Fermented vegetable oil, method for preparing the same, and use thereof

Abstract

The application discloses a fermented vegetable oil and a preparation method and application thereof. The preparation method of the fermented vegetable oil comprises the following steps: obtaining a seed solution containing Lip-DYH of Pseudozyma kudriavzevii; fermenting a fermentation tank containing the seed solution and a fermentation medium under preset conditions to obtain a fermentation liquor; and layering the fermentation liquor to collect an upper oil phase, so as to obtain the fermented vegetable oil; wherein the fermentation medium contains vegetable oil, and the vegetable oil comprises at least one of rice oil, peony seed oil, sacha inchi oil and Prinsepia uniflora oil. According to the embodiment of the application, the emulsifying capacity and hydrophilic moisturizing capacity of the vegetable oil can be improved.

Description

Technical Field

[0001] This application belongs to the field of fermentation technology, and in particular relates to a fermented vegetable oil, its preparation method and application. Background Technology

[0002] With the rapid development of the beauty industry, consumers have increasingly higher requirements for cosmetics, making the ingredients and performance of cosmetics receive more and more attention from the industry.

[0003] Plant oils are widely used in cosmetics, but their hydrophobic properties can impair their emulsification and film-forming properties on moist skin, reducing the transdermal absorption of nutrients and limiting their application in cosmetics. Summary of the Invention

[0004] This application provides a fermented vegetable oil, its preparation method, and its application, which can improve the emulsification ability and hydrophilic moisturizing ability of vegetable oil.

[0005] In a first aspect, embodiments of this application provide a method for preparing fermented vegetable oil, comprising: obtaining a seed liquid containing *Candida bumblebee* Lip-DYH; fermenting a fermenter containing the seed liquid and a fermentation medium under preset conditions to obtain a fermentation broth; allowing the fermentation broth to stand and separate into layers, collecting the upper oil phase to obtain the fermented vegetable oil; wherein the fermentation medium contains vegetable oil, and the vegetable oil includes at least one of rice bran oil, peony seed oil, sacha indica oil, and prickly pear fruit oil.

[0006] In any embodiment of this application, the step of obtaining a seed culture containing *Candida bumblebee* Lip-DYH includes: inoculating *Candida bumblebee* Lip-DYH or activated *Candida bumblebee* Lip-DYH into a liquid seed culture medium, and culturing it under shaking conditions at 26-32℃ and 200-220 rpm for 20-30 h to obtain a seed culture containing *Candida bumblebee* Lip-DYH.

[0007] In any embodiment of this application, the liquid seed culture medium comprises, by weight fraction: 2-5 parts yeast extract, 2-6 parts peptone, 1-2 parts ammonium sulfate, 6-15 parts glycerol, 10-100 parts vegetable oil, and 10-20 parts carbon source.

[0008] In any embodiment of this application, the carbon source includes at least one of molasses, glucose, and sucrose.

[0009] In any embodiment of this application, in the step of fermenting a fermenter containing seed liquid and fermentation medium under preset conditions, the amount of seed liquid added is 5%-10% of the total volume of fermentation medium.

[0010] In any embodiment of this application, in the step of fermenting a fermenter containing seed liquid and fermentation culture medium under preset conditions, the fermentation culture medium includes, by weight fraction: 2-5 parts yeast extract, 1-4 parts peptone, 2-5 parts soybean protein, 2-5 parts potassium dihydrogen phosphate, 5-10 parts dipotassium hydrogen phosphate, 1-2 parts ammonium sulfate, 6-15 parts glycerol, 300-500 parts vegetable oil, and 20-80 parts carbon source.

[0011] In any embodiment of this application, the carbon source includes at least one of molasses, glucose, and sucrose.

[0012] In any embodiment of this application, in the step of fermenting a fermenter containing seed liquid and fermentation culture medium under preset conditions, the preset conditions include: temperature of 26-32℃, time of 72-120h, aeration rate of 0.8-1.2vvm, stirring speed of 200-600rpm, pH of 5.0-7.0, and dissolved oxygen ≥10%.

[0013] In any embodiment of this application, the step of allowing the fermentation broth to stand and separate into layers, collecting the upper oil phase, and obtaining fermented vegetable oil includes: centrifuging and dehydrating the collected upper oil phase.

[0014] In any embodiment of this application, the centrifugation and dehydration steps include centrifuging the upper oil phase at 6000-10000 rpm for 5-15 min, collecting the oil phase, adding an adsorbent and stirring for adsorption to achieve dehydration.

[0015] Secondly, embodiments of this application provide a fermented vegetable oil, obtained using the above-described preparation method.

[0016] In any embodiment of this application, the fermented vegetable oil contains biosurfactants, fatty acid triglycerides, diglycerides, and monoglycerides.

[0017] In any embodiment of this application, the content of biosurfactant is 0.2%-1%.

