A composition for promoting skin collagen synthesis and its preparation method

Abstract

This invention belongs to the field of biotechnology. More specifically, it relates to compositions containing retinol with increased stability, which can be formulated into cosmetics beneficial for skin rejuvenation. The present invention describes a composition for promoting skin collagen synthesis, the composition comprising 0.05–2% retinol and *Acetobacter xylinum* fermentation product. This invention combines *Acetobacter xylinum* fermentation product with retinol, significantly improving the stability of retinol in different pH solutions and with high concentrations of Fe, without affecting the antioxidant activity of retinol. 3+ Stability in solution. In experiments promoting collagen synthesis in human skin, the combined retinol solution showed significantly better promoting effects than the single retinol.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. More specifically, it relates to compositions containing retinol with increased stability, which can be formulated into cosmetics beneficial for skin rejuvenation. Background Technology

[0002] Retinol has many positive effects on the skin, mainly including the following: ① Improving naturally aging skin: Retinol can stimulate fibroblasts, increasing collagen secretion; secondly, retinol can accelerate skin cell metabolism, promote the shedding of aged keratinocytes, and allow new cells to reach the skin surface more quickly; ② Whitening effect: Retinol can inhibit the activity of tyrosinase, a key enzyme in the process of melanin synthesis. By inhibiting its activity, melanin formation can be reduced, thereby preventing and alleviating problems such as age spots and dullness, achieving a whitening effect; in addition to inhibiting melanin production, it can also accelerate the decomposition and metabolism of melanin in melanocytes, allowing existing melanin to be excreted from the body more quickly, further brightening and evening out skin tone; ③ Oil control and acne treatment effect: Retinol can act on sebaceous glands and regulate the amount of sebum secreted. For oily skin, it can reduce sebum secretion, lower the oil content on the skin surface, thereby improving the oily condition of the skin and reducing the problem of enlarged pores caused by excessive sebum secretion; retinol also has a certain anti-inflammatory effect, which can reduce the inflammatory response of acne and relieve the redness and pain of pimples; ④ Improve the skin barrier function: retinol can increase and enhance the function of the stratum corneum, help maintain the normal structure and function of stratum corneum cells, enhance the barrier function of the stratum corneum, and enable the skin to better resist the damage of external harmful substances; in addition, retinol can also promote skin hydration. By promoting the metabolism of skin cells, they can enable skin cells to better retain moisture, improve the skin's moisturizing ability, make the skin more hydrated and plump, and reduce problems such as dryness and peeling.

[0003] However, retinol can cause skin irritation, manifesting as redness, itching, dryness, and peeling, a condition known as "retinol intolerance." This is related to the concentration, frequency of use, and skin type. Generally, sensitive skin is more prone to irritation; higher concentrations of products are more irritating, such as when the retinol concentration exceeds 1%, the risk of irritation increases significantly.

[0004] Secondly, retinol has poor stability and is easily affected by various factors. For example, retinol has a highly unsaturated structure, and the carbon-carbon double bonds in its molecule readily react with oxygen in the air, generating retinal, retinoic acid, and other substances upon oxidation, thus reducing its activity and efficacy. Ultraviolet and visible light can also damage retinol. Light exposure triggers photochemical reactions in the retinol molecule, altering its structure and leading to decreased stability. Retinol is unstable under both acidic and alkaline conditions. In acidic environments, retinol readily undergoes protonation, resulting in changes to its molecular structure; in alkaline environments, it may undergo saponification, affecting its stability. Therefore, the two major challenges for the application of retinol in this field are its low stability and high irritant properties.

[0005] Numerous studies both domestically and internationally have confirmed the antioxidant function of soybeans. The tocopherols, vitamin C, isoflavones, and phenolic compounds contained in soybeans all possess antioxidant activity. (Yamasaki et al.) [1] Substances extracted from fermented soy milk were found to possess antioxidant activity, according to Geng et al. [2 By optimizing the fermentation conditions of the lactic acid bacteria combination, the free radical scavenging rate of fermented soy milk can reach 84.3%; Xu Yin et al. [3 In vitro experiments demonstrated that fermented sour soybean milk had a higher DPPH scavenging rate and Fe content than before fermentation. 2+ The chelating capacity increased significantly, by 32.76% and 31.89%, respectively. Currently, there are no studies using *Acetobacter xylinum* to ferment soy milk and applying the resulting fermentation products to improve the stability of retinol while reducing its irritation.

[0006] [1] Yamasaki Y, Bakke M. Fermented soymilk extract and fermented hypocotyl extract: JP 2013082603[P].2014-06-19.

