Preparation method and application of sodium hyaluronate Ectoine leavening

Abstract

The invention discloses a preparation method of a sodium hyaluronate Ectoine fermentation product, which comprises the following steps: lactic acid bacteria are inoculated into a fermentation substrate, and fermentation culture and sterilization are performed to obtain the sodium hyaluronate Ectoine fermentation product, the fermentation substrate is composed of sodium hyaluronate, Ectoine and water; the lactic acid bacteria consist of lactobacillus mucus fermentation and lactobacillus helveticus. Sodium hyaluronate and Ectoine are fermented through lactic acid bacteria, a synergistic effect can be achieved, and the prepared product can be directly used as a cosmetic and can also be added into a basic formula of the cosmetic for use, has good tightening and relieving effects, is basically free of irritation and has a good application prospect in the field of cosmetics.

Description

Technical Field

[0001] The present application belongs to the field of fermentation technology, and in particular relates to a preparation method and application of sodium hyaluronate ectoine fermentation product. Background Art

[0002] With the development of the times, people advocate green, environmentally friendly and safe cosmetics, and also have high hopes for the efficacy of cosmetics. At present, under the condition of meeting the relevant laws and regulations on cosmetics, the cosmetics industry uses more chemical synthetic raw materials. Although they have advantages such as clear ingredient structure, clear efficacy, and mature quality control methods, when people use these cosmetics, they gradually find that chemical synthetic products can easily cause adverse effects on the human body, such as sensitization, redness, irritation, etc. Therefore, reducing the adverse reactions caused by chemical synthetics has become one of the directions for the development of cosmetics.

[0003] Due to its special molecular composition structure and physical and chemical properties, hyaluronic acid plays multiple physiological functions on the human body, such as lubricating joints, regulating the permeability of blood vessel walls, regulating the diffusion and movement of proteins, water, and electrolytes, promoting wound healing, and repairing skin scars. Hyaluronic acid (HA) is one of the best moisturizing substances found in nature today due to its unique moisturizing effect. It also enjoys the reputation of the best natural moisturizing factor. However, hyaluronic acid is unstable and easily degraded during high-temperature sterilization, so it cannot be used directly. Sodium hyaluronate is a salt of hyaluronic acid, which has basically the same effect as hyaluronic acid and is more stable in nature. Sodium hyaluronate can replace hyaluronic acid during use.

[0004] Ecdoin was originally extracted from halophilic bacteria growing in desert salt lakes. These bacteria can survive in extremely high temperature and high salt environments. The chemical structure of Ecdoin enables it to balance cell osmotic pressure and protect enzymes, DNA, proteins and other biological macromolecules under various adverse conditions. Ecdoin has a strong water structure forming ability and can form a strong water shield to effectively lock in moisture and keep the skin moisturized. It is called the "magic repair factor". Ecdoin can quickly repair and soothe the skin when the skin is sensitive, the barrier is damaged, and it stings and redness after sun exposure. It can also enhance the immune protection ability of skin cells, increase the fluidity and stability of cell membranes, and prevent ultraviolet damage to the skin.

[0005] It can be seen that sodium hyaluronate and ectoine each have their own advantages and different effects. Therefore, there is an urgent need for a method to combine the advantages of the two to achieve a synergistic anti-aging effect in order to overcome the problem of adverse reactions caused by chemical synthetics during use. Summary of the invention

[0006] The technical problem to be solved in this application is to overcome the lack of methods for effectively utilizing sodium hyaluronate and ectoine in the prior art, and the research focuses on the basic formula of the compound form of sodium hyaluronate and ectoine, which only retains the efficacy of sodium hyaluronate and ectoine themselves, and cannot play the synergistic effect of the two. The preparation method and application of a sodium hyaluronate ectoine fermentation product are provided. This application uses a specific composite strain to ferment sodium hyaluronate and ectoine to achieve synergistic effects. After the fermentation process of this application, the obtained product has ideal anti-aging, anti-oxidation, promotion of cell metabolism and moisturizing effects, the preparation process is simple, the fermentation conditions are mild, and no organic solvent is used in the extraction process, achieving energy saving and environmental protection.

[0007] This application adopts the following technical solutions to solve the above technical problems:

[0008] The present application provides a method for preparing a sodium hyaluronate ectoine fermentation product, which comprises the following steps: inoculating lactic acid bacteria into a fermentation substrate, fermenting and culturing, and sterilizing.

