Fermented camellia seed oil and preparation method thereof

Abstract

The invention provides fermented camellia-seed oil and a preparation method thereof, and the preparation method comprises the following steps: S1, culturing candida albicans to obtain candida albicans liquid; s2, carrying out solid-liquid separation on the bacterial liquid, collecting thalli, and washing to obtain clean resting cells; s3, suspending the resting cells in a water phase medium to form a cell suspension; s4, mixing camellia seed oil with the cell suspension, and performing resting cell enzymolysis fermentation under ventilation and stirring; and S5, after fermentation is finished, collecting an oil phase to obtain the fermented camellia seed oil.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a fermented camellia seed oil and its preparation method. Background Technology

[0002] "Oil-based skincare" has become a popular skincare concept in recent years, advocating the use of natural plant oils to replenish and strengthen the skin barrier function, balance oil and water levels, and improve skin texture. However, natural oils are lipophilic compounds formed by long-chain fatty acids and glycerol linked by ester bonds, often resulting in a "greasy" or "heavy" feel in cosmetic applications. Utilizing microorganisms or microbial enzymes to break down the triglycerides in natural plant oils, releasing free fatty acids and glycerol, can improve the skin feel and absorption of natural oils.

[0003] Several patents have disclosed technologies for fermenting natural plant oils using microorganisms. Patent CN107467207A discloses a process for fermenting mixed plant oils using Monascus purpureus, but this process mainly focuses on the synergistic skincare effects of amino acids, vitamins, and other functional substances produced by Monascus purpureus itself, as well as the mixture of various oil esters, without altering the oil structure. CN106420404B discloses a fermented plant oil mixture with antioxidant effects, using Basidiomycetes SY16 during fermentation. This patent increases the content of free fatty acids in the mixed plant oil through fermentation, thereby improving the oil's usability. However, the oil fermentation process in this patent involves directly adding the natural mixed oils into a culture medium rich in glucose, yeast extract, malt extract, peptone, and various salts from Basidiomycetes SY16. This process causes a large number of oil-soluble impurities in the culture medium to enter the oil phase during prolonged fermentation, affecting the color and odor of the final fermented oil, making it difficult to obtain a clean, clear fermented oil with a distinct plant oil aroma. The fermented oil preparation method disclosed in patent CN109010140A involves first obtaining *Mucor* through fermentation, then extracting glycerides from the *Mucor* mycelium using an organic solvent. These glycerides are then mixed with vegetable oil and fermented using *Foirurus*. The patent claims that the fermented oil obtained is safe, non-irritating, has a good skin feel, excellent antioxidant capacity and oxidative stability, and good skin repair ability. However, the patented preparation method requires the cultivation and fermentation of two different microorganisms, making the process complex and the product quality stability difficult to guarantee. Furthermore, the direct mixing of the culture medium and oil during the secondary fermentation process introduces a large number of oil-soluble impurities that are difficult to remove. Patent CN117821156A discloses a more complex fermentation oil process. This patent involves the co-fermentation of peony seed oil, Lactobacillus plantarum, Candida albicans, and Haematococcus pluvialis. The challenge of the process lies in how to satisfy the anaerobic and acidophilic requirements of Lactobacillus plantarum, as well as the aerobic and pH conditions required for Candida albicans production, while providing high-oxygen and high-light conditions for Haematococcus pluvialis. The complex product system resulting from the complex process places extremely high demands on subsequent extraction and purification. Therefore, this patented process requires extraction with three organic solvents, as well as further purification through distillation.

[0004] Chinese patents CN 117737137A and CN118975953 disclose a method for preparing natural plant oils by fermenting them with *Candida benjamina*. The advantages of using *Candida benjamina* to ferment natural plant oils are twofold: firstly, it hydrolyzes triglycerides to release diglycerides or monoglycerides; secondly, the fermentation process produces a small amount of glycolipid surfactants, thereby significantly improving the skin feel and emulsifying properties of the natural oils. However, both patents disclose methods that directly mix natural plant oils with yeast culture broth containing culture medium components for fermentation. As mentioned earlier, oil-soluble impurities in the culture medium components can enter the natural plant oil phase through the fermentation process, ultimately affecting the odor, color, purity, shelf life, and stability of the fermented natural plant oil.

