Preparation method and application of hyaluronic acid and salt thereof

Abstract

The invention relates to hyaluronic acid and a preparation method and application of a salt thereof. The preparation method of the hyaluronic acid comprises the following steps: adding glucose and the salt into an activation solution of a composite strain for fermentation, adjusting the pH value of a fermentation solution to 10-11, adding ethanol for precipitation, and filtering to obtain the hyaluronic acid, the composite strain comprises a combination of bifidobacterium longum, corynebacterium glutamicum, bacillus subtilis and lactococcus lactis. Composite strains are adopted for fermentation, all the strains are reasonably compounded, the yield of hyaluronic acid is jointly increased, and the obtained product is high in purity, low in molecular weight and beneficial to skin permeation and absorption, so that the active effects of improving wrinkles, delaying senescence, increasing elasticity and the like are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic raw materials, and in particular to a preparation method and application of hyaluronic acid and its salt. Background Art

[0002] Hyaluronic acid, also known as hyaluronic acid, has a molecular formula of (C 14 H 21 NO 11 )n, is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine, referred to as HA. Hyaluronic acid salts such as sodium hyaluronate and potassium hyaluronate are widely used in different fields, especially in health care, food manufacturing and cosmetics.

[0003] Hyaluronic acid and hyaluronate are both moisturizing ingredients in the cosmetics industry, but they have the following differences: 1. Hyaluronic acid exists in the form of acid, while hyaluronate exists in the form of salt; 2. Hyaluronic acid salts, such as sodium hyaluronate, are more stable, and the addition of sodium will turn them into non-Newtonian fluids, which are very good for exfoliation, while hyaluronic acid is not good for exfoliation; 3. The hyaluronic acid on the market is a long-chain macromolecule that cannot penetrate the skin to reach the dermis, so hyaluronic acid cannot achieve the effect of wrinkle removal and plumping. Through special extraction methods, sodium hyaluronate can penetrate into the dermis as a small molecule of hyaluronic acid.

[0004] Ordinary hyaluronic acid has a large molecular weight and high production cost, which affects the skin absorption effect and its application is also subject to certain restrictions. Therefore, a new preparation method of hyaluronic acid and its salt is developed to improve the yield and purity, and at the same time has better water solubility, absorbability, wrinkle removal and exfoliation effects, and has broad application prospects. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a preparation method and application of hyaluronic acid and its salts. In the preparation method of hyaluronic acid provided by the present invention, composite strains are used for fermentation, and the strains are reasonably compounded to jointly increase the yield of hyaluronic acid, and the obtained product has high purity and low molecular weight, which is conducive to skin penetration and absorption, thereby improving its active effects such as improving wrinkles, delaying aging, and increasing elasticity.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing hyaluronic acid, the preparation method comprising the following steps:

[0008] Glucose and salt are added to the activated liquid of the composite bacteria for fermentation, the pH of the fermentation liquid is adjusted to 10-11 (for example, it can be 10, 10.2, 10.4, 10.6, 10.8, 11, etc.), ethanol is added for precipitation, and hyaluronic acid is obtained after filtration; the composite bacteria include a combination of Bifidobacterium longum, Corynebacterium glutamicum, Bacillus subtilis and Lactococcus lactis.

[0009] The present invention adopts composite bacterial strains for fermentation, and the various bacterial strains are reasonably compounded and synergistically enhanced to jointly increase the yield of hyaluronic acid. The obtained product has high purity and low molecular weight, which is beneficial to skin penetration and absorption, thereby improving its active effects such as improving wrinkles, delaying aging, and increasing elasticity.

[0010] Preferably, the ratio of the number of live bacteria of Bifidobacterium longum, Corynebacterium glutamicum, Bacillus subtilis and Lactococcus lactis in the composite bacteria is 1:(0.5-1.5):(0.5-1.5):(0.5-1.5).

[0011] The numerical value in the above (0.5-1.5) can be, for example, 0.5, 0.8, 1, 1.2, 1.5, etc.

[0012] Preferably, during the fermentation, the amount of glucose added is 47-53% (for example, 47%, 48%, 49%, 50%, 51%, 52%, 53%, etc.) of the mass of the activation solution.

[0013] Preferably, during the fermentation, the amount of salt added is 0.8-1.8% (eg, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, etc.) of the mass of the activation solution.

