A fermented vegetable fat and a three-stage stepwise fermentation method for preparing the same

Abstract

The application discloses a kind of fermented vegetable oil and preparation method of three-stage step-by-step fermentation thereof, and relates to the technical field of oil fermentation, which includes sequentially carried out first-stage strain culture, second-stage induced fermentation and third-stage main fermentation;First-stage strain culture is used to carry out primary strain fermentation on yeast extract to obtain primary strain fermentation liquor, second-stage induced fermentation is used to carry out secondary strain fermentation on primary strain fermentation liquor to obtain secondary strain fermentation liquor, and third-stage main fermentation is used to ferment secondary strain fermentation liquor to obtain fermentation liquor.The application uses single strain, and the three-stage fermentation method of initial stage culture, second stage induction and third stage main fermentation is used to prepare fermented oil, improve the surface tension of vegetable oil, and the acid value, peroxide value and other indicators of the obtained fermented oil meet the requirements and are not easy to oxidize and deteriorate.

Description

Technical Field

[0001] This invention relates to a fermented vegetable oil and its three-stage stepwise fermentation preparation method, belonging to the field of oil fermentation technology. Background Technology

[0002] Damaged skin barrier has become a common skincare problem faced by modern people. Environmental pollution, ultraviolet radiation, over-cleansing, and stressful lifestyles can easily lead to damage to the stratum corneum structure and lipid loss. With the increasing popularity of the "oil-based skincare" concept, consumers have placed higher demands on the naturalness, safety, and functionality of cosmetic ingredients. Plant fermented oils, with their advantages of being gentle on the skin and rich in active ingredients, have become a research hotspot in the beauty industry.

[0003] Oils prepared through conventional processes such as cold pressing and extraction have several drawbacks. First, they are difficult to fully release the active ingredients in plant oils, and the resulting oils have a low content of functional substances, resulting in less significant efficacy. Second, unfermented plant oils are highly hydrophobic, have poor emulsification properties, feel sticky on the skin, are slow to absorb, and have low film-forming properties and transdermal absorption efficiency, which limits their application in high-end skincare products.

[0004] To address the bottlenecks in the efficacy and application of vegetable oils, microbial fermentation technology has been widely used in the field of oil modification. Through microbial metabolism, large-molecule triglycerides in oils can be degraded into more easily absorbed diglycerides and monoglycerides, while simultaneously generating additional active substances such as biosurfactants, flavonoids, and polyphenols, thereby improving the hydrophilicity, emulsification stability, and bioactivity of the oils.

[0005] However, existing fermented oil technology still has many shortcomings: First, the fermentation process lacks targeted optimization, and some processes use chemical reagents to assist, which can easily damage heat-sensitive active ingredients or even introduce risky substances; Second, existing methods for refining fermented oils have certain drawbacks. For example, current research uses activated carbon for purification after fermentation, but activated carbon has strong adsorption properties and may adsorb beneficial components after fermentation during decolorization. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fermented vegetable oil and its preparation method of a three-stage stepwise fermentation. The method uses a single strain of bacteria and a three-stage fermentation process of primary culture, secondary induced fermentation, and tertiary main fermentation to prepare fermented oil. This method improves the surface tension of the vegetable oil, so that the acid value, peroxide value and other indicators of the fermented oil meet the requirements and are not easily oxidized and deteriorated.

[0007] To achieve the above objectives, the present invention is implemented using the following technical solution: In a first aspect, the present invention provides a three-stage stepwise fermentation method, comprising a first-stage microbial culture, a second-stage induced fermentation, and a third-stage main fermentation performed sequentially. The first-stage culture is used to perform primary fermentation of yeast extract to obtain primary fermentation broth; the second-stage induced fermentation is used to perform secondary fermentation of primary fermentation broth to obtain secondary fermentation broth; and the third-stage primary fermentation is used to ferment secondary fermentation broth to obtain fermentation broth.

