Composition for promoting synthesis of skin collagen and preparation method thereof

Abstract

The invention belongs to the technical field of biology. The present invention relates to a composition comprising retinol and, more specifically, to a composition comprising retinol and having increased stability, which can be made into cosmetics beneficial for skin rejuvenation. The invention relates to a composition for promoting synthesis of skin collagen. The composition is prepared from 0.05 to 2 percent of retinol and an acetobacter xylinum fermentation product. The acetobacter xylinum fermentation product is compounded with the retinol, and the stability of the retinol in solutions with different pH values and solutions containing high-concentration Fe < 3 + > is remarkably improved on the premise that the antioxidant activity of the retinol is not affected. In a test for promoting synthesis of human skin collagen, the composite retinol solution shows a promoting effect which is obviously superior to that of a single retinol solution.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. More specifically, it relates to a composition with increased stability of retinol, which can be formulated into a cosmetic beneficial for skin rejuvenation. Background Art

[0002] Retinol has various positive effects on the skin, mainly including the following aspects: ① Improving naturally aged skin: Retinol can stimulate fibroblasts to increase the secretion of collagen. Secondly, retinol can accelerate the metabolism of skin cells, promote the shedding of aged keratinocytes, and enable newborn cells to reach the skin surface faster; ② Whitening effect: Retinol can inhibit the activity of tyrosinase, which is a key enzyme in the process of melanin synthesis. By inhibiting its activity, the formation of melanin can be reduced, thereby preventing and alleviating problems such as age spots and dullness, achieving a whitening effect. In addition to inhibiting melanin production, they can also accelerate the decomposition and metabolism of melanin in melanocytes, enabling the already formed melanin to be excreted from the body faster, further brightening the skin tone and evening out the skin color; ③ Oil control and acne treatment effect: Retinol can act on sebaceous glands to regulate the secretion of sebum. For oily skin, it can reduce sebum secretion, lower the oil content on the skin surface, thereby improving the greasy 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 acne inflammatory reaction and relieve the redness and pain of acne; ④ Improving the skin barrier effect: Retinol can enhance the function of the stratum corneum, help maintain the normal structure and function of stratum corneum cells, enhance the barrier effect of the stratum corneum, and enable the skin to better resist the invasion of external harmful substances. In addition, retinol can also promote skin moisturization. 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 moist and plump, and reduce problems such as dryness and flaking.

[0003] However, retinol may cause skin irritation, manifested as symptoms such as redness, itching, dryness, and flaking, i.e., "retinol intolerance". This is related to the use concentration, frequency, and skin type. Generally speaking, sensitive skin is more likely to show irritation reactions; products with higher concentrations are more irritating. For example, when the retinol concentration exceeds 1%, the irritation risk increases significantly.

[0004] Secondly, retinol has poor stability and is easily affected by various factors and undergoes changes. For example, retinol has a highly unsaturated structure, and the carbon-carbon double bonds in the molecule are prone to react with oxygen in the air. After being oxidized, substances such as retinal and retinoic acid are generated, thereby reducing its activity and efficacy. For example, both ultraviolet and visible light may have a destructive effect on retinol. Light can trigger a photochemical reaction of retinol molecules, causing changes in their structure and resulting in a decrease in stability. Retinol is unstable under both acidic and alkaline conditions. In an acidic environment, retinol is prone to undergo a protonation reaction, leading to a change in the molecular structure. In an alkaline environment, retinol may undergo a saponification reaction, etc., affecting its stability. Therefore, the two major problems in the application of retinol in this field are its low stability and high irritation.

[0005] Numerous research reports at home and abroad have confirmed the antioxidant function of soybeans. Tocopherols, vitamin C, isoflavones, and phenolic compounds contained in soybeans all have antioxidant activity. Yamasaki et al. [1] found its antioxidant activity in the substances extracted from fermented soy milk. Geng et al. [2 By optimizing the fermentation conditions of the lactic acid bacteria combination, the free radical scavenging rate of acidified soy milk can reach 84.3%; Xu Yin et al. [3 Proved through in vitro experiments that the DPPH scavenging rate and Fe 2+ chelating ability of fermented acidified soy milk are significantly increased compared with those before fermentation, reaching 32.76% and 31.89% respectively. At present, there is no research on fermenting soy milk with Acetobacter xylinum and applying the obtained fermentation product to improve the stability of retinol while reducing its irritation.