[0014] Preferably, in the fermentation, the salt comprises any one of sodium carbonate, potassium chloride, potassium carbonate, or sodium bicarbonate, or a combination of two thereof.

[0015] Preferably, the fermentation temperature is 35-37°C (for example, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, etc.), and the fermentation time is 18-26h (for example, 18h, 20h, 22h, 24h, 26h, etc.).

[0016] Preferably, the fermentation further includes a sterilization operation.

[0017] Preferably, the sterilization temperature is 85-90°C (for example, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, etc.), and the time is 25-35min (for example, 25min, 28min, 30min, 32min, 35min, etc.).

[0018] Preferably, the pore size of the filter membrane used for the filtration is 0.02-0.2 μm (for example, it can be 0.02 μm, 0.05 μm, 0.1 μm, 0.15 μm, 0.2 μm, etc.).

[0019] Preferably, the glucose is prepared by the following method, which comprises the following steps:

[0020] After squeezing the sugarcane juice, acid hydrolysis and enzymolysis are performed in sequence, the enzymolysis solution is filtered to obtain the filtrate, and the pH value is adjusted to 6.5-7.5 (for example, it can be 6.5, 6.8, 7, 7.2, 7.5, etc.), and after extraction and concentration, sterilization and activated carbon filtration, the glucose is cooled and crystallized to obtain glucose; the complex enzyme used in the enzymolysis includes glucoamylase and sucrase.

[0021] In the present invention, the preparation method of glucose extracted from sugarcane has high yield and purity, and reduces the use cost of glucose; the composite enzyme of glucoamylase and sucrase is used in the enzymatic hydrolysis process to convert starch and sugar substances in sugarcane juice into glucose, and the two enzymes have a synergistic effect, which jointly improves the efficiency of enzymatic hydrolysis.

[0022] Preferably, the acid used in the acid hydrolysis comprises dilute hydrochloric acid.

[0023] Preferably, the mass fraction of the dilute hydrochloric acid is 1.5-2.5% (for example, it can be 1.5%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.5%, etc.), and the mass ratio of the dilute hydrochloric acid to the sugarcane juice is 1:(8-12) (for example, it can be 1:8, 1:9, 1:10, 1:11, 1:12, etc.).

[0024] Preferably, the acid hydrolysis temperature is 80-90°C (for example, it can be 80°C, 82°C, 84°C, 86°C, 88°C, 90°C, etc.), the pressure is 0.6-1MPa (for example, it can be 0.6MPa, 0.7MPa, 0.8MPa, 0.MPa, 1MPa, etc.), and the time is 2-2.5h (for example, it can be 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, etc.).

[0025] Preferably, in the enzymatic hydrolysis, the amount of the complex enzyme used is 3-5% (eg, 3%, 3.5%, 4%, 4.5%, 5%, etc.) of the substrate mass.

[0026] Preferably, the mass ratio of glucoamylase to sucrase is 1:(0.5-1.5) (for example, it can be 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5, etc.).

[0027] Preferably, the enzymatic hydrolysis temperature is 50-55°C (for example, it can be 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, etc.), and the time is 24-48h (for example, it can be 24h, 32h, 40h, 48h, etc.).

[0028] In a second aspect, the present invention provides a method for preparing a hyaluronate, the preparation method comprising the following steps:

[0029] (1) preparing hyaluronic acid using the method for preparing hyaluronic acid described in the first aspect;

[0030] (2) Mixing and dissolving hyaluronic acid, salt and water, and subjecting the mixture to a primary complexation precipitation to obtain a crude hyaluronate; mixing and dissolving the crude hyaluronate, salt and water, and subjecting the mixture to a secondary complexation precipitation to obtain a hyaluronate; the salt includes a sodium salt or a potassium salt.

[0031] The preparation method of the hyaluronate provided by the present invention is simple to operate and has low cost, improves the activity, solubility and transdermal absorption effect of hyaluronic acid, has excellent moisturizing effect, and can be widely used in cosmetics.

[0032] Preferably, in step (2), in the solution of hyaluronic acid and salt, the mass fraction of hyaluronic acid is 10-15% (for example, it can be 10%, 11%, 12%, 13%, 14%, 15%, etc.), and the mass fraction of salt is 40-45% (for example, it can be 40%, 41%, 42%, 43%, 44%, 45%, etc.).

