A sodium hyaluronate seed solution and its preparation method

Abstract

This invention provides a sodium hyaluronate seed culture and its preparation method. It uses *Streptococcus vesicae* as the production strain and glucose as the carbon source. The culture medium contains 0.5%–1% monosodium glutamate (MSG), and the glucose is separately sterilized at a temperature lower than that of the culture medium. The sodium hyaluronate seed culture is prepared using the following steps: strain resuscitation, strain transfer, and seed tank culture, without any shake-flask culture. By omitting the shake-flask culture step, optimizing the culture medium ratio, and using glucose for separate low-temperature sterilization, the preparation cycle is shortened, the risk of contamination and carbon source loss is reduced, the cell concentration is increased, and the fermentation cost is reduced. This significantly improves the cell concentration of the seed culture and the subsequent fermentation yield, adapting to different scale production needs and effectively reducing the production cost of sodium hyaluronate fermentation.

Description

Technical Field

[0001] This invention relates to the field of seed liquid technology, and in particular to a sodium hyaluronate seed liquid and its preparation method. Background Technology

[0002] Sodium hyaluronate; chemical name: a sodium salt of a mucopolysaccharide composed of disaccharide units consisting of (1→4)-O-β-D-glucuronic acid and (1→3)-2-acetamido-2-deoxy-β-D-glucose; molecular formula: (C 14 H 20 NNaO 11 Sodium hyaluronate possesses excellent moisturizing properties, viscoelasticity, and biocompatibility, and is widely used in pharmaceuticals, cosmetics, and food. Currently, the main industrial method for producing sodium hyaluronate is through fermentation with Streptococcus vesicularis, and the preparation of the seed culture is a key step that determines the fermentation level, production cycle, and cost.

[0003] Existing fermentation methods for preparing sodium hyaluronate seed culture generally employ a multi-stage process involving frozen inoculum → slant activation → shake flask culture → seed tank scale-up culture, which presents the following prominent technical problems: The process is lengthy and carries a high risk of contamination: it requires a shake-flask culture step, involves many operation steps, multiple transfers, and long culture time, which significantly increases the probability of contamination by other bacteria and bacteriophages, affecting batch stability.

[0004] Inefficient use of carbon sources and severe losses: Glucose in culture media is usually sterilized at high temperature along with other components, which easily leads to Maillard reaction and decomposition, resulting in high carbon source loss rate, insufficient available carbon source for bacteria, and low proliferation efficiency.

[0005] The culture medium ratio was not optimized specifically: most processes use a composite carbon source or a universal nitrogen source system, which is not adapted to the metabolic characteristics of Streptococcus veterinaria. Insufficient nitrogen supply or low utilization efficiency leads to low concentration and poor activity of seed culture cells, which directly affects the subsequent fermentation yield.

[0006] High energy consumption and production costs: Multi-stage cultivation and high-temperature sterilization consume a lot of energy, and the waste of carbon sources and low yield further increase the cost of raw materials and manufacturing, reducing the product's market competitiveness.

[0007] While existing publicly available technologies have improved seed culture processes, they still have significant shortcomings: for example, some publicly available processes still retain the shake-flask step, failing to streamline the process; most processes do not perform separate low-temperature sterilization of glucose, failing to address the problem of high-temperature carbon source decomposition; and the culture medium formulation does not incorporate sodium glutamate for nitrogen source optimization, making it difficult to synergistically improve carbon source utilization and cell proliferation efficiency.

[0008] In summary, existing sodium hyaluronate seed culture preparation technologies suffer from long processes, high risk of bacterial contamination, significant carbon source loss, low bacterial concentration, and high production costs, failing to meet the demands for efficient, stable, and low-cost industrial production. Summary of the Invention

[0009] To address the aforementioned technical problems, the present invention aims to provide a sodium hyaluronate seed culture and its preparation method. This method omits the shake-flask culture step, optimizes the culture medium ratio, uses glucose for separate low-temperature sterilization, shortens the preparation cycle, reduces the risk of contamination and carbon source loss, increases cell concentration, and lowers fermentation costs. It significantly improves the cell concentration of the seed culture and the subsequent fermentation yield, adapts to different scale preparation needs, and can effectively reduce the production cost of sodium hyaluronate fermentation.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, the present invention provides a method for preparing sodium hyaluronate seed solution, using Streptococcus vesiculosus as the production strain and glucose as the carbon source. The glucose is sterilized separately at a low temperature lower than that of the culture medium, and the sterilization temperature of the glucose is 114-116°C. The preparation steps of the sodium hyaluronate seed solution are as follows: strain resuscitation, strain transfer and seed tank culture.

[0011] In some embodiments, the strain resuscitation includes the following steps: thawing the frozen Streptococcus veterinaria strain at 26–35°C until completely dissolved, transferring the strain to an eggplant slant culture medium, and culturing it statically at 35–38°C for 12–19 hours to obtain the resuscitated slant culture.

[0012] In some embodiments, the eggplant slant culture medium comprises, by weight, the following components: 0.5%–1% glucose, 1%–2% peptone, 0.5%–1% yeast extract, 0.1%–0.2% dipotassium hydrogen phosphate, 0.02%–0.05% magnesium sulfate, 1%–2% agar, with the remainder being purified water.

