Streptococcus zooepidemicus producing hyaluronic acid and application thereof

Abstract

The invention relates to streptococcus zooepidemicus for producing hyaluronic acid and application of the streptococcus zooepidemicus, and belongs to the technical field of bioengineering. The streptococcus zooepidemicus KFA018 resistant to high-content lactic acid and high in hyaluronic acid yield is obtained through ARTP mutagenesis and high-lactic acid tolerance domestication, and the problems that existing streptococcus zooepidemicus is low in hyaluronic acid yield, low in lactic acid tolerance and the like are effectively solved. The hyaluronic acid yield of the streptococcus zooepidemicus KFA018 disclosed by the invention is as high as 17-18g / L. Meanwhile, the lactic acid content of the streptococcus zooepidemicus KFA018 can reach 45 g / L, the streptococcus zooepidemicus KFA018 has the characteristic of high lactic acid resistance, and a very excellent strain selection is provided for production of hyaluronic acid.

Description

Technical Field

[0001] The invention relates to a strain of zooepidemic streptococcus producing hyaluronic acid and application thereof, belonging to the technical field of bioengineering. Background Art

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

[0003] According to the molecular weight of hyaluronic acid, it can be divided into ultra-high molecular weight (molecular weight greater than 3000KDa), high molecular weight (1000KDa-3000 KDa), medium molecular weight (100KDa-1000 KDa) and low molecular weight (less than 100KDa) hyaluronic acid. Hyaluronic acid of different molecular weights has 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 good moisturizing and anti-inflammatory functions, and is used as a viscoelastic agent and intra-articular injection therapeutic agent in ophthalmic surgery; medium molecular weight hyaluronic acid is widely used in the cosmetics field due to its superior moisturizing and lubricating effects; low molecular weight hyaluronic acid and its oligosaccharides have anti-tumor, accelerated wound healing, bone and angiogenesis promotion, immune regulation and other effects, and have good medical application prospects.

[0004] Microbial fermentation refers to the process of using hyaluronic acid production strains to ferment in a fermenter to obtain hyaluronic acid. Compared with the animal tissue extraction method, the production process of microbial fermentation is safer, the product is more uniform and the cost is lower. The process mainly includes strain activation, seed culture, fermentation in a fermenter and extraction of hyaluronic acid.

[0005] When Streptococcus zooepidemicus ferments to produce HA, about 80% of the carbon source is converted into lactic acid through the glycolysis pathway. On the one hand, the generated lactic acid will compete with HA synthesis for limited carbon sources, and on the other hand, lactic acid has a strong inhibitory effect on bacterial growth and HA synthesis. Therefore, if the inhibitory effect of lactic acid on bacterial growth and HA synthesis can be reduced, the HA yield will be increased to a certain extent. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a strain of Streptococcus zooepidemicus producing hyaluronic acid and its application. The present invention obtains a strain of Streptococcus zooepidemicus with a fermented hyaluronic acid yield of 17-18 g / L through ARTP mutagenesis screening and step-wise lactic acid acclimation, and the maximum tolerable lactic acid concentration is 45 g / L, and has achieved fermentation in a 5L system. The present invention effectively solves the problems of low hyaluronic acid yield and low lactic acid resistance of existing Streptococcus zooepidemicus.

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

[0008] A strain of Streptococcus zooepidemicus KFA018 was deposited in the China Center for Type Culture Collection on October 14, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20242194.

[0009] The zooepidemic streptococcus KFA018 described in the present invention is a strain with high hyaluronic acid production obtained through ARTP mutagenesis breeding and high-concentration lactic acid domestication. The strain also has high lactic acid tolerance, which enables the strain 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 above-mentioned Streptococcus zooepidemicus KFA018.

[0011] Application of the above-mentioned Streptococcus zooepidemicus KFA018 or bacterial agent in the preparation of hyaluronic acid.

[0012] A method for producing hyaluronic acid by fermentation using Streptococcus zooepidemicus KFA018 as a fermentation strain, comprising the following steps:

[0013] The zooepidemic streptococcus KFA018 is inoculated on a solid culture medium for activation culture, and the activated cultured strain is inoculated into a seed culture medium for seed culture; the cultured seed liquid is inoculated into a fermentation culture medium for fermentation culture to obtain a fermentation liquid containing hyaluronic acid.

