COMPOSITION OF MEDIUM FOR THE PREPARATION OF BOTULINUM TOXIN

Abstract

The present invention relates to a medium composition for the production of botulinum toxin and, more specifically, to a medium composition for the cultivation of Clostridium sp. strains capable of producing botulinum toxin. When a medium containing vegetable peptone, glucose, and yeast extracts, and containing no components of animal origin, is used according to the present invention for the cultivation of Clostridium botulinum strains, the growth rate of the strains increases compared to existing media, and the strains can be cultivated safely due to the absence of components of animal origin, thereby increasing the productivity of botulinum toxin.

Description

Medium composition for the production of botulinum toxin

[0001] The present invention relates to a medium composition for producing botulinum toxin, and more particularly, to a medium composition for culturing strains of the genus Clostridium capable of producing botulinum toxin. The medium composition of the present invention is characterized by comprising a vegetable peptone including wheat peptone and pea peptone, D-(+)-glucose, and yeast extract.

[0002]

[0003] A variety of Clostridium strains secreting neurotoxic toxins have been discovered since the 1890s, and the toxins secreted by these strains have been characterized over the past 70 years (Schant, EJ et al., Microbiol. Rev., 56:80, 1992).

[0004] Botulinum toxin, a neurotoxic toxin derived from the above Clostridium strains, is classified into seven types, A to G, based on their serological characteristics. Each toxin contains a toxic protein of approximately 150 kDa, which naturally forms a complex with several non-toxic proteins. The medium complex (300 kDa) consists of the toxic protein and the non-toxic non-hemagglutinin protein, while the large (450 kDa) and large-large (900 kDa) complexes are formed by the medium complex being bound to hemagglutinin (Sugiyama, H., Microbiol. Rev., 44:419, 1980). These non-toxic non-hemagglutinin proteins are known to function to protect the toxin from low pH and various proteolytic enzymes in the intestine.

[0005] The above toxin is synthesized as a single polypeptide with a molecular weight of approximately 150 kDa within the cell, and is then cleaved at a position one-third from the N-terminus by the action of intracellular proteases or artificial enzyme treatment such as trypsin, thereby dividing into two units, a light chain (L: light chain) (molecular weight: 50 kDa) and a heavy chain (H: heavy chain) (molecular weight: 100 kDa). The toxicity of the toxin divided in this way is significantly increased compared to when it is a single polypeptide. The two units are linked to each other by a disulfide bond, and each has a different function. The heavy chain binds to the receptor of the target cell (Park. MKet al., FEMS Microbiol. Lett., 72:243, 1990) and has the function of forming a channel by reacting with the biological membrane at low pH (pH4) (Mantecucco, C.et al., TIBS., 18:324, 1993), and the light chain has pharmacological activity and imparts permeability to the cell using a detergent or inhibits the secretion of neurotransmitters when introduced into the cell through electroporation (Poulain, B.et al., Proc. Natl. Acad. Sci. USA., 85:4090, 1988).

[0006] The toxin inhibits the exocytosis of acetylcholine at cholinergic presynapses of the neuromuscular junction, causing systemic weakness. Because even trace amounts of the toxin are toxic, it has been suggested that the toxin possesses some enzymatic activity (Simpson, L.L. et al., Ann. Rev. Pharmaeol. Toxicol., 26:427, 1986).

[0007] Recent studies have shown that the toxin possesses metallopeptidase activity, and its substrates are synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP25), which are unit proteins that form the exocytosis machinery complex. Each type of toxin uses one of these three proteins as a substrate, and types B, D, F, and G are known to cleave synaptobrevin at a specific site, types A and E to cleave SNAP25, and type C to cleave syntaxin at a specific site (Binz, T. et al., J. Biol. Chem., 265:9153, 1994).

[0008] In particular, botulinum toxin type A is known to be soluble in dilute aqueous solutions with a pH of 4.0 to 6.8. At pHs above approximately 7, the stabilized non-toxin protein dissociates from the neurotoxin, resulting in a gradual loss of toxicity, which is known to decrease particularly with increasing pH and temperature.

[0009] The above botulinum toxin is a toxin that can be used as one of the four major biological terrorism weapons, along with Bacillus anthracis, Yersinia pestis, and smallpox virus, because it is lethal to the human body in small amounts and easy to mass-produce. However, it has been revealed that type A botulinum toxin can paralyze local muscles at the injection site when injected in a dose below that which does not affect the human body systemically. This characteristic can be utilized in a wide range of applications, such as as a wrinkle remover, a treatment for spastic hemiplegia, and cerebral palsy. Therefore, demand is rapidly increasing, and research on methods for producing botulinum toxin is actively being conducted to meet this demand.

[0010] The currently representative commercialized product is BOTOX from Allergan, USA. ® (botulinum toxin type A purified neurotoxin complex), each BOTOX ® A 100-unit vial of botulinum toxin type A complex contains approximately 5 ng of purified botulinum toxin type A complex, 0.5 mg human serum albumin, and 0.9 mg sodium chloride, supplied in a vacuum-dried form and reconstituted with sterile saline without preservatives (0.9% sodium chloride injection). Another commercially available product is Dysport from Ipsen, UK. ® (Clostridium botulinum type A toxin hemagglutinin complex with lactose and human serum albumin in a botulinum toxin pharmaceutical composition, reconstituted with 0.9% sodium chloride before use) and MyoBloc from Solstice Neurosciences ® (a solution for injection containing botulinum toxin type B, human serum albumin, sodium succinate, and sodium chloride, pH 5.6).