[0013] In some embodiments, a strain passage step is further employed between the strain resuscitation and the strain transfer. The strain passage includes the following preparation steps: placing the slant after strain resuscitation at 26-35°C for 30-60 minutes, scraping the colonies on the slant and scratching it multiple times on the surface of several uninoculated slant culture media, and then culturing it at 35-38°C for 12-19 hours to obtain the eggplant-shaped slant after strain passage.

[0014] In some embodiments, the strain transfer includes the following steps: adding 20-30 ml of sterile physiological saline or liquid culture medium to the eggplant slant after strain passage, scraping out colonies to prepare a bacterial suspension, and transferring the bacterial suspension into 1500-2000 ml of liquid culture medium to obtain bottle-grade seed liquid.

[0015] In some embodiments, the liquid culture medium comprises, by weight, the following components: 1%–2% peptone, 0.5%–1% yeast extract, 0.1%–0.2% dipotassium hydrogen phosphate, 0.02%–0.05% magnesium sulfate, 0.5%–1% monosodium glutamate, with the remainder being purified water.

[0016] In some embodiments, the seed tank culture includes the following steps: transferring the prepared bottle-grade seed solution to the seed solution culture medium in the seed tank, wherein the seed tank culture conditions are: temperature 35–38°C, rotation speed 50–100 rpm, and aeration rate 5–25 m³ / h. 3 / h, pH 7.0~8.0, tank pressure 0.04~0.07MPa, fermentation time 15~20h.

[0017] In some embodiments, the seed culture medium comprises, by weight 100%, the following components: 2%–3% glucose, 1%–2% peptone, 0.5%–1% yeast extract, 0.1%–0.2% dipotassium hydrogen phosphate, 0.02%–0.05% magnesium sulfate, 0.5%–1% monosodium glutamate, 0.01%–0.05% defoamer, with the balance being purified water.

[0018] In some embodiments, the seed culture medium contains 0.5% to 1% sodium glutamate and uses glucose as a carbon source; the preparation of the seed culture medium includes the following steps: the seed culture medium is first adjusted to pH 6.0 to 7.0, then sterilized at 120 to 123°C, maintained at a pressure of 0.04 to 0.07 MPa, and cooled to below 40°C, wherein glucose is separately sterilized at 114 to 116°C and then added after cooling.

[0019] On the other hand, the present invention provides a sodium hyaluronate seed solution, which is prepared by any of the methods described above for preparing sodium hyaluronate seed solution, wherein the sodium hyaluronate seed solution has a specification of 100-1500L.

[0020] This invention provides a sodium hyaluronate seed solution and its preparation method, which has the following beneficial effects: 1) This invention provides a sodium hyaluronate seed liquid and its preparation method, which omits the shake flask culture step, optimizes the culture medium ratio, uses glucose for low-temperature sterilization, shortens the preparation cycle, reduces the risk of contamination and carbon source loss, increases cell concentration, reduces fermentation cost, significantly improves the cell concentration of the seed liquid and the subsequent fermentation yield, adapts to different scale preparation needs, and can effectively reduce the fermentation production cost of sodium hyaluronate.

[0021] 2) Specific mechanism: It should be noted that the four major technical features of this invention—(1) using Streptococcus vesicularis as the production strain; (2) using glucose as the carbon source and separately sterilizing it at a temperature lower than that of the culture medium; (3) adding 0.5% to 1% sodium glutamate to the culture medium; and (4) omitting shake-flask culture—are not simple combinations of existing technologies, but rather form a clear synergistic mechanism: First, low-temperature sterilization of glucose alone avoids the Maillard reaction and carbon source decomposition, preserving highly active reducing sugars. Second, monosodium glutamate (MSG), as a high-quality nitrogen source and growth factor, can rapidly activate the metabolic pathways of Streptococcus vesiculosus. The synergistic effect of these two factors allows the bacteria to quickly regain vitality and proliferate efficiently in the seed tank without the need for shake-flask acclimation. Using low-temperature sterilization alone without adding MSG (as in Comparative Example 2) results in a significant decrease in bacterial concentration; adding MSG alone while sterilizing glucose at high temperatures (as in Comparative Example 1) results in a carbon source loss rate as high as 12.8%. The above comparisons fully demonstrate that there is a synergistic relationship of mutual dependence and mutual enhancement among the technical features of this invention. By combining strain characteristics, carbon and nitrogen optimization, and separate sterilization, the shaking step was successfully omitted, significantly shortening the cycle and reducing the risk of contamination. This invention utilizes the high proliferation characteristics of Streptococcus vesicans, combined with glucose for low-temperature sterilization to ensure a stable carbon source supply and monosodium glutamate to enhance the nitrogen source supply. The three work synergistically to replace the traditional shake-flask domestication and propagation function, allowing direct entry into the seed tank for culture without the need for shaking flasks. This shortens the preparation cycle by 8-12 hours, reduces the number of transfer steps, significantly reduces the risk of contamination by miscellaneous bacteria and bacteriophages, and greatly improves process stability.

[0022] The combination of low-temperature sterilization of glucose alone and the addition of monosodium glutamate (MSG) reduced the carbon source loss rate from 12.8% to below 3.2%. Low-temperature sterilization of glucose alone at 114–116°C avoids high-temperature decomposition, while MSG promotes carbon source absorption and utilization. The synergistic effect of the two results in a glucose loss rate of only 3.0%–3.2%, which is far lower than the loss level of 12.0%–12.8% in traditional co-sterilization processes. The carbon source utilization rate is increased by more than 70%, an effect that cannot be achieved by a single method.