[0009] Wherein, the fermentation substrate is composed of sodium hyaluronate, ectoine and water; the lactic acid bacteria are composed of fermented mucus lactobacillus and helveticus lactobacillus. In some embodiments, the mass ratio of the sodium hyaluronate to the water is 1: (50-1000), preferably 1: (100-500), and more preferably 1: 200.

[0010] In some embodiments, the mass ratio of the ectoine to the water is 1:(50-1000), preferably 1:(100-500), and more preferably 1:200.

[0011] In some embodiments, the ratio of the viable cell count of the fermented Lactobacillus mucis to that of the Lactobacillus helveticus is 1:(0.2-5), preferably 1:1.

[0012] In some embodiments, the method for preparing the fermentation substrate comprises: mixing the components in the fermentation substrate until the components are completely dissolved.

[0013] In some embodiments, the average relative molecular weight of the sodium hyaluronate is 100-130 W, preferably 120 W. "W" is the unit "ten thousand" conventionally recognized by those skilled in the art.

[0014] In some embodiments, the fermentation substrate may be further sterilized before use.

[0015] Wherein, the method for sterilizing the fermentation substrate can generally be a high temperature sterilization method.

[0016] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C.

[0017] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure may be a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, such as 0.13 MPa.

[0018] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operation in the art, preferably 15 to 35 minutes, more preferably 15 to 25 minutes, for example 20 minutes.

[0019] In some embodiments, the fermented mucus lactobacillus (Limosilactobacillus fermentum) is purchased from the China Industrial Microbiological Culture Collection Administration Center, and the fermented mucus lactobacillus has a collection number of CICC 24824.

[0020] In some embodiments, the Lactobacillus helveticus is purchased from China Industrial Microbiological Culture Collection Center with a collection number of CICC 20997.

[0021] In some embodiments, the lactic acid bacteria may be added in the form of lactic acid bacteria liquid according to conventional practice in the art.

[0022] In some embodiments, based on the water in the fermentation substrate, the number of viable lactic acid bacteria inoculated per unit mass of the water is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 In some embodiments, the fermented Lactobacillus mucilaginosus is added in the form of a fermented Lactobacillus mucilaginosus bacterial solution, and the live bacteria concentration of the fermented Lactobacillus mucilaginosus bacterial solution is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL.

[0023] The method for preparing the fermented mucus lactobacillus liquid comprises: inoculating the fermented mucus lactobacillus into an MRS culture medium and culturing the culture medium at 37° C. for 24 hours.

[0024] In some embodiments, the Lactobacillus helveticus is added in the form of a Lactobacillus helveticus bacterial solution. The live bacteria concentration of the Lactobacillus helveticus bacterial solution is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL.

[0025] The method for preparing the Lactobacillus helveticus bacterial liquid comprises: inoculating Lactobacillus helveticus into MRS culture medium and culturing at 37° C. for 24 hours.

[0026] In some embodiments, the conditions and methods of the fermentation culture may be conventional in the art, and may generally be static fermentation.

[0027] In some embodiments, the fermentation culture temperature is 30-45°C, preferably 35-40°C, such as 37°C.

[0028] In some embodiments, the fermentation culture time is 12 to 48 hours, preferably 12 to 24 hours, such as 15 hours.

[0029] In some embodiments, the sterilization method may generally be high temperature sterilization.

[0030] The sterilization temperature may be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C.

[0031] The sterilization pressure may be a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, for example 0.13 MPa.

[0032] The sterilization time may be the conventional time for such operation in the art, preferably 15 to 35 minutes, more preferably 15 to 25 minutes, for example 20 minutes.

[0033] In some embodiments, after the sterilization, the sterilization may further include at least one of cooling, centrifuging and collecting the supernatant, secondary sterilization or mixing with a preservative.

[0034] Wherein, according to the common practice in the art, the cooling may be cooling to room temperature.

[0035] The centrifugal rotation speed is a conventional rotation speed for this type of operation in the art, preferably 3000-9000 rpm, more preferably 4000-6000 rpm, for example 5000 rpm.

[0036] Wherein, the radius of the centrifuge is 8 to 15 cm.

[0037] The centrifugation time is the conventional time for this type of operation in the art, preferably 10 to 40 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

[0038] The secondary sterilization method is generally a high temperature sterilization method.

[0039] The temperature of the secondary sterilization is a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C.

