A strain of Streptococcus vesiculosus producing hyaluronic acid and its application

Abstract

This invention relates to a hyaluronic acid-producing Streptococcus vesiculosus strain and its applications, belonging to the field of bioengineering technology. This invention obtains a high-lactate-tolerant and high-hyaluronic acid-producing Streptococcus vesiculosus strain KFA018 through ARTP mutagenesis and high lactate tolerance training, effectively solving the problems of low hyaluronic acid yield and low lactate tolerance in existing Streptococcus vesiculosus strains. The hyaluronic acid yield of the Streptococcus vesiculosus strain KFA018 of this invention is as high as 17-18 g / L. Simultaneously, the Streptococcus vesiculosus strain KFA018 of this invention can grow to lactate levels up to 45 g / L, exhibiting high lactate tolerance, providing an excellent strain selection for hyaluronic acid production.

Description

Technical Field

[0001] This invention relates to a strain of Streptococcus vesiculosus that produces hyaluronic acid and its applications, belonging to the field of bioengineering technology. Background Technology

[0002] Hyaluronic acid (HA) is a negatively charged non-sulfated glycosaminoglycan. It is a highly polymeric macromolecular viscous polysaccharide composed of UDP-N-acetylglucosamine and UDP-D-glucuronic acid linked by alternating β-1,3 and β-1,4 glycosidic bonds. It is found in the eyes, joints, and skin of vertebrates, where it can lubricate joints, repair damaged skin tissue, promote wound healing, and regulate inflammatory responses. In addition to vertebrates, some pathogenic microorganisms, such as Streptococcus group A and Pasteurella multocida, synthesize HA as a major component of their capsule to protect themselves from harsh external environments.

[0003] Hyaluronic acid can be classified into ultra-high molecular weight (molecular weight greater than 3000 kDa), high molecular weight (1000 kDa-3000 kDa), medium molecular weight (100 kDa-1000 kDa), and low molecular weight (less than 100 kDa) hyaluronic acid based on its molecular weight. Different molecular weights of hyaluronic acid possess different properties. Ultra-high molecular weight hyaluronic acid can inhibit cell migration, proliferation, differentiation, and phagocytosis, and can be used as a solid filler. High molecular weight hyaluronic acid has excellent moisturizing and anti-inflammatory functions, and is used in ophthalmic surgery as a viscoelastic agent and in intra-articular injection therapy. Medium molecular weight hyaluronic acid is widely used in cosmetics due to its superior moisturizing and lubricating properties. Low molecular weight hyaluronic acid and its oligosaccharides have anti-tumor, wound-healing, bone and angiogenesis-promoting, and immunomodulatory effects, showing promising medical application prospects.

[0004] Microbial fermentation refers to the process of obtaining hyaluronic acid by fermenting hyaluronic acid-producing strains in a fermenter. Compared with animal tissue extraction, microbial fermentation is safer, produces more uniform products, and is less expensive. The process mainly includes strain activation, seed culture, fermentation in a fermenter, and hyaluronic acid extraction.

[0005] When Streptococcus vesicae ferments to produce hyaluronic acid (HA), approximately 80% of the carbon source is converted into lactic acid via glycolysis. On the one hand, lactic acid competes with HA synthesis for a limited carbon source; on the other hand, lactic acid strongly inhibits bacterial growth and HA synthesis. Therefore, reducing the inhibitory effect of lactic acid on bacterial growth and HA synthesis will, to some extent, increase HA yield. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a hyaluronic acid-producing Streptococcus vesiculosus strain and its applications. This invention, through ARTP mutagenesis screening and stepwise lactic acid acclimatization, yields a Streptococcus vesiculosus strain with a fermentation hyaluronic acid yield of 17-18 g / L, with a maximum tolerated lactic acid concentration of 45 g / L. Fermentation has been successfully implemented in a 5L supertanker system. This invention effectively solves the problems of low hyaluronic acid yield and low lactic acid tolerance in existing Streptococcus vesiculosus strains.

[0007] The technical solution of the present invention is as follows:

[0008] A strain of Streptococcus zooepidemicus KFA018 was deposited at the China Center for Type Culture Collection (CCTCC) on October 14, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20242194.

[0009] The Streptococcus vesiculosus KFA018 described in this invention is a strain that produces high levels of hyaluronic acid, obtained through ARTP mutagenesis breeding and high-concentration lactic acid domestication. This strain also has high lactic acid tolerance, enabling it to survive in a high-concentration lactic acid environment, thereby solving the adverse effects of lactic acid accumulation on bacterial growth.

[0010] A bacterial agent comprising the aforementioned Streptococcus vesicularis KFA018.

[0011] The application of the above-mentioned Streptococcus vesiculosus KFA018 or its agent in the preparation of hyaluronic acid.

[0012] A method for producing hyaluronic acid by fermentation using Streptococcus vesiculosus KFA018 as the fermentation strain includes the following steps:

[0013] Streptococcus vesicularis KFA018 was inoculated onto a solid culture medium for activation culture, and the activated strain was inoculated into a seed culture medium for seed culture; the cultured seed liquid was inoculated into a fermentation culture medium for fermentation culture to obtain a fermentation broth containing hyaluronic acid.