[0011] The method for manufacturing botulinum toxin disclosed in Korean Patent No. 10-1339349 and the culture medium for Clostridium botulinum strains commonly used contain animal-derived ingredients. Therefore, if abnormal prion, a known cause of transmissible spongiform encephalopathy, is present in these animal-derived ingredients due to contamination, this could pose a problem in the botulinum toxin manufacturing process.

[0012] Transmissible spongiform encephalopathy (TSE) is a type of degenerative neurological disease that causes fatal neurodegenerative diseases. It occurs in humans and animals, such as bovine spongiform encephalopathy (BSE), scrapie, Creutzfeldt-Jacob disease (CJD), Gerstmann-Straussler-Scheinker syndrome, kuru, transmissible mink encephalopathy, chronic wasting disease of deer, and feline spongiform encephalopathy. In the case of bovine spongiform encephalopathy, it has been reported that it crosses the species barrier and occurs in humans as well.

[0013] The causative agent of transmissible spongiform encephalopathy is non-immunogenic and has a long incubation period. Postmortem analysis of the brains of cattle infected with bovine spongiform encephalopathy (BSE), or mad cow disease, reveals a distinct pattern of spongy vacuoles in the brain, caused by neuronal destruction and deposition of abnormal protein fibers.

[0014] The pathogen believed to cause transmissible spongiform encephalopathies is an infectious protein called abnormal prion. Unlike typical viruses, which require nucleic acid, abnormal prion is an infectious particle composed solely of protein and does not contain nucleic acid. Transmissible spongiform encephalopathies are known to develop when abnormal prion (PrPsc), an infectious agent, binds to normal prion (PrPc), transforming it into a pathogenic prion, which then accumulates in the brain (Prusiner SB, Alzheimer Dis Assoc Disord., 3:52-78, 1989).

[0015] Creutzfeldt-Jakob disease (CJD) is a rare neurodegenerative disorder of the human transmissible spongiform encephalopathy (TSE). The infectious agent is clearly an abnormal isoform of the prion protein. Individuals with CJD can progress from apparently healthy to akinetic mutism within six months. Therefore, administering pharmaceutical compositions containing biological agents, such as botulinum toxin derived from animal products, carries a risk of developing prion-mediated diseases, such as CJD. Therefore, manufacturing pharmaceuticals using bulk solutions produced using animal-derived components carries the risk of exposing patients to various pathogens or infectious agents.

[0016] Therefore, it has been reported that adding casein hydrolysate (e.g., TSE-Certificated casein hydrolysate) to APF (Animal Protein Free) medium improves the growth rate of strains and the production of botulinum toxin to prevent TSE infection (Korean Patent No. 17-1729251, etc.). However, since APF medium with casein hydrolysate added still contains casein, an animal-derived component, research is needed to achieve high growth rates and high toxin production of Clostridium botulinum strains in APF medium that does not contain animal-derived components.

[0017] Accordingly, the inventors of the present invention have made great efforts to develop a medium that contains plant-derived peptone without the risk of TSE infection when culturing Clostridium botulinum strains and does not contain animal-derived components in order to prevent the risk of acquiring the above-described prion-mediated disease, and as a result, have confirmed that the medium can improve the growth rate of Clostridium botulinum strains and toxin production compared to existing media, thereby completing the present invention.

[0018]

[0019] The purpose of the present invention is to provide a medium composition for culturing a Clostridium botulinum strain that does not contain animal-derived components.

[0020] Another object of the present invention is to provide a manufacturing method for improving the production amount of botulinum toxin by culturing Clostridium botulinum in the medium composition.

[0021]

[0022] To achieve the above purpose, the present invention provides a medium composition for culturing a Clostridium botulinum strain, which comprises vegetable peptone including wheat peptone and pea peptone, D-(+)-glucose and yeast extract, and does not contain animal-derived components.

[0023] The present invention also provides a method for producing botulinum toxin, comprising the steps of: (a) culturing Clostridium botulinum using the medium composition to produce botulinum toxin; and (b) recovering the produced botulinum toxin.

[0024]

[0025] When a medium containing vegetable peptone, glucose, and yeast extract according to the present invention and not containing animal-derived components is used for culturing a Clostridium botulinum strain, the strain growth rate increases compared to existing media, and since it does not contain animal-derived components, the strain can be safely cultivated, thereby increasing the productivity of botulinum toxin.

[0026]

[0027] Figure 1 shows the OD response using three plant-based peptones, peptone from pea, wheat peptone, and peptone from potatoes No. 2, as factors. 540nm This shows JMP's Custom Design to determine the appropriate ratio of plant-based peptones with a value of 3 or greater.

[0028] Figure 2 shows the Design Evaluation that confirmed the Power Analysis and Fraction of Design Space Plot of the Custom Design.

[0029] Figure 3 shows a medium containing APF medium components.

[0030] Figure 4a is a graph measuring the growth rate of Clostridium botulinum strains over time in APF medium with Dextrin as a carbon source, and Figure 4b is a graph measuring the growth rate of Clostridium botulinum strains over time in APF medium with Glucose as a carbon source.

[0031] Figure 5 is a graph measuring the growth rate of Clostridium botulinum strains over time in 16 types of APF media.