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

[0007] [2] Geng Y R, Li W J, Wang J M. Radical scavenging ability of soymilk fermented 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 Antioxidant Effects of Lactobacillus-fermented Soybean Milk in vitro and in vivo [J]. China Dairy Industry, 2012, 40(8): 16-19. Summary of the Invention

[0009] The present invention relates to Acetobacter xylinum, the product obtained by fermenting soybean milk with Acetobacter xylinum, the characteristics of this product and its applications. The present invention unexpectedly discovers that the product obtained by fermenting soybean milk and Atriplex nummularia L. juice with Acetobacter xylinum can significantly increase the stability of retinol and reduce the skin irritation caused by it.

[0010] In this article, stability refers to the stability of retinol in solutions with different pH values and solutions containing metal ions. Experiments show that the stability of retinol solution is relatively poor in solutions with pH 2.0 and pH 10.0. The test results show that the remaining rates of retinol are about 58% and 35% respectively after incubation at 37°C for 5 days, and it is relatively stable at pH 6.0 - 8.0. After adding the fermentation product of Acetobacter xylinum of the present invention, the retinol solution can exist stably in acidic and alkaline solutions (pH 2.0 - 10.0), and the remaining rate is above 85% after incubation at 37°C for 5 days. This indicates that the fermentation product of Acetobacter xylinum can improve the stability of retinol in acidic and alkaline solutions, but the specific mechanism of action remains to be further studied.

[0011] In addition, it has been proven that the presence of Fe 3+ relatively affects the stability of retinol, and as the concentration of Fe 3+ increases, the destructive effect on retinol becomes stronger. After incubation at 37°C for 5 days in a high-concentration Fe 3+ (10 mmol / L) solution, the remaining rate of retinol is only about 50%, and it is observed that the color of retinol turns brownish-yellow, which may be due to the oxidative degradation of retinol. After adding the fermentation product of Acetobacter xylinum, after incubation at 37°C for 5 days in a solution with the same concentration of Fe 3+ , the remaining rate of retinol is 90%. This indicates that the fermentation product of Acetobacter xylinum can significantly improve the stability of retinol in Fe 3+ solution, and there is a significant difference compared with the case without addition (P < 0.01).

[0012] Regrettably, the fermentation product of Acetobacter xylinum obtained in the present invention cannot improve the photo-stability of retinol.

[0013] The above object of the present invention is achieved by the following technical solutions:

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

[0015] In one embodiment of the present invention, in the composition, the weight ratio of the fermentation product of Acetobacter xylinum to retinol is 1:0.1 to 1. More preferably, the weight ratio of the fermentation product of Acetobacter xylinum 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 the fermentation product of Acetobacter xylinum to retinol is 1:0.5.

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

[0017] S1. Preparation of soymilk: Add deionized water to the cleaned soybeans, soak them in 0.1 - 0.5% NaHCO3 solution, then add deionized water and heat-mill for 10 - 30 min, sieve, homogenize, sterilize and cool to obtain soymilk;

[0018] S2. Preparation of Atriplex nummularia L. juice: Add deionized water to Atriplex nummularia L., extract it under ultrasonic waves, filter the extract and centrifuge to obtain the Atriplex nummularia L. juice;

[0019] S3. Fermentation: Add 10 - 35% by weight of soymilk and 3 - 10% by weight of Atriplex nummularia L. juice to the fermentation medium, inoculate Acetobacter xylinum for fermentation, collect the fermentation supernatant and filter to obtain the fermentation product of Acetobacter xylinum.

[0020] In the above, adding Atriplex nummularia L. juice is essential for achieving the purpose of the present invention. The above-mentioned "and this supernatant unexpectedly showed an unexpected effect of stabilizing retinol and reducing its skin irritation" is all based on the addition of Atriplex nummularia L. juice. Because the comparative test results show that the fermentation supernatant obtained without adding Atriplex nummularia L. juice showed the opposite effect.

[0021] In one embodiment of the present invention, the biological deposit number of the Acetobacter xylinum is: ATCC 23767. A large number of screening and comparative tests have also been carried out in the selection of strains. The fermentation products produced by over-fermentation of lactic acid bacteria (Lactobacillus acidophilus) and yeast (Saccharomyces boulardii) have no similar effects.

