Hyaluronic acid and its preparation method

Abstract

This invention proposes a hyaluronic acid and its preparation method, belonging to the field of microbial technology. The hyaluronic acid produced by *Rhizobium pusa* QY124aN in this invention solves the problem of high cultivation costs of existing strains and the lack of a technical method for producing high molecular weight hyaluronic acid using *Rhizobium pusa*. The hyaluronic acid powder prepared by this invention has a hyaluronic acid content of 57.68% and is safer than hyaluronic acid obtained from *Streptococcus vesiculosus* in existing technologies.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a method for preparing hyaluronic acid. Background Technology

[0002] Hyaluronic acid (HA), a natural linear glycosaminoglycan, is mainly composed of repeating units of β-(1,4)-glucuronic acid (GA) and β-(1,3)-acetylglucosamine (NAG), linked together by alternating β-1,3 and β-1,4 glycosidic bonds. Sodium hyaluronate is the sodium salt of hyaluronic acid. Based on molecular weight, HA is classified into three forms: low molecular weight HA (6-200 kDa), medium molecular weight HA (0.2-1.0 MDa), and high molecular weight HA (>1 MDa). The bioactivity of HA is closely related to its molecular weight; for example, adhesion and moisturizing properties are characteristics of high molecular weight HA. Medium-sized hyaluronic acid promotes wound healing. Low molecular weight HA exhibits good permeability, promotes angiogenesis, and has anti-apoptotic properties. In summary, research indicates that sodium hyaluronate (HA) possesses properties such as non-toxicity and non-immunogenicity. HA can be used in the research and development of products including moisturizers, injections, oral formulations, and bioactive drug delivery systems. It has found promising applications in facial wrinkle and fold correction, surgical wound regeneration, and drug delivery via various routes, making it a highly attractive biomaterial for industrial applications in cosmetics, pharmaceuticals, and medical devices. In January 2021, the National Health Commission of China approved sodium hyaluronate as a new food ingredient, allowing its use as an additive in ordinary foods. This further expands the application areas of HA, and the market demand for HA is expected to increase significantly.

[0003] Hyaluronic acid (HA) is typically extracted from animal tissues (e.g., bovine vitreous humor, rooster comb, and synovial fluid) or from the fermentation broth of microorganisms (e.g., Streptococcus equi, Bacillus subtilis, and Escherichia coli). Traditionally, HA production involves extraction from animal sources, a process that is difficult to control, costly, and results in low yields due to hyaluronidase degradation, affecting HA quality. Furthermore, animal-derived HA may contain host proteins and DNA, potentially triggering immune responses and allergies. Given these issues, current industrial-scale HA production methods utilize microbial fermentation, with the widely used strain being Streptococcus equi subsp. equine (Streptococcus equi). Streptococcus equi subsp. zooepidemicus ), to produce high molecular weight HA.

[0004] However, some problems still exist in the production of Streptococcus equi subsp. veterinaria. For example, the bacterial growth is slow, and there is substrate competition between bacterial growth and hyaluronic acid synthesis; the nutritional requirements are high, requiring the addition of yeast powder, peptone, and amino acids to the culture medium, which increases production costs; the fermentation broth has high viscosity, and the accumulation of lactic acid, a fermentation byproduct, inhibits cell growth and hyaluronic acid production. In particular, Streptococcus veterinaria carries the risk of bacterial endotoxins, which limits its application in biomedicine.

[0005] For the reasons mentioned above, developing new and safe HA production systems remains a key focus for the industry. Finding new hyaluronic acid production strains is an effective way to reduce production costs and expand application areas, in order to meet the growing market demand for high-quality HA products and to provide new ways to prepare HA of different molecular weights. Summary of the Invention

[0006] This invention addresses the high cultivation cost of existing strains by using hyaluronic acid produced by Rhizobium pusa QY12 4aN, and also solves the problem of the lack of technical methods for producing medium molecular weight hyaluronic acid from Rhizobium pusa.

[0007] A strain of Rhizobium pusa, namely Rhizobium pusa QY12 4aN, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29921 and deposit date of March 1, 2024.

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

[0009] The method is as follows:

[0010] Step 1: After reviving Rhizobium pusa QY12 4aN, inoculate it into YPD liquid medium to obtain bacterial culture;

[0011] Step 2: After centrifuging the bacterial culture, a precipitate is obtained. The precipitate is washed with physiological saline and a bacterial suspension is prepared.

[0012] Step 3: Inoculate the bacterial suspension into hyaluronic acid preparation medium and culture to obtain fermentation broth. Centrifuge the fermentation broth to obtain the supernatant.

