A method for producing a fermented vegetable oil

Abstract

This invention relates to a method for preparing fermented vegetable oil, characterized by the following steps: A) Using glucose as the carbon source and yeast extract as the nitrogen source, frozen yeast is activated. The frozen yeast is either *Candida shurata* or *Yarrowia lipolyticis*. Once activation is complete, a seed culture is obtained. B) The obtained seed culture is inoculated into malt extract liquid culture medium and fermented in shake flasks at 28°C and 100-150 rpm for 13-20 hours to obtain a primary fermentation broth. The malt extract liquid culture medium contains 130 g / L malt extract powder and 0.1 g / L chloramphenicol. C) The primary fermentation broth is used as the aqueous phase, and vegetable oil is used as the oil phase. The aqueous and oil phases are mixed at a volume ratio and fermented for another 1-4 days to obtain a secondary fermentation broth. D) The secondary fermentation broth obtained in step C is centrifuged at 4°C and 8000-13000 rpm for 5-20 minutes. After centrifugation, the upper oily liquid is collected to obtain the fermented vegetable oil. The obtained fermented vegetable oil exhibits good anti-inflammatory and antioxidant effects.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a method for preparing fermented vegetable oil. Background Technology

[0002] As we age, the structure and function of our skin undergo degenerative changes. In adverse environments such as high temperatures and dryness, the skin's health can also be accelerated. Aging skin experiences reduced lipid secretion, decreased keratinocyte renewal capacity, epidermal atrophy and thinning, weakened barrier function, and increased transepidermal water loss. Clinically, this manifests as pale, dry skin with fine flaking, roughness, and cracking.

[0003] Oils, as raw materials in cosmetics, can be classified as skin moisturizers based on their uses and properties. They can reduce skin moisture loss, protect the skin barrier, improve dry skin, significantly increase skin hydration and elasticity, prevent skin damage, and promote the repair of damaged skin.

[0004] Plant-based oils typically contain various unsaturated fatty acids, have low melting points, and are liquid at room temperature. Furthermore, most plant-based oils are rich in vitamin E, among other nutrients. These properties make them widely used as raw materials in skincare products and in the manufacture of cosmetics and skincare products. However, the fatty acid ratios of different plant-based oils vary, resulting in differences in their moisturizing power, skin feel, and oxidative stability.

[0005] In cosmetic production, plant oils are generally used in different types of dosage forms according to the formulation requirements. However, plant oils are natural high-molecular-weight compounds composed of fatty acids and glycerol linked by ester bonds. Because they are not composed of a single chemical structure, they exhibit different properties depending on the raw materials used in their preparation, making them difficult for human skin to absorb directly.

[0006] The main reason why plant oil skincare products have a poor skin feel is that plant oils contain a small number of large free molecular groups that are not easily absorbed by the skin. Therefore, there is a need to provide a way to break down the large free molecular groups in plant oils into smaller molecules so that the plant oils can be more easily absorbed by the skin.

[0007] Meanwhile, in cosmetic applications, the emulsifying properties of oils also affect the product's stability. An emulsion is a system formed by two immiscible liquid phases, generally oil and water, dispersed as tiny particles (droplets or liquid crystals) within another phase. To ensure the emulsion remains stable over a longer period, emulsifiers are usually added to reduce the interfacial tension of the dispersion, thus creating a relatively stable system. However, oil-water emulsions are inherently unstable, and their stability is generally determined by selecting suitable emulsifiers, reducing interfacial tension, and increasing the strength of the interfacial film.

[0008] On the other hand, oil oxidation has a great impact on its quality. This is mainly because oxides are produced during the oxidation process, which in turn produce small molecules such as aldehydes, ketones, and acids. When the concentration of these small molecules increases, they produce an irritating odor, commonly known as rancid or greasy smell.