[0007] [2] Geng YR, Li WJ, Wang J MR adical scavenging ability of soymilkfermented with compound lactic acid bacteria towards DPPH free radical[J]. Agricultural Science & Technology, 2015, 16(9): 2036-2039.

[0008] [3] Xu Yin, Huang Yujun, Chen Xia, et al. Study on the in vitro and in vivo antioxidant effects of lactic acid bacteria fermented soybean milk [J]. China Dairy Industry, 2012, 40(8): 16-19. Summary of the Invention

[0009] This invention relates to *Acetobacter xylinum*, products obtained from fermenting soy milk using *Acetobacter xylinum*, the properties of these products, and their applications. An unexpected discovery of this invention is that the product obtained from fermenting soy milk and Oceania quinoa juice using *Acetobacter xylinum* significantly increases the stability of retinol and reduces its skin irritation.

[0010] In this paper, stability refers to the stability of retinol in solutions with different pH values ​​and solutions containing metal ions. Experiments showed that retinol solutions exhibited poor stability at pH 2.0 and pH 10.0, with residual rates of approximately 58% and 35% after 5 days of incubation at 37°C, respectively. Retinol remained relatively stable at pH 6.0–8.0. However, after adding the *Acetobacter xylinum* fermentation product of this invention, the retinol solution remained relatively stable in both acidic and alkaline solutions (pH 2.0–10.0), with residual rates exceeding 85% after 5 days of incubation at 37°C. This indicates that the *Acetobacter xylinum* fermentation product can improve the stability of retinol in acidic and alkaline solutions, but the specific mechanism of action requires further investigation.

[0011] In addition, it has been proven that Fe 3+ The presence of Fe relatively affects the stability of retinol, and with the increase of Fe... 3+ The higher the concentration of Fe, the stronger its destructive effect on retinol. 3+ After incubation at 37°C for 5 days in a (10 mmol / L) solution, only about 50% of retinol remained. The retinol's color changed to brownish-yellow, likely due to oxidative degradation. However, after adding Acetobacter xylinum fermentation products, the retinol content increased significantly in the same Fe solution. 3+ After incubation at 37°C for 5 days in the solution, the remaining retinol content was 90%. This indicates that the fermentation product of *Acetobacter xylinum* can significantly increase the retinol content in Fe... 3+ The stability in solution was significantly different compared to that without the addition (P<0.01).

[0012] Unfortunately, the Acetobacter xylinum fermentation product obtained in this invention does not improve the photostability of retinol.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution:

[0014] The present invention provides a composition for promoting skin collagen synthesis, the composition comprising 0.05-2% retinol and Acetobacter xylinum fermentation product.

[0015] In one embodiment of the present invention, the weight ratio of Acetobacter xylinum fermentation product to retinol in the composition is 1:0.1 to 1. More preferably, the weight ratio of Acetobacter xylinum fermentation product to retinol is 1:0.1, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.5, 1:0.6, 1:0.65, 1:0.7, 1:0.8, 1:0.1, or 1:1. More preferably, the weight ratio of Acetobacter xylinum fermentation product to retinol is 1:0.5.

[0016] In one embodiment of the present invention, the Acetobacter xylinum fermentation product is prepared by the following steps:

[0017] S1. Preparation of soy milk: Add deionized water to cleaned soybeans, soak them in 0.1-0.5% NaHCO3 solution, then add deionized water and heat grind for 10-30 minutes. Sieve, homogenize, sterilize and cool to obtain soy milk.

[0018] S2. Preparation of Oceania quinoa juice: Add deionized water to Oceania quinoa, extract under ultrasonication, filter the extract, centrifuge to obtain Oceania quinoa juice.

[0019] S3. Fermentation: Add 10-35% by weight of soy milk and 3-10% by weight of quinoa juice to the fermentation medium, inoculate with Acetobacter xylophilus for fermentation, collect the fermentation supernatant, filter, and obtain the Acetobacter xylophilus fermentation product.

[0020] In the above description, the addition of Oceania quinoa juice is essential to achieving the purpose of this invention. The statement mentioned earlier that "this supernatant unexpectedly exhibits the effect of stabilizing retinol and reducing its skin irritation" is based on the addition of Oceania quinoa juice. This is because comparative test results show that the fermentation supernatant obtained without the addition of Oceania quinoa juice exhibits completely opposite effects.

[0021] In one embodiment of the present invention, the bioaccession number of the Acetobacter xylinum is ATCC 23767. Extensive screening and comparative experiments were conducted on the selection of strains, and the fermentation products produced after over-fermentation by lactic acid bacteria (Lactobacillus acidophilus) and yeast (Saccharomyces boulardii) showed no similar effects.