[0014] Preferably according to the present invention, the solid culture medium is composed of: 5-20 g / L glucose, 5-20 g / L peptone, 5-10 g / L yeast powder, 1-10 g / L dipotassium hydrogen phosphate, 0.1-2 g / L magnesium sulfate, and 15-20 g / L agar powder.

[0015] Preferably according to the present invention, the activation culture conditions are: culture at 35-37° C. for 20-50 h.

[0016] Preferably according to the present invention, the composition of the seed culture medium is: glucose 5-15 g / L, yeast powder 5-10 g / L, peptone 5-20 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.1-1 g / L, NaCl 15-20 g / L.

[0017] Preferably according to the present invention, the seed culture conditions are: 35-37° C., 200-220 rpm.

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

[0019] Preferably according to the present invention, the fermentation medium comprises: 50-100 g / L glucose, 5-10 g / L yeast powder, 5-20 g / L peptone, 1-5 g / L dipotassium hydrogen phosphate, and 0.1-1 g / L magnesium sulfate.

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

[0021] Preferably, the fermentation culture conditions are as follows: temperature of 35-37°C, stirring speed of 200-300 rpm, pH of 6.0-8.0 controlled by sodium hydroxide solution, and fermentation with ventilation for 20-30 hours.

[0022] Preferably, according to the present invention, the method further comprises the step of: during the fermentation culture process, when the viscosity of the fermentation broth increases to 5000-30000 mPa·s, adding hyaluronidase with a final concentration of 800-1200 U / mL to reduce the viscosity of the fermentation broth, and adding 5-6 times in total.

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

[0024] 1. Stabilize strain growth and metabolic activity: In the process of fermentation and production of hyaluronic acid, lactic acid is an important organic acid produced by bacterial metabolism. Improving lactic acid tolerance can help the production strain better cope with the high-concentration lactic acid environment and maintain stable growth and metabolic activities of the strain. 2. Reduce production costs: Fermentation under low pH conditions can reduce the use of neutralizers, thereby reducing production costs. 3. Strong adaptability: Streptococcus zooepidemicus grown under high lactic acid concentrations may have stronger adaptability and be able to cope with various environmental pressures that may be encountered during industrial production.

[0025] Beneficial effects:

[0026] The present invention obtains a strain of Streptococcus zooepidemicus KFA018 that is resistant to high-content lactic acid and high-yield hyaluronic acid through ARTP mutagenesis and high lactic acid tolerance domestication. In the process of microbial fermentation to produce hyaluronic acid, the large accumulation of lactic acid will cause the pH value of the fermentation liquid to decrease. When the pH value is lower than the pKa value of lactic acid, free lactic acid can penetrate the cell membrane into the cell, which will reduce the intracellular pH on the one hand and affect the stability of the cell membrane on the other hand. By domesticating the acid resistance of the hyaluronic acid production strain, the microbial strain can gradually adapt to a specific acidic environment and utilize the substrate more efficiently, thereby increasing the yield of the target product. The hyaluronic acid yield of the Streptococcus zooepidemicus KFA018 of the present invention is as high as 17-18g / L. At the same time, the Streptococcus zooepidemicus KFA018 of the present invention has a growth resistance to lactic acid content of up to 45g / L, has a high lactic acid resistance characteristic, and provides a very good strain selection for the production of hyaluronic acid. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0029] Figure 3 is the lethality curve of ARTP-induced strain;

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

[0031] Figure 5 The colony morphology of Streptococcus zooepidemicus mutant strain A1 and Streptococcus zooepidemicus KFA018;

[0032] Figure 6 OD of mutant strain A1 and KFA018 of Streptococcus zooepidemicus during fermentation 600 Value and residual sugar change curve. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0034] Unless otherwise specified, the raw materials and reagents in the examples of the present invention are commercially available products.

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

[0036] Plate culture medium and slant culture medium: 8g / L glucose, 15g / L peptone, 8g / L yeast powder, 4g / L potassium dihydrogen phosphate, 1g / L magnesium sulfate, 15g / L agar powder, prepared with sterile deionized water, adjusted to pH 7.4 with 1mol / L sodium hydroxide solution, sterilized at 121℃ for 20min. Pour the sterilized culture medium into a sterile culture dish or test tube and wait for it to cool and solidify.

