Preparation method of compound fermentation essence oil with anti-oxidation and moisturizing effects

Abstract

The invention discloses a preparation method of compound fermentation essence oil with anti-oxidation and moisturizing effects. The method comprises the following steps: respectively selecting single colonies of two strains of saccharomyces utilis and yarrowia lipolytica by using an inoculating loop, and respectively transferring the single colonies into an activation culture medium for culturing to obtain an activated saccharomyces utilis fermentation strain and an activated yarrowia lipolytica fermentation strain; mixing the two activated fermentation strains into a compound strain; mixing lucid ganoderma essence oil with wormwood essence oil to form composite essence oil, then adding fructose and trypsin extract powder, and performing sterilization treatment to obtain a composite essence oil culture solution; inoculating the compound strain into a compound essence oil culture solution for compound fermentation to obtain a compound fermentation solution; performing centrifugal treatment on the composite fermentation liquid, taking upper-layer composite essence oil, then adding cholesterol and vitamin E, and mixing, so as to obtain the lucid ganoderma and wormwood composite fermentation essence oil. The compound fermentation essence oil prepared by the preparation method disclosed by the invention has good antioxidant activity and moisturizing effect through implementation verification, and is a safe, comfortable and efficient nursing product.

Description

1. Technical field:

[0001] The invention belongs to the technical field of biological fermentation in health care products, and in particular relates to a method for preparing a composite fermented essential oil with antioxidant and moisturizing effects. 2. Background technology:

[0002] Essential oils are natural products. Traditional medicine has long used plant extracts and essential oils. Essential oils have a long history and good market prospects, especially in the field of cosmetics and personal care products. With the growing demand of consumers for healthy and natural products, the essential oil market continues to expand. Especially for the demand for personal care products, as consumers' demand for high-quality and personalized products increases, technological innovation will be an important factor in promoting the development of the essential oil industry, and the development of new products with diversified bioactive functions will be the focus of future research, and the essential oil market will develop in this direction.

[0003] Ganoderma lucidum is a famous medicinal and edible fungus with a history of thousands of years of medicinal use in China. It is known as the "immortal grass". Ganoderma lucidum spore oil is an oily substance extracted from Ganoderma lucidum spores. It is rich in active ingredients, such as triterpenoid ganoderic acid, unsaturated fatty acids and Ganoderma lucidum polysaccharides. These ingredients give Ganoderma lucidum spore essential oil a variety of pharmacological properties. Studies have shown that it can regulate skin microbiota, reduce inflammatory responses, and accelerate the healing process of skin wounds. In addition, the fatty acids and glycerides in Ganoderma lucidum spore oil have a protective effect on the skin, can improve external irritation to the skin, improve skin redness, prevent skin inflammation, reduce the occurrence of acne, and make the skin naturally shiny. Terpenoid compounds have anti-inflammatory, antioxidant and epidermal repair functions.

[0004] Artemisia argyi is a perennial herbaceous plant of the genus Artemisia in the Asteraceae family, with a long history and wide application value. It is widely distributed in China, among which Nanyang, Henan is a cluster area of ​​wormwood industry.

[0005] Wormwood essential oil is a volatile aromatic substance extracted from the leaves and stems of wormwood. It is usually light yellow or greenish yellow. Due to its unique chemical composition, such as triterpenes and polysaccharides, wormwood essential oil has multiple functions such as antibacterial, anti-inflammatory, antioxidant, and whitening. It can be used to treat skin inflammation and acne and promote skin repair. The moisturizing effect of wormwood essential oil can help skin care, skin rejuvenation, promote blood circulation, metabolize aging cells, fade spots, make the skin ruddy, and promote the regeneration of new cells, reduce wrinkles, and delay aging.

[0006] Faced with fierce market competition, the essential oil market is expected to continue to grow, especially in the fields of cosmetics and personal care products. Although plant essential oils such as Ganoderma lucidum and Artemisia argyi are rich in active ingredients, it is still a challenge to improve the body's absorption of these active ingredients to improve the skin's moisturizing ability and improve the antioxidant efficiency of active ingredients. In recent years, fermentation technology has continued to develop in the fields of daily chemical products and personal care products, and it has become possible to improve the low utilization rate of active ingredients in traditional essential oils through fermentation. 3. Summary of the invention:

[0007] The technical problem to be solved by the present invention is that although the plant essential oil based on Ganoderma lucidum and wormwood has diversified biological activities, the utilization rate in use is generally low. In order to improve the utilization rate of the composite essential oil of Ganoderma lucidum and wormwood, the present invention provides a preparation method of a composite fermented essential oil containing Ganoderma lucidum and wormwood with antioxidant and moisturizing effects. The present invention uses two excellent yeast strains (Torulopsis utilis and Yarrowia lipolytica) for composite fermentation, thereby enhancing the skin moisturizing ability of the composite essential oil of Ganoderma lucidum and wormwood, and biotransforms the active ingredients such as triterpenes and polysaccharides, thereby improving the antioxidant activity of the essential oil; in addition, the stability of the composite essential oil is further enhanced by supplementing vitamin E and other methods, providing a new way to develop natural antioxidants with high added value.