[0009] Chinese invention patent CN106619231B discloses a method for preparing fermented vegetable oil. This method achieves no impact on the acid value, saponification value, iodine value, and peroxide value of the vegetable oil itself through fermentation. Infrared spectral analysis of the fermented vegetable oil and the unfermented vegetable oil showed that the characteristic absorption peak of hydroxyl groups at 3500 cm⁻¹ almost disappeared after fermentation, and the peak value was significantly lower than before fermentation, proving that the free hydroxyl groups in olive oil were reduced after fermentation. This patent uses yeast or lactic acid as a fermenting agent, requiring multiple aeration processes to volatilize carbon dioxide, which can easily lead to contamination during fermentation. Frequent manual aeration is also necessary, making the preparation method complex and cumbersome.

[0010] Chinese patent CN109010140 A discloses a fermented oil, its preparation method, and its uses. By adding different proportions of vegetable oils and then mixing and fermenting them, the resulting fermented oil is safe, non-irritating, has a good skin feel, antioxidant capacity, good oxidative stability, and the ability to repair the skin barrier. This patent utilizes the fungus *Mucor*, which reacts with isopropanol to prepare an oily liquid. This liquid is then mixed with vegetable oil to obtain a mixed oil, which is then inoculated with *Fonotus strobilus* for fermentation. The oil requires preparation using *Mucor* before fermentation with *Fonotus strobilus*, making the process complex and time-consuming.

[0011] Korean Patent Publication No. 2012-0076170 discloses a "cosmetic oil composition prepared by fermenting a combination of vegetable oils in a culture medium". The process involves substances such as vegetable oils, vegetable butters, vegetable oil alcohols, and sterols, which leads to a complex composition of fermented oils and increases the factors that cause instability in product quality.

[0012] Chinese patent CN106420404A discloses a fermented plant oil cosmetic composition with antioxidant effects. This method utilizes several plant oils in different proportions fermented under the action of microorganisms of the genus *Pseudozyma* (Basidiomycetes) to obtain fermented plant oils suitable for use in cosmetics. The fermented oil prepared by this method exhibits improved antioxidant activity, emulsifying activity and emulsion stability, significantly increased free fatty acid content, and improved user experience and moisturizing power. However, the culture medium formulation in this patent is very complex, requiring the preparation of complex plant oil compositions for both the first and second fermentations. Furthermore, the first fermentation process using *Pseudozyma* microorganisms (KCTC 8950P) takes 30-60 hours, which is too time-consuming and results in low production efficiency.

[0013] Existing vegetable oil fermentation technology still has the following problems: the process of using mold to prepare fermented oil requires oxygen, and the intake and exhaust of air need to be balanced during the oxygen-assisted fermentation process. Mold or multiple bacteria participating in the preparation process are prone to cross-contamination, which is not conducive to production stability. Summary of the Invention

[0014] The technical problem to be solved by the present invention is to provide a method for preparing fermented vegetable oil, which has the advantages of simple process steps and high fermentation efficiency, and the resulting fermented vegetable oil has good anti-inflammatory and antioxidant effects.

[0015] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a method for preparing fermented vegetable oil, characterized by the following steps:

[0016] A. Using glucose as the carbon source of the culture medium and yeast extract as the nitrogen source of the culture medium, the frozen yeast was activated; the frozen yeast was Candida huhatana, with preservation number CGMCC 2.4317 or Yarrowia lipolytica, with preservation number CGMCC2.1291. Seed liquid was obtained after activation.

[0017] B. Inoculate the seed liquid obtained in step A into malt extract liquid culture medium at a volume ratio of 1%; ferment in a shake flask at 28℃ and 100-150 rpm for 13-20 hours to obtain the primary fermentation broth; the malt extract liquid culture medium contains 130 g / L malt extract powder and 0.1 g / L chloramphenicol.

[0018] C. Take the primary fermentation broth obtained in step B as the aqueous phase and vegetable oil as the oil phase. Mix the aqueous phase and oil phase according to the volume ratio, wherein the proportion of oil phase is 10-90%. Continue fermentation for 1-4 days after mixing. After fermentation is completed, the secondary fermentation broth is obtained.