[0014] According to a preferred embodiment of the present invention, the solid culture medium comprises: glucose 5-20 g / L, peptone 5-20 g / L, yeast extract 5-10 g / L, dipotassium hydrogen phosphate 1-10 g / L, magnesium sulfate 0.1-2 g / L, and agar powder 15-20 g / L.

[0015] According to a preferred embodiment of the present invention, the activation culture conditions are: culture at 35-37℃ for 20-50 hours.

[0016] According to a preferred embodiment of the present invention, the seed culture medium comprises: glucose 5-15 g / L, yeast extract 5-10 g / L, peptone 5-20 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.1-1 g / L, and NaCl 15-20 g / L.

[0017] According to a preferred embodiment of the present invention, the seed culture conditions are: culture at 35-37℃ and 200-220 rpm.

[0018] According to a preferred embodiment of the present invention, the OD of the seed solution 600 The value is 1.0-2.0.

[0019] According to a preferred embodiment of the present invention, the fermentation medium comprises: 50-100 g / L glucose, 5-10 g / L yeast extract, 5-20 g / L peptone, 1-5 g / L dipotassium hydrogen phosphate, and 0.1-1 g / L magnesium sulfate.

[0020] According to a preferred embodiment of the present invention, the inoculation amount of seed liquid in the fermentation medium is 5-10% by volume.

[0021] According to a preferred embodiment of the present invention, the fermentation conditions are as follows: temperature 35-37℃, stirring speed 200-300 rpm, pH controlled at 6.0-8.0 with sodium hydroxide solution, and aeration fermentation for 20-30 hours.

[0022] According to a preferred embodiment of the present invention, the method further includes the step of: during the fermentation process, when the viscosity of the fermentation broth increases to 5000-30000 mPa·s, adding hyaluronidase at a final concentration of 800-1200 U / mL to reduce the viscosity of the fermentation broth, and adding it a total of 5-6 times.

[0023] The Streptococcus vesiculosus KFA018 of the present invention, used for fermentation production of hyaluronic acid, has the following advantages:

[0024] 1. Stabilizes bacterial growth and metabolic activity: In the fermentation production of hyaluronic acid, lactic acid is an important organic acid produced by bacterial metabolism. Improving lactic acid tolerance helps the producing strain better cope with high-concentration lactic acid environments, maintaining stable growth and metabolic activity. 2. Reduces production costs: Fermentation under low pH conditions reduces the use of neutralizing agents, thereby lowering production costs. 3. Enhances adaptability: Streptococcus vesiculosus grown under high lactic acid concentrations may exhibit stronger adaptability, enabling it to cope with various environmental pressures encountered during industrial production.

[0025] Beneficial effects:

[0026] This invention yielded a strain of Streptococcus vesiculosus KFA018 that is resistant to high lactic acid content and produces high levels of hyaluronic acid through ARTP mutagenesis and high lactic acid tolerance acclimation. During the microbial fermentation production of hyaluronic acid, the accumulation of large amounts of lactic acid causes a decrease in the pH of the fermentation broth. When the pH value is lower than the pKa value of lactic acid, free lactic acid can permeate the cell membrane and enter the cell, lowering the intracellular pH and affecting cell membrane stability. By acclimating the hyaluronic acid-producing strain to acid tolerance, the microbial strain can gradually adapt to a specific acidic environment and utilize the substrate more efficiently, increasing the yield of the target product. The Streptococcus vesiculosus KFA018 of this invention produces a hyaluronic acid yield as high as 17-18 g / L. Simultaneously, the Streptococcus vesiculosus KFA018 of this invention exhibits high lactic acid tolerance, with a lactic acid content tolerance of up to 45 g / L, providing an excellent strain for hyaluronic acid production. Attached Figure Description

[0027] Figure 1 A standard curve for detecting hyaluronic acid content using high performance liquid chromatography;

[0028] Figure 2 This is a high-performance liquid chromatogram of a hyaluronic acid standard solution.

[0029] Figure 3 The lethality curve of ARTP-mutated strains;

[0030] Figure 4 The effect of different concentrations of sodium lactate on the growth of Streptococcus veterinaria mutant strain A1;

[0031] Figure 5 Colony morphology diagrams of Streptococcus vesiculosus mutant strain A1 and Streptococcus vesiculosus KFA018;

[0032] Figure 6 OD of Streptococcus pneumoniae mutant strain A1 and Streptococcus pneumoniae KFA018 during fermentation 600 The curves showing the changes in sugar content and residual sugar. Detailed Implementation

[0033] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0034] Unless otherwise specified, the raw materials and reagents used in the embodiments of this invention are all commercially available products.