[0008] In order to solve the above problems, the technical solution adopted by the present invention is:

[0009] The present invention provides a method for preparing a composite fermented essential oil with antioxidant and moisturizing effects, the preparation method comprising the following steps:

[0010] (1) using an inoculation loop to pick up single colonies of two strains of Torulopsis utilis and Yarrowia lipolytica respectively, and then transfer them to their respective activation culture media, adjust the initial pH to 6-7, place them in a shaker, and culture them at 28-30° C. and 100-120 rpm for 2-3 days, and after culture, obtain activated Torulopsis utilis fermentation strains and activated Yarrowia lipolytica fermentation strains;

[0011] (2) mixing the obtained activated Torulopsis utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain to prepare a composite strain;

[0012] (3) Ganoderma lucidum essential oil and wormwood essential oil are mixed to prepare a composite essential oil, fructose and trypsin extract powder are added to the composite essential oil, and then sterilized at 121° C. for 15 to 20 minutes, and cooled to room temperature after sterilization to obtain a composite essential oil culture solution;

[0013] (4) inoculating the composite strain obtained in step (2) into the composite essential oil culture solution obtained in step (3), and performing composite fermentation at 28-30° C. and 120-160 rpm for 4-7 days to obtain a composite fermentation solution;

[0014] (5) Centrifuging the composite fermentation liquid obtained in step (4), taking the upper composite essential oil after centrifugation, and then adding cholesterol and vitamin E to mix, thereby obtaining the composite fermentation essential oil of Ganoderma lucidum and Artemisia argyi.

[0015] According to the preparation method of the composite fermented essential oil with antioxidant and moisturizing effects, the preparation method of the activation culture medium in step (1) is: add 5-10g of peptone, 10-20g of sucrose, 2-5g of sodium chloride, add distilled water to 1L, sterilize at 121°C for 15-20min, and let it stand for use.

[0016] According to the preparation method of the composite fermented essential oil with antioxidant and moisturizing effects, the picking amount of single colonies of both Torulopsis utilis and Yarrowia lipolytica in step (1) is 0.05-0.1 g, and the transferred activation culture medium is 20-40 mL.

[0017] According to the preparation method of the composite fermented essential oil with antioxidant and moisturizing effects, the concentrations of the activated Torulopsis utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain obtained in step (1) are both 1×10 8 ~1×10 10 CFU / mL.

[0018] According to the method for preparing the composite fermented essential oil with antioxidant and moisturizing effects, in step (2), the volume ratio of the activated Torulopsis utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain when mixed is 1-3:1-3.

[0019] According to the method for preparing the composite fermented essential oil with antioxidant and moisturizing effects, the volume ratio of the Ganoderma lucidum essential oil and the Artemisia argyi essential oil mixed in step (3) is 1-3:1-3.

[0020] According to the preparation method of the composite fermented essential oil with antioxidant and moisturizing effects, the ingredients of the composite essential oil culture solution obtained in step (3) are: 5-10g of fructose, 5-10g of trypsin extract powder, 800mL of composite essential oil, and pure water is added to 1L.

[0021] According to the method for preparing the composite fermented essential oil with antioxidant and moisturizing effects, in step (4), the composite strain is inoculated into the composite essential oil culture solution at a volume ratio of 3-5%.

[0022] According to the above-mentioned method for preparing the composite fermented essential oil with antioxidant and moisturizing effects, the centrifugal conditions in step (5) are 5000-6000 rpm and the centrifugal time is 5-10 min.

[0023] According to the preparation method of the composite fermented essential oil with antioxidant and moisturizing effects, 0.1-0.2 g of cholesterol and 3-5 g of vitamin E are added to 1 L of the upper composite essential oil taken in step (5).

[0024] In the technical solution of the present invention, the ganoderma lucidum essential oil used is ganoderma lucidum spore essential oil, and the wormwood essential oil is wormwood leaf essential oil; the ganoderma lucidum spore essential oil is extracted from ganoderma lucidum spore powder, and is rich in triterpenoid ganoderma acid, unsaturated fatty acids, ganoderma lucidum polysaccharides, etc.; and the wormwood leaf essential oil is widely used in traditional Chinese medicine because of its unique herbal aroma and the effects of warming the meridians and stopping bleeding, dispelling cold and relieving pain. The combination of the two not only has the effects of nourishing and moisturizing, but also has the characteristics of anti-inflammatory and antioxidant.

[0025] The present invention adopts torula utilis and Yarrowia lipolytica as double yeast fermentation strains, and the growth rate of torula utilis is better than some yeasts, such as Saccharomyces cerevisiae. It can utilize pentose and hexose, does not produce alcohol under aerobic conditions, can use urea and nitric acid as nitrogen sources, and can grow without adding any growth factors in the culture medium. Yarrowia lipolytica is considered to be an ideal host for producing natural products such as terpenes, polyketides and flavonoids because it can utilize a variety of hydrophobic substrates, has high acid resistance, salt stress tolerance, and a high-throughput tricarboxylic acid cycle, providing sufficient acetyl-CoA precursors. Fermentation of the composite essential oil of Ganoderma lucidum and Artemisia argyi using these two dominant strains can not only improve the antioxidant activity of the composite essential oil, but also enhance the bioavailability of the composite essential oil and improve the absorption rate of the human body. It has significant advantages in the health aspects such as food and medicine care.

[0026] The positive beneficial effects of the present invention are:

[0027] 1. Synergistic effect of compound essential oil and superior strains: Based on the compound essential oil of Ganoderma lucidum and Artemisia argyi, yeast strains with high efficiency in converting the active ingredients of compound essential oil of Ganoderma lucidum and Artemisia argyi are selected: Torulopsis utilis and Yarrowia lipolytica, which can produce synergistic effects, enhance each other's beneficial effects, and jointly provide more comprehensive health benefits.