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

[0023] In one embodiment of the present invention, in the step S2, the ratio of Atriplex nummularia 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 the step S2, the filtration is usually carried out through filter paper, a filter screen or a filtration device, aiming to remove the solid residues therein to obtain the juice.

[0025] In one embodiment of the present invention, in the step S2, the centrifugation is to remove the small amount of tiny particles or suspended substances still remaining after filtration. Through centrifugation, the solid impurities can be precipitated to the bottom of the centrifuge tube to obtain a relatively clear juice. The rotation speed of the centrifugation can be 3000 to 10000 revolutions per minute, and the centrifugation time is 5 to 10 minutes.

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

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

[0028] In one embodiment of the present invention, in the step S3, the fermentation duration is 48 to 72 h. The fermentation time is very important for obtaining a fermentation product with ideal functional characteristics. Different fermentation durations will cause various changes in the nutritional components of the fermentation product. For example, certain components such as vitamins will be accumulated, and at the same time, some special metabolites may be produced. These changes endow the product with different new characteristics.

[0029] The present invention also provides a stable cosmetic for promoting skin collagen synthesis, comprising the said composition and auxiliaries available in the cosmetic field.

[0030] The cosmetics described in this article usually include conventional preparation forms in the field of cosmetics, including but not limited to: essence, cream, eye cream, essential oil, facial mask, essence milk, essence water, etc. The "auxiliaries" described in this article are usually determined by their preparation forms. In particular, the present invention is preferably made in the form of essence milk, and its auxiliaries generally include: solvent, humectant, emulsifier, thickener, emollient, preservative, pH regulator, etc.

[0031] In particular, a formulation suitable for the present invention is: retinol 0.05 - 2%; Gluconacetobacter xylinus fermentation product 1 - 4%; humectant 3 - 10%; emulsifier 1 - 3%; thickener 1 - 3%; emollient 3 - 10%; preservative 0.1 - 0.5%; pH regulator 0.1 - 0.5%; the balance being water.

[0032] In particular, in this formulation, the humectant can be selected from one or more of the combinations of glycerol, propylene glycol, 1,3 - propanediol, 1,2 - pentanediol, octyldecanol, sodium hyaluronate, chitosan acetylamine, allantoin, ceramide, phytosphingosine, murumuru butter, trehalose, ethylhexylglycerin.

[0033] In particular, in this formulation, the emulsifier can be selected from one or more of the combinations of octyldodecanol, cetearyl alcohol, polysorbate - 20, polysorbate - 80, sorbitan oleate, hexyl decanol, polyglyceryl - 3 methyl glucoside distearate, sucrose laurate, PEG - 40 hydrogenated castor oil.

[0034] In particular, in this formulation, the thickener can be selected from one or more of the combinations of acrylamide - acryloyldimethyltaurates copolymer, xanthan gum.

[0035] In particular, in this formulation, the emollient can be selected from one or more of the combinations of isocetane, dimethicone, phytosterol / octyldodecyl lauroyl glutamate, isotridecyl isononanoate, caprylic / capric triglyceride, PEG / PPG / polybutylene glycol - 8 / 5 / 3 glycerin.

[0036] In particular, in this formulation, the preservative is phenoxyethanol.

[0037] In particular, in this formulation, the pH regulator is sodium hydroxide.

[0038] In particular, 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] Gluconacetobacter xylinus fermentation product 1 - 4%;

[0042] 1 - 5% chitosanamine;

[0043] 0.5 - 2% allantoin;

[0044] 1 - 3% ceramide;

[0045] 0.1 - 1% octyldodecanol;

[0046] 0.1 - 1% cetearyl alcohol;

[0047] 0.1 - 0.5% polysorbate - 20;

[0048] 0.1 - 0.5% polysorbate - 80;

[0049] 1 - 3% acrylamide - sodium acryloyldimethyltaurate copolymer;

[0050] 1 - 3% polydimethylsiloxane;

[0051] 1 - 3% isopropyl myristate;

[0052] 1 - 3% caprylic / capric triglyceride;

[0053] 0.1 - 0.5% phenoxyethanol;

[0054] 0.1 - 0.5% sodium hydroxide; and

[0055] the balance being water.

