A double-yeast self-assembled nanoemulsion, a preparation method and application thereof

Abstract

The application discloses a double-yeast self-assembled nanoemulsion as well as a preparation method and application thereof. The preparation method comprises the following steps: step 1, seed liquid preparation; step 2, preliminary fermentation; step 3, oil splitting; step 4, secondary fermentation; step 5, self-assembly; step 6, desalting; and step 7, compounding. Through screening of specific strains, optimization of fermentation and purification processes, the double-yeast self-assembled nanoemulsion with good anti-wrinkle effect is obtained, and the product efficacy can be remarkably improved, so the double-yeast self-assembled nanoemulsion has a good application prospect in the daily chemical industry.

Description

TECHNICAL FIELD

[0001] The application discloses a fermentation product, in particular, a double yeast self-assembled nanoemulsion, and also discloses a preparation method and application of the double yeast self-assembled nanoemulsion, and belongs to the field of daily chemical raw materials. BACKGROUND

[0002] With the increasing demand for natural and efficient active ingredients in the cosmetic industry, microbial fermentation technology has become an important direction for raw material development due to its green and sustainable characteristics.

[0003] Currently, mainstream microbial cosmetic raw materials on the market rely on single strain fermentation, which has problems such as low content of active ingredients and weak efficacy. In addition, although traditional microencapsulation technology can improve the stability of active substances, the preparation process is complex and the cost is high, which makes it difficult to realize large-scale industrial production. In recent years, yeast fermentation has attracted attention due to its rich metabolic products and high safety, but single yeast fermentation cannot meet the demand for multifunctional raw materials. In the prior art, the combination of microencapsulation technology and fermentation technology is often difficult to promote due to complex process and poor cost control.

[0004] Therefore, developing a technology with simple process, controllable cost and significantly improved product efficacy has become an urgent problem in the industry. SUMMARY

[0005] In order to solve the existing problems, the first object of the present application is to provide a preparation method of a double yeast self-assembled nanoemulsion. The method selects specific strains, optimizes fermentation and purification processes, and obtains a double yeast self-assembled nanoemulsion with good anti-wrinkle effect, which can significantly improve the product efficacy.

[0006] To achieve the above object, the specific technical scheme is as follows: a preparation method of a double yeast self-assembled nanoemulsion, comprising the following steps:

[0007] Step 1, seed liquid preparation: the Rhodotorula and Candida are respectively streaked on yeast extract glucose agar medium to obtain pure single colonies, and the single colonies are respectively activated in yeast extract glucose medium to logarithmic phase;

[0008] Step 2, preliminary fermentation: the logarithmic phase Rhodotorula seed liquid is inoculated in nitrogen-limited medium for preliminary fermentation;

[0009] Step 3, oil splitting: the oil inside the Rhodotorula is released by acid heat method after fermentation;

[0010] Step 4, secondary fermentation: the Candida is inoculated for secondary fermentation;

[0011] Step 5, self-assembly: after the fermentation is completed, the yeast is inactivated, then low-temperature micro-jet is used for crushing and automatic wrapping treatment, and the yeast fragments are removed by centrifugation to obtain a nanometer powder-containing pink emulsion containing wrapped double-yeast active substances;

[0012] Step 6, desalination: the sample is added to a dialysis bag and dialyzed in a pure water pool, and the water is replaced at regular intervals to remove inorganic salts, thereby obtaining a pink self-assembled nanometer emulsion of yeast;

[0013] Step 7, compounding: 0.2-0.5% (W / W) p-hydroxyacetophenone and 0.5-1% sodium lactate (W / W) are added to compound the double-yeast self-assembled nanoemulsion.

[0014] Further, the Rhodotorula in step 1 is a self-researched strain, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Chaoyang District, Beijing, with a preservation date of July 15, 2024, and a number of CGMCC No. 31295, and is classified and named as Rhodotorula mucilaginosa MHT01 The Candida is a purchased strain, which is purchased from Shanghai Microbial Culture Collection Center, with a strain number of Candida bombicola ATCC22214.

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

[0016] Further, the initial fermentation parameters of the Rhodotorula in step 2 are a temperature of 28-30°C, a dissolved oxygen of 70-80%, and a fermentation time of 72 h.

