Culture medium composition for preparing botulinum toxin

Abstract

The present invention relates to a culture medium composition for producing botulinum toxin, and more particularly, to a culture medium composition for culturing Clostridium species capable of producing botulinum toxin. The culture medium composition of the present invention comprises at least one plant-derived proteose peptone selected from the group consisting of pea hydrolysate, cottonseed hydrolysate, and gluten hydrolysate. When Clostridium botulinum is cultured using the culture medium according to the present invention containing plant-derived proteose peptone and minerals, the growth rate of the bacteria in the culture medium is about 1.5-2 times higher than that of the bacteria in the currently used culture medium. In addition, when botulinum toxin is produced by culturing the bacteria in the culture medium, infection of transmissible spongiform encephalopathy (TSE) and the like is prevented by blocking the introduction of animal-derived components.

Description

TECHNICAL FIELD

[0001] The present invention relates to a culture medium composition for producing botulinum toxin, and more particularly, to a culture medium composition for culturing a Clostridium strain capable of producing botulinum toxin. The culture medium composition of the present invention comprises at least one plant-derived peptone selected from the group consisting of garden pea hydrolysate, cotton seed hydrolysate, and wheat gluten hydrolysate. BACKGROUND

[0003] Since the 1990's, various Clostridium sp. strains secreting neurotoxic toxins have been discovered, and characterization of toxins secreted from these bacteria has been conducted for the past 70 years (Schant, E.J. et al., Microbiol. Rev., 56:80, 1992).

[0004] Botulinum toxin derived from Clostridium sp. is classified into seven serotypes (serotypes A to G) according to its serological properties. Each toxin has a toxin protein having a molecular weight of about 150 kDa, and naturally contains a complex of several non-toxic proteins bound thereto. The medium complex (300 kDa) consists of a toxin protein and a non-toxic non-hemagglutinin protein, and the large complex (450 kDa) and the very large complex (900 kDa) consist of the medium-sized complex bound to hemagglutinin (Sugiyama, H., Microbiol. Rev., 44:419, 1980). Such non-toxic hemagglutinin proteins are known to function to protect the toxin from low pH in the intestine and various proteases.

[0005] The toxin is synthesized in a cell as a single polypeptide having a molecular weight of about 150 kDa, and then cleaved at a position 1 / 3 from the N-terminal end by the action of an intracellular protease or treatment with an artificial enzyme such as trypsin into two units: a light chain (L; molecular weight: 50 kDa) and a heavy chain (H; molecular weight: 100 kDa). The cleaved toxin has greatly increased toxicity compared to the single polypeptide. The two units are connected to each other by a disulfide bond and have different functions. The heavy chain binds to a receptor of a target cell (Park. M. K. et al., FEMS Microbiol. Lett., 72:243, 1990), and functions to interact with a biological membrane at low pH (pH 4) to form a channel (Mantecucco, C. et al., TIBS., 18:324, 1993), and the light chain has a pharmacological activity of interfering with neurotransmitter secretion when it is introduced into a cell or by electroporation, etc. (Poulain, B. et al., Proc. Natl. Acad. Sci. USA., 85:4090, 1988).

[0006] Toxins inhibit acetylcholine exocytosis at cholinergic presynaptic terminals of neuromuscular junctions to cause flaccidity. It has been considered that treatment with even a very small amount of toxin exhibits toxicity, which suggests that the toxin has any enzymatic activity (Simpson, L. L. et al., Ann. Rev. Pharmacol. Toxicol., 26: 427, 1986).

[0007] According to recent reports, toxins have metallopeptidase activity, and their substrates include synaptobrevin, syntaxin, 25 kDa synaptosomal-associated protein (SNAP25), etc., which are unit proteins of the exocytosis machinery complex. Each type of toxin uses one of the above three proteins as its substrate, and it is known that B, D, F, and G type toxins cleave synaptobrevin at specific sites, A and E type toxins cleave SNAP25 at specific sites, and C type cleaves syntaxin at specific sites (Binz, T. et al., J. Biol. Chem., 265: 9153, 1994).

[0008] In particular, it is known that A type botulinum toxin is soluble in a dilute aqueous solution at pH 4.0-6.8. It is known that the stable non-toxic protein is separated from the neurotoxin at a pH of about 7 or more, and thus the toxicity is gradually lost. In particular, it is known that the toxicity decreases as the pH and temperature increase.