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

[0057] The present invention combines the fermentation product of Gluconacetobacter xylinus with retinol, and significantly improves the stability of retinol in solutions with different pH values and solutions containing a high concentration of Fe3+ without affecting the antioxidant activity of retinol. In the test of promoting human skin collagen synthesis, the combined retinol solution shows a significantly better promoting effect than the single one. Description of the Drawings

[0058] Figure 1 It is a picture of Gluconacetobacter xylinus cells.

[0059] Figure 2 It is the change in the TEWL difference before and after the use of Gluconacetobacter xylinus fermentation product / retinol emulsion in each group;

[0060] Among them, compared with the blank control group, * P < 0.05, ** P < 0.01.

[0061] Figure 3 It is the comparison of the relative viability of fibroblasts of the essence milk in Application Example 1 and Comparative Example 1 with different concentrations,

[0062] Among them, compared with the blank control group, * P < 0.05, ** P < 0.01.

[0063] Figure 4 These are the detection results of the content of type I collagen.

[0064] Among them, compared with the blank control group, * P < 0.05, ** P < 0.01; compared with the model group, wP < 0.05, ## P < 0.01. Specific implementation manners

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

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

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

[0068] Example 1 Preparation of the fermentation product of Acetobacter xylinum

[0069] (1) Preparation of soymilk: After removing impurities and rinsing soybeans clean, add deionized water according to a solid-liquid ratio of 1:6, add 0.25% NaHCO3 solution and soak for 3 h, add deionized water according to a solid-liquid ratio of 1:10 and heat-mill at 100 °C for 30 min, pass through a 100-mesh sieve, homogenize at 25 MPa for 8 min, sterilize and cool to obtain soymilk;

[0070] (2) Preparation of Atriplex nummularia L. juice: Select fresh Atriplex nummularia L., wash its stems and leaves, crush them, add 6 times deionized water, place under ultrasonic wave for 30 minutes, the ultrasonic frequency is 60 kHz, the ultrasonic power is 150 W, and the temperature is controlled between 45 °C; after the ultrasonic wave ends, filter the obtained mixture, centrifuge at 3000 r / min for 5 minutes, and take the supernatant to obtain the Atriplex nummularia L. juice;

[0071] (3) Strain activation: In a laminar flow hood, use an inoculation loop to pick up a small amount of Acetobacter xylinum, and under sterile conditions, inoculate it into the prepared solid slant medium (HS medium + 1.5% agar, pH 5.5 - 6.0). Gently slide it on the slant to evenly distribute the strain on the slant. Place the inoculated medium in a constant temperature incubator and incubate at 28 - 30 °C for 24 h until obvious colonies appear on the medium, and collect the bacteria.

[0072] (4) Fermentation: Add 25 wt% soy milk and 8 wt% Atriplex nummularia juice to the HS medium to obtain a fermentation medium. Inoculate 5% Acetobacter xylinum into the fermentation medium and ferment for 48 - 72 h at a fermentation temperature of 28 °C; the fermentation pH is 5.5 - 6.0; the aeration rate is 1.0 vvm; the stirring speed is 150 r / min; the tank pressure is 0.1 MPa; collect the fermentation supernatant, filter it through a 0.22 μm ultrafiltration membrane, and collect the filtrate to obtain the Acetobacter xylinum fermentation product.

[0073] Example 2 Preparation of Acetobacter xylinum Fermentation Product

[0074] (1) Preparation of soy milk: After removing impurities and rinsing the soybeans thoroughly, add deionized water at a material-liquid ratio of 1:3, add 0.25% NaHCO3 solution and soak for 2 h. Then add deionized water at a material-liquid ratio of 1:10, heat-mill at 80 °C for 30 min, pass through a 100-mesh sieve, homogenize at 20 MPa for 10 min, sterilize and cool to obtain soy milk.

[0075] (2) Preparation of Atriplex nummularia juice: Select fresh Atriplex nummularia, wash its stems and leaves, crush them, add 3 times deionized water, place it under ultrasonic waves for 15 minutes, with an ultrasonic frequency of 860 kHz, an ultrasonic power of 200 W, and the temperature controlled at 50 °C. After the ultrasonic treatment, filter the obtained mixture, centrifuge at 3000 r / min for 10 min, and take the supernatant to obtain the Atriplex nummularia juice.