[0013] Step 4: Use D101 macroporous adsorption resin exchange column chromatography to separate the hyaluronic acid in the supernatant and obtain the fraction;

[0014] Step 5: Concentrate the fraction, precipitate with alcohol, dissolve and freeze-dry under vacuum to obtain hyaluronic acid lyophilized powder.

[0015] The culture medium described in step three consists of 1.0 g / L K₂HPO₄ and 0.5 g / L KCl. 0.5 g / L, 0.01 g / L, 0.02 g / L, Sterilize at 0.04 g / L glucose 5 g / L for 15 min at 121℃.

[0016] The cultivation conditions described in step three are: 30 ℃ and 180 r / min with shaking.

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

[0018] Beneficial effects

[0019] 1. The nutrients required for Rhizobium pusa QY12 4aN are simple and the culture cost is low.

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

[0021] 3. The production of medium molecular weight hyaluronic acid expands the application range of hyaluronic acid production by Psa rhizobium and also provides a new approach for the research of Psa rhizobium.

[0022] 4. The hyaluronic acid powder prepared by this invention has a hyaluronic acid content of 57.68%, and the yield and quality of hyaluronic acid are relatively 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 vesica in the prior art. Attached Figure Description

[0024] Figure 1 The colony morphology of Rhizobium pusa QY12 4aN on Ashube agar plates;

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

[0026] Figure 3 The cell morphology of Rhizobium pusa QY12 4aN ( );

[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 This is a blank HPLC chromatogram; Figure 5B HPLC chromatogram of acid-hydrolyzed N-acetyl-D-glucosamine standard sample; Figure 5C HPLC chromatogram of an acid-hydrolyzed sample of hyaluronic acid standard; Figure 5D This is the HPLC chromatogram of the lyophilized powder acid hydrolysis sample;

[0029] Figure 6 shows the infrared spectrum of hyaluronic acid produced by Rhizobium pusa QY12 4aN. Figure 6A Infrared spectrum of hyaluronic acid standard; Figure 6B This is the infrared spectrum of the freeze-dried powder. Detailed Implementation

[0030] Example 1.

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

[0032] The separation was performed using Ashby medium, the formulation of which is as follows: 0.2 g, 0.2 g of organic fertilizer from Heilongjiang Ruiyuan Biotechnology Co., Ltd. was inoculated onto Assoube agar plates and enriched at 39 ℃. Colonies were repeatedly streaked onto Assoube plates to isolate single colonies, which were then examined under a microscope to obtain pure cultures. These pure cultures were then inoculated onto Assoube agar plates and yeast extract peptone glucose (YPD) agar plates. The YPD medium formula was as follows: 5 g tryptone, 2.5 g yeast extract, 1 g glucose, 15 g agar, 1000 mL H2O, and sterilized at 121 ℃ for 15 min. Colony morphology is shown in the image. Figure 1 and Figure 2 As shown, its bacterial cell morphology is as follows Figure 3 As shown. Simultaneously, the pure culture was inoculated into YPD liquid medium and cultured with shaking. The bacterial cells were collected and 16S rRNA gene sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results, compared with BLAST, classified the strain as *Pulsatilla chinensis* (…). Rhizobium pusense ), named *Pusar rhizobium* QY12 4aN ( Rhizobium pusense The strain QY12 4aN was preserved using the glycerol preservation method at -80 °C. The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29921 on March 1, 2024.

[0033] Assumption medium is a nitrogen-free selective medium used for the enrichment and isolation of free-living nitrogen-fixing bacteria. Therefore, *Rhizobium pusa* QY12 4aN was isolated and purified from Assumption medium, as shown below. Figure 1 As shown, this indicates that *Pussa rhizobium* QY12 4aN can utilize free nitrogen in the air to synthesize nitrogen-containing organic matter it needs, i.e., it has a self-generating nitrogen fixation function.

[0034] Example 2.

[0035] Hyaluronic acid was prepared by fermentation of Rhizobium pusa QY12 4aN.

[0036] (1) Expansion culture of Rhizobium pusa QY12 4aN. QY12 4aN cells frozen at -80 ℃ were inoculated onto Assoube plates for revival. After single colonies grew, single colonies were picked and inoculated into YPD liquid medium. After continuous expansion culture, 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 physiological saline to prepare QY12 4aN bacterial suspension.

[0037] (2) Synthesis of hyaluronic acid. The prepared QY12 4aN bacterial suspension was inoculated into HA preparation medium. The medium formula was as follows: K2HPO4 1.0 g / L, KCl 0.5 g / L, 0.5 g / L, 0.01 g / L, 0.02 g / L, Sterilize at 121 °C for 15 min with 0.04 g / L glucose and 5 g / L. After inoculation, the final concentration of *Rhizobium pusa* QY12 4aN cells was 1.0 OD / mL. The mixture was then cultured at 30 °C and 180 r / min with shaking for 2 days, with samples taken at regular intervals (0 h, 24 h, and 48 h) to obtain the fermentation broth. After the culture was completed, the fermentation broth was centrifuged at 10000 r / min for 10 min, and the supernatant was collected.

