Animal-source-free culture medium as well as application and method of animal-source-free culture medium in production of plague bacillus F1 antigen

Abstract

The invention discloses an animal-source-free culture medium as well as application and a method of the animal-source-free culture medium in production of plague bacillus F1 antigens, and belongs to the technical field of culture mediums. The technical problem to be solved is that a special culture medium which is clear in component, free of animal sources and capable of efficiently and stably promoting plague bacillus growth and F1 antigen expression is lacked in the prior art. According to the key point of the technical scheme, the culture medium composition is provided, and the culture medium composition comprises 25-35 g / L of plant source peptone, 1.25-3.75 g / L of xylose, 5-7 g / L of yeast powder, 1.5-2.5 g / L of glycine, 1.5-4.5 g / L of sodium chloride and 0.5-1.5 g / L of disodium hydrogen phosphate. The culture medium composition does not contain animal source components, is clear in components, and has obvious effects in the aspects of culturing plague bacillus and fermenting to produce F1 antigen.

Description

Technical Field

[0001] This invention belongs to the field of culture medium technology, and relates to animal-free culture medium, specifically to an animal-free culture medium and its application and method in the production of Yersinia pestis F1 antigen. Background Technology

[0002] For understanding the technical content of this invention: Plague is a disease caused by Yersinia pestis (Yersinia pestis). Yersinia pestis Plague is a highly contagious zoonotic disease that poses a serious threat to human health. Although antibiotics can be used for treatment, prevention remains the preferred and most crucial strategy in combating plague due to its rapid onset, fast course, and high mortality rate. Vaccination is the core means of prevention. Traditional plague vaccines are mainly inactivated whole-cell vaccines and live attenuated vaccines (such as EV live vaccines). These vaccines are effective to some extent, but still have shortcomings. For example, inactivated vaccines have relatively weak immunogenicity and require multiple immunizations, while live attenuated vaccines have the potential safety risk of virulence reversion. With the development of biotechnology, "component vaccines" (or subunit vaccines) based on key protective antigens have become an important direction for next-generation vaccine development due to their clearly defined components, high safety, and controllable quality.

[0003] Among the many plague antigens, the F1 capsular antigen is widely recognized as one of the most crucial protective antigens. It forms an anti-phagocytic capsule structure on the bacterial surface, making it a key virulence factor for pathogen infection. Antibodies against the F1 antigen provide highly effective neutralizing protection. Therefore, the F1 antigen is not only an important biomarker for plague diagnosis but also an indispensable core component for constructing safe and effective plague component vaccines. However, achieving large-scale production and application of the F1 antigen, its low-cost, high-efficiency, and high-purity industrial preparation is currently the main technological bottleneck. The core of this bottleneck lies in the production culture medium. Currently, the culture media used for producing the F1 antigen mostly employ traditional Thick-Blood medium or its modified formulations. These media typically contain animal-derived components (such as beef extract, casein hydrolysate, etc.). Such media have the following inherent drawbacks: (1) Large batch-to-batch differences: The complex animal-derived components lead to unstable performance of culture media in different batches, which directly affects the uniformity of downstream F1 antigen yield and quality, posing a huge challenge to the standardized production of vaccines.

[0004] (2) High safety risk: There is a risk of potential contamination by exogenous factors (such as viruses and prions), which does not meet the strict requirements for the safety of raw materials in modern biological products, especially human vaccines.

[0005] (3) High cost and not in line with the trend: Animal-derived raw materials are expensive, and the entire production process does not conform to the modern biological product production safety trend of no animal-derived ingredients, which is not conducive to the global registration and promotion of the product.

[0006] While existing technologies have attempted to replace animal-derived components with plant-based peptones (such as soybean peptone and wheat peptone), these methods generally suffer from problems such as low F1 antigen expression, poor bacterial growth, or complex and insufficiently optimized culture medium composition. For example, direct replacement with some plant-based peptones fails to support sufficient bacterial density or effective F1 antigen expression in Yersinia pestis, resulting in insufficient production to meet the demands of industrial-scale production. Therefore, developing a dedicated culture medium with clearly defined components, free of animal sources, and capable of efficiently and stably promoting the growth of Yersinia pestis and F1 antigen expression is of paramount importance for overcoming the production bottleneck of F1 antigen and promoting the technological upgrading and industrialization of next-generation plague component vaccines.