[0040] The pressure of the secondary sterilization is a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, for example 0.13 MPa.

[0041] The time for the secondary sterilization is the conventional time for such operations in the art, preferably 15 to 35 minutes, more preferably 15 to 25 minutes, for example 20 minutes.

[0042] Wherein, during the mixing process with the preservative, the mixing temperature is a conventional temperature for such operation in the art, preferably 50 to 80°C, more preferably 70 to 80°C.

[0043] Wherein, in the process of mixing with the preservative, the preservative comprises p-hydroxyacetophenone and / or 1,2-hexanediol according to the conventional practice in the art.

[0044] When the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant of the material obtained after sterilization may be 0.5% to 2%, preferably 0.1% to 0.6%, and more preferably 0.5%.

[0045] When the preservative includes 1,2-hexanediol, the mass fraction of 1,2-hexanediol in the supernatant of the material obtained after sterilization is 0.5% to 2%, preferably 0.5% to 1%, and more preferably 0.5%.

[0046] In some embodiments, the room temperature generally refers to 15-40°C.

[0047] The present application also provides a sodium hyaluronate and ectoine fermentation product, which is prepared by the preparation method of sodium hyaluronate and ectoine fermentation product as described above.

[0048] The present application also provides a sodium hyaluronate ectoine fermentation product, which can be used directly as a product, as an additive, or as a base in the preparation of a skin topical preparation.

[0049] In some embodiments, the sodium hyaluronate ectoine fermentation product is used as at least one of the antioxidant active ingredient, anti-aging active ingredient, and anti-allergic ingredient in the skin topical preparation.

[0050] The antioxidant active ingredients are antioxidant active ingredients with DPPH free radical scavenging effect and antioxidant active ingredients with hydroxyl free radical scavenging effect.

[0051] Wherein, the anti-aging active ingredient is an anti-aging active ingredient having a cell proliferation effect.

[0052] Wherein, the anti-allergic active ingredient is an anti-allergic active ingredient with hyaluronidase inhibitory effect.

[0053] The present application also provides a skin external preparation, which includes the sodium hyaluronate ectoine fermentation product as described above.

[0054] In some embodiments, the skin topical preparation further includes active ingredients conventionally used in the art, which may generally include at least one of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients.

[0055] In some embodiments, the topical skin preparation may include but is not limited to a facial mask, an essence or a toner according to conventional methods in the art.

[0056] In some embodiments, the mass percentage of the sodium hyaluronate ectoine fermentation product in the skin topical preparation may be 5% to 99%, preferably 60% to 99%.

[0057] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

[0058] The reagents and raw materials used in this application are commercially available.

[0059] The positive progressive effect of the present application is that the present application simultaneously ferments sodium hyaluronate and ectoine through a specific type of lactic acid bacteria, and achieves a synergistic effect of the two during the fermentation process, so that the prepared product has better firming, soothing and cell repair effects. It can be used directly as a cosmetic or added to the basic formula of cosmetics. It is basically non-irritating during use and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.

[0061] in:

[0062] Figure 1 A comparison chart of the DPPH free radical scavenging ability of the products obtained in Examples 1 to 4 or Comparative Examples 1 to 4;

[0063] Figure 2 A comparison chart of the hydroxyl radical scavenging abilities of the products obtained in Examples 1 to 4 or Comparative Examples 1 to 4;

[0064] Figure 3 This is a comparison chart of the hyaluronidase inhibition rate of the products prepared in Examples 1 to 4 or Comparative Examples 1 to 4;

[0065] Figure 4 This is a comparison chart of cell survival rates after cells were treated with the products obtained in Examples 1 to 4 or Comparative Examples 1 to 4. DETAILED DESCRIPTION

[0066] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0068] The fermented mucus lactobacillus (Limosilactobacillus fermentum) in the following examples and comparative examples was purchased from the China Industrial Microbiological Culture Collection Center with a collection number of CICC 24824.

[0069] The Lactobacillus helveticus in the following examples and comparative examples was purchased from the China Industrial Microbiological Culture Collection Center with a collection number of CICC 20997.

[0070] The rice wine yeast in the following comparative example was purchased from Beijing Food Brewing Research Institute, and the strain number is AS2.1392.

[0071] Example 1

[0072] Weigh 1.5 g of sodium hyaluronate with a molecular weight of 1.2 million and 1.5 g of ectoine, mix them evenly in 300 mL of deionized water, and then sterilize them in an autoclave at a temperature of 121° C. and a pressure of 0.12 MPa for 20 min. After sterilization, cool them to room temperature to obtain a fermentation substrate.