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

[0039] The ARTP mutagenesis machine used in the embodiment: 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 hyaluronic acid content detection method used in the embodiment comprises the following steps:

[0041] (1) Draw a standard curve: Pipette 0.05mL, 0.1mL, 0.2mL, 0.5mL, 1.0mL and 2.0mL of 1.0mg / mL hyaluronic acid standard solution into a 15mL stoppered graduated test tube, add phosphate buffer (pH 7.4) to 9mL, then add 1mL hyaluronidase solution (1000IU / mL) and mix well. The final mass concentrations of hyaluronic acid standards are 0.005mg / mL, 0.010mg / mL, 0.020mg / mL, 0.050mg / mL, 0.100mg / mL and 0.200mg / mL, respectively. Perform enzymatic hydrolysis in a 37℃ water bath for 1h, and boil in a boiling water bath for 2min to terminate the reaction. After the reaction solution is cooled to room temperature, filter with a 0.22μm filter membrane and perform high performance liquid chromatography detection. Use the working mass concentration of the hyaluronic acid standard series as the horizontal axis and the peak area as the vertical axis to draw a standard curve, as shown in the figure. Figure 1As shown, the standard curve fitting equation is y = 25246x + 6.2447, R 2 =1; Figure 2 This is the HPLC chromatogram of hyaluronic acid standard solution.

[0042] Among them, the high performance liquid chromatography method is carried out in accordance with the records of the industry standard QB / T 4576-2023, wherein the chromatographic column: sulfonated cross-linked styrene divinylbenzene copolymer strong cation exchange chromatographic column; chromatographic conditions: flow rate 0.6mL / min, injection volume 20μL, column temperature 40°C, detection wavelength 232nm.

[0043] (2) Detection of test samples: Take the fermentation liquid to be tested, dilute it by an appropriate multiple, transfer 0.5 mL of the diluted fermentation liquid to be tested into a 15 mL stoppered graduated test tube, add 8.5 mL of phosphate buffer, then add 1.0 mL of hyaluronidase solution, mix well, enzymolyze in a 37°C water bath for 1 h, then boil in a boiling water bath for 2 min to terminate the reaction, cool the reaction liquid to room temperature, filter with a 0.22 μm filter membrane, and perform high performance liquid chromatography detection. Calculate the hyaluronic acid content in the fermentation liquid based on the standard curve.

[0044] In the embodiment, a visible ultraviolet spectrophotometer was used to detect OD 600 The values ​​were determined by using a biosensor analyzer to determine the glucose and sodium lactate contents; the viscosity was tested according to the "Pharmacopoeia 2015 Edition Part 4 General Rules 0633 Viscosity Determination Method Part 3 Rotational Viscometer Determination Method".

[0045] Embodiment 1:

[0046] The starting strain used in this experiment is a wild strain of Streptococcus zooepidemicus, which can produce hyaluronic acid. It was screened by Shandong Freda Pharmaceutical Group Co., Ltd. and is now preserved in the Synthetic Biology and Biomanufacturing Research Institute of Shandong Freda Pharmaceutical Group Co., Ltd. The strain was used for ARTP mutagenesis and screened using a plate culture medium containing 10% sodium lactate. The specific steps are as follows:

[0047] (1) Microbial culture: Streak wild Streptococcus zooepidemicus on a plate culture medium and culture at 35°C for 48 h;

[0048] (2) Microbial expansion: A single colony of the strain cultured on the plate medium was selected and inoculated into the seed medium for expansion. The culture was cultured at 35°C and 220 rpm until the logarithmic phase to obtain the seed solution;

[0049] (3) ARTP mutagenesis:

[0050] 1) Preparation of bacterial suspension: The seed liquid cultured to the logarithmic phase was centrifuged to collect the bacterial cells, and the bacterial cells were washed 3 times with sterile deionized water, and the OD was prepared with sterile deionized water. 600The bacterial suspension with a value between 0.6 and 0.8 was placed in a 1.5 mL EP tube;

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

[0052] 3) Pretreatment of mutagenesis: ARTP mutagenesis was used in advance to determine the lethality of wild Streptococcus zooepidemicus. A glass culture dish with a slide (containing bacterial suspension) was placed in an ARTP mutagenizer. The mutagenesis parameters were set for mutagenesis. The power and ventilation volume were 120 W and 10 SLM, respectively. The mutagenesis time was set to 30 s, 60 s, 90 s, 120 s, 150 s, and 180 s, respectively. Three parallels were set for each group of mutagenesis time. Wild Streptococcus zooepidemicus that was not mutated was used as a control to calculate the lethality of the strain. The lethality curve of the strain is shown in FIG. Figure 3 As shown in the figure, when the mutagenesis time is 120s, the lethality can reach more than 96%, and when the mutagenesis time increases to 180s, the lethality is close to 100%. Since it is easier to screen positive mutant strains in the mutation library when the lethality is high, and when the mutagenesis time is 120s, the lethality reaches 96.15%. The plasma mutagenesis dose under this lethality can not only ensure the number of samples in the mutation library, but also has a high mutation efficiency, so 120s is selected as the subsequent mutagenesis time;