[0023] The synergistic effect of glucose as the sole carbon source, monosodium glutamate as the nitrogen source, and strain compatibility increases the bacterial concentration by more than 60%. The efficient energy supply of glucose, the high-quality nitrogen source and growth factors provided by monosodium glutamate, and the metabolic compatibility with Streptococcus vesiculosus result in a seed culture bacterial concentration of 7.9–9.2 g / L, which is 56.4%–62.3% higher than the traditional process of 5.3–6.2 g / L. The bacterial culture OD600 reaches 1.72–1.93, and the bacterial activity is significantly enhanced.

[0024] The synergistic effect of the entire system increases the subsequent fermentation yield by more than 35% and significantly reduces costs. The high cell concentration and high-activity seed liquid, combined with low loss and high-efficiency process, enable the subsequent fermentation yield of sodium hyaluronate to reach 11.5-12.8 g / L, which is 35.2%-38.2% higher than the traditional process of 8.9-9.8 g / L. At the same time, the process is simplified, energy consumption is reduced, and raw material utilization is improved, resulting in a significant reduction in the overall fermentation production cost.

[0025] The synergistic process is stable and scalable, suitable for industrial production of 100L to 1500L scale. The nutrient system, sterilization method and culture process of this invention form a stable synergistic system, which can stably achieve a cell concentration of ≥7.9g / L and carbon source loss of ≤3.2% in seed tanks of 100L to 1500L. The pilot and industrial effects are consistent, there is no scale-up bottleneck, and it has strong industrial application value.

[0026] By combining a suitable nutrient system for Streptococcus veterinaria, separate low-temperature sterilization of glucose, and direct feeding into the fermentation tank without shaking flasks, carbon source loss is reduced from over 12% to 3.2%, cell concentration is increased by over 60%, and fermentation yield is increased by over 35%. This shortens the preparation cycle, reduces the risk of contamination and carbon source loss, increases cell concentration, and lowers fermentation costs. It significantly improves the cell concentration of the seed liquid and the subsequent fermentation yield, adapting to different scale preparation needs and effectively reducing the production cost of sodium hyaluronate fermentation. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. The Streptococcus veterinaria used in the present invention ( Streptococcus zooepidemicus The strain used is a commercially available standard strain, strain number CICC 23648, which can be purchased by the public from the China Industrial Microbiological Culture Collection Center (CICC) without the need for separate preservation. After long-term subculturing using the method of this invention, no changes in strain characteristics were detected. The strain contains peptone (biochemical grade tryptone, OXOID, catalog number LP0042), yeast extract (biochemical grade yeast extract, Angel Yeast Co., Ltd., catalog number FM802), agar (biochemical grade agar powder, Sinopharm Chemical Reagent Co., Ltd., catalog number 10000518), and defoamer (polyether defoamer, Shandong Luxin Chemical Co., Ltd., model GPE-30).

[0028] On one hand, the present invention provides a method for preparing sodium hyaluronate seed solution, using Streptococcus vesicularis as the production strain, glucose as the carbon source, and the culture medium containing 0.5% to 1% sodium glutamate. The glucose is sterilized separately at a temperature lower than that of the culture medium, specifically at 114 to 116°C. The sodium hyaluronate seed solution is prepared using the following steps: strain resuscitation, strain transfer, and seed tank culture, without any shake-flask culture involved in the entire preparation process.

[0029] Glucose is preferably sterilized at a low temperature of 114–116℃. Sterilization at 113℃ is incomplete (occasionally contamination may occur), while sterilization at 114–116℃ is stable with a glucose loss rate of ≤3.2%. At 117℃, the glucose loss rate increases to 4.5%. Therefore, 114–116℃ is the optimal temperature range that balances sterilization effectiveness and carbon source retention.

[0030] Preferably, the strain resuscitation includes the following steps: The Streptococcus veterinaria strain, frozen at -35.0℃ to -20.0℃, is thawed at 26~35℃ for 10~30 minutes until the solid in the preservation tube is completely dissolved. The strain solution is then transferred using a pipette to a sterilized, cooled, and solidified eggplant-shaped slant culture medium. An inoculation loop is used to draw 2~3 "S" shapes on the surface of the medium to facilitate strain dispersion. The medium is then statically cultured at 35~38℃ for 12~19 hours to obtain the resuscitated slant, which is then placed in a refrigerator at 2.0~8.0℃ for later use.

[0031] Preferably, the eggplant slant culture medium comprises, by weight, the following components: 0.5%–1% glucose, 1%–2% peptone, 0.5%–1% yeast extract, 0.1%–0.2% dipotassium hydrogen phosphate, 0.02%–0.05% magnesium sulfate, 1%–2% agar, with the remainder being purified water.

[0032] Preferably, the strain transfer includes the following steps: adding 20-30 ml of sterile physiological saline or liquid culture medium to the eggplant slant after strain passage, scraping colonies with an inoculation loop to prepare a bacterial suspension, and transferring the bacterial suspension into 1500-2000 ml of liquid culture medium to obtain bottle-grade seed liquid.

[0033] Preferably, the liquid culture medium comprises, by weight 100%, the following components: 1%–2% peptone, 0.5%–1% yeast extract, 0.1%–0.2% dipotassium hydrogen phosphate, 0.02%–0.05% magnesium sulfate, 0.5%–1% sodium glutamate, and the balance being purified water.

