Fermented vegetable oil and production method thereof

Abstract

The invention discloses fermented vegetable oil and a production method thereof. The invention belongs to a whole-cell enzymatic hydrolysis fermentation process of resting cells, which comprises the following steps: firstly, carrying out high-density fermentation to obtain a saccharomycetes liquid, then separating and washing to obtain clean high-activity saccharomycetes, and mixing the high-activity saccharomycetes, water and vegetable oil in proportion; vegetable oil is fermented by using resting yeast thalli through a whole-cell enzymolysis fermentation process under the conditions of specific temperature, oxygen supply and pH, and after fermentation is finished, secondary centrifugal filtration is performed to obtain the fermented vegetable oil. And compared with the fermented vegetable oil obtained by the conventional microbial fermentation process. The obtained fermented oil has the advantages of fewer impurities, lighter color, cleaner oil, low ash content, stronger oxidation stability, longer shelf life and the like, is rich in free fatty acids, flavonoids, polysaccharides and natural glycolipids compared with natural vegetable oil crude oil, has better skin moisturizing, repairing, soothing and anti-oxidation effects and the like, and is suitable for being used as a skin care product. Good economic values and application prospects are realized in the fields of beauty, skin care and washing.

Description

Technical Field

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

[0002] "Oil-based skincare" is a highly popular skincare concept in recent years, advocating the use of natural plant oils to supplement and strengthen the skin barrier function, balance water and oil, and improve skin texture.

[0003] Natural plant oils are generally obtained through physical pressing or organic solvent extraction, and contain tocopherols, carotenoids, etc. -Linolenic acid (GLA) - Linolenic acid (ALA) and polyphenols, among other natural herbal skincare ingredients, are common basic skincare oils in daily chemical products. However, because natural plant oils contain a large amount of high-molecular-weight triglycerides, they are not easily absorbed by the human skin. In addition, natural plant oils also have potential acne-causing and sensitizing risks.

[0004] Compared to natural plant oils, fermented plant oils are produced through microbial fermentation using yeast, lactic acid bacteria, and other microorganisms. These microorganisms break down large triglycerides into diglycerides, monoglycerides, and even smaller free fatty acids, significantly improving the skin's permeability while reducing the acne-causing and sensitizing properties of natural plant oils. Furthermore, the microbial fermentation process generates active ingredients not found in natural oils, or present in trace amounts, such as polysaccharides, organic acids, and glycolipids. These beneficial components give plant oils more efficacy, higher activity, and easier absorption by the skin, leading to their widespread use in moisturizing, whitening, firming, anti-aging, and anti-inflammatory repair skincare products.

[0005] 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 composition. 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 skin feel of the oil. 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.

[0006] Chinese patent CN109010140A discloses a method for preparing fermented oil by 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 again using *Foirurus*. The patent claims that the resulting fermented oil is safe, non-irritating, has a good skin feel, excellent antioxidant capacity and oxidative stability, and good skin repair ability. However, this 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. Chinese 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 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.

[0007] Chinese patents CN 117737137A and CN118975953 disclose a method for preparing natural plant oils by fermenting them with Candida albicans. The advantages of using Candida albicans to ferment natural plant oils are twofold: firstly, it hydrolyzes triglycerides to release them; secondly, the fermentation process produces a small amount of glycolipid surfactants, thus 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 oil phase through the fermentation process, ultimately affecting the odor, color, purity, shelf life, and stability of the fermented plant oil.

[0008] The aforementioned existing patents all involve fermenting natural plant oils together with microorganisms and microbial culture media. This results in oil-soluble components such as nitrogen sources, carbon sources, and inorganic salts from the culture media being incorporated into the final fermented plant oil, leading to a darker color or higher ash content in the final product, making it difficult to meet the application requirements of some high-end skincare products. Summary of the Invention

[0009] The purpose of this invention is to provide a fermented vegetable oil and its production method. The production method is a whole-cell catalytic fermentation process based on resting cells. First, yeast is inoculated into a high-density fermentation medium for cell expansion to obtain a high-density yeast culture. Then, the medium is separated and washed to remove impurities, resulting in clean, highly active wet yeast cells. The highly active yeast cells, physiological saline, and vegetable oil are mixed in a specific ratio, and the plant oil is subjected to resting cell enzymatic hydrolysis fermentation under specific temperature, oxygen supply, and pH conditions. A second centrifugation and filtration process yields the desired fermented camellia seed oil.

[0010] Compared with vegetable oils obtained through conventional microbial fermentation processes, the fermentation process of this invention pre-separates and washes the yeast strains before adding the vegetable oil, removing impurities (including metabolic byproducts produced during yeast growth and unused substrate) from the supernatant of the fermentation broth. Firstly, the process system and components are simpler, and the fermentation process is more controllable. Secondly, because impurities are removed beforehand, the difficulty of subsequent extraction processes is reduced, thus maximizing the retention of fermentation active products. Thirdly, the simple and controllable quantitative fermentation process maximizes the retention of active substances produced during fermentation, resulting in a final fermented vegetable oil with stronger oxidative stability and extended shelf life.