[0053] 4) Mutagenesis: Place the glass culture dish with the slide (containing bacterial suspension) in the ARTP mutagenesis instrument, set the mutagenesis parameters for mutagenesis, where the power and ventilation volume are 120 W and 10 SLM respectively; the mutagenesis time is 120 s;

[0054] (4) Plate coating: Dilute the bacterial suspension on the slide after ARTP mutagenesis with 990 μL of sterile saline and place it in a 1.5 mL EP tube. Then take 100 μL and evenly coat it on a plate medium containing 10% sodium lactate. Place it in a 35°C incubator and culture it for 48 h. Select the strains with good growth.

[0055] (5) Preliminary screening: According to the method of steps (1)-(2), the selected strains with good growth were cultured and expanded, and then the obtained seed liquid was inoculated into a shake flask containing fermentation medium at a volume ratio of 10%, and the fermentation medium was filled with 200mL / 1000mL. The culture was carried out in a constant temperature shaker at 35°C and 220rpm, and the OD of the fermentation liquid was measured at 4h and 8h respectively. 600 Value, select 4h and 8hOD 600 The strains with higher values ​​all showed rapid growth in the logarithmic phase and a long stable period.

[0056] (6) Rescreening: The strains selected in the initial screening were cultured and expanded according to the methods of steps (1)-(2), and then the seed liquid was inoculated into a fermentation tank containing a fermentation medium at an inoculum volume ratio of 10%, the fermentation medium volume was 3.5L / 5L, the fermentation temperature was 35°C, the ventilation volume 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 a strain corresponding to the highest hyaluronic acid content was selected;

[0057] (7) Stability test: The mutant strain selected by the rescreening in step (6) was subcultured for multiple times, and the subcultured strain was cultured and expanded according to the method of steps (1)-(2), and then the seed liquid was inoculated into a fermentation tank containing fermentation medium at an inoculum volume ratio of 10%, the fermentation medium liquid volume was 3.5L / 5L, the fermentation temperature was 35°C, the ventilation volume 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 broth was detected. The results are shown in Table 1.

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

[0059]

[0060] Compared with wild Streptococcus zooepidemicus (hyaluronic acid production of 14.5g / L), the hyaluronic acid production of the mutant strain of Streptococcus zooepidemicus is 15-16g / L, that is, after ARTP mutagenesis treatment, a mutant strain of Streptococcus zooepidemicus with further improved hyaluronic acid production and genetic stability was obtained, which was named A1.

[0061] The single colony of the mutant strain A1 of Streptococcus zooepidemicus screened above was inoculated into the seed culture medium for cultivation, and the sodium lactate concentration of the seed culture medium was set to 0, 10, 20, 40, and 60 g / L, respectively. The culture conditions were temperature 35°C, shaker speed 220 rpm, initial pH 7.4, culture time 32 h, and the OD of the seed solution was detected every 4 h. 600 Values, and draw the strain growth curve, such as Figure 4 As shown. Figure 4 It can be seen that as the concentration of sodium lactate increases, the growth of mutant strain A1 is more inhibited, affecting the production of hyaluronic acid. Therefore, mutant strain A1 was selected to continue the domestication of lactic acid tolerance.

[0062] Embodiment 2:

[0063] The mutant strain A1 of Streptococcus zooepidemicus screened in Example 1 was used for stepwise domestication of lactic acid tolerance in the plate culture stage, and the specific steps were as follows:

[0064] (1) Streak the mutant strain A1 of Streptococcus zooepidemicus on a plate culture medium, activate and culture at 35°C for 48 hours, and obtain a single colony with smooth colony edges and a regular circular shape;

[0065] (2) Pick a single colony from step (1) and streak it on a plate medium with a low sodium lactate content of 15 g / L, and culture it at 35° C. to screen out strains that have good growth and are resistant to low lactic acid content; inoculate these strains on a slant medium and culture it at 35° C., and inoculate the colonies grown on the slant medium into a fermentation medium with a low sodium lactate content (15 g / L) for shake flask fermentation, with the fermentation medium filling volume being 200 mL / 1000 mL, and culture it in a constant temperature shaker at 35° C. and 220 rpm for 24 h, and pick the strain with the highest hyaluronic acid production as the strain resistant to low sodium lactate content for preservation;