[0032] Figure 6 shows the distribution analysis of D-optimal Design Experiments, and it can be confirmed that the experimental results are generally evenly distributed.

[0033] Figure 7 shows the scatter plot matrix analysis of the D-optimal Design Results, and wheat peptone and pea peptone have OD 540nm And it can be confirmed that there is a weak positive correlation with the toxin concentration.

[0034] Figure 8 shows the residual analysis, and it can be confirmed that there are no outliers in the experimental results.

[0035] Fig. 9 is OD 540nm This shows the process for establishing a model. Since the VIF value does not exceed 10 and is at the level of 1, there is no problem with the model, and it can be confirmed that all Studentized Residuals results are included in the 95% confidence interval.

[0036] Fig. 10 is OD 540nm It shows the prediction formula for .

[0037] Fig. 11 is OD 540nm It shows the interaction profiles of .

[0038] Fig. 12 is OD 540nm This shows the process for establishing a model. Since the VIF value does not exceed 10 and is at the level of 1, there is no problem with the model, and it can be confirmed that all Studentized Residuals results are included in the 95% confidence interval.

[0039] Figure 13 shows a prediction formula for toxin concentration.

[0040] Figure 14 shows the interaction profile.

[0041] Figure 15 shows the OD based on the prediction formula for each response value. 540nm The process of finding the medium conditions for toxin concentration through Prediction Profiler is shown.

[0042] Figures 16a and 16b show the distribution of Y in Spec (Figure 16a) and Edge of failure analysis (Figure 16b) after performing 5000 simulations using the Simulator function to provide random variation to factors and model noise.

[0043] Figure 17 shows the capacity analysis of peas, with the range corresponding to 3σ set as NORs and the range corresponding to 4.5σ set as PARs.

[0044] Figure 18 shows the analysis of wheat capacity, with the range corresponding to 3σ set as NORs and the range corresponding to 4.5σ set as PARs.

[0045] Figure 19 shows the Contour Profiler results of the APF badge. When NORs (3σ, red line) and PARs (4.5σ, blue line) are displayed on the contour plot, it can be confirmed that they do not go beyond the white range, which is the range that satisfies the model.

[0046] Figure 20 shows the growth curve of botulinum strains by APF medium.

[0047] Figure 21 shows the amount of toxin expression by APF medium.

[0048]

[0049] Although the growth rate of Clostridium botulinum strains (growth rate) improved compared to conventional media using APF (Animal Protein Free) medium, there is a need to add medium components that further enhance strain growth while still eliminating the risk of infection with TSEs and other pathogens. Recently, casein hydrolysates (e.g., TSE-certified casein hydrolysate), for which no cases of TSE infection have been reported, have been added to APF medium to grow strains and produce toxins.

[0050] However, casein hydrolysate is a component extracted from milk, and casein accounts for approximately 80% of the total protein contained in milk. Although the APF medium with casein hydrolysate added does not contain animal-derived components such as fetal bovine serum (FBS), since casein hydrolysate itself is an animal-derived component, there is a need for a medium for culturing Clostridium botulinum strains that is safe and contains plant-based peptone without the addition of animal-derived components such as casein hydrolysate.

[0051] Accordingly, the present invention relates to a medium composition for culturing a Clostridium botulinum strain, comprising, in one aspect, a vegetable peptone including wheat peptone and pea peptone, D-(+)-glucose and yeast extract, and containing no animal-derived components. Here, the vegetable peptone refers to a peptone extracted from wheat or pea, preferably a commercially available Millipore TM 96174, Millipore TM It may be, but is not limited to, 93492-500G-F.

[0052] In the present invention, the existing medium refers to a medium containing animal-derived components such as casein hydrolysate, yeast extract, and thioglycollate medium. In addition, the APF medium (Animal Protein Free medium) refers to a medium that does not contain animal-derived proteins.

[0053] In one embodiment of the present invention, an APF medium containing various plant-based medium raw materials including Peptone from pea, Wheat peptone, Peptone from potatoes No. 2, Corn steep solid, Gluten from wheat, Potato peptone E210, Banana powder, Tomato juice, and Malt extract was prepared under TSE-free (TSE (Transmissible Spongiform Encephalopathy)-free) conditions without animal-derived components, and the growth rates of strains and toxin expression levels were compared. As a result, it was confirmed that the plant-based medium raw material for efficiently culturing Clostridium botulinum strains includes plant-based peptones including peptone from pea, wheat peptone, and peptone from potatoes No. 2, and in this case, the strain growth and toxin expression levels were excellent. Finally, as shown in Table 4, Table 5 and Figure 4, the vegetable peptones included in the final selected Clostridium botulinum strain culture medium composition were confirmed to be pea peptone, wheat peptone and potato peptone.

[0054] In another embodiment of the present invention, APF media containing plant-based peptones such as pea peptone, wheat peptone, and potato peptone in various composition ratios without animal-derived components under TSE-free (TSE (Transmissible Spongiform Encephalopathy)-free) conditions were prepared, and the growth rates and toxin expression levels of the strains were compared. As a result, the medium composition for efficiently culturing Clostridium botulinum strains was a case in which at least one plant-based peptone selected from the group consisting of wheat peptone and pea peptone was included, and a carbon source (e.g., glucose) and a nitrogen source (e.g., yeast extract) were added thereto, and in this case, it was confirmed that the strain growth and toxin expression levels were excellent. As a result, as shown in Tables 8 to 12 and Figures 5 to 19, the contents of the plant-based peptones included in the final selected medium composition for culturing Clostridium botulinum strains were determined to be 18 g / L of pea peptone and 7 g / L of wheat peptone.