[0011] The method for producing fermented vegetable oil in this invention includes the following steps:

[0012] Step 1. Yeast cell culture: Inoculate yeast cells into a high-density fermentation medium for cell expansion culture to obtain yeast culture solution;

[0013] Step 2. Aseptically centrifuge or filter the bacterial solution to obtain yeast cells. Wash the yeast cells with physiological saline, and centrifuge or filter again to obtain clean yeast cells.

[0014] Step 3. Add the clean yeast cells obtained in Step 2 to physiological saline to obtain a bacterial-water mixture;

[0015] Step 4. Add the vegetable oil to the bacterial water mixture from Step 3 in the correct proportion for secondary fermentation;

[0016] Step 5. After fermentation, centrifuge and filter the oil phase to obtain fermented vegetable oil.

[0017] Specifically, in step 1, the yeast cells are obtained by shaking flask culture, with an inoculum of 1%-12% and culture conditions of 20-40℃ for 16-48 hours.

[0018] In step 3, the bacterial cell (dry weight) content in the bacterial-water mixture is 20-50 g / kg.

[0019] More specifically, the conditions for yeast expansion in step 1 are as follows: fermentation at 28-32℃, stirring at 400-800 rpm, and aeration at 1.2-1.8 L / min for 30-60 hours to obtain OD. 600 It is a high-density yeast fermentation broth with a concentration of over 80%.

[0020] Preferably, fermentation is carried out at a temperature of 29-32℃, a stirring speed of 500-700 rpm, and an aeration rate of 1.4-1.6 L / min for 40-50 h to obtain OD. 600 The range is 90-120, specifically 100-118 for high-density yeast fermentation broth.

[0021] Step 2 is as follows: Take the cultured and cooled yeast fermentation broth and centrifuge it (e.g., 3000-5000 rpm, specifically 4000 rpm for 3-8 minutes, e.g., 5 minutes) to obtain wet yeast cells;

[0022] Add sterile physiological saline to the yeast wet cells at a weight ratio of 1-3 times (e.g., 2 times), stir and wash under low temperature conditions, and centrifuge under the same conditions to complete the first washing of yeast cells; optionally, wash the yeast wet cells obtained from the first washing a second time under the same operating conditions to obtain yeast wet cells.

[0023] In step 3, the obtained yeast cells are added to physiological saline to obtain a yeast suspension. The specific OD... 600 The range is 90-120, for example, 100-118, or more specifically, 107, 109, 112.

[0024] In step 4, the mass ratio of the bacterial aqueous solution to the vegetable oil is 1:4-4:1.

[0025] The secondary fermentation conditions in step 4 are: temperature 20-60℃, time 2h-120h;

[0026] Preferably, the secondary fermentation conditions are: temperature 25-50℃, time 8h-60h, for example 16-45h.

[0027] Specifically, the vegetable oils are selected from camellia seed oil, jojoba oil, perilla seed oil, peony seed oil, shea butter, coconut oil, Ganoderma lucidum spore oil, sea buckthorn fruit oil, almond oil, grape seed oil, sacha inchi oil, meadowfoam seed oil, rice bran oil, sunflower seed oil, coffee seed oil, flaxseed oil, kiwi seed oil, pomegranate seed oil, rosehip oil, Sapindus mukorossi oil, and safflower seed oil, with camellia seed oil, jojoba oil, perilla seed oil, shea butter, almond oil, sea buckthorn fruit oil, and flaxseed oil being the preferred choices.

[0028] In a specific embodiment, the yeast is selected from *Candida bombicola* BKSL02 (accession number CGMCC No. 34152) or *Candida bombicola* ATCC22214. *Candida bombicola* BKSL02 is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), dated April 9, 2025, accession number: CGMCC No. 34152, and classified as *Starmerella bombicola*.

[0029] The present invention also provides fermented vegetable oil obtained by the production method described above.

[0030] The fermented oil obtained by this invention has advantages such as fewer impurities, lighter color, cleaner oil, lower ash content, stronger oxidative stability, and longer shelf life. Compared with natural plant oil, it is rich in free fatty acids, flavonoids, polysaccharides, and natural glycolipids, and has better skin moisturizing, repairing, soothing, and antioxidant effects. It has great economic value and application prospects in the field of beauty, skin care, and personal care. Attached Figure Description

[0031] Figure 1 Example 2 and its crude oil DPPH removal rate (%) control;

[0032] Figure 2 Examples 2 and 1, and their crude oil color comparisons and color comparisons before and after being placed in a 45-degree oven for 6 months;

[0033] Figure 3 Examples 4, Comparative Example 3, and their crude oil color comparisons, as well as color comparisons before and after being placed in a 45-degree oven for 6 months;

[0034] Figure 4 Examples 6, Comparative Example 4, and their crude oil color comparisons, as well as color comparisons before and after being placed in a 45-degree oven for 6 months.

[0035] Information on the preservation of biological materials:

[0036] Starmerella bombicola BKSL02 is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​April 9, 2025, with accession number CGMCC No. 34152, and is classified as Starmerella bombicola. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0038] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0040] The plant oils described in this invention include, but are not limited to, camellia seed oil, jojoba oil, perilla seed oil, peony seed oil, shea butter, coconut oil, Ganoderma lucidum spore oil, sea buckthorn oil, almond oil, grape seed oil, sacha inchi oil, meadowfoam seed oil, rice bran oil, sunflower seed oil, coffee seed oil, flaxseed oil, kiwi seed oil, pomegranate seed oil, rosehip oil, Xanthoceras sorbifolium oil, and safflower seed oil. This invention uses camellia seed oil, flaxseed oil, almond oil, sea buckthorn oil, shea butter, jojoba oil, and perilla seed oil as examples for detailed description.