[0019] D. Centrifuge the secondary fermentation broth obtained in step C at 4℃ and 8000-13000 rpm for 5-20 minutes. After centrifugation, take the upper oily liquid to obtain fermented vegetable oil.

[0020] Furthermore, the vegetable oil is one of perilla seed oil, safflower seed oil, camellia oil, peony seed oil, or a mixture thereof in any proportion.

[0021] The unsaturated fatty acid content of the vegetable oil is greater than or equal to 60%.

[0022] This invention utilizes a single strain (Candida shurata or Yarrowia lipolyticis) to cultivate and ferment various plant oils, which can decompose triglycerides into fatty acids to reduce the molecular weight of oils and improve their multiple sensory properties; generate new polar lipids to improve their emulsification properties; and increase the flavonoid content of oils after fermentation. The resulting fermented plant oils have antioxidant and anti-inflammatory effects and can be directly applied to cosmetics.

[0023] The present invention underwent the following experiments and tests:

[0024] 1. Acid value test: The experimental method shall be performed in accordance with the QS-TM-16 standard.

[0025] 2. Sensory comparison test: Experimental method: The blank group (perilla seed oil), control group and fermented perilla seed oil were scored and compared in seven dimensions: spreadability, oiliness, softness, absorbency, gloss, smell and color. The higher the score, the better the positive performance.

[0026] 3. Emulsion stability test: Experimental method: Mix oil and water in a ratio of 1:9, then mix the four samples evenly with the same force, and observe the separation speed of each sample.

[0027] 4. Contact Angle Test: Experimental Method: Using a JY-82 contact angle meter, 50μL of different oils were dropped onto PMMA sheets (density approximately 1.2g / cm3). The contact angle was between 0 and 90 degrees. The smaller the contact angle, the better the wettability.

[0028] 5. Viscosity test: Experimental method: Refer to viscosity test standard GBT2794-2013.

[0029] Measurement temperature: 25℃; Sample container size (or rotor / sample cup geometry): 34# rotor; Original standard test container; Viscometer model: Brookfield DV2T; Test speed (or shear rate): 200 rpm; Measurement time: 30 minutes.

[0030] The experimental results are shown in Table 1 and Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 Among them, FP06 sample was fermented perilla seed oil prepared in Example 1, FP08 sample was prepared in Example 2, FP05 sample was blank group (which was untreated perilla seed oil), and FP07 was control group (a mixture of malt juice liquid culture medium and perilla seed oil, without the addition of seed liquid).

[0031] Table 1. Changes in acid value, sensory properties, emulsifying properties, contact angle, and viscosity of perilla seed oil before and after fermentation.

[0032]

[0033] According to Table 1 and Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 The results showed that the acid value of fermented perilla seed oil increased by approximately 60 times. This is because the microorganisms decomposed glycerides into free fatty acids, diglycerides, monoesters, or glycerol at the water-oil interface during fermentation, reducing the molecular weight of the oil. Simultaneously, the generated free fatty acids and sugars were esterified during fermentation, producing polar lipids such as glycolipids, altering the emulsification and sensory properties of the oil. Observations showed that the water-oil separation rate of fermented perilla seed oil was slow during the standing process after emulsification. The emulsification stability was observed in the following order: FP08 > FP06 > blank group = control group. Measurements showed that after fermentation by both microorganisms, the contact angle of perilla seed oil on PMMA substrates decreased to varying degrees, indicating improved wetting at the liquid-solid interface. The viscosity of the fermented perilla seed oil also decreased. These characteristics suggest that fermented perilla seed oil has greater potential for application in the cosmetics industry.

[0034] The present invention also conducted the following tests on fermented vegetable oils.