[0028] 2. Improving the antioxidant efficiency of active ingredients: The activity of natural antioxidant ingredients in Ganoderma lucidum and Artemisia argyi is enhanced through dominant fermentation strains and specific fermentation processes; in Example 9 of the present invention, the DPPH free radical scavenging rate reaches 92.58%, the ABTS free radical scavenging rate reaches 78.86%, and the iron ion reducing capacity reaches 2.68, thereby providing stronger antioxidant protection.

[0029] 3. Improve human absorption and enhance skin moisturizing ability: select yeast strains with efficient conversion ability, control fermentation conditions, enhance the bioconversion ability of the active ingredients of the Ganoderma lucidum and Artemisia argyi composite essential oil, efficiently decompose oil into glycerol and free fatty acids, and at the same time, the microbial metabolites produced during the fermentation process can help decompose plant cell walls, fully release these active ingredients, and make them easier to be used by the human body; in Example 9 of the present invention, the moisture content of the skin stratum corneum increased by 10.71%, and the epidermal water loss rate decreased by 8.11g / m 2 h, significant moisturizing effect.

[0030] 4. Improve economic benefits and promote product diversification: By selecting superior materials, matching strains and optimizing fermentation processes, it is not only possible to reduce production costs and increase output, but also to help develop a variety of health products, such as health products, cosmetics, etc., broaden the application areas, and thus enhance the market competitiveness of products.

[0031] 5. Safety and environmental protection: The preparation method of the composite fermented essential oil of Ganoderma lucidum and Artemisia argyi provided by the present invention uses probiotics and food-grade fermentation materials for fermentation operation, reduces or removes possible harmful substances, and improves the safety of the product. It helps to reduce the impact on the environment and meets the requirements of sustainable development. 4. Specific implementation methods:

[0032] In order to better explain the purpose, implementation scheme and technical advantages of the present invention, the present invention is further described below in conjunction with examples and comparative examples, but the scope of protection of the technical solution of the present invention is not limited.

[0033] The fermentation materials and strains used in the embodiments of the present invention: Ganoderma lucidum spore essential oil and wormwood essential oil are all commercially available products; reagents and consumables are all purchased from professional reagent or consumable companies; strains are all purchased from the Beina Biological Strain Collection Center.

[0034] In the following examples, the utilitarian yeast used is referred to as utilitarian, the Yarrowia lipolytica is referred to as lipolytica, the Saccharomyces cerevisiae is referred to as cerevisiae, the Ganoderma lucidum spore essential oil is referred to as Ganoderma lucidum, and the Artemisia argyi leaf essential oil is referred to as Artemisia argyi.

[0035] The mixing ratio parameters of the strains and the essential oils in the examples of the present invention and the comparative examples are shown in Table 1.

[0036] Table 1 Parameters of the mixing ratio of strains and essential oils in the examples of the present invention and the comparative examples

[0037]

[0038] Embodiment 1:

[0039] The preparation method of the composite fermented essential oil with antioxidant and moisturizing effects of the present invention comprises the following detailed steps:

[0040] (1) 0.1 g of a single colony of the utilis yeast strain and 0.1 g of a single colony of the Yarrowia lipolytica strain were picked up using an inoculation loop, and then transferred to 40 mL of activation culture medium, respectively. The initial pH was adjusted to 6, and the culture medium was placed in a shaker and cultured at 29°C and 120 rpm for 2 days. After culture, activated utilis yeast fermentation strains and activated Yarrowia lipolytica fermentation strains were obtained. The concentrations of the two fermentation strains were both 1×10 9 CFU / mL;

[0041] The preparation method of the activation medium is as follows: 5 g of peptone, 15 g of sucrose, and 3 g of sodium chloride are added with distilled water to 1 L, and sterilized at 121° C. for 20 min to obtain the activation medium;

[0042] (2) mixing the obtained activated Torula utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain in a volume ratio of 1:1 to prepare a composite strain;

[0043] (3) adding fructose and trypsin powder to the Ganoderma lucidum spore essence oil, sterilizing the mixture at 121° C. for 20 min, and cooling the mixture to room temperature to obtain a composite essence oil culture solution;

[0044] The ingredients of the compound essential oil culture solution are: 5g fructose, 5g trypsin extract powder, 800mL compound essential oil, and pure water added to 1L;

[0045] (4) inoculating the composite strain obtained in step (2) into the composite essential oil culture solution obtained in step (3) at a volume ratio of 3%, and performing composite fermentation at 29° C. and 140 rpm for 6 days to obtain a composite fermentation solution;

[0046] (5) The composite fermentation liquid obtained in step (4) was centrifuged at 6000 rpm for 7 min, and the upper layer of composite essential oil was taken after centrifugation. Then, 0.1 g / L cholesterol and 4 g / L vitamin E (to maintain its activity and stability) were added and mixed to obtain composite fermentation essential oil.

[0047] Embodiment 2:

[0048] The preparation method of the composite fermented essential oil with antioxidant and moisturizing effects of the present invention is basically the same as that of Example 1, except that:

[0049] In step (3): add fructose and trypsin powder to the mugwort essential oil.