[0007] Relevant patent documents retrieved: This document, published in Russia (publication number RU2844477C1) on July 31, 2025, discloses a method for producing an accumulating liquid culture medium for culturing Yersinia pestis EV vaccine strains. The method includes diluting enzymatic hydrolysates of casein and soybean with distilled water to an amine nitrogen content of 120 mg%. Then, the following substances are mixed: 89.0 g of casein hydrolysate, 89.0 g of soybean hydrolysate, and 10.0 g of dry enzymatically hydrolyzed protein. Ingredients: peptone, 0.2g magnesium sulfate, 3.0g sodium chloride, 4.0g disubstituted sodium phosphate dodecahydrate, 20.0g activated carbon. Add distilled water to 1L. Set the pH to 8.3±0.1 with 20% sodium hydroxide solution. Boil the culture medium for 3-5 minutes, then filter it through a belt filter and filter paper. Add growth promoters—0.3g sodium sulfite and 0.5g ammonium molybdate tetrahydrate—to the filtrate and stir vigorously. Add boiling distilled water to the initial volume and dilute with hydrochloric acid solution 1:1 to pH 7.2±0.1. Sterilize at (115±1)℃ for 20 minutes. This culture medium has a complex composition and high preparation cost.

[0008] Relevant non-patent literature retrieved: The journal or book title is *Advances in Microbiology and Immunology*, and the article title is "Optimization of Liquid Fermentation Medium for Yersinia pestis EV Strain using Response Surface Methodology," Volume 53, published in April 2025. This article discloses the optimization of the liquid fermentation medium for Yersinia pestis EV strain using response surface methodology to increase the yield of the target F1 antigen. In single-factor experiments, the highest bacterial density and F1 antigen expression were achieved under the conditions of 40.00 g / L Medtone and 6.00 g / L yeast extract. Xylose, sodium chloride, sodium dihydrogen phosphate, and glycine all promoted the growth of Yersinia pestis EV strain and the expression of F1 antigen to a certain extent. The optimized medium formulation after response surface methodology was: Medtone 30.00 g / L, yeast extract 6.00 g / L, and xylose 4.50 g / L. In the validation experiment in a 30 L bioreactor, the highest A600 nm value was 7.09, and the F1 antigen concentration in the harvest liquid was 383.36 μg / mL. Summary of the Invention

[0009] The purpose of this invention is to provide: A culture medium composition and related technologies are provided to address the technical problems in the prior art, such as the lack of a specific culture medium with clearly defined components, no animal origin, and capable of efficiently and stably promoting the growth of Yersinia pestis and the expression of F1 antigen, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] Definitions of standard chemical terms can be found in the reference "Handbook of Microbiological Media, CRC Press, Ronald M. Atlas, 4th edition (2010)".

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as the ELISA method, shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The term "cell culture medium," also known as cell culture medium, is not only the basic substance that supplies nutrients to cultured cells and promotes cell reproduction and proliferation, but also the living environment for the growth and reproduction of cultured cells.

[0016] The term "serum-free medium" refers to a synthetic culture medium that allows cells to grow and multiply in vitro for an extended period without the addition of serum. Serum-free medium generally consists of two main parts: a basal medium and added components. Cells used in biopharmaceutical and vaccine production typically exhibit adherent or facultative adherent growth when cultured in vitro; however, when grown in serum-free medium, they often grow in suspension.

[0017] The term "serum" refers to the pale yellow, transparent liquid separated from blood plasma after coagulation, by removing fibrinogen and certain clotting factors, or to plasma with fibrinogen removed. Its main functions are to provide essential nutrients, hormones and various growth factors, binding proteins, and factors that promote cell adhesion and prevent mechanical damage, as well as to provide some protection for cells in culture.