[0076] (3) Strain activation: In a laminar flow hood, use an inoculation loop to pick up a small amount of Acetobacter xylinum, and under sterile conditions, inoculate it into the prepared solid slant medium (HS medium + 1.5% agar, pH 5.5 - 6.0). Gently slide it on the slant to evenly distribute the strain on the slant. Place the inoculated medium in a constant temperature incubator and incubate at 28 - 30 °C for 24 h until obvious colonies appear on the medium, and collect the bacteria.

[0077] (4) Fermentation: Add 10 wt% soy milk and 10 wt% Atriplex nummularia juice to the HS medium to obtain a fermentation medium. Inoculate 3% Acetobacter xylinum into the fermentation medium and ferment for 48 h at a fermentation temperature of 28 °C; the fermentation pH is 5.5 - 6.0; the aeration rate is 1.0 vvm; the stirring speed is 150 r / min; the tank pressure is 0.1 MPa; collect the fermentation supernatant, filter it through a 0.22 μm ultrafiltration membrane, and collect the filtrate to obtain the Acetobacter xylinum fermentation product.

[0078] Example 3 Preparation of Acetobacter xylinum Fermentation Product

[0079] (1) Preparation of soy milk: After removing impurities and rinsing soybeans clean, add deionized water at a solid-liquid ratio of 1:10, add 0.25% NaHCO3 solution and soak for 12 h, add deionized water at a solid-liquid ratio of 1:10 and heat-mill at 100 °C for 30 min, sieve through a 100-mesh sieve, homogenize at 25 MPa for 5 min, sterilize and cool to obtain soy milk;

[0080] (2) Preparation of Atriplex nummularia juice: Select fresh Atriplex nummularia, wash its stems and leaves, crush them, add 5 times deionized water, place them under ultrasonic waves for 60 minutes, the ultrasonic frequency is 60 kHz, the ultrasonic power is 100 W, and the temperature is controlled between 35 °C; after ultrasonic treatment, filter the obtained mixture, centrifuge at 3000 r / min for 5 min, and take the supernatant to obtain the Atriplex nummularia juice;

[0081] (3) Strain activation: In a clean bench, use an inoculation loop to pick up a small amount of Acetobacter xylinum, and under sterile conditions, inoculate it into the prepared solid slant medium (HS medium + 1.5% agar, pH 5.5 - 6.0), gently slide it on the slant to make the strain evenly distributed on the slant; put the inoculated medium into a constant temperature incubator and culture at 28 - 30 °C for 24 h until obvious colonies appear on the medium, and collect the bacterial cells; (4) Fermentation: Add 35 wt% soy milk and 3 wt% Atriplex nummularia juice to the HS medium to obtain a fermentation medium. Inoculate 5% activated Acetobacter xylinum into the fermentation medium and ferment for 72 h at a fermentation temperature of 28 °C; the fermentation pH is 5.5 - 6.0; the aeration rate is 1.0 vvm; the stirring speed is 150 r / min; the tank pressure is 0.1 MPa; collect the fermentation supernatant, filter it through a 0.221 μm ultrafiltration membrane, and collect the filtrate to obtain the Acetobacter xylinum fermentation product.

[0082] Comparative Example 1: Different from Example 1, in the fermentation process of step (4), Atriplex nummularia juice is not added, and the other parameters are the same as those in Example 1.

[0083] Comparative Example 2: Different from Example 1, in the fermentation process of step (4), truffle extract (purchased from Shanghai Haotai Biotechnology Co., Ltd.) is added, and the other parameters are the same as those in Example 1.