[0035] Experimental method: The material used was a TLC Silica G 60 silica gel plate. The solvent phase was prepared as hexane:benzene:acetic acid (volume ratio 70:30:2). The mixture was placed in a chromatography tank and thoroughly mixed. The dried sample plate was then placed in the chromatography tank, and chromatography was started with the liquid level kept below the marked sample position. The chromatography was stopped when the liquid level of the developing phase reached the marked position on the glass plate. The silica gel plate was then removed, dried, and stained in a container containing iodine vapor for 5-10 minutes. After staining, the plate was removed for sample spot analysis.

[0036] The experimental results of TLC board analysis are as follows: Figure 9As shown, the blank is unfermented perilla seed oil, the control is a mixture of malt extract liquid culture medium and perilla seed oil, without the addition of seed liquid; sample FP06 was prepared in Example 1, and sample FP08 was prepared in Example 2. Figure 9 The comparison between the blank, control, and experimental groups clearly shows the generation of free fatty acids in the fermented oils (FP06 and FP08), indicating that the fermentation process hydrolyzed triglycerides; and two new polar lipid substances were found to be generated, indicating that the fermentation process produced new substances.

[0037] Experimental Method: Mix an equal volume (750 μL) of sample solution with DPPH solution and name the tube A1. Mix an equal volume (750 μL, or the sample solution solvent) with DPPH solution and name the tube A2. Mix an equal volume (750 μL) of sample solution with anhydrous ethanol and name the tube A3. Use vitamin C solution as a positive control and distilled water as a negative control. Perform three replicates for each sample group. After reacting in the dark for 30 min, measure the absorbance of A1, A2, and A3 at 517 nm using an ELISA reader.

[0038] Calculation of results: Free radical scavenging rate (%) = [(A2+A3)-A1] / A2

[0039] In this experiment, the oil sample was diluted before testing, with a dilution ratio of sample:DMSO = 1:9.

[0040] The experimental results are shown in Table 2.

[0041] Table 2 Comparative experimental data on the DPPH scavenging ability of fermented perilla seed oil

[0042]

[0043] According to the test results in Table 2, the DPPH scavenging ability of fermented perilla seed oil was improved. Among them, the DPPH scavenging ability of FP08 was slightly higher than that of FP06. However, after the perilla seed oil fermented by the two strains was diluted ten times with DMSO, there was no significant difference in the DPPH scavenging ability (p < 0.05).

[0044] Experimental method for flavonoid content determination: The relevant operations were performed according to the micro-method of the plant flavonoid content detection kit produced by Sangon Biotech (Shanghai) Co., Ltd. The experimental results are shown in Table 3.

[0045] Table 3 Comparative experimental data on flavonoid content of fermented perilla seed oil

[0046]

[0047] As shown in Table 3, the fermented perilla seed oil exhibited significant improvements compared to both the control and blank groups, with little difference between the two. The results obtained in this experiment show a similar trend to those obtained from the DPPH scavenging capacity test, suggesting that the antioxidant activity of the fermented perilla seed oil originates from the bacterial strain breaking down large flavonoid molecules in the oil into smaller flavonoid molecules during fermentation, thereby enhancing the oil's antioxidant activity.

[0048] This invention also conducted an anti-inflammatory efficacy experiment on fermented vegetable oil.

[0049] (1) Effects of four fermentation oil samples on the proliferation activity of Raw264.7 cells

[0050] Resuscitate the cells by thawing the frozen Raw264.7 cells and seeding them in DMEM medium containing 10% FBS to ensure cell status and passage number.

[0051] The samples were diluted to different concentrations using DMEM medium.

[0052] CCK-8 assay for cell proliferation activity of samples: After incubation for 24 h, 100 μL of CCK-8 working solution was added to each well, and the samples were incubated in a CO2 incubator for 30 min. The absorbance was then measured at 450 nm.

[0053] The experimental results were statistically analyzed using Excel.