[0066] (3) streaking the strains selected in step (2) onto a plate medium with a sodium lactate content of 30 g / L, culturing at 35° C., and screening out strains with good growth and tolerance to high lactic acid content; inoculating these strains onto a slant medium, culturing at 35° C., and inoculating the colonies grown on the slant medium into a fermentation medium with a sodium lactate content of 35 g / L for shake flask fermentation, the fermentation medium having a liquid volume of 200 mL / 1000 mL, culturing in a constant temperature shaker at 35° C. and 220 rpm for 24 h, and selecting the strain with the highest hyaluronic acid production as the strain resistant to medium sodium lactate content for preservation;

[0067] (4) streaking the strains selected in step (3) onto a plate medium with a high sodium lactate content of 45 g / L and culturing at 35° C. to screen out strains with good growth and tolerance to high lactic acid content; inoculating these strains onto a slant medium and culturing at 35° C., inoculating the colonies grown on the slant medium into a fermentation medium with a high sodium lactate content (45 g / L) for shake flask fermentation, the fermentation medium having a liquid volume of 200 mL / 1000 mL, culturing in a constant temperature shaker at 35° C. and 220 rpm for 24 h, selecting the strain with the highest hyaluronic acid production as a strain resistant to high sodium lactate content for preservation, and naming it B1;

[0068] (5) Inoculate the domesticated strain B1 on the slant culture medium and activate and culture it at 35°C for 24 hours; inoculate the activated cultured strain into a primary shake flask containing a seed culture medium for cultivation, the seed culture medium liquid volume is 100mL / 500mL, and the culture conditions are: culture in a shaker at 35°C and 220rpm for 15 hours; inoculate the cultured primary seed liquid into a secondary shake flask containing a seed culture medium at an inoculum volume of 10%, the seed culture medium liquid volume is 200mL / 1000mL, and the culture conditions are: culture in a shaker at 35°C and 220rpm until OD 600 The value is 1.2; the cultured secondary seed liquid is inoculated into a fermentation tank containing a fermentation medium at a volume ratio of 10% for fermentation culture, the fermentation medium is filled with 3.5L / 5L, and the culture conditions are: temperature 35°C, ventilation 1.0vvm, pH 7.0 is controlled by feeding 30% sodium hydroxide solution, stirring speed 300rpm, and fermentation for 24h. The same steps are used to ferment and produce hyaluronic acid using mutant strain A1.

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

[0070] Embodiment 3:

[0071] The mutant strain A1 of Streptococcus zooepidemicus screened in Example 1 was used for step-wise domestication of lactic acid tolerance in the seed culture stage, and the specific steps were as follows:

[0072] (1) Streak the mutant strain A1 of Streptococcus zooepidemicus on a plate culture medium and activate and culture at 35°C for 48 hours;

[0073] (2) Select a single colony with smooth edges and large size in step (1), streak it onto a slant culture medium, and culture it in a 35°C incubator for 24 h;

[0074] (3) inoculating the colonies grown on the slant medium in step (2) into a seed medium for shake flask culture, wherein the seed medium has a liquid volume of 200 mL / 1000 mL, and the culture is carried out in a shaker at 35° C. and 220 rpm for 24 h, and sodium lactate is added in the adjustment period, logarithmic period, stable period, and decay period of the strain growth, respectively, with a sodium lactate content of 15 g / L, to perform low-content lactic acid tolerance acclimation; inoculating the cultured seed liquid into a 5L fermentation tank containing a fermentation medium at an inoculum volume ratio of 10% for fermentation, wherein the fermentation medium has a liquid volume of 3.5 L / 5 L, the temperature is 35° C., the ventilation volume is 1.0 vvm, the pH is controlled to 7.0 by feeding 30% sodium hydroxide solution, the stirring speed is 300 rpm, the culture time is 24 h, and after the fermentation is completed, the hyaluronic acid content in the fermentation liquid is detected, and the strain with the highest hyaluronic acid content in the acclimated strains in each growth period is selected as the acclimated strain for preservation;