[0041] The microorganisms described in this invention include, but are not limited to, yeasts, lactic acid bacteria, Bacillus, and Aspergillus. This invention uses two strains, *Candida apigensis* BKSL02 and *Candida apigensis* ATC22214, as examples for detailed description.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] The camellia seed oil, almond oil, and perilla seed oil in the following examples are all commercially available products.

[0044] The Starmerella bombicola BKSL02 in the following examples was isolated from natural nectar by the Shanghai Laboratory of Biokoo Biotechnology and deposited at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, date of deposit: April 9, 2025, accession number: CGMCC No. 34152.

[0045] The Candida albicans ATCC22214 in the following examples is commercially available.

[0046] Example 1: Preparation of high-density Candida albicans solution

[0047] 1) Yeast strain: Starmerella Bombicola strain BKSL02, stored in glycerol cryovials at -80℃.

[0048] 2) Shake flask culture

[0049] Shake flask culture medium components: YPD medium was used.

[0050] Culture conditions: One glycerol tube of bacterial culture was inoculated into a 500 mL Erlenmeyer flask containing 150 g of sterilized seed culture medium and cultured at 25°C and 200 rpm for 48 hours to obtain OD. 600 It is a primary seed culture with a concentration of 32, used for high-density fermentation inoculation.

[0051] 3) High-density fermentation

[0052] Fermentation medium components: 0.8% glucose, 0.2% beef extract, and 0.6% inorganic salts (magnesium sulfate: potassium dihydrogen phosphate: disodium hydrogen phosphate: ammonium sulfate = 1:0.5:0.5:1). The fermentation medium was prepared after high-temperature sterilization and cooling.

[0053] 3 kg of the prepared fermentation medium was poured into a 5 L glass stirred fermenter and sterilized at 121 °C for 30 minutes. After cooling to 30 °C, 150 g of the pre-cultured primary seed culture was inoculated. Fermentation was carried out at 30 °C, stirring at 600 rpm, and aeration at 1.5 L / min for 45 h to obtain OD. 600 A portion of the high-density yeast fermentation broth (110 g / L) was cooled to 10°C and used to prepare a high-density, high-activity yeast suspension. The remaining portion was used for the direct fermentation of natural plant oils.

[0054] 4) Preparation of high-density, high-activity yeast suspension:

[0055] Take 1500g of high-density yeast fermentation broth that has been cultured and cooled, centrifuge at 4000rpm for 5 minutes to obtain wet yeast cells; add sterile physiological saline to the wet yeast cells at a ratio of 2 by weight, stir and wash at low temperature for 5 minutes, and centrifuge under the same conditions to complete the first washing of yeast cells; wash the wet yeast cells obtained from the first washing a second time under the same operating conditions to obtain 180g of wet yeast cells.

[0056] The obtained wet bacterial cells were added to 1.32 kg of physiological saline to obtain 1.5 kg of high-density, high-activity yeast suspension, OD 600 It is 107.

[0057] Example 2: Preparation of fermented camellia seed oil (bacterial to oil ratio 1:1)

[0058] Take 0.8 kg of the high-density, high-activity yeast culture obtained in Example 1, add 0.8 kg of crude camellia seed oil in a 1:1 ratio, and ferment the camellia seed oil in a 5L glass fermentation tank. The fermentation conditions are 30℃, flow rate 1L / min, stirring at 500 rpm, and fermentation for 28 hours. After the acid value reaches 11.5 mgKOH / g, the fermentation is stopped to obtain the fermented camellia seed oil broth. After separation and purification, fermented camellia seed oil is obtained.

[0059] Example 3: Preparation of fermented flaxseed oil (bacterial to oil ratio 1:3)

[0060] Take 0.4 kg of the high-density, high-activity yeast culture obtained in Example 1, add 1.2 kg of flaxseed crude oil at a ratio of 1:3, and ferment the flaxseed in a 5L glass fermentation tank. The fermentation conditions are 32℃, flow rate 1L / min, stirring at 500 rpm, and fermentation is carried out for 48 hours. When the acid value reaches 18.2 mgKOH / g, the fermentation is stopped to obtain flaxseed fermentation broth. After separation and purification, fermented flaxseed is obtained.

[0061] Example 4: Preparation of fermented almond oil (bacterial to oil ratio 1:4)

[0062] Take 0.3 kg of the high-density, high-activity yeast culture obtained in Example 1, add 1.2 kg of almond crude oil at a ratio of 1:4, and ferment the almond oil in a 5L glass fermentation tank. The fermentation conditions are 28℃, flow rate 1L / min, stirring at 500 rpm, and fermentation is carried out for 48 hours. When the acid value reaches 15.9 mgKOH / g, the fermentation is stopped to obtain almond oil fermentation broth. After separation and purification, fermented almond oil is obtained.

[0063] Example 5: Preparation of high-density Candida albicans solution

[0064] 1) Yeast strain: Starmerella Bombicola strain BKSL02, stored in glycerol cryovials at -80℃.