[0054] (2) CCK-8 assay to detect the effects of four fermentation oils on the proliferation of Raw264.7 cells

[0055] Resuscitate the cells by thawing the frozen Raw264.7 cells and seeding them in DMEM medium containing 10% FBS to ensure cell status and passage number.

[0056] The samples were diluted to 0.1% using DMEM medium and added to cell seeding plates. The following control groups were set up: blank control group (BC): normally cultured cells without any treatment; model control group (NC): cells induced by LPS; and positive control group (Dex): cells treated with LPS and dexamethasone. The cell culture plates were then incubated at 37 ℃ and 5% CO2 for 24 h, with 100 μL of culture medium per well.

[0057] After the culture is complete, NO is detected according to the NO reagent kit method.

[0058] The experimental results were statistically analyzed using Excel, and bar charts were created using Graph software.

[0059] The experimental results are shown in Figure 10 , Figure 11 , Figure 12, Figure 13 , Figure 10-13 A statistical graph showing the effects of four fermentation oils on the proliferation activity of Raw264.7 cells as detected by CCK-8 assay.

[0060] according to Figure 10 , Figure 11 , Figure 12 , Figure 13 The results show that, compared with the blank control group, sample FP05 showed no cytotoxicity at concentrations ≤0.4%; sample FP06 showed no cytotoxicity at concentrations ≤0.1%; sample FP07 showed no cytotoxicity at concentrations ≤0.4%; and sample FP08 showed no cytotoxicity at concentrations ≤0.2%. A concentration of 0.1% was consistently used in the formal experiments.

[0061] Compared with the control group, the LPS-induced group showed an increased NO release rate, indicating successful model establishment. Samples FP05, FP06, and FP08 significantly inhibited NO release; sample FP07 showed a NO inhibition rate of less than 20%, indicating no effect on inhibiting NO release, while the positive control dexamethasone showed a NO inhibition rate of 55.41%.

[0062] The experimental results are shown in Figure 14 And Table 4.

[0063] Table 4. NO inhibition rate of four samples

[0064]

[0065] According to Table 4 and Figure 14 As shown, samples FP06 and FP08 significantly inhibited NO release by more than 65%, which was higher than the inhibition effect of FP05 (27.25%); sample FP07 had a NO inhibition rate of less than 20% and no effect on inhibiting NO release; the positive control dexamethasone had a NO inhibition rate of 55.41%. In summary, the NO release inhibition rate of perilla seed oil was significantly improved after fermentation, and the fermentation process improved its anti-inflammatory activity.

[0066] In summary, the fermented plant oil prepared by this invention has significant improvements in multiple sensory properties such as spreadability, softness, absorbability, and gloss. It generates new polar lipids with good emulsifying properties; it has a high flavonoid content, thus exhibiting good antioxidant and anti-inflammatory effects, and can be directly applied to cosmetics.

[0067] This invention also tested the changes in flavonoid content in safflower seed oil, camellia oil, and peony seed oil before and after fermentation with Candida huhatana. The experimental results are shown in Table 5.

[0068] Table 5. Changes in flavonoid content before and after fermentation of different vegetable oils.

[0069]

[0070] As shown in Table 5, after fermentation of safflower seed oil, camellia seed oil, and peony seed oil by *Candida shurata*, the flavonoid content increased to varying degrees compared to the crude oil. Furthermore, in the example of fermenting perilla seed oil, the influence of the culture medium itself was excluded. It can be inferred that *Candida shurata*, after fermenting other plant oils including the aforementioned oils, can increase the original flavonoid content, thereby enhancing its anti-inflammatory activity.