[0033] Preferably, in the solution of the crude hyaluronate and the salt, the mass fraction of the crude hyaluronate is 10-15% (for example, 10%, 11%, 12%, 13%, 14%, 15%, etc.), and the mass fraction of the salt is 40-45% (for example, 40%, 41%, 42%, 43%, 44%, 45%, etc.).

[0034] Preferably, the sodium salt comprises sodium carbonate.

[0035] Preferably, the potassium salt comprises potassium chloride.

[0036] Preferably, the primary complexation and the secondary complexation are carried out under stirring conditions.

[0037] Preferably, the stirring rates are each independently 600-800 rpm (eg, 600 rpm, 650 rpm, 700 rpm, 750 rpm, 800 rpm, etc.).

[0038] Preferably, the temperatures of the primary complexation and the secondary complexation are independently 5-20°C (eg, 5°C, 10°C, 15°C, 20°C, etc.).

[0039] Preferably, after the secondary complex precipitation, the method further comprises: purifying by gel chromatography and spray drying to obtain hyaluronate.

[0040] In a third aspect, the present invention provides an application of the method for preparing hyaluronic acid according to the first aspect and the method for preparing a hyaluronate according to the second aspect in preparing cosmetics.

[0041] Compared with the prior art, the present invention has at least the following beneficial effects:

[0042] (1) In the preparation method of hyaluronic acid provided by the present invention, a composite bacterial strain is used for fermentation, and the various bacterial strains are rationally compounded and synergistically enhanced, thereby jointly increasing the yield of hyaluronic acid. The obtained product has high purity and low molecular weight, which is conducive to skin penetration and absorption, thereby improving its active effects such as improving wrinkles, delaying aging, and increasing elasticity.

[0043] (2) The preparation method of the hyaluronate provided by the present invention is simple to operate and has low cost, further improves the activity, solubility and transdermal absorption effect of hyaluronic acid, has excellent moisturizing effect, and can be widely used in cosmetics. DETAILED DESCRIPTION

[0044] For the convenience of understanding the present invention, the present invention lists the following embodiments. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0045] The sources of materials used in the following examples are as follows:

[0046] Glucoamylase was purchased from Ningxia Xiasheng Industry, model FDY-2223;

[0047] Sucrase was purchased from Zhengzhou Weifeng Biology, model: Sucrase;

[0048] Bifidobacterium longum was purchased from Dongchi Biotechnology with the strain number SHBCCD D24428;

[0049] Corynebacterium glutamicum was purchased from Shanghai Lianzu Biotechnology Co., Ltd., with the strain number ATCC14997;

[0050] Bacillus subtilis was purchased from Henan Zhengpintang, with the strain number CICC 10085;

[0051] Lactococcus lactis was purchased from Shaanxi Mufan Biology, with the strain number ATCC 15346;

[0052] Other raw materials are conventional commercially available products.

[0053] Preparation Example 1

[0054] This preparation example provides a method for preparing glucose, which comprises the following steps:

[0055] (1) After washing sugarcane, the juice was squeezed to obtain sugarcane juice, and 0.1 times (mass multiple) 2% (mass fraction) dilute hydrochloric acid was added, and acid hydrolysis was carried out at 85°C and 0.8MPa for 2.2 hours; then, a composite enzyme (glucoamylase and sucrase in a mass ratio of 1:1) of the substrate was added, and enzymolysis was carried out at 52°C for 36 hours to obtain an enzymolysis solution;

[0056] (2) Filter the enzymatic hydrolyzate, adjust the filtrate to neutrality with calcium hydroxide, and use an extraction concentrator for extraction purification, heating at 120°C for 7 minutes, static extraction for 2 minutes, and repeating twice; then sterilize at 80°C for 30 minutes; after sterilization, pass the filtrate through a plate and frame filter filled with activated carbon, control the temperature of the filtrate to 44°C, cool it to 30°C for crystallization after 50 hours, and obtain glucose after centrifugal separation.