[0065] 2) Shake flask culture

[0066] Shake flask culture medium components: YPD medium was used.

[0067] Culture conditions: One glycerol tube of bacterial culture was inoculated into a 500 mL Erlenmeyer flask containing 150 g of sterilized seed culture medium and cultured at 25°C and 200 rpm for 48 hours to obtain OD. 600 It is a 30% primary seed culture, used for high-density fermentation inoculation.

[0068] 3) High-density fermentation

[0069] Fermentation medium components: 1% glucose, 0.15% beef extract, and 0.8% inorganic salts (magnesium sulfate: potassium dihydrogen phosphate: disodium hydrogen phosphate: ammonium sulfate = 1:0.5:0.5:1). The fermentation medium was prepared after high-temperature sterilization and cooling.

[0070] 3 kg of the prepared fermentation medium was poured into a 5 L glass stirred fermenter and sterilized at 121 °C for 30 minutes. After cooling to 30 °C, 150 g of the pre-cultured primary seed culture was inoculated. Fermentation was carried out at 30 °C, stirring at 600 rpm, and aeration at 1.5 L / min for 48 h to obtain OD. 600 The high-density yeast fermentation broth is 115. A portion of the high-density yeast fermentation broth is cooled to 10℃ and used to prepare a high-density, high-activity yeast suspension. The other portion of the high-density yeast fermentation broth is used for direct fermentation of natural plant oils.

[0071] 4) Preparation of high-density, high-activity yeast suspension:

[0072] Take 1.5 kg of cooled high-density yeast fermentation broth, centrifuge at 4000 rpm for 5 minutes to obtain yeast wet cells; add sterile physiological saline to the yeast wet cells at a weight ratio of 2, stir and wash at low temperature for 5 minutes, and centrifuge under the same conditions to complete the first washing of yeast cells; wash the yeast wet cells obtained from the first washing a second time under the same operating conditions to obtain 191 g of yeast wet cells.

[0073] The obtained wet bacterial cells were added to 1.309 kg of physiological saline to obtain 1.5 kg of high-density, high-activity yeast culture. OD 600 It is 112.

[0074] Example 6: Preparation of fermented perilla seed oil (bacterial to oil ratio 1:2)

[0075] Take 0.5 kg of the high-density, high-activity yeast suspension obtained in Example 4, add 1 kg of crude perilla seed oil at a ratio of 1:2, and ferment the perilla seed oil in a 5L glass fermenter. The fermentation conditions are 32℃, flow rate 1L / min, stirring at 500 rpm, and fermentation for 45 h. When the acid value reaches 26.9 mgKOH / g, the fermentation is stopped, and the perilla seed oil fermentation broth is obtained. After separation and purification, fermented perilla seed oil is obtained.

[0076] Example 7: Preparation of fermented sea buckthorn fruit oil (bacterial to oil ratio 2:1)

[0077] Take 1 kg of the high-density, high-activity yeast suspension obtained in Example 4, add 0.5 kg of raw prickly ash oil at a ratio of 2:1, and ferment the prickly ash oil in a 5L glass fermenter. The fermentation conditions are 30℃, flow rate 1L / min, stirring at 500rpm, and fermentation for 16 hours. When the acid value reaches 28.9 mgKOH / g, the fermentation is stopped, and the prickly ash oil fermentation broth is obtained. After separation and purification, fermented prickly ash oil is obtained.

[0078] Example 8: Preparation of high-density Candida albicans solution

[0079] 1) Yeast strain: Starmerella Bombicola ATCC22214 strain, stored in glycerol cryovials at -80℃.

[0080] 2) Shake flask culture

[0081] Shake flask culture medium components: YPD medium was used.

[0082] Culture conditions: One glycerol tube of bacterial culture was inoculated into a 500 mL Erlenmeyer flask containing 150 g of sterilized seed culture medium and cultured at 25°C and 200 rpm for 48 hours to obtain OD. 600 It is a 30% primary seed culture, used for high-density fermentation inoculation.

[0083] 3) High-density fermentation

[0084] Fermentation medium components: 1% glucose, 0.15% beef extract, and 0.8% inorganic salts (magnesium sulfate: potassium dihydrogen phosphate: disodium hydrogen phosphate: ammonium sulfate = 1:0.5:0.5:1). The fermentation medium was prepared after high-temperature sterilization and cooling.

[0085] 3 kg of the prepared fermentation medium was poured into a 5 L glass stirred fermenter and sterilized at 121 °C for 30 minutes. After cooling to 30 °C, 150 g of the pre-cultured primary seed culture was inoculated. Fermentation was carried out at 30 °C, stirring at 600 rpm, and aeration at 1.5 L / min for 48 h to obtain OD. 600 The high-density yeast fermentation broth is 113. A portion of the high-density yeast fermentation broth is cooled to 10℃ and used to prepare a high-density, high-activity yeast suspension. The other portion of the high-density yeast fermentation broth is used for direct fermentation of natural plant oils.

[0086] 4) Preparation of high-density, high-activity yeast suspension:

[0087] Take 1.5 kg of cooled high-density yeast fermentation broth, centrifuge at 4000 rpm for 5 minutes to obtain yeast wet cells; add sterile physiological saline to the yeast wet cells at a weight ratio of 2, stir and wash at low temperature for 5 minutes, and centrifuge under the same conditions to complete the first washing of yeast cells; wash the yeast wet cells obtained from the first washing a second time under the same operating conditions to obtain 186 g of yeast wet cells.