[0009] Botulinum toxin is lethal to the human body even in a small amount, and is easily mass-produced. Thus, it constitutes one of the four biological terror weapons together with Bacillus anthracis, Yersinia pestis, and smallpox virus. However, it is found that when A type botulinum toxin is injected at a dose that does not systemically affect the human body, it can paralyze local muscles in the injection site. Based on this feature, A type botulinum toxin can be used for a wide range of applications, including a winkle removing agent, a medicament for treating spastic hemiplegia and cerebral palsy, etc. Thus, the demand for A type botulinum toxin has increased, and research on methods of producing botulinum toxin to meet the demand has been actively conducted.

[0010] The typical commercial product at present is BOTOX® (purified neurotoxin complex of A type botulinum toxin) available from Allergan, Inc., USA. (BOTOX® (purified neurotoxin complex of A type botulinum toxin) available from Allergan, Inc., USA. 100 units of BOTOX® is used for the treatment of 100 patients. The vial consists of about 5 ng of purified neurotoxin complex of botulinum toxin type A, 0.5 mg of human serum albumin, and 0.9 mg of sodium chloride, and is reconstituted using preservative-free sterile saline (0.9% sodium chloride injection). Other commercial products include BOTOX® (Botulinum Clostridium toxin type A toxin and hemagglutinin complex, which has lactose and human serum albumin in a pharmaceutical composition containing botulinum toxin, and is reconstituted using 0.9% sodium chloride before use), DYSPORT® (Botulinum Clostridium toxin type A toxin and hemagglutinin complex, which has lactose and human serum albumin in a pharmaceutical composition containing botulinum toxin, and is reconstituted using 0.9% sodium chloride before use), DYSPORT®

[0011] The culture medium for culturing Clostridium botulinum contains animal components, which is generally used in a method for producing botulinum toxin, as disclosed in Korean Patent No. 10-1339349. Therefore, if an animal abnormal prion, which is an agent causing transmissible spongiform encephalopathy, is included in the animal components due to contamination, it causes a problem in the process for producing botulinum toxin.

[0012] Transmissible spongiform encephalopathy (TSE) is called a neurodegenerative disease that causes severe degeneration of neurons, and examples thereof include bovine spongiform encephalopathy (BSE), Scrapie, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome, Kuru, transmissible mink encephalopathy, chronic wasting disease, feline spongiform encephalopathy, etc., which affect humans and animals. It is reported that BSE crosses the species barrier and even infects humans.

[0013] An agent causing transmissible spongiform encephalopathy (TSE) has a characteristic that it is not immunogenic and has a long incubation period. From the histopathological analysis of the brain tissue of a cow affected by BSE, it can be seen that special spongiform vacuoles are formed in the brain due to damage to neurons and deposition of abnormal protein fibers.

[0014] The cause of TSE is a proteinaceous infectious particle called abnormal prion. Unlike general viruses that require nucleic acid, abnormal prion is an infectious particle composed of a single protein without nucleic acid. In relation to TSE, it is known that when abnormal prion (PrPsc) as an infectious particle binds to normal prion (PrPc), it is converted into pathogenic prion, and then it is accumulated in the brain (Prusiner SB, Alzheimer Dis Assoc Disord., 3:52-78, 1989).

[0015] Creutzfeldt-Jakob disease is a neurodegenerative disease of a rare human transmissible spongiform encephalopathy (TSE) in which the infectious agent is apparently an abnormal isoform of the prion protein. Within six months, an individual with Creutzfeldt-Jakob disease can deteriorate from apparently full health to akinetic mutism. Thus, there can be a potential risk of prion-mediated disease (e.g., Creutzfeldt-Jakob disease) from the administration of a pharmaceutical composition containing a biological agent, such as botulinum toxin, obtained using animal-derived products. Therefore, if the pharmaceutical composition is prepared from a drug substance produced using animal-derived ingredients, it can expose patients to the potential risk of receiving various pathogens or infectious agents.

[0016] In this technical background, the present inventors have found that, when a medium containing a plant-derived peptone free of transmissible spongiform encephalopathy (TSE) and a mineral component is used for the culture of Clostridium botulinum to prevent the risk of the occurrence of the above-described prion-mediated disease, the risk of the occurrence of a prion-mediated disease that can exist in a currently used medium (initial medium) can be excluded, and the growth rate of Clostridium botulinum in the medium can be increased compared to the growth rate of Clostridium botulinum in the currently used medium, thereby accomplishing the present invention. SUMMARY

[0018] TECHNICAL PROBLEM

[0019] An object of the present invention is to provide a medium composition containing a plant-derived peptone free of the risk of infection of transmissible spongiform encephalopathy (TSE), and a method of producing botulinum toxin by culturing Clostridium botulinum in the medium composition to improve the production of botulinum toxin.