[0005] Camellia seed oil, also known as tea seed oil or tea tree oil, is extracted from the seeds of the camellia tree. Rich in oleic acid, linoleic acid, glycerides, and active ingredients such as tea saponins, tea polyphenols, flavonoids, and natural vitamin E, it possesses antioxidant, moisturizing, sun-protective, and anti-aging properties, making it frequently used in cosmetic creams, facial cleansers, body oils, and sunscreens. Besides the aforementioned patents related to fermented oils, there are also patents concerning fermented camellia seed oil. For example, patent CN117448212A discloses a moisturizing fermented camellia seed oil and its preparation method and application. This method utilizes Bacillus subtilis and camellia seed powder for solid-state fermentation, followed by air-drying and pressing the fermented product to obtain camellia oil with moisturizing, barrier-repairing, antioxidant, and skin-soothing effects. However, the fermentation process described in these patents is simple and rudimentary, lacking any quantitative control over the process, and the patents do not disclose quantitative indicators of the characteristic functional products in the final product. Summary of the Invention

[0006] The present invention relates specifically to a fermented camellia seed oil and its preparation method. Specifically, the present invention aims to provide a fermented camellia seed oil rich in monoglycerides, diglycerides, polysaccharides, and natural glycolipids, and its preparation method. The preparation method provided by the present invention employs a whole-cell catalytic fermentation process based on resting cells. First, *Candida albicans* is inoculated into a fermentation medium for high-density culture to obtain a high-density yeast broth. Subsequently, the broth undergoes solid-liquid separation and washing to thoroughly remove residual impurities in the culture medium and metabolic byproducts of the yeast cells, yielding highly active and clean resting yeast cells. These resting cells are then resuspended in an aqueous phase and mixed with crude camellia seed oil in a certain proportion. Enzymatic fermentation is carried out under controlled temperature, dissolved oxygen, and pH conditions. After fermentation, the oil is centrifuged, dehydrated, and filtered to obtain the fermented camellia seed oil.

[0007] The technical solution provided by this invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing fermented camellia seed oil, comprising the following steps: S1. Cultivate Candida beesiensis to obtain Candida beesiensis bacterial solution; S2. The bacterial solution is subjected to solid-liquid separation, the bacterial cells are collected and washed to obtain clean resting cells; S3. The resting cells are suspended in an aqueous medium to form a cell suspension; S4. The camellia seed oil is mixed with the cell suspension and subjected to resting cell enzymatic hydrolysis fermentation under aeration and stirring. S5. After fermentation, collect the oil phase to obtain the fermented camellia seed oil.

[0009] In step S1, the Candida albicans culture is obtained through a two-stage culture process including primary seed culture and high-density fermentation.

[0010] The *Candida albicans* yeast may optionally be *Candida bumblebee* ATCC 22214 (… Starmerella Bombicola ).

[0011] The specific steps of the primary seed culture are as follows: *Candida albicans* strain is inoculated into the primary seed culture medium and cultured with shaking at 28-30℃ and 180-220 rpm for 40-48 hours until the OD of the culture medium reaches 100°C. 600 Reach 30-40; obtain Grade 1 seed solution; The primary seed culture medium contains the following components: glucose 8.0-12.0 g / L, peptone 4.0-6.0 g / L, yeast extract 2.0-4.0 g / L, malt extract 2.0-4.0 g / L, and pH 6.0-6.4.

[0012] The high-density fermentation process is as follows: Primary seed culture is inoculated into the high-density fermentation medium at an inoculation rate of 3%-5% by volume. The medium is then cultured in a fermenter at 28-32°C, with a pH of 5.5-6.5, a stirring speed of 500-700 rpm, and an aeration rate of 0.5-2.0 vvm for 18-24 hours, until the culture medium reaches its OD value. 600 Reaching 50 or higher; yielding Candida albicans liquid.