[0088] The obtained wet bacterial cells were added to 1.314 kg of physiological saline to obtain 1.5 kg of high-density, high-activity yeast culture. OD 600 The value is 109.

[0089] Example 9: Preparation of fermented shea butter (bacterial to oil ratio 2:3)

[0090] Take 0.6 kg of the high-density, high-activity yeast suspension obtained in Example 4, add 0.9 kg of crude shea butter at a ratio of 2:3, and carry out shea butter fermentation in a 5L glass fermentation tank. The fermentation conditions are 35℃, flow rate 1L / min, stirring at 500 rpm, and fermentation is carried out for 48 hours. When the acid value reaches 26.9 mgKOH / g, fermentation is stopped to obtain shea butter fermentation broth. After separation and purification, fermented shea butter is obtained.

[0091] Example 10: Preparation of fermented jojoba oil (bacterial to oil ratio 3:2)

[0092] Take 0.9 kg of the high-density, high-activity yeast suspension obtained in Example 4, add 0.6 kg of crude jojoba oil at a ratio of 3:2, and carry out jojoba oil fermentation in a 5L glass fermenter. The fermentation conditions are 30℃, flow rate 1L / min, stirring at 500 rpm, and fermentation for 32 hours. When the acid value reaches 4.8 mgKOH / g, the fermentation is stopped, and the fermented jojoba oil broth is obtained. After separation and purification, fermented jojoba oil is obtained.

[0093] Comparative Example 1: Preparation of fermented camellia seed oil (bacterial to oil ratio 1:1)

[0094] Take 0.8 kg of the high-density, high-activity yeast fermentation broth remaining from step 3 of Example 1 (without centrifugation to remove supernatant), add 0.8 kg of camellia seed oil crude oil at a 1:1 water-to-oil ratio, and ferment the camellia seed oil under the following conditions: 30℃, flow rate 1 L / min, stirring at 500 rpm, for 28 h. When the acid value reaches 11.8 g KOH / g, stop the fermentation. After separation and purification, fermented camellia seed oil is obtained.

[0095] Comparative Example 2: Preparation of Fermented Flaxseed Oil (Bacterial to Oil Ratio 1:3)

[0096] Take 0.4 kg of the remaining high-density, high-activity yeast fermentation broth from step 3 of Example 1 (without centrifugation to remove supernatant), add 1.2 kg of flaxseed crude oil at a ratio of 1:3, and ferment the flaxseeds in a 5L glass fermentation tank. The fermentation conditions are 32℃, flow rate 1L / min, and stirring at 500 rpm. Fermentation is carried out for 48 hours. When the acid value reaches 17.9 mgKOH / g, fermentation is stopped to obtain flaxseed fermentation broth. After separation and purification, fermented flaxseeds are obtained.

[0097] Comparative Example 3: Preparation of Fermented Almond Oil (Bacterial to Oil Ratio 1:4)

[0098] Take 0.3 kg of the remaining high-density, high-activity yeast fermentation broth from step 3 of Example 1 (without centrifugation to remove supernatant), add 1.2 kg of crude almond oil at a water-to-oil ratio of 1:4, and ferment under the following conditions: 28°C, flow rate 1 L / min, stirring at 500 rpm for 48 h. When the acid value reaches 16.02 g KOH / g, stop fermentation to obtain almond oil fermentation broth; after separation and purification, obtain fermented almond oil.

[0099] Comparative Example 4: Preparation of Fermented Perilla Seed Oil (Bacterial to Oil Ratio 1:2)

[0100] Take 0.5 kg of the remaining high-density, high-activity yeast fermentation broth from step 3 of Example 1 (without centrifugation to remove supernatant), add 1 kg of perilla seed oil crude oil at a water-oil ratio of 1:2, and ferment under the following conditions: 32℃, 1 L / min flow rate, 500 rpm stirring, for 45 h. When the acid value reaches 27.52 g KOH / g, stop fermentation to obtain perilla seed oil fermentation broth; after separation and purification, obtain fermented perilla seed oil.

[0101] Comparative Example 5: Preparation of fermented sea buckthorn fruit oil (bacterial to oil ratio 2:1)

[0102] Take 1 kg of the remaining high-density, high-activity yeast fermentation broth from step 3 of Example 5 (without centrifugation to remove supernatant), add 0.5 kg of raw prickly ash fruit oil at a ratio of 2:1, and ferment the prickly ash fruit oil in a 5L glass fermentation tank. The fermentation conditions are 30℃, flow rate 1L / min, and stirring at 500rpm. Fermentation is carried out for 16 hours. When the acid value reaches 30.1 mgKOH / g, fermentation is stopped to obtain prickly ash fruit oil fermentation broth. After separation and purification, fermented prickly ash fruit oil is obtained.

[0103] Comparative Example 6: Preparation of Fermented Shea Butter (Bacterial to Oil Ratio 2:3)

[0104] Take 0.6 kg of the remaining high-density, high-activity yeast fermentation broth from step 3 of Example 8 (without centrifugation to remove supernatant), add 0.9 kg of crude shea butter at a ratio of 2:3, and ferment the shea butter in a 5L glass fermentation tank. The fermentation conditions are 35℃, flow rate 1L / min, and stirring at 500 rpm. Fermentation is carried out for 48 hours. When the acid value reaches 27.8 mgKOH / g, fermentation is stopped to obtain shea butter fermentation broth. After separation and purification, fermented shea butter is obtained.