[0050] Embodiment 3:

[0051] The preparation method of the composite fermented essential oil with antioxidant and moisturizing effects of the present invention is basically the same as that of Example 1, except that:

[0052] In step (3): Ganoderma lucidum essential oil and Artemisia argyi essential oil are mixed in a volume ratio of 1:1 to prepare a composite essential oil, and fructose and trypsin extract powder are added to the obtained composite essential oil.

[0053] Embodiment 4:

[0054] The preparation method of the composite fermented essential oil with antioxidant and moisturizing effects of the present invention is basically the same as that of Example 1, except that:

[0055] In step (3): Ganoderma lucidum essential oil and Artemisia argyi essential oil are mixed in a volume ratio of 3:1 to prepare a composite essential oil, and fructose and trypsin extract powder are added to the composite essential oil.

[0056] Embodiment 5:

[0057] The preparation method of the composite fermented essential oil with antioxidant and moisturizing effects of the present invention is basically the same as that of Example 1, except that:

[0058] In step (3): Ganoderma lucidum essential oil and Artemisia argyi essential oil are mixed in a volume ratio of 1:3 to prepare a composite essential oil, and fructose and trypsin extract powder are added to the obtained composite essential oil.

[0059] Embodiment 6:

[0060] The preparation method of the composite fermented essential oil with antioxidant and moisturizing effects of the present invention is basically the same as that of Example 1, except that:

[0061] In step (1), 0.1 g of a single colony of the utilis yeast strain was picked up with an inoculation loop, and then transferred to 40 mL of activation medium, the initial pH was adjusted to 6, and the culture was placed in a shaker at 29°C and 120 rpm for 2 days. After the culture, an activated utilis yeast fermentation strain was obtained, and the concentration of the obtained fermentation strain was 1×10 9 CFU / mL;

[0062] In step (2): omitted;

[0063] In step (3): Ganoderma lucidum essential oil and Artemisia argyi essential oil are mixed in a volume ratio of 3:1 to prepare a composite essential oil, and fructose and trypsin extract powder are added to the composite essential oil;

[0064] In step (4), the activated utilis fermentation strain obtained in step (1) is inoculated into the composite essential oil culture solution obtained in step (3) at a volume ratio of 3%, and composite fermentation is carried out at 29° C. and 140 rpm for 6 days to obtain a composite fermentation solution.

[0065] Embodiment 7:

[0066] The preparation method of the composite fermented essential oil with antioxidant and moisturizing effects of the present invention is basically the same as that of Example 6, except that:

[0067] In step (1), 0.1 g of a single colony of Yarrowia lipolytica was picked up with an inoculation loop, and then transferred to 40 mL of activation medium, the initial pH was adjusted to 6, and the culture was placed in a shaker at 29°C and 120 rpm for 2 days. After the culture, an activated Yarrowia lipolytica fermentation strain was obtained, and the concentration of the obtained fermentation strain was 1×10 9 CFU / mL;

[0068] In step (4): the activated Yarrowia lipolytica fermentation strain obtained in step (1) is inoculated into the composite essential oil culture solution obtained in step (3) at a volume ratio of 3%, and composite fermentation is carried out at 29° C. and 140 rpm for 6 days to obtain a composite fermentation solution.

[0069] Embodiment 8:

[0070] The preparation method of the composite fermented essential oil with antioxidant and moisturizing effects of the present invention is basically the same as that of Example 4, except that:

[0071] In step (2): the obtained activated Torulopsis utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain are mixed in a volume ratio of 3:1 to prepare a composite strain.

[0072] Embodiment 9:

[0073] The preparation method of the composite fermented essential oil with antioxidant and moisturizing effects of the present invention is basically the same as that of Example 4, except that:

[0074] In step (2): the obtained activated Torulopsis utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain are mixed in a volume ratio of 1:3 to prepare a composite strain.

[0075] Comparative Example 1:

[0076] A method for preparing a composite fermentation broth, the detailed steps of which are as follows:

[0077] (1) using an inoculation loop to pick up 0.1 g of a single colony of a utilis yeast strain and 0.1 g of a single colony of a Yarrowia lipolytica strain, respectively, and then transfer them to 40 mL of activation medium, respectively, adjust the initial pH to 6, place them in a shaker, and culture them at 29° C. and 120 rpm for 2 days, to obtain activated utilis yeast fermentation strains and activated Yarrowia lipolytica fermentation strains;

[0078] The preparation method of the activation medium is as follows: 5 g of peptone, 15 g of sucrose, and 3 g of sodium chloride are added with distilled water to 1 L, and sterilized at 121° C. for 20 min to obtain the activation medium;

[0079] (2) mixing the obtained activated Torula utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain in a volume ratio of 1:3 to prepare a composite strain;

[0080] (3) inoculating the composite strain obtained in step (2) into pure water at a volume ratio of 3%, and performing composite fermentation at 29° C. and 140 rpm for 6 days to obtain a composite fermentation liquid;

[0081] (4) The composite fermentation broth obtained in step (3) was centrifuged at 6000 rpm for 7 min, 0.1 g / L cholesterol and 4 g / L vitamin E were added to the supernatant, and the mixture was mixed to obtain a composite fermentation broth.