[0034] Preferably, the seed tank culture includes the following steps: transferring the prepared bottle-grade seed solution to the seed solution culture medium in the seed tank, wherein the seed tank culture conditions are: temperature 35-38℃, rotation speed 50-100 rpm, and aeration rate 5-25 m³ / h. 3 The fermentation conditions are as follows: pH 7.0–8.0, tank pressure 0.04–0.07 MPa, fermentation time 15–20 h; the endpoint criteria for seed tank culture are: cell concentration ≥7.5 g / L, OD600 ≥1.70, no contaminating bacteria and no bacteriophages, which are considered as qualified seed liquid. The seed tank is first cleaned and then sterilized in an air at 124–125 °C for 30 min.

[0035] Preferably, the seed culture medium contains 0.5% to 1% sodium glutamate and uses glucose as a carbon source; the preparation of the seed culture medium includes the following steps: the seed culture medium is first adjusted to pH 6.0 to 7.0, then sterilized at 120 to 123°C, maintained at a pressure of 0.04 to 0.07 MPa, and cooled to below 40°C, wherein glucose is separately sterilized at 114 to 116°C and then added after cooling.

[0036] Preferably, the seed culture medium comprises, by weight 100%, the following components: 2%–3% glucose, 1%–2% peptone, 0.5%–1% yeast extract, 0.1%–0.2% dipotassium hydrogen phosphate, 0.02%–0.05% magnesium sulfate, 0.5%–1% monosodium glutamate, 0.01%–0.05% defoamer, with the balance being purified water.

[0037] Preferably, the process between strain resuscitation and strain transfer includes a strain subculturing step, which comprises the following preparation steps: After resuscitation of the strain at 2.0–8.0°C, the slant culture is placed at 26–35°C and allowed to stand for 30–60 minutes. Colonies on the slant are scraped using an inoculation loop and traced 2–3 times in an "S" shape on the surface of multiple uninoculated slant culture media. The culture is then statically incubated at 35–38°C for 12–19 hours to obtain a subcultured eggplant-shaped slant culture. The culture is stored at 2.0–8.0°C and has a shelf life of 15 days. Strain subculturing can produce multiple slant cultures for repeated seed culture.

[0038] On the other hand, the present invention provides a sodium hyaluronate seed solution, which is prepared by any of the methods described above for preparing sodium hyaluronate seed solution, wherein the sodium hyaluronate seed solution has a specification of 100-1500L.

[0039] Example 1 The method for preparing sodium hyaluronate seed solution provided in Example 1 includes the following preparation steps: S1 strain resuscitation: Take the Streptococcus veterinaria strain frozen at -28℃, thaw at 30℃ for 20 minutes, and use a pipette to transfer the completely dissolved Streptococcus veterinaria strain solution in the preservation tube to an eggplant slant culture medium that has been sterilized and cooled to solidify at 121℃ (the eggplant slant culture medium formula consists of 0.8% glucose, 1.5% peptone, 0.8% yeast powder, 0.15% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 1.5% agar, and the remainder being purified water. Weigh out glucose, peptone, yeast powder, dipotassium hydrogen phosphate, and magnesium sulfate according to the formula, add purified water to dissolve, adjust the pH to 6.5, add agar and heat to melt, dispense into eggplant slant, sterilize at 121℃ and cool to solidify to obtain the eggplant slant culture medium), and incubate at 37℃ for 16 hours to obtain the eggplant slant culture after strain resuscitation, and store at 4℃ for later use; S2 strain subculturing: Take the eggplant-shaped slant after the strain has been revived from the 4℃ refrigerator, let it stand at 30℃ for 45 minutes, scrape the colonies on the slant with an inoculation loop and draw "S" shape 3 times on the surface of multiple uninoculated slant culture media, and then let it stand at 37℃ for 16 hours to obtain the eggplant-shaped slant after the strain has been subcultured, and store it at 4℃. S3 strain transfer: Add 25 mL of sterile physiological saline to the eggplant slant after strain passage, scrape colonies with an inoculation loop and shake to prepare a uniform bacterial suspension, then transfer to 1800 mL of liquid culture medium (the liquid culture medium consists of 1.5% peptone, 0.8% yeast extract, 0.15% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.8% monosodium glutamate, and purified water to a final volume of 1.8 L; weigh peptone, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate and monosodium glutamate according to the formula, add purified water to dissolve, adjust pH to 6.5, sterilize and set aside) to obtain bottle-grade seed culture; S4 Seed Tank Cultivation: After cleaning the 500L seed tank, sterilize it at 125℃ for 30 minutes. Prepare the seed culture medium (the seed culture medium consists of 2.5% glucose, 1.5% peptone, 0.8% yeast extract, 0.15% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.8% monosodium glutamate, 0.03% defoamer, and the remainder purified water). Weigh out the peptone, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate, monosodium glutamate, and defoamer according to the formula, add purified water to dissolve, and adjust the pH to 6.5. The culture medium was sterilized at 121℃ for 30 min, maintained at 0.07 MPa, and then cooled to 38℃. Glucose was sterilized separately at 115℃ for 30 min, cooled, and aseptically inoculated into the above culture medium to obtain the seed culture medium. The bottle-grade seed culture was aseptically inoculated into a seed tank containing the above seed culture medium. The culture temperature was controlled at 37℃, the stirring speed at 80 rpm, the aeration rate at 15 m³ / h, the pH at 7.5, and the tank pressure at 0.06 MPa. The culture was carried out for 18 h to obtain the sodium hyaluronate seed culture.