[0105] Comparative Example 7: Preparation of Fermented Jojoba Oil (Bacterial to Oil Ratio 3:2)

[0106] Take 0.9 kg of the remaining high-density, high-activity yeast fermentation broth from step 3 of Example 8 (without centrifugation to remove supernatant), add 0.6 kg of jojoba crude oil at a ratio of 3:2, and ferment the jojoba oil in a 5L glass fermentation tank. The fermentation conditions are 30℃, flow rate 1L / min, stirring at 500 rpm, and fermentation for 32 hours. When the acid value reaches 5.1 mg KOH / g, the fermentation is stopped, and the jojoba oil fermentation broth is obtained. After separation and purification, fermented jojoba oil is obtained.

[0107] Test Example 1: Determination of Physicochemical Parameters

[0108] 1) Test samples: Examples 2-4, Examples 6-7, Examples 9-10, Comparative Examples 1-7, and corresponding crude oil.

[0109] 2) Testing method:

[0110] 2.1) Ash content determination method, refer to GB / T 17375-2008 Determination of Ash Content in Animal and Vegetable Oils.

[0111] 2.2) Peroxide value determination method: Refer to "LS / T 6106-2012 Determination of Peroxide Value of Animal and Vegetable Oils by Automatic Titration Analyzer". Peroxide value (POV) is an important indicator for characterizing the content of hydroperoxides in oils and fats, and is suitable for measuring the degree of oxidation in the early stage of oil oxidation.

[0112] 2.3) Oxidative stability test method: Refer to GB / T 21121-2024 Determination of oxidative stability of animal and vegetable oils (accelerated oxidation test) .

[0113] 2.4) Accelerated stability test at 45 degrees: The sample is subjected to accelerated stability test at 45 degrees. ℃ The color and odor were examined by human sensory evaluation after being placed in a constant temperature oven for 6 months to assess its stability.

[0114] 2.5) Acid value determination method: Refer to QS-TM-16 standard;

[0115] 2.6) Flavonoid determination method: The flavonoid content was determined using the Shanghai Sangon Biotech Plant Flavonoid Content Detection Kit;

[0116] 2.7) Total polysaccharide determination method: anthrone-sulfuric acid method;

[0117] 2.8) Determination of glycolipid content: anthrone-sulfuric acid method;

[0118] 3) See Table 1 for test results.

[0119] As shown in Table 1, without centrifugation to remove the supernatant, some oil-soluble substances in the fermentation medium will enter the fermented oil. Pre-centrifugation to remove the supernatant from the fermentation broth removes impurities from the fermentation medium and metabolic byproducts of bacterial growth. Therefore, the fermented oil obtained after centrifugation has significant advantages compared to vegetable oil fermented without centrifugation, as detailed below:

[0120] 1) In terms of color: The data in Table 1 show that the color of the fermented oil in the examples is not much different from the corresponding crude oil color, and both are lighter than the corresponding comparative fermented oil, indicating that some oil-soluble pigments in the fermentation medium have dissolved into the fermented oil. Centrifugation to remove the supernatant before adding oil can effectively prevent the color of the vegetable oil from changing, which is particularly evident in the color contrast between Example 10 and its crude oil and Comparative Example 7.

[0121] 2) From the perspective of odor: The data in Table 1 show that the odor of the fermented oil in the examples is not much different from that of the corresponding crude oil, while the comparative fermented oils of some oils often have a unique fermented odor due to the long time the bacteria have been in the culture medium. The odor comparisons between Example 2 and its crude oil and Comparative Example 1, Example 3 and its crude oil and Comparative Example 2, and Example 4 and its crude oil and Comparative Example 3 are particularly obvious.

[0122] 3) From the perspective of surface tension and contact angle: The contact angle refers to the angle between the solid-liquid interface, through the liquid interior, and at the gas-liquid interface at the solid-liquid-gas three-phase junction. It is also called the wetting angle. The oil spreading angle (or oil displacement angle) is one of the important indicators for evaluating the performance of biosurfactants. This angle directly reflects the surfactant's ability to reduce liquid surface tension and promote oil-water interface spreading. As can be seen from Table 1, the surface tension and contact angle of the examples are lower than those of the corresponding crude oils, while the surface tension and contact angle of the fermented oils in the examples are not significantly different from those of the corresponding comparative fermented oils.

[0123] 4) From the perspective of ash content: Vegetable oil ash content refers to the total amount of non-flammable inorganic minerals remaining after vegetable oil is burned at high temperature (usually 550±25℃). The lower the ash content, the purer the substance. As can be seen from Table 1, the ash content of the fermented oils in all the examples increased by 1-1.5 times compared to the corresponding crude oil, while the ash content of the fermented oils from the comparative process increased by 42-61 times compared to the crude oil. This fully demonstrates that the purity of the fermented oil obtained by this invention (the fermented oil of the examples) is significantly higher than that of the fermented oil obtained by the corresponding comparative process.