[0082] Comparative Example 2:

[0083] A method for preparing a composite essential oil, the detailed steps of which are as follows:

[0084] (1) Ganoderma lucidum essential oil and wormwood essential oil are mixed in a volume ratio of 3:1 to prepare a composite essential oil, fructose and trypsin extract powder are added to the composite essential oil, and then sterilized at 121° C. for 20 min, and cooled to room temperature after sterilization to obtain a composite essential oil culture solution;

[0085] (2) inoculating the composite essential oil culture solution obtained in step (1) with purified water at a volume ratio of 3%, and then placing the solution in a shaker for composite fermentation at 29° C. and 140 rpm for 6 days to obtain a composite fermentation solution;

[0086] (3) The composite fermentation liquid obtained in step (2) was centrifuged at 6000 rpm for 7 min, and the upper layer of composite essential oil was taken after centrifugation. Then, 0.1 g / L cholesterol and 4 g / L vitamin E (to maintain its activity and stability) were added and mixed to obtain composite fermentation essential oil.

[0087] Comparative Example 3:

[0088] A method for preparing a composite essential oil is substantially the same as that of Example 6, except that:

[0089] In step (1), 0.1 g of a single colony of Saccharomyces cerevisiae strain was picked up with an inoculation loop, and then transferred to 40 mL of activation medium, the initial pH was adjusted to 6, and the culture was placed in a shaker at 29°C and 120 rpm for 2 days. After the culture, an activated Saccharomyces cerevisiae fermentation strain was obtained, and the concentration of the obtained fermentation strain was 1×10 9 CFU / mL;

[0090] In step (4): the activated Saccharomyces cerevisiae fermentation strain obtained in step (1) is inoculated into the composite essential oil culture solution obtained in step (3) at a volume ratio of 3%, and composite fermentation is carried out at 29° C. and 140 rpm for 6 days to obtain a composite fermentation solution.

[0091] Comparative Example 4:

[0092] A method for preparing a composite essential oil is substantially the same as that of Example 6, except that:

[0093] In step (1), 0.1 g of a single colony of the Saccharomyces cerevisiae strain and 0.1 g of a single colony of the Torulopsis utilis strain were picked up using an inoculation loop, and then transferred to 40 mL of activation medium, the initial pH was adjusted to 6, and the culture was placed in a shaker at 29° C. and 120 rpm for 2 days. After the culture, activated Saccharomyces cerevisiae fermentation strains and Torulopsis utilis fermentation strains were obtained, and the concentrations of the obtained fermentation strains were both 1×10 9 CFU / mL;

[0094] In step (2): the activated Saccharomyces cerevisiae fermentation strain and the activated Torulopsis utilis fermentation strain are mixed in a volume ratio of 1:1 to prepare a composite strain;

[0095] In step (4), the composite strain obtained in step (2) is inoculated into the composite essential oil culture solution obtained in step (3) at a volume ratio of 3%, and composite fermentation is carried out at 29° C. and 140 rpm for 6 days to obtain a composite fermentation solution.

[0096] Comparative Example 5:

[0097] A method for preparing a composite essential oil is substantially the same as that of Example 6, except that:

[0098] In step (1), 0.1 g of a single colony of Saccharomyces cerevisiae and 0.1 g of a single colony of Yarrowia lipolytica were picked up using an inoculation loop, and then transferred to 40 mL of activation medium, the initial pH was adjusted to 6, and the culture was placed in a shaker at 29° C. and 120 rpm for 2 days. After the culture, activated Saccharomyces cerevisiae fermentation strains and Yarrowia lipolytica fermentation strains were obtained, and the concentrations of the obtained fermentation strains were both 1×10 9 CFU / mL;

[0099] In step (2): the activated Saccharomyces cerevisiae fermentation strain and the activated Yarrowia lipolytica fermentation strain are mixed in a volume ratio of 1:1 to prepare a composite strain;

[0100] In step (4), the composite strain obtained in step (2) is inoculated into the composite essential oil culture solution obtained in step (3) at a volume ratio of 3%, and composite fermentation is carried out at 29° C. and 140 rpm for 6 days to obtain a composite fermentation solution.

[0101] Comparative Example 6:

[0102] A method for preparing a composite essential oil is substantially the same as that of Example 6, except that:

[0103] In step (1), 0.1 g of a single colony of Saccharomyces cerevisiae and 0.1 g of a single colony of Yarrowia lipolytica were picked up using an inoculation loop, and then transferred to 40 mL of activation medium, the initial pH was adjusted to 6, and the culture was placed in a shaker at 29° C. and 120 rpm for 2 days. After the culture, activated Saccharomyces cerevisiae fermentation strains and Yarrowia lipolytica fermentation strains were obtained, and the concentrations of the obtained fermentation strains were both 1×10 9 CFU / mL;

[0104] In step (2): the activated Saccharomyces cerevisiae fermentation strain and the activated Yarrowia lipolytica fermentation strain are mixed in a volume ratio of 3:1 to prepare a composite strain;

[0105] In step (4), the composite strain obtained in step (2) is inoculated into the composite essential oil culture solution obtained in step (3) at a volume ratio of 3%, and composite fermentation is carried out at 29° C. and 140 rpm for 6 days to obtain a composite fermentation solution.