[0035] The composition of the culture medium used in the examples:

[0036] Plate and slant agar media: glucose 8 g / L, peptone 15 g / L, yeast extract 8 g / L, dipotassium hydrogen phosphate 4 g / L, magnesium sulfate 1 g / L, agar powder 15 g / L. Prepare with sterile deionized water, adjust pH to 7.4 with 1 mol / L sodium hydroxide solution, and sterilize at 121℃ for 20 min. Pour the sterilized medium into sterile petri dishes or test tubes and allow it to cool and solidify.

[0037] Seed culture medium: glucose 15g / L, peptone 20g / L, yeast extract 4g / L, dipotassium hydrogen phosphate 3g / L, magnesium sulfate 1.5g / L, NaCl 15g / L, prepared with sterile deionized water, pH adjusted to 7.4 with 1mol / L sodium hydroxide solution, and sterilized at 121℃ for 20min.

[0038] Fermentation medium: 80 g / L glucose, 20 g / L peptone, 8 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, prepared with sterile deionized water, pH adjusted to 7.4 with 1 mol / L sodium hydroxide solution, and sterilized at 121℃ for 20 min.

[0039] The ARTP mutagenesis machine used in the examples: Product model: ARTP-M, manufacturer: Wuxi Yuanqing Tianmu Biotechnology Co., Ltd., discharge technology: atmospheric pressure uniform glow discharge, uniform and stable plasma jet, working gas: 99.999% and above high purity helium, gas volume control range: 0-15SLM (standard liters / minute), gas volume control accuracy ±1.0%FS (full scale).

[0040] The method for detecting hyaluronic acid content used in this example includes the following steps:

[0041] (1) Plotting the standard curve: Transfer 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, and 2.0 mL of 1.0 mg / mL hyaluronic acid standard solution to 15 mL stoppered graduated test tubes, respectively. Add phosphate buffer (pH 7.4) to a final volume of 9 mL, then add 1 mL of hyaluronidase solution (1000 IU / mL) and mix well. The final mass concentrations of the hyaluronic acid standards are 0.005 mg / mL, 0.010 mg / mL, 0.020 mg / mL, 0.050 mg / mL, 0.100 mg / mL, and 0.200 mg / mL, respectively. Incubate at 37°C for 1 h, then boil for 2 min to terminate the reaction. After cooling to room temperature, filter the solution through a 0.22 μm filter membrane and perform high-performance liquid chromatography (HPLC) detection. Plot the standard curve with the working mass concentration of the hyaluronic acid standard series as the x-axis and the peak area as the y-axis, as shown below. Figure 1As shown, the standard curve fitting equation is y = 25246x + 6.2447, R0 2 =1; Figure 2 This is a high-performance liquid chromatogram of a hyaluronic acid standard solution.

[0042] The high performance liquid chromatography method was performed in accordance with the industry standard QB / T 4576-2023. The chromatographic column was a sulfonated cross-linked styrene-divinylbenzene copolymer strong cation exchange column. The chromatographic conditions were: flow rate 0.6 mL / min, injection volume 20 μL, column temperature 40℃, and detection wavelength 232 nm.

[0043] (2) Detection of the test sample: Take the fermentation broth to be tested, dilute it appropriately, and transfer 0.5 mL of the diluted fermentation broth to a 15 mL stoppered graduated test tube. Add 8.5 mL of phosphate buffer, then add 1.0 mL of hyaluronidase solution, mix well, and incubate at 37°C for 1 h. Then, stop the reaction by boiling in a water bath for 2 min. Cool the reaction solution to room temperature, filter it through a 0.22 μm filter membrane, and perform high-performance liquid chromatography (HPLC) detection. Calculate the hyaluronic acid content in the fermentation broth based on the standard curve.

[0044] In this embodiment, a visible-ultraviolet spectrophotometer was used to detect OD. 600 The values ​​were determined using a biosensor analyzer to measure the glucose and sodium lactate content; the viscosity was measured according to the "Pharmacopoeia 2015 Edition, Part IV, General Chapter 0633, Viscosity Determination, Part III, Rotational Viscometer Determination Method".

[0045] Example 1:

[0046] The starting strain used in this experiment was wild-type Streptococcus vesicans, which can produce hyaluronic acid. It was obtained through screening by Shandong Freda Pharmaceutical Group Co., Ltd., and is currently deposited at the Institute of Synthetic Biology and Biomanufacturing of Shandong Freda Pharmaceutical Group Co., Ltd. This strain was used for ARTP mutagenesis, and screening was performed using agar plates containing 10% sodium lactate. The specific steps are as follows:

[0047] (1) Microbial culture: Wild Streptococcus veterinaria was streaked on a plate culture medium and cultured at 35°C for 48 hours;

[0048] (2) Microbial expansion: Single colonies of strains cultured on plate medium are picked and inoculated into seed medium for expansion culture. The culture is carried out at 35℃ and 220rpm to the logarithmic phase to obtain seed liquid.