[0124] 5) From the perspective of POV, POV is an important indicator of the hydroperoxide content in oils and fats, and is suitable for measuring the degree of oxidation in the early stage of oil oxidation. The higher the POV value, the higher the degree of primary oxidation. As can be seen from Table 1, the POV data of the fermented oils in the examples are all lower than those of the corresponding crude oils, and the POV of the fermented oils in the examples is not much different from that of the corresponding comparative fermented oils.

[0125] 6) From the perspective of OSI stability: OSI is mainly represented by the length of the oxidation induction time of the oil. The longer the oxidation induction time, the better the oxidation stability and the stronger the antioxidant capacity of the oil. As can be seen from Table 1, at 120 ℃, the OSI data of the fermented oils in the examples are higher than those of the corresponding crude oils, and the OSI of the fermented oils in the examples is not much different from that of the corresponding comparative fermented oils.

[0126] 7) Based on the color and odor observed in the accelerated drying experiment at 45℃ constant temperature oven: the fermented oil from the example showed more stable color and odor compared to the corresponding crude oil and the comparative fermented oil (among others) Figure 2 Examples 2 and 1, and their crude oil color comparisons and color comparisons before and after being placed in a 45-degree oven for 6 months; Figure 3 Examples 4, Comparative Example 3, and their crude oil color comparisons, as well as color comparisons before and after being placed in a 45-degree oven for 6 months; Figure 4 (This is a comparison of the color of Example 6, Comparative Example 4, and their crude oil, as well as a comparison of the color before and after being placed in a 45-degree oven for 6 months). The color stability comparisons of Example 2 and its crude oil with Comparative Example 1, Example 4 and its crude oil with Comparative Example 3, and Example 6 and its crude oil with Comparative Example 4 are particularly significant.

[0127] 8) In terms of acid value, all fermented oil processes show a significant increase in acid value compared to crude oil. Under the action of yeast, natural vegetable oils break down large-molecule triglycerides into diglycerides, monoglycerides, and even smaller free fatty acids, thus significantly increasing the acid value.

[0128] 9) In terms of flavonoids and polysaccharides, the flavonoid and polysaccharide contents of all fermented oil processes are significantly higher than those of crude oil. These components are released from natural plant oils on the one hand, and produced during the metabolism of yeast on the other.

[0129] 10) In terms of glycolipid content, all fermentation processes result in high glycolipid content. Glycolipids are synthesized from scratch by yeast using carbon sources during fermentation.

[0130] As described above, the fermented oil obtained by this invention has advantages such as fewer impurities, lighter color, cleaner fermented oil, lower ash content, stronger oxidative stability, and longer shelf life. Compared with natural plant oil, it contains more free fatty acids, flavonoids, polysaccharides, and natural glycolipids, and has better skin moisturizing, repairing, soothing, and antioxidant effects. It has great economic value and application prospects in the field of beauty, skin care, and personal care.

[0131]

[0132] Test Example 2: Antioxidant Efficacy Test

[0133] 1) Test sample: Example 2 and its crude oil;

[0134] Among all natural plant oils, camellia oil is rich in vitamin E and polyphenols, exhibiting a more effective neutralization of free radicals and prevention of oxidative stress damage to the skin compared to other plant oils. Therefore, this test case uses Example 2 and its crude oil for antioxidant efficacy testing.

[0135] 2) Testing method:

[0136] DPPH free radicals have a maximum absorption peak near 517 nm. When DPPH free radicals react with antioxidants, the absorbance at 517 nm decreases. The degree of decrease is quantitatively related to the number of electrons received (antioxidant free radical scavenging activity), and the reaction process can be easily monitored using a spectrophotometer. The formula for calculating DPPH free radical scavenging is generally as follows: A0 is the control, referring to the DPPH free radical absorbance without the sample added;

[0137] 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:

[0138] Clearance rate (%) = [(A0-A) / A0)] × 100%

[0139] In the formula, A0 represents the absorbance of the blank control, which refers to the DPPH free radical absorbance without the sample added;

[0140] A represents the sample, indicating the absorbance of DPPH free radicals after the sample is added and reacted.

[0141] For detailed test results, please see Figure 1 ;

[0142] Figure 1 The results showed that fermented camellia seed oil had a better DPPH free radical scavenging ability and better antioxidant properties than crude camellia seed oil.

[0143] Test Example 3: Human Body Moisturizing and Skin-Nourishing Efficacy Test

[0144] 1) Test sample: Example 9 and its crude oil;

[0145] Natural shea butter is actually a solid plant lipid containing 5-10% unsaponifiables, including phytosterols and vitamin E. These components not only prevent moisture loss but also repair the skin barrier, earning it the title of "moisturizing champion" in the cosmetics industry. Therefore, this test case uses Example 9 and its crude oil to test its moisturizing and skin-nourishing effects on the human body.

[0146] 2) Testing Method: Ten male and ten female volunteers aged 18-22 were recruited. The ambient temperature was controlled at 20-25℃ and the humidity at 40-70%. Participants were prohibited from using any cosmetics or skincare products, or coming into contact with irritants, for three days prior to the experiment. Before entering the laboratory, participants were required to avoid strenuous exercise and maintain a stable mood. Each participant's arm was divided into two areas, A and B. Shea butter crude oil was applied to area A, and fermented shea butter oil was applied to area B. Skin moisture, oiliness, and elasticity were measured using a skin moisture meter at the beginning of the experiment and 30 minutes later.