[0008] Furthermore, the first-stage bacterial culture includes: Yeast extract was inoculated into the first culture medium and fermented for 18-24 hours to obtain the primary culture fermentation broth. The first culture medium after inoculation with yeast extract contains, by weight percentage, 0.2-0.4% yeast extract, 0.2-0.4% malt extract, 0.4-0.6% peptone, 0.8-1.2% glucose, 0.08-0.12% urea, 4-6% vegetable oil, and the remainder is water.

[0009] Furthermore, the second-stage induced fermentation includes: The primary fermentation broth was transferred to a second culture medium for secondary fermentation for 18-24 hours to obtain the secondary fermentation broth. The second culture medium, by weight percentage, includes 0.2–0.4% yeast extract, 0.2–0.4% malt extract, 0.4–0.6% peptone, 1.8–2.2% glycerol, 0.3–0.6% betaine, 0.08–0.12% urea, 0.05–0.5% Tween 80, 4–6% vegetable oil, with the remainder being water.

[0010] Furthermore, the third-stage primary fermentation includes: The secondary bacterial fermentation broth was transferred to a third culture medium and fermented for 90-100 hours to obtain the fermentation broth; The third culture medium, by weight percentage, includes 1.8–2.2% glycerol, 2.2–2.8% K₂HPO₄, 0.6–0.8% KH₂PO₄, 0.2–0.4% NH₄NO₃, 0.15–0.25% MgSO₄, 0.8–1.2% yeast extract, 0.3–0.6% betaine, 0.05–0.5% Tween 80, 45–55% vegetable oil, and the remainder is water.

[0011] Furthermore, the first-stage microbial culture and the second-stage induced fermentation were carried out at a temperature of 20-30℃, a rotation speed of 160-200 rpm, and an aeration rate of 0.5-1.5 vvm. The third-stage main fermentation was carried out at a temperature of 20-30℃, a rotation speed of 320-400 rpm, and an aeration rate of 1.0-2.0 vvm.

[0012] Furthermore, the vegetable oil is one of olive oil, grape seed oil, and rice germ oil.

[0013] Secondly, the present invention also provides a three-stage stepwise fermentation method for preparing fermented vegetable oil, comprising: The fermentation broth was prepared by the three-stage stepwise fermentation method as described in any of the first aspects; Fermented vegetable oil was obtained by sequentially performing preliminary purification and deep purification on the fermentation broth. The preliminary purification is used to remove moisture, microorganisms and some impurities, while the deep purification is used to remove fatty acids and remaining impurities.

[0014] Furthermore, the preliminary purification includes: taking the oil layer after the fermentation broth has been left to stand and separate, adding anhydrous MgSO4 to it, stirring at a speed of 200-500 rpm for 1.5-2.5 hours, centrifuging and separating to obtain the preliminarily purified oil layer.

[0015] Furthermore, the deep purification includes: filtering the initially purified oil layer through a 0.45µm membrane for initial separation, then refining it through a chromatography column, and obtaining fermented vegetable oil after filtration, wherein the chromatography column is packed with magnesium silicate.

[0016] Thirdly, the present invention also provides a fermented vegetable oil, which is prepared by a three-stage stepwise fermentation preparation method for fermented vegetable oil as described in any of the second aspects.

[0017] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: The strain used in this invention is *Candida hydrolyticus*. A three-stage fermentation method is employed, consisting of a single strain, initial culture, second-stage induction, and third-stage main fermentation, to prepare fermented oil. In the first-stage culture phase, *Candida hydrolyticus* rapidly proliferates, obtaining a highly active inoculum. During the second-stage glycerol-induced fermentation, the cells are directed by the carbon source (glycerol and lipids), secreting large amounts of key hydrolytic enzymes such as lipases. These enzymes, acting as biocatalysts, specifically recognize and bind to triglyceride molecules in the lipids, providing a prerequisite for subsequent efficient hydrolysis. This "induced enzyme production" step is the core foundation for achieving efficient lipid conversion. In the third stage, lipases act on the ester bonds between the glycerol backbone and fatty acid chains, gradually breaking down triglycerides to generate more diglycerides, monoglycerides, and fatty acids. These fatty acids are further utilized by *Candida hydrolyticus* for growth, reproduction, and the synthesis of related enzymes. During the metabolism of *Candida hydrolyticus*, some amphiphilic biolipids are also generated, ultimately leading to significant changes in the physicochemical properties of the fermented oil.