[0040] This embodiment does not involve shake-flask culture at all.

[0041] Example 2 The method for preparing sodium hyaluronate seed solution provided in Example 2 includes the following preparation steps: S1 strain resuscitation: Take the Streptococcus veterinaria strain frozen at -35℃, thaw at 26℃ for 30 minutes, and use a pipette to transfer the completely dissolved Streptococcus veterinaria strain solution in the preservation tube to an eggplant slant culture medium that has been sterilized and cooled to solidify at 121℃ (the eggplant slant culture medium formula consists of 0.5% glucose, 1% peptone, 0.5% yeast powder, 0.1% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 1% agar, and the remainder being purified water. Weigh out glucose, peptone, yeast powder, dipotassium hydrogen phosphate, and magnesium sulfate according to the formula, add purified water to dissolve, adjust the pH to 6.0, add agar and heat to melt, dispense into eggplant slant, sterilize at 121℃ and cool to solidify to obtain the eggplant slant culture medium), and incubate at 35℃ for 19 hours to obtain the eggplant slant culture after strain resuscitation. Store at 4℃ for later use. S2 strain subculturing: Take the eggplant slant after the strain has been revived from the 4℃ refrigerator, let it stand at 26℃ for 60 min, scrape the colonies on the slant with an inoculation loop and draw "S" shape 3 times on the surface of multiple uninoculated slant culture media, and then let it stand at 35℃ for 19 h to obtain the eggplant slant after the strain has been subcultured, and store it at 4℃. S3 strain transfer: Add 20 mL of sterile physiological saline to the eggplant slant after strain passage, scrape colonies with an inoculation loop and shake to prepare a uniform bacterial suspension, then transfer to 1500 mL of liquid culture medium (the liquid culture medium consists of 1% peptone, 0.5% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 0.5% monosodium glutamate, and purified water to a final volume of 1.5 L; weigh peptone, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate and monosodium glutamate according to the formula, add purified water to dissolve, adjust pH to 6.0, sterilize and set aside), to obtain bottle-grade seed culture; S4 Seed Tank Cultivation: After cleaning the 100L seed tank, sterilize it at 124℃ for 30 minutes. Prepare the seed culture medium (the seed culture medium consists of 2% glucose, 1% peptone, 0.5% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 0.5% monosodium glutamate, 0.01% defoamer, and the remainder purified water). Weigh out the peptone, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate, monosodium glutamate, and defoamer according to the formula, add purified water to dissolve, and adjust the pH to 6.0. Sterilize at 21℃ for 30 min, maintain pressure at 0.04 MPa, and cool to 37℃; separately sterilize glucose at 114℃ for 30 min, cool and aseptically inoculate into the above culture medium to obtain seed culture medium. Inoculate the bottle-grade seed culture into a seed tank containing the above seed culture medium under aseptic conditions, control the culture temperature at 35℃, stirring speed at 50 rpm, aeration rate at 5 m³ / h, pH at 7.0, tank pressure at 0.05 MPa, and culture for 20 h to obtain sodium hyaluronate seed culture.

[0042] This embodiment does not involve shake-flask culture at all.

[0043] Example 3 The method for preparing sodium hyaluronate seed solution provided in Example 3 includes the following preparation steps: S1 strain resuscitation: Take the Streptococcus vesicae strain frozen at -20℃, thaw at 35℃ for 10 min, and use a pipette to transfer the completely dissolved Streptococcus vesicae strain solution in the preservation tube to an eggplant slant culture medium that has been sterilized and cooled to solidify at 121℃ (the eggplant slant culture medium formula consists of 0.9% glucose, 1.8% peptone, 0.9% yeast powder, 0.18% dipotassium hydrogen phosphate, 0.04% magnesium sulfate, 1.8% agar, with the remainder being purified water. Weigh out glucose, peptone, yeast powder, dipotassium hydrogen phosphate, and magnesium sulfate according to the formula, add purified water to dissolve, adjust the pH to 7.0, add agar and heat to melt, dispense into eggplant slant, sterilize at 121℃ and cool to solidify to obtain the eggplant slant culture medium), and incubate at 37℃ for 12 h to obtain the eggplant slant culture after strain resuscitation, and store at 4℃ for later use; S2 strain subculturing: Take the eggplant-shaped slant after the strain has been revived from the 4℃ refrigerator, let it stand at 35℃ for 30 minutes, scrape the colonies on the slant with an inoculation loop, and draw "S" shape twice on the surface of multiple uninoculated slant culture media. Incubate at 37℃ for 12 hours to obtain the eggplant-shaped slant after strain subculturing, and store at 4℃. S3 strain transfer: Add 30 mL of sterile physiological saline to the eggplant slant after strain passage, scrape colonies with an inoculation loop and shake to prepare a uniform bacterial suspension, then transfer to 2000 mL of liquid culture medium (the liquid culture medium consists of 1.8% peptone, 0.9% yeast extract, 0.18% dipotassium hydrogen phosphate, 0.04% magnesium sulfate, 0.9% monosodium glutamate, and purified water to a final volume of 2 L; weigh peptone, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate and monosodium glutamate according to the formula, add purified water to dissolve, adjust pH to 7.0, sterilize and set aside) to obtain bottle-grade seed culture; S4 Seed Tank Cultivation: After cleaning the 1500L seed tank, sterilize it at 125℃ for 30 minutes. Prepare the seed culture medium (the seed culture medium consists of 2.8% glucose, 1.8% peptone, 0.9% yeast extract, 0.18% dipotassium hydrogen phosphate, 0.04% magnesium sulfate, 0.9% monosodium glutamate, 0.04% defoamer, and the remainder being purified water). Weigh out the peptone, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate, monosodium glutamate, and defoamer according to the formula, add purified water to dissolve, and adjust the pH to 7.0. Sterilize at 122℃ for 30 min, maintain pressure at 0.06 MPa, and then cool to 38℃; separately sterilize glucose at 116℃ for 30 min, cool and aseptically inoculate into the above culture medium to obtain seed culture medium. Inoculate the bottle-grade seed culture into a seed tank containing the above seed culture medium under aseptic conditions, and control the culture temperature at 38℃, stirring speed at 100 rpm, aeration rate at 25 m³ / h, pH at 8.0, tank pressure at 0.07 MPa, and culture for 15 h to complete the preparation of sodium hyaluronate seed culture.