[0106] Comparative Example 7:

[0107] A method for preparing a composite essential oil is substantially the same as that of Example 6, except that:

[0108] In step (1), 0.1 g of a single colony of Saccharomyces cerevisiae and 0.1 g of a single colony of Yarrowia lipolytica were picked up using an inoculation loop, and then transferred to 40 mL of activation medium, the initial pH was adjusted to 6, and the culture was placed in a shaker at 29° C. and 120 rpm for 2 days. After the culture, activated Saccharomyces cerevisiae fermentation strains and Yarrowia lipolytica fermentation strains were obtained, and the concentrations of the obtained fermentation strains were both 1×10 9 CFU / mL;

[0109] In step (2): the activated Saccharomyces cerevisiae fermentation strain and the activated Yarrowia lipolytica fermentation strain are mixed in a volume ratio of 1:3 to prepare a composite strain;

[0110] In step (4), the composite strain obtained in step (2) is inoculated into the composite essential oil culture solution obtained in step (3) at a volume ratio of 3%, and composite fermentation is carried out at 29° C. and 140 rpm for 6 days to obtain a composite fermentation solution.

[0111] In order to further illustrate that the present invention can achieve good technical effects, the following tests and questionnaires were conducted on the composite fermented essential oil products prepared in each embodiment and comparative example:

[0112] 1. In vitro antioxidant activity test

[0113] The example groups involved in the test are: Examples 1 to 9 and Comparative Examples 1 to 7.

[0114] (1) DPPH free radical scavenging rate detection (DPPH free radical scavenging ability detection kit)

[0115] Test method: Pipette 100 μL sample and add 900 μL extract, vortex to mix, centrifuge at room temperature 10000 rpm for 10 min, take the supernatant and place on ice for testing. 空白 ): Extraction solution + DPPH working solution, determination tube (A 测定 ): supernatant + DPPH working solution, control tube (A 对照 ): supernatant + anhydrous ethanol, positive control tube (A 阳性对照 ): Vitamin C + DPPH working solution. Vortex to mix, stand at room temperature away from light for 30 minutes, preheat the spectrophotometer for more than 30 minutes, and adjust the wavelength to OD 515 , anhydrous ethanol was used to adjust the zero value, and all experiments were repeated three times.

[0116] The DPPH free radical scavenging rate was calculated by the following formula:

[0117] Sample: DPPH free radical scavenging rate (%) = [[A 空白 -(A 测定 -A 对照 )]÷A 空白 ]×100%.

[0118] Positive control: DPPH free radical scavenging rate (%) = [(A 空白 -A 阳性对照 )÷A 空白 ]×100%.

[0119] (2) ABTS free radical scavenging rate test (determined by ABTS free radical scavenging ability test kit)

[0120] Test method: Pipette 100 μL sample and add 900 μL extract, vortex to mix, centrifuge at 10000 rpm for 10 min at room temperature, take the supernatant and place on ice for testing. 空白 ): distilled water + assay working solution + ABTS working solution, assay tube (A 测定): supernatant + assay working solution + ABTS working solution, control tube (A 对照 ): supernatant + anhydrous ethanol, positive control tube (A 阳性对照 ): Vitamin C + ABTS working solution. Vortex to mix, stand at room temperature away from light for 30 minutes, preheat the spectrophotometer for more than 30 minutes, and adjust the wavelength to OD 405 , anhydrous ethanol was used to adjust the zero value, and all experiments were repeated three times.

[0121] The ABTS free radical scavenging rate was calculated by the following formula:

[0122] Sample: ABTS free radical scavenging rate (%) = [[A 空白 -(A 测定 -A 对照 )]÷A 空白 ]×100%.

[0123] Positive control: ABTS free radical scavenging rate (%) = [(A 空白 -A 阳性对照 )÷A 空白 ]×100%.

[0124] (3) Determination of iron ion reducing ability

[0125] Test method: After mixing phosphate buffer (0.2 mol / L) and potassium ferricyanide solution (1%) at a ratio of 1:1 (v:v), take 2 ml and add it to 1 ml of 0.5 mg / ml sample, incubate at 50°C for 20 min, cool in an ice bath, take 1 ml of trichloroacetic acid (10%) and mix thoroughly, centrifuge at 3000 rpm for 10 min, add 0.6 ml of ferric chloride (0.1%) to the supernatant and mix well, let stand for 10 min, adjust to zero with distilled water, and measure OD 700 The absorbance values ​​were measured (the blank control had no sample solution and the positive control was vitamin C). All experiments were repeated three times.

[0126] The in vitro antioxidant activity test results of the products prepared in the above Examples 1-9 and Comparative Examples 1-7 are shown in Table 2.

[0127] Table 2 In vitro antioxidant activity test results of the products prepared in Examples 1-9 and Comparative Examples 1-7

[0128]

[0129] (*: p<0.05) The result is significant.

[0130] It can be concluded from the data in Table 2 that by comparing the test results between Examples 1 to 9 and Comparative Examples 1 to 7, it can be seen that the composite fermented essential oil of Ganoderma lucidum and Artemisia argyi with antioxidant and moisturizing effects prepared by the present invention has significant antioxidant effects. Among them, under the joint fermentation of Toruloides utilis and Yarrowia lipolytica, the antioxidant activity is higher than that of single strain fermentation and Saccharomyces cerevisiae fermentation. In addition, the antioxidant effect of the mixed essential oil of Ganoderma lucidum and Artemisia argyi is higher than that of a single essential oil, and the antioxidant activity is highest under the implementation conditions where the ratio of Ganoderma lucidum essential oil to Artemisia argyi essential oil in the mixed essential oil of Ganoderma lucidum and Artemisia argyi is 3:1, such as in Example 9: the DPPH free radical scavenging rate is 92.58%, the ABTS free radical scavenging rate is 78.86%, and the iron ion reducing ability is 2.68.

