Hyaluronic acid and preparation method thereof

Abstract

The invention provides hyaluronic acid and a preparation method thereof, and belongs to the technical field of microorganisms. According to the invention, hyaluronic acid is generated by rhizobium pustatae QY124aN, so that the problems of high strain culture cost in the prior art and no technical method for producing medium-molecular-weight hyaluronic acid from rhizobium pustatae are solved. The content of hyaluronic acid in the hyaluronic acid dry powder prepared by the method is 57.68%, and the hyaluronic acid dry powder is safer than hyaluronic acid prepared by streptococcus zooepidemicus in the prior art.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a method for preparing hyaluronic acid. Background Art

[0002] Hyaluronic acid (HA), as a natural linear glycosaminoglycan, is mainly composed of repeating units of β-(1,4)-glucuronic acid (GA) and β-(1,3)-acetylglucosamine (NAG), which are linked together by alternating β-1,3 and β-1,4 glycosidic bonds. Sodium hyaluronate is the sodium salt form of hyaluronic acid. According to the molecular weight, HA is divided into three forms: low molecular weight HA (6-200kDa), medium molecular weight HA (0.2-1.0MDa) and high molecular weight HA (>1MDa). The biological activity of HA is closely related to its molecular weight. For example, adhesion and moisturizing are characteristics of high molecular weight HA. Medium-sized hyaluronic acid promotes wound repair. Low molecular weight HA has good permeability, promotes angiogenesis and resists cell apoptosis. In summary, studies have shown that HA has properties such as non-toxicity and non-immunogenicity. HA can be used in the research and development of products such as moisturizers, injections, oral preparations, and bioactive drug delivery systems. It has been well applied in the correction of facial folds and wrinkles, surgical wound regeneration, and drug delivery through various routes of administration. It has become an extremely attractive biomaterial for industrial applications such as cosmetics, pharmaceuticals, and medical devices. In January 2021, the National Health Commission approved sodium hyaluronate as a new food raw material that can be used as an additive in ordinary foods. The application field of HA has been further expanded, and the market demand for HA will increase significantly.

[0003] HA is usually extracted from animal tissues (e.g., bovine vitreous, cockscomb, and synovial fluid, etc.) or fermentation broth of microorganisms (e.g., Streptococcus zooepidemicus, Bacillus subtilis, and Escherichia coli). Traditionally, HA is produced by extracting it from animal sources. The extraction process is difficult to control, the extraction cost is high, and the yield is low due to degradation by hyaluronidase, which affects the quality of HA. In particular, the presence of other substances such as host proteins and DNA in HA from animal sources may induce immune reactions and allergies. In view of the above problems, the currently developed method for industrial production of HA is to use microbial fermentation. The widely used production strain is Streptococcus equi subsp. zooepidemicus, which produces high molecular weight HA.

[0004] However, there are still some problems in the production process of Streptococcus equi subspecies zooepidemicus, such as slow bacterial growth, substrate competition between bacterial growth and hyaluronic acid synthesis; high nutritional requirements, the need to add yeast powder, peptone and amino acids to the culture medium, resulting in increased production costs; high viscosity of the fermentation broth, and the accumulation of fermentation by-product lactic acid leads to inhibition of cell growth and hyaluronic acid production, etc., especially for Streptococcus zooepidemicus, there is a risk of bacterial endotoxins, which limits its application in biomedicine.

[0005] In view of the above reasons, the development of new and safe HA production systems remains one of the focuses of industry attention. Finding new hyaluronic acid production strains is an effective way to reduce production costs and expand application areas to meet the market's growing demand for high-quality HA products. It also provides a new way to prepare HA of different molecular weights. Summary of the invention

[0006] The hyaluronic acid produced by Rhizobium pusa QY12 4aN solves the problem that the strain culture cost in the prior art is high, and solves the problem that there is no technical method for producing medium molecular weight hyaluronic acid by Rhizobium pusa.

[0007] A strain of Rhizobium pusa, wherein the Rhizobium pusa QY12 4aN is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.29921 and a deposit date of March 1, 2024.

[0008] A method for preparing hyaluronic acid, wherein the hyaluronic acid is prepared by using Rhizobium pusa QY12 4aN.