[0022] In one embodiment of the present invention, in step S1, the ratio of soybeans to deionized water is 1:3 to 10; NaHCO3 soaking time is 1 to 12 hours; hot grinding temperature is 80 to 100°C; homogenization pressure is 10 to 30 MPa; and homogenization time is 5 to 10 minutes.

[0023] In one embodiment of the present invention, in step S2, the ratio of Oceania schreberi to deionized water is 1:3 to 6; the extraction time is 10 to 60 minutes; the ultrasonic frequency is 60 to 80 kHz; the ultrasonic power is 100 to 200 W; and the temperature is 30 to 50 °C.

[0024] In one embodiment of the present invention, in step S2, filtration is typically performed using filter paper, a filter screen, or a filtration device to remove solid residues and obtain juice.

[0025] In one embodiment of the present invention, in step S2, centrifugation is performed to remove any small amount of tiny particles or suspended matter remaining after filtration. Centrifugation allows solid impurities to settle to the bottom of the centrifuge tube, resulting in a clearer liquid. The centrifugation speed can be 3000–10000 rpm, and the centrifugation time is 5–10 minutes.

[0026] In one embodiment of the present invention, in step S3, during the fermentation process, the inoculum amount of Acetobacter xylinum is 3-10%; the fermentation temperature is 25-30℃; the fermentation pH is 5.0-6.0; the aeration rate is 0.5-1.5 vm; the stirring speed is 100-300 r / min; and the tank pressure is 0.05-0.15 MPa.

[0027] In one embodiment of the present invention, in step S3, the fermentation medium is Hestrin-Schramm (HS) medium. It contains 20 g / L glucose, 5 g / L peptone, 5 g / L yeast extract, 2.7 g / L potassium dihydrogen phosphate, and 0.2 g / L magnesium sulfate heptahydrate, with a pH of 5.5–6.0.

[0028] In one embodiment of the present invention, in step S3, the fermentation time is 48–72 hours. Fermentation time is crucial for obtaining fermentation products with ideal functional properties. Different fermentation times result in various changes in the nutritional composition of the fermentation product; for example, certain components, such as vitamins, may accumulate, and some special metabolites may also be produced. These changes endow the product with unique new properties.

[0029] The present invention also provides a stable cosmetic that promotes the synthesis of collagen in the skin, comprising the aforementioned composition and an adjuvant available in the cosmetic field.

[0030] The cosmetics described herein generally include conventional formulations in the cosmetic field, including but not limited to: serums, face creams, eye creams, facial oils, face masks, serum lotions, and serum waters. The term "auxiliary agent" as used herein is usually determined by its formulation form. In particular, this invention is preferably formulated as a serum lotion, and its auxiliary agents generally include: solvents, humectants, emulsifiers, thickeners, moisturizers, preservatives, pH adjusters, etc.

[0031] Specifically, a suitable formulation for this invention is: 0.05-2% retinol; 1-4% Acetobacter xylinum fermentation product; 3-10% humectant; 1-3% emulsifier; 1-3% thickener; 3-10% skin moisturizer; 0.1-0.5% preservative; 0.1-0.5% pH adjuster; and the balance being water.

[0032] Specifically, the moisturizer in this formula may be selected from one or more combinations of glycerin, propylene glycol, 1,3-propanediol, 1,20-pentanediol, caprylyl glycol, sodium hyaluronate, acetyl glucosamine, allantoin, ceramide, phytosphingosine, palmetto fruit oil, trehalose, and ethylhexylglycerin.

[0033] Specifically, in this formulation, the emulsifier may be selected from one or more combinations of octyl dodecanol, cetearyl alcohol, polysorbate-20, polysorbate-80, sorbitan oleate, hexyl decyl alcohol, polyglycerol-3-methyl glucoside distearate, sucrose laurate, and PEG-40 hydrogenated castor oil.

[0034] Specifically, in this formulation, the thickener may be selected from one or more combinations of acrylamide-acryloyldimethyl taurate copolymer and xanthan gum.

[0035] Specifically, in this formulation, the emollient may be selected from one or more combinations of isohexadecane, dimethicone, phytosterol / octyldodecyl lauroyl glutamate, isotretinoate, caprylic / capric triglyceride, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerin.

[0036] Specifically, the preservative in this formula is phenoxyethanol.

[0037] Specifically, in this formula, the pH adjuster is sodium hydroxide.

[0038] Specifically, in this formulation, the solvent is water.