[0018] The innovations in the fermentation and refining processes of this invention are as follows: 1. Betaine and glycerol are added as fermentation promoters and osmotic pressure protectants, helping microorganisms survive and function in unfavorable environments such as high salt or high sugar, and protecting the activity of microorganisms and enzymes. Betaine also acts as a methyl donor, participating in the synthesis of some important metabolites, while glycerol can also serve as a direct carbon source, promoting the fermentation process. 2. Tween-80 is added to increase the oil-water interface, improve the contact between microorganisms, enzymes, and oils at the interface, enhance the conversion and utilization of oils, and increase the degree of fermentation modification of oils. 3. In the third fermentation stage, the addition of the surfactant Tween-80 and the increase in oxygen aeration offset the inhibition of microbial growth and metabolism caused by the decrease in dissolved oxygen after the addition of high-concentration oils. 4. The deep refining process employs a combination of membrane filtration and magnesium silicate chromatography column filtration, which can better control the acid value, polar substance content, water content, and color of the fermented oils.

[0019] After the three-stage fermentation process of this invention, the molecular structure and composition of the plant oil are optimized: large-molecule triglycerides are moderately hydrolyzed, forming a mixed system mainly composed of monoglycerides, diglycerides, and free fatty acids of specific chain lengths. These free fatty acids are utilized by hydrolyzed Candida albicans for lipase synthesis, generating bioactive lipids synthesized by the hydrolyzed Candida albicans. The resulting fermented oil exhibits lower surface tension, lower acid value, lower irritation, and higher stability. The average molecular weight of the fermented oil is reduced, improving its permeability and efficacy. The fermented oil has a more layered skin feel, being light and easy to spread initially, with a moderate moisturizing sensation during application, resulting in better skin affinity and hydration. It can be used alone as a moisturizer in formulations, avoiding the need for compounding with small-molecule synthetic esters in conventional formulations to reduce the greasiness and spreadability of plant oils, aligning with the current trend towards pure and natural beauty products. The fermented oil is also easier to emulsify, and due to the enrichment of amphiphilic bioglycolipids from fermentation, the amount of emulsifier required during emulsification can be reduced, making it more suitable as a cosmetic ingredient. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the principle of a three-stage stepwise fermentation method in one embodiment of the present invention; Figure 2 This is a schematic diagram comparing the emulsifying ability of fermented rice germ oil prepared in Example 3 of the present invention before and after fermentation. The left side shows the emulsifying ability of fermented rice germ oil prepared in Example 3, and the right side shows the emulsifying ability of fermented rice germ oil prepared in Example 3 before fermentation (ordinary rice germ oil). Figure 3 This is a schematic diagram showing the comparison of surface tension of fermented vegetable oils prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention before and after fermentation; Figure 4 This is a schematic diagram of the static contact angle of the fermented rice germ oil prepared in Example 3 of the present invention before and after fermentation. In this diagram, A is a schematic diagram of the static contact angle of the fermented rice germ oil prepared in Example 3, and B is a schematic diagram of the static contact angle of the fermented rice germ oil prepared in Example 3 before fermentation (ordinary rice germ oil). Figure 5 This is a schematic diagram showing the fibroblast toxicity test results of the fermented vegetable oils prepared in Examples 1-3 of the present invention. Figure 6 This is a schematic diagram showing the antioxidant test results of fermented rice germ oil prepared in Example 3 of the present invention at different concentrations; Figure 7 This is a schematic diagram illustrating the sensory evaluation criteria of fermented olive oil and unfermented olive oil prepared in Example 1 and Comparative Example 1 of the present invention during the skin feel test. Figure 8This is a schematic diagram illustrating the sensory evaluation criteria of fermented olive oil and unfermented olive oil prepared in Example 1 and Comparative Example 1 of the present invention after 5 minutes of use in a skin feel test. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0022] like Figure 1 As shown, this embodiment of the invention provides a three-stage distributed fermentation method for preparing fermented vegetable oils, specifically including the following steps: First-stage bacterial culture: Yeast extract was inoculated into the first culture medium and primary fermentation was carried out for 24 hours at 25°C, 180 rpm, and an aeration rate of 1.0 vvm to obtain the primary fermentation broth. To optimize the cell growth and metabolic processes of the microorganisms, the first culture medium after inoculation with yeast extract included the following components by mass percentage: 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, 0.1% urea, 5% vegetable oil, and 92.8% distilled water.