[0018] Thirdly, embodiments of this application provide a cosmetic product comprising fermented plant oil obtained by the above preparation method or the above-described fermented plant oil; the fermented plant oil also includes a skin conditioning agent.

[0019] The fermented vegetable oil, its preparation method, and its application provided in this application utilize *Starmerella bombicola* Lip-DYH to ferment vegetable oil, which degrades some of the triglycerides in the vegetable oil into diglycerides and monoglycerides. Due to the breakage of hydrophobic groups (fatty acids) in the triglyceride structure, the proportion of diglycerides and monoglycerides in the fermented oil increases, thereby improving the hydrophilicity of the fermented vegetable oil. Simultaneously, the broken fatty acids can further combine with sugars to synthesize bio-glycolipid surfactants and with sphingosine to synthesize ceramides, thereby improving the emulsifying ability of the fermented vegetable oil. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments of this application will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The HPLC chromatogram of the reference oil in Example 2 of this application is shown.

[0022] Figure 2 The diagram illustrates the effects of fermented sacha inchi oil and unfermented sacha inchi oil (provided in Example 3 of this application) on scratch repair in human fibroblasts.

[0023] Figure 3 This diagram illustrates a comparison of the cosmetic emulsification stability of fermented sacha in Sacha Intense Oil and unfermented sacha intense Oil provided in Example 4 of this application.

[0024] Figure 4 This diagram illustrates the comparative effect of fermented vegetable oils (fermented peony seed oil, fermented rice bran oil) and non-fermented vegetable oils (unfermented peony seed oil, fermented butter) provided in Example 5 of this application on hydrophilic diffusion ability.

[0025] Figure 5 The diagram shows the effect of fermented vegetable oils (fermented sacha inchi oil, fermented peony seed oil, fermented prickly pear oil, and fermented rice bran oil) provided in Example 6 of this application on improving the DPPH free radical scavenging ability.

[0026] Figure 6 This diagram illustrates the improved moisturizing effect of the fermented vegetable oil provided in Example 7 of this application. Ferm-Lip67 is fermented peony seed oil, and CK is unfermented peony seed oil.

[0027] Figure 7This diagram illustrates the improved oil control effect of the fermented vegetable oil provided in Embodiment 7 of this application. Ferm-Lip67 represents fermented peony seed oil, and CK represents unfermented peony seed oil.

[0028] Figure 8 A schematic diagram illustrating the enhanced anti-inflammatory and soothing effects of fermented peony seed oil in Example 8 of this application is shown. Detailed Implementation

[0029] To better understand the above-mentioned objectives, features, and advantages of this application, the solution of this application will be further described below. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0030] Numerous specific details are set forth in the following description in order to provide a full understanding of this disclosure, but this application may also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of this application, and not all embodiments.

[0031] The following detailed description, with appropriate reference to the accompanying drawings, discloses embodiments of the fermented vegetable oil of this application, its preparation method, and its application. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of essentially identical structures may be omitted. This is to avoid unnecessarily lengthy descriptions and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided for the purpose of enabling those skilled in the art to fully understand this application and are not intended to limit the subject matter of the claims.

[0032] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0033] Unless otherwise specified, all embodiments and optional embodiments of this application may be combined with each other to form new technical solutions, and such technical solutions should be considered to be included in the disclosure of this application.

[0034] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions, and such technical solutions shall be deemed to be included in the disclosure of this application.

[0035] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0036] Unless otherwise specified, all raw materials used in the embodiments of this application were purchased through commercial channels.

[0037] Unless otherwise specified, this application uses conventional testing methods or testing methods recommended by the instrument.

[0038] Existing research on Candida bumblebee mainly focuses on the genetic level. Currently, there is no technical solution that utilizes Candida bumblebee to ferment plant oils, thereby increasing the active substances in the fermented plant oils and improving the performance of cosmetics.

[0039] This application describes the fermentation of specific plant oils by bumblebee Candida albicans to obtain fermented oils containing a variety of active substances such as biosurfactants, fatty acid triglycerides, diglycerides, and monoglycerides. By utilizing the synergistic effect between these active substances, the hydrophilicity and emulsifying ability of the fermented plant oils are improved, thereby further enhancing the performance of cosmetics.

[0040]