[0131] 2. Moisturizing and repairing test

[0132] (1) Testing of stratum corneum moisture content and epidermal water loss rate (testing by stratum corneum moisture content tester / skin moisture tester)

[0133] The example groups involved in the test are: Examples 1 to 9 and Comparative Examples 1 to 7.

[0134] Test method: 64 subjects aged 25 to 45 were selected and randomly divided into 16 groups to participate in the test, corresponding to 9 examples and 7 comparative examples. The room temperature was adjusted to 22 to 26°C and the relative humidity was 40 to 50%. 0.08 mL of the sample was taken and evenly applied with a latex fingertip for 30 seconds. The specified application area was the middle of the left and right palm forearms, and the sample dosage was 2.0 to 2.2 mg / cm 2 , and rinse with water after 5 minutes. The moisture content and moisture loss rate of the stratum corneum of the skin in the application area were measured by a skin stratum corneum moisture content tester and a skin moisture tester before (A) and 2 hours after (B) sample application. The increase in the moisture content of the stratum corneum of the skin and the decrease in the epidermal moisture loss rate after the sample was used were calculated using the following formula (the average value was taken for statistical data results):

[0135] Improvement value = B 皮肤角质层水分含量 -A 皮肤角质层水分含量

[0136] Drop value = A 表皮水分流失率 -B 表皮水分流失率

[0137] The moisturizing and repairing test results of the products prepared in the above Examples 1-9 and Comparative Examples 1-7 are shown in Table 3.

[0138] Table 3 Test results of stratum corneum moisture content and epidermal water loss rate of the products prepared in Examples 1-9 and Comparative Examples 1-7

[0139]

[0140] (*: p<0.05) The result is significant.

[0141] From the data in Table 3, it can be seen that by comparing the test results of Examples 1 to 9 and Comparative Examples 1 to 7, it can be seen that the products prepared by the method of the present invention have the advantage of significantly improving the ability to moisturize the skin. The specific data are as follows: after using the fermented samples, the moisture content of the stratum corneum of the skin is increased to varying degrees, and the epidermal water loss rate is reduced. Among them, the overall moisturizing effect of the examples is higher than that of the comparative examples, especially in Example 9, the moisture content of the stratum corneum of the skin is increased by 10.71%, and the epidermal water loss rate is reduced by 8.11g / m 2 ·h.

[0142] (2) Self-evaluation of skin moisture and comfort

[0143] The embodiment groups participating in the questionnaire survey are: Embodiments 1 to 9 (due to the obvious advantages of the embodiments compared with the same period last year, only the embodiments are surveyed).

[0144] Questionnaire method: 64 subjects aged 25 to 45 years old were selected for the questionnaire survey. The room temperature was adjusted to 22 to 26°C, the relative humidity was 40 to 50%, 0.08 mL of the sample was taken, and the sample was evenly applied with a latex finger cot for 1 minute. The specified application area was the middle of the left and right palm forearms, and the sample dosage was 3.0 to 3.2 mg / cm 2 , and rinse with water after 10 minutes. Self-evaluation of the skin moisture and comfort of the application area 2 hours after sample application was conducted, and the sample was evaluated for its moisturizing and repairing effect based on the proportion of subjects in each level of moisturizing evaluation and the percentage of subjects who felt good in the comfort evaluation level. The self-evaluation results are detailed in Table 4.

[0145] The good rate calculation formula is as follows:

[0146]

[0147] Table 4 Results of self-evaluation of skin moisturizing and comfort of the products obtained in Examples 1-9

[0148]

[0149] From the data in Table 4, it can be seen that by comparing the questionnaire results between Examples 1 to 9, Example 9 has good moisturizing effect on the skin, and the subjects generally believe that Example 9 has good comfort, among which the good rate is the best, reaching 90%. This also fully verifies that the product prepared by the present invention has a good moisturizing effect.

[0150] 3. Human skin test

[0151] The example groups involved in the test: Examples 1 to 9.

[0152] This trial complies with the human skin patch test method in the "Technical Specifications for Safety of Cosmetics" revised in 2023, and 40 subjects are selected according to the subject inclusion criteria.

[0153] (1) Skin occlusive patch test

[0154] Test method: The selected area shall not exceed 50mm 2 , a qualified spot tester with a depth of about 1mm. Place the sample in the small chamber of the spot tester, with a dosage of about 0.02mL. Apply the spot tester with scalp care solution to the curved side of the subject's forearm with hypoallergenic tape, and gently press it with the palm of your hand to evenly apply it to the skin for 24 hours. Observe the skin reaction according to the skin reaction evaluation criteria 30 minutes (after the indentation disappears), 24 hours and 48 hours after removing the test object spot tester, and record the observation results.

[0155] (2) Repeated open smear test

[0156] Test method: The flexed side of the forearm is used as the test site, with an area of ​​3×3cm 2 The test area should be kept dry and avoid contact with other topical preparations. Take about 0.05 mL of the sample and apply it evenly to the test area twice a day for 7 consecutive days. Observe according to the skin reaction evaluation criteria and record the results.

[0157] The human skin safety test results of the products obtained in the above Examples 1-9 are shown in Table 5. Table 6 shows the criteria for judging skin reactions.

