Double-yeast self-assembled nano-emulsion as well as preparation method and application thereof

Abstract

The invention discloses a double-yeast self-assembled nano-emulsion as well as a preparation method and application thereof. The preparation method comprises the following steps: step 1, preparing a seed solution; step 2, primary fermentation; step 3, grease cracking; step 4, performing secondary fermentation; step 5, carrying out self-assembly; step 6, desalting; and step 7, compounding. By screening specific strains and optimizing fermentation and purification processes, the double-yeast self-assembled nano-emulsion with a good anti-wrinkle effect is obtained, the effect of a product can be remarkably improved, and the double-yeast self-assembled nano-emulsion has a good application prospect in the daily chemical industry.

Description

Technical Field

[0001] This invention discloses a fermentation product, specifically a dual-yeast self-assembled nanoemulsion, and also discloses the preparation method and application of the dual-yeast self-assembled nanoemulsion, belonging to the field of daily chemical raw materials. Background Art

[0002] As the cosmetics industry continues to demand natural and highly effective active ingredients, microbial fermentation technology, with its green and sustainable characteristics, has become an important direction for raw material development.

[0003] Currently, most mainstream microbial cosmetic raw materials on the market rely on single-strain fermentation, resulting in low levels of active ingredients and weak efficacy. Furthermore, while traditional microencapsulation technology can improve the stability of active ingredients, its complex preparation process and high cost make large-scale industrial production difficult. In recent years, yeast fermentation has attracted much attention due to its rich metabolites and high safety; however, single-strain yeast fermentation cannot meet the demand for multifunctional raw materials. Existing attempts to combine microencapsulation and fermentation technologies often face difficulties in widespread adoption due to cumbersome processes and poor cost control.

[0004] Therefore, developing a technology that is simple to implement, cost-controllable, and can significantly improve the efficacy of raw materials has become an urgent problem for the industry. Summary of the Invention

[0005] To address the shortcomings of current methods, the first objective of this invention is to provide a method for preparing a dual-yeast self-assembled nanoemulsion. This method, through screening specific strains and optimizing fermentation and purification processes, yields a dual-yeast self-assembled nanoemulsion with excellent anti-wrinkle effects, significantly enhancing product efficacy.

[0006] To achieve the above objectives, the specific technical solution is as follows: A method for preparing a dual-yeast self-assembled nanoemulsion, comprising the following steps:

[0007] Step 1, Seed culture preparation: Red yeast and Candida albicans were streaked on yeast extract glucose agar medium to obtain pure single clones, and the single clones were activated to the logarithmic phase in yeast extract glucose medium.

[0008] Step 2, Preliminary fermentation: Inoculate the logarithmic phase red yeast seed culture into a nitrogen-limited medium for preliminary fermentation;

[0009] Step 3, Lipid Decomposition: After fermentation, the lipids inside the red yeast are released using an acid-heat method;

[0010] Step 4, Secondary Fermentation: After inoculating with Candida albicans, carry out secondary fermentation;

[0011] Step 5, Self-assembly: After fermentation, the yeast is inactivated and then crushed and automatically encapsulated by low-temperature microfluidic jet. The cell fragments are removed by centrifugation to obtain a nano-pink emulsion containing encapsulated dual yeast active substances.

[0012] Step 6, Desalination: The sample is placed in a dialysis bag and dialyzed in a pure water tank. The water is changed regularly to remove inorganic salts and obtain pink yeast self-assembled nanoemulsion.

[0013] Step 7, Compounding: Add 0.2-0.5% (W / W) p-hydroxyacetophenone and 0.5-1% sodium lactate (W / W) to compound and obtain a dual-yeast self-assembled nanoemulsion.

[0014] Furthermore, the red yeast in step 1 is a self-developed strain, preserved at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a preservation date of July 15, 2024, and the collection number CGMCC No. 31295, classified and named as follows: Rhodotorula mucilaginosa MHT01 The Candida albicans strain was purchased from Shanghai Baocang Microbial Co., Ltd., and the strain number is [strain number missing]. Candida bombicola ATCC22214.