[0023] Second-stage induced fermentation: 100 mL of the primary fermentation broth was transferred to 1 L of secondary culture medium and fermented for 24 hours at 28 °C, 180 rpm and an aeration rate of 1.0 vvm to obtain the secondary fermentation broth.

[0024] The second culture medium used comprises the following components by weight percentage: 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 2% glycerol, 0.1% urea, 0.4% betaine, 0.1% Tween 80, 5% vegetable oil, and 91.3% distilled water.

[0025] Third stage primary fermentation: 1 L of secondary bacterial culture broth was transferred to 10 L of tertiary culture medium and fermented for 96 hours at 28 °C, 300 rpm and an aeration rate of 1.5 vvm to obtain the fermentation broth.

[0026] The third culture medium used consisted of the following components by weight percentage: 2% glycerol, 2.5% K2HPO4, 0.7% KH2PO4, 0.3% NH4NO3, 0.2% MgSO4, 1% yeast extract, 0.4% betaine, 0.3% Tween 80, 42.6% distilled water, and 50% vegetable oil.

[0027] Preliminary purification: After the fermentation broth was left to stand for 30 minutes, the upper oil layer was taken and heated to 121°C and held for 20 minutes for sterilization. Then, the temperature was lowered to 50°C, and 2% anhydrous MgSO4 was added to the oil layer. The mixture was stirred at 350 rpm, just enough to allow the liquid surface to tumble slightly. The mixture was stirred for 2 hours to remove excess water from the oil layer. The mixture was then centrifuged for 10 minutes to remove any residue and anhydrous MgSO4 from the oil layer.

[0028] Deep purification: The pre-purified oil layer was passed through a chromatography column packed with a 0.45µm filter membrane and magnesium silicate to adsorb and filter out fatty acids and impurities generated during enzymatic hydrolysis, and finally fermented vegetable oil was prepared. Example 1

[0029] Based on the above preparation method, fermented olive oil was prepared using olive oil as the vegetable oil. Example 2

[0030] Based on the above preparation method, fermented grape seed oil was prepared using grape seed oil. Example 3

[0031] Based on the above preparation method, fermented rice germ oil was prepared using rice germ oil as the vegetable oil.

[0032] Comparative Example 1: The second-stage induced fermentation in the above preparation method is omitted, and olive oil is used as the vegetable oil to prepare fermented olive oil.

[0033] Comparative Example 2: Based on the above preparation method, glycerol in the second culture medium used in the secondary fermentation was replaced with glucose, and betaine and Tween 80 were not added. Olive oil was used as the vegetable oil to prepare fermented olive oil.

[0034] The culture medium formulations of Examples 1-3 and Comparative Examples 1 and 2 are shown in Table 1.

[0035] Table 1: Culture medium ratios for Examples 1-3 and Comparative Examples 1 and 2

[0036] The fermented vegetable oils prepared in Examples 1-3 and Comparative Examples 1 and 2 were tested and analyzed below.

[0037] Emulsifying ability and surface tension testing: To determine emulsifying activity, 10 mL of untreated rice germ oil and fermented rice germ oil prepared in Example 3 were dissolved in 50 mL of pH 7 buffer solution. After homogenization and emulsification for 5 min without the addition of emulsifiers or surfactants, the mixtures were allowed to stand for 20 min, and their emulsifying abilities were observed and compared. The results are as follows: Figure 2 As shown.

[0038] from Figure 2 It can be seen that the fermented rice germ oil prepared in Example 3 can form a uniform emulsion system, while the untreated rice germ oil has obvious stratification. This shows that the emulsification ability of the fermented rice germ oil is significantly stronger than that of the untreated rice germ oil.