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

[0085] Comparative Example 4: The difference from Example 1 is that Saccharomyces boulardii is used instead of Acetobacter xylinum. The fermentation parameters are as follows: The culture medium is YPD medium, fermentation is carried out on a shaker at 30 °C, the pH is 5.0 - 6.0, the aeration rate is 1.0 vvm, the shaker speed is 1500 r / min, and the other 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 are prepared by using phosphate buffer solutions with different pH values and 100% DMSO solution. An appropriate amount of retinol is accurately weighed and dissolved in the above solutions with different pH values respectively to prepare retinol solutions with the same concentration (1%) to ensure that the retinol is completely dissolved. The fermentation products of Acetobacter xylinum from Example 1 and Comparative Examples 1 - 4 are added to the above retinol solutions respectively to ensure that the weight ratio of the fermentation products of Acetobacter xylinum to retinol is 1:0.5, and they are mixed evenly. The pH of each group of sample solutions is calibrated (the addition of the fermentation products of Acetobacter xylinum will cause a deviation in the pH of the solution). The sample tubes are placed in an incubator (37 °C) for 5 days. Then the samples are taken out, and the content of retinol in each sample tube is determined by high performance liquid chromatography (HPLC). Each sample is replicated 6 times. The retinol solution without the addition of the fermentation products of Acetobacter xylinum is used as the control group, and the remaining rate of retinol at different pH values is calculated. The results are shown in Table 1 below.

[0088] Remaining 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 has poor stability in solutions with pH 2.0, 4.0, and pH 10.0, and the remaining rates are about 58%, 62%, and 35% respectively. After adding the fermentation product of Acetobacter xylinum in Example 1, the stability of retinol in solutions with different pH values has been significantly improved. Especially under acidic and alkaline conditions, the remaining rate of retinol is above 85%, and the highest can reach 96%. Analysis of the results of other comparative examples shows that the fermentation product of Acetobacter xylinum prepared without adding the juice of Atriplex nummularia and the fermentation product of Acetobacter xylinum prepared by replacing Acetobacter xylinum with Lactobacillus acidophilus showed opposite effects to those in Example 1, and the remaining rate of retinol was significantly lower than that of the control group (P<0.05); the fermentation product of Acetobacter xylinum prepared by adding truffle extract had no obvious effect on the stability of retinol, but did not show the opposite effect either; the fermentation product of Acetobacter xylinum prepared by replacing Acetobacter xylinum with Saccharomyces boulardii had a certain improvement effect on stabilizing retinol in acidic and alkaline solutions, but the intensity was not as good as that in Example 1.

[0093] Metal ion influence test

[0094] Prepare the solution: Prepare a 1% ethanol solution of retinol, divide it into 18 equal parts and randomly divide it into 3 groups, with 6 parts in each group, corresponding to three concentrations of ferric chloride solutions (0.1 mmol / L, 1 mmol / L, and 10 mmol / L). Add the fermentation products of Acetobacter xylinum in Example 1 and Comparative Examples 1-4 to 6 parts of each group respectively, and leave 1 part as the blank control group. Mix evenly to obtain the mixed solution. Add different concentrations of FeCl3 solutions to the three groups of mixed solutions so that their final concentrations are 0.1 mmol / L, 1 mmol / L, and 10 mmol / L respectively. After incubating each group of samples at 37°C for 5 days, detect the retinol content by HPLC and calculate the remaining rate of retinol. 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] As can be seen from Table 2, retinol has poor stability in a 10.0 mmol / L iron ion solution, and only 50% of retinol remains after incubation at 37°C for 5 days, with a damage rate reaching 50%. However, after adding the Acetobacter xylinum fermentation product of Example 1, the stability of retinol in iron ion solutions with different concentrations is significantly improved, and the loss rate in a 10.0 mmol / L iron ion solution is less than 10%. From the results of other comparative examples, it can be seen that the Acetobacter xylinum fermentation product prepared by adding black truffle extract has a certain effect on stabilizing retinol in iron ion solutions with concentrations of 0.1 mmol / L and 1 mmol / L, but a similar effect cannot be observed in high-concentration iron ion solutions. The Acetobacter xylinum fermentation product prepared without adding Atriplex nummularia juice has no obvious effect on stabilizing retinol in low-concentration iron ion solutions, but the opposite effect is observed in high-concentration iron ion solutions. No similar improvement or opposite effect is observed for the Acetobacter xylinum fermentation products prepared by replacing Acetobacter xylinum with Lactobacillus acidophilus and Saccharomyces boulardii in iron ion solutions with different concentrations.