[0018] To achieve the above objectives, the present invention provides the following technical solution: On one hand, the present invention provides a culture medium composition comprising 25-35 g / L of plant-derived peptone, 1.25-3.75 g / L of xylose, 5-7 g / L of yeast extract, 1.5-2.5 g / L of glycine, 1.5-4.5 g / L of sodium chloride, and 0.5-1.5 g / L of disodium hydrogen phosphate, with the balance being water.

[0019] Peptone is a core nitrogen source in bacterial culture media, produced by enzymatic or acid hydrolysis of animal and plant proteins (such as casein, gelatin, and fishmeal). It contains abundant polypeptides, amino acids, nitrogenous bases, and small amounts of carbohydrates and minerals. It provides comprehensive nitrogen nutrition for bacterial growth and reproduction, meeting the needs of bacteria in synthesizing proteins, nucleic acids, enzymes, and other nitrogenous biomolecules. Simultaneously, the small peptides and free amino acids in peptone can be directly absorbed and utilized by bacteria without additional decomposition, making it suitable for the growth of various heterotrophic bacteria, especially crucial for facultative anaerobes or aerobic bacteria with high nutritional requirements. Furthermore, peptone can regulate the osmotic pressure and pH buffering capacity of the culture medium, enhancing its stability, and is an indispensable component of general-purpose culture media (such as LB broth and nutrient broth).

[0020] Xylose is a pentose, a five-carbon monosaccharide primarily used as a selective or differential carbon source in bacterial culture media. As a carbon source, it can be broken down and utilized by specific xylose-metabolizing bacteria (such as certain Enterobacteriaceae and Pseudomonas), providing them with energy and a carbon skeleton for the synthesis of cell walls, cell membranes, and other carbohydrate derivatives. Bacteria unable to utilize xylose cannot grow in media where xylose is the sole carbon source. Therefore, xylose is often used in selective media (such as media for isolating enteropathogenic bacteria) to screen for target strains. In differential media, the breakdown of xylose produces organic acids, causing a decrease in pH. Combined with acid-base indicators (such as bromocresol purple), this color change distinguishes xylose-fermenting bacteria from non-fermenting bacteria, for example, differentiating Shigella (which does not ferment xylose) from Escherichia coli (which ferments xylose). Furthermore, the metabolic characteristics of xylose can also be used for bacterial biochemical identification and to aid in bacterial classification.

[0021] Yeast powder (also known as yeast extract) is a complex produced from brewer's yeast or baker's yeast through autolysis, filtration, concentration, and drying. It is rich in B vitamins (such as vitamins B1, B2, B6, folic acid, and niacin), nucleotides, amino acids, trace elements (such as magnesium, iron, and zinc), and a small amount of carbohydrates. In culture media, it primarily acts as a growth factor supplement, providing essential growth factors for nutritionally demanding bacteria (such as lactic acid bacteria, streptococci, and mycoplasma)—bacteria that cannot synthesize certain vitamins or coenzymes and must obtain them from external sources. Yeast powder can meet this need, promoting rapid bacterial growth and proliferation. Simultaneously, the amino acids and peptides in yeast powder can assist in nitrogen supply, increasing the nutrient concentration of the culture medium; its buffering substances can also slightly regulate the pH of the culture medium, reducing drastic pH fluctuations during bacterial metabolism. Yeast powder is often used in combination with peptone (such as LB medium) to form a nutritionally complete composite culture medium suitable for high-density cultivation of various bacteria.