Preparation Method

[0041] A method for preparing fermented vegetable oil includes: obtaining a seed culture containing *Starmerella bombicola* Lip-DYH; fermenting a fermenter containing the seed culture and a fermentation medium under preset conditions to obtain a fermentation broth; allowing the fermentation broth to stand and separate into layers, collecting the upper oil phase to obtain the fermented vegetable oil; wherein the fermentation medium contains vegetable oil, including at least one selected from rice bran oil, peony seed oil, sacha inchi oil, and prickly pear fruit oil. The preservation number of *Starmerella bombicola* Lip-DYH is CGMCC No. 25797. The preservation date of the above-mentioned *Starmerella bombicola* Lip-DYH is September 26, 2022, the depository institution is the China Culture Collection Center for Microbial Cultures, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. Rice bran oil refers to the oil obtained by extracting and separating the fat from rice through processes such as extraction. Rice oil is divided into two categories: rice bran oil and rice germ oil. Rice bran oil is extracted from rice bran (the protective outer layer of rice, including the pericarp, seed coat, and endosperm), while rice germ oil is extracted from rice germ (the purified product of rice germ and aleurone layer after precise milling in a clean production workshop). Peony seed oil, also known as peony oil, is an oil extracted from peony seeds, a woody nut plant. It is made from peony seed kernels through processes such as pressing, crude oil filtration, decolorization, deodorization, dewaxing, and precision filtration. Sacha inchi oil is derived from sacha inchi seeds and is a pale yellow, transparent oily liquid produced through processes such as shelling, crushing, pressing, and filtration. Prickly pear oil comes from the prickly pear fruit.

[0042] In some embodiments, the step of obtaining a seed culture containing *Candida bumblebee* Lip-DYH includes: inoculating *Candida bumblebee* Lip-DYH or activated *Candida bumblebee* Lip-DYH into a liquid seed culture medium, and culturing it with shaking at 26-32°C and 200-220 rpm for 20-30 hours to obtain a seed culture containing *Candida bumblebee* Lip-DYH. Activation of *Candida bumblebee* Lip-DYH includes: inoculating *Candida bumblebee* Lip-DYH onto a seed culture medium, and culturing it with shaking at 26-32°C and 200-220 rpm for 20-30 hours. The seed culture medium, by weight fraction, comprises: 2-5 parts yeast extract, 2-6 parts peptone, 1-2 parts ammonium sulfate, 6-15 parts glycerol, 10-100 parts vegetable oil, and 10-20 parts glucose. When each of the above portions is 1g, add pure water to 1L to prepare the seed culture medium, sterilize at 121℃ for 20min, and adjust the pH to 6.8-7.0.

[0043] In some embodiments, the liquid seed culture medium comprises, by weight fraction: 2-5 parts yeast extract, 2-6 parts peptone, 1-2 parts ammonium sulfate, 6-15 parts glycerol, 10-100 parts vegetable oil, and 10-20 parts carbon source. When each of the above components is 1g, pure water is added to 1L to prepare the liquid seed culture medium, which is then sterilized at 121°C for 20min and the pH is adjusted to 6.8-7.0.

[0044] In some embodiments, the carbon source in the liquid seed culture medium includes at least one of molasses, glucose, and sucrose.

[0045] In some embodiments, in the step of fermenting a fermenter containing seed culture and fermentation medium under preset conditions, the amount of seed culture added is 5%-10% of the total volume of the fermentation medium. The amount of seed culture added, i.e., the inoculation amount, is generally calculated based on the fermentation volume (or weight). Before inoculation, the mixture, including the fermentation medium, is sterilized, cooled to obtain a slurry, and then the seed culture is inoculated into the slurry for fermentation; alternatively, a carbon source can be added to the slurry before inoculation with the seed culture containing fermentation bacteria.

[0046] In some embodiments, in the step of fermenting a fermenter containing seed culture and fermentation medium under preset conditions, the fermentation medium, by weight fraction, comprises: 2-5 parts yeast extract, 1-4 parts peptone, 2-5 parts soybean protein, 2-5 parts potassium dihydrogen phosphate, 5-10 parts dipotassium hydrogen phosphate, 1-2 parts ammonium sulfate, 6-15 parts glycerol, 300-500 parts vegetable oil, and 20-80 parts carbon source. When each of the above components is 1g, pure water is added to 1L to prepare the fermentation medium. The medium is then sterilized at 121°C for 20min and the pH is adjusted to 6.8-7.0.

[0047] In some embodiments, the carbon source in the fermentation medium includes at least one of molasses, glucose, and sucrose.

[0048] In some embodiments, in the step of fermenting a fermenter containing seed culture and fermentation medium under preset conditions, the preset conditions include: a temperature of 26-32°C, a time of 72-120 h, an aeration rate of 0.8-1.2 vvm, a stirring speed of 200-600 rpm, a pH of 5.0-7.0, and dissolved oxygen ≥10%. The fermentation time can be controlled by adjusting the dissolved oxygen content. During fermentation, the dissolved oxygen (DO) concentration is affected and constrained by various factors in the fermenter. Generally, we control the DO in the fermenter through aeration, stirring, and feeding.

[0049] In some embodiments, the step of allowing the fermentation broth to stand and separate into layers, collecting the upper oil phase, and obtaining fermented vegetable oil includes: centrifuging and dehydrating the collected upper oil phase. Centrifugation and dehydration can remove impurities from the oil phase.

[0050] In some embodiments, the centrifugation and dehydration steps include: centrifuging the upper oil phase at 6000-10000 rpm for 5-15 min, collecting the oil phase, and then adding an adsorbent for stirring and adsorption to achieve dehydration. The amount of adsorbent added can be 2%-5%, and the adsorbent can be at least one of activated carbon, kaolin, anhydrous magnesium sulfate, and diatomaceous earth.