[0049] (3) ARTP mutagenesis:

[0050] 1) Preparation of bacterial suspension: Centrifuge the seed culture cultured to the logarithmic growth phase to collect bacterial cells. Wash the bacterial cells three times with sterile deionized water, and then prepare OD using sterile deionized water. 600Bacterial suspensions with a pH value between 0.6 and 0.8 were placed in 1.5 mL EP tubes;

[0051] 2) Sample preparation: In a clean bench, use a pipette to draw 10 μL of bacterial suspension and place it on a sterile slide for mutagenesis. Spread the bacterial suspension evenly with the pipette tip, and then place the slide in a sterile glass culture dish.

[0052] 3) Pretreatment with mutagenesis: The lethality of wild-type Streptococcus vesicularis was determined beforehand using ARTP mutagenesis. Glass culture dishes containing bacterial suspension were placed in an ARTP mutagenesis apparatus, and the mutagenesis parameters were set as follows: power and aeration rate of 120W and 10 SLM, respectively. Mutagenesis times were set to 30s, 60s, 90s, 120s, 150s, and 180s, with three replicates for each mutagenesis time. Unmutated wild-type Streptococcus vesicularis was used as a control. The lethality of the strains was calculated. The lethality curves are shown below. Figure 3 As shown in the figure, when the mutagenesis time is 120s, the lethality rate can reach over 96%, and when the mutagenesis time is increased to 180s, the lethality rate approaches 100%. Since a higher lethality rate makes it easier to screen for positive mutant strains in the mutant library, and the lethality rate reaches 96.15% at a mutagenesis time of 120s, the plasma mutagenesis dose at this lethality rate can ensure the number of samples in the mutant library while also having a high mutation efficiency. Therefore, 120s was selected as the subsequent mutagenesis time.

[0053] 4) Mutagenesis treatment: Place the glass culture dish containing the slide (containing bacterial suspension) in the ARTP mutagen, set the mutagenesis parameters, and carry out mutagenesis. The power and aeration rate are 120W and 10SLM, respectively; the mutagenesis time is 120s.

[0054] (4) Plate spreading: Dilute the bacterial suspension after ARTP mutagenesis on the slide with 990 μL of sterile physiological saline and place it in a 1.5 mL EP tube. Then take 100 μL and spread it evenly on a plate medium containing 10% sodium lactate. Incubate at 35℃ for 48 h and select well-growing strains.

[0055] (5) Initial screening: Following the methods in steps (1)-(2), the selected well-growing strains were cultured and expanded. The resulting seed culture was then inoculated into shake flasks containing fermentation medium at a volume ratio of 10%. The volume of fermentation medium was 200 mL / 1000 mL. The flasks were cultured in a constant temperature shaker at 35°C and 220 rpm. The OD of the fermentation broth was then measured at 4 h and 8 h. 600 Values, select the 4th and 8th hour OD. 600 Strains with higher values ​​showed rapid growth and long stability during the logarithmic phase.

[0056] (6) Secondary screening: The strains selected in the initial screening were cultured and expanded according to the method of steps (1)-(2). Then, the seed liquid was inoculated into the fermenter containing fermentation medium at a volume ratio of 10%. The volume of fermentation medium was 3.5L / 5L, the fermentation temperature was 35℃, the aeration rate was 1.0vvm, the pH was 7.0, the stirring speed was 300rpm, and the culture time was 24h. After the culture was completed, the hyaluronic acid content in the fermentation liquid was detected, and the strain corresponding to the highest hyaluronic acid content was selected.

[0057] (7) Stability test: The mutant strains selected in step (6) were subcultured multiple times, and the subcultured strains were cultured and expanded according to the method of steps (1)-(2). Then, the seed liquid was inoculated into the fermenter containing fermentation medium at a volume ratio of 10%. The volume of fermentation medium was 3.5L / 5L, the fermentation temperature was 35℃, the aeration rate was 1.0vvm, the pH was 7.0, the stirring speed was 300rpm, and the culture time was 24h. After the culture was completed, the hyaluronic acid content in the fermentation liquid was detected. The results are shown in Table 1.

[0058] Table 1. Hyaluronic acid content in fermentation broth of passaged Streptococcus vesiculosus mutant strains

[0059]

[0060] Compared with the wild-type Streptococcus vesicae (hyaluronic acid production of 14.5 g / L), the hyaluronic acid production of the mutant Streptococcus vesicae strain was 15-16 g / L. After ARTP mutagenesis, a genetically stable mutant Streptococcus vesicae strain with further increased hyaluronic acid production was obtained, which was named A1.

[0061] Single colonies of the selected Streptococcus vesiculosus mutant strain A1 were inoculated into seed culture medium and cultured. The sodium lactate concentrations of the seed culture medium were set at 0, 10, 20, 40, and 60 g / L. The culture conditions were: temperature 35℃, shaker speed 220 rpm, initial pH 7.4, and culture time 32 h. The OD of the seed culture was measured every 4 h. 600 Values ​​were calculated, and growth curves of the strains were plotted, such as... Figure 4 As shown. (Through) Figure 4 It can be seen that the growth of mutant strain A1 is more inhibited with increasing sodium lactate concentration, thus affecting hyaluronic acid production. Therefore, mutant strain A1 was selected for further lactate tolerance acclimatization.