[0039] More specifically, an optimal formulation of the present invention comprises the following components in weight fractions:

[0040] Retinol 0.05–2%;

[0041] 1-4% of Acetobacter xylinum fermentation products;

[0042] Acetyl glucosamine 1-5%;

[0043] Allantoin 0.5-2%;

[0044] Ceramide 1-3%;

[0045] Octyldodecyl alcohol 0.1-1%;

[0046] Cetearyl alcohol 0.1-1%;

[0047] Polysorbate - 200.1% to 0.5%;

[0048] Polysorbate - 800.1% to 0.5%;

[0049] Acrylamide-acryloyldimethyl taurate copolymer 1-3%;

[0050] 1-3% polydimethylsiloxane;

[0051] Isotridecyl isonononate 1–3%;

[0052] Caprylic / capric triglycerides 1-3%;

[0053] Phenoxyethanol 0.1–0.5%;

[0054] Sodium hydroxide 0.1–0.5%; and

[0055] The remaining water.

[0056] The present invention has the following beneficial effects:

[0057] This invention combines Acetobacter xylinum fermentation products with retinol, significantly improving the stability of retinol in solutions with different pH levels and in solutions containing high concentrations of Fe3+ without affecting its antioxidant activity. In experiments promoting collagen synthesis in human skin, the combined retinol solution showed a significantly better promoting effect than the single retinol. Attached Figure Description

[0058] Figure 1 Image of Acetobacter xylinum cells.

[0059] Figure 2 The changes in TEWL values ​​before and after the use of Acetobacter xylinum fermentation products / retinol emulsions in each group;

[0060] Compared with the blank control group, * P < 0.05 ** P < 0.01.

[0061] Figure 3 To compare the relative fibroblast activity of different concentrations of the serum in Application Example 1 and Comparative Example 1,

[0062] Compared with the blank control group, * P < 0.05 ** P < 0.01.

[0063] Figure 4 This is the result of the type I collagen content test.

[0064] Compared with the blank control group, * P < 0.05 ** P < 0.01; compared with the model group, wP < 0.05. ## P<0.01. Detailed Implementation

[0065] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0066] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0067] The HS culture medium described in the article contains 20 g / L glucose, 5 g / L peptone, 5 g / L yeast extract, 2.7 g / L potassium dihydrogen phosphate, and 0.2 g / L magnesium sulfate heptahydrate, with a pH of 5.5–6.0.

[0068] Example 1: Preparation of fermentation products of Acetobacter xylinum

[0069] (1) Preparation of soy milk: After cleaning the soybeans, add deionized water at a ratio of 1:6, soak in 0.25% NaHCO3 solution for 3 hours, add deionized water at a ratio of 1:10, heat grind at 100℃ for 30 minutes, pass through a 100-mesh sieve, homogenize at 25MPa for 8 minutes, sterilize and cool to obtain soy milk.

[0070] (2) Preparation of Oceania quinoa juice: Select fresh Oceania quinoa, take its stems and leaves, wash them, crush them, add 6 times the amount of deionized water, place it under ultrasound for 30 minutes, the ultrasound frequency is 60kHz, the ultrasound power is 150W, and the temperature is controlled at 45℃; after ultrasound, filter the resulting mixture, centrifuge at 3000 rpm for 5 minutes, take the supernatant, and obtain the Oceania quinoa juice.

[0071] (3) Activation of bacterial strain: In a clean bench, pick up a small amount of Acetobacter xylinum with an inoculation loop and inoculate it into the prepared solid slant medium (HS medium + 1.5% agar, pH 5.5-6.0) under aseptic conditions. Gently slide the medium on the slant to distribute the bacterial strain evenly. Place the inoculated medium in a constant temperature incubator and incubate at 28-30℃ for 24 hours until obvious colonies appear on the medium. Collect the bacterial cells.

[0072] (4) Fermentation: 25 wt% soybean milk and 8 wt% Oceania quinoa juice were added to the HS medium to obtain the fermentation medium. 5% Acetobacter xylophilus was inoculated into the fermentation medium and fermented for 48-72 h. The fermentation temperature was 28℃, the fermentation pH was 5.5-6.0, the aeration rate was 1.0 vvm, the stirring speed was 150 r / min, and the tank pressure was 0.1 MPa. The fermentation supernatant was collected, filtered through a 0.22 μm ultrafiltration membrane, and the filtrate was collected to obtain the Acetobacter xylophilus fermentation product.

[0073] Example 2: Preparation of fermentation products of Acetobacter xylinum

[0074] (1) Preparation of soy milk: After cleaning the soybeans, add deionized water at a ratio of 1:3 and soak them in 0.25% NaHCO3 solution for 2 hours. Add deionized water at a ratio of 1:10 and heat grind at 80℃ for 30 minutes. Pass through a 100-mesh sieve, homogenize at 20MPa for 10 minutes, sterilize and cool to obtain soy milk.