[0055] In another embodiment of the present invention, an experiment was performed to compare the growth pattern and toxin concentration of Clostridium botulinum strains when grown in APF medium containing the final selected plant peptone of the present invention and when grown in medium (Comparative Example 7) described in Korean Patent Publication No. 10-2022-0140696 published prior to the present invention. As a result, as shown in Tables 13 to 15 and Figures 20 and 21, in the case of the medium of the present invention, OD at 28 hours after culturing the Clostridium botulinum strain 540nm The OD value was 7.1021, and after that, the OD value gradually decreased, and after 48 hours, the OD 540nmThe OD of the Clostridium botulinum toxin supernatant was 5.5636, and the toxin concentration in the supernatant of Clostridium botulinum toxin was 588.8 ㎍ (10 mL of culture medium) after 48 hours of culture. Meanwhile, in the case of the medium of Comparative Example 7, as shown in Tables 13 to 15 and Figures 20 and 21, the OD of the Clostridium botulinum strain after 28 hours of culture 540nm The concentration of toxin in the supernatant of Clostridium botulinum toxin was 6.7739, and the concentration of toxin in the supernatant of Clostridium botulinum toxin was 263.7 ㎍ (10 mL of culture medium) after 48 hours of culture, confirming that the amount of toxin expression was reduced by 55.2% compared to the medium of the present invention.

[0056] In another embodiment of the present invention, when the composition component of the medium described in Korean Patent Publication No. 10-2022-0140696, L-cysteine ​​hydrochloride monohydrate and / or medical antifoam c emulsion (Dow Corning®) were additionally included in the medium of the present invention (Comparative Examples 1 to 3), an experiment was performed to compare the growth pattern and toxin concentration of Clostridium botulinum strains. As a result, as shown in Tables 13 to 15 and Figures 20 and 21, Comparative Examples 1 to 3 showed OD 540nm It can be confirmed that the value is lower than that of the present invention and that the amount of toxin expression after 48 hours of culture is reduced by 14.9 to 76.6% compared to that of the present invention.

[0057] In another embodiment of the present invention, when soy peptone was added instead of pea peptone and / or wheat peptone in the medium of the present invention, and L-cysteine ​​hydrochloride monohydrate and medical antifoam c emulsion (Dow Corning®) were additionally included (Comparative Examples 4 to 6), an experiment was performed to compare the growth pattern and toxin concentration of Clostridium botulinum strains. As a result, as shown in Tables 13 to 15 and Figures 20 and 21, Comparative Examples 4 to 6 showed OD 540nm It can be confirmed that the value is significantly lower than that of the present invention, and that the amount of toxin expression after 48 hours of culture is reduced by 87.0 to 94.9% compared to that of the present invention.

[0058] In the present invention, the medium composition for culturing the Clostridium botulinum strain may include one or more plant-derived ingredients selected from the group consisting of peptone from pea, wheat peptone, peptone from poatatoes No. 2, corn steep solid, gluten from wheat, potato peptone E210, banana powder, tomato juice, and malt extract, and preferably includes peptone from pea, wheat peptone, and peptone from poatatoes No. 2, and more preferably includes peptone from pea and wheat peptone, but is not limited thereto.

[0059] As used herein, the terms "vegetable peptone" or "vegetable hydrolysate" refer to a decomposition product of a protein obtained from a plant. For example, wheat peptone (wheat hydrolysate) refers to a product obtained by decomposing total protein obtained from wheat. Additionally, "casein hydrolysate" refers to a decomposition product of casein protein.

[0060] The decomposition of the above vegetable protein or casein protein is preferably carried out by partial digestion. The decomposition of the protein is preferably carried out by decomposition by acid treatment, alkali treatment, enzymatic treatment, high-pressure treatment, heat treatment, or physical treatment. More preferably, the vegetable peptone or casein hydrolysate may be characterized as having been enzymatically treated. The physical treatment is, for example, grinding.

[0061] The vegetable peptone or casein hydrolysate of the present invention is a product of partial decomposition of protein, and is in the form of a mixture containing not only single amino acids but also peptides composed of several to several dozen amino acids and complete protein molecules.

[0062] In the present invention, the content of the vegetable peptone may be characterized as being 0.1 to 10 w / v% (1 to 100 g / L), preferably 0.2 to 5 w / v% (2 to 50 g / L), and more preferably 0.5 to 2 w / v% (5 to 20 g / L).

[0063] In the present invention, the content of the wheat peptone may be 0.1 to 4.0 w / v% (1 to 40 g / L), preferably 0.45 to 0.95 w / v% (4.5 to 9.5 g / L), preferably 0.48 to 0.92 w / v% (4.8 to 9.2 g / L), preferably 0.55 to 0.85 w / v% (5.5 to 8.5 g / L), and more preferably 0.7 w / v% (7 g / L).

[0064] In the present invention, the content of the pea peptone may be 0.1 to 3.5 w / v% (1 to 35 g / L), preferably 1.55 to 2.05 w / v% (15.5 to 20.5 g / L), preferably 1.58 to 2.03 w / v% (15.8 to 20.3 g / L), preferably 1.65 to 1.95 w / v% (16.5 to 19.5 g / L), and more preferably 1.8 w / v% (18 g / L).