[0062] Example 2:

[0063] Lactic acid tolerance was acclimated stepwise in plate culture using the Streptococcus vesiculosus mutant strain A1 screened in Example 1. The specific steps are as follows:

[0064] (1) Streptococcus vesicular mutant strain A1 was streaked on a plate medium and activated at 35°C for 48 h to obtain single colonies with smooth edges and regular round shape.

[0065] (2) Select a single colony from step (1) and streak it onto a plate medium with a low sodium lactate content of 15 g / L. Incubate at 35°C and screen out strains that are tolerant to low lactate content and grow well. Inoculate these strains onto slant medium and incubate at 35°C. Inoculate the colonies that grow on the slant medium into a fermentation medium with a low sodium lactate content (15 g / L) and carry out shake flask fermentation. The volume of the fermentation medium is 200 mL / 1000 mL. Incubate at 35°C and 220 rpm for 24 h in a constant temperature shaker. Select the strain with the highest hyaluronic acid production as the strain tolerant to low sodium lactate content for preservation.

[0066] (3) The strains selected in step (2) were streaked onto a plate medium with a sodium lactate content of 30 g / L and cultured at 35°C to screen out strains that were tolerant to high lactic acid content and grew well. These strains were then inoculated onto slant medium and cultured at 35°C. The colonies that grew on the slant medium were then inoculated into a fermentation medium with a sodium lactate content of 35 g / L and fermented in shake flasks. The volume of the fermentation medium was 200 mL / 1000 mL. The culture was carried out in a constant temperature shaker at 35°C and 220 rpm for 24 h. The strain with the highest hyaluronic acid production was selected as the strain tolerant to medium sodium lactate content and preserved.

[0067] (4) The strains selected in step (3) were streaked onto a plate medium with a high sodium lactate content of 45 g / L and cultured at 35°C to screen out strains that were resistant to high lactic acid content and grew well. These strains were then inoculated onto slant medium and cultured at 35°C. The colonies that grew on the slant medium were then inoculated into a fermentation medium with a high sodium lactate content (45 g / L) for shake-flask fermentation. The volume of the fermentation medium was 200 mL / 1000 mL. The culture was carried out in a constant temperature shaker at 35°C and 220 rpm for 24 h. The strain with the highest hyaluronic acid production was selected as the strain resistant to high sodium lactate content and preserved. It was named B1.

[0068] (5) Inoculate the domesticated strain B1 onto an agar slant and activate it at 35°C for 24 hours; inoculate the activated strain into a primary shake flask containing seed culture medium (100 mL / 500 mL) and culture it at 35°C and 220 rpm for 15 hours; inoculate the activated primary seed culture into a secondary shake flask containing seed culture medium (200 mL / 1000 mL) at a volume ratio of 10% and culture it at 35°C and 220 rpm until OD. 600 The value was 1.2. The cultured secondary seed culture was inoculated into a fermenter containing fermentation medium at a volume ratio of 10%. The fermentation medium volume was 3.5L / 5L. The culture conditions were: temperature 35℃, aeration rate 1.0 vvm, pH controlled at 7.0 by adding 30% sodium hydroxide solution, stirring speed 300 rpm, and fermentation time 24 h. The same steps were used to produce hyaluronic acid using the mutant strain A1.

[0069] The hyaluronic acid content in the fermentation broth was tested. The results showed that the final fermentation broth viscosity of the Streptococcus vesicular mutant strain A1 reached 54000 mPa·s, and the hyaluronic acid content in the fermentation broth was 15.5 g / L; the final fermentation broth viscosity of the Streptococcus vesicular domesticated strain B1 was 58000 mPa·s, and the hyaluronic acid content in the fermentation broth was 16.2 g / L.

[0070] Example 3:

[0071] Lactic acid tolerance was acclimated stepwise during the seed culture stage using the Streptococcus vesiculosus mutant strain A1 screened in Example 1. The specific steps are as follows:

[0072] (1) Streptococcus veterinaria mutant strain A1 was streaked on a plate medium and activated at 35°C for 48h.

[0073] (2) Select single colonies with smooth edges and large volume from step (1) and streak them onto slant culture medium. Incubate them in a 35℃ incubator for 24 hours.

[0074] (3) The colonies grown on the slant culture medium in step (2) were inoculated into seed culture medium for shake flask culture. The seed culture medium volume was 200mL / 1000mL. The culture was carried out in a shaker at 35℃ and 220rpm for 24h. Sodium lactate was added at the adjustment phase, logarithmic phase, stationary phase and death phase of the strain growth, respectively. The sodium lactate content was 15g / L to acclimate the strain to low lactic acid content. The cultured seed liquid was inoculated into a 5L fermenter containing fermentation medium at a volume ratio of 10% for fermentation. The fermentation medium volume was 3.5L / 5L, the temperature was 35℃, the aeration rate was 1.0vvm, the pH was controlled by adding 30% sodium hydroxide solution to 7.0, the stirring speed was 300rpm, and the culture time was 24h. After the fermentation was completed, the hyaluronic acid content in the fermentation liquid was detected. The strain with the highest hyaluronic acid content in each growth phase was selected as the acclimatized strain for preservation.

