Chondroitin fermentation medium and fermentation method

Abstract

This invention relates to the field of microbiology, and more particularly to a chondroitin fermentation medium and fermentation method. The invention provides a chondroitin fermentation medium comprising the following components: 20-30 g / L tryptone, 20-30 g / L yeast extract, 8-16 g / L urea, 5-12 g / L ammonium sulfate, 1-5 g / L potassium dihydrogen phosphate, 8-16 g / L dipotassium hydrogen phosphate, 1-4 g / L sodium chloride, 0.5-3 g / L anhydrous magnesium sulfate, 30-50 g / L sucrose, and trace elements. Through optimization of the specialized culture medium, this invention achieves a significant increase in chondroitin yield and stable production, demonstrating significant advantages in product quality, process scalability, and operational economy. It provides a practical industrial-scale technical solution for the safe, efficient, and large-scale production of chondroitin using Bacillus subtilis.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more particularly to chondroitin fermentation culture medium and fermentation method. Background Technology

[0002] Chondroitin sulfate (ChS / CS) is a naturally derived, bioactive macromolecule widely found in mammals and invertebrates, profoundly influencing various physiological processes, including antioxidant and anti-inflammatory effects, inhibition of tumor progression and metastasis, and participation in angiogenesis. Due to these significant bioactivities, chondroitin sulfate has gained recognition in the medical field and is now recommended by the European League Against Rheumatism (EULAR) as SYSADOA (Symptomatic Slow Acting Drug for OA) for the treatment of knee osteoarthritis. It is also recommended for the treatment of osteoarthritis in the knee, hip, and hand. As an important bioactive substance, the choice of commercial-scale industrial production methods for chondroitin sulfate is crucial for product quality, cost, and sustainability.

[0003] Currently, common production methods include animal tissue extraction, chemical synthesis, and enzymatic hydrolysis. However, these traditional methods all have limitations in practical applications, such as safety issues, high costs, environmental pollution, and low production efficiency. To seek safer, more controllable, and sustainable production methods, microbial fermentation has become a key research and development direction in the industry.

[0004] Microbial fermentation utilizes natural microbial strains or genetically engineered strains under highly efficient fermentation processes. It offers advantages such as no raw material limitations, low cost, and minimal environmental pollution, effectively meeting the needs of industrial production. In microbial fermentation, the composition of the culture medium is crucial for microbial growth and metabolism, and is one of the core factors determining the yield, conversion rate, and production cost of the target product. For extracellular polysaccharides like chondroitin, the culture medium not only needs to provide the basic nutrients required for cell growth but also requires a precise balance of carbon sources, nitrogen sources, inorganic salts, and inducing factors to maximize the metabolic flow towards chondroitin synthesis and secretion.

[0005] Currently, numerous studies have utilized strains such as *Bacillus megaterium* (CN107151685), recombinant *Escherichia coli* K4 (CN103228781B), and recombinant *Escherichia coli* BL21(DE3) (Reference 1: Zhao C, Li X, Guo L, Gao C, Song W, Wei W, Wu J, Liu L, Chen X. Reprogramming Metabolic Flux in *Escherichia Coli* to Enhance Chondroitin Production. Adv Sci (Weinh). 2024 Mar;11(10):e2307351.) to produce chondroitin or its derivatives via microbial fermentation. However, these reports primarily focus on increasing chondroitin yield through genetic modification of the strains or macroscopic control of the fermentation process. Nevertheless, these studies typically employ conventional culture media for fermentation production, and there is a lack of in-depth research and public reporting on the systematic optimization of culture media, particularly high-yield culture medium formulations suitable for industrial-scale scaling up of microbial fermentation technology.

[0006] Especially for Bacillus subtilis, a recognized safe (GRAS) host platform, research on dedicated culture media for high-yield chondroitin fermentation is still lacking. Because the growth characteristics, nutritional requirements, and metabolic regulatory networks of Bacillus subtilis differ significantly from those of strains such as Escherichia coli, directly applying culture media from other strains or conventional culture medium formulations fails to realize its production potential. This has led to generally low yields of chondroitin produced by Bacillus subtilis in existing technologies, and key bottlenecks remain for its industrial-scale production.

[0007] Lack of specificity: Existing studies mostly use laboratory-optimized culture media rich in expensive components such as yeast extract, peptone, and special growth factors. While these media can support cell growth and product synthesis in small-scale cultures, they are difficult to directly apply to large-scale industrial fermentation, resulting in problems such as high raw material costs, poor batch-to-batch stability, and loss of metabolic flux regulation after scale-up. In addition, existing general-purpose culture media are not specifically designed for the metabolic characteristics of the chondroitin synthesis pathway, which can easily lead to problems such as insufficient precursor supply or metabolic imbalance.

[0008] Cost and efficiency imbalance: Most of the culture media commonly used in existing technologies are laboratory-optimized formulations. Although they can achieve a certain yield, they use expensive or complex components, which is not conducive to cost control in industrial production. On the other hand, if a culture medium with a simple formulation is used, the yield is often low and cannot meet the needs of economical production.

[0009] Weak synergy with fermentation process: Conventional culture medium design often fails to fully consider the synergistic optimization with key fermentation process parameters such as subsequent feeding strategies, pH control, and dissolved oxygen regulation, which can easily lead to performance degradation and poor production stability during scale-up.

[0010] Therefore, developing a high-yield culture medium specifically for Bacillus subtilis that has a scientific nutrient ratio, is highly targeted, cost-effective, and can work closely with efficient fermentation processes has become a key prerequisite for unleashing the industrialization potential of Bacillus subtilis engineered strains in the production of chondroitin sulfate. Summary of the Invention

[0011] In view of this, the present invention provides a chondroitin fermentation medium and fermentation method. The present invention provides a high-yield chondroitin fermentation medium and fermentation process, particularly relating to a medium for the efficient production of chondroitin by engineered Bacillus subtilis, and a fermentation production process for chondroitin production by engineered Bacillus subtilis. This aims to fill the research gap in dedicated culture media for high-yield chondroitin fermentation, increase the yield of chondroitin produced by engineered Bacillus subtilis fermentation, overcome the production bottleneck of the Bacillus subtilis platform, and lay a solid foundation for large-scale industrial production.

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] This invention provides a chondroitin fermentation medium, comprising the following components:

[0014] Tryptone 20-30 g / L, yeast extract 20-30 g / L, urea 8-16 g / L, ammonium sulfate 5-12 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 8-16 g / L, sodium chloride 1-4 g / L, anhydrous magnesium sulfate 0.5-3 g / L, sucrose 30-50 g / L, and trace elements;

[0015] The trace elements include: ferrous sulfate heptahydrate, manganese sulfate tetrahydrate, calcium chloride, zinc sulfate heptahydrate, copper sulfate pentahydrate, ammonium molybdate tetrahydrate, and sodium tetraborate decahydrate.