[0020] TECHNICAL SOLUTION

[0021] To achieve the above object, the present invention provides a medium composition for culturing Clostridium botulinum, the medium composition comprising: at least one plant-derived peptone selected from the group consisting of pea hydrolysate, cottonseed hydrolysate, and wheat gluten hydrolysate.

[0022] The present application also provides a method for producing botulinum toxin, comprising the steps of: (a) culturing Clostridium botulinum using the above-mentioned culture medium composition to produce botulinum toxin; and (b) recovering the produced botulinum toxin. SUMMARY

[0024] Figure 1 Growth of Clostridium botulinum in a medium containing plant-derived peptone (APF medium) is shown.

[0025] Figure 2 Growth of Clostridium botulinum in a medium containing plant-derived peptone, minerals, amino acids, and vitamins is shown.

[0026] Figure 3 Results of checking whether a precipitate is formed after sterilization of a medium containing plant-derived peptone, minerals, amino acids, and vitamins are shown.

[0027] Figure 4 Results of checking whether a precipitate is formed after sterilization of a medium containing plant-derived peptone and minerals are shown.

[0028] Figure 5 Growth of Clostridium botulinum in a medium obtained by additionally adding vitamins, amino acids, and "BD Recharge TM " without glucose and L-glutamine to a medium for culturing bacteria containing plant-derived peptone and minerals is shown.

[0029] Figure 6 Growth of Clostridium botulinum in a medium for culturing bacteria containing various kinds of plant-derived peptone is shown.

[0030] FIG. 7 shows FFD contour plots and response optimization for mineral screening. Figure 7a Contour plot for high setting; Figure 7b Contour plot for middle setting; Figure 7c Contour plot for low setting; and Figure 7d Response optimization for maximum OD.

[0031] FIG. 8 shows FFD contour plots and response optimization for mineral screening. Figure 8a Contour plot for high setting; Figure 8b Contour plot for middle setting; Figure 8c Contour plot for low setting; and Figure 8d Response optimization for maximum OD.

[0032] FIG. 9 shows contour plots and response optimization for plant peptone screening. Figure 9a Contour plot for middle setting; Figure 9b Contour plot for low setting; andFigure 9c Response optimization of maximum OD.

[0033] Figure 10 The growth curve of C. botulinum in the finally selected APF medium, and the change in toxin concentration, are shown.

[0034] BEST MODE FOR CARRYING OUT THE INVENTION

[0035] In the present invention, a medium further increasing the growth rate of C. botulinum compared to the currently used medium (initial medium), and having no risk of infection with TSE or the like, was attempted to be prepared. Therefore, an animal protein-free (APF) medium containing a plant-derived proteose peptone was used, and the growth of bacteria in the APF medium was examined. As a result, the APF medium showed an increased bacterial growth rate compared to the currently used medium. Therefore, if the APF medium is used, a high concentration of botulinum toxin can be produced by culturing bacteria in a safe manner under TSE-free conditions.

[0036] As used herein, the term "currently used medium or initial medium" means a medium comprising casein hydrolysate, yeast extract, and thioglycollate medium, which are medium ingredients of animal origin. The term "APF medium (animal protein-free medium)" means a medium not containing animal-derived proteins, and containing a plant-derived proteose peptone, minerals, and glucose.

[0037] In the examples of the present invention, in order to produce botulinum toxin by culturing C. botulinum under TSE-free conditions, an APF medium containing a TSE-free plant-derived proteose peptone was prepared, and compared with the currently used medium (containing animal ingredients). As a result, it could be seen that the optimal medium composition for culturing C. botulinum was a medium comprising a plant-derived proteose peptone, at least one mineral selected from KH2PO4, K2HPO4, and Na2HPO4, and a carbon source (e.g., glucose), and the optimal growth of bacteria in this medium was found. As a result, as shown in Table 13, the optimal content of plant-derived proteose peptone in the finally selected medium composition for culturing C. botulinum was determined to be 5 g / L Hy-Pea TM 7404, 10 g / L UltraPep TM Cotton and 5 g / L HyPep TM 4601N, and the optimal content of minerals in the medium composition was 5.5 g / L K2HPO4and 3 g / L Na2HPO4.