[0057] Preparation Example 2

[0058] This preparation example provides a method for preparing glucose, which comprises the following steps:

[0059] (1) After washing sugarcane, the juice was squeezed to obtain sugarcane juice, and 0.08 times (mass multiple) of 2.5% (mass fraction) of dilute hydrochloric acid was added, and acid hydrolysis was carried out at 80°C and 0.6MPa for 2.5 hours; then, 3% of the substrate mass of the composite enzyme (glucoamylase and sucrase in a mass ratio of 1:1) was added, and enzymolysis was carried out at 50°C for 48 hours to obtain an enzymolysis solution;

[0060] (2) Filter the enzymatic hydrolyzate, adjust the filtrate to neutrality with calcium hydroxide, and use an extraction concentrator for extraction purification, heating at 120°C for 7 minutes, static extraction for 2 minutes, and repeating twice; then sterilize at 85°C for 30 minutes; after sterilization, pass the filtrate through a plate and frame filter filled with activated carbon, control the temperature of the filtrate to 46°C, cool it to 22°C for crystallization after 70 hours, and obtain glucose after centrifugal separation.

[0061] Preparation Example 3

[0062] This preparation example provides a method for preparing glucose, which comprises the following steps:

[0063] (1) Wash the sugarcane and squeeze the juice to obtain sugarcane juice, add 0.12 times (mass multiple) of 1.5% (mass fraction) dilute hydrochloric acid, and acid hydrolyze at 90°C and 1 MPa for 2 hours; then add 5% of the substrate mass of the composite enzyme (glucoamylase and sucrase in a mass ratio of 1:1), and enzymolyze at 55°C for 24 hours to obtain an enzymolysis solution;

[0064] (2) Filter the enzymatic hydrolyzate, adjust the filtrate to neutrality with calcium hydroxide, and use an extraction concentrator for extraction purification, heating at 120°C for 7 minutes, static extraction for 2 minutes, and repeating twice; then sterilize at 82°C for 30 minutes; after sterilization, pass the filtrate through a plate and frame filter filled with activated carbon, control the temperature of the filtrate to 45°C, cool it to 25°C for crystallization after 60 hours, and obtain glucose after centrifugal separation.

[0065] Preparation Example 4

[0066] This preparation example provides a method for preparing glucose, which differs from Preparation Example 1 only in that glucoamylase is not added in step (1), and its amount is allocated to sucrase. Other details are as in Preparation Example 1.

[0067] Preparation Example 5

[0068] This preparation example provides a method for preparing glucose, which differs from Preparation Example 1 only in that invertase is not added in step (1), and its amount is allocated to glucoamylase. For other matters, refer to Preparation Example 1.

[0069] Test Example 1

[0070] The yield and purity information of glucose in the preparation methods provided in Preparation Examples 1-5 are shown in Table 1.

[0071] Table 1

[0072]

[0073]

[0074] The test results show that the method for extracting glucose from sugarcane provided by the present invention has a high yield and purity, and significantly reduces the use cost of glucose. By comparing Preparation Example 1 with Preparation Examples 4-5, it can be seen that the present invention has a better enzymolysis effect by rationally compounding glucoamylase and sucrase, and further improves the yield of glucose.

[0075] Example 1

[0076] This example provides a method for preparing hyaluronic acid and its salts. The glucose used in this example comes from Preparation Example 1;

[0077] The preparation method of hyaluronic acid and its salt comprises the following steps:

[0078] (1) Inoculating a composite bacterial strain (Bifidobacterium longum, Corynebacterium glutamicum, Bacillus subtilis and Lactococcus lactis at a live bacterial count ratio of 1:1:1:1) into a liquid culture medium at an inoculum rate of 10%, stirring and activating the culture at 36°C for 14 hours to obtain a bacterial strain activation liquid; the components of the liquid culture medium include 15 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 5 g / L beef extract, and KH 2 PO 4 1.5g / L;

[0079] (2) adding 50% of the system weight of glucose and 1.5% of sodium carbonate to the activated bacterial solution, maintaining a constant temperature of 36° C., maintaining a ventilation rate of 0.4 vvm (ventilation rate per minute relative to unit fermentation volume), stirring and fermenting at 120 rpm for 24 h, and adjusting the pH of the fermentation liquid to maintain at 6.5-7 during the fermentation process;

[0080] (3) After the fermentation is completed, the temperature is raised to 86° C., sterilized at the constant temperature for 30 min, then the temperature is lowered to 33° C., the pH is adjusted to 10, 3 volumes of 95% ethanol are added for precipitation, and then filtered with a filter membrane to obtain hyaluronic acid;

[0081] (4) A 35% by mass aqueous solution of hyaluronic acid and a 65% by mass aqueous solution of sodium carbonate were mixed and dissolved in a mass ratio of 7:13, and a primary complexation was performed at 700 rpm and 15° C. to obtain a crude sodium hyaluronate; a 35% by mass aqueous solution of crude sodium hyaluronate and a 65% by mass aqueous solution of sodium carbonate were mixed and dissolved in a mass ratio of 7:13, and a secondary complexation was performed at 700 rpm and 15° C. The precipitate was liquid purified by gel chromatography (gel model: LH-20), and sodium hyaluronate was obtained after spray drying.