[0075] (4) activating and culturing the strain selected in step (3) according to the method of steps (1)-(2), and acclimating and fermenting the strain to medium-content lactic acid according to step (3), wherein the sodium lactate content in the seed culture medium is increased to 30 g / L during the adjustment period, logarithmic period, stable period or decay period of the growth of the corresponding strain; and selecting the strain with the highest hyaluronic acid content in the acclimated strains in each growth period as the acclimated strain for preservation;

[0076] (5) subjecting the strain selected in step (4) to high-content lactic acid tolerance acclimation and fermentation according to step (4), wherein the sodium lactate content in the seed culture medium is increased to 45 g / L during the adjustment period, logarithmic period, stable period, and decay period of the growth of the corresponding strain, and the strain with the highest hyaluronic acid content among the acclimated strains in each growth period is selected as the acclimated strain for preservation;

[0077] (6) Inoculate the strain selected in step (5) on a slant culture medium and activate and culture at 35°C for 24 hours; inoculate the activated cultured strain into a primary shake flask containing a seed culture medium for culture, the seed culture medium liquid volume is 100mL / 500mL, and the culture conditions are: culture in a shaker at 35°C and 220rpm for 15 hours; inoculate the cultured primary seed liquid into a secondary shake flask containing a seed culture medium at an inoculum volume of 10%, the seed culture medium liquid volume is 200mL / 1000mL, and the culture conditions are: culture in a shaker at 35°C and 220rpm until OD 600The value is 1.2; the cultured secondary seed liquid is inoculated at a volume ratio of 10% into a fermentation tank containing a fermentation medium for fermentation culture. The fermentation medium is filled with 3.5L / 5L, and the culture conditions are: temperature 35°C, ventilation 1.0vvm, pH 7.0 is controlled by adding 30% sodium hydroxide solution, stirring speed 300rpm, fermentation for 24h, and the hyaluronic acid content in the fermentation liquid is detected. The strain with the highest hyaluronic acid content is preserved as an domesticated strain and named B2.

[0078] After the mutant strain A1 of Streptococcus zooepidemicus was subjected to step-wise acclimation of lactic acid tolerance in the seed culture stage, the test results of the viscosity of the fermentation broth and the content of hyaluronic acid are shown in Table 2.

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

[0080]

[0081] It can be seen from Table 2 that the domesticated strain B2 obtained by step-wise domestication of lactic acid tolerance in the logarithmic phase of strain growth at the seed culture stage had a fermentation liquid viscosity of 62000 mPa·s and a HA content of 17.2 g / L after 24 h of ventilation fermentation.

[0082] Embodiment 4:

[0083] The mutant strain A1 of Streptococcus zooepidemicus screened in Example 1 was used for stepwise domestication of lactic acid tolerance in the fermentation culture stage, and the specific steps were as follows:

[0084] (1) Streak the mutant strain A1 of Streptococcus zooepidemicus on a plate culture medium and activate and culture at 35°C for 48 hours;

[0085] (2) Select a single colony with smooth edges and large size in step (1), streak it onto a slant culture medium, and culture it in a 35°C incubator for 24 h;

[0086] (3) inoculating the colonies grown on the slant medium in step (2) into a seed medium for shake flask culture, wherein the seed medium is filled with 200 mL / 1000 mL and cultured in a shaker at 35° C. and 220 rpm for 20 h to obtain a seed solution;

[0087] (4) The seed liquid cultured in step (3) was inoculated into a 5L fermentation tank containing a fermentation medium at an inoculum volume ratio of 10% for fermentation. The fermentation medium was filled with 3.5L / 5L of liquid, the temperature was 35°C, the ventilation volume was 1.0vvm, the pH was controlled to 7.0 by adding 30% sodium hydroxide solution, the stirring speed was 300rpm, and the culture time was 24h. During the fermentation process, sodium lactate was added in the adjustment period, logarithmic period, stable period, and decay period of the strain growth, and the sodium lactate content was 15g / L to carry out low-content lactic acid tolerance domestication; the strain with the highest hyaluronic acid content in the domesticated strains in each growth period was selected as the domesticated strain for preservation;

[0088] (5) activating and culturing the strain selected in step (4) according to the methods of steps (1) to (3), and taming and fermenting the strain according to step (4), wherein the sodium lactate content in the fermentation culture is increased to 30 g / L during the adjustment period, logarithmic period, stable period, and decay period of the growth of the corresponding strain; and selecting the strain with the highest hyaluronic acid content in the tamed strains in each growth period as the tamed strain for preservation;

[0089] (6) performing strain domestication and fermentation according to step (5) on the strain selected in step (5), wherein the sodium lactate content in the fermentation medium is increased to 45 g / L during the adjustment period, logarithmic period, stable period, and decay period of the growth of the corresponding strain; and selecting the strain with the highest hyaluronic acid content among the domesticated strains in each growth period as the domesticated strain for preservation;

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

[0091] After the mutant strain A1 of Streptococcus zooepidemicus was subjected to step-wise acclimation of lactic acid tolerance during the fermentation culture stage, the test results of the viscosity of the fermentation broth and the hyaluronic acid content are shown in Table 3.