[0044] This embodiment does not involve shake-flask culture at all.

[0045] Comparative Example 1 (Glucose and culture medium were sterilized together) The method for preparing sodium hyaluronate seed culture provided in Comparative Example 1 differs from that in Example 1 in that: during the seed tank culture process in step S4, glucose is not sterilized at low temperature separately, but is sterilized together with the seed culture medium at 121°C for 30 minutes. The remaining steps, process parameters and culture medium formulation are the same as in Example 1.

[0046] This comparative example did not involve shake-flask culture throughout the entire process.

[0047] Comparative Example 2 (without added monosodium glutamate) The method for preparing sodium hyaluronate seed culture provided in Comparative Example 2 differs from that in Example 1 in that sodium glutamate is not added to the liquid culture medium in step S3 and the seed culture medium in step S4. The remaining steps, process parameters and culture medium formulations are the same as those in Example 1.

[0048] This comparative example did not involve shake-flask culture throughout the entire process.

[0049] Comparative Example 3 (Traditional Preparation Process) The method for preparing sodium hyaluronate seeds before fermentation provided in Comparative Example 3 includes the following steps: S1 strain resuscitation: Take the Streptococcus veterinaria strain frozen at -28℃, thaw at 30℃ for 20 min, and inoculate it into a nightshade slant medium (formula: starch 0.8%, sucrose 0.4%, peptone 1.2%, yeast powder 0.6%, dipotassium hydrogen phosphate 0.12%, magnesium sulfate 0.03%, agar 1.5%, the remainder being purified water), and culture statically at 37℃ for 16 h, then refrigerate at 4℃ until use; S2 strain subculture: Take out the slant and let it stand at 30℃ for 45 min, then streak it to a new eggplant slant medium, and incubate at 37℃ for 16 h. Store at 4℃. S3 strain transfer: Add 25 mL of sterile physiological saline to the passage slant, scrape off the colonies to make a bacterial suspension, and transfer it into 1800 mL of liquid culture medium (formula: starch 0.8%, sucrose 0.4%, peptone 1.2%, yeast extract 0.6%, dipotassium hydrogen phosphate 0.12%, magnesium sulfate 0.03%, purified water 1.8 L, without sodium glutamate) to obtain bottle-grade seed culture; S4 shake flask culture: The above bottle-grade seed solution was dispensed into 500mL shake flasks (each flask contains 100mL of solution), and cultured on a shaker at 37℃ and 150rpm for 8h to obtain shake flask seed solution; S5 Seed Tank Cultivation: A 500L seed tank was sterilized at 125℃ for 30 minutes. Seed culture medium (formula: starch 1%, sucrose 0.5%, peptone 1.5%, yeast powder 0.8%, dipotassium hydrogen phosphate 0.15%, magnesium sulfate 0.03%, defoamer 0.03%, the remainder being purified water, without adding monosodium glutamate) was added. The pH was adjusted to 6.5. The carbon source (starch + sucrose) and culture medium were sterilized together at 121℃ for 30 minutes. The pressure was maintained at 0.05MPa and the temperature was lowered to 38℃. The seed culture from the shake flask was aseptically inoculated. The temperature was controlled at 37℃, rotation speed 80rpm, aeration rate 15m³ / h, pH 7.5, and tank pressure 0.06MPa. The culture was carried out for 12 hours to obtain the pre-fermentation seed of sodium hyaluronate.

[0050] This comparative example includes a shake-flask culture step and is compared with Example 1.

[0051] Comparative Example 4 (MSG addition 0.3%) The method for preparing sodium hyaluronate seed solution provided in Comparative Example 4 differs from that in Example 1 in that only the amount of sodium glutamate added is changed to 0.3% in steps S3 and S4, while the remaining steps, process parameters and culture medium formulation are the same as in Example 1.

[0052] This comparative example did not involve shake-flask culture throughout the entire process.

[0053] Comparative Example 5 (monosodium glutamate added 1.2%) The method for preparing sodium hyaluronate seed solution provided in Comparative Example 5 differs from that in Example 1 in that only the amount of sodium glutamate added is changed to 1.2% in steps S3 and S4, while the remaining steps, process parameters and culture medium formulation are the same as in Example 1.

[0054] This comparative example did not involve shake-flask culture throughout the entire process.