[0065] In the present invention, the medium composition for culturing the Clostridium botulinum strain is characterized in that it does not contain L-cysteine ​​hydrochloride monohydrate and / or an antifoaming agent.

[0066] As used herein, the term "antifoaming agent" refers to a substance that completely or partially prevents the formation of foam, and is used interchangeably with "antifoaming agent." The antifoaming agent may include, but is not limited to, medical-grade antifoaming agents, including Dow Corning®.

[0067] In the present invention, the medium composition for culturing the Clostridium botulinum strain may include a carbon source and a nitrogen source.

[0068] In the present invention, the carbon source may be a monosaccharide (e.g., glucose, fructose, etc.), a disaccharide (e.g., maltose, sucrose, etc.), an oligosaccharide; a polysaccharide (e.g., dextrin, cyclodextrin, starch, etc.), a sugar alcohol (e.g., xylitol, sorbitol, erythritol, etc.), and preferably D-(+)-glucose, but is not limited thereto.

[0069] In the present invention, the content of the carbon source may be 0.1 to 1.5 w / v% (1 to 15 g / L), preferably 0.25 to 1.5 w / v% (2.5 to 15 g / L), preferably 1.0 w / v% (10 g / L), but is not limited thereto.

[0070] In this specification, “nitrogen source” means an organic or inorganic substance containing nitrogen atoms essential for the synthesis of proteins, purines, pyrimidines, and polysaccharides such as chitin for the growth of a strain, and the nitrogen source is preferably a yeast extract, but is not limited thereto.

[0071] In the present invention, the nitrogen source may be yeast extract, polypeptone, tryptone, malt extract, corn steep liquor, soytone, and preferably yeast extract, but is not limited thereto.

[0072] In the present invention, the content of the nitrogen source may be 0.5 to 2.0 w / v% (5 to 20 g / L), preferably 0.5 to 1.0 w / v% (5 to 10 g / L), preferably 1.0 w / v% (10 g / L), but is not limited thereto.

[0073] From another aspect, the present invention relates to a method for producing botulinum toxin, comprising the steps of (a) culturing Clostridium botulinum using the medium composition to produce botulinum toxin; and (b) recovering the produced botulinum toxin.

[0074] In the present invention, the culturing may be characterized as being performed under anaerobic conditions, the culturing may be characterized as being performed under conditions of 33 to 37°C, the culturing may be characterized as being performed for 20 to 48 hours, and the toxin may be characterized as being selected from the group consisting of botulinum toxins A, B, C, D, E, F, and G.

[0075]

[0076] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0077]

[0078] Example 1: Selection of plant-based peptones

[0079] 1-1: Composition of CYG medium used for culture (control group)

[0080] CYG medium (Casein, Yeast extract, and Glucose) for culturing Clostridium botulinum was prepared by dissolving 2% casein hydrolysate [20 g / L], 1% yeast extract [10 g / L], and 1% glucose [10 g / L] in 90 mL of distilled water, adjusting the final volume to 100 mL, and dispensing 10 mL of the prepared medium into 15 mL culture tubes. The medium was then autoclaved at 121°C for 30 minutes and maintained under anaerobic conditions in an anaerobic incubator.

[0081] 1-2: Composition of APF medium used for culture

[0082] As shown in Tables 1 and 2 below, 9 candidate 2% plant peptones (Sigma) were added to 1% yeast extract [10 g / L], 1% dextrin or glucose [10 g / L] TM C8160-500G, Millipore TM 96174, Millipore TM 93492-500G-F, Sigma-Aldrich TM G5004-500G, Millipore TM 38143-500G, Organotechnie TM 19425, Sigma-AldrichTM B4032-500G, Sigma-Aldrich TM 17218-500G, Merck TM After adding 1.05391.0500)[10g / L], the final volume was adjusted to 100 mL, the pH of each medium was adjusted to 7.3, and the prepared medium was dispensed into 15 mL culture tubes in 10 mL portions. After that, the medium was autoclaved at 121°C for 30 minutes and maintained in an anaerobic state in an anaerobic incubator.

[0083]

[0084]

[0085] 1-3: Cultivation of Clostridium botulinum strains

[0086] Culture tubes containing media components 1 to 18 of Tables 1 and 2 were inoculated with 20 μL of Clostridium botulinum (Korea Centers for Disease Control and Prevention Management No.: 4-029-CBB-MF-2020001), and the cultures were incubated under anaerobic conditions at 35°C±1°C for 20, 24, 28, and 48 h, and then sampled. Afterwards, the OD of the culture fluid sampled at each hour was measured using a UV spectrophotometer. 540nm The growth rate was confirmed by measuring the value, and the blank was used as a culture medium. The measured OD 540nm Based on the values, the plant peptone candidates for the medium were selected by comparing them with the control group.

[0087] 1-4: Strain growth rate

[0088] Among the plant-based peptone candidates, corn steep liquor solids, banana powder, and wheat gluten produce sediment after sterilization of the medium, making it difficult to perform the filtration process in the future and obtaining accurate OD. 540nmBecause it was difficult to measure the values, the medium components including the plant peptone candidates were excluded from the plant peptone candidates, as shown in Table 3 below:

[0089]

[0090] OD of culture fluid sampled at each time for the media containing media components 1 to 12 of Table 3 and two control media (CYG media) 540nm The values ​​were measured (Table 4), and the OD of each medium's carbon source (Dextrin or Gluccose) at each time point 540nm The value graphs were compared (Fig. 4).