[0016] In some embodiments of the present invention, the content of each component of the trace elements in the above-mentioned chondroitin fermentation culture medium is as follows:

[0017] Ferrous sulfate heptahydrate 8~12 g / L, manganese sulfate tetrahydrate 0.3~0.7 g / L, calcium chloride 1~3 g / L, zinc sulfate heptahydrate 2~2.4 g / L, copper sulfate pentahydrate 0.8~1.2 g / L, ammonium molybdate tetrahydrate 0.08~0.12 g / L, and sodium tetraborate decahydrate 0.01~0.03 g / L.

[0018] In some embodiments of the present invention, the content of each component in the trace elements (mother liquor) in the above-mentioned chondroitin fermentation culture medium is as follows:

[0019] Ferrous sulfate heptahydrate, 8~12 g / L (e.g. 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L), preferably 9~11 g / L, more preferably 10 g / L;

[0020] Manganese sulfate tetrahydrate, 0.3~0.7 g / L (e.g. 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L), preferably 0.4~0.6 g / L, more preferably 0.5 g / L;

[0021] Calcium chloride, 1~3 g / L (e.g. 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L), preferably 1.5~2.5 g / L, more preferably 2 g / L;

[0022] Zinc sulfate heptahydrate, 2~2.4 g / L (e.g. 2 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L), preferably 2.1~2.3 g / L, more preferably 2.2 g / L;

[0023] Copper sulfate pentahydrate, 0.8~1.2 g / L (e.g. 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L), preferably 0.9~1.1 g / L, more preferably 1 g / L;

[0024] Ammonium molybdate tetrahydrate, 0.08~0.12 g / L (e.g. 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.11 g / L, 0.12 g / L), preferably 0.09~0.11 g / L, more preferably 0.1 g / L;

[0025] Sodium tetraborate decahydrate, 0.01~0.03 g / L (e.g. 0.01 g / L, 0.015 g / L, 0.02 g / L, 0.025 g / L, 0.03 g / L), preferably 0.015~0.025 g / L, more preferably 0.02 g / L.

[0026] In some embodiments of the present invention, the content of the trace elements in the above-mentioned chondroitin fermentation culture medium is 8~12 mL / L.

[0027] In some embodiments of the present invention, the content of trace elements in the chondroitin fermentation medium is 8-12 mL / L (e.g., 8 mL / L, 9 mL / L, 10 mL / L, 11 mL / L, 12 mL / L); preferably, the content of trace elements in the fermentation medium is 9-11 mL / L; more preferably, the content of trace elements in the fermentation medium is 10 mL / L.

[0028] In some embodiments of the present invention, when the content of trace elements in the above-mentioned chondroitin fermentation medium is 10 mL / L, the chondroitin fermentation medium comprises the following components:

[0029] Tryptone 20-30 g / L, yeast extract 20-30 g / L, urea 8-16 g / L, ammonium sulfate 5-12 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 8-16 g / L, sodium chloride 1-4 g / L, anhydrous magnesium sulfate 0.5-3 g / L, sucrose 30-50 g / L, ferrous sulfate heptahydrate 100 mg / L, manganese sulfate tetrahydrate 5 mg / L, calcium chloride 20 mg / L, zinc sulfate heptahydrate 22 mg / L, copper sulfate pentahydrate 10 mg / L, ammonium molybdate tetrahydrate 1 mg / L, and sodium tetraborate decahydrate 0.2 mg / L.

[0030] In some embodiments of the present invention, the chondroitin fermentation medium comprises the following components:

[0031] Tryptone 24 g / L, yeast extract 24 g / L, urea 12 g / L, ammonium sulfate 8 g / L, potassium dihydrogen phosphate 3 g / L, dipotassium hydrogen phosphate 13 g / L, sodium chloride 2 g / L, anhydrous magnesium sulfate 1.5 g / L, sucrose 40 g / L, ferrous sulfate heptahydrate 100 mg / L, manganese sulfate tetrahydrate 5 mg / L, calcium chloride 20 mg / L, zinc sulfate heptahydrate 22 mg / L, copper sulfate pentahydrate 10 mg / L, ammonium molybdate tetrahydrate 1 mg / L, and sodium tetraborate decahydrate 0.2 mg / L; or

[0032] Tryptone 20 g / L, yeast extract 20 g / L, urea 8 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 8 g / L, sodium chloride 1 g / L, anhydrous magnesium sulfate 0.5 g / L, sucrose 50 g / L, ferrous sulfate heptahydrate 100 mg / L, manganese sulfate tetrahydrate 5 mg / L, calcium chloride 20 mg / L, zinc sulfate heptahydrate 22 mg / L, copper sulfate pentahydrate 10 mg / L, ammonium molybdate tetrahydrate 1 mg / L, and sodium tetraborate decahydrate 0.2 mg / L; or

[0033] Tryptone 30 g / L, yeast extract 30 g / L, urea 16 g / L, ammonium sulfate 12 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 16 g / L, sodium chloride 4 g / L, anhydrous magnesium sulfate 3 g / L, sucrose 30 g / L, ferrous sulfate heptahydrate 100 mg / L, manganese sulfate tetrahydrate 5 mg / L, calcium chloride 20 mg / L, zinc sulfate heptahydrate 22 mg / L, copper sulfate pentahydrate 10 mg / L, ammonium molybdate tetrahydrate 1 mg / L, and sodium tetraborate decahydrate 0.2 mg / L.

[0034] The present invention also provides a seed culture medium comprising the following components:

[0035] Tryptone 20-30 g / L, yeast extract 20-30 g / L, urea 8-16 g / L, ammonium sulfate 5-12 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 8-16 g / L, sodium chloride 1-4 g / L, anhydrous magnesium sulfate 0.5-3 g / L, and sucrose 30-50 g / L.

[0036] In some embodiments of the present invention, the anhydrous magnesium sulfate and the sucrose in the above-mentioned seed culture medium can be prepared and used immediately.

[0037] In some embodiments of the present invention, the above-mentioned seed culture medium includes the following components:

[0038] Tryptone 24 g / L, yeast extract 24 g / L, urea 12 g / L, ammonium sulfate 8 g / L, potassium dihydrogen phosphate 3 g / L, dipotassium hydrogen phosphate 13 g / L, sodium chloride 2 g / L, anhydrous magnesium sulfate 1.5 g / L, and sucrose 40 g / L; or

[0039] Tryptone 20 g / L, yeast extract 20 g / L, urea 8 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 8 g / L, sodium chloride 1 g / L, anhydrous magnesium sulfate 0.5 g / L, and sucrose 50 g / L; or

[0040] Tryptone 30 g / L, yeast extract 30 g / L, urea 16 g / L, ammonium sulfate 12 g / L, potassium dihydrogen phosphate 5 g / L, dipotassium hydrogen phosphate 16 g / L, sodium chloride 4 g / L, anhydrous magnesium sulfate 3 g / L, and sucrose 30 g / L.

[0041] The present invention also provides the application of the above-mentioned chondroitin fermentation medium in the fermentation of Bacillus subtilis to produce chondroitin.