[0022] Glycine is the simplest amino acid, and its role in bacterial culture varies depending on the strain and culture objective: ① Selective inhibitor: High concentrations of glycine (usually 10-20 g / L) can inhibit the growth of Gram-positive bacteria because it interferes with the synthesis of peptidoglycan in the cell wall of Gram-positive bacteria (glycine replaces alanine residues in peptidoglycan, destroying the cross-linking structure of peptidoglycan, leading to cell wall defects). Gram-negative bacteria, whose cell walls contain an outer membrane, are more tolerant to glycine. Therefore, glycine is often used in selective culture media to isolate Gram-negative bacteria (such as Enterobacteriaceae); ② Nutrient additive: Low concentrations of glycine (0.1-1 g / L) can serve as a nitrogen source or amino acid source, providing raw materials for bacterial protein synthesis, especially suitable for auxotrophic strains that cannot synthesize glycine; ③ Inducer: In the culture of some genetically engineered strains, glycine can induce bacteria to secrete exogenous proteins or promote increased cell membrane permeability, facilitating product extraction; ④ pH adjustment aid: Glycine has both acidic and basic groups (zwitterions), and can act as a weak buffer to stabilize the pH of the culture medium.

[0023] The core role of sodium chloride (NaCl) in bacterial culture media is to regulate osmotic pressure. Bacterial cells possess a certain osmotic pressure, and an appropriate amount of NaCl in the culture medium (usually 0.5–1 g / L, such as 10 g / L in LB medium) maintains the osmotic pressure balance between the inside and outside of the cell, preventing bacterial death due to excessively high osmotic pressure (water loss and shrinkage) or excessively low osmotic pressure (water absorption and rupture), thus ensuring normal bacterial morphology and physiological function. Furthermore, NaCl increases the ionic strength of the culture medium, enhances its stability, and reduces the damage to the cell membrane caused by bacterial metabolites. In selective media, high concentrations of NaCl (such as 7.5% NaCl) can inhibit the growth of most salt-intolerant bacteria, allowing only halophilic bacteria (such as Staphylococcus aureus) to grow, achieving selective separation. Simultaneously, sodium ions in NaCl act as activators of certain bacterial enzymes, participating in bacterial material transport and energy metabolism processes, and have a slight promoting effect on bacterial growth.

[0024] Disodium hydrogen phosphate (Na₂HPO₄) is a commonly used buffer in culture media. It is often combined with potassium dihydrogen phosphate (KH₂PO₄) or sodium dihydrogen phosphate (NaH₂PO₄) to form a phosphate buffer system, maintaining the pH of the culture medium within a stable range (typically pH 6.5–7.5, suitable for the growth of most bacteria). Bacterial metabolism produces organic acids (such as lactic acid and acetic acid) or alkaline substances, causing changes in the pH of the culture medium. The phosphate buffer system can mitigate these changes by using HPO₄²⁻. - With H2PO4 -Disodium hydrogen phosphate neutralizes acids or bases produced during metabolism, preventing drastic pH fluctuations from affecting bacterial enzyme activity and growth. Furthermore, it provides phosphorus and sodium: phosphorus is an important component of bacterial nucleic acids, phospholipids, ATP, and coenzymes, participating in energy metabolism and genetic material synthesis; sodium participates in bacterial ion balance and substance transport. Disodium hydrogen phosphate also slightly increases the ionic strength of the culture medium, enhancing its stability and preventing nutrient precipitation.

[0025] Preferably, the plant-derived peptone is wheat peptone or soybean peptone.

[0026] Preferably, the plant-derived peptone is wheat peptone.

[0027] In some specific embodiments, the wheat peptone is selected from one or more of Kerry HyPep 4601N, Angel wheat peptone, and Aoboxin wheat peptone.

[0028] In some specific embodiments, the culture medium composition comprises the following components: 30.6 g / L plant-derived peptone, 5.5 g / L yeast extract, 3 g / L sodium chloride, 2.1 g / L glycine, 1 g / L disodium hydrogen phosphate, and 2.5 g / L xylose.

[0029] On the other hand, the present invention provides the application of the above-described culture medium composition in the culture of Yersinia pestis.

[0030] On the other hand, the present invention provides a liquid fermentation culture method for Yersinia pestis, the liquid fermentation culture method comprising inoculating Yersinia pestis into the above-mentioned culture medium composition for culture.