[0013] The culture medium used in the high-density fermentation contains a carbon source, a nitrogen source, and inorganic salts; the carbon source is glucose, and the nitrogen source includes one or more of yeast extract, peptone, and malt extract.

[0014] In one embodiment of the present invention, the high-density fermentation medium comprises the following components: glucose: 30-50 g / L, yeast extract: 2-5 g / L, peptone: 3-10 g / L, malt extract: 2-5 g / L, potassium dihydrogen phosphate: 0-2 g / L; dipotassium hydrogen phosphate: 0-2 g / L; magnesium sulfate heptahydrate: 0-1 g / L In step S2, the solid-liquid separation is centrifugation or filtration; the washing is washing the bacterial cells at least once with sterile physiological saline.

[0015] In step S2, the steps of performing solid-liquid separation on the bacterial culture, collecting the bacterial cells, washing, and obtaining clean resting cells include: S21. Low-temperature centrifugation: Centrifuge the Candida bee-derived yeast culture prepared in step S1 at 4-10℃ at a speed of 3000-5000 rpm for 3-10 minutes and collect the wet cells. S22. Low-temperature washing: The wet bacterial cells are washed with sterile physiological saline solution, the amount of which is 1-3 times the weight of the wet bacterial cells, the washing temperature is 4-10℃, and the washing time is 3-10 minutes. S23. Separation again: After washing, the system is centrifuged at 3000-5000 rpm for 3-10 minutes at 4-10℃ to collect the clean resting cells; Among them, steps S22 and S23 are repeated at least once.

[0016] In step S3, the aqueous medium is sterile physiological saline. The mass ratio of the resting yeast cells to the aqueous medium is 1:5 to 1:10.

[0017] In step S4, the mass ratio of the resting cell suspension to the camellia seed oil is 1:1 to 2:1.

[0018] In step S4, the conditions for the enzymatic hydrolysis and fermentation of resting cells are: temperature 28-32℃, stirring speed 400-600rpm, and aeration rate 0.5-1.5 L / min.

[0019] In step S4, the acid value of the fermentation system is used as the indicator for determining the fermentation endpoint; preferably, fermentation is stopped when the acid value reaches 4.0-10.0 g KOH / g.

[0020] In step S5, the oil phase is collected by centrifugation at a speed of 5000-8000 rpm for 5-15 minutes. Preferably, the collected oil phase undergoes post-treatment steps of dehydration and filtration sterilization; more preferably, the dehydration is carried out at 70-85°C under vacuum for 1-3 hours; the filtration sterilization is performed using a filter membrane with a pore size of 0.1-0.45μm.

[0021] Compared with existing fermentation processes, this invention pre-cultivates, separates, and washes the microbial cells before introducing camellia seed oil, thus avoiding contamination of the oil phase by oil-soluble impurities and fermentation byproducts in the culture medium. On the one hand, the process system and components are simpler, and the fermentation process is more controllable; on the other hand, because impurities are removed beforehand, the extraction process after fermentation is simpler, thereby maximizing the retention of active fermentation products; furthermore, through a simple and controllable quantitative fermentation process, the retention rate of active substances during fermentation is significantly improved. The resulting fermented camellia seed oil is rich in monoglycerides, diglycerides, polysaccharides, and natural glycolipids, possessing excellent application value and prospects in the fields of beauty, skincare, personal care, and healthcare.

[0022] Compared with the prior art, the effective effects of the present invention are as follows: 1. This invention removes oil-soluble impurities (such as pigments and extraneous proteins) and byproducts of cell growth and metabolism from the yeast cells by separating and washing them before fermentation, which may be introduced into the culture medium in traditional fermentation methods. This results in a pure and bright fermented camellia seed oil that retains its inherent fresh aroma, has a longer shelf life, and better stability, effectively overcoming the problems of dark color, mixed odor, and numerous difficult-to-remove impurities in existing technologies.