[0042] The present invention also provides the application of the above-mentioned seed culture medium in the cultivation of Bacillus subtilis to produce chondroitin.

[0043] In some embodiments of the present invention, in the above applications, the Bacillus subtilis was deposited on December 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 36960, and classified as Bacillus subtilis.

[0044] The present invention also provides a fermentation method for chondroitin, wherein after obtaining the seed liquid of Bacillus subtilis, it is inoculated into a fermentation medium and fermented to obtain chondroitin;

[0045] The fermentation medium is the chondroitin fermentation medium described above.

[0046] In some embodiments of the present invention, the above fermentation method uses a seed culture medium to obtain the seed liquid.

[0047] In some embodiments of the present invention, the anhydrous magnesium sulfate and the sucrose in the above fermentation method are prepared and used immediately.

[0048] In some embodiments of the present invention, in the above-described fermentation method, the dissolved oxygen content during fermentation is 15-30%;

[0049] When the dissolved oxygen level is below 20%, the dissolved oxygen level is increased by sequentially increasing the rotation speed and increasing the ventilation rate.

[0050] When the dissolved oxygen level is greater than 30%, the dissolved oxygen level is reduced by successively decreasing the ventilation rate and the rotation speed.

[0051] In some embodiments of the present invention, in the above fermentation method, when the dissolved oxygen content is less than 20%, the rotation speed is increased by 25 rpm each time until the rotation speed is 1000 rpm; if the dissolved oxygen content is still less than 20%, the aeration rate is increased by 0.25 vvm each time until the aeration rate is 2.5 vvm.

[0052] When the dissolved oxygen level is greater than 30%, reduce the ventilation rate by 0.25 vvm each time until it reaches 1 vvm. If the dissolved oxygen level is still greater than 30%, reduce the ventilation rate by 25 rpm each time until it reaches 30%.

[0053] In some embodiments of the present invention, in the above fermentation method, when the dissolved oxygen content is below 20%, the dissolved oxygen content is increased to 30% by sequentially increasing the rotation speed, increasing the aeration rate, and increasing the tank pressure, wherein:

[0054] The increase in rotational speed includes: increasing the speed by 150 rpm each time to a speed of 900 rpm;

[0055] The increase in ventilation volume includes increasing it by 0.25 vvm each time until the ventilation volume reaches 2 vvm;

[0056] The increase in tank pressure includes: from 0.01 MPa to a tank pressure of 0.06 MPa;

[0057] When the dissolved oxygen level is higher than 30%, the dissolved oxygen level is reduced to 20% by sequentially decreasing the tank pressure, decreasing the ventilation rate, and decreasing the rotation speed.

[0058] The reduction of tank pressure includes: reducing the pressure by 0.01 MPa each time until the tank pressure is 0.01 MPa;

[0059] The reduction in ventilation volume includes: reducing the ventilation volume by 0.25 vvm each time until the ventilation volume is 1 vvm;

[0060] The reduction in rotation speed includes reducing the speed by 25 rpm each time until the dissolved oxygen level is 30%.

[0061] In some embodiments of the present invention, the above-described fermentation method further includes a feed addition step; the feed addition includes ammonia, sucrose, xylose, and an antifoaming agent.

[0062] In some embodiments of the present invention, in the above fermentation method, when the concentration of sucrose drops to 1 g / L during fermentation, the sucrose is added in batches, and the residual sucrose concentration in the fermentation environment is controlled to be 0~2 g / L and the residual glucose concentration to be 0~1 g / L.

[0063] In some embodiments of the present invention, in the above fermentation method, the xylose is added for 2 to 12 hours, the final concentration is 18 g / L, and the addition is performed at least once.

[0064] In some embodiments of the present invention, the xylose is added at 2.5 h and 12 h, or 4 h and 12 h, in the above fermentation method.

[0065] In some embodiments of the present invention, the xylose is added once or twice in the above fermentation method.

[0066] In some embodiments of the present invention, in the above fermentation method, the fermentation temperature is 37°C, the pH value is 7.0±0.05, and the time is 48~60h.

[0067] In some embodiments of the present invention, in the above fermentation method, the feed includes 25 wt% ammonia water, 750 g / L sucrose, xylose with a final concentration of 18 g / L, and 0.1 g / L defoamer.

[0068] In some embodiments of the present invention, the fermentation method described above further includes a purification step after fermentation.

[0069] Description of Biological Deposit: *Bacillus subtilis* was deposited with the General Microbiological Center of the China Microbiological Culture Collection Management Committee on December 9, 2025, with the deposit address in Beijing, China. The deposit number is CGMCC No. 36960, and the taxonomic designation is *Bacillus subtilis*.

[0070] The beneficial effects of the present invention include:

[0071] (1) Medium optimization significantly improves cell growth performance and product yield, breaking through the yield bottleneck of the platform.

[0072] With the fermentation medium of the present invention, the yield of chondroitin produced by *Bacillus subtilis* engineered strain DH003 reaches 11.29 ± 0.27 g / L at a 5 L fermentation scale. Compared with the control example using conventional medium and process (yield of 8.01 ± 0.16 g / L), the yield is significantly increased by about 41%. Meanwhile, the cells supported by the present invention grow more vigorously, with the maximum OD600 reaching 121.2 ± 2.4, which is approximately twice that of the control example (61.1 ± 0.7). This fully proves that through targeted nutrient ratio, the present invention effectively promotes cell growth and efficiently directs metabolic flux to chondroitin synthesis.

[0073] (2) The process has excellent scalability and stability, laying a foundation for industrial production.

[0074] The fermentation process supporting the present invention ensures the stability of the fermentation process and the sustainability of product synthesis. The process of the present invention exhibits good stability and reproducibility during scale-up from laboratory scale (5 L) to pilot scale (50 L). In a 50 L fermenter, the chondroitin yield is further increased to 13.50 ± 0.67 g / L, and the common yield attenuation phenomenon during scale-up does not occur. This proves that the process control strategy of the present invention has good scalability and can meet the requirements for process stability in large-scale production.

[0075] (3) The synergistic effect of the medium and the process maximizes the yield.

[0076] When the medium and the fermentation process of the present invention are used in combination, the synergistic effect is significant: at the 5 L fermentation scale, the chondroitin yield of Example 1 reaches 11.29 g / L, which is 27% higher than that obtained when only the medium of the present invention is used (Control Example 3, 8.88 g / L), and 34% higher than that obtained when only the process of the present invention is used (Control Example 2, 8.45 g / L). This indicates that the present invention works synergistically from the two dimensions of nutrient supply and process control, fully releasing the production potential of the *Bacillus subtilis* engineered strain and achieving a substantial increase in yield.

[0077] (4) The product quality is clear and uniform, and meets the standards for subsequent applications.