[0031] Preferably, the culture conditions are: culture at 35-38°C with aeration and stirring for 24-72 hours.

[0032] On the other hand, the present invention provides the use of the above-described culture medium composition in the preparation of Yersinia pestis F1 antigen.

[0033] On the other hand, the present invention provides a method for preparing Yersinia pestis F1 antigen, comprising culturing Yersinia pestis using the above-mentioned culture medium composition and producing F1 antigen from Yersinia pestis.

[0034] The present invention has at least the following beneficial effects: 1. This invention provides a completely animal-free, well-defined, and highly efficient culture medium solution, successfully overcoming the technical bottleneck of low and unstable Yersinia pestis F1 antigen yield in plant-derived systems. Specifically: (1) Shift in technical approach: The traditional approach that must rely on complex animal-derived components has been abandoned, and a new animal-free culture medium design scheme has been established with plant-derived peptone as the core, combined with specific carbon sources (xylose) and key amino acids (glycine).

[0035] (2) Scientific optimization method: The system optimization was carried out using Design of Experiments (DOE) and Response Surface Methodology (RSM), which revealed the interaction between the three core components, peptone, yeast powder and glycine, and determined their optimal ratio range.

[0036] (3) Core component discovery: It was verified that xylose, as a carbon source, has a unique promoting effect on the expression of F1 antigen when combined with other components of the present invention. This discovery is crucial for increasing yield.

[0037] 2. This invention has achieved clear and beneficial technical effects through laboratory shake flask and 30L fermentation tank verification: (1) Safety has been fundamentally improved This eliminates the risk of exogenous factors that may arise from animal-derived components, meeting the stringent safety requirements for raw materials in biopharmaceutical production. It ensures that the product's biosafety performance meets the stringent domestic and international regulatory requirements for raw materials used in human vaccines.

[0038] (2) The production of F1 antigen meets the application requirements. At shake-flask scale, the F1 antigen yield (159.08-237.08 μg / mL) was comparable to or better than that of conventional Thick-Style medium (149.33-197.47 μg / mL).

[0039] At a 30L fermenter scale, the F1 antigen yield reached 400.4 μg / mL, demonstrating that the formulation has good scalability and can meet the needs of industrial production.

[0040] (3) Simultaneously reduce costs and improve production efficiency By using inexpensive plant-based raw materials, particularly validating the suitability of domestically produced wheat peptone, production costs were effectively reduced. While lowering costs, the yield of F1 antigen reached a high level, improving overall production efficiency.

[0041] (4) Better consistency between production batches Because the raw materials have clear composition and stable sources, the inherent batch-to-batch differences of animal-derived raw materials are overcome, which helps to ensure the stability of F1 antigen production and quality, and provides a guarantee for the standardized production of vaccines.

[0042] (5) Enhanced supply chain stability The formula's applicability to multiple peptone sources reduces reliance on a single imported raw material and enhances the resilience and self-control of the supply chain. Attached Figure Description

[0043] Figure 1 This is a contour plot showing the effect of the peptone-yeast interaction on the 48-hour F1 antigen concentration obtained from response surface optimization in Example 4 of the present invention.

[0044] Figure 2 This is a contour plot showing the effect of the peptone-glycine interaction on the 48-hour F1 antigen concentration obtained from response surface optimization in Example 4 of the present invention.

[0045] Figure 3 This is a contour plot of the effect of the interaction between glycine and yeast powder on the 48-hour F1 antigen concentration obtained by response surface optimization in Example 4 of the present invention.

[0046] Figure 4 This is a graph showing the change in F1 antigen yield over time in a 30L fermenter using the optimized culture medium of the present invention in Example 5 of the present invention. Detailed Implementation

[0047] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0049] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0050] Experimental materials The strains used in all embodiments of this invention are Yersinia pestis EV vaccine strains. This strain is a standard attenuated strain known to those skilled in the art for use in plague vaccine production. All the following embodiments use this strain for verification, but it is for illustrative purposes only. It should be understood that the culture medium compositions provided by this invention are also applicable to other plague strains that require culturing.