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

[0093]

[0094] It can be seen from Table 3 that the domesticated strain B3 obtained by step-wise domestication of lactic acid tolerance in the logarithmic phase of strain growth during the fermentation culture stage had a fermentation liquid viscosity of 55500 mPa·s and a hyaluronic acid content of 15.2 g / L after 24 h of ventilation fermentation.

[0095] In Examples 2, 3, and 4 of the present invention, sodium lactate is used to perform step-by-step domestication of the mutant strain A1 of Streptococcus zooepidemicus with lactic acid tolerance in the plate culture stage, the seed culture stage, and the fermentation culture stage, respectively. Among them, the domesticated strain B2 obtained after the step-by-step domestication of lactic acid tolerance in the logarithmic phase of strain growth in the seed culture stage has the highest hyaluronic acid yield, the final fermentation broth viscosity is 62000 mPa·s, and the hyaluronic acid content is 17.2 g / L.

[0096] The domesticated strain B2 is Streptococcus zooepidemicus KFA018, which was deposited in the China Center for Type Culture Collection on October 14, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20242194. This strain is the strain that has undergone ARTP mutagenesis and then lactic acid domestication, and the domestication result is the best.

[0097] Embodiment 5:

[0098] The colony morphology of the mutant strain A1 of Streptococcus zooepidemicus and Streptococcus zooepidemicus KFA018 on the plate culture medium is as follows Figure 5 As shown in the figure, the colony morphology of the mutant strain A1 of Streptococcus zooepidemicus before lactic acid acclimation is relatively small, and some colonies have irregular morphology; the colony edge of Streptococcus zooepidemicus KFA018 after lactic acid acclimation is smooth and regular round, and there is a large transparent capsule around the colony, which has a "raindrop" shape. Usually, the hyaluronic acid production is proportional to the volume of the transparent capsule.

[0099] The mutant strain A1 of Streptococcus zooepidemicus and Streptococcus zooepidemicus KFA018 were used for seed culture. The seed solution (OD 600=1.2) inoculated into a 5L fermentation tank containing a fermentation medium at a volume ratio of 10%, the fermentation medium was filled with 3.5L / 5L, the temperature was 35°C, the ventilation volume was 1.0vvm, the pH was controlled at 7.0 by adding 30% sodium hydroxide solution, the stirring speed was 300rpm, and the culture time was 24h. The OD of the fermentation liquid was detected during the fermentation culture process. 600 Value and residual sugar (in terms of glucose) content, the results are as follows Figure 6 As shown in the figure, it can be seen that the domesticated Streptococcus zooepidemicus KFA018 grows faster, enters the logarithmic growth phase earlier and lasts longer, and the OD 600 The value is higher than that of the mutant strain A1 of Streptococcus zooepidemicus before domestication. Streptococcus zooepidemicus is a semi-coupled fermentation, and there is an indirect relationship between the growth of microorganisms and the production of products; and the domesticated Streptococcus zooepidemicus KFA018 consumes more glucose during the fermentation process, which improves the utilization rate of glucose.

[0100] Embodiment 6:

[0101] By adding hyaluronidase to the fermentation broth, the viscosity of the fermentation broth is reduced and the yield of hyaluronic acid is increased. The specific steps are as follows:

[0102] The mutated and lactic acid-acclimated Streptococcus zooepidemicus 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 an OD 600 The value is 1.2 seed liquid, and the seed liquid is inoculated into a 5L fermentation tank containing 3.5L fermentation medium according to the inoculum amount of 10% volume ratio for ventilation fermentation culture, the temperature is 35 ° C, the ventilation volume is 1.0vvm, and the pH is controlled by adding 30% sodium hydroxide solution 7.0, the stirring speed is 300rpm, and the fermentation time is 24h. During the fermentation process, when the viscosity of the fermentation broth increases to 5000, 10000, 20000, and 30000mPa·S, a final concentration of 1000U / mL hyaluronidase is added to reduce the viscosity of the fermentation broth, and a total of 5 times are added. The hyaluronidase is derived from the intracellular enzyme produced by Enterobacter fermentation, which is recorded in patent document CN110923173A (application number 201911365498.0). Finally, the fermentation broth at which the fermentation is terminated in the fermentation tank is tested for viscosity and hyaluronic acid content, and the results are shown in Table 4.