[0078] Analysis of the 50 L fermentation product showed that the mass spectrometry information of the disaccharide units obtained by enzymatic hydrolysis was completely consistent with the theoretical value of standard chondroitin disaccharide, confirming the correct product structure. Molecular weight distribution analysis showed that the weight-average molecular weight (Mw) of its main components was concentrated at approximately 40 kDa, indicating that the process can produce high-quality chondroitin with uniform molecular weight and well-defined structure, providing quality assurance for its application in the food, health product, and pharmaceutical fields. Attached Figure Description

[0079] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0080] Figure 1 Fermentation curves for Example 1 are shown; where: (a) represents OD600 and chondroitin yield; (b) represents residual sucrose and residual glucose concentrations;

[0081] Figure 2 Fermentation curves for Example 2 are shown; where: (a) represents OD600 and chondroitin yield; (b) represents residual sucrose and residual glucose concentrations;

[0082] Figure 3 Fermentation curves for Example 3 are shown; where: (a) represents OD600 and chondroitin yield; (b) represents residual sucrose and residual glucose concentrations;

[0083] Figure 4 Fermentation curves for Example 4 are shown; where: (a) represents OD600 and chondroitin yield; (b) represents residual sucrose and residual glucose concentrations;

[0084] Figure 5 HPLC chromatogram of chondroitin disaccharide after enzymatic hydrolysis of chondroitin sulfate sodium standard is shown.

[0085] Figure 6 The HPLC analysis chromatogram of chondroitin disaccharide after enzymatic hydrolysis of the fermentation product in Example 4 is shown.

[0086] Figure 7 The mass spectrum of chondroitin disaccharide after enzymatic hydrolysis of the fermentation product in Example 4 is shown.

[0087] Figure 8 The molecular weight analysis spectrum of the fermentation products in Example 4 is shown.

[0088] Figure 9 Fermentation curves for control example 1 are shown; where: (a) represents OD600 and chondroitin yield; (b) represents residual sucrose and residual glucose concentrations;

[0089] Figure 10The fermentation curves for control example 2 are shown below; where: (a) represents OD600 and chondroitin yield; (b) represents residual sucrose and residual glucose concentrations.

[0090] Figure 11 The fermentation curves of control example 3 are shown; where: (a) represents OD600 and chondroitin yield; (b) represents the concentrations of residual sucrose and residual glucose. Detailed Implementation

[0091] This invention discloses a chondroitin fermentation culture medium and fermentation method.

[0092] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0093] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0094] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0095] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0096] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0097] This invention provides a high-yield chondroitin fermentation medium and fermentation process, specifically relating to a medium for the efficient production of chondroitin by engineered Bacillus subtilis, and a fermentation production process for chondroitin by engineered Bacillus subtilis. The aim is to fill the research gap in dedicated culture media for high-yield chondroitin fermentation, increase the yield of chondroitin produced by engineered Bacillus subtilis fermentation, overcome the production bottleneck of the Bacillus subtilis platform, and lay a solid foundation for large-scale industrial production.

[0098] Specifically, the present invention provides a chondroitin fermentation culture medium comprising the following components: tryptone, yeast extract, urea, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, anhydrous magnesium sulfate, and sucrose.

[0099] Furthermore, the content of tryptone in the above-mentioned chondroitin fermentation medium is 20-30 g / L, preferably 24 g / L.

[0100] Furthermore, the yeast extract content in the chondroitin fermentation medium is 20-30 g / L, preferably 24 g / L.

[0101] Furthermore, the urea content in the chondroitin fermentation medium is 8-16 g / L, preferably 12 g / L.

[0102] Furthermore, the ammonium sulfate content in the chondroitin fermentation medium is 5-12 g / L, preferably 8 g / L.

[0103] Furthermore, the potassium dihydrogen phosphate content in the chondroitin fermentation medium is 1~5 g / L, preferably 3 g / L.

[0104] Furthermore, the content of dipotassium hydrogen phosphate in the above-mentioned chondroitin fermentation medium is 8~16 g / L, preferably 13 g / L.

[0105] Furthermore, the sodium chloride content in the chondroitin fermentation medium is 1~4 g / L, preferably 2 g / L.

[0106] Furthermore, the content of anhydrous magnesium sulfate in the above-mentioned chondroitin fermentation medium is 0.5~3 g / L, preferably 1.5 g / L.

[0107] Furthermore, the sucrose content in the chondroitin fermentation medium is 30-50 g / L, preferably 40 g / L.

[0108] Furthermore, the chondroitin fermentation medium mentioned above also contains trace elements.

[0109] Furthermore, the trace elements include ferrous sulfate heptahydrate, manganese sulfate tetrahydrate, calcium chloride, zinc sulfate heptahydrate, copper sulfate pentahydrate, ammonium molybdate tetrahydrate, and sodium tetraborate decahydrate.

[0110] Furthermore, the content of each component in the above-mentioned trace elements (mother liquor) is as follows:

[0111] Ferrous sulfate heptahydrate, 8~12 g / L (e.g. 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L), preferably 9~11 g / L, more preferably 10 g / L;

[0112] Manganese sulfate tetrahydrate, 0.3~0.7 g / L (e.g. 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L), preferably 0.4~0.6 g / L, more preferably 0.5 g / L;

[0113] Calcium chloride, 1~3 g / L (e.g. 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L), preferably 1.5~2.5 g / L, more preferably 2 g / L;

[0114] Zinc sulfate heptahydrate, 2~2.4 g / L (e.g. 2 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L), preferably 2.1~2.3 g / L, more preferably 2.2 g / L;

[0115] Copper sulfate pentahydrate, 0.8~1.2 g / L (e.g. 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L), preferably 0.9~1.1 g / L, more preferably 1 g / L;

[0116] Ammonium molybdate tetrahydrate, 0.08~0.12 g / L (e.g. 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.11 g / L, 0.12 g / L), preferably 0.09~0.11 g / L, more preferably 0.1 g / L;

[0117] Sodium tetraborate decahydrate, 0.01~0.03 g / L (e.g. 0.01 g / L, 0.015 g / L, 0.02 g / L, 0.025 g / L, 0.03 g / L), preferably 0.015~0.025 g / L, more preferably 0.02 g / L;

[0118] Furthermore, the trace element content in the chondroitin fermentation medium is 8~12 mL / L (e.g., 8 mL / L, 9 mL / L, 10 mL / L, 11 mL / L, 12 mL / L); preferably, the trace element content in the fermentation medium is 9~11 mL / L; more preferably, the trace element content in the fermentation medium is 10 mL / L.

[0119] When the content of trace elements in the fermentation medium is 10 mL / L, the fermentation medium contains:

[0120] The content of the ferrous sulfate heptahydrate is 100 mg / L;

[0121] The content of the manganese sulfate tetrahydrate is 5 mg / L;

[0122] The calcium chloride content is 20 mg / L;

[0123] The content of zinc sulfate heptahydrate is 22 mg / L;

[0124] The content of copper sulfate pentahydrate is 10 mg / L;

[0125] The content of the ammonium molybdate tetrahydrate is 1 mg / L;

[0126] The content of sodium tetraborate decahydrate is 0.2 mg / L;

[0127] Furthermore, the pH of the chondroitin fermentation medium is 7.0.