[0009] The method is:

[0010] Step 1: Resuscitate Rhizobium pusa QY12 4aN and inoculate into YPD liquid culture medium to obtain bacterial liquid;

[0011] Step 2: Centrifuge the bacterial solution to obtain a precipitate, wash the precipitate with saline and prepare a bacterial suspension;

[0012] Step 3: inoculating the bacterial suspension into a hyaluronic acid preparation medium for cultivation to obtain a fermentation liquid, and centrifuging the fermentation liquid to obtain a supernatant;

[0013] Step 4: Separate the hyaluronic acid in the supernatant using D101 macroporous adsorption resin exchange column chromatography to obtain fractions;

[0014] Step 5: Concentrate, precipitate with alcohol, dissolve and freeze-dry the fractions in a vacuum to obtain hyaluronic acid freeze-dried powder.

[0015] The culture medium in step 3 is K2HPO4 1.0 g, KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O0.01 g, MnSO4·H2O 0.02 g, ZnSO4·7H2O 0.04 g, glucose 5 g, 121°C, sterilized for 15 min.

[0016] The culture conditions in step 3 are 30° C. and shaking culture at 180 r / min.

[0017] A hyaluronic acid dry powder is prepared by the above method.

[0018] Beneficial Effects

[0019] 1. The nutrients required by Rhizobium Pusha QY12 4aN are simple and the cultivation cost is low.

[0020] 2. The hyaluronic acid produced by Rhizobium pusa QY12 4aN is a medium molecular weight hyaluronic acid, which solves the problem that the existing Rhizobium pusa cannot produce medium molecular weight hyaluronic acid.

[0021] 3. The medium molecular weight hyaluronic acid produced expands the application scope of hyaluronic acid produced by Rhizobium pusa and also provides a new approach for the research of Rhizobium pusa.

[0022] 4. The content of hyaluronic acid in the hyaluronic acid dry powder prepared by the present invention is 57.68%, and the yield and quality of hyaluronic acid are high.

[0023] 5. The present invention discloses that the hyaluronic acid prepared by Rhizobium pusa QY12 4aN is safer than the hyaluronic acid prepared by Streptococcus zooepidemicus in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The colony morphology of Rhizobium pusa QY12 4aN on the Axubei medium plate;

[0025] Figure 2 The colony morphology of Rhizobium pusa QY12 4aN on YPD medium plate;

[0026] Figure 3 The bacterial morphology of Rhizobium pusa QY12 4aN (10×100);

[0027] Figure 4 It is hyaluronic acid after alcohol precipitation;

[0028] Figure 5 shows the detection results of N-acetyl-D-glucosamine in hyaluronic acid of Rhizobium pusa QY12 4aN. Figure 5A is a blank HPLC spectrum; Figure 5BThis is the HPLC spectrum of the acid hydrolysis sample of N-acetyl-D-glucosamine standard; Figure 5C This is the HPLC spectrum of the acid hydrolysis sample of hyaluronic acid standard; Figure 5D HPLC spectrum of the lyophilized powder acid hydrolysis sample;

[0029] FIG6 is an infrared spectrum of hyaluronic acid produced by Rhizobium pusa QY12 4aN, Fig. 6A is the infrared spectrum of hyaluronic acid standard; Figure 6B This is the infrared spectrum of the freeze-dried powder. DETAILED DESCRIPTION

[0030] Example 1.

[0031] Isolation and identification of Rhizobium pusa QY12 4aN.

[0032] Ashby medium was used for separation, and its formula is as follows: KH2PO4 0.2g, MgSO4·7H2O0.2g, NaCl 0.2g, CaCO3 5.0g, mannitol 10.0g, CaSO4 0.1g, agar 15g, H2O 1000mL, sterilized at 121℃ for 15min. The organic fertilizer of Heilongjiang Ruiyuan Biotechnology Co., Ltd. was inoculated on Ashby plates, enriched and cultured at 39℃, and colonies were picked and repeatedly streaked on Ashby plates to obtain single colonies, and single colonies were observed under a microscope to obtain pure cultures. The pure cultures were inoculated on Ashby plates and yeast extract peptone glucose (YPD) plates for culture, and the formula of YPD medium was as follows: tryptone 5g, yeast extract 2.5g, glucose 1g, agar 15g, H2O1000mL, sterilized at 121℃ for 15min. Colony morphology Figure 1 and Figure 2 As shown, the bacterial morphology is Figure 3 As shown. At the same time, the pure culture was inoculated into YPD liquid medium for shaking culture, and the bacteria were collected and commissioned to Sangon Biotech (Shanghai) Co., Ltd. for 16s rRNA gene sequencing. The sequencing results were compared by Blast, and the strain was assigned to Rhizobium pusense and named Rhizobium pusense QY12 4aN. The glycerol preservation method was used and stored in a -80°C refrigerator. Rhizobium pusense QY12 4aN was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a preservation number of CGMCC NO.29921 and a preservation date of March 1, 2024.