[0103] Table 4. Viscosity and hyaluronic acid content of the fermentation broth of Streptococcus zooepidemicus KFA018 Viscosity of the fermentation broth when hyaluronidase was added (mPa·S) Hyaluronic acid content at 24h (g / L) Viscosity of the fermentation broth at 24h (mPa·S)

[0104]

[0105] From the data in the table above, it can be seen that when the viscosity of the fermentation broth increases to 10000mPa·S, the addition of hyaluronidase reduces the viscosity of the fermentation broth, and the hyaluronic acid content in the fermentation broth is the highest. After 24 hours of fermentation, the viscosity of the fermentation broth is 3200mPa·S, and the hyaluronic acid content is 18.5g / L. Compared with Streptococcus zooepidemicus KFA018 without the addition of hyaluronidase, after 24 hours of ventilation fermentation, the hyaluronic acid content is 17.2g / L and the viscosity of the fermentation broth is 62000mPa·s.

[0106] After lactic acid acclimation, the addition of hyaluronidase to the zooepidemic Streptococcus KFA018 can effectively solve the problems of increased viscosity of the fermentation broth and decreased dissolved oxygen in the fermentation broth in the late fermentation period, increase the dissolved oxygen effect, increase the metabolic activity of the strain, and increase the production of hyaluronic acid.

[0107] In summary, the present invention adopts a lactic acid tolerance step-by-step acclimation treatment during the logarithmic phase of strain growth in the seed culture stage to increase the strain's tolerance to lactic acid and reduce the damage of lactic acid to the strain; at the same time, hyaluronidase is added according to the viscosity of the fermentation broth to reduce the viscosity of the fermentation broth, improve the mass transfer effect, promote the metabolic efficiency of the strain, and increase the hyaluronic acid yield.

Claims

1. A strain of Streptococcus zooepidemicus KFA018, deposited on October 14, 2024 in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M20242194.

2. A bacterial agent comprising the zooepidemic Streptococcus KFA018 according to claim 1.

3. Use of the zooepidemic streptococcus KFA018 described in claim 1 or the bacterial agent described in claim 2 in the preparation of hyaluronic acid.

4. A method for producing hyaluronic acid by fermenting Streptococcus zooepidemicus KFA018 according to claim 1 as a fermentation strain, characterized in that: The steps include: The zooepidemic streptococcus KFA018 is inoculated on a solid culture medium for activation culture, and the activated cultured strain is inoculated into a seed culture medium for seed culture; the cultured seed liquid is inoculated into a fermentation culture medium for fermentation culture to obtain a fermentation liquid containing hyaluronic acid.

5. The method according to claim 4, characterized in that The solid culture medium is composed of: 5-20 g / L glucose, 5-20 g / L peptone, 5-10 g / L yeast powder, 1-10 g / L dipotassium hydrogen phosphate, 0.1-2 g / L magnesium sulfate, and 15-20 g / L agar powder; Preferably, the activation culture conditions are: culturing at 35-37° C. for 20-50 h.

6. The method according to claim 4, characterized in that The seed culture medium is composed of: 5-15 g / L glucose, 5-10 g / L yeast powder, 5-20 g / L peptone, 1-5 g / L dipotassium hydrogen phosphate, 0.1-1 g / L magnesium sulfate, and 15-20 g / L NaCl; Preferably, the seed culture conditions are: 35-37°C, 200-220rpm.

7. The method according to claim 4, characterized in that The OD of the seed solution 600 The value is 1.0-2.

0.

8. The method according to claim 4, characterized in that The fermentation medium is composed of: 50-100 g / L glucose, 5-10 g / L yeast powder, 5-20 g / L peptone, 1-5 g / L dipotassium hydrogen phosphate, and 0.1-1 g / L magnesium sulfate; Preferably, the fermentation culture conditions are: temperature of 35-37° C., stirring speed of 200-300 rpm, pH of 6.0-8.0 controlled by sodium hydroxide solution, and fermentation with ventilation for 20-30 hours.

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

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

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