[0091]

[0092] As a result, as shown in Table 4 and Figure 4, it was confirmed that the strains commonly showed high growth rates in media 1, 2, 3, 7, 8, and 9 using pea, wheat, and potato-derived components.

[0093] In addition, even when using the same plant-based peptones derived from peas, wheat, and potatoes, it was confirmed that the strains in media 7, 8, and 9 using glucose as a carbon source showed higher growth rates than in media 1, 2, and 3 using dextrin. In particular, in the case of wheat-derived components, the strain showed a growth trend even after 48 hours of inoculation in media 2 using dextrin (Fig. 4), and since this may unnecessarily delay the time of the future raw liquid production process, it was confirmed that it would be appropriate to use glucose as a carbon source in the medium.

[0094] 1-5: Botulinum toxin concentration

[0095] The toxin protein amounts of media 1, 2, 3, 7, 8, and 9, which showed high growth rates of the strains in Examples 1-4, were measured. Since toxins were recovered during the death phase in previous studies, the results of quantitative testing of botulinum toxin type A culture fluid at 28 and 48 hours in the latter half of the culture, measured using an ELISA test method, are shown in Table 5 below (ICH guideline, Q2(R1)).

[0096] The antitoxin was diluted and dispensed into each well of a 96-well plate and coated for more than 16 hours. The contents of the plate were removed, and blocking solution was dispensed into each well for blocking, followed by washing the plate three times with wash buffer. The standard and culture medium were diluted and dispensed into each well in triplicate for reaction, and the plate was washed three times with wash buffer. The primary antibody dilution solution was dispensed into each well for reaction, and the plate was washed three times with wash buffer. The secondary antibody dilution solution was dispensed into each well for reaction, and the plate was washed three times with wash buffer. The substrate solution was dispensed into each well for color development, and the reaction was stopped by dispensing stop solution into each well. The absorbance was measured at a wavelength of 450–540 nm.

[0097]

[0098] As a result, as shown in Table 5, the toxin expression levels were highest in the order of peas, potatoes, and wheat. In addition, when the same plant-based peptone was used, the toxin expression levels in the medium using glucose as a carbon source were confirmed to be higher than in the medium using dextrin, and it was confirmed that using glucose as a carbon source in the medium for toxin production of Clostridium botulinum strains is more suitable.

[0099] Based on the experimental results of Example 1, three plant-based peptones were selected as final plant-based peptone candidates: Peptone from pea, Wheat peptone, and Peptone from potatoes No. 2.

[0100]

[0101] Example 2: Selection of the ratio of vegetable peptone

[0102] 2-1: Method for selecting the ratio of vegetable peptone

[0103] In order to select the ratio of the three final candidate plant-based peptones selected in Examples 1-4 and 1-5, the response OD was used using JMP's Custom Design. 540nm The value was set to 3 or more, and the ratio of the three plant-based peptones as factors was designed to be within 2%. In addition, similar to Examples 1-1 and 1-2, the carbon source and yeast extract were fixed at a ratio of 1%, and the experiment was conducted in duplicate per medium condition (Fig. 1; Peptone 1: pea peptone; Peptone 2: wheat peptone; Peptone 3: potato peptone).

[0104] For design evaluation, Power Analysis and Fraction of Design Space Plot were confirmed (Fig. 2; Peptone 1: pea peptone; Peptone 2: wheat peptone; Peptone 3: potato peptone)).

[0105] OD according to the vegetable peptone ratio obtained through Custom Design 540nm The experimental design of the values ​​is as shown in Table 6 below, and the toxin concentration was measured after completion of culture for each condition and added to the analysis items (Peptone 1: pea peptone; Peptone 2: wheat peptone; Peptone 3: potato peptone).

[0106]

[0107] 2-2: Composition of APF medium used for culture

[0108] As shown in Table 7 below, 1% glucose [10 g / L], 1% yeast extract [10 g / L] as a carbon source, and 3 kinds of 2% plant-based peptone candidates [10 g / L] were added, dissolved in 90 mL of water for injection, and the final volume was adjusted to 100 mL. The prepared medium was dispensed into 15 mL culture tubes in 10 mL portions. After that, the medium was autoclaved at 121°C for 30 minutes and maintained under anaerobic conditions in an anaerobic incubator.

[0109]

[0110] 2-3: Cultivation of Clostridium botulinum strains

[0111] 20 μL of Clostridium botulinum (Korea Centers for Disease Control and Prevention Management No.: 4-029-CBB-MF-2020001) was inoculated into culture tubes containing media from Table 7, and the cultures were incubated under anaerobic conditions at 35°C±1°C for 20, 24, 28, and 48 h, and then sampled. After that, the OD of the culture solution sampled at each hour was measured using a UV spectrophotometer. 540nm The growth rate was confirmed by measuring the value, and the blank was used as a culture medium. The measured OD 540nm Based on the values, the addition ratio of vegetable peptone to the medium was selected.

[0112] 2-4: Strain growth rate

[0113] The OD540nm value of the culture solution sampled at each time point for the media components 1 to 16 in Table 7 was measured (Table 8), and the OD at each time point for each media 540nm The value graphs were compared (Fig. 5).