[0128] In some specific embodiments, the chondroitin fermentation medium comprises the following components: 24 g / L tryptone, 24 g / L yeast extract, 12 g / L urea, 8 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 13 g / L dipotassium hydrogen phosphate, 2 g / L sodium chloride, 1.5 g / L anhydrous magnesium sulfate, 40 g / L sucrose, 100 mg / L ferrous sulfate heptahydrate, 5 mg / L manganese sulfate tetrahydrate, 20 mg / L calcium chloride, 22 mg / L zinc sulfate heptahydrate, 10 mg / L copper sulfate pentahydrate, 1 mg / L ammonium molybdate tetrahydrate, 0.2 mg / L sodium tetraborate decahydrate, and pH 7.0.

[0129] Furthermore, the fermentation medium described in any of the above is intended for use in the fermentation production of chondroitin by engineered Bacillus subtilis bacteria.

[0130] The present invention also provides a method for producing chondroitin by fermenting Bacillus subtilis engineered bacteria using the above-mentioned fermentation medium, wherein the production method includes the following steps: transferring Bacillus subtilis engineered bacteria that have undergone necessary strain activation and seed culture into a fermenter containing any of the above-mentioned fermentation medium for fermentation to obtain a fermentation broth containing chondroitin.

[0131] Furthermore, in the above production method, the initial fermentation medium filling amount in the fermenter is 40% to 50% of the fermenter volume.

[0132] Furthermore, the inoculation amount of the engineered Bacillus subtilis strain in the above production method is 5% (v / v).

[0133] Furthermore, the fermentation process temperature is controlled at 37°C in the above production method.

[0134] Furthermore, the fermentation time in the above production method is 48-60 hours.

[0135] Furthermore, in the above production method, the pH of the fermentation process is controlled at 7.0±0.05.

[0136] Preferably, in the above production method, the pH is controlled at 7.0±0.05 using 25 wt% ammonia water during the fermentation process.

[0137] Furthermore, in the above production method, dissolved oxygen is controlled at 15%~30% during the fermentation process.

[0138] Furthermore, in the above production method, during fermentation, when dissolved oxygen is <20%, the rotation speed is sequentially increased to 1000 rpm, increasing by 25 rpm each time; when dissolved oxygen is still below 20% after the rotation speed reaches 1000 rpm, the aeration is sequentially increased to 2.5 vvm, increasing by 0.25 vvm each time; when dissolved oxygen is greater than 30%, the aeration is sequentially decreased to 1 vvm, decreasing by 0.25 vvm each time; when dissolved oxygen is still greater than 30% after the aeration reaches 1 vvm, the rotation speed is sequentially decreased by 25 rpm each time until dissolved oxygen stabilizes at 30%.

[0139] Furthermore, in the above production method, when the initial sucrose concentration drops to 1 g / L, sucrose is used for feeding to control the residual sucrose concentration to be 0-10 g / L and the residual glucose concentration to be 0-5 g / L; preferably, the residual sucrose concentration is controlled to be 0-5 g / L and the residual glucose concentration to be 0-2 g / L; more preferably, the residual sucrose concentration is controlled to be 0-2 g / L and the residual glucose concentration to be 0-1 g / L.

[0140] Preferably, in the above production method, when the initial sucrose concentration drops to 1 g / L, 50%-75% (w / v) sucrose is used for feeding.

[0141] Furthermore, in the above production method, xylose is added during the fermentation process to achieve a final concentration of 18 g / L, and the addition is done once, twice, or multiple times; preferably, xylose is added to achieve a final concentration of 18 g / L during fermentation from 2 h to 12 h, and the addition is done twice.

[0142] Furthermore, the production method also includes recovery and purification: the obtained fermentation broth is separated into solid and liquid components by centrifugation or filtration, the filtrate is collected, and ultrafiltration is performed using a 50 K roll-up membrane until the conductivity of the filtrate is reduced to 2-3.5 ms / cm; the filtrate is transferred to an acid hydrolysis tank, heated to 85-90°C, and the pH is adjusted to 2.3-2.5 until a large amount of precipitate appears in the liquid. The filtrate is collected by filtration, the pH of the liquid is adjusted to neutral, and ultrafiltration is performed using a 30 K plate ultrafiltration membrane until the conductivity of the filtrate is reduced to 0.9 ms / cm; the filtrate is precipitated with alcohol, dehydrated, and then filtered to collect the chondroitin solid. The chondroitin powder is collected by vacuum drying and stored.

[0143] Furthermore, the weight-average molecular weight (Mw) of chondroitin described in any of the above items is approximately 40 kDa.

[0144] The method for detecting chondroitin yield involved in this invention can be found in the analytical methods on page 1595 of Part II of the Chinese Pharmacopoeia 2020. Specifically, the method for detecting chondroitin yield involved in this invention can be performed as follows: Take 10 mL of fermentation broth into a 50 mL centrifuge tube, add pure glacial acetic acid to adjust the pH to pH 3.8, place in an 85℃ water bath for acid hydrolysis for 4 h, after acid hydrolysis, cool to room temperature, adjust the pH to between 7.0 and 8.0 using 6 M sodium hydroxide (NaOH), and transfer to a 25 mL volumetric flask for final volume adjustment. Take 100 μL of the acid-hydrolyzed sample and mix it with 100 μL of chondroitin sulfate ABC enzyme (SIGMA-Aldrich, C2905-10UN). Add 800 μL of tris(hydroxymethyl)chloromethane buffer, mix thoroughly, and place in a 37℃ water bath for 1 h. After the enzymatic digestion is completed, boil in a water bath for 5 minutes, cool with cold water, centrifuge, and take 20 μL of the supernatant. Load the sample onto a Spherisorb SAX Column (Waters) for HPLC analysis at a detection wavelength of 232 nm. Quantification was performed using the external standard method. The sum of the peak areas of ΔDi-0S, ΔDi-4S, and ΔDi-6S after enzymatic hydrolysis of 10 g / L chondroitin sulfate sodium standard (China National Institutes for Food and Drug Control, 4386 / 10 / 12, batch number 140792-202003) was used as the peak area of ​​the reference standard. The peak area of ​​ΔDi-0S after enzymatic hydrolysis of the sample was used as the peak area of ​​the sample, and the chondroitin content was calculated as (sample peak area / reference peak area × standard concentration 10 g / L × dilution factor 2.5).

[0145] The method for determining the molecular weight of chondroitin involved in this invention can be based on the analytical method of "Method for Determining the Molecular Weight of Low Molecular Weight Heparin", and uses a TSKgel G3000SWXL column to determine the molecular weight of chondroitin produced by fermentation.

[0146] The glucose determination method of the present invention is as follows: centrifuge the fermentation broth at 10,000 rpm for 5 min, take the supernatant, dilute it 20 times, and then measure it using a biosensor analyzer M-100 (Silman Technology).