[0039] Surface tension was tested using the platinum plate suspension method. An A101 fully automatic surface tension meter was used to measure the surface tension of the fermented vegetable oils prepared in Examples 1-3, Comparative Examples 1 and 2, and their corresponding untreated vegetable oils. The test results are as follows: Figure 3 As shown.

[0040] Combination Figure 3 It can be seen that the vegetable oils in Comparative Examples 1, 2, and Example 1 are all olive oils. However, Example 1 shows the most significant change in surface tension before and after fermentation, and also exhibits the lowest surface tension. This is because the three-stage distributed fermentation method of the present invention facilitates oil fermentation through three stages of inoculum cultivation, and the replacement of glucose with glycerol in the second-stage induced fermentation can induce enzymatic hydrolysis, resulting in a better improvement in surface tension. Among Examples 1-3, Example 3 shows the greatest improvement in surface tension before and after fermentation, indicating that the three-stage distributed fermentation method is preferably suitable for rice germ oil.

[0041] Then, contact angle tests were performed on untreated rice germ oil and fermented rice germ oil prepared in Example 1. The test results are as follows: Figure 4 As shown, fermented rice germ oil has a smaller contact angle, which proves that fermented rice germ oil has lower surface tension and better wettability than untreated rice germ oil.

[0042] Acid value determination: The method for determining acid value is based on the first method in the national standard GB5009.229 <<National Food Safety Standard - Determination of Acid Value in Food>>: cold solvent indicator titration method.

[0043] The acid values ​​of the fermented vegetable oils prepared in Examples 1-3 and Comparative Example 1, and their corresponding untreated vegetable oils, are shown in Table 2. Table 2: Acid values ​​of fermented vegetable oils prepared in Examples 1-3 and Comparative Example 1 before and after fermentation

[0044] As shown in Table 2, the acid value of Examples 1-3 did not increase significantly before and after fermentation, indicating that the fermentation process of various vegetable oils has little impact on their acid value. However, the acid value of Comparative Example 1 after fermentation was 12.34 mg KOH / g, which was more than 17 times higher than before fermentation. The acid value of normal cosmetics is generally controlled within 5 mg KOH / g. High acid value will lead to easy oxidation and rancidity of oils, and they will develop a rancid smell if left for too long. This indicates that the fermented oil obtained by the two-step fermentation has the problem of excessively high acid value.

[0045] Peroxide value test: The test method was based on Method 1: Indicator Titration Method in GB 5009.227 "National Food Safety Standard - Determination of Peroxide Value in Food". The peroxide value test results of the fermented vegetable oils prepared in Examples 1-3 before and after fermentation are shown in Table 3.

[0046] Table 3: Peroxide values ​​of fermented vegetable oils prepared in Examples 1-3 before and after fermentation

[0047] As can be seen from the data in Table 3, the peroxide value after fermentation was slightly higher than that before fermentation, but it was still lower than the cosmetic requirement of 20 meq / kg. The higher the peroxide value, the more severe the rancidity. The peroxide values ​​of the fermented vegetable oils prepared in Examples 1 to 3 all met the requirements.

[0048] Cytotoxicity assay: The fermented plant oils prepared in Examples 1-3 were subjected to fibroblast toxicity tests, and the specific steps are as follows: Human fibroblasts were distributed at a density of 1 × 10⁶ cells per well. 4 Each inoculum was inoculated into a 96-well culture plate and cultured for 24 hours in an incubator at 37°C and 5% CO2.

[0049] After culturing, cells were first subjected to serum starvation for 2 hours, followed by treatment with different concentrations (0-1%) of plant fermentation oil, and cultured for another 48 hours. To determine cell viability, 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT reagent) was added after removing the culture medium, and the cells were cultured for another 2 hours. After culturing, each well was dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using a microplate reader.

[0050] Test results are as follows Figure 5 As shown, the fermented vegetable oils prepared in Examples 1-3 all exhibited high fibroblast survival at a concentration of 0.5%, and the cell survival rate remained above 80% even when the concentration was increased to 1.0%, indicating that the fermented vegetable oils did not show significant cytotoxicity at concentrations below 1.0%.