[0051] Fermented vegetable oil

[0052] A fermented vegetable oil is obtained using the preparation method described above.

[0053] In some embodiments, the fermented vegetable oil contains biosurfactants, fatty acid triglycerides, diglycerides, and monoglycerides. The fatty acid triglycerides, diglycerides, and monoglycerides can improve the hydrophilicity and emulsifying ability of the fermented vegetable oil.

[0054] In some embodiments, the content of biosurfactant is 0.2%-1%. Biosurfactants can enhance the antioxidant capacity of fermented vegetable oils.

[0055] Biosurfactants, fatty acid triglycerides, diglycerides, and monoglycerides can synergistically enhance the hydrophilicity and emulsifying ability of fermented vegetable oils.

[0056]

cosmetic

[0057] A cosmetic product comprises fermented plant oil obtained by the above-described preparation method or the aforementioned fermented plant oil; the fermented plant oil also includes skin conditioning agents. The fermented plant oil contains biosurfactants, fatty acid triglycerides, diglycerides, and monoglycerides, which can improve the emulsification stability, antioxidant capacity, anti-aging capacity, barrier repair function, and film-forming properties (skin feel) of the cosmetic product on moist skin. In particular, different types of plant oils result in different active substances in the fermented plant oil, which can bring different effects to the cosmetic product. When the plant oil is peony seed oil, the fermented plant oil can be used in moisturizing, oil-controlling, soothing, and anti-inflammatory cosmetics.

[0058] Example 1

[0059] Preparation of fermented vegetable oil using bumblebee Candida albicans Lip-DYH:

[0060] The activated Candida bumblebee Lip-DYH was inoculated into liquid seed culture medium and cultured at 30℃ and 200-220 rpm for 24 h to obtain liquid seed (seed liquid containing Candida bumblebee Lip-DYH).

[0061] The prepared liquid seed was transferred into a fermenter containing fermentation medium. The inoculation amount of liquid seed was 5% of the volume of fermentation medium. The dissolved oxygen was controlled at ≥10% under the conditions of 1.0 vvm aeration rate and 200-600 rpm stirring speed. After fermentation culture for 120 h, the fermentation broth was obtained.

[0062] The obtained fermentation broth was sterilized by moist heat at 105℃ for 10 min to inactivate microorganisms. After cooling, it was allowed to stand and separate into layers. The upper oil phase was collected and centrifuged at 8000 rpm for 10 min to remove precipitate and aqueous phase. The upper liquid was collected to obtain crude fermented oil. 2% activated carbon and 1% anhydrous magnesium sulfate were added to the obtained crude fermented oil, and the mixture was stirred and adsorbed at room temperature for 60 min. After filtration, fermented sacha inchi oil was obtained.

[0063] Liquid seed culture medium (g / L): 2g yeast extract, 4g peptone, 1g ammonium sulfate, 10g glycerol, 50g vegetable oil, added to pure water to a volume of 1L. The pH is 6.8-7.0. Sterilize at 121℃ for 20min.

[0064] Fermentation medium (g / L): 2g yeast extract, 2g peptone, 2g soy protein, 3g potassium dihydrogen phosphate, 10g dipotassium hydrogen phosphate, 1g ammonium sulfate, 6g glycerol, 500g vegetable oil, 40g glucose, add pure water to a volume of 1L, pH value of 6.8-7.0, sterilize at 121℃ for 30min; the above vegetable oil is unfermented sacha inchi oil purchased from Shanghai Hongtu Trading Co., Ltd.

[0065] Example 2

[0066] Comparison of oil composition between fermented and unfermented sacha inchi oil:

[0067] Take the fermented sacha in Sacha Ingredient Oil and the unfermented sacha in Sacha Ingredient Oil from Example 1.

[0068] Other reference standards: glyceryl monooleate, glyceryl dioleate, and trioleate were all purchased from Shanghai Sinopharm Group.

[0069] The analytical method was based on the liquid chromatographic analysis of glycerides by Zhong Nanjing et al., and the specific conditions are as follows:

[0070] Chromatographic conditions: Waters 2424-ELSD, reversed-phase liquid chromatography column: C18 (250×4.6mm, 5μm), ELSD parameters: drift tube temperature 85℃, nitrogen flow rate 2.0L / min. Gradient elution conditions are shown in Table 1, detection column temperature 40℃.

[0071] Table 1 Gradient elution conditions for RP-HPLC-ELSD

[0072]

[0073]

[0074] Fermented and unfermented sacha inchi oil were analyzed by HPLC (high performance liquid chromatography) to determine the proportions of triglycerides (TAG), diglycerides (DAG), and monoglycerides (MAG) in the two oils. The results are shown in Table 2.

[0075] Reference standards: The detection results for triglycerides (TAG), diglycerides (DAG), and monoglycerides (MAG) are as follows: Figure 1 As shown.