[0051] Example 1: Basic preparation method of culture medium composition 1.1 The culture medium composition of this embodiment is prepared through the following steps: Weigh each component according to the concentration range shown in Table 1: Table 1

[0052] 1.2 Dissolve the above components (except xylose and calcium chloride) in purified water, adjust the pH to 7.0-7.2 with 1 M NaOH or HCl solution, and bring the volume to the target level. Autoclave at 115°C for 30 minutes. After the culture medium has cooled to room temperature, add the xylose stock solution and calcium chloride stock solution (if added) that have been sterilized by filtration through a 0.22 μm membrane under aseptic conditions, and mix well for later use.

[0053] 1.3 A non-animal-derived culture medium composition within the concentration range described in this invention has been successfully prepared and can be used for subsequent liquid fermentation culture of Yersinia pestis.

[0054] Example 2: Applicability verification of peptones from different plant sources The purpose of this embodiment is to verify that the culture medium formulation provided by the present invention is universally applicable to wheat peptone from different manufacturers and can achieve efficient F1 antigen production.

[0055] The culture medium composition used a uniform basic formula: 30 g / L wheat peptone, 6 g / L yeast extract, 3 g / L sodium chloride, 2 g / L glycine, 1 g / L disodium hydrogen phosphate, and 2.5 g / L xylose. Three different sources of wheat peptone were used as nitrogen sources: Group A: Kerry wheat peptone (purchased from Kerry Company, USA, product number HyPep 4601N); Group B: Angel Wheat Peptone (purchased from Angel Yeast, product number Q / AQJM 2287); Group C: Aoboxin wheat peptone (purchased from Beijing Aoboxin Biotechnology, product number 01-992); Traditional Houshi culture medium was used as a control (Group D), and the main component was Houshi beef digestive fluid, which was prepared by our company.

[0056] Culture medium was prepared according to the method in Example 1. After inoculation with Yersinia pestis EV strain, the culture was incubated in shake flasks at 37°C and 200 rpm for 48 hours. The culture supernatant was collected, and the concentration of F1 antigen was quantitatively detected by ELISA. Specifically, the Yersinia pestis F1 antigen detection kit (enzyme-linked immunosorbent assay) was used for detection. The kit was manufactured by Lanzhou Institute of Biological Products Co., Ltd., Medical Device Manufacturing Enterprise License No.: Gansu Food and Drug Administration Medical Device Production Permit 20120006.

[0057] The results are shown in Table 2. Under the unified formulation provided by the present invention, wheat peptone from three different sources can support the production of high levels of F1 antigen by Yersinia pestis, and the yields are comparable to those of Thick's medium, and in some cases even better.

[0058] Table 2

[0059] *Note: The experiment was repeated 3 times in parallel. The data is the average value (low value - high value).

[0060] The above results lead to the conclusion that the culture medium formulation of this invention is a highly efficient "platform technology" with broad applicability to plant-derived peptones (especially wheat peptone) produced by different manufacturers. Using domestically produced Angel wheat peptone, which has lower costs, the average F1 antigen yield can reach 161.19 μg / mL, comparable to that of Thick's medium. Furthermore, using optimized imported or domestically produced wheat peptone can achieve even higher yields, thus solving the technical problem of unstable performance in the application of domestic raw materials.

[0061] Example 3: Discovery and Verification of Synergistic Effects of Key Components The purpose of this embodiment is to systematically verify the individual and synergistic promoting effects of xylose, calcium chloride, and sodium thiosulfate on the expression of Yersinia pestis F1 antigen in basal culture medium No. 9.