[0038] In another example of the present application, the growth pattern and toxin concentration of Clostridium botulinum in the final selected APF medium comprising plant-derived peptone and minerals were measured. The results, as shown in Table 12 and Figure 10 540nm 600nm 540nm 600nm The OD values then gradually decreased and at 48 hours of incubation, the medium showed an OD

[0039] Based on this, in one aspect, the present application relates to a medium composition for culturing Clostridium botulinum, said medium composition comprising: at least one plant-derived peptone selected from the group consisting of pea hydrolysate, cottonseed hydrolysate, and wheat gluten hydrolysate.

[0040] As used herein, the term "plant-derived peptone" refers to a peptone extracted from pea, cottonseed, or wheat gluten. Preferably, the plant-derived peptone can be commercially available Hy-Pea TM 7404, UltraPep TM Cotton, HyPep TM 7504, or HyPep TM 4601N, but not limited thereto.

[0041] As used herein, the term "plant-derived peptone" or "plant-derived hydrolysate" means a product obtained by degrading a protein isolated from a plant. For example, pea peptone (pea hydrolysate) means a product obtained by degrading total protein isolated from pea.

[0042] The degradation of the plant-derived protein is preferably performed by partial digestion. The degradation of the protein is preferably performed by acid treatment, alkali treatment, enzyme treatment, high pressure treatment, heat treatment, or physical treatment. More preferably, the plant-derived peptone can be obtained by enzyme treatment. The physical treatment is, for example, grinding.

[0043] The plant-derived peptone used in the present application is a partially degraded product of a plant-derived protein, which is a mixture containing not only amino acids as single molecules, but also peptides consisting of several to several tens of amino acids, and intact protein molecules. ​​​​

[0044] In the present application, the content of the plant-derived peptone in the medium composition can be 0.1-10 w / v% (1-100 g / L), preferably 0.2-5 w / v% (2-50 g / L), and more preferably 0.5-2 w / v% (5-20 g / L).

[0045] In the present application, the medium composition comprises all of the pea hydrolysate, cottonseed hydrolysate, and wheat gluten hydrolysate, and the content ratio of the pea hydrolysate, cottonseed hydrolysate, and wheat gluten hydrolysate in the medium composition can be 1:0.24-43.62:0.01-50.57 by weight, preferably 1:0.68-14.46:0.09-9.87 by weight, and more preferably 1:1.6-2.4:0.6-1.4 by weight.

[0046] In the present application, the medium composition for culturing Clostridium botulinum can further contain a carbon source and at least one mineral selected from the group consisting of K2HPO4 (dipotassium hydrogen phosphate), Na2HPO4 (sodium dihydrogen phosphate), and KH2PO4 (potassium dihydrogen phosphate).

[0047] Herein, examples of the carbon source include, but are not limited to, monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, starch, etc.), sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.).

[0048] In the present application, the content of the mineral in the medium composition can be 0.05-3.5 w / v% (0.5-35 g / L), preferably 0.1-1.75 w / v% (1-17.5 g / L), and more preferably 0.25-0.7 w / v% (2.5-7 g / L).

[0049] In another aspect, the present application relates to a method for producing botulinum toxin, comprising the steps of: (a) culturing Clostridium botulinum using the above-mentioned medium composition to produce botulinum toxin; and (b) recovering the produced botulinum toxin.

[0050] In the present application, the culturing can be performed under anaerobic conditions, and the botulinum toxin can be selected from the group consisting of botulinum toxin types A, B, C, D, E, F, and G. EXAMPLE

[0051] Hereinafter, the present application will be described in more detail with reference to Examples. It will be obvious to those skilled in the art that these Examples are for illustrative purposes only, and should not be construed to limit the scope of the present application. The true scope of the present application is defined by the appended claims and their equivalents.

[0052] Example 1 : Culturing Clostridium botulinum in a plant-derived peptone medium

[0053] 1-1 : Composition of the medium currently used for culturing

[0054] Reagents and media components used in the present application were purchased from Sigma (USA), Kerry Inc. (USA), BD Biosciences (USA), Gibco Life Technologies (USA) and Quest (USA).