[0076] Table 2 Comparison of glycerol fatty acid ester composition in vegetable oils and fermented vegetable oils

[0077]

[0078] Table 2 shows that the triglyceride content in fermented Sacha inchi oil is significantly lower than that in unfermented Sacha inchi oil, while the content of diglycerides and monoglycerides is higher. This indirectly verifies the utilization of triglycerides by *Candida bumblebee* yeast. This is because *Candida bumblebee* DYH-01 can degrade some triglycerides into diglycerides and monoglycerides after fermentation. Due to the breakage of hydrophobic groups (fatty acids) in the triglyceride structure, the proportion of diglycerides and monoglycerides in the fermented vegetable oil increases, ultimately increasing the hydrophilicity of the fermented vegetable oil and weakening its hydrophobic properties, making it more hydrophilic and skin-friendly. This results in a good lightweight and skin-adhering effect in cosmetics.

[0079] Example 3

[0080] Improvement of human fibroblast scratch repair by fermented vegetable oil: A comparison of in vitro cell scratch repair experiments between fermented vegetable oil prepared by fermentation of *Candida bacillus* and the original vegetable oil.

[0081] Take the fermented sacha in Sacha Ingredient Oil and the unfermented sacha in Sacha Ingredient Oil from Example 1.

[0082] Human fibroblast scratch assay method:

[0083] (1) Cells: Human fibroblasts HDF-α;

[0084] (2) Sample concentration: 2.5% fermented sacha inchi oil, 2.5% unfermented sacha inchi oil;

[0085] (2) Collect cells in the logarithmic growth phase and seed them into 24-well culture plates at a certain cell density;

[0086] (3) After culturing in an incubator (37℃, 5% CO2) for 24h, use a 200μL pipette tip to draw "damage" in a 24-well plate, wash the cells 3 times with PBS and remove the drawn cells;

[0087] (4) Load the samples according to Table 3 below. Incubate in an incubator (37℃, 5% CO2) for 24 hours, with 3 replicates per group;

[0088] (5) Use an inverted microscope to photograph each group of migrating cells.

[0089] Table 3. Experimental Design for Cell Migration

[0090] Fermented Safflower Oil FBS-free culture medium 2.5％ HDF-α Unfermented tamarisk oil FBS-free culture medium 2.5％ HDF-α

[0091] Fermented and unfermented sacha inchi oil were added separately to culture dishes with scratched cells, and cultured for another 48 hours. Cell migration was then observed, and the results were as follows: Figure 2 As shown. By Figure 2It can be seen that the cell scratch migration rate in the 2.5% fermented sacha inchi oil group was significantly faster than that in the 2.5% unfermented sacha inchi oil group, indicating that fermented plant oil has a stronger ability to promote cell repair than plant oil. This shows that after fermentation, plant oil has a better barrier repair function for human skin cells and can be better applied in skin care products.

[0092] Example 4

[0093] Improvement of cosmetic oil performance: A comparison of the emulsification stability of fermented vegetable oil prepared by bumblebee Candida albicans fermentation with that of raw vegetable oil in cosmetics.

[0094] Fermented and unfermented sacha inoara oil from Example 1 were used to test the emulsification stability of the two oils. The procedure was as follows: 5g of fermented and unfermented sacha inoara oil, 0.1g of Tween 80, and 44.9g of deionized water were accurately weighed into two test tubes respectively. The mixture was emulsified at 12000rpm for 5 minutes, and the emulsification stability was observed after standing. The results are as follows: Figure 3 As shown.

[0095] Figure 3 The left image shows the initial state after emulsification, and the right image shows the state after standing for 20 minutes. Comparing the two images, the cosmetic with fermented sacha inchi oil did not show obvious stratification after 20 minutes of emulsification, while the cosmetic with unfermented sacha inchi oil showed obvious stratification after 20 minutes. This indicates that fermented sacha inchi oil can effectively improve the emulsification stability of cosmetic emulsions.

[0096] Example 5

[0097] Improvement of the performance of cosmetic oils: A comparison of the diffusion capacity of fermented vegetable oil prepared by fermentation of bumblebee Candida albicans and the original vegetable oil.

[0098] The preparation method is the same as in Example 1, except that the vegetable oil used is unfermented rice bran oil or unfermented peony seed oil, which is fermented to obtain fermented rice bran oil and fermented peony seed oil.