[0015] Further, the nitrogen-limiting culture medium in step 2 consists of 50.0-100 g / L glucose, 0.5-1.5 g / L yeast extract, 0.3-0.5 g / L (NH4)2SO4, 0.5-2.0 g / L KH2PO4, 1.0-1.5 g / L MgSO4·7H2O, 0.1-0.2 g / L CaCl2, 0.02-0.05 g / L FeCl3, 0.01-0.03 g / L ZnSO4, 0.001-0.002 g / L CuSO4, 0.001-0.005 g / L MnSO4, 0.01-0.02 mg / L biotin, and 1-2 g / L citric acid.

[0016] Furthermore, the initial fermentation parameters for the red yeast in step 2 are: temperature 28-30℃, dissolved oxygen 70-80%, and fermentation time 72 h.

[0017] Furthermore, in step 3, the acid-heat method involves adjusting the pH to 2.0-3.5 with a 2-4% (V / V) hydrochloric acid solution to create an acidic environment, which enhances cell wall breaking efficiency and oil release. The temperature of the fermenter is then raised to 65-75°C and maintained for 30 minutes before being cooled to below 30°C.

[0018] Furthermore, the secondary fermentation parameters for Candida albicans in step 4 are: temperature 30°C, dissolved oxygen 70-80%, and fermentation time 48 hours.

[0019] Furthermore, the self-assembly process in step 5 is as follows: low-temperature microjet pressure 2500-3000 psi, temperature 4-10℃, high-pressure homogenization for 45-60 min, followed by centrifugation at 6000-12000 rpm for 30-45 min.

[0020] Furthermore, the desalination process in step 6 is as follows: the sample is added to a dialysis bag of 100-150 Da specification, placed in a pure water tank for dialysis, and the water is changed once every 8-24 hours, for a total of 3-5 water changes.

[0021] Further, the compounding process in step 7 is as follows: add 0.2-0.5% (W / W) p-hydroxyacetophenone and 0.5-1% (W / W) sodium lactate, mix evenly, and compound to obtain a dual-yeast self-assembled nanoemulsion.

[0022] The second technical solution provided by the present invention is: a dual-yeast self-assembled nanoemulsion, prepared according to the above preparation method.

[0023] The third technical solution provided by this invention is: the application of the above-mentioned dual-yeast self-assembled nanoemulsion in cosmetic preparation.

[0024] Furthermore, the cosmetic is a topical skin preparation made with the aforementioned dual-yeast self-assembled nanoemulsion as the active ingredient, plus conventional pharmaceutical or cosmetic excipients or auxiliary ingredients.

[0025] Furthermore, the cosmetics include one or more of the following: aqueous solutions, creams, cleansers, serums, masks, and mud masks.

[0026] Furthermore, the amount of dual-yeast self-assembled nanoemulsion added to the cosmetic is 0.1% - 10%.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] In situ co-fermentation of two yeasts reduces substrate costs. Plant oils are a common substrate for preparing sophorolipids. Compared with microbial oils, plants have a longer growth cycle, and the costs of planting and extracting oils are higher. Through culture medium optimization: using a nitrogen-limiting culture medium with a low C / N ratio, the yield of red yeast oil is significantly increased.

[0029] Timing control: 0-72h Red yeast dominates oil production, 72-120h Candida albicans utilizes Red yeast oil to synthesize sophorolipids;

[0030] Self-assembly mechanism: After high-pressure homogenization, the active substances released by the two yeasts self-assemble with sophorolipid molecules through hydrophobic interactions; the permeability and efficacy of the raw materials are significantly enhanced.