[0071] In summary, the present invention has the advantages of simple process steps and high fermentation efficiency, and the fermented vegetable oil obtained has good anti-inflammatory and antioxidant effects. Attached Figure Description

[0072] Figure 1 Photograph of fermented vegetable oil before it is mixed with water;

[0073] Figure 2 Photograph taken after fermenting vegetable oil and water for 1 minute;

[0074] Figure 3 Photograph taken after fermenting vegetable oil and water for 30 minutes;

[0075] Figure 4 Photograph of fermented vegetable oil mixed with water for 2 days;

[0076] Figure 5 Photograph of the contact angle test results for sample FP05;

[0077] Figure 6 Photograph of the contact angle test results for sample FP07;

[0078] Figure 7 Photograph of the contact angle test results for sample FP06;

[0079] Figure 8 Photographs showing the contact angle test results for sample FP08;

[0080] Figure 9 Comparison of components of perilla seed oil before and after fermentation for TLC plate analysis;

[0081] Figure 10 This is a statistical graph showing the effect of sample FP05 on cell proliferation activity;

[0082] Figure 11 This is a statistical graph showing the effect of sample FP06 on cell proliferation activity.

[0083] Figure 12 This is a statistical graph showing the effect of sample FP07 on cell proliferation activity;

[0084] Figure 13 This is a statistical graph showing the effect of sample FP08 on cell proliferation activity.

[0085] Figure 14 This is a schematic diagram showing the experimental results of the inhibitory effect of samples FP05, FP06, FP07 and FP08 on NO. Detailed Implementation

[0086] Example 1: A method for preparing fermented vegetable oil, comprising the following steps:

[0087] The frozen Candida shchuata (CGMCC 2.4317) was activated using glucose as the carbon source in the culture medium at a concentration of 40 g / L and yeast extract as the nitrogen source in the culture medium at a concentration of 17 g / L to obtain the seed culture.

[0088] The seed culture was inoculated into malt extract liquid medium at an inoculation rate of 1%, and fermented in shake flasks at 28°C and 150 rpm for 13 hours to obtain the primary fermentation broth.

[0089] The primary fermentation broth was transferred to a culture medium containing perilla seed oil for further fermentation, with a water-to-oil ratio of 4:6 after transfer. The secondary fermentation conditions were the same as the primary fermentation, and the fermentation time was 3 days, yielding a water-oil mixture in the secondary fermentation broth.

[0090] The mixed liquid obtained from the secondary fermentation was centrifuged at 8000 rpm and kept at 4℃ for 20 min to separate the upper oily liquid; the sample obtained was fermented perilla seed oil and named FP06.

[0091] Example 2: A method for preparing fermented vegetable oil, comprising the following steps:

[0092] Using glucose as the carbon source in the culture medium at a concentration of 40 g / L and yeast extract as the nitrogen source in the culture medium at a concentration of 8 g / L, the frozen Yeast lipolytica (CGMCC 2.1291) was activated to obtain the seed culture.

[0093] The seed culture was inoculated into malt extract liquid medium at an inoculation rate of 1%, and fermented in shake flasks at 28°C and 120 rpm for 17 hours to obtain the primary fermentation broth.

[0094] The primary fermentation broth was transferred to a culture medium containing perilla seed oil for further fermentation, with a water-to-oil ratio of 1:7 after transfer. The secondary fermentation conditions were the same as the primary fermentation, and the fermentation time was 4 days, yielding a water-oil mixture in the secondary fermentation broth.

[0095] The mixed liquid obtained from the secondary fermentation was centrifuged at 10,000 rpm and kept at 4°C for 15 min to separate the upper oily liquid; the sample obtained was fermented perilla seed oil and named FP08.

[0096] Example 3: A method for preparing fermented vegetable oil, comprising the following steps:

[0097] Using glucose as the carbon source in the culture medium at a concentration of 25 g / L and yeast extract as the nitrogen source in the culture medium at a concentration of 10 g / L, the frozen Yeast lipolyticum (CGMCC 2.1291) was activated to obtain the seed culture.

[0098] The seed culture was inoculated into malt extract liquid medium at an inoculation rate of 1%, and fermented in shake flasks at 28°C and 120 rpm for 24 hours to obtain the primary fermentation broth.