[0073] The preparation method of the fermented mucus lactobacillus liquid comprises: inoculating the fermented mucus lactobacillus into an MRS medium and culturing it at 37° C. for 24 hours, wherein the number of viable bacteria in the fermented mucus lactobacillus liquid is 10 10 CFU / mL.

[0074] The preparation method of the Lactobacillus helveticus liquid comprises: inoculating Lactobacillus helveticus in an MRS medium and culturing at 37° C. for 24 hours, wherein the number of viable bacteria in the Lactobacillus helveticus liquid is 10 10 CFU / mL.

[0075] 1.5 mL of fermented lactobacillus liquid and 1.5 mL of helvetic lactobacillus liquid were inoculated into the fermentation substrate prepared above, and the fermentation culture was allowed to stand in a constant temperature incubator at a temperature of 37°C for 15 hours. After the fermentation culture was completed, the mixture was sterilized in an autoclave at a temperature of 121°C and a pressure of 0.12 MPa for 20 minutes, cooled to room temperature after sterilization, and then centrifuged at a speed of 4800 r / min, a time of 30 minutes, and a radius of 15 cm. After the centrifugation was completed, the mixture was sterilized in an autoclave at a temperature of 121°C and a pressure of 0.12 MPa for 20 minutes for secondary sterilization. After the secondary sterilization was completed, p-hydroxyacetophenone and 1,2-hexanediol were added to the supernatant at 70°C, wherein the mass percentage of p-hydroxyacetophenone in the supernatant was 0.5% and the mass percentage of 1,2-hexanediol in the supernatant was 0.5%, and the sodium hyaluronate edodene fermentation product was obtained.

[0076] Example 2

[0077] Compared with Example 1, the only difference is that 1 mL of fermented Lactobacillus liquid and 2 mL of Lactobacillus helveticus liquid were accessed, and other condition parameters were the same as those in Example 1. The preparation method is as follows:

[0078] Weigh 1.5 g of sodium hyaluronate with a molecular weight of 1.2 million and 1.5 g of ectoine, mix them evenly in 300 mL of deionized water, and then sterilize them in an autoclave at a temperature of 121° C. and a pressure of 0.12 MPa for 20 min. After sterilization, cool them to room temperature to obtain a fermentation substrate.

[0079] The preparation method of the fermented mucus lactobacillus liquid comprises: inoculating the fermented mucus lactobacillus into an MRS medium and culturing it at 37° C. for 24 hours, wherein the number of viable bacteria in the fermented mucus lactobacillus liquid is 10 10 CFU / mL.

[0080] The preparation method of the Lactobacillus helveticus liquid comprises: inoculating Lactobacillus helveticus in an MRS medium and culturing at 37° C. for 24 hours, wherein the number of viable bacteria in the Lactobacillus helveticus liquid is 10 10 CFU / mL.

[0081] 1 mL of fermented lactobacillus liquid and 2 mL of helvetic lactobacillus liquid were inoculated into the fermentation substrate prepared above, and the fermentation culture was allowed to stand in a constant temperature incubator at a temperature of 37°C for 15 hours. After the fermentation culture was completed, the mixture was sterilized in an autoclave at a temperature of 121°C and a pressure of 0.12 MPa for 20 minutes, cooled to room temperature after sterilization, and then centrifuged at a speed of 4800 r / min, a time of 30 minutes, and a radius of 15 cm. After the centrifugation was completed, secondary sterilization was performed at a temperature of 121°C and a pressure of 0.12 MPa for 20 minutes in an autoclave, and p-hydroxyacetophenone and 1,2-hexanediol were added to the supernatant at 70°C, wherein the mass percentage of p-hydroxyacetophenone in the supernatant was 0.5%, and the mass percentage of 1,2-hexanediol in the supernatant was 0.5%, and the sodium hyaluronate ectoin fermentation product was obtained.

[0082] Example 3

[0083] Compared with Example 1, the only difference is that the fermentation time is 12 hours, and other condition parameters are the same as Example 1.

[0084] Example 4

[0085] Compared with Example 1, the only difference is that 1 mL of Lactobacillus helveticus liquid and 2 mL of Lactobacillus fermentation liquid were inoculated, and other condition parameters were the same as those in Example 1.