[0158] Table 5 Human skin safety test of the products obtained in Examples 1-9

[0159] Test items 24h 48h 7d Example 1 Level 0 Level 0 Level 0 Example 2 Level 0 Level 0 Level 0 Example 3 Level 0 Level 0 Level 0 Example 4 Level 0 Level 0 Level 0 Example 5 Level 0 Level 0 Level 0 Example 6 Level 0 Level 0 Level 0 Example 7 Level 0 Level 0 Level 0 Example 8 Level 0 Level 0 Level 0 Example 9 Level 0 Level 0 Level 0

[0160] Table 6 Skin reaction evaluation criteria

[0161]

[0162] It can be seen from the data obtained in Table 5 that the data obtained through the human skin safety test show that the Ganoderma lucidum and Artemisia argyi composite fermented essential oil with antioxidant and moisturizing effects prepared by the present invention will not affect the safety of human skin under various implementation methods and is a safe care product.

[0163] In summary, through the above in vitro antioxidant activity test, moisturizing and repairing test, moisturizing and comfort questionnaire, and human skin safety test, these tests are comprehensively analyzed to show that in the preparation method of the present invention, the composite fermented essential oil obtained by mixing two strains of toruloides utilis and Yarrowia lipolytica in a volume ratio of 1:3 and the essential oil mixed with fermented ganoderma and wormwood in a volume ratio of 3:1 has the best data. Comprehensively considering the four aspects of in vitro antioxidant activity, moisturizing and repairing, moisturizing and comfort, the scheme of Example 9 is the best. Therefore, the composite fermented essential oil of ganoderma and wormwood with antioxidant and moisturizing effects prepared by the present invention has good antioxidant activity and moisturizing effects through implementation verification, and is a safe, comfortable and efficient care product.

Claims

1. A method for preparing a composite fermented essential oil with antioxidant and moisturizing effects, characterized in that: The preparation method comprises the following steps: (1) using an inoculation loop to pick up single colonies of two strains of Torulopsis utilis and Yarrowia lipolytica respectively, and then transfer them to their respective activation culture media, adjust the initial pH to 6-7, place them in a shaker, and culture them at 28-30° C. and 100-120 rpm for 2-3 days, and after culture, obtain activated Torulopsis utilis fermentation strains and activated Yarrowia lipolytica fermentation strains; (2) mixing the obtained activated Torulopsis utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain to prepare a composite strain; (3) Ganoderma lucidum essential oil and wormwood essential oil are mixed to prepare a composite essential oil, fructose and trypsin extract powder are added to the composite essential oil, and then sterilized at 121° C. for 15 to 20 minutes, and cooled to room temperature after sterilization to obtain a composite essential oil culture solution; (4) inoculating the composite strain obtained in step (2) into the composite essential oil culture solution obtained in step (3), and performing composite fermentation at 28-30° C. and 120-160 rpm for 4-7 days to obtain a composite fermentation solution; (5) Centrifuging the composite fermentation liquid obtained in step (4), taking the upper composite essential oil after centrifugation, and then adding cholesterol and vitamin E to mix, thereby obtaining the composite fermentation essential oil of Ganoderma lucidum and Artemisia argyi.

2. The method for preparing the composite fermented essential oil with antioxidant and moisturizing effects according to claim 1, characterized in that: The preparation method of the activation culture medium in step (1) is as follows: add 5-10 g of peptone, 10-20 g of sucrose, and 2-5 g of sodium chloride, add distilled water to 1 L, sterilize at 121° C. for 15-20 min, and let stand for use.

3. The method for preparing the composite fermented essential oil with antioxidant and moisturizing effects according to claim 1, characterized in that: In step (1), the picking amount of single colonies of both Torulopsis utilis and Yarrowia lipolytica is 0.05-0.1 g, and the activated culture medium used for transfer is 20-40 mL.

4. The method for preparing the composite fermented essential oil with antioxidant and moisturizing effects according to claim 1, characterized in that: The concentrations of the activated Torulopsis utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain obtained in step (1) were both 1×10 8 ~1×10 10 CFU / mL.

5. The method for preparing the composite fermented essential oil with antioxidant and moisturizing effects according to claim 1, characterized in that: In step (2), the volume ratio of the activated Torulopsis utilis fermentation strain and the activated Yarrowia lipolytica fermentation strain when mixed is 1-3:1-3.

6. The method for preparing the composite fermented essential oil with antioxidant and moisturizing effects according to claim 1, characterized in that: In step (3), the volume ratio of the ganoderma lucidum essential oil to the wormwood essential oil is 1-3:1-3.

7. The method for preparing the composite fermented essential oil with antioxidant and moisturizing effects according to claim 1, characterized in that: The ingredients of the composite essential oil culture solution obtained in step (3) are: 5-10 g of fructose, 5-10 g of trypsin extract powder, 800 mL of composite essential oil, and pure water is added to 1 L.

8. The method for preparing the composite fermented essential oil with antioxidant and moisturizing effects according to claim 1, characterized in that: In step (4), the composite strain is inoculated into the composite essential oil culture solution at a volume ratio of 3-5%.

9. The method for preparing the composite fermented essential oil with antioxidant and moisturizing effects according to claim 1, characterized in that: The centrifugal conditions in step (5) are 5000-6000 rpm and the centrifugal time is 5-10 min.

10. The method for preparing the composite fermented essential oil with antioxidant and moisturizing effects according to claim 1, characterized in that: Add 0.1-0.2 g of cholesterol and 3-5 g of vitamin E to 1 L of the upper composite essential oil taken in step (5).

Images