[0031] Improved economic efficiency: Eliminating the step of purchasing oil from outside sources reduces raw material costs; integrated crushing and packaging reduces energy consumption and shortens the process cycle;

[0032] Enhanced efficacy: The dual-yeast co-fermented nanoemulsion contains a high concentration of natural yeast small molecule peptides, provitamin A, yeast pigments, dual-yeast polysaccharides, bioglycolipids, nucleotides, and amino acids, among other natural skin-care factors. It has advantages such as synergistic effects and enhanced penetration, resulting in a more significant anti-wrinkle effect than single-yeast fermentation. Detailed Implementation

[0033] The claims of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention. Any limited modifications made by any person within the scope of protection of the claims of the present invention are still within the scope of protection of the claims of the present invention.

[0034] Example 1

[0035] Step 1, Seed culture preparation: Prepare red yeast... Rhodotorula mucilaginosa MHT01 and Candida albicans Candida bombicola ATCC22214 was streaked onto yeast extract glucose agar to obtain pure single clones, which were then activated to the logarithmic growth phase in yeast extract glucose agar.

[0036] Step 2, Fermentation: The logarithmic phase red yeast seed culture was inoculated into a nitrogen-limited medium at an inoculation rate of 5%. The nitrogen-limited medium formula was as follows: glucose 50.0 g / L, yeast extract 1.5 g / L, (NH4)2SO4 0.3 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 1.5 g / L, CaCl2 0.1 g / L, FeCl3 0.04 g / L, ZnSO4 0.01 g / L, CuSO4 0.002 g / L, MnSO4 0.005 g / L, biotin 0.02 mg / L, and citric acid 2 g / L. Fermentation conditions were as follows: temperature controlled at 28℃, dissolved oxygen at 80%, fermentation time at 72h. After fermentation, the pH was adjusted to 3.5 with 3% hydrochloric acid solution to create an acidic environment, which enhanced cell wall breaking efficiency and oil release. The temperature of the fermentation tank was raised to 70℃ and maintained for 30 min, then cooled to 30℃. Logarithmic Candida yeast was inoculated into the fermentation tank at an inoculation rate of 5%, and glucose was added to bring the concentration to 50 g / L. Dissolved oxygen was maintained at 80%, and fermentation was carried out at 30℃ for 48 h.

[0037] Step 3, Purification: After fermentation, the yeast was inactivated at 70℃ for 30 min. The fermentation broth was homogenized under low temperature microfluidic pressure of 3000psi and temperature of 4℃ for 45 min. After that, it was centrifuged at 8000 rpm for 30 min to remove cell fragments and obtain a pink emulsion.

[0038] Step 4, Desalination: Add the sample to a 100 Da dialysis bag and place it in a pure water bath for dialysis. Change the water every 24 hours for a total of 4 times to remove inorganic salts and obtain pink dual-yeast self-assembled nanoemulsion.

[0039] Example 2

[0040] Step 1, Seed culture preparation: Prepare red yeast... Rhodotorula mucilaginosa MHT01 and Candida albicans Candida bombicola ATCC22214 was streaked onto yeast extract glucose agar to obtain pure single clones, which were then activated to the logarithmic growth phase in yeast extract glucose agar.

[0041] Step 2, Fermentation: The logarithmic phase red yeast seed culture was inoculated into a nitrogen-limited medium at an inoculation rate of 3%. The nitrogen-limited medium formula was as follows: glucose 100.0 g / L, yeast extract 1 g / L, (NH4)2SO4 0.3 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 1.5 g / L, CaCl2 0.1 g / L, FeCl3 0.02 g / L, ZnSO4 0.03 g / L, CuSO4 0.001 g / L, MnSO4 0.005 g / L, biotin 0.02 mg / L, and citric acid 1 g / L. Fermentation conditions were as follows: temperature controlled at 28℃, dissolved oxygen at 70%, fermentation time at 72 h, pH adjusted to 3.5 with 3% hydrochloric acid solution at the end of fermentation to create an acidic environment, enhance cell wall breaking efficiency and oil release, the temperature of the fermenter was raised to 70℃ and maintained for 30 min, then cooled to 30℃, and Candida albicans in the logarithmic phase was inoculated into the fermenter at an inoculation amount of 3%, glucose was added to 100 g / L, dissolved oxygen was 80%, and fermentation was carried out at 30℃ for 48 h.