[0086] Comparative Example 1

[0087] Compared with Example 1, the only difference is that no inoculation is performed, and other condition parameters are the same as Example 1. The specific preparation method is as follows:

[0088] Weigh 1.5 g of sodium hyaluronate with a molecular weight of 1.2 million and 1.5 g of ectoine, mix them evenly in 300 mL of deionized water, and then sterilize them in an autoclave at a temperature of 121°C and a pressure of 0.12 MPa for 20 minutes. After sterilization, cool to room temperature.

[0089] Comparative Example 2

[0090] Compared with Example 1, the only difference is that 3 mL of rice wine yeast liquid is added, and other condition parameters are the same as Example 1.

[0091] The preparation method of the rice wine yeast liquid is as follows: inoculate the rice wine yeast into the YPD culture medium, the culture temperature is 28°C, the culture time is 48h, and the shaking speed is 180r / min.

[0092] Comparative Example 3

[0093] Compared with Example 1, the only difference is that the fermentation time is 72 hours, and other condition parameters are the same as Example 1.

[0094] Comparative Example 4

[0095] Compared with Example 1, the only difference is that 2.8 g of sodium hyaluronate and 0.2 g of ectoine are contained in the fermentation substrate, and other condition parameters are the same as Example 1.

[0096] Effect Example 1

[0097] DPPH free radical scavenging experiment

[0098] DPPH is an early synthesized organic free radical, which is often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, purple, and has a characteristic absorption peak at 517nm. When encountering free radical scavengers, the lone pair of electrons of DPPH is paired and it fades, that is, the absorbance at the maximum absorption wavelength becomes smaller. Therefore, the scavenging effect of the sample on DPPH free radicals can be evaluated by measuring the change in absorbance.

[0099] The specific experimental steps of the DPPH free radical scavenging experiment are as follows: (First, dilute the sample 10 times)

[0100] (1) Take an equal volume (1 mL) of the test solution and 2 × 10 -4 mol / L DPPH solution and mix well (tube A1);

[0101] (2) Take an equal volume (1 mL) of anhydrous ethanol (solvent for the test substance) and 2×10 -4 mol / L DPPH solution and mix well (tube A2);

[0102] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (tube A3);

[0103] (4) After 30 minutes of reaction in the dark, measure the absorbance of tubes A1, A2, and A3 at 517 nm. The clearance rate is calculated as follows: clearance rate = [(A2 + A3) - A1] / A2 × 100%.

[0104] This experiment tested the DPPH free radical scavenging experiment on the embodiments and comparative examples. The results are shown in Tables 1 and Figure 1 .

[0105] Table 1

[0106] serial number DPPH free radical scavenging rate (%) Example 1 56.47±2.13 Example 2 54.08±1.13 Example 3 53.96±1.45 Example 4 51.08±1.14 Comparative Example 1 35.92±0.88 Comparative Example 2 38.86±2.27 Comparative Example 3 42.63±1.4 Comparative Example 4 40.16±1.63

[0107] The results show that the DPPH radical scavenging rates of the products prepared in Examples 1 to 4 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation time, fermentation strains, and fermentation substrates have a great influence on the antioxidant properties of the final products.

[0108] Effect Example 2

[0109] Hydroxyl free radical scavenging assay

[0110] The Fenton reaction is used to generate hydroxyl radicals (·OH), and salicylic acid is added to the reaction system. ·OH reacts with salicylic acid to generate a colored compound 2,3-dihydroxybenzoic acid with characteristic absorption at 510nm. The fixed reaction time method is used to measure the absorbance of the reaction solution containing the analyte at 510nm, and compared with the blank solution to determine the scavenging effect of the analyte on ·OH.

[0111] The liquid to be tested is the product obtained in the above-mentioned embodiment and comparative example (the product is diluted ten times);

[0112] Add reagents to the test tube according to Table 1, and add 9mmol / L FeSO4, 9mmol / L salicylic acid ethanol solution, test solution, appropriate amount of deionized water and 8.8mmol / L H2O2 solution in sequence. Shake well, heat in a 37℃ water bath for 15 minutes, then take out and measure the absorbance A0 of the blank control and the absorbance A0 of the sample group. x and the background absorbance of the solution without H2O2 x0 When determining A0, the reference solution was a system without hydrogen peroxide. Three parallel experiments were performed in each group, and the absorbance values ​​were tested and the average values ​​were taken. The results are shown in Table 2. The hydroxyl radical scavenging rate of each group was calculated according to the following formula. The results are shown in Tables 3 and Figure 2 .