[0099] Hydrophilic diffusion experiments were conducted on the fermented rice bran oil, fermented peony seed oil, unfermented peony seed oil, and commercially available fermented butter prepared above. The unfermented rice bran oil, unfermented peony seed oil, and fermented butter were all purchased from Shanghai Hongtu Trading Co., Ltd. The specific procedure was as follows: the surface of a G254 silicone plate was evenly sprayed with pure water until moist. Simultaneously, 50 μl of the aforementioned oils were dripped onto the silicone plate using a spray gun. After diffusion for 2 minutes, the size of the diffusion circle was observed and measured. The results are as follows: Figure 4 As shown, Figure 4The image above shows the diffusion rings and diameters (mm) of unfermented peony seed oil, fermented peony seed oil, fermented rice bran oil, and commercially available fermented butter on a G254 humidity plate. Figure 4 Below is the droplet thickness of the aforementioned oil, according to Figure 4 The results showed that fermented peony seed oil and fermented rice bran oil had higher diffusion abilities on the humidity plate than unfermented peony seed oil and commercially available fermented butter. Fermented peony seed oil, obtained after fermentation, exhibited a 30.4% increase in hydrophilic diffusion ability, indicating that fermentation significantly improves the hydrophilic diffusion of plant oils, enhancing their film-forming properties and skin-adhering feel on moist skin, and expanding their application scenarios in cosmetics.

[0100] Example 6 (Increase in DPPH before and after fermentation of various oils)

[0101] Improved antioxidant effects of fermented vegetable oils: A comparison of the in vitro DPPH free radical scavenging rates between fermented vegetable oils prepared by fermentation with *Candida bacillus* and raw vegetable oils.

[0102] The preparation method is the same as in Example 1, except that the vegetable oil used is unfermented rice bran oil, unfermented peony seed oil, or unfermented sea buckthorn fruit oil. After fermentation, fermented rice bran oil, fermented peony seed oil, and fermented sea buckthorn fruit oil are obtained. All of the above vegetable oils were purchased from Shanghai Hongtu Trading Co., Ltd.

[0103] The DPPH free radical scavenging rates of the fermented rice bran oil, fermented peony seed oil, fermented prickly pear fruit oil, and fermented sacha indicum oil prepared in Example 1, as well as the vegetable oils before fermentation, were tested.

[0104] Antioxidant test

[0105] DPPH radical scavenging test: 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) radical is a stable, long-lived free radical. Its ethanol solution is deep purple and exhibits strong absorption near 515 nm. In the presence of a DPPH radical scavenger, the light absorption of the DPPH ethanol solution is reduced due to the pairing of its unpaired electrons. The degree of fading of the DPPH ethanol solution is linearly related to the number of electrons it accepts. This allows evaluation of the test sample's ability to scavenge free radicals, i.e., its antioxidant activity. The sample addition process is shown in Table 3.

[0106] Table 3. DPPH Experimental Sample Addition Table

[0107]

[0108] The formula for calculating the scavenging rate of DPPH free radicals is as follows:

[0109]

[0110] In the formula:

[0111] A - Absorbance value of the control group, i.e., the absorbance value of the solution after the sample reacts with DPPH;

[0112] B - Zeroing group, solvent and absorbance;

[0113] C - Absorbance of experimental group, sample group;

[0114] D - Absorbance value of the sample's background color;

[0115] E-positive control;

[0116] The final concentration of the sample in the reaction system after dilution with DMSO was 5%, and the final concentration of the reference standard EGCG (epigallocatechin gallate) was 20 ug / ml.

[0117] The results are as follows Figure 5 As shown, compared with fermented plant oils, unfermented sacha inchi oil, unfermented peony seed oil, unfermented prickly pear oil, and unfermented rice bran oil, fermented plant oils all showed improved DPPH free radical scavenging rates. In particular, the DPPH free radical scavenging rate of unfermented rice bran oil increased by 13.48% after fermentation. This indicates that the fermented plant oils of this application can effectively improve the antioxidant capacity of the original plant oils and promote the application of fermented plant oils in skin care cosmetics.

[0118] Implementing Column 7

[0119] Improvements in moisturizing and oil control by fermented plant oils: A comparison of the moisturizing and oil-controlling abilities of fermented plant oils prepared by fermentation with bumblebee Candida albicans with those of raw plant oils.

[0120] The preparation method is the same as in Example 1, except that the vegetable oil used is unfermented peony seed oil. All of the above vegetable oils were purchased from Shanghai Hongtu Trading Co., Ltd., and fermented peony seed oil was obtained after fermentation.

[0121] Tests on human efficacy:

[0122] Healthy Chinese male subjects aged 20-40 years were selected. Sebum secretion on the forehead skin was measured before use and at 0h, 0.5h, 1h, 1.5h, and 2h after product use (half-face test sample, half-face control sample). The difference in sebum secretion before and after product use was compared. Simultaneously, facial skin moisture content and transepidermal water loss were measured to test the moisturizing performance of the samples.

[0123] sample:

[0124] CK: Peony seed oil (unfermented vegetable oil)

[0125] Ferm-Lip67: Fermented Peony Seed Oil

[0126] Test environment conditions: Temperature: 21±1℃; Humidity: 50±5%

[0127] Baseline skin values ​​of subjects were tested before using the sample:

[0128] Laboratory technicians used Sebumeter, Corneometer, and Tewameter to test the subjects' facial skin oil, skin moisture content, and transepidermal water loss rate (measured once on each side of the face).