[0023] 2. The use of "resting cell catalysis" instead of the traditional "direct mixing and fermentation of fermentation broth and oil" simplifies the process and avoids complex multi-strain co-cultivation or secondary fermentation. Because impurities are removed pre-processed, post-processing eliminates the need for complex purification steps such as organic solvent extraction and distillation. Only simple centrifugation, dehydration, and filtration are required to obtain high-purity products, significantly reducing the complexity of the production process and equipment costs, and better meeting green and environmental protection requirements.

[0024] 3. Maximum retention of active ingredients: By precisely controlling the fermentation conditions, the triglycerides in camellia seed oil are hydrolyzed to generate triglycerides and monoglycerides rich in diglycerides and monoglycerides. At the same time, the active ingredients such as natural glycolipids and polysaccharides produced by yeast metabolism are efficiently retained in the oil phase.

[0025] 4. The entire fermentation process does not involve the continued growth of the microorganisms, the reaction system is simple, and it is easy to achieve precise quantitative control and large-scale scale-up. This avoids batch-to-batch differences caused by uncertainties in microbial growth and complex culture media, ensuring a high degree of consistency and stability in the composition and efficacy of the final product. Attached Figure Description

[0026] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1The surface tension and contact angle of fermented camellia seed oil and crude camellia seed oil; Figure 2 The images show crude oil samples of fermented oil and camellia seed oil used in the examples. Detailed Implementation

[0027] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0028] Example 1: Preparation of high-density resting yeast cells Yeast strain: Bumblebee Candida ATCC 22214 ( Starmerella Bombicola ), store in glycerol cryovials at -80°C.

[0029] 1. Microbial culture Primary seed culture medium components: peptone: 5.0 g / L, malt extract: 3.0 g / L, yeast extract: 3.0 g / L, glucose: 10.0 g / L, pH: 6.2 ± 0.2; Culture conditions: Thaw the glycerol tubes at room temperature, and transfer 1 ml of the bacterial culture from the glycerol tubes into a 500 ml Erlenmeyer flask containing 100 ml of sterile seed culture medium. Incubate at 28°C. o C. OD was obtained by incubating at 200 rpm for 48 hours. 600 It is a primary seed culture with a concentration of 32, used for high-density fermentation inoculation.

[0030] 2. High-density fermentation High-density fermentation medium: glucose: 30 g / L, yeast extract: 3 g / L, peptone: 5 g / L, malt extract: 3 g / L, potassium dihydrogen phosphate: 1 g / L; dipotassium hydrogen phosphate: 1 g / L; magnesium sulfate heptahydrate: 0.5 g / L 2.5L of the prepared culture medium was poured into a 5L glass stirred fermenter (Intelli-Ferm A G3 5L, Dibier Biotechnology Shanghai Co., Ltd.), sterilized at 121°C for 30 minutes, cooled to 30°C, and then 100ml of the pre-cultured primary seed culture was inoculated. o C. Fermentation was carried out for 22 hours under the conditions of stirring at 600 rpm and aeration at 1.5 L / min to obtain OD. 600 The high-density yeast culture solution with a concentration of 60 is cooled to 10°C before use.

[0031] 3. Preparation of high-activity, high-density yeast cells: 3.1 Low-temperature centrifugation: Centrifuge the high-density yeast culture from step 2 at 10°C at 4000 rpm for 5 minutes and collect the wet yeast cells; 3.2 Low-temperature washing: Add sterile physiological saline at twice the weight of the wet bacterial weight, and wash at low temperature (10℃) with stirring for 5 minutes; 3.3 Separation again: After washing, the system was centrifuged at 4000 rpm for 5 minutes at 10°C to collect the clean resting cells; repeat steps 3.2 and 3.3 to finally obtain 300g of resting yeast cells.