[0114]

[0115] 2-5: Analysis of Design of Experiment (DoE) Results

[0116] For the media of the media components 1 to 16 in Table 7, the OD of 24 hours, which is the first seed culture time of the existing Clostridium botulinum study results 540nm The values ​​were used for analysis, and since the toxin was recovered during the death period, the amount of toxin protein was measured using ELISA values ​​at 48 hours (Table 9), and the results were statistically analyzed using JMP based on this.

[0117]

[0118] Through distribution analysis of the conditional results in [Table 9], it was confirmed that the experimental design was D-optimal, and the experimental results were confirmed to be generally evenly distributed (Fig. 6).

[0119] According to scatterplot matrix analysis, the correlation values ​​were all less than 0.75, indicating a weak correlation, and the R value between the X variables (peas, wheat, and potatoes) was a very weak correlation close to 0, so there was no interaction, and the Y variable (OD 540nm , Toxin concentration), peas and Y variables, wheat and OD 540nm It was confirmed that all values ​​showed a weak positive correlation (Fig. 7), and outlier analysis confirmed that there were no outliers in the experimental results (Fig. 8).

[0120] 2-6: OD according to plant-based peptone type 540nm Value Analysis

[0121] OD according to plant peptone type 540nmBased on the value analysis results, a fit model was established. The model was established using the effect leverage method using least squares, and the X variable was analyzed using the response surface method. To select the optimal model, the R square and RMSE values ​​were considered together, and insignificant terms were deleted after checking the effect summary and the p-value of the effect test.

[0122] As a result of checking the VIF value of the optimally established model, it was confirmed that there was no problem with the model as the value did not exceed 10 and was at the level of 1. In addition, in the residual analysis, it was confirmed that each point was evenly distributed above and below, and in the studentized residuals results, it was confirmed that all were included within the 95% confidence interval (Fig. 9).

[0123] As a result of checking the factors that have a great influence on the model through the effect test results, it was found that peas and wheat had an influence on the model (Table 10), and through this model, OD 540nm A prediction formula was obtained (Fig. 10), and the main effects and interaction profiles were examined, and it was confirmed that there was an interaction between peas and potatoes, and wheat and potatoes, but no interaction between the remaining factors (Fig. 11).

[0124]

[0125] 2-7: Analysis of toxin expression levels according to plant-based peptone types

[0126] A fit model was established based on the ELISA analysis results. The model was constructed using the effect leverage method using least squares, and the X variable was analyzed using the response surface method. To select the optimal model, the R square and RMSE values ​​were considered together, and insignificant terms were deleted after checking the effect summary and the p-value of the effect test.

[0127] As a result of checking the VIF value of the optimally established model, it was confirmed that there was no problem with the model as the value did not exceed 10 and was at the level of 1. In addition, in the residual analysis, it was confirmed that each point was evenly distributed above and below, and in the studentized residuals results, all were included within the 95% confidence interval (Fig. 12).

[0128] As a result of checking the factors that have a great influence on the model through the effect test results, it was found that peas had a significant influence on the model (Table 11), and through this model, OD 540nm A prediction formula was obtained (Fig. 13), and the main effects and interaction profiles were examined, and it was confirmed that there was an interaction between peas and potatoes, and wheat and potatoes, but no interaction between the remaining factors (Fig. 14).

[0129]

[0130] 2-8: Robust Optimization

[0131] Based on the prediction formula established for each response value, an appropriate OD 540nm In order to determine the medium conditions that can obtain the maximum toxin concentration, the Robust Optimization Conditions were determined using Profiler. In the actual culture process, OD540nm For the value, the cell state that can continuously grow after passage while maintaining the exponential phase is selected. Therefore, the minimum cell concentration for passage within the exponential phase is OD 540nm Based on the established model formula, OD is calculated based on the value 3 or more and 7 or less. 540nm The value was set to Match Target: 5 to explore appropriate conditions. For toxin concentration, the ELISA value was set to Maximize to identify the composition that produced the highest concentration of toxin. An analysis that maximized desirability for both response values ​​confirmed the use of only peas and wheat, and potatoes were excluded from subsequent analyses (Fig. 15).

[0132] After performing 5,000 simulations using the Simulator function to randomly vary factors and model noise (Fig. 16a), the simulation results were presented using edge-of-failure analysis. It was confirmed that all but one of the 5,000 responses fell within the spec limits (Fig. 16b).

[0133] To obtain results under appropriate conditions, the acceptable ranges of each component were determined, and the range corresponding to 3σ was set as NORs (the range representing 99.7% of the actual result distribution), and the range corresponding to 4.5σ was set as PARs (Figs. 17 and 18). The acceptable ranges for each component obtained based on this are as shown in Table 12.

[0134]

[0135] For the convenience of operators when applying to the actual process, the set points were set to 18 g / L for pea peptone and 7 g / L for wheat peptone. To confirm the range satisfying the model, NORs (3σ, red line) and PARs (4.5σ, blue line) were displayed on a contour plot, and as a result, they did not go beyond the white range satisfying the process conditions. This confirmed that the NORs and PARs set through this test and statistical analysis were appropriate (Fig. 19).

[0136]

[0137] Example 3: Comparative measurement of absorbance and toxin expression by APF medium

[0138] 3-1: Composition of the medium used for culture

[0139] To the APF medium for culturing Clostridium botulinum, pea peptone and wheat peptone were added in the amounts selected in Example 2-8, and D-(+)-glucose as a carbon source and yeast extract as a nitrogen source were added in the same amounts as in Example 1-2.