[0042] Step 3, Purification: After fermentation, the yeast was inactivated at 65℃ for 30 min. The fermentation broth was homogenized under low temperature microfluidic pressure of 3000psi and temperature of 4℃ for 45 min. After that, it was centrifuged at 6000 rpm for 30 min to remove cell fragments and obtain a pink emulsion.

[0043] Step 4, Desalination: Add the sample to a 150 Da dialysis bag and place it in a pure water tank for dialysis. Change the water every 12 hours for a total of 5 times to remove inorganic salts and obtain pink dual-yeast self-assembled nanoemulsion.

[0044] Example 3

[0045] Step 1, Seed culture preparation: Prepare red yeast... Rhodotorula mucilaginosa MHT01 and Candida albicans Candida bombicola ATCC22214 was streaked onto yeast extract glucose agar to obtain pure single clones, which were then activated to the logarithmic growth phase in yeast extract glucose agar.

[0046] Step 2, Fermentation: Inoculate the logarithmic phase red yeast seed culture into a nitrogen-limited medium at an inoculation rate of 10%. The nitrogen-limited medium formula is as follows: glucose 80.0 g / L, yeast extract 1 g / L, (NH4)2SO4 0.3 g / L, KH2PO4 2.0 g / L, MgSO4·7H2O 1.5 g / L, CaCl2 0.15 g / L, FeCl3 0.02 g / L, ZnSO4 0.01 g / L, CuSO4 0.002 g / L, MnSO4 0.002 g / L, biotin 0.01 mg / L, and citric acid 2 g / L. Fermentation conditions were as follows: temperature controlled at 28℃, dissolved oxygen at 80%, fermentation time at 72 h, pH adjusted to 3.5 with 3% hydrochloric acid solution at the end of fermentation to create an acidic environment, enhance cell wall breaking efficiency and oil release, the temperature of the fermenter was raised to 70℃ and maintained for 30 min, then cooled to 30℃, and logarithmic Candida yeast was inoculated into the fermenter at an inoculation amount of 10%, glucose was added to 80 g / L, dissolved oxygen was maintained at 80%, and fermentation was carried out at 30℃ for 48 h.

[0047] Step 3, Purification: After fermentation, the yeast was inactivated at 70℃ for 30 min. The fermentation broth was homogenized under low temperature microfluidic pressure of 3000psi and temperature of 4℃ for 45 min. After that, it was centrifuged at 6000 rpm for 30 min to remove cell fragments and obtain a pink emulsion.

[0048] Step 4, Desalination: Add the sample to a 100 Da dialysis bag and place it in a pure water bath for dialysis. Change the water every 12 hours for a total of 5 times to remove inorganic salts and obtain pink dual-yeast self-assembled nanoemulsion.

[0049] Comparative Example 1

[0050] The difference between this comparative example and Example 1 is that only red yeast was inoculated for single-strain fermentation, and a blank culture medium inoculated with Candida albicans, yeast extract powder, and glucose broth was used as a control. All other parts were the same as in Example 1.

[0051] Comparative Example 2

[0052] The difference between this comparative example and Example 1 is that only Candida albicans was inoculated for single-strain fermentation, and the blank culture medium inoculated with Rhodotorula rubra was used as a control, while the other parts were the same as in Example 1.

[0053] Efficacy test

[0054] 1. Anti-wrinkle efficacy test

[0055] Test groups: Three cosmetic raw materials prepared in Comparative Example 1, Comparative Example 2 and Example 1 were selected and added to the base emulsion at a dosage of 2% respectively. They were set up as control group 1, control group 2 and sample group, with 30 people in each group. The changes in the occupancy rate of periorbital wrinkles, skin firmness R0 value and elasticity R2 value of the three raw materials were compared to evaluate the anti-wrinkle and firming effects of the raw materials.

[0056] Testing Method: Ninety volunteers were recruited and randomly divided into three groups. The product was used twice daily, morning and evening. After cleansing, two pumps of lotion were applied to the entire face.