[0032] The obtained 300g of resting yeast cells were added to 2.2kg of sterile physiological saline to obtain 2.5kg of high-density, high-viability resting cell suspension.

[0033] Example 2: Preparation of fermented camellia seed oil A (bacterial oil mass ratio 1:1) 1) Fermentation Take 0.8 kg of the high-density, high-viability resting cell suspension obtained in Example 1, and add 0.8 kg of crude camellia seed oil (Shanghai Qitan Biotechnology Co., Ltd.) at a mass ratio of 1:1. Ferment the camellia seed oil in a 5L glass fermentation tank at a fermentation temperature of 30°C. o C. Fermentation was stopped when the acid value reached 6.07 g KOH / g under the stirring conditions of 1 L / min and 500 rpm, and camellia seed oil fermentation liquid was obtained. 2) Separation and extraction: Camellia seed oil fermentation broth was centrifuged at 6000 rpm for 10 min to obtain the oil phase. o After vacuum dehydration under C conditions, fermented camellia seed oil A was obtained by filtration through a 0.2µm filter membrane.

[0034] Example 3: Preparation of fermented camellia seed oil B (bacterial-to-oil ratio 1:1.25) Take 0.8 kg of the high-density, high-viability resting cell suspension from Example 1, add 1 kg of crude camellia seed oil at a ratio of 1:1.25, and ferment the camellia seed oil in a 5L glass fermentation tank at a fermentation temperature of 30°C. o C. Under the conditions of 1 L / min flow rate and 500 rpm stirring, the fermentation was stopped when the acid value reached 4.37 g KOH / g, and camellia seed oil fermentation liquid was obtained. 2) Separation and extraction: Camellia seed oil fermentation broth was centrifuged at 6000 rpm for 10 min to obtain the oil phase. o After vacuum dehydration under C conditions, fermented camellia seed oil B is obtained by filtration through a 0.2-micron filter membrane.

[0035] Example 4: Preparation of fermented camellia seed oil C (bacterial-to-oil ratio 2:1) Take 0.8 kg of the high-density, high-viability resting cell suspension from Example 1, add 1.2 kg of crude camellia seed oil at a ratio of 1:1.5, and ferment the camellia seed oil in a 5L glass fermentation tank at a fermentation temperature of 30°C. o C, with a flow rate of 1 L / min and a stirring speed of 500 rpm, fermentation was carried out for 82 hours until the acid value reached 9.49 g KOH / g. Fermentation was then stopped to obtain camellia seed oil fermentation liquid. 2) Separation and extraction: Camellia seed oil fermentation broth was centrifuged at 6000 rpm for 10 min to obtain the oil phase. o After vacuum dehydration under C conditions, fermented camellia seed oil C is obtained by filtration through a 0.2-micron filter membrane.

[0036] Figure 2 The images show crude oil samples of fermented oil and camellia seed oil used in the examples.

[0037] Comparative Example 1 This comparative example uses crude camellia seed oil.

[0038] Comparative Example 2 The difference between this comparative example and Example 2 is that the brewer's yeast is replaced with Candida albicans to prepare fermented camellia seed oil; The brewing yeast is: Candida albicans PRO-LQX001, whose Latin name is Starmerella bombicola, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCCNo.29573.

[0039] Test Example 1 Samples of fermented camellia seed oil AC obtained in Examples 2 to 4 were taken, and their acid value, diglycerides, total polysaccharides, and glycolipid content were measured. The results were compared with those of Comparative Example 1, as shown in the table below: Table 1. Comparison of components between fermented camellia seed oil and Comparative Example 1

[0040] The data in the table show that, after fermentation with Candida albicans, Comparative Example 1 can significantly increase the content of acid value, diglycerides, total polysaccharides and glycolipids.

[0041] Test Example 2 The surface tension, contact angle with water, antioxidant properties, high-temperature stability at 45°C, and skin moisturizing properties of the fermented camellia seed oil obtained in Example 2 were measured and compared with those of Comparative Example 1.