[0100] Irritation test

[0101] Preparation of retinol emulsion: Weigh 0.35 g of carbomer into a 200 mL beaker, add 7.5 g of glycerol, and then add 79.55 g of deionized water. Heat to 75 - 80°C and stir to obtain Phase A. Weigh 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol into a small beaker, stir until completely dissolved under heating conditions at 60 - 70°C to obtain Phase B. Add retinol and the Acetobacter xylinum fermentation product of Example 1 (the weights of the two are 1% and 2% respectively), stir evenly at 75 - 80°C until completely dissolved to obtain Phase C. Add Phase C to Phase A at 75 - 80°C, disperse for 5 - 10 min, then cool down to 40°C, add Phase B, and adjust the pH to about 6.0 to obtain the sample emulsion of Example 1. Prepare the sample emulsions of Comparative Examples 1 - 4 according to the above method, and use the retinol emulsion without adding the Acetobacter xylinum fermentation product as the blank control group.

[0102] Screening of retinol-sensitive population: Apply 1 g of the emulsion sample containing only retinol on the inner forearm of the subject, smear evenly in a circular motion 20 times, apply once in the morning and once in the evening for 2 consecutive days, and select the subjects who give feedback of stinging, burning, and pain. A total of 30 subjects meeting this standard are selected for the irritation test.

[0103] Irritation test procedure: Thirty subjects were randomly divided into six groups of five. Before the experiment, the inner anterior wall of each group of subjects' arms was rinsed with water, wiped clean, and left to stand for 30 min in a constant temperature and humidity environment (temperature 20 - 22 °C, humidity 40% - 60%). The inner side of the left forearm of each subject was marked into six square areas, each with a size of 2×2 cm, and the TEWL value of each area before applying the sample was measured using a Tewameter RTM Hex. The sample emulsion was applied to the six square areas respectively. 1 g of the sample was taken and applied evenly by circling 20 times, once in the morning and once in the evening for 2 consecutive days. On the third day, the TEWL value of the applied area was measured. The collected data was sorted out, and the differences were analyzed by statistical methods. The results are shown in Table 3 and Figure 2 as follows.

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

[0105] Table 3

[0106] Group Change in average TEWL value before and after use 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 follows. After using only the retinol solution, the TEWL of the subjects increased, indicating that the transdermal water loss of the skin increased after using retinol, and the skin barrier was significantly damaged; while after applying the emulsion samples containing the fermentation products of Gluconacetobacter xylinus in Example 1 and Comparative Example 2, the TEWL values of the subjects' skin became smaller, indicating that they had little irritation to the skin.

[0108] Retinol essence milk for Application Examples 1 - 3 (mass fraction)

[0109]

[0110]

[0111] Preparation process:

[0112] Add deionized water to the reaction kettle, start stirring, and set the rotation speed to 100 revolutions per minute; sequentially add allantoin, acrylamide-sodium acryloyldimethyltaurate copolymer, and sodium hydroxide to the reaction kettle, heat to 70 - 80 °C, and stir until completely dissolved to obtain the aqueous phase; in another reaction kettle, sequentially add octyldodecanol, cetearyl alcohol, polysorbate-20, polysorbate-80, polydimethylsiloxane, isoisotridecyl isononanoate, and caprylic / capric triglyceride, heat to 60 - 80 °C, stir at 100 revolutions per minute for 15 minutes to obtain the oil phase; slowly add the oil phase to the aqueous phase, stir at 200 revolutions per minute for 30 min, after cooling to room temperature, add retinol, Gluconacetobacter xylinus fermentation product, N-acetylglucosamine, ceramide, and phenoxyethanol, stir for 10 min, and then homogenize at 20 MPa for 2 min to obtain the essence milk.

[0113] Comparative Example 1: The difference compared with Application Example 1 is that the Gluconacetobacter xylinus fermentation product is not added.

