude oil (1.25% concentration); Example 2, Comparative Example 2 and jojoba oil crude oil (5% concentration);

[0135] 2. Testing Method:

[0136] 1) Cytotoxicity Screening Concentration: Based on keratinocyte cytotoxicity assay, the MTT assay was used to detect the toxic effects of different concentrations of samples on keratinocytes, and the safe working concentration range of the samples was screened for subsequent efficacy evaluation experiments. Seven concentrations were selected for MTT assay, and then one sample concentration with cell viability ≥90% was selected for subsequent efficacy evaluation.

[0137] 2) Experimental Groups: Refer to the experimental groups; for specific dosing regimens, see Table 9:

[0138] Table 9 Dosing Regimen

[0139]

[0140] 3) Adjust cell density to 5 10 5 / ml, place a 6-well plate on it and incubate for 24 hours.

[0141] 4) Scratching: When the cell deposition rate in the 6-well plate reaches approximately 80%, scratch the sample group and control group using a 200μL sterile pipette tip. After scratching, gently wash the cells once with PBS to remove dead cells, observe under an inverted microscope, and take photos at fixed points (4X), with the photos taken at 0h.

[0142] 5) Drug administration: Based on the experimental groups, administer drugs to the selected groups, choosing one sample concentration with cell viability ≥90% and three parallel samples, following the above procedure. The sample volume per well is 0.5 mL. After addition, incubate in an incubator for 24 hours. After incubation, gently wash the cells three times with PBS, observe and photograph them under an inverted microscope (4X) at the previously recorded locations.

[0143] 6) Analysis: Transferred images were analyzed using ImageProPlus software. Cell healing capacity (%) = (initial scratch area - scratch area after 24h) / initial scratch area × 100%.

[0144] 7) Experimental results: See details Figure 3-5 .

[0145] from Figure 3 and Figure 4 As can be seen, Example 1 exhibits a higher cell healing rate and more significant cell migration ability compared to Comparative Example 1 and camellia seed oil crude oil, thus demonstrating a more significant barrier repair effect. Figure 5 As can be seen, Example 2 has a higher cell healing rate and more significant cell migration ability than Comparative Example 2 and jojoba oil crude oil, and therefore has a more significant barrier repair effect.

[0146] Experiment 4: Anti-aging and anti-wrinkle efficacy test

[0147] 1. Test samples: Example 1 and its crude oil (2.5% concentration), Example 2 and its crude oil (7.5% concentration);

[0148] 2. Positive control: Vitamin C;

[0149] 3. Test reagents: collagenase solution, TESC, buffer solution, FALGP solution;

[0150] 4. Experimental instruments: analytical balance, enzyme-linked immunosorbent assay (ELISA) reader, constant temperature reactor, pipette;

[0151] 5. Test methods:

[0152] 1) For each experimental sample, add each component according to the table below. For the experimental results, add 200 μL of the reaction solution to a 96-well plate and read the value at 335 nm using an ELISA reader.

[0153] Table 10 Component Addition Table

[0154]

[0155] 2) Experimental results: See details Figure 6 .

[0156] from Figure 6 It can be seen that Example 1 has a significant collagen-inhibiting effect compared to camellia seed oil crude oil and Example 2 has a significant anti-aging effect compared to jojoba oil crude oil.

[0157] Experiment 5: Anti-inflammatory and soothing trial

[0158] 1. Test samples: Example 1 and its crude oil (0.625% concentration), Example 2 and its crude oil (7.5% concentration);

[0159] 2. Experimental Methods: The inflammatory factor IL-1 was measured using macrophage ELISA. The inhibition rate;

[0160] 3. Experimental Results: See details Figure 7 .

[0161] from Figure 7 It can be seen that Examples 1 and 2 have significantly higher levels of the inflammatory factor IL-1 compared to their corresponding crude oils. It has an inhibitory effect, thus exhibiting more significant anti-inflammatory and soothing effects.

[0162] Experiment 6: Acne Bacterium Inhibition Test

[0163] Test samples: Example 1, Comparative Example 1, rapeseed oil crude oil; Example 2, Comparative Example 2, jojoba oil crude oil.

[0164] Referring to WS / T650—2019, the inhibition zone test was used. Fresh 24-hour slant culture of *Propionibacterium acnes* was washed down with PBS and diluted to 5.0 × 10⁻⁶. 5 CFU / mL - 5.0 × 10 6 CFU / mL available for use.

[0165] Preparation of antibacterial tablets: Cut filter paper with a diameter of 5 mm and a thickness of no more than 4 mm, and soak 4 tablets in 10 mL of each of Example 1, Comparative Example 1, Camellia seed oil, Example 2, Comparative Example 2 and Jojoba oil respectively.

[0166] Preparation of negative control specimens: Take specimens of the same material and make round pieces (blocks) of the same size as those in the test group, and soak them in sterile water.