[0062] (1) Method Using soybean peptone basal medium No. 9 (30 g / L peptone, 6 g / L yeast extract, 3 g / L sodium chloride, 2 g / L glycine, 1 g / L disodium hydrogen phosphate) as a control, the following experimental groups were set up: Experimental group 1: basal culture medium + xylose 5 g / L; Experimental group 2: basal culture medium + calcium chloride 0.5 g / L; Experimental group 3: basal culture medium + xylose 5 g / L + calcium chloride 0.5 g / L; Experimental group 4: basal culture medium + xylose 5 g / L + calcium chloride 0.5 g / L + sodium thiosulfate 0.5 g / L; Positive control: Thick's medium; The culture medium was prepared according to the method in Example 1 and inoculated with Yersinia pestis EV strain. After culturing in shake flasks at 37°C for 48 hours, the supernatant was taken for quantitative detection of F1 antigen by ELISA.

[0063] (2) Results As shown in Table 3, adding xylose or calcium chloride alone can improve F1 yield to some extent, but adding xylose and calcium chloride simultaneously can significantly increase the yield, indicating a synergistic effect between the two. When xylose, calcium chloride, and sodium thiosulfate are added together, the F1 antigen yield reaches the highest value, significantly better than the other groups, proving that there is a higher-level synergistic enhancement effect among the three components.

[0064] Table 3

[0065] *Note: The experiment was repeated 3 times in parallel. The data is the average value (low value - high value).

[0066] Based on the above results, this experiment clearly reveals that xylose, calcium chloride, and sodium thiosulfate have a significant synergistic and enhancing effect on promoting F1 antigen expression in plant-derived culture media. This fundamental discovery provides a crucial theoretical basis and ample room for component optimization in the formulation design of this invention. Although the core optimal formulation tended to be simplified in subsequent in-depth optimization of wheat peptone No. 5, this systematic discovery of the synergistic effect is a crucial cornerstone of the entire invention process.

[0067] Example 4: Response Surface Methodology Optimization and Confirmation of Optimal Formulation The purpose of this embodiment is to, based on the verified synergistic effect of additives such as xylose and calcium chloride, use experimental design methods to accurately locate the core component that contributes the most to the F1 antigen yield from among many influencing factors, and determine its optimal ratio.

[0068] (1) Method Based on Example 3, xylose, calcium chloride, and sodium thiosulfate were demonstrated to have a clear promoting or synergistic effect on F1 yield. To construct a streamlined, efficient, and easily industrialized core formulation, this study first focused on screening for the most influential basic nutrient factors.

[0069] First, a component factorial assay was used to screen the six basic components of the culture medium: peptone (A), yeast extract (B), sodium chloride (C), glycine (D), xylose (E), and disodium hydrogen phosphate (F). In this screening, calcium chloride and sodium thiosulfate, which have proven effective, were included as fixed additives in the basal medium.

[0070] The results are as follows Figures 1-3As shown, peptone (A), yeast extract (B), and glycine (D) are the three most critical factors dominating F1 yield, with their effects significantly higher than other factors. Subsequently, the concentration of xylose (E) was fixed at the central point level (2.5 g / L), and the addition of calcium chloride and sodium thiosulfate was omitted. Instead, a central composite design was used to conduct response surface methodology for these three key factors (A, B, D), establishing a quadratic polynomial model, which was optimized using the 48-hour F1 antigen concentration as the response value.

[0071] (2) Results Analysis of variance showed that the established model was highly significant (p<0.001) and exhibited a strong curvature effect. The optimal formulation determined analytically by the model is: Peptone (Angel Wheat Peptone): 30.6 g / L; Yeast powder: 5.5 g / L; Glycine: 2.1 g / L; (Other ingredients: sodium chloride 3 g / L, xylose 2.5 g / L, disodium hydrogen phosphate 1 g / L).

[0072] Using this optimal formulation (without the addition of calcium chloride and sodium thiosulfate), a validation experiment was conducted. After 48 hours of shake-flask incubation, the F1 antigen concentration remained stable at a high level of 159.08-237.08 μg / mL.

[0073] Based on the above results, the core formulation of this invention was successfully optimized and confirmed using response surface methodology. This process revealed that the ratio of peptone, yeast extract, and glycine in the culture medium system is the most critical lever for regulating F1 antigen yield. The finally determined optimal formulation achieved extremely high F1 antigen yield without the addition of calcium chloride and sodium thiosulfate, demonstrating that by precisely optimizing the core nutritional framework, optimal production results can be achieved, thus making the formulation more streamlined and efficient.