[0017] Further, the acid heat method in step 3 is to adjust the pH to 2.0-3.5 by using 2-4% (V / V) hydrochloric acid solution to create an acidic environment, thereby enhancing the efficiency of cell wall rupture and the release of oil and fat. The temperature of the fermentation tank is raised to 65-75°C for 30 min and then cooled to below 30°C.

[0018] Further, the Candida secondary fermentation parameters in step 4 are temperature of 30 DEG C, dissolved oxygen of 70-80%, and fermentation for 48h.

[0019] Further, the self-assembly process in step 5 is low-temperature micro-jet pressure of 2500-3000 psi, temperature of 4-10 DEG C, high-pressure homogenization for 45-60 min, and then centrifugation at 6000-12000 rpm for 30-45 min.

[0020] Further, the desalination process in step 6 is: the sample is added into a dialysis bag with a size of 100-150 Da, and dialysis is performed in a pure water pool, and the water is changed once every 8-24h, and the water is changed for 3-5 times.

[0021] Further, the compounding process in step 7 is: 0.2-0.5% (W / W) of p-hydroxyacetophenone and 0.5-1% (W / W) of sodium lactate are added and uniformly mixed to obtain the double-yeast self-assembled nanoemulsion.

[0022] The second technical solution provided by the application is a double-yeast self-assembled nanoemulsion prepared according to the preparation method.

[0023] The third technical solution provided by the application is an application of the double-yeast self-assembled nanoemulsion in the preparation of cosmetics.

[0024] Further, the cosmetic is a skin external preparation prepared from the double-yeast self-assembled nanoemulsion as an active component, and pharmaceutically or cosmetically conventional adjuvants or auxiliary components.

[0025] Further, the cosmetic includes one or more of water agent, cream, face wash, essence, mask, and mud mask.

[0026] Further, the addition amount of the double-yeast self-assembled nanoemulsion in the cosmetic is 0.1%-10%.

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

[0028] The double-yeast in-situ co-fermentation reduces the substrate cost, the plant oil is a commonly used substrate for preparing sophorolipids, compared with microbial oil, the growth cycle of plants is long, and the cost of planting and extracting oil is higher, through medium optimization: the oil yield of red yeast is significantly improved by using a low C / N ratio nitrogen-limited medium;

[0029] Timing control: 0-72h of red yeast dominated oil production, 72-120h of Candida utilizing red yeast oil to synthesize sophorolipids;

[0030] Self-assembly mechanism: the active substances released by the two yeasts after high-pressure homogenization self-assemble with sophorolipid molecules through hydrophobic forces; the raw material penetration and efficacy are significantly enhanced;

[0031] Economic improvement: the step of purchasing oil is omitted, the raw material cost is reduced; the crushing and wrapping are integrated, the process energy consumption is reduced and the process cycle is shortened;

[0032] Enhanced efficacy: the double yeast co-fermented nanoemulsion contains a variety of natural skin care factors such as high-concentration yeast small molecule polypeptide, vitamin A precursor, yeast pigment, double yeast polysaccharide, biological glycolipid, nucleic acid salt and amino acid, has the advantages of efficacy synergy and penetration promotion, and exhibits more significant anti-wrinkle effect than single yeast fermentation. DETAILED DESCRIPTION

[0033] The claims of the present application will be further described in detail in conjunction with specific embodiments, but do not constitute any limitation on the present application, and any limited number of modifications made by anyone within the scope of protection of the claims of the present application are still within the scope of protection of the claims of the present application.

[0034] Example 1

[0035] Step 1, seed liquid preparation: red yeast Rhodotorula glutinis and Candida tropicalis ATCC22214 were respectively streaked on yeast extract glucose agar medium to obtain pure single clones, and the single clones were respectively activated to the logarithmic phase in yeast extract glucose medium. Rhodotorula mucilaginosa MHT01 and Candida tropicalis Candida bombicola ATCC22214 were respectively streaked on yeast extract glucose agar medium to obtain pure single clones, and the single clones were respectively activated to the logarithmic phase in yeast extract glucose medium.