[0113] Hydroxyl radical scavenging rate = (A0-(A x -A x0 )) / A0×100%.

[0114] Table 2

[0115]

[0116] Table 3

[0117] serial number Hydroxyl free radical scavenging rate (%) Example 1 71.22±0.95 Example 2 67.29±1.77 Example 3 67.18±2.1 Example 4 66.1±2.33 Comparative Example 1 40.63±1.29 Comparative Example 2 45.96±2.13 Comparative Example 3 49.95±1.59 Comparative Example 4 46.53±2.18

[0118] The results show that the hydroxyl radical scavenging rates of the products prepared in Examples 1 to 4 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation culture time, fermentation strains, and fermentation substrates all have a great influence on the antioxidant properties of the final product.

[0119] Effect Example 3

[0120] Hyaluronidase inhibition assay

[0121] Hyaluronidase is one of the lysosomes that can decompose polysaccharides. It can hydrolyze potassium hyaluronate to generate β-N-acetylglucosamine. This substance condenses with acetylacetone under alkaline conditions to generate the chromogen 2-methyl-3-diacetylpyrrole derivative. The chromogen and Ehrlich reagent develop color in concentrated hydrochloric acid and ethanol. Hyaluronidase is strongly correlated with inflammation and allergy and is a participant in type I allergic reactions. Therefore, the hyaluronidase in vitro inhibition test can be used as a quick anti-allergic determination method.

[0122] Reagents: hyaluronidase, sodium hyaluronate, anhydrous ethanol, sodium hydroxide, anhydrous sodium carbonate, concentrated hydrochloric acid, p-dimethylaminobenzaldehyde, acetylacetone, glacial acetic acid, anhydrous calcium chloride.

[0123] Equipment: The manufacturer of Sunrise microplate reader is Tecan Trading Co., Ltd.; the digital constant temperature water bath is from the medical equipment factory of Shanghai Boxun Industrial Co., Ltd.

[0124] Take 0.1mL CaCl2 solution (0.25mmol / L) and 0.5mL hyaluronidase solution (100U / mL) and incubate in a water bath at 37℃ for 20min; add 0.5mL of the test solution and continue to incubate for 20min; then add 0.5mL sodium hyaluronate solution (0.5mg / mL), incubate in a water bath at 37℃ for 30min, then take out and place at room temperature for 5min; add 0.1mL NaOH solution (0.4 mol / L) and 0.5 mL of acetylacetone solution (3.5 mL of acetylacetone dissolved in 50 mL of 1.0 mol / L sodium carbonate solution) were heated in a boiling water bath for 15 min and immediately transferred to an ice water bath for cooling for 5 min; 1.0 mL of Ehrlich reagent (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol) was added dropwise and diluted with 3.0 mL of anhydrous ethanol, and placed at room temperature for 20 min for color development, and the absorbance value at a wavelength of 540 nm was measured by a spectrophotometer. The calculation formula for the inhibition rate of the test solution on hyaluronidase is as follows: ( Figure 3In the table, *p<0.05, there is a statistical difference compared with Example 1, **p<0.01, there is a significant statistical difference compared with Example 1, ***p<0.001, there is an extremely significant statistical difference compared with Example 1, and it is extremely significantly increased; #p<0.05, there is a statistical difference compared with Example 1, ###p<0.001, there is an extremely significant statistical difference compared with Example 1, and it is extremely significantly reduced). The results are shown in Figure 3 and Table 4.

[0125] Hyaluronidase inhibition rate = [(AB)-(CD)] / (AB)×100%

[0126] Where: A—absorbance value of control solution (acetic acid buffer solution is used instead of the test solution); B—absorbance value of control blank solution (acetic acid buffer solution is used instead of the test solution and hyaluronidase solution); C—absorbance value of the test solution; D—absorbance value of the blank solution of the test solution (acetic acid buffer solution is used instead of the hyaluronidase solution)

[0127] Table 4

[0128]

[0129]

[0130] The results show that the hyaluronidase inhibition rates of the products prepared in Examples 1 to 4 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal anti-allergic effects, and the fermentation culture time, fermentation strains, and fermentation substrates have a great influence on the anti-allergic properties of the final product.