[0055] Seed culture and main culture of Clostridium botulinum for production of botulinum toxin was carried out using the currently used medium having a composition comprising 2% casein hydrolysate (20 g / L), 1% yeast extract (10 g / L), 1% glucose (10 g / L) and 0.5% thioglycollate medium (5 g / L). The 5 g thioglycollate medium per liter of the currently used medium was composed of 2.52 g enzymatic digest of casein, 0.84 g yeast extract, 0.925 g dextrose, 0.085 g sodium thioglycollate, 0.42 g NaCl, 0.085 g L-cysteine, 0.00014 g Resazurin and 0.125 g bacteriological agar.

[0056] 1-2: Composition of the APF medium used in the culturing

[0057] The negative control medium was prepared by removing casein hydrolysate, yeast extract and thioglycollate medium from the medium currently used for culturing Clostridium botulinum (initial medium) and the animal protein free (APF) medium was prepared by adding four plant derived peptone candidates (Hy-Pea TM 7404, UltraPep TM Cotton, HyPep TM 7504, and HyPep TM 4601N) to the negative control medium (Table 1).

[0058] Table 1 shows the composition of the APF medium containing plant derived peptone for culturing Clostridium botulinum as compared to the currently used medium.

[0059] Table 1

[0060]

[0061] 1-3: Seed culturing of Clostridium botulinum

[0062] CBB-IS-001) was inoculated into a culture tube containing 10 ml of sterile medium having each composition described in Examples 1-1 and 1-2, and primary seed culture (stationary culture) was performed under anaerobic conditions at 35°C for 22-30 hours. When the bacterial growth in the primary seed culture was confirmed, 8 ml of the primary seed culture was inoculated into a 1 liter culture flask containing 800 ml of sterile medium having the same medium composition, and secondary seed culture (stationary culture) was performed under anaerobic conditions at 35°C for 8-15 hours.

[0063] 1-4: Main culturing of Clostridium botulinum

[0064] In order to produce botulinum toxin by culturing Clostridium botulinum, main culture of bacteria was performed. Specifically, 9.3 L of medium having each composition described in Examples 1-1 and 1-2 was prepared and placed in a 10 liter culture chamber, and then the medium was sterilized. Nitrogen was supplied to create anaerobic conditions, and growth of bacteria was performed at a temperature of 35°C and a stirring speed of 50 rpm.

[0065] The secondary seed culture in the 1 liter culture flask in Example 1-3 was inoculated into a 10 liter culture chamber through an inoculation line connected to an inoculation port of the 10 liter culture chamber. Clostridium botulinum in the 10 liter culture chamber was cultured at 35°C and 50 rpm, and the set culture conditions were monitored and recorded. When the bacteria were cultured for 100 hours or more, the main culture was terminated.

[0066] Growth of Clostridium botulinum in the animal protein-free (APF) medium prepared by adding four plant-derived peptone candidates (Hy-Pea TM 7404, UltraPep TM Cotton, HyPep TM 7504 and HyPep TM 4601N) to the animal protein-free (APF) medium prepared in the negative control was compared with the bacterial growth in the negative control medium prepared by removing casein hydrolysate, yeast extract, and thioethanolate medium from the currently used medium (initial medium) (Table 1).

[0067] Therefore, as shown in Table 1 and Figure 1 Clostridium botulinum did not grow in the negative control medium, but started to grow in the initial medium (currently used medium) 24 hours after inoculation of bacteria, and started to grow in the medium containing plant-derived peptone 30 hours after inoculation of bacteria.

[0068] Example 2: Culturing Clostridium botulinum in a medium containing plant-derived peptone, minerals, amino acids and vitamins Table 2

[0069] Since the growth of Clostridium botulinum in the medium prepared by adding four kinds of plant-derived peptones in Example 1 was slower than that of Clostridium botulinum in the initial medium, a solution thereof is provided as follows.

[0070] 1) To examine the effect of thioethanolate, which functions to create anaerobic conditions, the thioethanolate was removed from the initial medium (the currently used medium), and the change in the growth rate of bacteria in the thioethanolate-free medium was analyzed.

[0071] 2) Since the slower growth rate can be due to nitrogen source deficiency, the concentration of peptone in the medium for bacterial culture was increased by 2 times.

[0072] 3) The growth of Clostridium botulinum in the medium obtained by adding minerals, amino acids, and vitamins to the medium containing plant-derived peptone was compared with that of Clostridium botulinum in the APF medium (Table 2) disclosed in U.S. Patent No. 8,012,716 (Allergan).