[0075] (2) Preparation of Oceania quinoa juice: Select fresh Oceania quinoa, take its stems and leaves, wash them, crush them, add 3 times the amount of deionized water, place it under ultrasound for 15 minutes, the ultrasound frequency is 860kHz, the ultrasound power is 200W, and the temperature is controlled at 50℃; after ultrasound, filter the resulting mixture, centrifuge at 3000 rpm for 10 minutes, take the supernatant, and obtain the Oceania quinoa juice.

[0076] (3) Activation of bacterial strain: In a clean bench, pick up a small amount of Acetobacter xylinum with an inoculation loop and inoculate it into the prepared solid slant medium (HS medium + 1.5% agar, pH 5.5-6.0) under aseptic conditions. Gently slide the medium on the slant to distribute the bacterial strain evenly. Place the inoculated medium in a constant temperature incubator and incubate at 28-30℃ for 24 hours until obvious colonies appear on the medium. Collect the bacterial cells.

[0077] (4) Fermentation: 10 wt% soybean milk and 10 wt% Oceania quinoa juice were added to the HS medium to obtain the fermentation medium. 3% Acetobacter xylophilus was inoculated into the fermentation medium and fermented for 48 h at a fermentation temperature of 28℃. The fermentation pH was 5.5-6.0, the aeration rate was 1.0 vvm, the stirring speed was 150 r / min, and the tank pressure was 0.1 MPa. The fermentation supernatant was collected, filtered through a 0.22 μm ultrafiltration membrane, and the filtrate was collected to obtain the Acetobacter xylophilus fermentation product.

[0078] Example 3: Preparation of fermentation products of Acetobacter xylinum

[0079] (1) Preparation of soy milk: After cleaning and rinsing soybeans, add deionized water at a ratio of 1:10 and soak in 0.25% NaHCO3 solution for 12 hours. Add deionized water at a ratio of 1:10 and heat grind at 100℃ for 30 minutes. Pass through a 100-mesh sieve, homogenize at 25MPa for 5 minutes, sterilize and cool to obtain soy milk.

[0080] (2) Preparation of Oceania quinoa juice: Select fresh Oceania quinoa, take its stems and leaves, wash them, crush them, add 5 times the amount of deionized water, place it under ultrasound for 60 minutes, the ultrasound frequency is 60kHz, the ultrasound power is 100W, and the temperature is controlled at 35℃; after the ultrasound is completed, filter the resulting mixture, centrifuge at 3000 rpm for 5 minutes, take the supernatant, and obtain the Oceania quinoa juice.

[0081] (3) Activation of strain: In a clean bench, pick up a small amount of Acetobacter xylophilus with an inoculation loop and inoculate it into a prepared solid slant culture medium (HS medium + 1.5% agar, pH 5.5-6.0) under aseptic conditions. Gently slide it on the slant to distribute the strain evenly. Place the inoculated culture medium in a constant temperature incubator and incubate at 28-30℃ for 24h until obvious colonies appear on the culture medium. Collect the bacterial cells. (4) Fermentation: Add 35wt% soybean milk and 3wt% Oceania quinoa juice to the HS medium to obtain a fermentation culture medium. Inoculate 5% activated Acetobacter xylophilus into the fermentation culture medium and ferment for 72h at a fermentation temperature of 28℃, a fermentation pH of 5.5-6.0, an aeration rate of 1.0 vvm, a stirring speed of 150 r / min, and a tank pressure of 0.1 MPa. Collect the fermentation supernatant, filter it through a 0.221 μm ultrafiltration membrane, collect the filtrate, and obtain the Acetobacter xylophilus fermentation product.

[0082] Comparative Example 1 differs from Example 1 in that Oceania quinoa juice is not added during the fermentation process in step (4), while the other parameters are the same as in Example 1.

[0083] Comparative Example 2 differs from Example 1 in that black truffle extract (purchased from Shanghai Haotai Biotechnology Co., Ltd.) is added during the fermentation process in step (4), while the other parameters are the same as in Example 1.

[0084] Comparative Example 3 differs from Example 1 in that Lactobacillus acidophilus is used instead of Acetobacter xylinum. The fermentation parameters are as follows: MRS medium, fermentation at 37°C on a shaker, pH 5.5–6.5, shaker speed 150 r / min, aeration rate 1.0 vvm, and other parameters are the same as in Example 1.

[0085] Comparative Example 4 differs from Example 1 in that Saccharomyces boulardii is used instead of Acetobacter xylinum. The fermentation parameters are as follows: YPD medium, fermentation at 30°C on a shaker, pH 5.0–6.0, aeration rate of 1.0 vvm, and shaker speed of 1500 r / min. The remaining parameters are the same as those in Example 1.