[0114] Cell experiment

[0115] 1. MTT assay: Mouse embryonic fibroblasts (3T3 cells, purchased from BeiNa Biotechnology Co., Ltd.) were used. Cells were seeded into 96-well plates at a density of 1×10 5 cells / well, placed in an incubator at 37 °C and 5% CO2 for overnight incubation. The experiment was set up with a blank control group and experimental groups. In the experimental groups, the samples were set at 3 volume fraction gradients (0.5%, 1%, 3%), and 3 parallel experiments were set up for each gradient. According to the experimental design, test substances with different contents were prepared with DMEM basal medium. After 24 h of cell culture, the 96-well plates were taken out, the old medium was discarded. In the blank group, 100 μL of DMEM culture medium was added to each well, and in the experimental groups, 100 μL of DMEM culture medium containing the corresponding content of the sample was added to each well. The zero-adjustment group had no cell seeding and only 100 μL of DMEM culture medium was added. After dosing, the plates were returned to the incubator for continued culture. After 24 h, the supernatant was discarded, and MTT working solution was added. After incubating at 37 °C in the dark for 4 h, the supernatant was discarded, 100 μL of DMSO was added to each well, and the OD value was read at 490 nm. The relative viability of the cells in the blank control group was marked as 100%.

[0116] 2. Experiment on the effect of fibroblasts on the secretion of type I collagen: After the cells were seeded and incubated overnight, the cells were washed with PBS. According to the experimental grouping (see Table 4), for the groups with UVA irradiation, 30 J / cm 2UVA irradiation. According to the cell viability test results obtained from the MTT assay, an appropriate sample mass concentration was selected as the test concentration and the sample solution to be tested was prepared. Using TGF-β1 (100 ng / mL) as the positive control, 100 μL of the sample was added to each well, and 3 replicate wells were set up for each group. After the administration, the cells were incubated in the dark for 24 h. The cell supernatants corresponding to each sample group were collected into centrifuge tubes respectively. After centrifugation, the supernatants were collected and placed in 1.5 mL centrifuge tubes, and the test was carried out according to the instructions of the ELISA kit.

[0117] Table 4 Experimental grouping

[0118]

[0119] 3. Data statistics and analysis

[0120] The data were analyzed using SPSS software. Measurement data were expressed as mean ± standard deviation. One-way analysis of variance was used for comparison among multiple groups. A P value < 0.05 was considered statistically significant.

[0121] 4 Results

[0122] From Figure 3 and 4 it can be seen that after the cells were intervened with Application Example 1 at volume fractions of 0.5%, 1%, and 3%, there was no cytotoxic effect. 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 Comparative Example 1 without the addition of the fermentation product of Acetobacter xylinum.

[0123] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A composition for promoting the synthesis of skin collagen, characterized in that, The composition contains 0.05 to 2% of retinol and the fermentation product of Acetobacter xylinum.

2. The composition according to claim 1, wherein In the composition, the weight ratio of the fermentation product of Acetobacter xylinum to retinol is 1:0.1 to 1.

3. The composition according to claim 1 or 2, characterized in that, The fermentation product of Acetobacter xylinum is prepared by the following steps: S1. Preparation of soy milk: Add deionized water to the cleaned soybeans, soak them in a 0.1 - 0.5% NaHCO3 solution, then add deionized water and heat - mill for 10 - 30 min, sieve, homogenize, sterilize and cool to obtain soy milk; S2. Preparation of Atriplex nummularia L. juice: Add deionized water to Atriplex nummularia L., extract it under ultrasonic waves, filter and centrifuge the extract to obtain the Atriplex nummularia L. juice; S3. Fermentation: Add 10 - 35% by weight of soy milk and 3 - 10% by weight of Atriplex nummularia L. juice to the fermentation medium, inoculate Acetobacter xylinum for fermentation, collect the fermentation supernatant, and filter to obtain the fermentation product of Acetobacter xylinum.

4. The composition according to claim 3, characterized in that, In step S1, the ratio of soybeans to deionized water is 1:3 to 10; the soaking time in NaHCO3 is 1 - 12 h; the heat - milling temperature is 80 - 100 °C; the homogenization pressure is 10 - 30 MPa; the homogenization time is 5 - 10 min.

5. The composition according to claim 4, characterized in that, In step S2, the ratio of Atriplex nummularia L. to deionized water is 1:3 to 6; the extraction time is 10 - 60 minutes; the ultrasonic frequency is 60 - 80 kHz; the ultrasonic power is 100 - 200 W; the temperature is 30 - 50 °C.

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

7. The composition according to claim 6, characterized in that, In step S3, the fermentation duration is 48 - 72 h.

8. A stable cosmetic for promoting skin collagen synthesis, characterized in that, Contains the composition according to any one of claims 1 - 7 and auxiliaries usable in the cosmetic field.

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