[0082] Example 2

[0083] This example provides a method for preparing hyaluronic acid and its salts. The glucose used in this example comes from Preparation Example 2;

[0084] The preparation method of hyaluronic acid and its salt comprises the following steps:

[0085] (1) Inoculating a composite bacterial strain (Bifidobacterium longum, Corynebacterium glutamicum, Bacillus subtilis and Lactococcus lactis with a live cell count ratio of 1:1.5:0.5:1.5) into a liquid culture medium at an inoculum size of 8%, stirring and activating the culture at 35°C for 16 hours to obtain a bacterial strain activation liquid; the components of the liquid culture medium include 15 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 5 g / L beef extract, and KH 2 PO 4 1.5g / L;

[0086] (2) adding 47% glucose and 0.8% potassium chloride by mass to the activated bacterial solution, maintaining a constant temperature of 35° C., maintaining a ventilation rate of 0.3 vvm (ventilation rate per minute relative to unit fermentation volume), stirring and fermenting at 120 rpm for 18 h, and adjusting the pH of the fermentation liquid to maintain at 6.5-7 during the fermentation process;

[0087] (3) After the fermentation is completed, the temperature is raised to 90° C., sterilized at the constant temperature for 30 min, then the temperature is lowered to 35° C., the pH is adjusted to 11, 3 volumes of 95% ethanol are added for precipitation, and then filtered with a filter membrane to obtain hyaluronic acid;

[0088] (4) A 35% by mass aqueous solution of hyaluronic acid and a 65% by mass aqueous solution of sodium carbonate were mixed and dissolved in a mass ratio of 7:16, and a primary complexation was performed at 600 rpm and 20° C. to obtain a crude sodium hyaluronate; a 35% by mass aqueous solution of crude sodium hyaluronate and a 65% by mass aqueous solution of sodium carbonate were mixed and dissolved in a mass ratio of 7:16, and a secondary complexation was performed at 600 rpm and 20° C. The precipitate was liquid purified by gel chromatography (gel model: LH-20), and sodium hyaluronate was obtained after spray drying.

[0089] Example 3

[0090] This example provides a method for preparing hyaluronic acid and its salts. The glucose used in this example comes from Preparation Example 3;

[0091] The preparation method of hyaluronic acid and its salt comprises the following steps:

[0092] (1) Inoculating a composite bacterial strain (Bifidobacterium longum, Corynebacterium glutamicum, Bacillus subtilis and Lactococcus lactis at a live cell count ratio of 1:0.5:1.5:0.5) into a liquid culture medium at an inoculum rate of 12%, stirring and activating the culture at 37°C for 12 hours to obtain a bacterial strain activation liquid; the components of the liquid culture medium include 15 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 5 g / L beef extract, and KH 2 PO 4 1.5g / L;

[0093] (2) adding 53% of the system weight of glucose and 1.8% of sodium carbonate to the activated bacterial solution, maintaining a constant temperature of 37° C., maintaining a ventilation volume of 0.5 vvm (ventilation volume per minute relative to unit fermentation volume), stirring and fermenting at 120 rpm for 26 h, and adjusting the pH of the fermentation liquid to maintain at 6.5-7 during the fermentation process;

[0094] (3) After the fermentation is completed, the temperature is raised to 86° C., sterilized at the constant temperature for 30 min, then the temperature is lowered to 34° C., the pH is adjusted to 10, 3 volumes of 95% ethanol are added for precipitation, and then filtered with a filter membrane to obtain hyaluronic acid;

[0095] (4) A 35% hyaluronic acid aqueous solution and a 65% potassium chloride aqueous solution were mixed and dissolved in a mass ratio of 8:13, and a primary complexation was performed at 800 rpm and 5° C. to obtain a crude potassium hyaluronate; a 35% crude potassium hyaluronate aqueous solution and a 65% potassium carbonate aqueous solution were mixed and dissolved in a mass ratio of 7:13, and a secondary complexation was performed at 800 rpm and 5° C. The precipitate was liquid purified by gel chromatography (gel model: LH-20), and potassium hyaluronate was obtained after spray drying.