[0075] (4) The strains selected in step (3) are activated and cultured according to the methods in steps (1)-(2), and the strains are domesticated and fermented to tolerate medium lactic acid according to step (3). During the adjustment phase, logarithmic phase, stationary phase or death phase of the corresponding strains, the sodium lactate content in the seed culture medium is increased to 30 g / L respectively. The strains with the highest hyaluronic acid content among the domesticated strains in each growth phase are selected as domesticated strains for preservation.

[0076] (5) The strains selected in step (4) are subjected to high lactic acid tolerance acclimatization and fermentation according to step (4). During the adjustment period, logarithmic period, stationary period and death period of the corresponding strains, the sodium lactate content in the seed culture medium is increased to 45 g / L respectively. The strains with the highest hyaluronic acid content in each growth period are selected as acclimatized strains for preservation.

[0077] (6) Inoculate the strains selected in step (5) onto slant culture medium and activate them at 35℃ for 24 hours; inoculate the activated strains into a primary shake flask containing seed culture medium (100 mL / 500 mL) and culture them at 35℃ and 220 rpm for 15 hours; inoculate the activated primary seed culture into a secondary shake flask containing seed culture medium at a volume ratio of 10% (200 mL / 1000 mL) and culture them at 35℃ and 220 rpm until OD. 600The value was 1.2. The cultured secondary seed liquid was inoculated into a fermenter containing fermentation medium at a volume ratio of 10% for fermentation culture. The volume of fermentation medium was 3.5L / 5L. The culture conditions were: temperature 35℃, aeration rate 1.0 vvm, pH controlled at 7.0 by adding 30% sodium hydroxide solution, stirring speed 300 rpm, fermentation for 24h, and the hyaluronic acid content in the fermentation broth was detected. The strain with the highest hyaluronic acid content was selected as the domestication strain and preserved, named B2.

[0078] The results of detecting the viscosity and hyaluronic acid content of the fermentation broth of Streptococcus vesiculosus mutant strain A1 after lactic acid tolerance stepwise acclimatization during the seed culture stage are shown in Table 2.

[0079] Table 2. Viscosity and hyaluronic acid content of fermentation broth after domestication of Streptococcus vesiculosus mutant strain A1

[0080]

[0081] As shown in Table 2, the domesticated strain B2, obtained by stepwise acclimatization to lactic acid tolerance during the logarithmic growth phase of the strain in the seed culture stage, had a viscosity of 62000 mPa·s and an HA content of 17.2 g / L after 24 hours of aerated fermentation.

[0082] Example 4:

[0083] Lactic acid tolerance was acclimated stepwise during the fermentation culture stage using the Streptococcus vesiculosus mutant strain A1 screened in Example 1. The specific steps are as follows:

[0084] (1) Streptococcus veterinaria mutant strain A1 was streaked on a plate medium and activated at 35°C for 48h.

[0085] (2) Select single colonies with smooth edges and large volume from step (1) and streak them onto slant culture medium. Incubate them in a 35℃ incubator for 24 hours.

[0086] (3) Inoculate the colonies that grow on the slant culture medium in step (2) into seed culture medium and culture in shake flasks. The volume of seed culture medium is 200mL / 1000mL. Culture in a shaker at 35℃ and 220rpm for 20h to obtain seed liquid.

[0087] (4) The seed culture prepared in step (3) was inoculated into a 5L fermenter containing fermentation medium at a volume ratio of 10% for fermentation. The volume of fermentation medium was 3.5L / 5L, the temperature was 35℃, the aeration rate was 1.0 vvm, the pH was controlled at 7.0 by adding 30% sodium hydroxide solution, the stirring speed was 300 rpm, and the culture time was 24h. During the fermentation process, sodium lactate was added at the adjustment phase, logarithmic phase, stationary phase, and death phase of the strain growth, with a sodium lactate content of 15g / L, to acclimate the strain to low lactic acid content. The strain with the highest hyaluronic acid content among the acclimatized strains at each growth stage was selected as the acclimatized strain for preservation.

[0088] (5) The strains selected in step (4) are activated and cultured according to the methods in steps (1)-(3), and the strains are domesticated and fermented according to step (4). In the process, the sodium lactate content in the fermentation culture is increased to 30 g / L during the adjustment phase, logarithmic phase, stationary phase and death phase of the corresponding strains. The strains with the highest hyaluronic acid content in each growth phase are selected as domesticated strains for preservation.

[0089] (6) The strains selected in step (5) are domesticated and fermented according to step (5). During the adjustment phase, logarithmic phase, stationary phase and death phase of the corresponding strains, the sodium lactate content in the fermentation medium is increased to 45 g / L respectively. The strain with the highest hyaluronic acid content among the domesticated strains in each growth phase is selected as the domesticated strain for preservation.