[0038] Example 3.

[0039] The fermentation broth obtained in Example 2 was centrifuged, and the supernatant was analyzed to determine its pH, viscosity, and the contents of polysaccharides, glucuronic acid, and hyaluronic acid. The specific analytical methods were as follows:

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

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

[0042] (3) Determination of water-soluble polysaccharide content in fermentation broth. Take 1 mL of supernatant, add 3 times the volume of anhydrous ethanol, mix well, let stand at 4 ℃ for 2 h, centrifuge at 8000 r / min for 5 min, discard the supernatant, dissolve the precipitate in 4 mL of purified water, centrifuge again, collect the supernatant, dilute appropriately, and determine the content using the sulfuric acid-phenol method. Use glucose as a standard to plot 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, add 3 times the volume of anhydrous ethanol, mix well, let stand at 4 ℃ for 2 h, centrifuge at 8000 r / min for 5 min, discard the supernatant, dissolve the precipitate in 4 mL of purified water, centrifuge again, take the supernatant, dilute appropriately, and determine by sulfuric acid-carbazole method. Use glucuronic acid as a standard to plot 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 hyaluronic acid content was determined using the spectrophotometric method-sulfuric acid-carbazole method. A standard curve was plotted using glucuronic acid as the standard. The hyaluronic acid content in the supernatant was calculated based on the glucuronic acid standard curve using the following formula:

[0045]

[0046] In the formula:

[0047] C HA Hyaluronic acid content in the sample, expressed in grams per liter (g / L).

[0048] c: Based on 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 the sample solution;

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

[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. Composition analysis of the fermentation broth of Rhizobium pusa QY12 4aN.

[0054]

[0055] Example 4.

[0056] The supernatant was prepared using the method in Example 2, and separated using D101 macroporous adsorption resin exchange column chromatography. The supernatant was mixed with deionized water at a 1:1 (v / v) ratio and loaded onto the supernatant. The elution flow rate was 2 mL / min. During elution, the eluent was monitored online at 220 nm using a UV detector. The distribution of total sugars and glucuronic acid in the eluent was tracked using a microplate method. The glucuronic acid fraction was collected and appropriately concentrated using a rotary evaporator. The concentrated fraction was then subjected to alcohol precipitation. Three volumes of anhydrous ethanol were added, mixed, and allowed to stand at 4°C for 2 h. A precipitate formed, which was picked up with a glass rod. Figure 4 As shown, the precipitate was collected by centrifugation at 8000 r / min for 5 min, and then dissolved in deionized water. The alcohol precipitation-deionized water dissolution step was repeated twice. The precipitate was then freeze-dried under vacuum to obtain lyophilized powder.

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

[0058] (1) Determination of glucuronic acid content. The sulfuric acid-carbazole method was used for determination. A standard curve was plotted using glucuronic acid as a standard. 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 water bath at 30 ℃ for 1 h, vortexing every 10 min to mix. After the reaction is complete, add 14 mL of deionized water and acid hydrolyze at 100 ℃ for 4 h. Cool to room temperature and adjust the pH of the hydrolysate to 6.0 with CaCO3. Centrifuge and retain the supernatant. Mix the supernatant with acetonitrile 2:8 (v / v), filter through a 0.22 μm microporous membrane, and then perform HPLC analysis. Hyaluronic acid standard and N-acetyl-D-glucosamine standard were treated with the same acid hydrolysis method and used as sample controls. HPLC conditions: An Aglent 20RBA×NH2 (4.6×250 mm) amino column was used, with a detection wavelength of 210 nm, a column temperature of 40℃, a mobile phase of acetonitrile:water = 8:2 (v / v), a flow rate of 1.0 mL / min, and an injection volume of 20 μL. The detection results are shown in Figure 5.

[0060] (3) Determination of the infrared spectrum of the lyophilized powder sample. The functional groups of hyaluronic acid were determined using a Bruker EQUINOX 55 infrared spectrometer. An appropriate amount of lyophilized powder sample was mixed with KBr powder at a mass ratio of 1:100, ground evenly in a mortar, and then compressed into tablets. The hyaluronic acid was measured at 4000-400 cm⁻¹. -1 Spectral scanning was performed within the wavenumber range with a resolution of 4 cm⁻¹. -1 The scan was performed 32 times against an air background. Sodium hyaluronate standard (catalog number: 53747, Sigma-Aldrich) was used as a control. The results are shown in Figure 6.