[0036] Step 2, fermentation: the logarithmic phase red yeast seed liquid was inoculated in a nitrogen-limited medium at an inoculation amount of 5%, and 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. The fermentation conditions were as follows: the temperature was controlled at 28℃, the dissolved oxygen was 80%, the fermentation time was 72 h, the pH was adjusted to 3.5 at the end of fermentation using 3% hydrochloric acid solution to create an acidic environment, enhance the efficiency of cell wall rupture and oil release, the temperature of the fermentation tank was increased to 70℃ for 30 min, cooled to 30℃, and the logarithmic phase Candida tropicalis was inoculated into the fermentation tank at an inoculation amount of 5%, and glucose was added to 50 g / L, the dissolved oxygen was 80%, and the fermentation was carried out at 30℃ for 48 h.

[0037] Step 3, purification: after the fermentation is completed, the yeast is inactivated at 70°C for 30 min, and the fermentation liquor is subjected to high-pressure homogenization at a low-temperature microjet pressure of 3000 psi and a temperature of 4°C for 45 min. Then, the bacterial debris is removed by centrifugation at 8000 rpm for 30 min, and a pink emulsion is obtained.

[0038] Step 4, desalination: the sample is added to a dialysis bag with a molecular weight cut-off of 100 Da, and dialysis is performed in a pure water tank. The water is changed every 24 h, and a total of 4 times of water change are performed to remove inorganic salts, thereby obtaining a pink double-yeast self-assembled nanoemulsion.

[0039] Example 2

[0040] Step 1, preparation of seed liquid: red yeast Rhodotorula mucilaginosa MHT01 and Candida Candida bombicola ATCC22214 are respectively streaked on yeast extract glucose agar medium to obtain pure single clones, and the single clones are respectively activated to the logarithmic phase in yeast extract glucose medium.

[0041] Step 2, fermentation: the logarithmic phase red yeast seed liquid is inoculated in a nitrogen-limited medium at a inoculation amount of 3%. The formula of the nitrogen-limited medium is as follows: glucose 100.0 g / L, yeast extract 1 g / L, (NH4)2SO40.3 g / L, KH2PO42.0 g / L, MgSO4·7H2O 1.5 g / L, CaCl20.1 g / L, FeCl30.02 g / L, ZnSO40.03 g / L, CuSO40.001 g / L, MnSO40.005 g / L, biotin 0.02 mg / L, and citric acid 1 g / L. The fermentation conditions are as follows: the temperature is controlled at 28°C, the dissolved oxygen is 70%, the fermentation time is 72 h, the pH is adjusted to 3.5 by using 3% hydrochloric acid solution to create an acidic environment, thereby enhancing the efficiency of cell wall breaking and the release of oil, the temperature of the fermentation tank is increased to 70°C and maintained for 30 min, the temperature is then cooled to 30°C, the logarithmic phase Candida is inoculated in the fermentation tank at a inoculation amount of 3%, and glucose is supplemented to 100 g / L, the dissolved oxygen is 80%, and the fermentation is performed at 30°C for 48 h.

[0042] Step 3, purification: after the fermentation is completed, the yeast is inactivated at 65°C for 30 min, and the fermentation liquor is subjected to high-pressure homogenization at a low-temperature microjet pressure of 3000 psi and a temperature of 4°C for 45 min. Then, the bacterial debris is removed by centrifugation at 6000 rpm for 30 min, and a pink emulsion is obtained.

[0043] Step 4, desalination: the sample is added to a dialysis bag with a molecular weight cut-off of 150 Da, and dialysis is performed in a pure water tank. The water is changed every 12 h, and a total of 5 times of water change are performed to remove inorganic salts, thereby obtaining a pink double-yeast self-assembled nanoemulsion.

[0044] Example 3

[0045] Step 1, seed liquid preparation: red yeast Rhodotorula glutinis and Candida tropicalis ATCC22214 were respectively inoculated on yeast extract glucose agar medium to obtain pure single colonies, and the single colonies were respectively activated to the logarithmic phase in yeast extract glucose medium. Rhodotorula mucilaginosa MHT01 and Candida tropicalis Candida bombicola ATCC22214 were respectively inoculated on yeast extract glucose agar medium to obtain pure single colonies, and the single colonies were respectively activated to the logarithmic phase in yeast extract glucose medium.