[0055] Experimental methods and testing standards 1. Reducing sugar content before sterilization: determined by DNS colorimetric method, referring to GB / T 5009.7-2016 "National Food Safety Standard - Determination of Reducing Sugars in Food"; 2. Reducing sugar content after sterilization: determined by DNS colorimetric method, referring to GB / T 5009.7-2016 "National Food Safety Standard - Determination of Reducing Sugars in Food"; 3. Carbon source loss rate: Calculated according to the formula "(reducing sugar content before sterilization - reducing sugar content after sterilization) / reducing sugar content before sterilization × 100%"; 4. Bacterial suspension OD value: After the seed culture is completed, the bacterial suspension is diluted with sterile physiological saline to an appropriate multiple (usually 10-20 times). Using sterile physiological saline as a blank control, the absorbance is measured at a wavelength of 600 nm using a UV spectrophotometer. The recorded value is multiplied by the dilution factor to obtain the final OD600 value. Each batch of samples is measured in triplicate, and the average value is taken. 5. Final cell concentration: determined by gravimetric method. The bacterial solution was centrifuged (8000 r / min, 10 min), the cells were collected, dried to constant weight, and then weighed for calculation. 6. Subsequent fermentation yield: After the seed liquid is introduced into the fermenter, the sodium hyaluronate content in the fermentation broth is determined by the phenol-sulfuric acid colorimetric method after fermentation, which is the subsequent fermentation yield.

[0056] The performance data of Examples 1-3 and Comparative Examples 1-5 tested using the above experimental methods and testing standards are shown in Table 1 below: Table 1. Performance comparison experimental data of Examples 1-3 and Comparative Examples 1-5

[0057] Note: 1. Carbon source loss rate = (reducing sugar content before sterilization - reducing sugar content after sterilization) / reducing sugar content before sterilization × 100%; The reducing sugar content before and after sterilization in Comparative Example 3 is an estimated value after starch + sucrose is converted into glucose equivalent, which is used to calculate the total carbon source loss rate. 2. The OD value and cell concentration of the bacterial culture were measured after the seed culture was completed; 3. The subsequent fermentation yield is the final fermentation yield of sodium hyaluronate after the seed liquid is transferred to the fermenter; All the above experiments were replicated in parallel with 3 batches, and the RSD was <5%, indicating good batch stability. Scale-up verification in a 5000L production tank showed that the bacterial concentration was ≥7.9g / L and the carbon source loss was ≤3.5%, demonstrating reproducible results.

[0058] From Table 1, we can draw the following experimental conclusions: 1. Glucose was sterilized separately at 114–116°C, which significantly reduced carbon source loss and improved cell growth efficiency. Comparing Example 1 and Comparative Example 1 (only the glucose sterilization method differed), Example 1 used separate sterilization at 115°C, resulting in a glucose loss rate of only 3.2%, far lower than the 12.8% of Comparative Example 1 (glucose and culture medium were sterilized together at 121°C). This indicates that this separate low-temperature sterilization method can effectively avoid high-temperature decomposition of glucose, maximize the retention of effective carbon source components, and reduce carbon source waste. At the same time, the OD value of the bacterial culture in Example 1 (1.85), the final cell concentration (8.6 g / L), and the subsequent fermentation yield (12.3 g / L) were increased by 52.9%, 56.4%, and 35.2% respectively compared to Comparative Example 1. This confirms that this sterilization method can significantly improve the reproduction efficiency of Streptococcus vesiculosus by stabilizing the carbon source supply, optimize the seed culture quality, and lay a good foundation for subsequent fermentation (reducing sugar content was determined according to GB / T5009.7-2016).

[0059] 2. Adding 0.5%–1% monosodium glutamate (MSG) to the culture medium can significantly improve the cell growth status and subsequent fermentation performance. Comparing Example 1 and Comparative Example 2 (different only in terms of whether MSG was added), both groups were sterilized at low temperature with glucose alone. After adding 0.8% MSG in Example 1, the OD value (1.85), final cell concentration (8.6 g / L), and subsequent fermentation yield (12.3 g / L) of the bacterial culture were increased by 34.1%, 38.7%, and 25.5% respectively compared with Comparative Example 2 (1.38, 6.2 g / L, 9.8 g / L) without MSG. This indicates that MSG can accurately meet the nutritional requirements of Streptococcus veterinaria, providing sufficient nitrogen source and growth factors for cell reproduction, and effectively improving the cell concentration and fermentation potential of the seed culture.

[0060] 3. The amount of monosodium glutamate (MSG) added should be controlled within the range of 0.5% to 1%. Exceeding this range will reduce the effectiveness. Comparing Example 1 with Comparative Examples 4 and 5 (which differ only in the amount of MSG added), the OD value of the bacterial culture (1.45), the final cell concentration (6.5 g / L), and the subsequent fermentation yield (10.2 g / L) of Comparative Example 4 (addition amount of 0.3%) decreased by 21.6%, 24.4%, and 17.1% respectively compared to Example 1. The glucose loss rate (3.6%) of Comparative Example 5 (addition amount of 1.2%) increased by 12.5% ​​compared to Example 1, while the OD value of the bacterial culture (1.78), the final cell concentration (8.2 g / L), and the subsequent fermentation yield (11.8 g / L) decreased by 3.8%, 4.7%, and 4.1% respectively compared to Example 1. This confirms that too low an amount of MSG added cannot meet the growth requirements of the cells, while too high an amount will increase carbon source loss and reduce the process advantages. 0.5% to 1% is the optimal addition range.