[0147] The Bacillus subtilis provided by this invention was deposited on December 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with accession number CGMCC No. 36960, and classified as Bacillus subtilis.

[0148] The Bacillus subtilis engineered strain DH003 involved in this invention is Bacillus subtilis, which was deposited on December 9, 2025 at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with accession number CGMCC No. 36960, and classified as Bacillus subtilis.

[0149] In Examples 1 to 4 and Comparative Examples 1 to 3 of the present invention, the raw materials and reagents used can all be purchased from the market.

[0150] The present invention will be further illustrated below with reference to the embodiments:

[0151] Example 1: Production of chondroitin by fermentation of engineered Bacillus subtilis strain DH003 in a 5 L fermenter

[0152] Strain activation: Take a loopful of Bacillus subtilis DH003 (Bacillus subtilis, deposited on December 9, 2025 at the China General Microbiological Culture Collection Center, Beijing, China, accession number CGMCC No. 36960, classified as Bacillus subtilis) from a glycerol tube, streak it onto solid LB medium, and incubate at 37°C until a single colony grows. Pick one single colony and inoculate it into LB liquid medium, and activate it overnight at 37°C.

[0153] Preparation of fermentation medium: Weigh the required materials according to the fermentation medium ratio shown in Table 1. Add an appropriate amount of deionized water to the mixing tank, pour the weighed materials (except anhydrous magnesium sulfate, sucrose, and trace elements) into the mixing tank, stir to completely dissolve, continue to add deionized water to the required volume, adjust the pH to 7.0, then add trace elements and defoamer according to the required volume, control the pressure at 0.06 MPa, the temperature at 115℃, and keep warm for 15 min. After sterilization, introduce sterile air, turn on the cooling water to cool down, and maintain pressure until inoculation. Prepare anhydrous magnesium sulfate stock solution (75 g / L) and sucrose stock solution (500 g / L) separately, control the pressure at 0.06 MPa, the temperature at 115℃, and keep warm for 15 min. Before inoculation, add the anhydrous magnesium sulfate stock solution and sucrose stock solution according to the required volumes to make their final concentrations 1.5 g / L and 40 g / L, respectively.

[0154] Table 1 Fermentation medium components

[0155]

[0156] Preparation of Seed Culture Medium: Since the main function of the seed culture medium is to cultivate and propagate microorganisms, providing sufficient cell volume for the next fermentation step, and enabling the inoculum to rapidly proliferate and metabolize into the target product after entering the fermenter, this embodiment also prepares a corresponding seed culture medium based on the above-mentioned fermentation medium. Weigh the required materials according to the seed culture medium ratio shown in Table 2. Add an appropriate amount of deionized water to the seed bottle or seed tank. Pour the weighed materials (except anhydrous magnesium sulfate and sucrose) into the seed bottle or seed tank, stir until completely dissolved, and continue adding deionized water to the required volume. Adjust the pH to 7.0, control the pressure at 0.06 MPa, and the temperature at 115℃, and incubate for 15 min. Prepare anhydrous magnesium sulfate stock solution (75 g / L) and sucrose stock solution (500 g / L) separately, control the pressure at 0.06 MPa, and the temperature at 115℃, incubating for 15 min. Before inoculation, add the anhydrous magnesium sulfate stock solution and sucrose stock solution according to the required volumes, making their final concentrations 1.5 g / L and 40 g / L, respectively.

[0157] Table 2 Seed Culture Medium Components

[0158]

[0159] Seed culture: Inoculate 5% (v / v) into a 500 mL shake flask containing 100 mL of the seed culture medium shown in Table 2, and incubate at 37℃ and 220 rpm for 4.5 h. OD600>12.

[0160] Fermentation culture: The seed culture was transferred at a 5% inoculum (v / v) into a 5 LT&J Intelli-FermA benchtop fermenter (purchased from Shanghai Dibier Company) containing 1.8 L of the fermentation medium shown in Table 1, and 0.1 g / L of defoamer was added. Fermentation was carried out for 48 h according to the control process shown in Table 3. After fermentation, the fermentation broth was collected, and the fermentation curve is shown in Table 3. Figure 1 The yield of chondroitin was determined by HPLC, and the results are shown in Table 13.

[0161] Table 3.5 L-tank fermentation control process

[0162]

[0163] Example 2: Production of chondroitin by fermentation of engineered Bacillus subtilis strain DH003 in a 5 L fermenter

[0164] After activating the engineered Bacillus subtilis strain DH003 according to the method in Example 1, it was inoculated at a 5% inoculum (v / v) into a 500 mL shake flask containing 100 mL of the seed culture medium shown in Table 4, and cultured at 37℃ and 220 rpm for 4.5 h, with an OD600 > 12. The seed culture was then transferred at a 5% inoculum (v / v) into a 5 L T&J Intelli-FermA benchtop fermenter (purchased from Shanghai Dibier Company) containing 1.8 L of the fermentation culture medium shown in Table 5, and 0.1 g / L of antifoaming agent was added. Fermentation was carried out for 48 h according to the control process shown in Table 3 of Example 1. This process was repeated three times. After fermentation, the fermentation broth was collected, and the fermentation curve is shown in [reference needed]. Figure 2 The yield of chondroitin was determined by HPLC, and the results are shown in Table 13.

[0165] Table 4 Seed Culture Medium Components

[0166]

[0167] Table 5 Fermentation medium components

[0168]

[0169] Example 3: Production of chondroitin by fermentation of engineered Bacillus subtilis strain DH003 in a 5 L fermenter

[0170] After activating the engineered Bacillus subtilis strain DH003 according to the method in Example 1, it was inoculated at a 5% inoculum (v / v) into a 500 mL shake flask containing 100 mL of the seed culture medium shown in Table 6, and cultured at 37℃ and 220 rpm for 4.5 h, with an OD600 > 12. The seed culture was then transferred at a 5% inoculum (v / v) into a 5 L T&J Intelli-FermA benchtop fermenter (purchased from Shanghai Dibier Company) containing 1.8 L of the fermentation culture medium shown in Table 7, and 0.1 g / L of antifoaming agent was added. Fermentation was carried out for 48 h according to the control process shown in Table 3 of Example 1. This process was repeated three times. After fermentation, the fermentation broth was collected, and the fermentation curve is shown in [reference needed]. Figure 3 The yield of chondroitin was determined by HPLC, and the results are shown in Table 13.

[0171] Table 6 Seed Culture Medium Components

[0172]

[0173] Table 7 Fermentation medium components

[0174]

[0175] Example 4: Production of chondroitin by fermentation of engineered Bacillus subtilis strain DH003 in a 50 L fermenter

[0176] Strain activation: The engineered Bacillus subtilis strain DH003 was activated according to the method in Example 1.