[0074] Example 5: Scale Validation of Fermentation Tanks The purpose of this embodiment is to verify the feasibility and superiority of the culture medium of the present invention in culturing Yersinia pestis and producing F1 antigen in industrial-scale fermenters.

[0075] (1) Method The optimized culture medium of this invention (formulation same as the optimal formulation in Example 4) was prepared in a 30L fermenter, with a working volume of 15L. After inoculation with Yersinia pestis EV strain, the culture was carried out at 37°C with dissolved oxygen controlled at above 20% by adjusting the stirring speed and aeration rate for 48 hours. During the process, samples were taken regularly to monitor bacterial density, and the supernatant was used to detect the F1 antigen concentration.

[0076] (2) Results like Figure 4As shown, the cell growth was normal during the fermentation process. The F1 antigen was detected by ELISA. The F1 antigen was continuously expressed and accumulated during the culture process. At the time of harvest after 48 hours, the concentration of F1 antigen reached 400.4 μg / mL.

[0077] Based on the above results, the culture medium of the present invention performs excellently on a 30L fermenter scale, supporting high-density growth of Yersinia pestis and high-level F1 antigen expression. Its yield is significantly better than that of traditional Thick's medium on the same scale, which fully demonstrates the feasibility and superiority of the culture medium of the present invention in industrial-scale production.

[0078] Comparative Example 1 The control culture medium shown in Table 4 was prepared according to the method of Example 4, and the F1 antigen yield was detected by fermentation. It was compared with Thick's medium and the culture medium of Example 4 of the present invention. Each group was repeated 3 times in parallel. The results are shown in Table 4. It can be found that the different components and concentrations in the culture medium composition provided by the present invention work synergistically to significantly increase the F1 antigen yield.

[0079] Table 4

[0080] *Note: For data in the same row, the same letter indicates that there is no significant difference between the data (P>0.05), while different letters indicate that there is a significant difference between the data (P<0.05).

[0081] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A culture medium composition, characterized in that, The culture medium composition comprises 25-35 g / L of plant-derived peptone, 1.25-3.75 g / L of xylose, 5-7 g / L of yeast extract, 1.5-2.5 g / L of glycine, 1.5-4.5 g / L of sodium chloride, and 0.5-1.5 g / L of disodium hydrogen phosphate, with the balance being water.

2. The culture medium composition according to claim 1, characterized in that, The plant-derived peptone is wheat peptone or soybean peptone.

3. The culture medium composition according to claim 2, characterized in that, The plant-derived peptone is wheat peptone.

4. The culture medium composition according to claim 3, characterized in that, The wheat peptone is selected from one or more of KerryHyPep 4601N, Angel wheat peptone, and Aoboxin wheat peptone.

5. The culture medium composition according to any one of claims 1-4, characterized in that, The culture medium composition consists of the following components: 30.6 g / L plant-derived peptone, 5.5 g / L yeast extract, 3 g / L sodium chloride, 2.1 g / L glycine, 1 g / L disodium hydrogen phosphate, and 2.5 g / L xylose.

6. The use of the culture medium composition according to any one of claims 1-5 in culturing Yersinia pestis.

7. A liquid fermentation culture method for Yersinia pestis, characterized in that, The liquid fermentation culture method includes inoculating Yersinia pestis into the culture medium composition according to any one of claims 1-5 for culture.

8. The liquid fermentation culture method according to claim 7, characterized in that, The culture conditions are as follows: culture at 35-38℃ with aeration and stirring for 24-72 hours.

9. The use of the culture medium composition according to any one of claims 1-5 in the preparation of Yersinia pestis F1 antigen.

10. A method for preparing Yersinia pestis F1 antigen, characterized in that, This includes culturing Yersinia pestis using the culture medium composition according to any one of claims 1-5, and producing F1 antigen from Yersinia pestis.

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