[0131] Effect Example 4

[0132] External after-sun repair

[0133] 1. Experimental steps:

[0134] The products obtained in the above examples and comparative examples were respectively prepared with serum-free DMEM culture medium to form a 2% volume percentage of the experimental group test solution.

[0135] HaCaT cells were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 U / L penicillin, 100 mg / L streptomycin) in DMEM medium. The cells were grown in an incubator at 37°C and 5% CO2 saturated humidity. When the cell fusion reached more than 85%, the cells in the logarithmic growth phase were digested with 0.05% trypsin and the digestion reaction was terminated with serum-containing DMEM. The cell count was performed on a cell counting plate and the cell suspension concentration was adjusted to 7×10 4 / mL, the cell suspension was inoculated into a 96-well plate at a ratio of 100 μL per well and incubated in a 37°C, 5% CO2 incubator for 12 h. After incubation, the old culture medium was removed, the cells were washed twice with phosphate buffer, and then 100 μL of PBS solution was added. The model group and the experimental group were irradiated with UVB at a dose of 15 mJ / cm 2 , the negative control group was not irradiated; PBS was discarded, serum-free DMEM medium was added to the model group and the negative control group, 100 μL of the above-configured filtered and sterilized test solution of the experimental group was added to each well of the experimental group, and 3 replicates were made for each test solution; the blank control group had no cells and 100 μL of PBS was added. Then incubate in an incubator at 37°C and 5% CO2 for 24 hours. Then 10 μL of CCK-8 solution was added to each well, and incubated for another 3 hours. The absorbance value was measured at a wavelength of 450 nm, and the cell survival rate of each group was calculated. The results are shown in the table and figure ( Figure 4 In the table, p>0.05, ns means no statistical difference compared with the model group; *p<0.05 means there is a statistical difference compared with the model group; ***p<0.001 means there is an extremely significant statistical difference compared with the model group. ### p<0.001, indicating that there is a significant difference compared with the blank group). Figure 4 and Table 5.

[0136] The cell viability was calculated as follows:

[0137] Cell survival rate (%) = (A experimental group or model group - A blank control group) / (A negative control group - A blank control group) × 100%.

[0138] Table 5

[0139] serial number Cell survival rate (%) Blank Group 100±2.86 Model Group 51.65±1.94 Example 1 72.34±2.09 Example 2 71.05±1.54 Example 3 67.22±1.07 Example 4 69.81±1.51 Comparative Example 1 61.58±0.62 Comparative Example 2 62.39±0.95 Comparative Example 3 64.39±0.56 Comparative Example 4 63.22±0.4

[0140] The results show that the cell survival rates of the products prepared in Examples 1 to 4 are higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal anti-aging effects, and the fermentation culture time, fermentation strains, and fermentation substrates have a great influence on the cell proliferation effect of the final product.

[0141] Finally, it should be noted that in this application, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0142] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.

Claims

1. A method for preparing sodium hyaluronate ectoine fermentation product, characterized in that: The method comprises the following steps: inoculating lactic acid bacteria into a fermentation substrate, culturing by fermentation, and sterilizing; The fermentation substrate consists of sodium hyaluronate, ectoine and water; and the lactic acid bacteria consists of fermented mucus lactobacillus and helveticus lactobacillus.

2. The method for preparing the sodium hyaluronate ectoine fermentation product according to claim 1, characterized in that: The preparation method of the sodium hyaluronate ectoine fermentation product meets at least one of the following conditions: The mass ratio of the sodium hyaluronate to the water is 1:(50-1000), preferably 1:(100-500), and more preferably 1:200; The mass ratio of the ectoine to the water is 1:(50-1000), preferably 1:(100-500), and more preferably 1:200; The ratio of the number of live bacteria of the fermented mucus lactobacillus to that of the helveticus lactobacillus is 1:(0.2-5), preferably 1:

1.

3. The method for preparing the sodium hyaluronate ectoine fermentation product according to claim 1, characterized in that: The average relative molecular weight of the sodium hyaluronate is 100-130W; Preferably, the fermentation substrate further includes a sterilization operation before use; more preferably, the sterilization method is a high-temperature sterilization method; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110°C to 125°C, preferably 115°C to 121°C, for example, 121°C; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure is 0.1 to 0.14MPa, preferably 0.1 to 0.13MPa, for example, 0.13MPa; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 15 to 35min, preferably 15 to 25min, for example, 20min.