[0096] Comparative Example 1

[0097] This comparative example provides a method for preparing hyaluronic acid, which is different from Example 1 only in that the composite bacteria do not contain Bifidobacterium longum, and the number of its live bacteria is distributed according to the proportion of the remaining bacteria. The rest is referred to Example 1.

[0098] Comparative Example 2

[0099] This comparative example provides a method for preparing hyaluronic acid, which is different from Example 1 only in that the composite bacteria do not contain Corynebacterium glutamicum, and the number of its live bacteria is distributed according to the proportion of the remaining bacteria. The rest is referred to Example 1.

[0100] Comparative Example 3

[0101] This comparative example provides a method for preparing hyaluronic acid, which is different from Example 1 only in that the composite bacteria does not contain Bacillus subtilis, and the number of its live bacteria is distributed according to the proportion of the remaining bacteria. The rest is referred to Example 1.

[0102] Comparative Example 4

[0103] This comparative example provides a method for preparing hyaluronic acid, which is different from Example 1 only in that the composite bacteria do not contain Lactococcus lactis, and the number of live bacteria thereof is distributed according to the proportion of the remaining bacteria. The rest is referred to Example 1.

[0104] Comparative Example 5

[0105] This comparative example provides a method for preparing hyaluronic acid, which differs from Example 1 only in that sodium carbonate is not added in step (2). The rest is the same as Example 1.

[0106] Test Example 2

[0107] The quality information of hyaluronic acid and its salts obtained by the preparation methods provided in Examples 1-3 and Comparative Examples 1-5 is shown in Table 2.

[0108] Table 2

[0109]

[0110]

[0111] The test results show that:

[0112] (1) It can be seen from Examples 1-3 that the present invention uses composite bacterial strains for fermentation, and the various bacterial strains are reasonably compounded to jointly increase the yield of hyaluronic acid, and the resulting product has high purity and low molecular weight, which is beneficial to skin penetration and absorption; at the same time, the preparation method of hyaluronate provided by the present invention is simple to operate and has a high yield and purity.

[0113] (2) By comparing Example 1 with Comparative Examples 1-4, it can be seen that the composite bacterial species used in the present invention have a synergistic technical effect. When any one of them is missing, the yield of hyaluronic acid is reduced and the molecular weight is increased.

[0114] By comparing Example 1 with Comparative Example 5, it can be seen that the present invention adds specific salts during the fermentation process, which can adjust the osmotic pressure and pH, which is beneficial to maintaining the stability of the fermentation environment and increasing the yield of hyaluronic acid.

[0115] Test Example 3

[0116] Moisture test

[0117] Test Method

[0118] (1) Place color-changing silica gel at the bottom of the desiccator, place a glass plate with 3M breathable adhesive in the desiccator one day in advance, and weigh its weight M. 0 ;

[0119] (2) According to the size of the tape, 2mg±0.1mg / cm 2 Weigh the corresponding samples and reference substances, spread them evenly on the tape, and weigh their weight M 1 ;

[0120] (3) Place the glass plate coated with the test sample in a desiccator and accurately weigh its mass M after leaving it for 30 min, 1 h, 2 h, and 4 h;

[0121] (4) Calculate the moisturizing rate and relative moisturizing rate, analyze the data, and draw the moisturizing rate curve.

[0122] The sample groups used the hyaluronic acid and sodium hyaluronate provided in Example 1 and the potassium hyaluronate provided in Example 3 respectively; the positive control group used glycerol; and the blank control group used deionized water.

[0123] Standard judgment:

[0124] Below 0-50% is low moisturizing performance, 50-100% is medium moisturizing performance, 100-150% is high moisturizing performance, and above 150% is good moisturizing performance.

[0125] The test results of hyaluronic acid and sodium hyaluronate prepared in Example 1 are shown in Table 3.

[0126] Table 3

[0127]

[0128]

[0129] The test results show that the hyaluronic acid, sodium hyaluronate and sodium hyaluronate prepared by the preparation method provided by the present invention have moisturizing effects. The moisturizing rates of the test samples hyaluronic acid, sodium hyaluronate and potassium hyaluronate at each test time point (30min / 1h / 2h / 4h) are higher than those of the blank control and the positive. The relative moisturizing rates of the test samples at the test time points (30min / 1h / 2h / 4h) are all above 150%, with good moisturizing performance; and there are significant differences at the test points of 30min / 1h / 2h / 4h (p<0.05).