[0086] pH stability test

[0087] Solutions with pH values ​​of 2, 4, 6, 8, and 10 were prepared using phosphate buffer solutions with different pH values ​​and 100% DMSO solution. Appropriate amounts of retinol were accurately weighed and dissolved in these solutions at different pH values ​​to prepare retinol solutions of the same concentration (1%), ensuring complete dissolution of the retinol. The *Acetobacter xylinum* fermentation products from Examples 1 and Comparative Examples 1-4 were added to the above retinol solutions, ensuring a weight ratio of *Acetobacter xylinum* fermentation products to retinol of 1:0.5. The mixture was thoroughly mixed, and the pH of each sample solution was calibrated (the addition of *Acetobacter xylinum* fermentation products caused a slight pH deviation). Each sample tube was incubated in a constant temperature incubator (37°C) for 5 days. The samples were then removed, and the retinol content in each sample tube was determined using high-performance liquid chromatography (HPLC). Six replicates were performed for each sample, with the retinol solution without *Acetobacter xylinum* fermentation products serving as the control group. The residual retinol rate at different pH values ​​was calculated, and the results are shown in Table 1 below.

[0088] Residual rate = (Retinol content after incubation - Retinol content before incubation) / Retinol content before incubation × 100%

[0089] Table 1

[0090]

[0091] Note: Compared with the control group, * P<0.05, ** P < 0.01.

[0092] Analysis of Table 1 shows that retinol exhibits poor stability in solutions with pH 2.0, 4.0, and 10.0, with residual rates of approximately 58%, 62%, and 35%, respectively. However, the addition of the *Acetobacter xylinum* fermentation product from Example 1 significantly improves the stability of retinol in solutions at different pH levels. Particularly under acidic and alkaline conditions, the residual rate of retinol is above 85%, reaching a maximum of 96%. Analysis of other comparative examples reveals that the *Acetobacter xylinum* fermentation product prepared without the addition of *Acer buergerianum* juice and the *Acetobacter xylinum* fermentation product prepared using *Lactobacillus acidophilus* instead of *Acetobacter xylinum* showed the opposite effect to Example 1, with a significantly lower residual rate of retinol compared to the control group (P<0.05). The *Acetobacter xylinum* fermentation product prepared with the addition of black truffle extract had no significant effect on the stability of retinol, but also did not show the opposite effect. The *Acetobacter xylinum* fermentation product prepared using *Saccharomyces boulardii* instead of *Acetobacter xylinum* had a certain improving effect on the stability of retinol in acidic and alkaline solutions, but the effect was not as strong as that of Example 1.

[0093] Metal ion influence test

[0094] Solution preparation: Retinol was prepared into a 1% ethanol solution, divided into 18 equal portions, and then randomly divided into 3 groups of 6 portions each, corresponding to three concentrations of ferric chloride solution (0.1 mmol / L, 1 mmol / L, and 10 mmol / L). The fermentation products of *Acetobacter xylinum* from Example 1 and Comparative Examples 1–4 were added to each of the 6 portions in each group, with the remaining portion serving as a blank control. All samples were mixed thoroughly to obtain a mixed solution. Different concentrations of FeCl3 solution were added to the three mixed solutions to achieve final concentrations of 0.1 mmol / L, 1 mmol / L, and 10 mmol / L, respectively. After incubating each group of samples at 37°C for 5 days, the retinol content was detected by HPLC, and the remaining retinol rate was calculated. The results are shown in Table 2 below.

[0095] Table 2

[0096]

[0097]

[0098] Note: Compared with the control group, * P < 0.05 ** P < 0.01.

[0099] Analysis of Table 2 shows that retinol has poor stability in a 10.0 mmol / L iron ion solution. After incubation at 37°C for 5 days, only 50% of the retinol remained, indicating a damage rate of 50%. However, after adding the *Acetobacter xylinum* fermentation product from Example 1, the stability of retinol in iron ion solutions of different concentrations was significantly improved, with a loss rate of less than 10% in a 10.0 mmol / L iron ion solution. Analysis of other comparative examples shows that the *Acetobacter xylinum* fermentation product prepared with the addition of black truffle extract showed good stability in iron ion solutions at concentrations of 0.1 mmol / L and 1 mmol / L. The fermentation product of Acetobacter xylophilus has a certain stabilizing effect on retinol in the solution, but no similar effect was observed in the high concentration of iron ions. The fermentation product of Acetobacter xylophilus prepared without the addition of Oceania stellaria juice has no obvious effect on the stabilization of retinol in the low concentration of iron ions, but the opposite effect was observed in the high concentration of iron ions. The fermentation products of Acetobacter xylophilus prepared by replacing Acetobacter xylophilus with Lactobacillus acidophilus and the fermentation products of Acetobacter xylophilus prepared by replacing Acetobacter xylophilus with Saccharomyces boulardii were not observed to have similar enhancing or opposite effects in the iron ion solutions of different concentrations.