[0073] Table 2 shows the composition of the medium for culturing Clostridium botulinum, which contains plant-derived peptone, minerals, amino acids, and vitamins.

[0074] Figure 2

[0075]

[0076] As a result, as shown in Tables 2 and Table 3 , the growth rate of bacteria in the medium was slower than that in the thioethanolate-containing medium when the bacteria were cultured in the currently used medium not containing thioethanolate, indicating that thioethanolate affects the growth rate of bacteria. When the concentration of peptone in the medium was increased by 2 times, the bacteria did not grow in the medium. When the mineral component, amino acids, and vitamins were added to the medium containing peptone, the growth rate of bacteria was similar to that in the currently used medium, but a precipitate was formed after sterilization of the medium. In addition, it was seen that the growth rate of bacteria in the APF medium of Allergan was similar to that in the currently used medium.

[0077] Example 3: Precipitate formation by sterilization of a medium containing plant-derived peptone, minerals, amino acids, and vitamins

[0078] In Example 2, it was observed that the growth rate of C. botulinum in the medium containing plant-derived peptone, minerals, amino acids, and vitamins in the medium candidates 2 to 4 shown in Table 2 was similar to that in the currently used medium. However, the formation of precipitates occurred after sterilization of the medium, and thus the reason therefor was examined (Table 3).

[0079] Table 3 shows the components of the medium for culturing C. botulinum using and containing plant-derived peptone, minerals, amino acids, and vitamins in sterilization.

[0080] Figure 3

[0081]

[0082] As shown in Table 3 and Example 4: Formation of a precipitate by sterilizing a medium containing plant-derived peptone and minerals , precipitates were formed only in the case where the medium containing plant-derived peptone was added with minerals after sterilization of the medium, indicating that the main cause of the formation of precipitates was minerals. This is considered to be because the mineral components interact with each other under high temperature and high pressure conditions during sterilization of the medium.

[0083] Table 4

[0084] In order to identify the mineral components involved in the formation of precipitates caused by sterilization as confirmed in Example 3, various combinations of different components were added to the medium, and then sterilized (Table 4).

[0085] Table 4 shows the components of the medium for culturing C. botulinum containing plant-derived peptone and minerals, and the results of sterilization of the medium.

[0086] Figure 4

[0087]

[0088] As shown in Table 4 and Example 5: Culturing Clostridium botulinum under conditions in which no precipitate is formed in the APF medium , among the medium containing plant-derived peptone and minerals, the medium containing MgSO4·7H2O and K2HPO4 and the medium containing MgSO4·7H2O and Na2HPO4 formed precipitates after sterilization.

[0089] Table 5

[0090] When vitamins and amino acids were additionally added to the APF medium of Example 4 containing plant-derived peptone and minerals, experiments were performed to determine whether cultivation of Clostridium botulinum was possible. In addition, experiments were performed to examine whether cultivation of the bacteria was possible in a medium not containing plant-derived peptone and minerals and containing vitamins, amino acids and / or "BD Recharge TM " (product catalog number 670002, BD Bioscience) (yeast extract-based medium component not containing glucose and L-glutamine) (Table 5).

[0091] Table 5 shows the medium components obtained by additionally adding vitamins, amino acids and "BD Recharge TM " not containing glucose and L-glutamine to the medium for cultivating Clostridium botulinum containing plant-derived peptone and minerals, and the growth rate of the bacteria in the medium.

[0092] Figure 5

[0093]

[0094] As a result, as shown in Table 5 and Table 6 , Clostridium botulinum grew within 24 hours after bacterial inoculation only in the case of the medium containing plant-derived peptone and a combination of two or more minerals of KH2PO4, K2HPO4 and Na2HPO4, and further containing vitamins and amino acids. In addition, the bacteria grew within 48 hours after bacterial inoculation in the case of the medium not containing plant-derived peptone and minerals and containing vitamins, amino acids and "BD Recharge TM ". In summary, the most suitable medium composition for cultivating Clostridium botulinum includes plant-derived peptone, KH2PO4, K2HPO4, Na2HPO4, amino acids and vitamins.

[0095] Example 6: Cultivation of Clostridium botulinum in a medium containing different plant-derived proteins

[0096] Experiments were performed to examine whether cultivation of Clostridium botulinum was possible when different combinations of plant-derived peptone were added to the APF medium of Example 5.