[0061] As can be seen from the test results of (1) above, the freeze-dried powder sample contains glucuronic acid, with a content of 29.52%.

[0062] The detection results of (2) above are shown in Figure 5. After acid hydrolysis, the N-acetyl-D-glucosamine standard was detected by HPLC, and peak 1 appeared in the HPLC spectrum. Figure 5A ), and N-acetyl-D-glucosamine standard ( Figure 5B ) and hyaluronic acid standard ( Figure 5C Comparison of HPLC spectra of acid-hydrolyzed samples of *Rhizobium pusa* QY12 4aN lyophilized powder ( Figure 5D On the HPLC chromatogram of the acid-hydrolyzed sample, N-acetyl-D-glucosamine was also detected at 5.60 min. This indicates that the lyophilized powder contains N-acetyl-D-glucosamine monomer from hyaluronic acid.

[0063] The above test results indicate that the freeze-dried powder obtained by precipitation with 75% ethanol contains glucuronic acid and N-acetyl-D-glucosamine, which are components of hyaluronic acid, indicating that the freeze-dried powder contains hyaluronic acid.

[0064] The detection results of (3) above are shown in Figure 6. The infrared spectrum is at 3416 cm⁻¹. -1 A strong peak is observed at 2921 cm⁻¹, indicating the presence of OH tensile vibration. -1 The peak values ​​at 1726 and 1637.00 cm⁻¹ correspond to the presence of the extended symmetrical methyl group CH in glucuronic acid; -1 The peak value at 1566 cm⁻¹ indicates the presence of C=O carboxylamide I extension. -1 The peak values ​​at 1406 and 1375 cm⁻¹ indicate the presence of bending vibrations in NH₃; peak values ​​at 1406 and 1375 cm⁻¹ further indicate the presence of bending vibrations in NH₃. -1 The peaks at 1163, 1073, and 1045 cm⁻¹ indicate the presence of CO groups combined with C=O; -1 The peaks at 902 and 608 cm⁻¹ indicate the presence of COC, CO, and COH extension;-1 The peak at this point indicates the presence of COC extension. In summary, the infrared spectrum of the lyophilized powder ( Figure 6B ) exhibits characteristic peaks in the infrared spectrum of hyaluronic acid, and compares them with the infrared spectrum of hyaluronic acid standards ( Figure 6A Similar to this, it can be further determined that the main component of the above-mentioned freeze-dried powder is hyaluronic acid.

[0065] Example 5.

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

[0067] (1) Determination of polysaccharide content. The lyophilized powder obtained in Example 4 was prepared into a solution of appropriate concentration and the content was determined by the sulfuric acid-phenol method. A standard curve was plotted using glucose as a standard. The content of water-soluble polysaccharides in the supernatant was calculated based on the glucose standard curve.

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

[0069] (3) Determination of the average relative molecular weight of hyaluronic acid from Rhizobium pusa QY12 4aN. According to the People's Republic of China Light Industry Standard QB / T4416-2012, sodium hyaluronate for cosmetic use, the molecular weight was measured using an Ubbelohde viscometer.

[0070] The detection results of (1)-(2) above are shown in Table 2. The freeze-dried powder of fermentation product produced by Rhizobium pusa QY12 4aN contains 86.13% polysaccharide 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 lyophilized powder

[0072]

[0073] The results of the above (3) test show that the molecular weight of hyaluronic acid produced by Rhizobium pusa QY12 4aN is: Da is a medium molecular weight hyaluronic acid.

Claims

1. A Bradyrhizobium sp. characterized in that, The Psorospermi QY12 4aN is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.29921, and the preservation date is March 1, 2024.

2. A method for producing hyaluronic acid, characterized by, The method is prepared by using the Psorospermi QY12 4aN in claim 1.

3. The method of claim 2, wherein, The method is: Step one: inoculate the Psorospermi QY12 4aN after resuscitation into YPD liquid medium to obtain a bacterial liquid; Step two: centrifuge the bacterial liquid to obtain a precipitate, wash the precipitate with physiological saline, and prepare a bacterial suspension; Step three: inoculate the bacterial suspension into a hyaluronic acid preparation medium for culture to obtain a fermentation liquid, centrifuge the fermentation liquid to obtain a supernatant; Step four: use D101 macroporous adsorption resin exchange column chromatography to separate the hyaluronic acid in the supernatant to obtain a fraction; Step five: concentrate, alcohol precipitation, dissolution and vacuum freeze-drying of the fraction to obtain hyaluronic acid freeze-dried powder.

4. The method of claim 3, wherein, The medium of step three is , , , , , , , , , glucose 5 g / L, 121 °C, 15 min sterilization.

5. The method of claim 3, wherein, The culture condition in step three is 30 DEG C, 180 r / min under the condition of shaking culture.

Images