[0167] Dip a sterile cotton swab into Propionibacterium acnes suspension with a concentration of 5.0×10 5 CFU / mL - 5.0×10 6 CFU / mL, and evenly smear it on the surface of a suitable culture medium plate 3 times. Each time it is smeared, the plate should be rotated 60°. Finally, smear the cotton swab around the edge of the plate once. Cover the petri dish and let it dry at room temperature for 5 min. For each test, place 1 contaminated plate, and stick 4 test specimens and 1 negative control specimen on each plate, a total of 5 specimens. Use sterile forceps to pick up the specimens and stick them on the surface of the plate. Stick the negative control specimen in the center of the plate, and stick the test specimens around. After sticking, gently press the specimens with sterile forceps to make them adhere tightly to the surface of the plate. The distance between the centers of each specimen should be more than 25 mm, and the distance from the periphery of the plate should be more than 15 mm. Cover the plate and incubate it at 36°C ± 1°C for 16 h - 18 h to observe the results. Repeat the test 3 times. Use a vernier caliper to measure the diameter of the inhibition zone (including the patch) and record it. When measuring the inhibition zone, select a uniform and completely sterile growth inhibition zone for measurement, and measure its diameter with the outer edge of the inhibition zone as the boundary.

[0168] No inhibition zone should be produced for the negative control specimen. If the diameter of the inhibition zone of the test sample is > 7 mm, it is judged to have an antibacterial effect; if the diameter of the inhibition zone is ≤ 7 mm, it is judged to have no antibacterial effect. If all 3 repeated tests (a total of 12 specimens) have an antibacterial effect, it is judged to be qualified. The specific results are shown in Table 8 below.

[0169] Table 11 Results of Propionibacterium acnes inhibition test

[0170]

[0171] The results show that: in terms of antibacterial performance, the antibacterial property of Example 1 is weaker than that of Comparative Example 1, and is significantly better than crude camellia seed oil; the antibacterial property of Example 2 is significantly better than the corresponding crude oil.

[0172] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a fermented plant oil with multiple effects including whitening, anti-aging, anti-inflammatory, soothing, repairing, and antibacterial properties, characterized in that, include: 1) Inoculate the bacterial cells into a basic culture medium for fermentation to obtain a basic fermentation broth rich in active substances; 2) Centrifuge and filter the basic fermentation broth to obtain cell-free basic fermentation broth; 3) Fermenting vegetable oils using cell-free basic fermentation broth and natural vegetable oils; 4) Centrifugation, oil phase extraction, and filtration yield fermented vegetable oil; The basic fermentation medium consists of the following components by mass percentage: 0.6%–1.8% carbon source, 0.1%–0.5% nitrogen source, 0.45%–1.2% inorganic salts, and the remainder is deionized water.

2. The preparation method according to claim 1, characterized in that, Before the basic fermentation culture of the bacteria in step 1), the process also includes preparing a seed culture medium and inoculating the bacteria into the seed culture medium to obtain a seed solution.

3. The preparation method according to claim 2, characterized in that, The cultivation conditions include: a cultivation temperature of 25-30℃ and a cultivation time of 30-48h.

4. The preparation method according to claim 1, characterized in that, The natural plant oil mentioned is camellia seed oil. A blend of any one or more of the following oils in any proportion: jojoba oil, perilla seed oil, shea butter, peony seed oil, coconut oil, Ganoderma lucidum spore oil, sea buckthorn oil, almond oil, grape seed oil, sacha inchi oil, rice bran oil, sunflower seed oil, and flaxseed oil.

5. The preparation method according to claim 1, characterized in that, The bacterial cells are selected from one of the following: yeast, lactic acid bacteria, Bacillus, and Aspergillus.

6. The preparation method according to claim 4, characterized in that, In step 3), the fermentation temperature is 20-60℃, the fermentation time is 4-48h, and the ratio of cell-free basic fermentation broth to natural plant oil is 1:0.25-6. Preferably, in step 3), the fermentation temperature is 26-32℃, the fermentation time is 24-34h, and the ratio of cell-free basic fermentation broth to natural plant oil is 1:0.5-5.

7. The preparation method according to claim 6, characterized in that, For camellia seed oil, in step 3), the fermentation temperature is 27-29℃, the fermentation time is 25-27h, and the ratio of cell-free basic fermentation liquid to camellia seed oil is 1:3-5; more preferably, the fermentation temperature is 28℃, the fermentation time is 26h, and the ratio of cell-free basic fermentation liquid to camellia seed oil is 1:

4.

8. The preparation method according to claim 6, characterized in that, For jojoba oil, in step 3), the fermentation temperature is 29-31℃, the fermentation time is 30-34h, and the ratio of cell-free basic fermentation broth to jojoba oil is 3:1-3; more preferably, in step 3), the fermentation temperature is 30℃, the fermentation time is 32h, and the ratio of cell-free basic fermentation broth to jojoba oil is 3:

2. In step 4), the fermented liquid is centrifuged, the upper oily liquid is taken, and then filtered to obtain fermented vegetable oil. Specifically, the centrifugation speed is 2000-10000 r / min and the centrifugation time is 5-60 min.

9. A fermented vegetable oil, characterized in that, Prepared according to the preparation method according to any one of claims 1-8; Specifically, the fermented camellia seed oil prepared has an acid value of <15mgKOH / g and a sugar and lipid content of >0.04%; the fermented jojoba seed oil prepared has an acid value of <8mgKOH / g and a sugar and lipid content of >0.03%.

10. The application of the fermented vegetable oil according to claim 9 in the preparation of daily chemical products, specifically, in the preparation of beauty and skin care products.

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