[0177] Seed culture: Inoculate 5% of the seed culture medium in a shake flask containing the seed culture medium shown in Table 2 of Example 1, and incubate at 37°C and 220 rpm for 4.5 h. OD600 > 12 to obtain primary seed culture. Then, inoculate 5% of the primary seed culture into a 15 L seed tank containing the seed culture medium shown in Table 2 of Example 1, and incubate at 37°C for 2.5 h. Dissolved oxygen is controlled at 30-40%. When dissolved oxygen is below 20%, the rotation speed, airflow, and tank pressure are increased sequentially until OD600 > 12, yielding secondary seed culture.

[0178] Fermentation culture: The secondary seed culture prepared above was transferred at an inoculum rate of 5% into a 50 L fermenter containing the fermentation medium shown in Table 1 of Example 1, with an initial liquid volume of 25 L. Fermentation was carried out for 48 h according to the control process shown in Table 8, then the fermentation broth was collected.

[0179] Table 8. Fermentation Control Process for 50 L Tanks

[0180]

[0181] Its fermentation curve is shown in [reference]. Figure 4The highest OD600 reached 120±3.5. The chondroitin content in the fermentation broth was detected by HPLC. The HPLC chromatogram of chondroitin disaccharide after enzymatic hydrolysis of chondroitin sulfate standard is shown below. Figure 5 As shown, the HPLC chromatogram of chondroitin disaccharide after enzymatic hydrolysis of the fermentation product is as follows. Figure 6 As shown, the chondroitin content in the fermentation broth was calculated to be 13.50 ± 0.67 g / L.

[0182] In this embodiment, the chondroitin produced in the 50 L fermenter was further identified by mass spectrometry (LC-MS) and its molecular weight was determined. The mass spectrum of chondroitin disaccharide after enzymatic hydrolysis is shown below. Figure 7 As shown, its mass-to-charge ratio is 378, which is similar to the theoretical molecular weight (C) of chondroitin disaccharide (ΔDi-OS). 14 H 21 NO 11 The [MH]⁻ m / z = 378.103 values ​​are completely consistent, indicating that the chondroitin structure obtained through the fermentation process provided by this invention is correct. The molecular weight detection results of chondroitin are as follows: Figure 8 As shown in Table 9, the weight-average molecular weight of chondroitin produced by fermentation is 40.101 kDa for 71.11% of the product, 12.984 kDa for 19.04% of the product, and 6.411 kDa for 9.85% of the product.

[0183] Table 9. Results of chondroitin molecular weight analysis

[0184]

[0185] Comparative Example 1: Chondroitin production by fermentation of engineered Bacillus subtilis strain DH003 in a 5 L fermenter.

[0186] After activating the engineered Bacillus subtilis strain DH003 according to the method in Example 1, it was inoculated at a 5% inoculum (v / v) into a 500 mL shake flask containing 100 mL of the seed culture medium shown in Table 10, and cultured at 37℃ and 220 rpm for 4.5 h, with an OD600 > 12. The seed culture was then transferred at a 5% inoculum (v / v) into a 5 L T&J Intelli-FermA benchtop fermenter (purchased from Shanghai Dibier Company) containing 1.8 L of the fermentation culture medium shown in Table 11, and 0.1 g / L of antifoaming agent was added. Fermentation was carried out according to the control process shown in Table 12 for 48 h. This process was repeated three times. After fermentation, the fermentation broth was collected, and the fermentation curve is shown below. Figure 9 As shown in Table 13, the yield of chondroitin was determined by HPLC.

[0187] Table 10 Seed Culture Medium Formulation

[0188]

[0189] Table 11 Fermentation medium formulation

[0190]

[0191] Table 12.5 L-tank fermentation control process

[0192]

[0193] Comparative Example 2: Chondroitin production by fermentation of engineered Bacillus subtilis strain DH003 in a 5 L fermenter.

[0194] After activating the engineered Bacillus subtilis strain DH003 according to the method in Example 1, it was inoculated at a 5% inoculum (v / v) into a 500 mL shake flask containing 100 mL of the seed culture medium shown in Table 10 of Control Example 1. The flask was incubated at 37°C and 220 rpm for 4.5 h, with an OD600 > 12. The seed culture was then transferred at a 5% inoculum (v / v) into a 5 LT&J Intelli-FermA benchtop fermenter (purchased from Shanghai Dibier Company) containing 1.8 L of the fermentation medium shown in Table 11 of Control Example 1. 0.1 g / L of antifoaming agent was added, and fermentation was carried out according to the control process shown in Table 3 of Example 1 for 48 h. This process was repeated three times. After fermentation, the fermentation broth was collected, and the fermentation curve is shown below. Figure 10 As shown in Table 13, the yield of chondroitin was determined by HPLC.

[0195] Comparative Example 3: Chondroitin production by fermentation of engineered Bacillus subtilis strain DH003 in a 5 L fermenter.

[0196] After activating the engineered Bacillus subtilis strain DH003 according to the method in Example 1, it was inoculated at a 5% inoculum (v / v) into a 500 mL shake flask containing 100 mL of the seed culture medium shown in Table 2 of Example 1, and cultured at 37℃ and 220 rpm for 4.5 h, with an OD600 > 12. The seed culture was then transferred at a 5% inoculum (v / v) into a 5 L T&J Intelli-FermA benchtop fermenter (purchased from Shanghai Dibier Company) containing 1.8 L of the fermentation culture medium shown in Table 1 of Example 1, and 0.1 g / L of antifoaming agent was added. Fermentation was carried out for 48 h according to the control process shown in Table 12 of Control Example 1. This process was repeated three times. After fermentation, the fermentation broth was collected, and the fermentation curve is shown below. Figure 11 As shown in Table 13, the yield of chondroitin was determined by HPLC.

[0197] Table 13 Results of Chondroitin Fermentation in 5 L Tanks

[0198]

[0199] Based on a comparative analysis of the specific experimental data provided in Examples 1-3 and Comparative Examples 1-3 of the present invention, the technical advantages of the present invention over the prior art are mainly reflected in the following aspects:

[0200] (1) Significantly increased yield: Under the same 5 L fermentation scale, the chondroitin produced by the engineered Bacillus subtilis strain using the culture medium and process of the present invention (Examples 1-3) reached a yield of 9.62-11.29 g / L; while the same strain using the control culture medium and process (Control Example 1) had a yield of 8.01 g / L, the same strain using the control culture medium and process of the present invention (Control Example 2) had a yield of 8.45 g / L, and the same strain using the culture medium and process of the present invention had a yield of 8.88 g / L (Control Example 3). The yield of the method of the present invention is about 20%-41% higher than that of the control method (Control Example 1), the culture medium of the present invention is about 14%-34% higher than that of the control culture medium (Control Example 2), and the process of the present invention is about 8%-27% higher than that of the control process (Control Example 3). The above results show that the optimized culture medium formulation and fermentation process of the present invention have a significant effect on improving the yield of chondroitin: the yield of the culture medium of the present invention (Comparative Example 3) is 11% higher than that of the control culture medium (Comparative Example 1), and the yield of the process of the present invention (Comparative Example 2) is 5% higher than that of the control process (Comparative Example 1); when the two are used in combination (Examples 1-3), the yield is further increased to 9.62-11.29 g / L, which is 8%-27% higher than that of the culture medium of the present invention alone (Comparative Example 3) and 14%-34% higher than that of the process of the present invention alone (Comparative Example 2), which fully demonstrates the complementarity and combined advantages of the culture medium and the process.