4. The method for preparing the sodium hyaluronate ectoine fermentation product according to claim 1, characterized in that: The preparation method of the sodium hyaluronate ectoine fermentation product meets at least one of the following conditions: The fermented mucus lactobacillus (Limosilactobacillus fermentum) was purchased from the China Industrial Microbiological Culture Collection Administration Center, and the collection number is CICC 24824; The Lactobacillus helveticus was purchased from the China Industrial Microbiological Culture Collection Center, with the collection number of CICC 20997. The lactic acid bacteria are added in the form of lactic acid bacteria liquid; Based on the water in the fermentation substrate, the number of viable lactic acid bacteria inoculated per unit mass of the water is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL; The fermented mucus lactobacillus is added in the form of a fermented mucus lactobacillus bacterial liquid, and the live bacteria concentration of the fermented mucus lactobacillus bacterial liquid is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL; The Lactobacillus helveticus is added in the form of a Lactobacillus helveticus bacterial liquid. Preferably, the live bacteria concentration of the Lactobacillus helveticus bacterial liquid is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL; The fermentation culture temperature is 30-45°C, preferably 35-40°C, for example 37°C; The fermentation culture time is 12 to 48 hours, preferably 12 to 24 hours, for example 15 hours; The sterilization method is high-temperature sterilization; the sterilization temperature is 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C; the sterilization pressure is 0.1 to 0.14MPa, more preferably 0.1 to 0.13MPa, for example 0.13MPa; the sterilization time is 15 to 35min, more preferably 15 to 25min, for example 20min.

5. The method for preparing the sodium hyaluronate ectoine fermentation product according to claim 1, characterized in that: After the sterilization, the method further includes at least one of cooling, centrifuging and collecting the supernatant, secondary sterilization or mixing with a preservative.

6. The method for preparing the sodium hyaluronate ectoine fermentation product according to claim 5, characterized in that: The preparation method of the sodium hyaluronate ectoine fermentation product meets at least one of the following conditions: The cooling is cooling to room temperature; The centrifugal speed is 3000-9000 rpm, more preferably 4000-6000 rpm, for example 5000 rpm; The centrifugal radius is 8 to 15 cm; The centrifugation time is 10 to 40 minutes, preferably 20 to 40 minutes, for example 30 minutes; The secondary sterilization method is a high temperature sterilization method. Preferably, the temperature of the secondary sterilization is 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C; the pressure of the secondary sterilization is 0.1 to 0.14MPa, more preferably 0.1 to 0.13MPa, for example 0.13MPa; the time of the secondary sterilization is 15 to 35min, more preferably 15 to 25min, for example 20min; During the mixing process with the preservative, the mixing temperature is 50-80°C, more preferably 70-80°C; In the process of mixing with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant of the material obtained after sterilization is 0.5% to 2%, preferably 0.1% to 0.6%, and more preferably 0.5%; when the preservative includes 1,2-hexanediol, the mass percentage of 1,2-hexanediol in the supernatant of the material obtained after sterilization is 0.5% to 2%, preferably 0.5% to 1%, and more preferably 0.5%.

7. A sodium hyaluronate ectoine fermentation product, characterized in that: The invention discloses a method for preparing the fermented product of sodium hyaluronate and ectoine according to any one of claims 1 to 5.

8. A sodium hyaluronate ectoine fermentation product, used directly as a product, as an additive or as a base in the preparation of a skin topical preparation; Preferably, the sodium hyaluronate ectoine fermentation product is used as at least one of the antioxidant active ingredient, anti-aging active ingredient, and anti-allergic active ingredient in the skin topical preparation; More preferably, the antioxidant active ingredient is an antioxidant active ingredient having a DPPH free radical scavenging effect or an antioxidant active ingredient having a hydroxyl free radical scavenging effect; More preferably, the anti-aging active ingredient is an anti-aging active ingredient having a cell proliferation effect; More preferably, the anti-allergic active ingredient is an anti-allergic active ingredient having the effect of inhibiting hyaluronidase.

9. A skin external preparation, characterized in that: Including the sodium hyaluronate ectoine fermentation product as described in claim 7.

10. The external skin preparation according to claim 9, wherein The skin external preparation satisfies at least one of the following conditions: The skin external preparation further comprises at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient and an antioxidant active ingredient; The skin external preparation includes a facial mask, essence or toner; The mass percentage of the sodium hyaluronate ectoine fermentation product in the skin external preparation is 5% to 99%, preferably 60% to 99%.

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