[0090] (7) Inoculate the strains selected in step (6) onto slant culture medium and activate them at 35°C for 24 hours; inoculate the activated strains into a primary shake flask containing seed culture medium at a volume of 100 mL / 500 mL, and culture them at 35°C and 220 rpm for 15 hours; inoculate the prepared primary seed culture into a secondary shake flask containing seed culture medium at a volume ratio of 10%, with a seed culture medium volume of 200 mL / 1000 mL, and culture them at 35°C and 220 rpm until OD. 600 The value was 1.2. The cultured secondary seed liquid was inoculated into a fermenter containing fermentation medium at a volume ratio of 10% for fermentation culture. The volume of fermentation medium was 3.5L / 5L. The culture conditions were: temperature 35℃, aeration rate 1.0 vvm, pH controlled at 7.0 by adding 30% sodium hydroxide solution, stirring speed 300 rpm, fermentation for 24h, and the hyaluronic acid content in the fermentation broth was detected. The strain with the highest hyaluronic acid content was selected as the domestication strain and preserved, named B3.

[0091] The results of detecting the viscosity and hyaluronic acid content of the fermentation broth after the Streptococcus vesiculosus mutant strain A1 underwent a stepwise acclimatization process to lactic acid tolerance during the fermentation culture stage are shown in Table 3.

[0092] Table 3. Viscosity and hyaluronic acid content of fermentation broth after domestication of Streptococcus vesiculosus mutant strain A1

[0093]

[0094] As shown in Table 3, the domesticated strain B3, obtained by stepwise domestication of lactic acid tolerance during the logarithmic growth phase of the strain in the fermentation culture stage, had a viscosity of 55500 mPa·s and a hyaluronic acid content of 15.2 g / L after 24 hours of aerated fermentation.

[0095] In Examples 2, 3, and 4 of this invention, sodium lactate was used to induce lactic acid tolerance in a stepwise manner for the mutant strain A1 of Streptococcus vesiculosus during the plate culture, seed culture, and fermentation culture stages, respectively. Among them, the domesticated strain B2, obtained after undergoing stepwise lactic acid tolerance acclimatization during the logarithmic growth phase of the strain in the seed culture stage, had the highest hyaluronic acid yield, with a final fermentation broth viscosity of 62000 mPa·s and a hyaluronic acid content of 17.2 g / L.

[0096] The aforementioned domesticated strain B2 is *Streptococcus zooepidemicus* KFA018, which was deposited on October 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242194. This strain is the one that underwent ARTP mutagenesis followed by lactic acid domestication, achieving the best domestication results.

[0097] Example 5:

[0098] The colony morphology of Streptococcus vesiculosus mutant strain A1 and Streptococcus vesiculosus KFA018 on plate culture medium is as follows: Figure 5 As shown in the figure, the colony morphology of Streptococcus vesiculosus mutant strain A1 before lactic acid domestication is relatively small, and some colonies are irregular in shape; after lactic acid domestication, the colony edge of Streptococcus vesiculosus KFA018 is smooth, presenting a regular round shape, and the colony is surrounded by a large transparent capsule with a "raindrop" shape. Usually, the production of hyaluronic acid is directly proportional to the volume of the transparent capsule.

[0099] Seed culture was performed using Streptococcus vesica mutant strain A1 and Streptococcus vesica KFA018. The resulting seed culture (OD) was then analyzed. 600=1.2) Fermentation was carried out by inoculating the culture medium into a 5L fermenter containing fermentation medium at a volume ratio of 10% (3.5L / 5L). The temperature was 35℃, the aeration rate was 1.0 vvm, the pH was controlled to 7.0 by adding 30% sodium hydroxide solution, the stirring speed was 300 rpm, and the incubation time was 24 h. The OD of the fermentation broth was measured during the fermentation process. 600 Value and residual sugar (calculated as glucose) content, results are as follows Figure 6 As shown in the figure, the domesticated Streptococcus vesicanthii KFA018 grows faster, enters the logarithmic growth phase earlier and for a longer period, and has a higher OD value. 600 The value is higher than that of the undomesticated Streptococcus pneumoniae mutant strain A1. Streptococcus pneumoniae is a semi-coupled fermenter, and there is an indirect relationship between the growth of microorganisms and the production of products. Furthermore, the domesticated Streptococcus pneumoniae KFA018 consumes more glucose during fermentation, thus improving the utilization rate of glucose.

[0100] Example 6:

[0101] The viscosity of the fermentation broth can be reduced and the yield of hyaluronic acid increased by adding hyaluronidase. The specific steps are as follows:

[0102] Mutagenized and lactic acid-acclimated Streptococcus vesiculosus KFA018 was inoculated into a 500 mL shake flask containing 100 mL of seed culture medium and cultured at 35 °C and 220 rpm to obtain OD. 600 A seed culture with a pH of 1.2 was inoculated at a 10% volume ratio into a 5L fermenter containing 3.5L of fermentation medium for aerated fermentation. The temperature was 35℃, the aeration rate was 1.0 vvm, the pH was controlled at 7.0 by adding 30% sodium hydroxide solution, the stirring speed was 300 rpm, and the fermentation time was 24 hours. During fermentation, when the viscosity of the fermentation broth increased to 5000, 10000, 20000, and 30000 mPa·s, hyaluronidase at a final concentration of 1000 U / mL was added to reduce the viscosity, for a total of 5 additions. This hyaluronidase was an intracellular enzyme produced by Enterobacter fermentation, described in patent document CN110923173A (application number 201911365498.0). Finally, the viscosity and hyaluronic acid content of the fermentation broth after fermentation in the fermenter were measured, and the results are shown in Table 4.