[0046] Step 2, fermentation: the logarithmic phase red yeast seed liquid was inoculated in a nitrogen-limited medium at a 10% inoculation amount, and the nitrogen-limited medium formula was 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. The fermentation conditions were as follows: the temperature was controlled at 28°C, the dissolved oxygen was 80%, the fermentation time was 72 h, the pH was adjusted to 3.5 at the end of fermentation using 3% hydrochloric acid solution to create an acidic environment, enhance the efficiency of cell wall breaking and oil release, the temperature of the fermentation tank was increased to 70°C for 30 min, and then cooled to 30°C. The logarithmic phase Candida tropicalis was inoculated in the fermentation tank at a 10% inoculation amount, and the glucose was supplemented to 80 g / L, the dissolved oxygen was 80%, and the fermentation was carried out at 30°C for 48 h.

[0047] Step 3, purification: after the fermentation was completed, the yeast was inactivated at 70°C for 30 min, and the fermentation broth was subjected to high-pressure homogenization at a low-temperature microfluid pressure of 3000 psi and a temperature of 4°C for 45 min. Then, the bacterial fragments were removed by centrifugation at 6000 rpm for 30 min, and a pink emulsion was obtained.

[0048] Step 4, desalination: the sample was added to a dialysis bag with a molecular weight cut-off of 100 Da, and dialyzed in a pure water tank. The water was changed every 12 h for a total of 5 times to remove inorganic salts, and a pink double-yeast self-assembled nanoemulsion was obtained.

[0049] Comparative Example 1

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

[0051] Comparative Example 2

[0052] The difference between the present comparative example and Example 1 is that only Candida is inoculated for single strain fermentation, and the blank medium yeast extract glucose broth inoculated with Rhodotorula is used as a control, and the other parts are the same as in Example 1.

[0053] Efficacy test

[0054] 1. Anti-wrinkle efficacy test

[0055] Test grouping: Select three cosmetic raw materials prepared in Comparative Example 1, Comparative Example 2 and Example 1 respectively, and add them to the base emulsion at an amount of 2%, respectively, to set up control group 1, control group 2 and sample group, each with 30 people; compare the changes of the eye wrinkle coverage value, skin tightness R0 value and elasticity R2 value of the three raw materials to evaluate the anti-wrinkle and firming efficacy of the raw materials.

[0056] Test method: 90 volunteers were recruited and randomly divided into 3 groups. The product was used once a day in the morning and evening, and 2 pumps of emulsion were used after facial cleansing every day.

[0057] Test index: Wrinkle coverage value: this value represents the proportion of wrinkle number, the smaller the value, the fewer the wrinkles. This value is obtained by analyzing the VISIA-CR image of the facial image analyzer using ImageProplus analysis software.

[0058] Elasticity R0 value: a representation of skin tightness. The smaller the R0 value, the tighter the skin.

[0059] Elasticity R2 value: total elastic-plasticity of the rebound part / total elastic-plasticity of the stretched part, the closer the value to 1 (100%) curve, the greater the elastic force; represents the total elasticity of the skin.

[0060] Inclusion and exclusion criteria:

[0061] Inclusion criteria: (1) healthy women aged 33-45 years; (2) dry skin and obvious periorbital fine lines; (3) able to use the sample as required, fill out the relevant questionnaire and follow up; (4) not received other product testing within 1 month;

[0062] Exclusion criteria: (1) use of hormone drugs and immunosuppressants within 1 month; (2) pregnant or lactating women and women within 6 months after childbirth;

[0063] Test date: February 2025 to March 2025;

[0064] Data analysis: SPSS 19.0 statistical software was used for data analysis. Before statistical analysis, the normal distribution of each parameter was tested. The values before and after use were compared, and T test or rank sum test was used for significance analysis. p <0.05 (*) indicates that the difference is statistically significant; p< 0.01 (**), < 0.01 (**), p < 0.005 (***), < 0.005 (***), p < 0.001 (****) indicates that the difference is extremely statistically significant.