[0100] Stimulation test

[0101] Preparation of retinol emulsion: Weigh 0.35g of carbamoyl ether into a 200mL beaker, add 7.5g of glycerol, and then add 79.55g of deionized water. Heat to 75-80℃ and stir to obtain phase A. Weigh 0.5g of p-hydroxyacetophenone and 0.5g of 1,2-hexanediol into a small beaker and stir at 60-70℃ until completely dissolved to obtain phase B. Add retinol and the *Acetobacter xylinum* fermentation product from Example 1 (1% and 2% by weight, respectively), and stir at 75-80℃ until completely dissolved to obtain phase C. Add phase C to phase A at 75-80℃, disperse for 5-10 minutes, cool to 40℃, add phase B, and adjust the pH to approximately 6.0 to obtain the sample emulsion of Example 1. Comparative sample emulsions 1-4 were prepared according to the above method, and the retinol emulsion without *Acetobacter xylinum* fermentation product was used as a blank control group.

[0102] Screening of retinol-sensitive individuals: 1g of a retinol-containing lotion sample was applied to the inner forearm of the subjects, spreading it evenly in circular motions 20 times. This was done twice daily, morning and evening, for two consecutive days. Subjects who reported stinging, burning, or pain were selected. A total of 30 subjects meeting this criterion were selected for irritation testing.

[0103] Irritation test procedure: Thirty subjects were randomly divided into six groups of five. Before the experiment, the inner forearm of each subject was rinsed with water and dried. The subjects were then placed in a constant temperature and humidity environment (temperature 20–22℃, humidity 40%–60%) for 30 minutes. Six square areas (2×2cm) were marked on the inner forearm of each subject. The TEWL value of each area before sample application was measured using a Tewameter RTM Hex. Sample emulsion (1g) was applied to each of the six square areas, swirling 20 times to ensure even distribution. This was done twice daily (morning and evening) for two consecutive days. The TEWL value was measured on the third day. The collected data were compiled and statistically analyzed. The results are shown in Table 3. Figure 2 As shown.

[0104] TEWL difference = TEWL value after use - TEWL value before use

[0105] Table 3

[0106] Blank control group 1.35 Example 1 -0.44 Comparative Example 1 0.89 Comparative Example 2 -0.12 Comparative Example 3 1.67 Comparative Example 4 0.53

[0107] The results are shown in Table 3 and Figure 2 As shown, the subjects' TEWL increased after using a solution containing only retinol, indicating that the transepidermal water loss of the skin increased after using retinol and the skin barrier was significantly damaged; while the subjects' skin TEWL values ​​decreased after applying the emulsion samples containing the fermentation products of Acetobacter xylinum in Example 1 and Comparative Example 2, indicating that it had less skin irritation.

[0108] Application Example 1 – Retinol Serum (mass fraction)

[0109]

[0110]

[0111] Preparation process:

[0112] Deionized water was added to the reaction vessel, and stirring was started at 100 rpm. Allantoin, acrylamide-acryloyldimethyl taurate copolymer, and sodium hydroxide were added to the reaction vessel in sequence. The mixture was heated to 70-80°C and stirred until completely dissolved to obtain the aqueous phase. In another reaction vessel, octyl dodecanol, cetearyl alcohol, polysorbate-20, polysorbate-80, polydimethylsiloxane, isotretinoinate, and caprylic / capric triglycerides were added in sequence. The mixture was heated to 60-80°C and stirred at 100 rpm for 15 minutes to obtain the oil phase. The oil phase was slowly added to the aqueous phase and stirred at 200 rpm for 30 minutes. After cooling to room temperature, retinol, Acetobacter xylinum fermentation product, acetyl glucosamine, ceramide, and phenoxyethanol were added. The mixture was stirred for 10 minutes and then homogenized at 20 MPa for 2 minutes to obtain the essence emulsion.

[0113] The difference between Comparative Example 1 and Application Example 1 is that no Acetobacter xylinum fermentation products were added.