[0033] Ashubei medium is a selective medium without nitrogen source, which has the function of enriching and isolating self-generated nitrogen-fixing bacteria. Therefore, Rhizobium pusa QY12 4aN was isolated and purified from Ashubei medium. Figure 1 As shown, it shows that Rhizobium spp. QY12 4aN can use the free nitrogen in the air to synthesize the nitrogen-containing organic matter it needs, that is, it has the function of autogenous nitrogen fixation.

[0034] Example 2.

[0035] Preparation of hyaluronic acid by fermentation of Rhizobium spp. QY12 4aN.

[0036] (1) Cultivation of Rhizobium pusa QY12 4aN. The QY12 4aN cells frozen at -80°C were inoculated onto an Axubei plate for recovery. After a single colony grew out, a single colony was picked and inoculated into a YPD liquid medium. After continuous cultivation, a bacterial suspension was obtained. The bacterial suspension was centrifuged at 6000 r / min for 5 min, the supernatant was discarded, and the precipitate was washed twice with sterile saline to prepare a QY12 4aN bacterial suspension.

[0037] (2) Synthesis of hyaluronic acid. The prepared QY12 4aN bacterial suspension was inoculated into the HA preparation medium. The medium formula was as follows: K2HPO4 1.0 g, KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.02 g, ZnSO4·7H2O 0.04 g, glucose 5 g, 121°C, sterilized for 15 min. After inoculation, the final concentration of the Pusha rhizobium QY12 4aN bacteria was 1.0 OD / mL. The culture was shaken at 30°C and 180 r / min for 2 days. Samples were taken at regular intervals (0 h, 24 h, 48 h) to obtain the fermentation broth. After the culture was completed, the fermentation broth was centrifuged at 10,000 r / min for 10 min and the supernatant was taken.

[0038] Example 3.

[0039] The fermentation liquid obtained in Example 2 was centrifuged, and the supernatant was taken for component analysis to detect the pH, viscosity, polysaccharide, glucuronic acid and hyaluronic acid content of the fermentation liquid. The specific analysis method is:

[0040] (1) Determination of pH of fermentation broth: The pH was determined using a SevenExcellence multi-parameter tester (Mettler-Toledo, Switzerland).

[0041] (2) Determination of the viscosity of the fermentation broth: The viscosity was determined using a DV-II+pro viscometer (Brookfield, USA).

[0042] (3) Determination of water-soluble polysaccharide content in fermentation broth. Take 1 mL of supernatant and add 3 times the volume of anhydrous ethanol, mix well, let stand at 4°C for 2 h, centrifuge at 8000 r / min for 5 min, discard the supernatant, dissolve the precipitate with 4 mL of pure water, centrifuge and take the supernatant, dilute it appropriately, and determine it using the sulfuric acid-phenol method. Use glucose as the standard and draw a standard curve. Calculate the water-soluble polysaccharide content in the supernatant based on the glucose standard curve.

[0043] (4) Determination of glucuronic acid content in fermentation broth. Take 1 mL of supernatant and add 3 times the volume of anhydrous ethanol, mix well, let stand at 4°C for 2 h, centrifuge at 8000 r / min for 5 min, discard the supernatant, dissolve the precipitate with 4 mL of pure water, centrifuge and take the supernatant, dilute appropriately, and determine by sulfuric acid-carbazole method. Use glucuronic acid as the standard and draw a standard curve. Calculate the glucuronic acid content in the supernatant based on the glucuronic acid standard curve.

[0044] (5) Determination of hyaluronic acid content in fermentation broth. Referring to the China Food Industry Association group standard T / CNFIA155-2022 Sodium Hyaluronate Beverage, the spectrophotometer method-sulfuric acid-carbazole method was used to determine the content of hyaluronic acid. Glucuronic acid was used as the standard to draw a standard curve. The content of hyaluronic acid in the supernatant was calculated according to the glucuronic acid standard curve, and the calculation formula is as follows:

[0045] C HA =c×N×1.9542×10 -3

[0046] Where:

[0047] C HA : The content of hyaluronic acid in the sample, in grams per liter (g / L);

[0048] c: According to the absorbance of the sample, find the corresponding glucuronic acid concentration from the standard curve, in micrograms per milliliter (μg / mL);

[0049] N: dilution factor of sample solution;

[0050] 1.9542: Ratio of the relative molecular mass of the repeating disaccharide unit of hyaluronic acid (379.3) to the relative molecular mass of glucuronic acid (194.1).