[0103] Table 4. Viscosity and Hyaluronic Acid Content of Fermentation Broth of Streptococcus vesiculosus KFA018 | Viscosity of Fermentation Broth (mPa·S) at 24h with Hyaluronidase Addition | Hyaluronic Acid Content (g / L) | Viscosity of Fermentation Broth (mPa·S) at 24h

[0104]

[0105] The data in the table above shows that when the viscosity of the fermentation broth increased to 10000 mPa·s, adding hyaluronidase to reduce the viscosity resulted in the highest hyaluronic acid content in the broth. After 24 hours of fermentation, the viscosity of the broth was 3200 mPa·s, and the hyaluronic acid content was 18.5 g / L. In contrast, without the addition of hyaluronidase, after 24 hours of aerated fermentation of Streptococcus vesiculosus KFA018, the hyaluronic acid content was 17.2 g / L, and the broth viscosity was 62000 mPa·s.

[0106] After lactic acid domestication, the Streptococcus vesicularis KFA018 can effectively solve the problems of increased viscosity and decreased dissolved oxygen in the fermentation broth during the later stage of fermentation by adding hyaluronidase, thereby increasing dissolved oxygen, increasing the metabolic activity of the strain, and increasing the yield of hyaluronic acid.

[0107] In summary, this invention employs a stepwise acclimatization treatment of lactic acid tolerance during the logarithmic growth phase of the strain in the seed culture stage, thereby increasing the strain's tolerance to lactic acid and reducing the damage caused by lactic acid. Simultaneously, hyaluronidase is added according to the viscosity of the fermentation broth to reduce the viscosity, improve the mass transfer effect, promote the metabolic efficiency of the strain, and increase the yield of hyaluronic acid.

Claims

1. A strain of Streptococcus veterinaria ( Streptococcus zooepidemicus KFA018 was deposited on October 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20242194.

2. A bacterial agent comprising Streptococcus vesiculosus KFA018 as described in claim 1.

3. The application of the Streptococcus vesiculosus KFA018 of claim 1 or the bacterial agent of claim 2 in the preparation of hyaluronic acid.

4. A method for producing hyaluronic acid by fermentation using *Streptococcus vesiculosus* KFA018 as the fermentation strain according to claim 1, characterized in that, Includes the following steps: Streptococcus vesicularis KFA018 was inoculated onto a solid culture medium for activation culture, and the activated strain was inoculated into a seed culture medium for seed culture; the cultured seed liquid was inoculated into a fermentation culture medium for fermentation culture to obtain a fermentation broth containing hyaluronic acid.

5. The method as described in claim 4, characterized in that, The solid culture medium consists of: glucose 5-20 g / L, peptone 5-20 g / L, yeast extract 5-10 g / L, dipotassium hydrogen phosphate 1-10 g / L, magnesium sulfate 0.1-2 g / L, and agar powder 15-20 g / L.

6. The method as described in claim 4, characterized in that, The activation culture conditions are: 35-37℃ for 20-50 hours.

7. The method as described in claim 4, characterized in that, The seed culture medium consists of: glucose 5-15 g / L, yeast extract 5-10 g / L, peptone 5-20 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.1-1 g / L, and NaCl 15-20 g / L.

8. The method as described in claim 4, characterized in that, The seed culture conditions are: 35-37℃ and 200-220 rpm.

9. The method as described in claim 4, characterized in that, The OD of the seed liquid 600 The value is 1.0-2.

0.

10. The method as described in claim 4, characterized in that, The fermentation medium consists of: 50-100 g / L glucose, 5-10 g / L yeast extract, 5-20 g / L peptone, 1-5 g / L dipotassium hydrogen phosphate, and 0.1-1 g / L magnesium sulfate.

11. The method as described in claim 4, characterized in that, The fermentation conditions are as follows: temperature 35-37℃, stirring speed 200-300 rpm, pH controlled at 6.0-8.0 with sodium hydroxide solution, and fermentation with ventilation for 20-30 h.

12. The method as described in claim 4, characterized in that, The inoculation amount of seed liquid in the fermentation medium is 5-10% by volume.

13. The method as described in claim 4, characterized in that, The method also includes the step of: during the fermentation process, when the viscosity of the fermentation broth increases to 5000-30000 mPa·s, adding hyaluronidase at a final concentration of 800-1200 U / mL to reduce the viscosity of the fermentation broth, and adding it 5-6 times in total.

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