[0201] (2) Significantly enhanced cell growth: At the end of fermentation, the highest OD600 of the cells in Examples 1-3 reached 101.2-125.8, while that in Control Example 1 was only 61.1, in Control Example 2 it was 70.1, and in Control Example 3 it was 116. The cell biomass supported by the method of the present invention is about 1.6-2 times that of the control method (Control Example 1), the cell biomass supported by the culture medium of the present invention is about 1.4-1.8 times that of the control method (Control Example 2), and the cell biomass supported by the process of the present invention is about 0.9-1.1 times that of the control method (Control Example 3). The cell density of Examples 1 and 2 is higher than that of Control Example 3, indicating that the process of the present invention can still effectively maintain the metabolic activity of the cells under high-density culture conditions, laying the foundation for high yield.

[0202] Based on the fermentation experimental data of Example 1 (5 L tank) and Example 4 (50 L tank), it can be seen that the special fermentation medium provided by this invention successfully achieved efficient and stable production of chondroitin by the engineered Bacillus subtilis strain DH003 during the scale-up process from small-scale (5 L) to pilot-scale (50 L). Specifically:

[0203] 1) High-yield performance was validated and scaled up: In a 5 L tank, the chondroitin yield reached 11.29 ± 0.27 g / L; after scaling up to a 50 L tank, the yield further increased to 13.50 ± 0.67 g / L. This not only confirmed the high efficiency of the culture medium formulation and process, but more importantly, no yield decline occurred during the scale-up process. Instead, it demonstrated the potential for process optimization and broke through the yield bottleneck of chondroitin production from Bacillus subtilis.

[0204] 2) The product quality is well-defined and homogeneous: In-depth analysis of the product from the 50 L tank showed that the mass spectrometry information of the disaccharide unit produced after enzymatic hydrolysis of the obtained polymer was completely consistent with the standard chondroitin disaccharide, confirming that the product is chondroitin with the correct structure. Molecular weight distribution analysis showed that the weight average molecular weight (Mw) of its main components was approximately 40 kDa, and the distribution was relatively concentrated, indicating that the fermentation process can produce high-quality products with well-defined structure and homogeneous molecular weight.

[0205] In summary, this invention develops a highly efficient culture medium specifically for the fermentation production of chondroitin by engineered Bacillus subtilis bacteria. Through a precisely designed culture medium formulation (containing specific ratios of tryptone, yeast extract, a complex nitrogen source, a high-concentration phosphate buffer pair, and trace elements), it effectively solves the problems of insufficient specificity of the culture medium, crude process control, and unstable scale-up performance in existing technologies. This provides a sufficient nutritional foundation for the growth (even high-density growth) and efficient synthesis of the engineered bacteria. Experimental data fully verify the significant advantages of this invention: at the same 5 L fermentation scale, the technology of this invention can achieve a chondroitin yield of 11.29 g / L, compared to 8.01 g / L in the control example, representing an increase of approximately 41%. Simultaneously, bacterial growth is more vigorous, with a maximum OD600 reaching 121.2, approximately twice that of the control example (61.1), providing sufficient bacterial quantity for efficient synthesis. This culture medium also exhibits excellent scalability; after scaling up to a 50 L scale, the yield stabilizes at 13.50 g / L without any decline. Furthermore, analysis of the product confirmed that the obtained chondroitin has the correct structure, with the weight-average molecular weight of its main components concentrated at approximately 40 kDa, exhibiting uniform mass. In addition, this invention also provides a refined fermentation process specifically for the fermentation production of chondroitin using engineered Bacillus subtilis bacteria. Through key control strategies such as staged dissolved oxygen linkage control, precise residual sugar regulation, and batch-feeding of xylose, it effectively solves the problems of extensive process control and unstable scale-up performance in existing technologies, providing a reliable guarantee for the stability of the fermentation process and the continuity of product synthesis.

[0206] In summary, the specialized culture medium and supporting fermentation process provided by this invention work separately or synergistically from the two dimensions of nutrient supply and process control. This not only significantly improves the yield and efficiency of chondroitin production on the Bacillus subtilis platform, but also ensures the quality of the product and the stability of the process, providing a reliable and competitive solution for the industrial biomanufacturing of chondroitin.

[0207] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fermentation method for chondroitin, characterized in that, After obtaining the seed culture of Bacillus subtilis, it was inoculated into a fermentation medium and fermented to obtain chondroitin. The fermentation medium comprises the following components: Tryptone 20-30 g / L, yeast extract 20-30 g / L, urea 8-16 g / L, ammonium sulfate 5-12 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 8-16 g / L, sodium chloride 1-4 g / L, anhydrous magnesium sulfate 0.5-3 g / L, sucrose 30-50 g / L, and trace elements; The trace elements include: ferrous sulfate heptahydrate, manganese sulfate tetrahydrate, calcium chloride, zinc sulfate heptahydrate, copper sulfate pentahydrate, ammonium molybdate tetrahydrate, and sodium tetraborate decahydrate. The content of each component in the trace element mother liquor is as follows: ferrous sulfate heptahydrate 10 g / L, manganese sulfate tetrahydrate 0.5 g / L, calcium chloride 2 g / L, zinc sulfate heptahydrate 2.2 g / L, copper sulfate pentahydrate 1 g / L, ammonium molybdate tetrahydrate 0.1 g / L, and sodium tetraborate decahydrate 0.02 g / L; the content of the trace elements is 10 mL / L. The Bacillus subtilis strain was deposited on December 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 36960, and classified as Bacillus subtilis. Bacillus subtilis ; The dissolved oxygen level during fermentation is 15-30%. When the dissolved oxygen level is below 20%, the dissolved oxygen level is increased by sequentially increasing the rotation speed and increasing the ventilation rate. When the dissolved oxygen level is greater than 30%, the dissolved oxygen level is reduced by successively decreasing the ventilation rate and decreasing the rotation speed. When the sucrose concentration drops to 1 g / L during fermentation, the sucrose is added continuously, and the residual sucrose concentration in the fermentation environment is controlled to be 0~2 g / L and the residual glucose concentration is 0~1 g / L. The fermentation process also includes a fed-batch feeding step; the fed-batch feeding includes ammonia, sucrose, xylose, and an antifoaming agent; The xylose is added over a period of 2 to 12 hours, to a final concentration of 18 g / L, and at least once.

2. The fermentation method as described in claim 1, characterized in that, The anhydrous magnesium sulfate and the sucrose are prepared and used immediately.

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