[0051] 10 -3 : Conversion factor between micrograms per milliliter (μg / mL) and grams per liter (g / L).

[0052] The test results of (1)-(5) above are shown in Table 1.

[0053] Table 1 Component analysis of fermentation broth of Rhizobium spp. QY12 4aN.

[0054]

[0055] Example 4.

[0056] Prepare the supernatant using the method of Example 2, and separate the supernatant using D101 macroporous adsorption resin exchange column chromatography. Mix the above supernatant and deionized water in a ratio of 1:1 (v / v) and apply the mixture. The elution flow rate is 2 mL / min. During the elution process, use an ultraviolet detector to detect the eluate online at 220 nm. Use a microplate method to track the distribution of total sugar and glucuronic acid in the eluate. Collect the glucuronic acid fraction, and use a rotary evaporator to appropriately concentrate the collected fraction. Then, precipitate the concentrated fraction with alcohol, add 3 times the volume of anhydrous ethanol, mix well, and let stand at 4°C for 2 hours to produce a precipitate. Use a glass rod to pick up the precipitate, such as Figure 4 As shown, the precipitate was collected by centrifugation at 8000 r / min for 5 min, and then the precipitate was dissolved in deionized water. The alcohol precipitation-deionized water dissolution step was repeated twice, and the precipitate was vacuum freeze-dried to obtain a lyophilized powder.

[0057] Hyaluronic acid is mainly composed of repeating units of β-(1,4)-glucuronic acid (GA) and β-(1,3)-acetylglucosamine (NAG). In order to prove that the freeze-dried powder contains hyaluronic acid, the freeze-dried powder is first analyzed for glucuronic acid and N-acetyl-D-glucosamine. The analysis and verification are as follows:

[0058] (1) Determination of glucuronic acid content. The glucuronic acid content was determined by the sulfuric acid-carbazole method. Glucuronic acid was used as the standard substance and a standard curve was drawn. The glucuronic acid content in the supernatant was calculated based on the glucuronic acid standard curve.

[0059] (2) Detection of N-acetyl-D-glucosamine in hyaluronic acid. Weigh 50 mg of the above lyophilized powder and add 500 μL of 72% sulfuric acid solution. React in a 30°C water bath for 1 h and vortex to mix once every 10 min. After the reaction is completed, add 14 mL of deionized water, acid hydrolyze at 100°C for 4 h, cool to room temperature, and adjust the pH of the hydrolyzate to 6.0 with CaCO3. Centrifuge and retain the supernatant, mix the supernatant with acetonitrile at 2:8 (v / v), filter through a 0.22 μm microporous filter membrane, and then perform HPLC analysis. Hyaluronic acid standard and N-acetyl-D-glucosamine standard were treated with the same acid hydrolysis method as sample controls. HPLC conditions: Aglent 20RBA×NH2 (4.6×250 mm) amino column, detection wavelength of 210 nm, column temperature of 40°C, mobile phase of acetonitrile: water = 8:2 (v / v), flow rate of 1.0 mL / min, injection volume of 20 μL. The above test results are shown in Figure 5.

[0060] (3) Determination of infrared spectrum of freeze-dried powder sample. The functional groups of hyaluronic acid were determined using a Bruker EQUINOX 55 infrared spectrometer. An appropriate amount of freeze-dried powder sample was mixed with KBr powder at a mass ratio of 1:100, ground evenly in a mortar, and pressed into tablets. -1 Spectral scanning is performed within the wavenumber range with a resolution of 4 cm -1 , scanned 32 times, with air as the background. Sodium hyaluronate standard (Cat. No. 53747, Sigma-Aldrich) was used as a control, and the results are shown in FIG6 .

[0061] The test results of (1) above show that the freeze-dried powder sample contains glucuronic acid, and its content is 29.52%.

[0062] The detection result of the above (2) is shown in FIG5 . After acid hydrolysis of the N-acetyl-D-glucosamine standard, peak 1 ( Figure 5A ), and N-acetyl-D-glucosamine standard ( Figure 5B ) and hyaluronic acid standard ( Figure 5C ) of the acid hydrolyzed samples, in the lyophilized powder sample of Rhizobium pusa QY12 4aN ( Figure 5D ), N-acetyl-D-glucosamine was also detected at 5.60 min in the HPLC spectrum of the acid hydrolyzed sample, indicating that the freeze-dried powder contained N-acetyl-D-glucosamine monomers in hyaluronic acid.