[0129] After using the sample, the subject's baseline skin values ​​were tested:

[0130] Laboratory technicians used Sebumeter, Corneometer, and Tewameter to test the skin oil, skin moisture content, and transepidermal water loss of the subjects' facial areas at 0h, 0.5h, 1h, 1.5h, and 2h after sample application. Each area was measured five times in parallel (Note: the same instrument and the same operator were used for each subject's test, and the probes were cleaned between measurements). The results of the moisturizing efficacy test are as follows: Figure 6 As shown in the figure, the results of the oil-control efficacy test are as follows: Figure 7 As shown. Figure 6 As shown in Figure A, the facial skin moisture content decreased after using CK (peony seed oil), while it slightly increased after using Ferm-Lip67 (fermented peony seed oil). Furthermore, after 1.5 hours and 2 hours of using Ferm-Lip67 fermented oil, the rate of change in facial skin moisture content showed an increasing trend compared to before using unfermented peony seed oil. Figure 6 As shown in Figure B, this indicates that fermented peony seed oil has a certain short-term moisturizing ability. (For example...) Figure 7 As shown in Figure A, compared to before using both unfermented and fermented peony seed oils, skin oil levels increased after using both oils. However, after 2 hours of using Ferm-Lip67 fermented oil, compared to 1.5 hours after using Ferm-Lip67 fermented oil, facial and forehead oil levels significantly decreased (p < 0.05). Furthermore, after 2 hours of using Ferm-Lip67 fermented oil, the rate of change in skin oil levels showed a decreasing trend, indicating that fermented peony seed oil has good oil-controlling ability, exhibiting a smaller rate of change in skin oil levels and more stable oil-controlling ability compared to unfermented peony seed oil.

[0131] Implementing Column 8

[0132] Improved soothing and anti-inflammatory effects of fermented vegetable oils: A comparison of the soothing and anti-inflammatory effects of fermented vegetable oils prepared by fermentation with bumblebee Candida albicans and raw vegetable oils.

[0133] The preparation method is the same as in Example 1, except that the vegetable oil used is unfermented peony seed oil, which was purchased from Shanghai Hongtu Trading Co., Ltd., and fermented peony seed oil was obtained after fermentation.

[0134] Soothing and anti-inflammatory test method:

[0135] (1) LPS treatment of HaCaT cells

[0136] 1. Immortalized keratinocytes were cultured in a solution containing 10% fetal bovine serum and 1% penicillin-dextrose antibody (1×10⁻⁶). 5 Cells were cultured in DMEM medium containing 100 mg / L penicillin and 100 mg / L streptomycin. Cells were grown at 37°C in a 5% CO2 incubator until cell confluence reached 85%-95%.

[0137] 2. Digest logarithmically growing cells with 0.05% trypsin, and terminate the digestion reaction with DMEM medium containing 10% serum;

[0138] 3. Count the cells using a cell counting chamber, seed the appropriate number of cells into 6-well plates, and incubate at 37°C and 5% CO2 for a certain period of time until the cell confluence reaches 60%-70%;

[0139] 4. Replace the original culture medium in the six-well plate with serum-free medium, starve the plate for two hours, add the corresponding active ingredient for four hours, and then add 1 ug / ml LPS for 24 hours of stimulation.

[0140] 5. Add Trizol and collect the cells.

[0141] (2) RNA extraction

[0142] 1. Cell sampling (operation on ice): Wash cells cultured in 6-well plates twice with pre-cooled sterile PBS, add 500 μl of TRIzol to each well, gently pipette and collect the cell lysate into 1.5 ml EP tubes.

[0143] 2. Pre-cool the product in a high-speed centrifuge at 4°C, centrifuge at 12,000 rpm for 10 min, transfer the supernatant to a new RNase-FREE 1.5 ml EP tube, and discard the tissue precipitate.

[0144] 3. Add 200ml of chloroform to the clear liquid, invert and shake 15 times to mix the liquid thoroughly, keep the cap closed and let it stand at room temperature for 5 minutes to allow it to separate naturally.

[0145] 4. Centrifuge at 4℃ and 12000rpm for 15min. Carefully aspirate the supernatant aqueous phase into a new RNase-free 1.5ml EP tube. Take special care not to touch the central precipitate to avoid protein contamination.

[0146] 5. Add 500 μl of isopropanol to the supernatant, gently invert to mix, keep the cap closed and let stand at room temperature for 5 min to allow the mRNA to be fully extracted.

[0147] 6. Centrifuge at 4℃ and 12000rpm for 10min, discard the supernatant and add 75% ethanol solution prepared with DEPC water, then gently invert to wash the mRNA precipitate.

[0148] 7. Centrifuge at 4℃ and 7500rpm for 5 minutes, discard the supernatant and wash again with 75% ethanol solution.