[0057] Test metric: Wrinkle percentage: This value represents the proportion of wrinkles; the smaller the value, the fewer wrinkles. This value is obtained by analyzing facial image analyzers (VISIA-CR) using ImageProplus analysis software.

[0058] Elasticity R0 value: This is a measure of skin firmness. The smaller the R0 value, the firmer the skin.

[0059] Elasticity R2 value: Total elasticity of the rebound portion / Total elasticity of the stretched portion. The closer the value is to 1 (100%), the greater the elasticity of the curve; it characterizes the total elasticity of the skin.

[0060] Inclusion and exclusion criteria:

[0061] Inclusion criteria: (1) Healthy women aged 33 to 45; (2) Dry skin with obvious fine lines around the eyes; (3) Able to use the sample as required, fill in the relevant questionnaire and follow up; (4) Have not been tested on other products within 1 month;

[0062] Exclusion criteria: (1) Those who have used systemic hormone drugs and immunosuppressants within 1 month; (2) Women who are pregnant or lactating or within 6 months postpartum;

[0063] Testing period: February to March 2025;

[0064] Data Analysis: SPSS 19.0 statistical software was used for data analysis. Normality tests were performed on each parameter before statistical analysis. Before-and-after values ​​were compared, and significance analysis was performed using t-tests or rank-sum tests. p < 0.05 (*) indicates that the difference is statistically significant; p< 0.01 (**) p < 0.005 (***) p < 0.001 (****) indicates that the difference is statistically significant.

[0065] Experimental Results and Analysis:

[0066] 1. Skin firmness R0 value:

[0067] Table 1. Results of Skin Tightness R0 Value

[0068]

[0069] Table 1 shows that, compared with the initial values, the R0 values ​​of skin firmness in the volunteers decreased significantly after 2 and 4 weeks of product use. The R0 values ​​of skin firmness in the control group using single-strain red yeast fermentation active ingredients improved by an average of 13.93% and 23.38% after 2 and 4 weeks of product use, respectively. The R0 values ​​of skin firmness in the control group using single-strain Candida fermentation active ingredients improved by an average of 6.03% and 10.05% after 2 and 4 weeks of product use, respectively. The R0 values ​​of skin firmness in the control group using dual-strain fermentation active ingredients improved by an average of 28.28% and 36.36% after 2 and 4 weeks of product use, respectively. This indicates that the nano-self-assembled emulsion prepared by dual-strain co-fermentation has a more significant skin firming effect.

[0070] 2. Skin elasticity R2 value:

[0071] Table 2. Results of Skin Elasticity R2 Values

[0072]

[0073] Table 2 shows that after 2 and 4 weeks of product use, the skin elasticity R2 values ​​of volunteers in control group 2 and sample group 3 significantly increased compared to the initial values, while the skin elasticity R2 value of volunteers in control group 1 initially increased and then decreased, with a relatively small rate of change. The skin elasticity R2 values ​​of volunteers in the control group using single-strain red yeast fermentation active ingredients improved by an average of 13.93% and 23.38% after 2 and 4 weeks of product use, respectively. The skin elasticity R2 values ​​of volunteers in the control group using single-strain Candida fermentation active ingredients improved by an average of 6.03% and 10.05% after 2 and 4 weeks of product use, respectively. The skin elasticity R2 values ​​of volunteers in the control group using dual-strain fermentation active ingredients improved by an average of 8.06% and 11.29% after 2 and 4 weeks of product use, respectively, significantly higher than those in control groups 1 and 2. This indicates that adding dual-strain synergistic fermentation raw materials can significantly enhance the anti-wrinkle effect of the product, making the skin firmer and more elastic.

[0074] 3. Wrinkle occupancy rate:

[0075] Table 3. Results of Wrinkle Prevalence Values

[0076]

[0077] As shown in Table 3, after 4 weeks of product use, the wrinkle occupancy rate of the three groups of test volunteers gradually decreased, and the decrease was highly significant relative to the initial value (P < 0.01). This indicates that all three raw materials can improve wrinkles to varying degrees. However, after 2 weeks of use, only the wrinkle occupancy rate of the sample group showed a highly significant difference compared to the initial value, indicating that the dual-yeast fermented raw material prepared in this invention exhibits a more superior wrinkle-reducing effect.