[0063] The above test results show that the freeze-dried powder obtained by 75% ethanol precipitation contains glucuronic acid and N-acetyl-D-glucosamine that constitute hyaluronic acid, indicating that the freeze-dried powder contains hyaluronic acid.

[0064] The detection results of the above (3) are shown in Figure 6. The infrared spectrum at 3416cm -1 A strong peak is shown at 2921 cm -1 The peaks at 1726 and 1637.00 cm-1 correspond to the presence of the symmetrical methyl CH stretching in glucuronic acid; -1 The peak at 1566 cm -1 The peaks at 1406 and 1375 cm -1 The peaks at 1163, 1073 and 1045 cm -1 The peaks at 902 and 608 cm -1A peak appears at , indicating the presence of COC stretching. In summary, the infrared spectrum of the freeze-dried powder ( Figure 6B ) shows the characteristic peaks of the infrared spectrum of hyaluronic acid, and is consistent with the infrared spectrum of the hyaluronic acid standard ( Fig. 6A ), it can be further determined that the main component of the above freeze-dried powder is hyaluronic acid.

[0065] Example 5.

[0066] The hyaluronic acid freeze-dried powder obtained in Example 4 was subjected to component analysis and molecular weight determination.

[0067] (1) Determination of polysaccharide content. The freeze-dried powder obtained in Example 4 was prepared into a solution of appropriate concentration and determined by the sulfuric acid-phenol method. Glucose was used as the standard substance to draw a standard curve. The water-soluble polysaccharide content in the supernatant was calculated based on the glucose standard curve.

[0068] (2) Determination of hyaluronic acid content: The method of Example 3 (5) was used for determination.

[0069] (3) Determination of the average relative molecular weight of Rhizobium pusa QY12 4aN hyaluronic acid: According to the light industry standard QB / T4416-2012 of the People's Republic of China, sodium hyaluronate, a raw material for cosmetics, was measured using an Ubbelohde viscometer.

[0070] The test results of (1)-(2) above are shown in Table 2. The freeze-dried powder of the fermentation product produced by Rhizobium pusa QY12 4aN contains 86.13% polysaccharides and 57.68% hyaluronic acid. It is speculated that it may also contain other polysaccharides.

[0071] Table 2 Determination of polysaccharides and hyaluronic acid in freeze-dried powder

[0072] sample Polysaccharide content (%, w / w) Hyaluronic acid content (%, w / w) Hyaluronic acid freeze-dried powder 86.13％ 57.68％

[0073] The results of the above (3) test showed that the molecular weight of hyaluronic acid produced by Rhizobium pusa QY12 4aN was 5.6-8.3×10 5 kDa, medium molecular weight hyaluronic acid.

Claims

1. A strain of Rhizobium pusa, characterized in that: The Pusha rhizobium QY124aN is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.29921 and the deposit date March 1, 2024.

2. A method for preparing hyaluronic acid, characterized in that: The method is to prepare the product using the Rhizobium spp. QY124aN described in claim 1.

3. The method according to claim 2, characterized in that: The method is: Step 1: Resuscitate Rhizobium pusa QY124aN and inoculate into YPD liquid culture medium to obtain bacterial solution; Step 2: Centrifuge the bacterial solution to obtain a precipitate, wash the precipitate with saline and prepare a bacterial suspension; Step 3: inoculating the bacterial suspension into a hyaluronic acid preparation medium for cultivation to obtain a fermentation liquid, and centrifuging the fermentation liquid to obtain a supernatant; Step 4: Separate the hyaluronic acid in the supernatant using D101 macroporous adsorption resin exchange column chromatography to obtain fractions; Step 5: Concentrate, precipitate with alcohol, dissolve and freeze-dry the fractions in a vacuum to obtain hyaluronic acid freeze-dried powder.

4. The method according to claim 3, characterized in that: The culture medium in step 3 is K2HPO4 1.0 g, KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, MnSO4·H2O 0.02 g, ZnSO4·7H2O 0.04 g, glucose 5 g, 121°C, sterilized for 15 min.

5. The method according to claim 3, characterized in that: The culture conditions in step 3 are 30° C. and shaking culture at 180 r / min.

6. A hyaluronic acid dry powder, characterized in that: The hyaluronic acid dry powder is prepared according to any one of claims 2-5.

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