[0097] Table 6 shows the components of the medium for cultivating Clostridium botulinum containing different plant-derived peptones, and the results of examining whether the bacteria grew in the medium.

[0098] Figure 6

[0099]

[0100] As shown in Tables 6 and Example 7: Experiment for selecting two of the three types of minerals contained in the medium , even when only one or two of the four plant-derived peptones are added to the culture medium, it is possible to culture Clostridium botulinum.

[0101] In view of the results of Examples 5 and 6, it can be seen that at least one plant-derived peptone should be contained in the culture medium, and the plant-derived peptone cannot be replaced with "BD Recharge TM " (product catalog number 670002, BD Bioscience) (a yeast extract-based culture medium component not containing glucose and L-glutamine).

[0102] Table 7

[0103] In Examples 1 to 7, the APF culture medium composition for Clostridium botulinum culture was determined to contain glucose, sodium chloride (NaCl), four plant-derived peptones, three minerals, amino acids, and vitamins. Among these culture medium components, the culture medium components that did not have a significant effect on bacterial growth were removed to reduce the number of culture medium components. Thus, it was judged that the amino acids and vitamins did not have a significant effect on the growth of Clostridium botulinum, and on the basis of this judgment, the amino acids and vitamins were removed from the culture medium components. In addition, in order to select two from among the three types of minerals, bacteria were cultured using the culture medium compositions shown in Table 7, and the OD (540 nm and 600 nm) values at 24 hours and 48 hours after inoculation of the bacteria were measured and compared.

[0104] Table 7 shows the composition of the culture medium resulting from the first stage selection of minerals and the growth of Clostridium botulinum in the culture medium.

[0105] Figures 7a to 7d

[0106]

[0107] As a result, as shown in Table 7, 24 hours after inoculation of the bacteria, the currently used culture medium showed an OD (540 nm) value of 0.942, and the APF culture medium containing K2HPO4 and Na2HPO4 showed the highest OD (540 nm) value of 4.964 in the APF culture medium. In addition, 48 hours after inoculation of the bacteria, the APF culture medium containing KH2PO4 and Na2HPO4 showed the highest OD value and active bacterial growth.

[0108] Meanwhile, as Table 8As shown in FIG. 4, contour plots showing high main effects of K2HPO4 and Na2HPO4 were drawn. Accordingly, as the concentrations of K2HPO4 and Na2HPO4 increase, the OD value increases. And, when minerals are added to the medium at the concentrations of KH2PO4 = 0 g / L, K2HPO4 = 5.5 g / L, and Na2HPO4 = 5 g / L, Clostridium botulinum shows the highest growth.

[0109] Meanwhile, in order to confirm the bacterial culture results according to more precise mineral addition, a second stage experiment was performed using a response surface method. Since the medium composition cannot have a negative value, the experiment was planned using a CCF (Central Composite Face) design, and was performed by culturing bacteria in the medium composition shown in Table 8. Then, the experimental results were combined with the results of the FFD performed previously and statistical analysis was performed.

[0110] Table 8 shows the composition of the medium obtained through the second stage selection of minerals and the growth of Clostridium botulinum in the medium.

[0111] Figures 8a to 8d

[0112]

[0113] Contour plots were drawn and used for comparison. As shown in FIG. 5, as the KH2PO4 concentration decreases, the OD value increases. When the optimal conditions are compared, the results are different from those of the FFD due to the curvature effect, and the value of K2HPO4 is the same, but the value of Na2HPO4 changes from 5 g / L to 3.1313 g / L. Accordingly, it was confirmed through statistical analysis that the optimal mineral conditions of the medium are 5.5 g / L K2HPO4 and 3 g / L Na2HPO4. Example 8: Experiment for selecting plant-derived peptone contained in the medium

[0114] Table 9 As shown in Tables 9 and 10, plant-derived peptone was combined according to a mixture design, and the growth of Clostridium botulinum in a medium containing the combined plant-derived peptone was examined.

[0115] Table 9 shows the composition of the medium obtained through the first stage selection of plant-derived peptone and the growth of Clostridium botulinum using the medium.

[0116]

[0117] Table 10

[0118] Table 10 shows the composition of the medium obtained through the second stage selection of plant-derived peptone and the growth of Clostridium botulinum using the medium.