[0051] Skin patch test: The test included 31 participants, using samples with an area not exceeding 50 mm². 2 A qualified spot test apparatus with a depth of approximately 1 mm was used. The fermented vegetable oil prepared in Examples 1-3 was added to the spot test apparatus, with a volume of approximately 0.020-0.025 mL. The spot test apparatus containing the test substance and negative control was applied to the flexor side of the subject's forearm, and removed after 24 hours. Skin reactions were observed and recorded according to the standards shown in Table 4 at 30 minutes (after the indentation disappeared), 24 hours, and 48 hours after removal of the test substance and spot test apparatus.

[0052] Table 4: Grading Criteria for Skin Reaction in Closed Patch Tests

[0053] The test results are shown in Table 5.

[0054] Table 5: Skin patch test results of fermented vegetable oils prepared in Examples 1-3

[0055] As can be seen from the data in Table 5, the fermented vegetable oils prepared in Examples 1-3 showed no skin reaction in any of the 31 subjects at 0.5h, 24h, and 48h after the removal of the test sample.

[0056] Antioxidant test: The fermentation oil prepared in Example 3 was diluted with hexane to a concentration of 1–10%, and 20 L of the diluted solution was added to a 96-well plate. 180 μL of DPPH reagent (Sigma) was added to each well, and the plate was incubated at room temperature for 30 minutes. The optical density was measured at 517 nm.

[0057] S = (1 - A1 / A2) × 100% In the formula, A1 is the absorbance value of the sample group, A2 is the absorbance value of the blank group, and S is the optical density.

[0058] Test results are as follows Figure 6 As shown, the fermented rice germ oil prepared in Example 3 has antioxidant effects. With the addition of up to 10%, the antioxidant effect is better as the concentration increases. When 10% is added, the antioxidant effect reaches 68%. Free radicals are the main cause of skin aging and oxidative stress, which effectively shows that it can scavenge free radicals and has antioxidant activity.

[0059] Skin feel test: Thirty-one female subjects aged 20 to 50 years were selected to conduct skin feel evaluations of the fermented and unfermented olive oils prepared in Example 1 and Comparative Example 1. Scores were given for six dimensions: spreadability, smoothness, absorption speed during use, and gloss, smoothness, and stickiness after 5 minutes of use. Each dimension was scored out of 15, with scent satisfaction scored out of 10. The average score was calculated and rounded to one decimal place. The scoring details are as follows: Figure 7 and Figure 8 As shown.

[0060] The test results are shown in Table 6.

[0061] Table 6: Skin feel evaluation results of fermented olive oil and unfermented olive oil prepared in Example 1 and Comparative Example 1

[0062] The data in the table shows that the fermented olive oil prepared in Example 1 outperformed Comparative Example 1 in terms of odor, spreadability, smoothness, and absorption speed during application. Comparative Example 1 was superior to unfermented olive oil. After 5 minutes of use, Example 1 also showed the best results in terms of shine, smoothness, and stickiness. This demonstrates that the fermented olive oil prepared in Example 1 has a less pungent odor, better shine, is refreshing and non-sticky, and has significantly improved absorption. It penetrates the skin much more quickly after application. During fermentation, the olive oil's hydrophobicity is reduced, and its affinity for highly polar substances is enhanced, thereby reducing surface tension and improving skin absorption, spreadability, and smoothness.

[0063] Efficacy test results: The fermented rice germ oil prepared in Example 3 was mixed with squalane to obtain a skin care product, wherein the fermented rice germ oil prepared in Example 3 had a mass fraction of 5%. Skin condition tests were conducted on 30 people before and after two weeks of use, and the average value was taken. The results are shown in Table 7.