[0042] 1) Surface tension and contact angle with water: Compared with Comparative Example 1, the surface tension of fermented camellia seed oil decreased and the wetting angle with water was close to 0. Figure 1 The surface tension and contact angle of fermented camellia seed oil and crude camellia seed oil.

[0043] 2) Antioxidant efficacy test DPPH free radicals have a maximum absorption peak near 517 nm. When DPPH free radicals react with antioxidants, the absorption value at 517 nm decreases. The degree of decrease is quantitatively related to the number of electrons received (the antioxidant's free radical scavenging activity). The reaction process can be easily monitored by a spectrophotometer.

[0044] Take 2.00 mL of the test sample solution of different mass concentrations into nine centrifuge tubes containing 2.00 mL of DPPH solution, making the total volume 4.00 mL. Vortex thoroughly to mix, and react at room temperature in the dark for 30 min. Measure the absorbance (517 nm) using a 1.00 cm quartz cuvette with a UV spectrophotometer. Repeat the measurement three times for each concentration sample and take the average value. The clearance rate is calculated using the following formula: Clearance rate (%) = [(A0-A) / A0)] 100% In the formula, A0 represents the absorbance of the blank control, which refers to the absorbance of DPPH free radicals without the addition of the sample; A is the absorbance of the sample, which refers to the absorbance of DPPH free radicals after the reaction with the addition of the sample.

[0045] The results showed that Example 2 had a better DPPH radical scavenging ability and better antioxidant properties than Comparative Example 1.

[0046] Table 1. Comparison of antioxidant properties between fermented camellia seed oil and Comparative Example 1

[0047] Test Example 3: High Temperature Stability Test Examples 2-4 and Comparative Examples 1-2 were placed at 45°C. o The high-temperature stability of the product was observed after 6 months in a C oven. The results are shown in the table below. Table 2 High Temperature Stability Test Results

[0048] Test Example 4: Moisturizing and Skin-Nourishing Efficacy Test Recruit 10 male and 10 female volunteers aged 18-22, and maintain an ambient temperature of 20-25 degrees Celsius. o C, humidity 40-70% oC. Subjects must not use any cosmetics or skincare products, or come into contact with irritants, for 3 days prior to the experiment. At the start of the experiment, subjects are required to refrain from strenuous exercise and maintain a calm mood before entering the laboratory. Each subject's arm will be divided into two areas, A and B. Crude camellia seed oil will be applied to area A, and fermented camellia seed oil will be applied to area B. Skin moisture, oiliness, and elasticity will be measured using a skin moisture meter at the start of the experiment and 30 minutes later. The test results are as follows: Table 3. Results of Moisturizing Efficacy Tests of Fermented Camellia Seed Oil and Comparative Example 1

[0049] Test results show that fermented camellia seed oil has better moisturizing and skin-softening effects than control ratio 1, and is also more refreshing and non-greasy.

[0050] Test Example 3: In Vitro Efficacy Test 1) Repair efficacy test comparison - cell scratch (keratinocytes) Epidermal keratinocytes are essential cells that make up the epidermal layer. When the skin surface is damaged, keratinocytes are stimulated to migrate and repair the damaged area. When cells grow to a monolayer in vitro, an artificially created blank area is formed on the monolayer. Cells at the edge of the scratch gradually enter the blank area, causing the scratch to heal, which to some extent simulates the in vivo cell migration process. The repair efficacy of the two methods was compared by measuring the cell migration rate after treatment in Example 2 and Comparative Example 1. The results are shown in Table 3.

[0051] Table 4 Results of in vitro repair efficacy tests for Examples and Comparative Example 1

[0052] 2) Repair Efficacy Test Comparison - Anti-wrinkle Test Elastase catalyzes the hydrolysis of N-succinyl-Ala-Ala-Ala-p-nitroaniline to generate nitroaniline, and the production of the enzymatic reaction product shows a good linear relationship with time. Nitroaniline is a colored product; this property is utilized to determine the amount of nitroaniline produced. The absorbance at 410 nm was measured using a microplate reader to reflect the elastase activity. The method followed the guidelines in "Q / YTFE 005--2022 Cosmetic Firming and Anti-wrinkle Efficacy Test - Elastase Inhibition Experiment". The results are shown in Table 4.