[0078] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a dual-yeast self-assembled nanoemulsion, characterized in that: It consists of the following steps: Step 1, Seed culture preparation: Red yeast and Candida albicans were streaked on yeast extract glucose agar medium to obtain pure single clones, and the single clones were activated to the logarithmic phase in yeast extract glucose medium. Step 2, Preliminary fermentation: Inoculate the logarithmic phase red yeast seed culture into a nitrogen-limited medium for preliminary fermentation; Step 3, Lipid Decomposition: After fermentation, the lipids inside the red yeast are released using an acid-heat method; Step 4, Secondary Fermentation: After inoculating with Candida albicans, carry out secondary fermentation; Step 5, Self-assembly: After fermentation, the yeast is inactivated and then crushed and automatically encapsulated by low-temperature microfluidic jet. The cell fragments are removed by centrifugation to obtain a nano-pink emulsion containing encapsulated dual yeast active substances. Step 6, Desalination: The sample is placed in a dialysis bag and dialyzed in a pure water tank. The water is changed regularly to remove inorganic salts and obtain pink yeast self-assembled nanoemulsion. Step 7, Compounding: Add 0.2-0.5% (W / W) p-hydroxyacetophenone and 0.5-1% sodium lactate (W / W) to compound and obtain a dual-yeast self-assembled nanoemulsion.

2. The preparation method according to claim 1, characterized in that: The red yeast strain number in step 1 is: Rhodotorula mucilaginosa MHT01, the strain number of Candida albicans is... Candida bombicola ATCC22214.

3. The preparation method according to claim 1, characterized in that: The nitrogen-limiting culture medium in step 2 consists of 50.0-100 g / L glucose, 0.5-1.5 g / L yeast extract, 0.3-0.5 g / L (NH4)2SO4, 0.5-2.0 g / L KH2PO4, 1.0-1.5 g / L MgSO4·7H2O, 0.1-0.2 g / L CaCl2, 0.02-0.05 g / L FeCl3, 0.01-0.03 g / L ZnSO4, 0.001-0.002 g / L CuSO4, 0.001-0.005 g / L MnSO4, 0.01-0.02 mg / L biotin, and 1-2 g / L citric acid.

4. The preparation method according to claim 1, characterized in that: The initial fermentation parameters for the red yeast in step 2 are a temperature of 28-30℃ and dissolved oxygen of 70-80%.

5. The preparation method according to claim 1, characterized in that: The acid-heat method in step 3 involves adjusting the pH to 2.0-3.5 with a 2-4% (V / V) hydrochloric acid solution to create an acidic environment, which enhances cell wall breaking efficiency and oil release. The temperature of the fermenter is then raised to 65-75℃ and maintained for 30 minutes before being cooled to below 30℃.

6. The preparation method according to claim 1, characterized in that: The secondary fermentation parameters for Candida albicans in step 4 are: temperature 30°C and dissolved oxygen 70-80%.

7. The preparation method according to claim 1, characterized in that: The self-assembly process in step 5 is as follows: low temperature microjet pressure 2500-3000 psi, temperature 4-10℃, high pressure homogenization for 45-60 min, followed by centrifugation at 6000-12000 rpm for 30-45 min.

8. The preparation method according to claim 1, characterized in that: The desalination process in step 6 is as follows: the sample is placed in a dialysis bag with a specification of 100-150 Da, and then placed in a pure water tank for dialysis. The water is changed once every 8-24 hours, for a total of 3-5 water changes.

9. A dual-yeast self-assembled nanoemulsion, characterized in that, Prepared by the preparation method according to any one of claims 1-8.

10. An application of a dual-yeast self-assembled nanoemulsion, characterized in that, The dual-yeast self-assembled nanoemulsion is used in cosmetic preparation.

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