[0099] The primary fermentation broth was transferred to a culture medium containing oat kernel oil for further fermentation, with a water-to-oil ratio of 1:9 after transfer. The secondary fermentation conditions were the same as the primary fermentation, and the fermentation time was 4 days, yielding a water-oil mixture in the secondary fermentation broth.

[0100] The mixed liquid obtained from the secondary fermentation was centrifuged at 13,000 rpm and kept at 4°C for 15 min to separate the upper oily liquid; the sample obtained was fermented oat kernel oil.

[0101] Example 4: A method for preparing fermented vegetable oil, comprising the following steps:

[0102] The frozen Candida shchuata (CGMCC 2.4317) was activated using glucose as the carbon source in the culture medium at a concentration of 30 g / L and yeast extract as the nitrogen source in the culture medium at a concentration of 15 g / L to obtain the seed culture.

[0103] The seed culture was inoculated into malt extract liquid medium at an inoculation rate of 1%, and fermented in shake flasks at 28°C and 120 rpm for 22 hours to obtain the primary fermentation broth.

[0104] The primary fermentation broth was transferred to a culture medium containing Moringa seed oil for further fermentation, with a water-to-oil ratio of 9:1 after transfer. Secondary fermentation conditions were maintained identical to the primary fermentation, with a fermentation time of 4 days, yielding a water-oil mixture of the secondary fermentation broth.

[0105] The mixed liquid obtained from the secondary fermentation was centrifuged at 10,000 rpm and kept at 4°C for 20 min to separate the upper oily liquid; the sample obtained was fermented Moringa seed oil.

[0106] Application Example 1: #1 Essential Oil

[0107] Manufacturing process: (Note: Ensure all equipment and containers are clean and dry)

[0108] A. Put all the raw materials of phase A into the operating pot and stir until completely transparent;

[0109] B. The material should be clear and transparent. If a filter is required, a dry 0.45-micron filter must be used.

[0110] Table 6. Formula for #1 Essential Oil

[0111]

[0112] Application Example 2: #2 Essential Oil

[0113] Manufacturing process: (Note: Ensure all equipment and containers are clean and dry)

[0114] A. Put all the raw materials of phase A into the operating pot and stir until completely transparent;

[0115] B. The material should be clear and transparent. If a filter is required, a dry 0.45-micron filter must be used.

[0116] Table 7. Formula for #2 essential oil

[0117]

Claims

1. A method for preparing fermented vegetable oil, characterized in that: The following steps are adopted: A. Using glucose as the carbon source and yeast extract as the nitrogen source, the frozen yeast was activated. The frozen yeast was *Candida shurata*. Scheffersomyces shehatae The seed solution was obtained after activation, with the preservation number CGMCC 2.4317. B. Inoculate the seed liquid obtained in step A into malt extract liquid culture medium at a volume ratio of 1%; ferment in a shake flask at 28℃ and 100-150 rpm for 13-20 hours to obtain the primary fermentation broth; the malt extract liquid culture medium contains 130 g / L malt extract powder and 0.1 g / L chloramphenicol. C. Take the primary fermentation broth obtained in step B as the aqueous phase and vegetable oil as the oil phase. Mix the aqueous phase and oil phase in a volume ratio, wherein the proportion of the oil phase is 10-90%. Continue fermentation for 1-4 days after mixing. After fermentation is completed, a secondary fermentation broth is obtained. The vegetable oil is one of perilla seed oil, safflower seed oil, camellia oil, and peony seed oil or any mixture of them in any proportion. D. Centrifuge the secondary fermentation broth obtained in step C at 4℃ and 8000-13000 rpm for 5-20 minutes. After centrifugation, take the upper oily liquid to obtain fermented vegetable oil.

2. The method for preparing fermented vegetable oil according to claim 1, further characterized in that: The unsaturated fatty acid content of the vegetable oil is greater than or equal to 60%.

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