[0140] Meanwhile, to determine the effect of L-cysteine ​​hydrochloride monohydrate or antifoam emulsion on the APF medium, 0.2 g / L L-cysteine ​​hydrochloride monohydrate and / or 0.24 g / L medical antifoam c emulsion (Dow Corning®) were additionally added to the APF medium. In addition, to determine the effect of soy peptone on the APF medium, 18 g / L soy peptone was added instead of the pea peptone and / or wheat peptone of the APF, and 0.2 g / L L-cysteine ​​hydrochloride monohydrate and 0.24 g / L medical antifoam c emulsion (Dow Corning®) were additionally added.

[0141] The above media were prepared by dispensing 10 mL each into 15 mL culture tubes. The media were then autoclaved at 121°C for 30 minutes and maintained under anaerobic conditions in an anaerobic incubator. The APF media for culturing Clostridium botulinum and the specific composition of the media are shown in Table 13.

[0142]

[0143] 3-2: Culturing of Clostridium botulinum strains

[0144] A culture tube containing the medium in Table 13 was inoculated with 20 μL of Clostridium botulinum (Korea Centers for Disease Control and Prevention Management Number: 4-029-CBB-MF-2020001) and cultured under anaerobic conditions at 35°C±1°C for 20, 24, 28, and 48 h, and then sampled. Afterwards, the OD of the culture fluid sampled at each hour was measured using a UV spectrophotometer. 540nm The growth rate was confirmed by measuring the value, and the blank was used as a culture medium.

[0145] 3-3: Strain growth rate and toxin expression level

[0146] OD of culture medium sampled at each time in Table 13 540nm The values ​​and toxin expression levels were measured after 48 hours of culture (Table 14, Table 15, Figures 20 and 21).

[0147]

[0148]

[0149] As a result, as shown in Table 14, Figures 20 and 21, when Clostridium botulinum was cultured in the APF medium of the present invention, the growth of APF was the best in the 20 to 28 hour period, and the toxin expression level was also the highest in the 48 hour period after strain growth.

[0150] Meanwhile, in Comparative Example 1, in which only L-cysteine ​​HCl monohydrate was additionally added to the APF medium of the present invention, Comparative Example 2, in which only medical antifoam C emulsion was additionally added to the APF medium of the present invention, and Comparative Example 3, in which L-cysteine ​​HCl monohydrate and medical antifoam C emulsion were additionally added to the APF medium of the present invention, it was confirmed that the strain growth was lower and the toxin expression amount was reduced by 23.2%, 76.6%, and 14.9%, respectively, compared to the APF medium of the present invention. Therefore, it can be confirmed that the APF medium composition of the present invention is superior in strain growth and toxin expression amount even when L-cysteine ​​HCl monohydrate and / or medical antifoam C emulsion are additionally added to the APF medium composition of the present invention.

[0151] Meanwhile, in Comparative Example 4, in which soybean peptone was added instead of pea peptone and wheat peptone to the APF medium of the present invention, Comparative Example 5, in which soybean peptone was added instead of pea peptone to the APF medium of the present invention, Comparative Example 6, in which soybean peptone was added instead of pea peptone to the APF medium of the present invention and additionally L-cysteine ​​HCl monohydrate and medical antifoam C emulsion were added, and Comparative Example 7, which has the same composition as that of the prior art Korean Patent Publication No. 10-2022-0140696, it was confirmed that the strain growth was lower and the toxin expression level was reduced by 87.2%, 87.0%, 94.9%, and 55.2%, respectively, compared to the APF medium of the present invention. Therefore, considering that the time required until the death phase at which the toxin can be recovered is expected to be more than 72 hours, it can be confirmed that the APF medium composition of the present invention is superior in strain growth and toxin expression level.

Claims

1. A medium composition for culturing a Clostridium botulinum strain, comprising vegetable peptones including wheat peptone and pea peptone; D-(+)-glucose; and yeast extract.

2. A medium composition for culturing a Clostridium botulinum strain, characterized in that it does not contain animal-derived components in the first paragraph.

3. A medium composition for culturing a Clostridium botulinum strain, characterized in that the animal-derived component in the second paragraph is a casein hydrolyzate.

4. A medium composition for culturing a Clostridium botulinum strain, characterized in that the content of the wheat peptone in the first paragraph is 0.1 to 4.0 w / v%.

5. A medium composition for culturing a Clostridium botulinum strain, characterized in that the content of the pea peptone in the first paragraph is 0.1 to 3.5 w / v%.

6. A medium composition for culturing a Clostridium botulinum strain, characterized in that the contents of the D-(+)-glucose and the yeast extract in the first paragraph are each 0.005 to 2 w / v%.

7. A method for producing botulinum toxin comprising the following steps: (a) a step of culturing Clostridium botulinum using a medium composition of any one of claims 1 to 6 to produce botulinum toxin; and (b) A step of recovering the above-mentioned generated botulinum toxin.

8. A method for producing botulinum toxin, characterized in that in paragraph 7, the culturing is performed under anaerobic conditions.

9. A method for producing botulinum toxin, characterized in that in paragraph 7, the culturing is performed under conditions of 33 to 37°C.

10. A method for producing botulinum toxin, characterized in that in paragraph 7, the culturing is performed for 20 to 48 hours.

11. A method for producing botulinum toxin, characterized in that in paragraph 7, the toxin is selected from the group consisting of botulinum toxins A, B, C, D, E, F and G.