[0053] Table 5. Results of in vitro anti-wrinkle efficacy tests for Examples and Comparative Example 1

[0054] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing fermented camellia seed oil, characterized in that, Includes the following steps: S1. Cultivate Candida beesiensis to obtain Candida beesiensis bacterial solution; S2. The bacterial solution is subjected to solid-liquid separation, the bacterial cells are collected and washed to obtain clean resting cells; S3. The resting cells are suspended in an aqueous medium to form a cell suspension; S4. The camellia seed oil is mixed with the cell suspension and subjected to resting cell enzymatic hydrolysis fermentation under aeration and stirring. S5. After fermentation, the oil phase is collected to obtain the fermented camellia seed oil.

2. The preparation method according to claim 1, characterized in that, In step S1, the Candida albicans broth is obtained through a two-stage culture including primary seed culture and high-density fermentation; The specific steps of the primary seed culture are as follows: *Candida albicans* strain is inoculated into the primary seed culture medium and cultured with shaking at 28-30℃ and 180-220 rpm for 40-48 hours until the OD of the culture medium reaches 100°C. 600 Reach 30-40; obtain Grade 1 seed solution; The high-density fermentation process is as follows: the primary seed liquid is inoculated into the high-density fermentation medium at an inoculation rate of 3%-5% by volume, and cultured for 18-24 hours at 28-32℃, with pH controlled at 5.5-6.5, stirring speed at 500-700 rpm, and aeration rate at 0.5-2.0 vvm to obtain the Candida beescens liquid.

3. The preparation method according to claim 2, characterized in that, In step S1, the culture medium used for high-density fermentation contains a carbon source, a nitrogen source, and inorganic salts; the carbon source is glucose, and the nitrogen source includes one or more of yeast extract, peptone, and malt extract.

4. The preparation method according to claim 1, characterized in that, In step S2, the steps of performing solid-liquid separation on the bacterial culture, collecting the bacterial cells, washing, and obtaining clean resting cells include: S21. Low-temperature centrifugation: Centrifuge the Candida albicans culture prepared in step S1 at 4-10℃ at a speed of 3000-5000 rpm for 3-10 minutes and collect the wet cells. S22. Low-temperature washing: The wet bacterial cells are washed with sterile physiological saline solution, the amount of which is 1-3 times the weight of the wet bacterial cells, the washing temperature is 4-10℃, and the washing time is 3-10 minutes. S23. Separation again: After washing, the system is centrifuged at 3000-5000 rpm for 3-10 minutes at 4-10℃ to collect the clean resting cells; Among them, steps S22 and S23 are repeated at least once.

5. The preparation method according to claim 1, characterized in that, In step S3, the aqueous medium is sterile physiological saline; the mass ratio of the resting yeast cells to the aqueous medium is 1:5-1:

10.

6. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of the resting cells to the camellia seed oil is 1:1 to 2:

1.

7. The preparation method according to claim 1, characterized in that, In step S4, the conditions for the enzymatic hydrolysis and fermentation of resting cells are: temperature 28-32℃, stirring speed 400-600 rpm, and aeration rate 0.5-1.5 L / min.

8. The preparation method according to claim 1, characterized in that, In step S4, the acid value of the fermentation system is used as the indicator for determining the fermentation endpoint; fermentation is stopped when the acid value reaches 4.0-10.0 g KOH / g.

9. The preparation method according to claim 1, characterized in that, In step S5, the oil phase is collected by centrifugation; the collected oil phase also undergoes post-processing steps of dehydration and filtration for sterilization.

10. A fermented camellia seed oil prepared by the method described in any one of claims 1-9.

Images