[0149] 8. Centrifuge at 4℃ and 7500rpm for 5 minutes, discard the supernatant and remove as much residual liquid as possible, and let stand at room temperature for 30-60 minutes with the lid open to allow residual ethanol to evaporate.

[0150] 9. Add an appropriate amount of DEPC water to dissolve the mRNA, determine the concentration, and perform reverse transcription according to the instructions of the reverse transcription kit.

[0151] (3) Reverse transcription PCR

[0152] System (20 μl): 10× buffer 2 μl, 2.5 mM dNTP 2 μl, DNA polymerase (Taq) 0.5 μl, primer 2 μl, template (cDNA obtained from reverse transcription diluted 10 times as template) 2-10 μl, ultrapure water to make up to 20 μl.

[0153] Reaction conditions: Pre-denaturation 94℃, 5 min; 30 cycles: denaturation 94℃, 30 s, annealing 60℃, 30 s, extension 72℃, 30 s; post-extension 72℃, 10 min.

[0154] (4) Quantitative Real-Time PCR (QPCR) experiment

[0155] System (20 μl): 10 μl of 2×SYBR, 2-5 μl of template (cDNA obtained by reverse transcription diluted 10 times as template), 1-2 μl of primer, and ultrapure water to make up to 20 μl.

[0156] Reaction conditions: Pre-denaturation 94℃, 5 min; 40 cycles: denaturation 94℃, 30 s, annealing 60℃, 30 s, extension 72℃, 30 s (real-time fluorescence photography); melting curve 94℃, 30 s, 60℃, 30 s, 72℃, 1 s (real-time fluorescence photography during heating process).

[0157] The expression levels of inflammatory factors at the mRNA level were determined in unfermented peony seed oil and fermented peony seed oil, respectively. The results are as follows: Figure 8As shown, fermented peony seed oil significantly inhibited the expression of LPS-induced inflammatory factors IL-6 and IL-8 compared to unfermented peony seed oil, indicating that fermented plant oils have significant soothing and anti-inflammatory effects, greatly expanding the application of fermented oils in cosmetics.

[0158] The above description is merely a specific implementation of this application. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems, modules, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here. It should be understood that the protection scope of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the protection scope of this application.

Claims

1. A method for preparing fermented vegetable oil, characterized in that, include: Obtain Lip-DYH containing bumblebee Candida albicans ( Starmerella bombicola The seed culture of the bumblebee Candida Lip-DYH is described in the CGMCC No. 25797. The fermenter containing the seed liquid and fermentation culture medium is fermented under preset conditions to obtain fermentation broth; The fermentation broth was allowed to stand and separate into layers. The upper oil phase was collected and centrifuged and dehydrated to obtain the fermented vegetable oil. The fermentation medium contains vegetable oil, which includes at least one of rice bran oil, peony seed oil, sacha inchi oil, and prickly pear fruit oil.

2. The preparation method according to claim 1, characterized in that, The steps for obtaining a seed culture containing *Candida bumblebee* Lip-DYH include: Inoculate liquid seed culture medium with either *Candida baccata* Lip-DYH or activated *Candida baccata* Lip-DYH and culture at 26-32℃ and 200-220 rpm for 20-30 h to obtain seed culture containing *Candida baccata* Lip-DYH.

3. The preparation method according to claim 2, characterized in that, The liquid seed culture medium comprises, by weight fraction: 2-5 parts yeast extract, 2-6 parts peptone, 1-2 parts ammonium sulfate, 6-15 parts glycerol, and 10-100 parts vegetable oil.

4. The preparation method according to claim 1, characterized in that, In the step of fermenting the fermenter containing the seed liquid and the fermentation medium under preset conditions, the amount of seed liquid added is 5%-10% of the total volume of the fermentation medium.

5. The preparation method according to claim 1, characterized in that, In the step of fermenting the fermenter containing the seed liquid and the fermentation medium under preset conditions, the fermentation medium comprises, by weight fraction: 2-5 parts yeast extract, 1-4 parts peptone, 2-5 parts soybean protein, 2-5 parts potassium dihydrogen phosphate, 5-10 parts dipotassium hydrogen phosphate, 1-2 parts ammonium sulfate, 6-15 parts glycerol, 300-500 parts vegetable oil, and 20-80 parts carbon source.

6. The preparation method according to claim 5, characterized in that, The carbon source includes at least one of molasses, glucose, and sucrose.

7. The preparation method according to claim 1, characterized in that, In the step of fermenting the fermenter containing the seed liquid and fermentation medium under preset conditions, the preset conditions include: The temperature is 26-32 ℃. The time is 72-120 hours. Ventilation rate: 0.8-1.2 vvm Stirring speed 200-600 rpm, pH=5.0-7.0, Dissolved oxygen ≥10%.

8. The preparation method according to claim 1, characterized in that, The centrifugation and dehydration steps include: After centrifuging the upper oil phase at 6000-10000 rpm for 5-15 min, the oil phase is collected, and then an adsorbent is added for stirring and adsorption to dehydrate the oil.

Images