[0061] 4. The technical solution of this invention has significant advantages over the traditional process of Comparative Example 3, and can effectively solve the pain points of existing technologies. Comparing Example 1 (the solution of this invention) and Comparative Example 3 (the traditional process), under the same strain and basic process conditions, the optimized combination of "glucose as the sole carbon source + glucose alone at low temperature sterilization + addition of monosodium glutamate" in this invention reduces the carbon source loss rate from 12.0% to 3.2%, a reduction of 73.3%; the OD value of the bacterial solution increases from 1.15 to 1.85, an increase of 60.9%; the final cell concentration increases from 5.3 g / L to 8.6 g / L, an increase of 62.3%; and the subsequent fermentation yield increases from 8.9 g / L to 12.3 g / L, an increase of 38.2%. The above data fully demonstrate that the solution of this invention is significantly superior to the traditional process in shortening the preparation process, reducing carbon source loss, increasing cell concentration and fermentation yield, and effectively solves the technical pain points of long seed liquid preparation cycle, high risk of contamination, large carbon source loss and high production cost in existing technologies.

[0062] In summary, this invention, by omitting the shake-flask culture step, optimizing the culture medium ratio (using glucose as the sole carbon source and adding 0.5%–1% monosodium glutamate), and employing separate low-temperature sterilization of glucose at 114–116°C, effectively shortens the preparation cycle of sodium hyaluronate seed culture, reduces the risk of contamination and carbon source loss, significantly improves the cell concentration of the seed culture and the subsequent fermentation yield, and is suitable for large-scale preparation of 100–1500L. It can effectively reduce the fermentation production cost of sodium hyaluronate and has good prospects for industrial application.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing sodium hyaluronate seed solution, characterized in that, Streptococcus vesicularis was used as the production strain, and glucose was used as the carbon source. The glucose was sterilized separately at a low temperature lower than that of the culture medium, and the sterilization temperature of the glucose was 114-116°C. The sodium hyaluronate seed solution was prepared by the following steps: strain resuscitation, strain transfer and seed tank culture.

2. The method for preparing sodium hyaluronate seed solution according to claim 1, characterized in that, The strain resuscitation includes the following steps: thawing the frozen Streptococcus veterinaria strain at 26-35°C until completely dissolved, transferring the liquid to an eggplant slant culture medium, and culturing it statically at 35-38°C for 12-19 hours to obtain the resuscitated slant.

3. The method for preparing sodium hyaluronate seed solution according to claim 2, characterized in that, The eggplant slant culture medium, by weight, comprises the following components: 0.5%–1% glucose, 1%–2% peptone, 0.5%–1% yeast extract, 0.1%–0.2% dipotassium hydrogen phosphate, 0.02%–0.05% magnesium sulfate, 1%–2% agar, with the remainder being purified water.

4. The method for preparing sodium hyaluronate seed solution according to claim 1, characterized in that, The process between strain resuscitation and strain transfer also involves a strain passage step, which includes the following preparation steps: placing the resuscitated slant at 26-35℃ for 30-60 minutes, scraping the colonies on the slant onto the surface of multiple uninoculated slant culture media and swiping them multiple times, and then incubating at 35-38℃ for 12-19 hours to obtain the passaged eggplant-shaped slant.

5. The method for preparing sodium hyaluronate seed solution according to claim 4, characterized in that, The strain transfer includes the following steps: adding 20-30 ml of sterile physiological saline or liquid culture medium to the eggplant slant after the strain has been passaged, scraping off colonies to prepare a bacterial suspension, and transferring the bacterial suspension into 1500-2000 ml of liquid culture medium to obtain bottle-grade seed liquid.

6. The method for preparing sodium hyaluronate seed solution according to claim 5, characterized in that, The liquid culture medium, by weight, comprises the following components: 1%–2% peptone, 0.5%–1% yeast extract, 0.1%–0.2% dipotassium hydrogen phosphate, 0.02%–0.05% magnesium sulfate, 0.5%–1% monosodium glutamate, with the remainder being purified water.

7. The method for preparing sodium hyaluronate seed solution according to claim 1, characterized in that, The seed tank culture includes the following steps: The prepared bottle-grade seed culture was transferred to the seed culture medium in a seed tank. The seed tank culture conditions were: temperature 35–38℃, rotation speed 50–100 rpm, and aeration rate 5–25 m³ / h. 3 / h, pH 7.0~8.0, tank pressure 0.04~0.07MPa, fermentation time 15~20h.

8. The method for preparing sodium hyaluronate seed solution according to claim 7, characterized in that, The seed culture medium, by weight, comprises the following components: 2%–3% glucose, 1%–2% peptone, 0.5%–1% yeast extract, 0.1%–0.2% dipotassium hydrogen phosphate, 0.02%–0.05% magnesium sulfate, 0.5%–1% monosodium glutamate, 0.01%–0.05% defoamer, with the remainder being purified water.

9. The method for preparing sodium hyaluronate seed solution according to claim 8, characterized in that, The seed culture medium contains 0.5%–1% sodium glutamate and uses glucose as a carbon source. The preparation of the seed culture medium includes the following steps: the seed culture medium is first adjusted to pH 6.0–7.0, then sterilized at 120–123°C, maintained at a pressure of 0.04–0.07 MPa, and cooled to below 40°C. In this case, glucose is separately sterilized at 114–116°C and then added after cooling.

10. A sodium hyaluronate seed solution, characterized in that, It was prepared using the method for preparing sodium hyaluronate seed solution as described in any one of claims 1 to 9.

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