[0119]

[0120] ​Figures 9a to 9c

[0121]

[0122] Results, as Table 11 shown in Table 2, were plotted as contour maps and used for comparison. HyPep TM 7504 had the lowest impact on the growth of C. botulinum. Based on this assay, HyPep TM 7504 was excluded from the medium components. In summary, the composition of the final selected plant-derived peptone to be contained in the medium was determined to comprise 5 g / L Hy-Pea TM 7404, 10 g / L UltraPep TM cotton, and 5 g / L HyPep TM 4601N.

[0123] Example 9: Cultivation of C. botulinum in medium containing or not containing NaCl

[0124] The medium composition used in Examples 1 to 8 contained a small amount (0.5 g / L) of NaCl. In order to examine the growth of C. botulinum according to the concentration of NaCl, the content of NaCl in the medium was adjusted to a range of 0 to 1 g / L, and then the bacteria were cultivated in the medium.

[0125] Table 11 shows the composition of the medium containing NaCl for cultivating C. botulinum and the growth of C. botulinum in the medium.

[0126] Example 10: Measurement of the growth pattern and toxin concentration of Clostridium botulinum in the finally selected APF medium

[0127]

[0128] Therefore, as shown in FIG. 11, there was no difference in the growth of the bacteria regardless of whether the medium contained NaCl or not. Thus, NaCl was excluded from the final APF medium composition.

[0129] Table 12

[0130] C. botulinum was inoculated into the final selected medium for C. botulinum (10 g / L glucose, 5 g / L Hy-Pea TM 7404, 10 g / L UltraPep TM cotton, 5 g / L HyPep TM 4601N, 5.5 g / L K2HPO4, and 3 g / L Na2HPO4) determined based on the results of Examples 1 to 9, and then the growth pattern of the bacteria and the toxin concentration were measured.

[0131] Table 12 shows the time-dependent OD values ​​and toxin concentrations of Clostridium botulinum grown in the final selected APF medium.

[0132] Figure 10

[0133]

[0134] Therefore, as shown in Table 12 and Table 13 As shown, the OD value began to increase after culturing Clostridium botulinum for 12 hours, and the culture medium showed an OD value of 3.5465 after 24 hours of culture. 540nm and OD of 3.0695 600nm Then, the OD value gradually decreased, and at 48 hours of culture, the culture medium showed an OD of 0.792. 540nm and 0.7224 OD 600nm The toxin concentration in the Clostridium botulinum supernatant began to increase after 24 hours of incubation, reaching a final value of 31.41 μg / ml. When the toxin concentration was measured after bacterial rupture, toxin production began after 5 hours of incubation, and the toxin concentration continued to increase, remaining at a consistent level after 28 hours of incubation, reaching a final value of 38.39 μg / ml.

[0135] In summary, Table 13 summarizes the final selected APF (animal protein-free culture medium) composition determined based on the results of Examples 1 to 10.

[0136] ​

[0137]

[0138] Industrial applicability

[0139] As described above, when the culture medium according to the invention, containing plant-derived peptone and minerals, is used for culturing Clostridium botulinum, the growth rate of bacteria in the culture medium is approximately 1.5 to 2 times higher than that of bacteria in currently used culture media. Furthermore, when producing botulinum toxin by culturing bacteria in the culture medium, infections such as infectious spongiform encephalopathy (TSE) can be prevented by blocking the introduction of animal-derived components.

[0140] Although the invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this specification is directed only to preferred embodiments and does not limit the scope of the invention. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

Claims

1. A culture medium composition for culturing Clostridium botulinum, said culture medium composition comprising: 10 g / L glucose; Plant-derived peptone, wherein the plant-derived peptone comprises 5 g / L of pea hydrolysate, 10 g / L of cottonseed hydrolysate and 5 g / L of wheat gluten hydrolysate; 5.5 g / L of K2HPO4; and 3g / L Na2HPO4.

2. The culture medium composition according to claim 1, wherein the plant-derived peptone is subjected to enzyme treatment.

3. A method for producing botulinum toxin, comprising the following steps: (a) Culturing Clostridium botulinum using the culture medium composition of claim 1 or 2 to produce botulinum toxin; and (b) Recovering the produced botulinum toxin.

4. The method of claim 3, wherein the culture is carried out under anaerobic conditions.

5. The method according to claim 3, wherein the botulinum toxin is selected from the group consisting of botulinum toxin serotypes A, B, C, D, E, F, and G.

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