[0130] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing hyaluronic acid, characterized in that: The preparation method comprises the following steps: Glucose and salt are added to the activated liquid of the composite bacteria for fermentation, the pH of the fermentation liquid is adjusted to 10-11, ethanol is added for precipitation, and hyaluronic acid is obtained after filtration; the composite bacteria include a combination of Bifidobacterium longum, Corynebacterium glutamicum, Bacillus subtilis and Lactococcus lactis.

2. The preparation method according to claim 1, characterized in that: The ratio of the number of live bacteria of Bifidobacterium longum, Corynebacterium glutamicum, Bacillus subtilis and Lactococcus lactis in the composite bacteria is 1:(0.5-1.5):(0.5-1.5):(0.5-1.5).

3. The preparation method according to claim 1 or 2, characterized in that: In the fermentation, the amount of glucose added is 47-53% of the mass of the activation solution; Preferably, during the fermentation, the amount of salt added is 0.8-1.8% of the mass of the activation solution; Preferably, in the fermentation, the salt comprises any one or a combination of at least two of sodium carbonate, potassium chloride, potassium carbonate or sodium bicarbonate; Preferably, the fermentation temperature is 35-37°C and the fermentation time is 18-26h; Preferably, the fermentation further includes a sterilization operation; Preferably, the pore size of the filter membrane used for the filtration is 0.02-0.2 μm.

4. The preparation method according to any one of claims 1 to 3, characterized in that The glucose is prepared by the following method, which comprises the following steps: After squeezing the sugarcane juice, acid hydrolysis and enzymolysis are performed in sequence, the enzymolysis solution is filtered to obtain the filtrate, the pH value is adjusted to 6.5-7.5, and after extraction and concentration, sterilization and activated carbon filtration, the glucose is cooled and crystallized to obtain glucose; the complex enzyme used in the enzymolysis includes glucoamylase and sucrase.

5. The preparation method according to claim 4, characterized in that: The acid used in the acidolysis includes dilute hydrochloric acid; Preferably, the mass fraction of the dilute hydrochloric acid is 1.5-2.5%, and the mass ratio of the dilute hydrochloric acid to the sugarcane juice is 1:(8-12); Preferably, the acid hydrolysis temperature is 80-90°C, the pressure is 0.6-1 MPa, and the time is 2-2.5 hours.

6. The preparation method according to claim 4 or 5, characterized in that: In the enzymatic hydrolysis, the dosage of the complex enzyme is 3-5% of the substrate mass; Preferably, the mass ratio of glucoamylase to sucrase is 1:(0.5-1.5); Preferably, the enzymatic hydrolysis temperature is 50-55°C and the time is 24-48h.

7. A method for preparing a hyaluronate, characterized in that: The preparation method comprises the following steps: (1) preparing hyaluronic acid by the method for preparing hyaluronic acid according to any one of claims 1 to 6; (2) Mixing and dissolving hyaluronic acid, salt and water, and subjecting the mixture to a primary complexation precipitation to obtain a crude hyaluronate; mixing and dissolving the crude hyaluronate, salt and water, and subjecting the mixture to a secondary complexation precipitation to obtain a hyaluronate; the salt includes a sodium salt or a potassium salt.

8. The preparation method according to claim 7, characterized in that: In step (2), in the solution of hyaluronic acid and salt, the mass fraction of hyaluronic acid is 10-15%, and the mass fraction of salt is 40-45%; Preferably, in the solution of the crude hyaluronate and the salt, the mass fraction of the crude hyaluronate is 10-15%, and the mass fraction of the salt is 40-45%; Preferably, the sodium salt comprises sodium carbonate; Preferably, the potassium salt comprises potassium chloride.

9. The preparation method according to claim 7 or 8, characterized in that: In step (2), the primary complexation and the secondary complexation are carried out under stirring; Preferably, the stirring rate is independently 600-800 rpm; Preferably, the temperature of the primary complexation and the secondary complexation is independently 5-20°C; Preferably, after the secondary complex precipitation, the method further comprises: purifying by gel chromatography and spray drying to obtain hyaluronate.

10. Use of the method for preparing hyaluronic acid according to any one of claims 1 to 6 or the method for preparing hyaluronate according to any one of claims 7 to 9 in preparing cosmetics.

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