[0114] Cell experiments

[0115] 1. Tetramethylazothiazolium blue colorimetric method: Mouse embryonic fibroblasts (3T3 cells, purchased from Beina Biotechnology Co., Ltd.) were used at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded into 96-well plates at a density of cells / well and incubated overnight at 37°C with 5% CO2. A blank control group and an experimental group were set up. In the experimental group, three volume fraction gradients of the sample were prepared (0.5%, 1%, and 3%), with three replicates for each gradient. Different concentrations of the test substance were prepared using DMEM basal medium according to the experimental design. After 24 hours of cell culture, the 96-well plates were removed, the old medium was discarded, and 100 μL of DMEM medium was added to each well of the blank group. 100 μL of DMEM medium containing the corresponding sample was added to each well of the experimental group. No cells were seeded in the zero group; only 100 μL of DMEM medium was added. After drug administration, the plates were returned to the incubator for further culture. After 24 hours, the supernatant was discarded, and MTT working solution was added. After incubation at 37°C in the dark for 4 hours, the supernatant was discarded, and 100 μL of DMSO was added to each well. The OD value was read at 490 nm, and the relative cell viability of the blank control group was marked as 100%.

[0116] 2. Effect of UVA on type I collagen secretion by fibroblasts: After overnight incubation, cells were washed with PBS and then subjected to UVA irradiation at 30 J / cm² according to the experimental groups (see Table 4). 2The cells were subjected to UVA irradiation. Based on the cell viability test results obtained from the MTT assay, an appropriate sample concentration was selected as the test concentration, and the sample solution was prepared accordingly. TGF-β1 (100 ng / mL) was used as a positive control, with 100 μL added to each well, and three replicates were set up for each group. After drug administration, the cells were incubated in the dark for 24 h. The cell supernatant of each sample group was collected into centrifuge tubes, and after centrifugation, the supernatant was collected into 1.5 mL centrifuge tubes and tested according to the ELISA kit instructions.

[0117] Table 4 Experimental Groups

[0118]

[0119] 3. Data Statistics and Analysis

[0120] Data were analyzed using SPSS software. Quantitative data were expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0121] 4 Results

[0122] Depend on Figure 3 and 4 It can be seen that the application of 0.5%, 1%, and 3% volume fractions of the product in Example 1 did not have any cytotoxic effect on the cells. On the contrary, compared with the control group (100%), they all significantly stimulated the growth of HSF cells and the secretion of type I collagen, and this stimulating effect was stronger than that of the control group without the addition of Acetobacter xylinum fermentation product.

[0123] 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 composition for promoting collagen synthesis in the skin, characterized in that, The composition comprises 0.05-2% retinol and 1-4% Acetobacter xylinum fermentation product; the weight ratio of Acetobacter xylinum fermentation product to retinol in the composition is 1:0.1-1; the Acetobacter xylinum fermentation product is prepared by the following steps: S1. Preparation of soy milk: Add deionized water to cleaned soybeans, soak them in 0.1~0.5% NaHCO3 solution, then add deionized water and heat grind for 10~30 minutes. Sieve, homogenize, sterilize and cool to obtain soy milk. S2. Preparation of Oceania quinoa juice: Add deionized water to Oceania quinoa, extract under ultrasonication, filter the extract, centrifuge to obtain Oceania quinoa juice. S3. Fermentation: Add 10-35% by weight of soybean milk and 3-10% by weight of quinoa juice to the fermentation medium, inoculate with Acetobacter xylophilus for fermentation, collect the fermentation supernatant, filter, and obtain the Acetobacter xylophilus fermentation product; the biological preservation number of the Acetobacter xylophilus is: ATCC 23767.

2. The composition according to claim 1, characterized in that, In step S1, the ratio of soybeans to deionized water is 1:3~10; the soybeans are soaked in NaHCO3 solution for 1~12 hours; the hot grinding temperature is 80~100℃; the homogenization pressure is 10~30MPa; and the homogenization time is 5~10 minutes.

3. The composition according to claim 1, characterized in that, In step S2, the ratio of Oceania schlegelii to deionized water is 1:3~6; the extraction time is 10~60 minutes; the ultrasonic frequency is 60~80kHz; the ultrasonic power is 100~200W; and the temperature is 30~50℃.

4. The composition according to claim 1, characterized in that, In step S3, during the fermentation process, the inoculum amount of Acetobacter xylinum is 3-10%; the fermentation temperature is 25-30℃; the fermentation pH is 5.0-6.0; the aeration rate is 0.5-1.5 vvm; the stirring speed is 100-300 r / min; and the tank pressure is 0.05-0.15 MPa.

5. The composition according to claim 1, characterized in that, In step S3, the fermentation time is 48~72 hours.

6. A stable cosmetic that promotes collagen synthesis in the skin, characterized in that, It includes the composition according to any one of claims 1 to 5 and the adjuvants available in the cosmetics field.

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