[0064] Table 7: Skin texture test results of skincare products

[0065] As can be seen from the data in Table 7, the fermented rice germ oil prepared in Example 3 has significant moisturizing, repairing, brightening, anti-wrinkle and firming effects.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A three-stage stepwise fermentation method, characterized in that, This includes the sequential first-stage microbial culture, the second-stage induced fermentation, and the third-stage main fermentation. The first-stage culture is used to perform primary fermentation of yeast extract to obtain primary fermentation broth; the second-stage induced fermentation is used to perform secondary fermentation of primary fermentation broth to obtain secondary fermentation broth; and the third-stage primary fermentation is used to ferment secondary fermentation broth to obtain fermentation broth.

2. The three-stage stepwise fermentation method according to claim 1, characterized in that, The first-stage bacterial culture includes: Yeast extract was inoculated into the first culture medium and fermented for 18-24 hours to obtain the primary culture fermentation broth. The first culture medium after inoculation with yeast extract contains, by weight percentage, 0.2-0.4% yeast extract, 0.2-0.4% malt extract, 0.4-0.6% peptone, 0.8-1.2% glucose, 0.08-0.12% urea, 4-6% vegetable oil, and the remainder is water.

3. The three-stage stepwise fermentation method according to claim 2, characterized in that, The second stage of induced fermentation includes: The primary fermentation broth was transferred to a second culture medium for secondary fermentation for 18-24 hours to obtain the secondary fermentation broth. The second culture medium, by weight percentage, includes 0.2–0.4% yeast extract, 0.2–0.4% malt extract, 0.4–0.6% peptone, 1.8–2.2% glycerol, 0.08–0.12% urea, 0.3–0.6% betaine, 0.05–0.5% Tween 80, 4–6% vegetable oil, with the remainder being water.

4. The three-stage stepwise fermentation method according to claim 3, characterized in that, The third-stage primary fermentation includes: The secondary bacterial fermentation broth was transferred to a third culture medium and fermented for 90-100 hours to obtain the fermentation broth; The third culture medium, by weight percentage, includes 1.8–2.2% glycerol, 2.2–2.8% K₂HPO₄, 0.6–0.8% KH₂PO₄, 0.2–0.4% NH₄NO₃, 0.15–0.25% MgSO₄, 0.8–1.2% yeast extract, 0.3–0.6% betaine, 0.05–0.5% Tween 80, 45–55% vegetable oil, and the remainder is water.

5. The three-stage stepwise fermentation method according to claim 4, characterized in that, The first-stage culture and the second-stage induced fermentation were carried out at a temperature of 20-30℃, a rotation speed of 160-200 rpm, and an aeration rate of 0.5-1.5 vvm. The third-stage main fermentation was carried out at a temperature of 20-30℃, a rotation speed of 320-400 rpm, and an aeration rate of 1.0-2.0 vvm.

6. The three-stage stepwise fermentation method according to any one of claims 2 to 4, characterized in that, The vegetable oil is one of olive oil, grapeseed oil, and rice germ oil.

7. A three-stage, stepwise fermentation method for preparing fermented vegetable oil, characterized in that, include: Fermentation broth was prepared by the three-stage distributed fermentation method as described in any one of claims 1 to 6; Fermented vegetable oil was obtained by sequentially performing preliminary purification and deep purification on the fermentation broth. The preliminary purification is used to remove moisture, microorganisms and some impurities, while the deep purification is used to remove fatty acids and remaining impurities.

8. The three-stage stepwise fermentation method for preparing fermented vegetable oil according to claim 7, characterized in that, The preliminary purification process includes: allowing the fermentation broth to stand to extract the oil layer, adding anhydrous MgSO4 to it, stirring at a speed of 200-500 rpm for 1.5-2.5 hours, centrifuging and separating to obtain the preliminarily purified oil layer.

9. The three-stage stepwise fermentation method for preparing fermented vegetable oil according to claim 8, characterized in that, The deep purification includes: filtering the initially purified oil layer through a 0.45µm membrane for initial separation, then refining it through a chromatography column, and obtaining fermented vegetable oil after filtration, wherein the chromatography column is filled with magnesium silicate.

10. A fermented vegetable oil, characterized in that, It is prepared by the three-stage stepwise fermentation method for preparing fermented vegetable oil as described in any one of claims 7 to 9.

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