[0147] 3) Test results are detailed in Table 2:

[0148] Table 2. Results of the moisturizing efficacy test of fermented shea butter and its crude oil.

[0149]

[0150] Test results show that, compared with crude oil, the fermented shea butter in Example 3 has better moisturizing and skin-softening effects, and is more refreshing and non-greasy.

[0151] Test Example 4: Human Anti-inflammatory Efficacy Test

[0152] 1) Test Sample: Example 6 and its crude oil

[0153] Flaxseed oil and perilla seed oil are both rich in alpha-linolenic acid. According to literature, alpha-linolenic acid can be converted into anti-inflammatory mediators in the body, thus giving them excellent anti-inflammatory and soothing effects. Therefore, this test case uses Example 6 and its crude oil to determine their anti-inflammatory efficacy in humans.

[0154] 2) Testing method:

[0155] Participants: Thirty volunteers aged 20-30 years with healthy skin, regardless of gender, and a positive complex lactic acid stinging test were selected. Participants voluntarily participated, signed informed consent, and were able to complete the test as required during the testing period. They were randomly assigned to groups of 10 participants each.

[0156] Test ambient temperature: (21±1)℃; Test ambient humidity: (50±10)%.

[0157] Test method: The inside of the subject's arm was washed with water and dried with cool air for 15-20 minutes. 20 μL of 1% histamine solution was applied to a patch and adhered to the inside of the volunteer's arm. After 30 minutes, the patch was removed, the patch location was marked with a marker, and different test samples were applied. The time for the redness, pain, and itching at the patch location was recorded. No sample was applied to the blank control group.

[0158] 3) The test results are detailed in Table 3:

[0159] Table 3. Test on the patch anti-inflammatory efficacy of fermented perilla seed oil.

[0160]

[0161] As shown in Table 3, compared with crude oil, the fermented perilla seed oil of Example 6 has significant anti-inflammatory and soothing effects.

Claims

1. A method for producing fermented plant oil, comprising the steps of:

1. inoculating yeast cells into a high-density fermentation medium to expand the cells, thereby obtaining a yeast cell suspension; 2. centrifuging or filtering the cell suspension to obtain yeast cells, washing the yeast cells with physiological saline, and centrifuging or filtering the washed yeast cells to obtain clean yeast cells; 3. adding the clean yeast cells obtained in step 2 into physiological saline to obtain a mixture of the yeast cells and the physiological saline; 4. adding plant oil into the mixture of step 3 in a certain proportion to perform secondary fermentation; and 5. centrifuging the mixture after the fermentation to obtain the fermented plant oil. 2.The method of claim 1, wherein the yeast cells in step 1 are obtained by shake flask culture, and the culture conditions are 1%-12% inoculation amount, 20-40℃, and 16-48h. 3.The method of claim 1, wherein the secondary fermentation in step 4 is performed under the following conditions: 20-60℃, and 2h-120h. 4.The method of claim 1, wherein the mixture of step 3 contains 20-50g of yeast cells (dry weight) per kg of the mixture. 5.The method of claim 1, wherein the centrifugation in step 2 is performed at 3000-5000rpm, preferably 4000rpm, for 3-8min, preferably 5min. 6.The method of claim 1, wherein the mass ratio of the mixture of step 3 to the plant oil in step 4 is 1:4-4:

1. 7.The method of claim 1, wherein the secondary fermentation in step 4 is performed under the following conditions: 20-60℃, and 2h-120h. 8.The method of claim 1, wherein the plant oil is selected from the group consisting of camellia seed oil, jojoba oil, perilla seed oil, peony seed oil, shea butter, coconut oil, ganoderma spore oil, Prinsepia uniflora oil, apricot kernel oil, grape seed oil, babassu oil, white pool flower seed oil, rice oil, sunflower seed oil, coffee seed oil, flaxseed oil, kiwifruit seed oil, pomegranate seed oil, rose fruit oil, schizocarpy fruit oil, safflower seed oil, preferably camellia seed oil, jojoba oil, perilla seed oil, apricot kernel oil, Prinsepia uniflora oil, flaxseed oil. 9.The method of claim 1, wherein the yeast is selected from the group consisting of Candida bombicola BKSL02 and Candida bombicola ATCC22214. The bacteria body of step 1 is expanded under the following conditions: fermentation culture at 28-32℃, stirring at 400-800rpm, ventilation at 1.2-1.8L / min for 30-60h, and the OD 600 of the high-density yeast fermentation liquid is above 80. Preferably, the fermentation culture is carried out at a temperature of 29-32°C, with stirring at 500-700 rpm, and ventilation at 1.4-1.6 L / min for 40-50 h, to obtain a high-density yeast fermentation broth with OD 600 of 90-120, specifically 100-118. 10.Fermented plant oil produced by the method of any one of claims 1-9. ​ ​ ​ ​ In step 3, the obtained yeast is added to physiological saline to obtain a yeast suspension, and the OD 600 is 90-120, such as 100-118, more specifically 107, 109, 112. ​ ​ ​ ​ ​ 8. The production method according to any one of claims 1 to 7, characterized by, ​ 9. The production method according to any one of claims 1 to 7, wherein ​ ​

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