[0065] Experimental results and analysis:

[0066] 1. Skin tightness R0 value:

[0067] Table 1, skin tightness R0 value result table

[0068]

[0069] From Table 1, the test results of the three groups show that compared with the initial value, the skin tightness R0 value of the volunteers in the two control groups and the sample group decreased after using the product for 2 weeks and 4 weeks, and all had significant differences. The skin tightness R0 value of the volunteers in the control group using the active material of single strain fermentation of Rhodotorula rubra improved by an average of 13.93% and 23.38% after using the product for 2 weeks and 4 weeks respectively, the skin tightness R0 value of the volunteers in the control group using the active material of single strain fermentation of Candida utilis improved by an average of 6.03% and 10.05% after using the product for 2 weeks and 4 weeks respectively, and the skin tightness R0 value of the volunteers in the sample group using the active material of double yeast fermentation improved by an average of 28.28% and 36.36% after using the product for 2 weeks and 4 weeks respectively, indicating that the nano self-assembled emulsion prepared by double yeast co-fermentation has a more significant effect of tightening the skin.

[0070] 2. Skin elasticity R2 value:

[0071] Table 2, skin elasticity R2 value result table

[0072]

[0073] From Table 2, the test results of the three groups show that after using the product for 2 weeks and 4 weeks, the skin elasticity R2 value of the volunteers in the control group 2 and the sample group has a significant increase compared with the initial value, and the skin elasticity R2 value of the volunteers in the control group 1 first increases and then decreases, and the change rate is smaller. The skin elasticity R2 value of the volunteers in the control group using the active material of single strain fermentation of Rhodotorula rubra improved by an average of 1.54% and -3.08% after using the product for 2 weeks and 4 weeks respectively, the skin elasticity R2 value of the volunteers in the control group using the active material of single strain fermentation of Candida utilis improved by an average of 3.17% and 6.35% after using the product for 2 weeks and 4 weeks respectively, and the skin elasticity R2 value of the volunteers in the sample group using the active material of double yeast fermentation improved by an average of 8.06% and 11.29% after using the product for 2 weeks and 4 weeks respectively, which is significantly higher than that of the control group 1 and the control group 2, indicating that the addition of double yeast co-fermentation raw materials can significantly improve the anti-wrinkle effect of the product, making the skin tight and elastic.

[0074] 3. Wrinkle coverage value:

[0075] Table 3, wrinkle coverage value result table

[0076]

[0077] From Table 3, it can be seen that after using the product for 4 weeks, the wrinkle coverage values of the three groups of test volunteers gradually decreased, and there was a very significant decrease compared with the initial value (P<0.01), indicating that the three raw materials can improve wrinkles to different degrees, but only the sample group has a very significant difference in wrinkle coverage value compared with the initial value after using for 2 weeks, indicating that the raw material prepared by double yeast fermentation in the present application shows more excellent wrinkle lightening effect.

[0078] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

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: Inoculate with Candida albicans activated to the logarithmic phase for 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% (w / w) sodium lactate to obtain a dual-yeast self-assembled nanoemulsion. The red yeast strain number in step 1 is: Rhodotorula mucilaginosa MHT01, the strain number of Candida albicans is... Candida bombicola ATCC22214, The nitrogen-limiting culture medium in step 2 consists of: glucose 50.0-100 g / L, yeast extract 0.5-1.5 g / L, (NH4)2SO4 0.3-0.5 g / L, KH2PO4 0.5-2.0 g / L, MgSO4·7H2O 1.0-1.5 g / L, CaCl2 0.1-0.2 g / L, FeCl3 0.02-0.05 g / L, ZnSO4 0.01-0.03 g / L, CuSO4 0.001-0.002 g / L, MnSO4 0.001-0.005 g / L, biotin 0.01-0.02 mg / L, and citric acid 1-2 g / L. The acid-thermal 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, enhancing cell wall disruption efficiency and oil release. The fermentation tank temperature is then raised to 65-75℃ and maintained for 30 minutes before cooling to below 30℃. 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.

2. 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%.

3. 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%.

4. 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.

5. A dual-yeast self-assembled nanoemulsion, characterized in that, Prepared by the preparation method according to any one of claims 1-4.

6. An application of the dual-yeast self-assembled nanoemulsion according to claim 5, characterized in that, The dual-yeast self-assembled nanoemulsion is used in cosmetic preparation.

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